https://doi.org/10.1093/plcell/koac263 THE PLANT CELL 2023: 35: 67–108 Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress Paul E. Verslues ,1,* Julia Bailey-Serres ,2 Craig Brodersen ,3 Thomas N. Buckley ,4 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Lucio Conti ,5 Alexander Christmann ,6 José R. Dinneny ,7 Erwin Grill ,6 Scott Hayes ,8 Robert W. Heckman ,9 Po-Kai Hsu ,10 Thomas E. Juenger ,9 Paloma Mas ,11,12 Teun Munnik ,13 Hilde Nelissen ,14,15 Lawren Sack ,16 Julian I. Schroeder ,10 Christa Testerink ,8 Stephen D. Tyerman ,17 Taishi Umezawa 18 and Philip A. Wigge 19,20 Review 1 Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan 2 Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California 92521, USA 3 School of the Environment, Yale University, New Haven, Connecticut 06511, USA 4 Department of Plant Sciences, University of California, Davis, California 95616, USA 5 Department of Biosciences, University of Milan, Milan 20133, Italy 6 School of Life Sciences, Technical University Munich, Freising-Weihenstephan 85354, Germany 7 Department of Biology, Stanford University, Stanford, California 94305, USA 8 Laboratory of Plant Physiology, Plant Sciences Group, Wageningen University and Research, Wageningen 6708 PB, The Netherlands 9 Department of Integrative Biology, University of Texas at Austin, Austin, Texas 78712, USA 10 Department of Cell and Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA 11 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Barcelona 08193, Spain 12 Consejo Superior de Investigaciones Cientıficas (CSIC), Barcelona 08028, Spain 13 Department of Plant Cell Biology, Green Life Sciences Cluster, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam NL-1098XH, The Netherlands 14 Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent 9052, Belgium 15 VIB Center for Plant Systems Biology, Ghent 9052, Belgium 16 Department of Ecology and Evolutionary Biology, Institute of the Environment and Sustainability, University of California, Los Angeles, California 90095, USA 17 ARC Center Excellence, Plant Energy Biology, School of Agriculture Food and Wine, University of Adelaide, Adelaide, South Australia 5064, Australia 18 Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 6708 PB, Japan 19 Leibniz-Institut für Gemüse- und Zierpflanzenbau, Großbeeren 14979, Germany 20 Institute of Biochemistry and Biology, University of Potsdam, Potsdam 14476, Germany *Author for correspondence: paulv@gate.sinica.edu.tw Authors are listed alphabetically (with exception of the coordinating editor), article sections are arranged alphabetically based on first author of each section. All authors contributed to writing the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) is: Paul E. Verslues (paulv@gate.sinica.edu.tw). Abstract We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this Received May 19, 2022. Accepted August 21, 2022. Advance access publication August 26, 2022 C The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists. V Open Access This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 68 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus de- fense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechano- sensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adapta- tion involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpre- dictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better de- fining what constitutes beneficial stress resistance in different contexts and making connections between genes and pheno- types, and between laboratory and field observations, are overarching challenges. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Introduction to keep pace with, and make best use of, the ever-increasing amount of genomic data. How can this growing body of ge- (By Paul E. Verslues, editor) nomics data help us to understand gene and protein func- “Before now, you just needed to know the answers to the tion and, ultimately, deploy that knowledge for plant questions you were given; now you need to know questions improvement or understanding natural systems? Despite which you have not been given and for which there is no advances such as automated plant phenotyping and image answer.” This is the advice that many mentors have given, analysis systems, this “phenotype gap” (Miflin, 2000) contin- in one form or another, to those new to research. Here, we ues to grow larger as -omics data accumulate. In the process put ourselves to this test and present what several groups of closing the phenotype gap, one needs to decide what of scientists working in plant abiotic stress biology consider phenotype(s) to measure and how to interpret the results. to be big questions for future research. This is a timely topic Readers of this article may also get a sense that there are as climate change will bring about not just a general warm- several alternative meanings of “stress resistance” (this is per- ing but also instability and extreme weather events of many haps most pronounced for drought where the term types. Thus, climate change can increase the frequency and “drought tolerance” is often broadly used for both avoidance severity of single, combined, and even sequential abiotic of water depletion and true tolerance of low water stresses including drought, salinity, flooding, and even freez- ing. Rising carbon dioxide (CO2) levels can also directly influ- potentials). Does increased resistance (often referred to as ence how plants respond to these stresses. A common increased tolerance, regardless of whether avoidance mecha- theme of plant stress research is that we are trying to un- nisms are involved) mean the ability to better survive a derstand how plants respond to excesses: too hot, too cold, near-lethal stress or the ability to remain more productive too little water, too much water, too much light, too little during a moderate severity stress? Those approaching plant light, or too much salt. These excesses are gradually (or not abiotic stress from an agronomic versus ecophysiology per- so gradually in some cases) becoming more of the norm for spective can have differing views of this question. Several plants in many parts of the world. In this article, we focus types of data indicate that the mechanisms plants use to on questions of fundamental plant biology and how stress survive severe stress only partially overlap with mechanisms affects physiological and molecular processes and adapta- enabling greater productivity at more moderate stress severi- tion, rather than climate change mitigation strategies dis- ties. Thus, there is a need to clearly state and define which cussed by our colleagues. Ultimately, we want to use our view of stress resistance/stress tolerance one is applying physiological and molecular knowledge to both predict the when interpreting data. In addition, the challenges of con- effect of climate change on plants and intervene to improve necting phenotypes observed in the laboratory to real differ- those outcomes, particularly in terms of ecosystem resiliency ences in a field environment (and vice versa the challenge of or crop yield. Thus, perhaps one overriding challenge is the achieving a mechanistic understanding of quantitative traits question of scale and how to move our knowledge from related to yield and stress resistance) demand a certain level one scale to another. How does the opening and closing of of circumspection from those working at all levels of plant a membrane channel that occurs at a time scale of seconds stress biology. (or less) influence growth responses that occur over days? For cellular studies of abiotic stresses such as drought and How do those growth responses affect yield or reproductive temperature stress, we have a particularly challenging ques- fitness which is the culmination of months, or more, of the tion of how plants perceive the stress at the molecular scale. plant life cycle? How does knowing where a gene is Such abiotic stress perception does not follow the familiar expressed at the cellular scale, or where a protein is localized receptor–ligand paradigm many of us learned in biochemis- at the subcellular scale, help us understand coordination of try class (back when it was enough just to know the root and shoot responses at the whole plant scale? answers to the instructor’s questions). Without knowing the Another common theme that emerges from our big ques- beginning, how the plant perceives a change in its environ- tions is the challenge for measurements of stress phenotypes ment, it is much harder to understand the downstream Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 69 responses at any scale. One emerging area of interest is the use and acquiring less carbon but maximizing water use effi- plasma membrane, and its interfaces with the cell wall and ciency (WUE; amount of carbon acquired per unit of water cytoskeleton, as a logical place for plants to sense environ- lost via transpiration). Which strategy is better for a species mental signals and control water and solute transport while depends not only on the environment but also on which also initiating down-stream signaling and induction of signal- other plants share that environment. Saving water in the ing intermediates such as abscisic acid (ABA). Yet we know soil for later is less effective if your neighbor spends it first. relatively little about how the cell wall–plasma membrane– An important question for research is whether these trade- cytoskeleton interface acts in stress sensing and signaling offs, or other ecologically important tradeoffs, can be broken and what the key molecular players in stress sensing are. (high WUE and rapid growth, for example). If we scale up to the whole plant level, there are long- In the sections below, we consider these and related ques- standing questions about how plants control the movement tions with the hope that other researchers will be informed of water through the soil–plant–atmosphere continuum and motivated to add, and answer, many other questions and how stress responses may be coordinated across differ- about plant abiotic stress that we do not yet know to exist. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 ent tissues. For drought stress, it is sometimes assumed that the initial sensing events occur in roots because they are di- Can plasticity in traits beneficial in both wet rectly exposed to drying soil. But this need not be the case and dry soils be recognized and used to limit as the whole plant is hydraulically connected and changes in crop yield loss? water potential at the root will be quickly propagated (By Julia Bailey-Serres) through the plant. Conversely, one could make an equally Increased climate variability is responsible for excessive wet logical hypothesis that water limitation is first sensed in and dry soil conditions that affect irrigated and rain-fed agri- leaves as these are the site of water loss to the atmosphere culture. From this, there arises a question of whether there and the site where stomata must quickly respond to restrict are genes and traits, or sets of genes and traits, associated water loss when water supply from the roots and vascular with greater plant resilience in both of these extreme soil system is disrupted. There is also a question of whether environments. One can find support for this notion in spe- there are nonhydraulic, chemical signals that move from cies that thrive in ephemeral wetlands. These possess consti- root to shoot, presumably in the xylem, to communicate a tutive adaptive traits or display plastic acclimation strategies change in water status. The type of signal that this could be that facilitate survival in areas that undergo a seasonal rise remains uncertain, although peptide signaling has recently and ebb of the water table that inundates root systems and received increased attention (Takahashi et al., 2018; can partially or completely submerge aerial tissues. More of- Reichardt et al., 2020). These hypotheses of root versus ten than not, wet and dry cycles occur in succession, neces- shoot sensing and nonhydraulic signaling versus hydraulic sitating traits that are plastic or beneficial under both signaling are not mutually exclusive and it seems likely that extremes. Few crops withstand water-saturated soil (water- something as important for the plant as sensing changes in logging), let alone submergence for more than several days. water availability (or changes in salinity, temperature, or Rice is an exception, surviving by accelerating or dampening CO2 levels) is likely to have multiple mechanisms which op- underwater growth. Flooding escape of seedlings is aided by erate in both distinct and overlapping ways. Under severe ANAEROBIC GERMINATION 1, encoding a trehalose 6- water limitation, disruption of water transport via xylem phosphate phosphatase, that increases sink strength of the embolism becomes more likely and there is ongoing debate snorkel-like coleoptile, allowing the germinating seedling on how and when (or whether) refilling of xylem and recov- access to air (Kretzschmar et al., 2015). Deepwater rice ery of hydraulic conductance can occur. There is also debate can outgrow a seasonal rise in paddy depth of over 3 m. on how much loss of vascular function and hydraulic con- Within submerged stems, ethylene activates a gene suite ductivity is lethal, either to specific tissue which becomes (SNORKEL1/2, SEMIDWARF1, and ACCELERATOR1) that cut off from water supply or to the plant as a whole. amplifies cell division at stem node meristems and subse- The concept of tradeoffs, and how they are regulated, is quent internode elongation (Hattori et al., 2009; Kuroha et al., also a recurring theme of plant stress research. The concept 2018; Nagai et al., 2020). In contrast to this adaptive strategy, of a “growth-versus-defense” trade-off is now frequently the submergence tolerance regulator SUBMERGENCE1A mentioned in molecular studies (including sometimes in (SUB1A), encoding an ethylene-responsive transcriptional studies that have data for only one side of the proposed regulator factor subfamily VII (ERF-VII), limits the exhaus- tradeoff). While the “defense” side of the tradeoff may refer tion of leaf carbohydrate in leaf elongation (Fukao et al., to pathogen defenses, which often cause obvious disruption 2006) and minimizes postsubmergence ROS and water def- of plant growth, this has gradually broadened to include icit (Fukao et al., 2011). This transient tolerance protects many types of stress responses that may, either directly or semi-dwarf paddy rice from deep but short-term flash indirectly, affect growth. Another trade-off example is the floods. While these studies focus on rice, there are species concept that plants can take a “water spender” strategy of in all major crop families that are adapted to transient wet maximizing carbon acquisition even at the cost of high wa- zones (Oryza and Zea in the Poaceae, Lotus in the ter use versus a “water saver” strategy of restricting water Fabaceae, Solanum dulcamara in the Solanaceae, and 70 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. Rorippa in the Brassicaceae). These, along with rice and in domesticated maize cultivars (Abiko et al., 2012). The flooding tolerant Arabidopsis (Arabidopsis thaliana), pro- mapping and marker-assisted breeding of genetic determi- vide insight into plastic survival strategies lost during crop nants of these plastic traits are important for crop improve- domestication or selection for production agriculture. ment. Promising examples include a locus associated with Roots perceive subtle changes in soil moisture including adventitious rooting upon waterlogging in soybean that flooding, which restricts diffusion of gases, elevating ethylene enhances yield stability (Ye et al., 2018) and loci determining and depleting O2. Can the discovery of regulatory mecha- aeration traits in teosinte that have been pyramided into nisms accelerate improvement of waterlogging resilience in maize cultivars (Mano and Omori, 2013; Mano and crops without a yield penalty? Might this be accomplished Nakazono, 2021). even if flooding is followed by water deficit? Can root aeration traits be beneficial in dry soils? Water Let us consider root system traits that are associated with deficit also activates the formation of aerenchyma, a strategy survival of waterlogged and anaerobic soils. When roots of that purportedly reduces metabolic costs of deep water- diverse crops (e.g. rice, tomato, and Medicago) become O2- seeking roots (Lynch, 2015). It also reinforces the exodermal Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 deprived, a conserved low-O2 gene regulatory network suberin barrier to limit water loss by diffusion between the (GRN) is activated by SUB1A-like ERFs that are stabilized as root tip zone and distal regions. In contrast to the shallow- O2 levels fall (van Dongen and Licausi, 2015; Reynoso et al., angle roots of wetland rice, constitutive deep rooting is 2019). The genes with conserved ERF-VII cis-regulation en- characteristic of drought-resilient crops. In upland rice, a code enzymes of anaerobic metabolism, turnover of ERF-VIIs functional DRO1 allele promotes auxin-mediated gravitrop- upon reoxygenation, and ABA perception. Without aeration, ism resulting in a deep root angle (Uga et al., 2013). Yet as in root meristems of fully submerged rice, DNA synthesis plants with shallow root systems that allow greater access and the cell cycle are attenuated until shoots are re-aerated to phosphate, nitrogen and other nutrients can display hy- (Reynoso et al., 2022). drotropism (growth toward moisture through gradient sens- Aeration in flooded soils is enhanced by conditional prolif- ing) and hydropatterning (Bao et al., 2014; Robbins and eration of roots with a shallow angle. In rice, this is aug- Dinneny, 2018; Lind et al., 2021), discerned as auxin- mented by loss-of-function of SOIL SURFACE ROOTING1, a mediated but ABA-limited lateral branching on the moist homolog of DEEPER ROOTING1 (DRO1) (Kitomi et al., 2020), side of a root in Arabidopsis (Orosa-Puente et al., 2018). limiting auxin-mediated gravitropism. Also important are These traits, along with the prioritization of elongation of shoot-borne (adventitious) roots that emerge near the air– established crown (nodal) or other roots with access to water interface, capturing dissolved O2 and nutrients of moisture, may provide sufficient plasticity for survival of a floodwaters (Lin et al., 2021). Their emergence is auxin- wet to dry transition (Figure 1). But a challenge may be mediated, triggered by ethylene, and localized production of whether spatial or temporal perception of ABA promotes or reactive oxygen species (ROS). Aeration is enhanced by in- inhibits root growth in the specific environmental context ternal or external passageways called aerenchyma that pro- and species. Another consideration is the consequence of vide a low resistance path for the diffusion of O2 and other waterlogging on beneficial plant–microbe interactions. gasses between aerated shoots and waterlogged roots Rhizobial nitrogen fixation in legumes and mycorrhizal fun- (Pedersen et al., 2021). Aerenchyma form within the cortex gal interactions are limited by waterlogging, yet both recover of rice and maize roots through ethylene- and ROS-triggered as soils dry (Justino and Sodek, 2013; Groen et al., 2022). programmed cell death (He et al., 1996; Yamauchi et al., Comparative genomics, systems biology at the tissue and 2016, 2017); auxin signaling is also implicated in rice cell level, and genome editing have expanded opportunities (Yamauchi et al., 2019). Another characteristic of water- to address these questions to overcome the urgent chal- logged roots is the accumulation of suberin lamellae, a lay- lenge to increase water extreme resilience in crops. ered polyester of poly(phenolic) and poly(aliphatic) fatty How do plants sense and communicate water acids, in the apoplasm of the exterior side of the outermost cortical layer (exodermis) or periderm (epidermis of older deficit? roots and stems) (Pedersen et al., 2021). Accumulation of (By Alexander Christmann and Erwin Grill) the suberin lamellae is mediated by ABA in rice (Shiono Homeostasis of water status is a major challenge for plants. et al., 2022). The extension of this gas and water imperme- The gradient of water potential (ww) from the soil to the able barrier toward the root tip limits the outward diffusion plant and subsequently to the atmosphere generates a hy- of O2 en route to root meristems. draulic continuum that efficiently mobilizes soil-borne water Aeration traits can be constitutive, as observed in paddy via stomata transpiration into the air. Water availability and weeds (i.e. Echinochloa species; Ejiri and Shiono, 2019) and the water-conducting capacity of plants impose constraints Amazonian Oryzae (Ejiri et al., 2020), or induced by water- on this water flux. Regulatory processes governing long-term logging or prolonged (stagnant) flooding. Both cortical aer- adjustments and stomata responses to changes in water sta- enchyma and exodermal suberin are constitutive in the tus are well understood on a molecular level and involve wetland teosinte Zea nicaraguensis, but induced by ethylene the phytohormone ABA (Yoshida et al., 2019a, 2019b; Zhang Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 71 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 1 Characteristics of shoot and root growth plasticity associated with variation in soil moisture using semi-dwarf rice as the model. Roots in dry soils grow toward water reserves and limit water loss by deposition of a pronounced suberin barrier, particularly surrounding the exterior side of the exodermis. In moist soils, root architecture is more compact, crown root initiation and elongation are active, and exodermal suberization is limited. Waterlogged roots containing aerenchyma proliferate near the soil surface and exodermal suberin prevents oxygen loss by radial diffusion. In fully submerged plants, cell division activity in root systems is rapidly switched off and leaf elongation is enhanced, promoting escape to the air. Key phytohormones, their relationships and genes mentioned in the main text are shown. et al., 2022). However, the molecular components of sensing Reduction of stomatal aperture by ABA signaling readjusts water deficit and communicating these cues within the the water balance by lowering transpiration. Stomatal plant remain largely speculative. responses to high water vapour deficit are regulated in a largely guard cell-autonomous manner but with pavement Effects of water deficit cell–guard cell interaction. Signaling elements of this re- Uptake of soil water by plants requires a root ww more neg- sponse include several protein kinases, Raf-like proteins, and ative than the surrounding soil, which is achieved predomi- the receptor-like kinase GHR1, which act upstream of the nantly by osmotic adjustment and negative hydraulic ABA response mediator Open Stomata 1 (OST1) (Hsu et al., pressure due to transpiration. A negative water balance (i.e. 2021). Improved water uptake via increases in root hydraulic water uptake lower than its release by transpiration) imme- conductance (Maurel et al., 2016) and osmotic adjustment diately changes a number of parameters within the plant. In recover leaf gas exchange at the expense of increasingly neg- the substomatal cavity, the major evaporation site, ww of ative plant ww. These adjustments are incremental and oc- the apoplastic fluid becomes more negative, causing neigh- cur even under nonstress water conditions. boring cells to lose water, and consequently causing their turgor and volume to decrease (Figure 2). Simultaneously, How is the change in ww in the plant perceived? the hydraulic tension increases within the xylem. These wa- Changes of tissue ww relative to its surroundings affect cellu- ter deficit-induced changes are relayed within the tissue and, lar water flux, osmolarity, and mechanical forces including depending on the extent of water loss and water capaci- turgor acting on the cell wall, biomembranes (e.g. plasma tance of the plant tissue, possibly to other organs. Changes membrane, tonoplast, and endomembranes), and the cyto- in hydraulic parameters serve as a fast long-distance signal skeleton. There is mounting evidence for a mechano- (440 cm min–1) (Christmann et al., 2013). Suppression of sensitive mechanism that detects these changes. Classical the hydraulic signal from desiccating roots blocked the paradigms for mechano-sensitive and ww change-mediated ABA-induced leaf response, that is, stomatal closure and plant responses are touch-induced leaf closure of the Venus ABA-dependent gene expression (Christmann et al., 2007). flytrap and tendril coiling of Bryonia that are very sensitive 72 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. receptor Ca2 + channel GLR3.6 and hyperosmolality-induced [Ca2 + ]-increase (OSCA)-family channels. Members of the OSCA family are mechanically activated and ion nonselec- tive (Murthy et al., 2018). Touch-induced depolarization of the sensory cells is mediated by MSL10 and initiates action potentials that propagate via GLR3.6 and OSCAs. MSL10 and GLR3.6 are known components of electrical signal prop- agation upon wounding (Toyota et al., 2018; Farmer et al., 2020; Moe-Lange et al., 2021). In the case of wounding, hy- draulic pressure waves are initiated that propagate through the xylem and trigger an electrical and Ca2 + wave in the vasculature. The waves are relayed (10 cm min–1; Farmer et al., 2020) by MSL10 and may require downstream-acting Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 GLRs localized to endomembranes, namely the phloem- expressed GLR3.3 and GLR3.6, which are also highly expressed in xylem-contacting cells (Nguyen et al., 2018; Moe-Lange et al., 2021). Hence, mechano-sensing in the Venus flytrap and wounding response utilize many of the same molecular components and also share a signal propa- gation mechanism that includes a wave of increased intra- cellular Ca2 + . Endomembrane compartments also play a critical role in Ca2 + release during mechano-stimulated responses (Klusener et al., 1995). Mechanical forces acting from outside the cell can be relayed to these intracellular cell compartments by the cytoskeleton and cytoskeleton- Figure 2 Water flux from the xylem vessel to the evaporation site in a associated proteins (Bhaskara et al., 2017; Hamant et al., schematic leaf cross section. Water transported through the xylem (X) 2019) or by the loss of water causing osmotic disequilibrium of a small leaf vein passes through bundle sheath (BS) cells to meso- phyll and epidermal cells, which surround guard cells (GCs), the sites between intracellular compartments which may activate of transpiration. GCs are symplastically isolated (have no plasmodes- stretch related sensing on intracellular membranes in addi- mata) and water supply occurs from the apoplast. Transpiration ex- tion to the plasma membrane. The involvement of endo- ceeding water replenishing at the evaporation sites results in a membrane signaling in stress response is also indicated by reduced water potential of the apoplastic fluid (wwa). This key event observations that loss of the chloroplast-localized mechano- triggers a number of changes: a reduction of turgor (T) and cell vol- sensitive channels MSL2 and MSL3 leads to osmotic imbal- ume within the mesophyll, and an increase in the hydraulic tension ance between chloroplast and cytosol and constitutive (hT) within the vasculature. The change in hT serves as fast long-dis- activation of low ww response in unstressed plants (Wilson tance signal and there might be parallels with mechanical sensing and et al., 2014). relay of touch and wounding. The hydraulic signal is converted into The electrical wave-induced cell depolarization involves the chemical signal ABA to adjust water conductance and stomatal aperture. Mechano-sensitive components are likely involved in all cell additional components shared with ABA responses such as types but have not been unequivocally identified. voltage-dependent anion and cation channels, NADPH- oxidase, and the proton ATPase driving re-polarization (Farmer et al., 2020; Iosip et al., 2020). While wounding to external force, at least as sensitive as human touch. results in a sudden relaxation of the hydraulic tension at the (Klusener et al., 1995; Escalante-Perez et al., 2011) Thus, severed xylem, water deficit increases this tension. The in- touch-sensitive signaling offers a good model to conceptual- creased xylem tension translates into a stronger pulling force ize the types of mechanisms that could be involved in low acting on the xylem-contacting cells. A sudden change of ww perception and initial signaling. this force might distort and stretch domains at the plasma The Venus flytrap uses multicellular trigger hairs to sense membrane. Pulling forces of –0.1 bar resulted in half- prey by converting mechanical forces exerted by the insect maximum MSL10 activation in the Venus flytrap (Procko into a turgor-driven snap. The response to insect touch is et al., 2021) and even relatively mild water deficit could be relayed to neighboring cells by electrical signaling involving a expected to generate similar or stronger forces. Touch- and calcium (Ca2 + ) wave within the leaf lobe (Suda et al., 2020). wound-activated responses also induce chemical signals in- The sensory cells of the trigger hair are enriched in ion cluding oxo-phytodienoic acid and jasmonate for subse- channels associated with mechano-perception and Ca2 + en- quent phytohormone signaling (Escalante-Perez et al., 2011; try (Iosip et al., 2020; Procko et al., 2021). These include Farmer et al., 2020). We currently do not know the extent homologs of the Arabidopsis mechano-sensitive-like channel to which touch- and wound-induced signaling mechanisms 10 (MSL10) (Basu and Haswell, 2020), the glutamate overlap with the mechanisms used to detect and respond Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 73 to water-deficit. If such mechanisms are involved in water yield penalty needed for crops of the future (Yang et al., deficit signaling they would be expected to induce ABA ac- 2019). cumulation as a key signaling factor to turn on further downstream stress responses. How, where, and when are water deficit Genetic screens for Arabidopsis mutants impaired in regu- signals integrated during floral transition? lating ABA-responsive reporter expression under hyperos- (By Lucio Conti) motic stress have failed so far in identifying water-deficit Plant physiology textbooks emphasize how plants are con- sensing receptors (Wang et al., 2011; personal experience). tinuously challenged by their surrounding environment and Genetic and functional redundancy provides an explanation, how this triggers developmental adjustments. But what hap- in which receptors act in parallel pathways that converge on pens if the environment deteriorates to the point of threat- ABA. This might be the case considering the multiple cellu- ening survival? Stressors like water deficit can pose such a lar effects of leaf transpiration exceeding water uptake. Use challenge for plants and one survival strategy relies on the of Arabidopsis natural variation in low ww-induced ABA ac- high flexibility of the flowering program. The switch to flow- Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 cumulation identified candidate loci affecting ABA accumu- ering is a crucial decision for plants, determining the cessa- lation (Kalladan et al., 2017); however, the possible role of tion of the vegetative phase and initiation of reproductive these candidate loci in stress-signaling needs to be validated development. It occurs at the shoot apex upon receipt of and further studied. The increased tension of xylem water environmental and endogenous signals and precedes shoot may activate MSL-type channels of contact cells or mechani- elongation, specification, and outgrowth of floral organs. cal force-sensing Ca2 + channels as part of long-distance Depending on the ecological context appropriate timing of communication. These contact cells and the surrounding the floral transition positively influences inflorescence parenchyma of the vasculature play a prominent role as spe- growth, architecture, and the number of flowers produced cific sites of ABA biosynthesis (Endo et al., 2008). Cells lining (as these processes rely on carbon assimilation from vegeta- the substomatal evaporation sites are particularly challenged tive leaves). Several studies describe the rapid natural and ar- with water efflux and turgor decrease. A decrease in cell vol- tificial selection for early flowering phenotypes to evade ume and the concomitant reduction in plasma membrane harsh summer drought scenarios (Franks, 2011; Kenney surface (Sack et al., 2018) could be sensed similar to yeast et al., 2014; Monroe et al., 2018; Groen et al., 2020), a strat- target of rapamycin (TOR) complex2 that balances plasma egy referred to as drought escape (DE). DE permits the com- membrane constraints with membrane lipid level (Riggi pletion of the life cycle before water deficit conditions et al., 2019). Turgor-sensing and—controlling mechanisms become extreme, even if at the expense of fitness. Flowering need to be activated to re-establish water balance. However, time regulation is also extremely plastic in the face of unpre- bona fide turgor sensors of plants are not known yet. In dictable environmental constraints (Blackman, 2017). yeast, the histidine-kinase SLN1 senses turgor and controls Similarly, DE can also be adaptive (Figure 3A) which means the hyperosmolarity response (Reiser et al., 2003). that upon experiencing water deficit, some species can acti- Interestingly, several structurally related histidine kinases of vate an earlier floral transition, although significant varia- Arabidopsis, including AHK1 and the cytokinin receptors tions in genotype by water deficit interaction exist (Franks, AHK2, AHK3, and AHK4, can complement the turgor- 2011; Kenney et al., 2014). sensing function of SLN1 either alone or in the presence of Where does DE originate? Flowering is regulated through cytokinin for AHK4 (Reiser et al., 2003; Tran et al., 2007). a network of genes that perceive environmental/endogenous However, it is unclear whether AHKs act as water stress sen- signals and genes that integrate these signals to regulate flo- sors in plants (Kumar et al., 2013). RAF-like protein kinases ral induction. Florigen genes are important integrators involved in osmotic adjustments are promising candidates expressed in the vascular tissue of vegetative leaves. They for downstream-acting signaling components (Lin et al., encode a class of small globular proteins that move long- 2020; Soma et al., 2020). distance through phloem vessels at the shoot apex to cause Components that maintain or monitor cell wall integrity transcriptional reprogramming of meristem cells and floral are critical in stressful conditions exerted by high turgor specification of new primordia. While florigen’s transcrip- pressures. Such components include pectate lyase (Chen tional activation usually occurs upon perception of critical et al., 2021) and several plasma membrane-localized protein daylengths, studies in Arabidopsis, rice, and tomato further kinases like FERONIA, required to avoid root cell burst in re- point to the contribution of water deficit signals (Riboni sponse to salt stress (Feng et al., 2018), CrRLK1L/BUPS1 et al., 2013; Du et al., 2018; Chong et al., 2022). (Zhou et al., 2021), THESEUS (Bacete et al., 2022), and Transcriptional upregulation of florigen genes in response to STRUBBELIG (Chaudhary et al., 2021). water deficit appears to be necessary and sufficient to cause In summary, water deficit responses are activated during DE in these species, despite their evolutionary separation. increasing transpirational demand to restore plant water ho- The phytohormone ABA emerges as a conserved molecule meostasis and sustain photosynthesis. Understanding how regulating florigen expression, although different mecha- plants sense and communicate water deficit on a molecular nisms are involved (Figure 3B). In tomato, ABA stimulates level provides a promising tool to increase WUE without phosphorylation and subsequent nuclear shuttling of the 74 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 3 Known and uncharacterized signals causing DE and their sites of action. A, wild-type Arabidopsis plant photographed at 3 weeks from germination. Plants were grown under normal or low-watering (NW and LW, respectively) regimes. LW plants exhibit DE. B, Cartoon outlining a leaf cross-section. Water deficit signals (WD) are transmitted through the vasculature to stimulate ABA production. ABA positively contributes to florigen transcriptional activation in leaves in response to water deficit. ABA and WD promote expression of many other flowering time genes reg- ulating guard cell activity and drought tolerance traits. C, Besides florigen proteins, different signals converge at the shoot apical meristem to relay water deficit information and regulate floral transition accordingly. Despite the ability of the shoot meristem cells to integrate these cues, it is still unclear how water deficit signals (WD) can reach the apex and if significant ABA translocation occurs. transcription factor VASCULAR PLANT ONE-ZINC FINGER be distributed across different tissues (Kuromori et al., 2018; 1 to activate the florigen SINGLE FLOWER TRUSS (Chong Daszkowska-Golec, 2022). While measuring ABA cellular et al., 2022). In Arabidopsis and rice, ABA orchestrates differ- concentrations in specific shoot cells remains challenging, ent transcriptional and posttranscriptional mechanisms, there is evidence that florigen proteins can directly control with key contribution of a class of ABA-regulated basic the expression of different ABA signaling genes at the shoot leucine-zipper (bZIP) transcription factors that, in apex (Zhu et al., 2020). ABA and its signaling cascade regu- Arabidopsis, are incorporated into multimeric protein com- late leaf emergence rates, suggesting a direct influence on plexes to activate (indirectly) florigen expression (Yoshida meristem cell activity, possibly mediated by regulation of et al., 2014; Hwang et al., 2019). primary metabolism (Yoshida et al., 2019b). These findings Are florigens unique in relaying water deficit information could set the stage for a better understanding of ABA- at the shoot apex? Considering that ABA is an important regulated cell fate reprogramming in response to, or in par- driver of DE, its redistribution at the shoot could also influ- allel with, florigen mobilization at the shoot in response to ence the flowering process (Figure 3C). Phosphoproteomics water deficit. Additionally, this would stimulate further ques- studies in Arabidopsis reveal several flowering time regula- tions about the role of ABA accumulation and signaling in tors as substrates of ABA-activated signaling, suggesting mul- flowering time regulation under optimal irrigation condi- tiple and spatially separate points of control of the floral tions and its conservation across species. For example, re- network (Wang et al., 2013a). Is there ABA translocation at ducing ABA sensitivity of rice plants by mutations in a clade the shoot apex or are shoot meristem cells capable of de of ABA receptors causes late flowering, a phenotype that is novo ABA production in response to water deficit signals? not observed in Arabidopsis (Miao et al., 2018). Can ABA directly affect cell fates at this site? These ques- Many questions remain concerning the number of signals tions remain largely unsolved. In Arabidopsis, ABA biosyn- elicited in response to water deficit, their integration in re- thesis occurs in roots and vascular bundles of leaves, largely productive development, and the role of flowering time overlapping with the main sites of florigen expression genes in conferring drought protection (Figure 3, B and C). (Kuromori et al., 2014). ABA is loaded into the phloem to Water deficit-stimulated ABA production inhibits shoot Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 75 growth, thereby delaying the appearance of floral structures insight into the major innovations plants have evolved to (LeNoble et al., 2004). Thus, water deficit may lead to survive in different environments and will also inform strate- uncoupling chronological time to flower (the appearance of gies for introducing such mechanisms into a broader range floral organs) from floral transition per se. Furthermore, un- of crop plants. known signals can influence shoot meristem function, flow- Much of the past emphasis on investing in molecular ge- ering time, and florigen expression, depending on the level netic model systems such as Arabidopsis was based on the of water deficit imposed (Galbiati et al., 2016; Du et al., historic limitation in the availability of genomic resources. 2018). How cells can distinguish between varying levels of Furthermore, these concerted efforts established a critical water deficit is unclear. ABA also activates negative regula- mass of researchers focused on determining a baseline un- tors of flowering, including FLOWERING LOCUS C (FLC) in derstanding of plant molecular biology (Provart et al., 2016); Arabidopsis (Wang et al., 2013b). FLC contributes to delay- however, this is no longer applicable. The Kew Royal Botanic ing flowering, which is most apparent when water deficit Gardens recently estimated that 374,000 plant species occurs under noninductive daylength conditions (i.e. when have been discovered (Christenhusz and Byng, 2016) and re- Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 florigen expression is repressed) (Riboni et al., 2013). Because cent efforts have led to genome sequences being available FLC antagonizes the expression of multiple floral genes in for over 350 species. Botanic gardens have made efforts to leaves and at the shoot apical meristem, its activation may sequence the genomes of their collections and the Ruili be required to curb excess florigen signaling and finetune in- Botanic Gardens in China has done so for 689 species (Liu florescence development according to stress intensity. et al., 2019). The 10,000 Plant Genomes Project run by the Concurrent regulation of antagonistic flowering mechanisms Beijing Genome Institute promises to expand this list of may also generally impact drought tolerance traits. For ex- available sequences far beyond what is currently available ample, plants carrying functional alleles of FLC and its up- (https://db.cngb.org/datamart/plant/DATApla1/). stream regulator FRIGIDA (FRI), display strong reductions in Furthermore, transcriptomic profiling of species across the water use (Mckay et al., 2003). SHORT VEGETATIVE PHASE, green plant lineage (Leebens-Mack et al., 2019), or occurring a floral repressor related to FLC, promotes ABA accumula- in specific ecological niches, such as the Atacama Desert tion (Wang et al., 2018b). In contrast, the florigen gene (Eshel et al., 2021), has clarified phylogenetic relationships FLOWERING LOCUS T (FT) regulates stomatal opening, fa- and illuminated physiological adaptations of plants to arid voring transpiration (Kinoshita et al., 2011). Considering the climates. In short, the book of life for plants is being importance of the duration of crop cycles on yield and the revealed at an extraordinary pace. role of ABA in reducing water loss, identification of DE mo- Despite this rapid progress, the pace of studies that func- lecular mechanisms and their natural genetic variations tionally explore this glut of genomic data has not kept pace. could offer targeted strategies to balance flowering time and The bottlenecks that limit our ability to functionalize ge- drought tolerance traits. nome sequences and discover the molecular mechanisms How do we incorporate plant diversity into governing adaptations to the environment are three-fold. The first limitation is our understanding of the diverse physi- our molecular understanding of ological adaptations that plants use to survive environmen- environmental stress adaptation? tal challenges. The second is the paucity of methods (By José R. Dinneny) available in nonmodel plants to enable the functional char- It is an obvious fact, but worth reiterating, that plants have acterization of a plant’s genomic sequence. Finally, limita- evolved to occupy nearly every environment on the earth’s tions in the ability to manipulate the genomes of a diverse surface (Corlett, 2016). Furthermore, through agriculture, array of species through genetic engineering prevent hypoth- humans have bred plants that are now cultivated across esis testing about genotype–phenotype relationships and 12.6% of the total terrestrial landscape (Global Cropland the application of this knowledge. Extent Product at 30 m (GCEP30) (Thenkabail et al., 2021). The lack of understanding of the diversity of physiological The ability of plants to occupy this breadth of environments adaptations to the environment is particularly apparent for involved the evolution or breeding of plant physiological the root system, which by its nature has remained hidden mechanisms to meet the diverse environmental challenges behind a veil of soil. Roots can be considered the sustainabil- that are faced in each ecosystem and agricultural manage- ity organ system (Lynch, 2007). They function to provide ment system. Despite this clear abundance of physiological the plant anchorage and prevent displacement from a fixed diversity, the majority of mechanistic research in plants is position in the ground (Hostetler et al., 2021b), but other still focused on a small collection of stress-sensitive model processes are also relevant. Roots are the major conduit for systems. This is not to say that such discoveries are the absorption of water and nutrients and roots engage in a unimportant or limited in impact, however, we have a metabolic bartering system with soil microbes, which patchy understanding as to whether such studies will iden- facilitates nutrient uptake in exchange for the products of tify broadly relevant principles, or rather species-specific photosynthesis (Fitzpatrick et al., 2018). Plants also commu- details. Addressing the question of how plant–environmen- nicate with each other through their roots and this can af- tal responses are diversified across the kingdom will provide fect the density and diversity of local communities 76 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. (Mommer et al., 2016). Despite these varied functions, very pathways, diametric changes in response to ABA are possi- little molecular insight into how roots perform these func- ble. Further exploration of the diversification of stress re- tions has been described beyond in a few model species. sponse pathways will help to reveal the principles behind Furthermore, of the tissues and cell types that are thought the tuning of such pathways during evolution. to compose most roots, little is understood as to whether Methodological innovations are beginning to provide plant species have evolved innovations in cellular function functional insight into nonmodel plants (Figure 4, B and C). that allow them to survive in the vast array of soil types Single-cell RNA sequencing (scRNA-seq) now allows cell and terrains on earth. Innovations in the preparation of type-specific transcriptional profiles to be obtained from a plant tissue for light-based microscopy, such as ClearSee diverse range of species (Tarashansky et al., 2021). This pro- (Ursache et al., 2018), and in the use of other imaging mo- vides insight into the diversification of cell type functions dalities, such as microscopic computed tomography and the potential discovery of new cell types. DNA affinity (microCT) (Mairhofer et al., 2011; Morris et al., 2017), have purification and sequencing (DAP-seq), which allows for the opened up opportunities for the quantitative exploration of in vitro reconstitution of transcription factor–genome inter- Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 diverse plant anatomical structures and of root systems actions (O’Malley et al., 2016), enables the determination of grown in soil. GRNs in nonmodel species (Sun et al., 2022). Comparisons Specific plant families such as the Brassicaceae (Koenig of GRN architecture between related species provide a and Weigel, 2015), Solanaceae (Fernandez-Pozo et al., means of understanding how the evolution of genomic se- 2015), and Poaceae (Buell, 2009) have emerged as models quence leads to the rewiring of GRNs and the regulation of for comparative genomic studies. Encompassing 3,630 spe- downstream physiological processes important for stress ac- cies, the Brassicaceae family is home to plant species used in climation. These advances, together with improvements in agriculture including oilseed crops Brassica napus (canola) gene editing and plant transformation (Nadakuduti and and Camelina sativa, salt-tolerant halophytes species Enciso-Rodrıguez, 2020; Anjanappa and Gruissem, 2021) dra- Eutrema salsugineum and Schrenkiella parvula, and the well- matically brighten the horizon for studies in nonmodel characterized model molecular-genetic plant Arabidopsis species. (Figure 4A). Arabidopsis provides a nested model system within the Brassicaceae family for exploring the diversifica- How do plants integrate climate signals? tion of stress responses. The sequencing of over 1,000 acces- sions facilitated the identification of genetic loci under (By Scott Hayes and Christa Testerink) selection, and identified the ABA signaling pathway as being Integrating stress signals: from field observations to important (1001 Genomes Consortium, 2016). ABA, which understanding cellular events is induced under drought and salinity stress (Cutler et al., Over the past two decades, several canonical environmental 2010), suppresses root growth in Arabidopsis, particularly at signaling pathways have been established. These pathways concentrations above 1 mM, while in other species like S. trace environmental signals from perception to transduction parvula, an extremophyte plant living at the edge of a and finally an altered transcriptional and phenotypic re- hyper-saline lake, ABA accelerates growth (Sun et al., 2022). sponse. These advances are a notable achievement. Studying These data suggest that even for well-characterized signaling “clean” responses to a single stress has proved a sensible Figure 4 The Brassicaceae family contains species with diverse physiological traits and agronomic value. A, Phylogeny of species in the Brassicaceae family with sequenced genomes. Stars indicate different physiological or agronomic properties of interest. Figure courtesy of Dong-Ha Oh. B, DAP-seq facilitates the rapid determination of transcription factor–genome interaction landscapes. GRN architecture can be compared be- tween species to identify rewiring events. C, scRNA-seq enables cell-type functions to be explored between species and for new functions to be uncovered. Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 77 and effective approach to identifying sensors and signaling characterized. And while in-depth phenotypic and transcrip- pathways (Lamers et al., 2020). What is becoming increas- tomic analysis has led to the identification of several genes ingly clear, however, is that many of these signaling path- involved in the cross-talk between nutrient deficiency stress ways are context-dependent. Environmental cues are often (Kellermeier et al., 2014), we still lack a coherent framework transduced through overlapping molecular components, for understanding this interaction. leading to highly contextual molecular responses. While many agronomical and crop science studies have already ex- Progress toward understanding the mechanisms of signal tensively addressed naturally occurring combinations of en- integration vironmental stress factors (Rivero et al., 2022), the molecular Several studies have started to probe environmental signal mechanisms underpinning these interactions are often integration on a mechanistic and cellular level. For example, obscure. low levels of soil salinity suppress shade avoidance (Hayes To understand plant growth in complex environments, et al., 2019). Soil salinity acts through the ABA pathway to we must improve our understanding of how different cues suppress brassinosteroid-activated transcription factors, thus Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 are integrated into plant development. In this section, we limiting shade-induced growth. There have also been signifi- focus on the cellular pathways governing the integration of cant advances in our understanding of how light and tem- abiotic signals, with a focus on water availability and tem- perature signals are integrated (Hayes et al., 2021). In several perature as relevant climate change-related cues, that often Arabidopsis accessions, simulated neighbor shade triggers an coincide (Livneh and Hoerling, 2016). We highlight impor- increase in petiole elongation at 22 C, but not at 16 C. This tant factors that need to be taken into consideration when striking, temperature-dependent response to shade involves studying signal integration, and we put forward conceptual the receptor-like kinase ERECTA (Patel et al., 2013). More re- frameworks through which to study these processes. cently, it has become clear that shoot temperature percep- tion is heavily integrated with light signaling pathways. Genome-wide studies identify interesting patterns Phytochrome and phototropin photoreceptors revert more Phenotypic and transcriptomic studies have offered the first quickly to their inactive forms at warm temperatures (Legris clues to the molecular mechanisms involved in signal inte- et al., 2016; Jung et al., 2016a; Fujii et al., 2017), with the re- gration. Pioneering studies found that the transcriptomic sult that light signaling is suppressed in these conditions. and metabolic response of plants treated with both drought Additionally, the light-suppressed transcription factor and heat stress differed dramatically from when these PHYTOCHOME INTERACTING FACTOR 7 (PIF7) exhibits stresses were applied in isolation (Rizhsky et al., 2004). enhanced translation at warm temperatures due to changes Proline accumulated in response to drought, but not in re- in its mRNA structure (Chung et al., 2020). In accordance sponse to a combination of drought and heat stress. Instead with the antagonistic relationship between light and warm of proline, sucrose was produced as an osmoprotectant in temperature, shade-avoidance is more aggressive at high am- these conditions (Rizhsky et al., 2004). More in-depth stud- bient temperatures (Romero-Montepaone et al., 2021). ies, using up to six combinations of five stresses (cold, high light, salinity, heat, or flagellin) found that around 60% of The future outlook transcriptional responses could not be predicted from the Recently, data obtained by Prasch and Sonnewald (2013) response to single stresses alone (Rasmussen et al., 2013). were re-analyzed and used by (Azodi et al., 2020) to train Sewelam et al. (2014) tested the transcriptional response to machine learning models to predict cis-regulatory elements salt, mannitol, heat stress, and combinations of the three required for the synergistic response to multiple stresses. and also found that transcripts in the combined treatment Novel approaches using computational models including could not be accounted for by the data for single stresses cellular and functional structural modeling are a promising alone (Sewelam et al., 2014). Most studies into transcrip- avenue to capture and predict interactions. In addition, tional signal integration have opted for severe stress levels, Morales et al. (2021) reported detailed phenotypes of and one could argue that the unexplained transcriptional re- Arabidopsis plants in response to drought and temperature sponse was caused by tissue damage. However, Prasch and stress in combination with recovery from flooding. Together Sonnewald (2013) found that relatively moderate soil these studies provide a starting point to investigate the drought (30% of field capacity) substantially altered the mechanisms underlying consequences of combined stress transcriptional response to warm temperature (32 C/28 C). on plant development and resilience. Several other -omics approaches have expanded our knowl- A major limitation of most studies on signal integration is edge by documenting the responses to combined abiotic the use of single stress intensities. Many stresses have a non- stresses, but few have shed light on the molecular and cellu- linear effect on plant traits, dependent on intensity lar mechanisms involved in signal integration (Zandalinas (Figure 5A). It is likely that different signaling networks act et al., 2021). Genome-wide association studies have allowed at different intensities of the same stress. When investigating for the identification of genomic loci important for the in- the integration of two stresses, it may be useful to plot traits teraction between nutrient deficiency signaling and salt in a matrix of severity (Figure 5B). Comparing heat map ma- stress (Kawa et al., 2016), but these loci remain to be further trices of mutants and wild types may eventually allow us to 78 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. A B Control Stress 2 Trait Trait Test Stress Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Stress 1 C Figure 5 How do plants integrate climate signals? A, Most environmental cues do not have linear effects on plant development. When investigat- ing the molecular basis for a particular cue, it is important to consider that different intensities of the same stress likely act through different mo- lecular pathways. B, Representing gradients of two stresses as a heat map could be a potential way to conceptualize complex interactions. Note that the intensity of Stress 1 affects the way plants respond to the intensity of Stress 2. C, Through taking a step back and studying the interac- tions between signaling pathways in their relevant environmental context, we will come closer to understanding the molecular basis for plant de- velopment in natural environments. place signaling networks within specific environmental con- factors (Figure 5C) will ultimately allow us to understand texts. It will also highlight environmental contexts in which plant responses to abiotic stress in a realistic, complex phenotypes cannot be explained by known developmental environment. regulators and offer potential avenues for future research. Transitioning to gradients of environmental conditions How do trade-offs impact abiotic stress (rather than one, often severe stress condition) will require responses and climate adaptation? conceptual and computational advances. It should also be noted that environmental integration is likely to be different (By Robert W. Heckman and Thomas E. Juenger) for different organs, tissues, or even cell types. Temperature Trade-offs occur when a phenotype that confers an advan- signaling in the roots, for example, can act independently of tage in one context also confers a disadvantage, whether in the light signaling components in the shoot (Bellstaedt the same or a different context (Agrawal, 2020). A classic et al., 2019; Ludwig et al., 2021). Providing spatial and trade-off is between carbon gain and water loss during pho- dose-dependent context of other relevant environmental tosynthesis: when stomata open to absorb CO2, they lose Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 79 H2O (Schulze and Hall, 1982). Numerous ways of mitigating between root and shoot biomass is commonly invoked, be- this trade-off have evolved in plants, from minor adapta- cause when resources are fixed, any allocation to root bio- tions like changes in stomatal behavior or development to mass must come at the expense of allocation to shoot major innovations like C4 and CAM photosynthesis. From biomass (Shipley and Meziane, 2002). But, in nature, many an evolutionary perspective, trade-offs impose costs that plants with large root systems also have large shoots, sug- constrain the ability of plant populations to evolve in re- gesting that no trade-off exists. This failure to detect an allo- sponse to selection (Roff and Fairbairn, 2007; Donovan et al., cation trade-off occurs when failing to account for 2011). These constraints may prevent populations from differences in resource acquisition. reaching adaptive peaks, particularly in new environments, Context is also key when considering attributes of wild which can limit species’ ranges and the distribution of habi- species in agronomic environments. Often, the characteris- tats suitable for particular crops (Blows and Hoffmann, 2005; tics that make wild plants successful become liabilities in Shaw and Etterson, 2012; Dwivedi et al., 2021). Trade-offs crops. In wild plants, many stress-response strategies ensure can originate from different biological processes, like the spe- plant survival via reduced growth rates or stress tolerance Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 cialization of ecotypes in different habitats (Agrawal, 2020). strategies, including slow growth, leaf abscission, or early These differences in the origins of trade-offs can impact how flowering (Fang and Xiong, 2015). In agronomic conditions, plants acclimate, and how plant populations evolve, in re- where rapid growth and high yield are prized, these stress sponse to climate change. responses are often maladaptive (Maggio et al., 2018). This Here, we focus on trade-offs that occur within species as may make extremely well-adapted, stress-tolerant wild they are most likely to be the subject of ongoing evolution relatives (or their adaptive strategies) poor targets for do- that can drive adaptation. Trade-offs within a species or mestication or crop improvement. Instead, biologists should population can result from pleiotropy and genetic linkage re-examine some ideas about what makes plants successful and the degree of standing genetic variation (i.e. genetic ar- in resource-rich, agronomic environments. For instance, chitecture) (Saltz et al., 2017). Pleiotropy occurs when the domestication simultaneously increased growth rates and re- same gene encodes multiple traits; linkage occurs when duced drought tolerance in Helianthus annuus (Koziol et al., genes encoding different traits are located in close physical 2012). Traits like stay-green sorghum and maize circumvent proximity on a chromosome, reducing recombination and the plant’s natural stress response (Zheng et al., 2009; Jordan resulting in coinheritance (Lynch and Walsh, 1998; Mackay, et al., 2012) and are usually beneficial in an agronomic set- 2001). In the short term, trade-offs can constrain evolution ting. Reducing other natural stress responses like high water- by linking particular trait combinations and making other use efficiency, which often leads plants to leave water in the trait combinations less likely (Walsh and Blows, 2009). soil at the expense of potential growth, could be targets of Trade-offs can also be reinforced by correlational selection, similar innovation (Leakey et al., 2019). which occurs when certain combinations of traits, rather When trade-offs occur within species and there is stand- than traits in isolation, are advantageous (Sinervo and ing genetic variation in performance or yield, biologists can Svensson, 2002). Trade-offs can also occur among popula- leverage the possibility of continued evolutionary change to tions within species. These trade-offs often arise due to local break trade-offs and remove conditionally deleterious var- adaptation, which occurs when plants perform better in iants. In most cases, trade-offs can be broken by consistent their home environment than plants from a different envi- selection orthogonal to the direction of the trade-off or by ronment (Kawecki and Ebert, 2004). Local adaptation results crossing locally adapted genotypes to break up linkage from multiple factors, including antagonistic pleiotropy, (Conner, 2003; Agrawal et al., 2010) and through multi-trait where an allele at a genetic locus leads to high relative per- genomic prediction-based breeding for large mega- formance in one environment and low relative performance environments (Wallace et al., 2018). The trade-offs among in a contrasting environment, and conditional neutrality, locally adapted genotypes that result from conditional neu- where an allele confers high (or low) relative performance in trality are an ideal target. Since there is no cost of condition- one environment and has no impact on performance in a ally neutral alleles in a disfavored environment (Anderson contrasting environment (Anderson et al., 2011). et al., 2011), breeding that focuses on combining many con- Understanding how and when trade-offs operate will help ditionally neutral alleles may quickly generate generalist biologists to gain the benefits of a trade-off while avoiding ideotypes, avoiding trade-offs altogether. A similar approach the costs. To do this, context is often critical. Trade-offs in can be used in a molecular context: Kudo et al. (2019) allocation to different functions may be hidden when plants found that drought tolerant rice plants over-express stress- grow under benign conditions, such as those in many agro- inducible genes, like DREB1A, which reduces growth. By nomic and laboratory settings (Roff and Fairbairn, 2007). breeding plants that co-expressed growth-promoting genes, When plants grow under more natural, stressful conditions, like GA5 and PHYTOCHROME INTERACTING FACTOR4 the trade-offs may be exposed, leading to unexpectedly sub- (PIF4), this trade-off could be eliminated (Kudo et al., 2019). optimal performance (MacTavish and Anderson, 2020). This The consequences of failing to break trade-offs can be se- can occur because benign conditions often increase the total vere, especially when trade-offs constrain the ability of plant resource acquisition by plants. For example, a trade-off populations to adapt to keep pace with ongoing global 80 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. change (Chapman et al., 2012). Left alone, species that can- connected with the circadian clock is ample and includes not adapt to rapid global change will need to acclimate or drought, heat, cold, or redox imbalance (Grundy et al., will likely go locally extinct (Hoffmann and Sgrò, 2011; 2015). Interestingly, the circadian clock not only regulates Christmas et al., 2016). In these cases, biologists can also use daily or seasonal oscillatory stresses such as heat stress dur- our understanding of trade-offs to move species with desir- ing the day or severe cold during the night, but also contin- able attributes to matching environments (e.g. targeting uous stresses with no obvious oscillations, like high salinity crop species to the most suitable environments or combin- (Park et al., 2016). The anticipation provided by the circa- ing certain species to enhance ecosystem services) (Willi and dian clock may enable plants to prepare in advance against Van Buskirk, 2022). stresses that diurnally or seasonally oscillate, and thus con- Trade-offs are, to some extent, inevitable in biology. But, ferring an adaptive advantage. However, the regulation of trade-offs do not need to hamstring our ability to respond constant stresses may rely on specific clock components to climate change. As the climate becomes more variable that act independently of their function within the clock. It and moves further away from historical averages, the target is also possible that the constant stress is related to other Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 environments for crop breeding will become less obvious stresses and pathways in which circadian timing is relevant. (Chapman et al., 2012). Thus, the specialized strategies Based on the pervasive role of the circadian clock in the exhibited by locally adapted crops may become less valuable. regulation of abiotic stress responses, the focus of attention A major challenge will be to identify the trade-offs that are is directed toward the use of the circadian system for im- most limiting to desired outcomes (e.g. increased produc- proved tolerance to a broad combination of stresses without tion or resilience to climatic extremes) and use our biologi- imposing detrimental pleiotropic effects such as growth ar- cal insight and engineering principles to break, alleviate, or rest, or reduced yield. To that end, multiple strategies can circumvent those trade-offs. Gene stacking to produce gen- be adopted, but particularly interesting are those related to eralist ideotypes and artificial selection in the direction or- a central function of the circadian system, known as thogonal to a trade-off are promising approaches that may “gating,” whereby the clock differentially regulates the mag- be feasible in the short term. In the longer term, more ex- nitude of the plant response to environmental signals treme interventions, like genetically engineering crops to use depending on the time-of-day (Seo and Mas, 2015). The cir- C4 photosynthesis or to become perennials, may be required cadian peak of expression of many genes involved in stress to break or alleviate some of the most recalcitrant trade- responses coincides with the recurrent peak time of the offs. Given the importance of trade-offs for so many aspects stress (Bonnot et al., 2021), and so, a large proportion of of biology, their study can be a promising approach for en- the heat- and cold-responsive transcriptome is gated by the hancing the resilience of our agricultural and natural systems clock (Covington et al., 2008; Blair et al., 2019). Interestingly, in an increasingly variable world. the clock is also able to gate stress responses depending on the time of the year (Lee and Thomashow, 2012). For exam- How does the circadian clock “gate” plant ple, key factors involved in cold responses oscillate with responses to abiotic stress? higher amplitude under short-day conditions, providing (By Paloma Mas) improved tolerance to cold conditions during the winter Severe drought, extreme temperatures, and changes in salin- time. Thus, using circadian gating might be advantageous ity all disturb plant cellular homeostasis and cause deleteri- for plants to restrict their response only to the most appro- ous effects on crop growth and productivity. Throughout priate and needed time, as opposed to a constitutive evolution, plants have developed a battery of responses to response normally associated with high energy demand and reach a cellular status that is tolerant or compatible with with the penalty on growth and yield. harsh conditions (Markham and Greenham, 2021). But, what are the mechanisms behind the gating function Understanding the array of responses triggered by abiotic and regulation? Although most studies have focused on stress can provide useful information to obtain crop varieties transcriptional control, posttranscriptional regulation can be adapted to stress (Iannacone et al., 2012; González-Guzmán also gated by the clock. For example, alternative splicing has et al., 2022). Among the different signaling pathways in- been associated with heat stress responses (Ling et al., 2021), volved in plant responses to stress, the circadian clock and Splicing Factor 30 could be one of the many plausible stands out as a main cellular mechanism able to measure candidates linking the clock with alternative splicing and time and to coordinate key biological processes in synchrony heat stress (Bonnot and Nagel, 2021). Identifying all the with the environment (Sanchez and Kay, 2016). The proper components of the splicing machinery gated by the clock, function of the circadian clock enables plants to anticipate and the alternative splicing isoforms functionally relevant the daily changes in the environment, controlling the timing under single or combined stress conditions is an interesting of growth, development, and responses to biotic and abiotic area of ongoing and future research. Similarly, studies show- stresses (Sanchez and Kay, 2016). ing the connection of alternative polyadenylation with the Mutation of clock components disturbs the ability of clock (Yang et al., 2020), and with stress (Yang et al., 2021) plants to adapt to environmental stress conditions (Seo and open the way for functional studies on the circadian gating Mas, 2015; Bonnot et al., 2021). The number of stresses of alternative polyadenylation. New areas of study can Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 81 specifically focus on the circadian coupling of transcription proteins essential for membrane energization, signal detec- with polyadenylation, splicing, or RNA modification (N6- tion and transduction, as well as primary and secondary me- methyladenosine, 5-methylcytosine, or pseudouridine) (Yang tabolism (Munnik et al., 2021). Phospholipids are et al., 2021). The clock also selectively redefines the pool of indispensable for cell function (Noack and Jaillais, 2020). mRNAs to be translated under heat stress, controlling about Besides a structural function, phospholipids have also one-third of the circadian- and heat-dependent translated emerged as crucial signaling molecules, either as precursors proteins (Bonnot and Nagel, 2021). Expanding these studies of signaling molecules or as lipid second messengers them- to other abiotic stresses will provide a global view of how selves. The best examples come from polyphosphoinositides circadian clock gating impacts the abiotic stress-related (PPIs), which are inositol-containing phospholipids that can translatome. The key role of posttranslational modifications be phosphorylated at the D-3, D-4, and/or D-5 position of on abiotic stress responses (Hashiguchi and Komatsu, 2016) the inositol ring, thus creating five distinct molecular species also paves the way for future studies on how circadian gat- in plants (i.e. PI3P, PI4P, PI5P, PI(3,5)P2, and PI(4,5)P2) as well ing controls other posttranslational modifications such as as two more that are present in metazoans but have not Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 phosphorylation, ubiquitination, glycosylation, etc. to regu- been detected in plants [PI(3,4)P2 and PI(3,4,5)P3] (Munnik late protein function and localization in response to abiotic and Testerink, 2009; Gerth et al., 2017). PPIs are typically low stress (Figure 6). abundance lipids that escape detection by common mass Changes in DNA methylation and histone modifications spectrometry methods, and turn over rapidly. They act as result in epigenetic variation that provides phenotypic plas- biochemical and biophysical landmarks that contribute to ticity and plant adaptation to changing environments membrane identity, signaling, and compartment morphody- (Miryeganeh, 2021). As the circadian clock is also closely namics, with each PPI species accumulating in different set connected with chromatin remodeling (Chen and Mas, of endomembranes thus helping to define membrane iden- 2019), it would be interesting to fully explore the circadian tity (Gerth et al., 2017; Dubois and Jaillais, 2021). gating of epigenetic responses to stress. Research could focus In the world of PPI signaling in animals there has been on identifying the full array of chromatin “readers” and something of a revolution over the last decade that has “erasers” controlled by the clock and their connection with manifested itself in an increasing appreciation of PI(4,5)P2 as the stress-responsive loci. The fact that epigenetic marks can a regulator of cellular events in its own right. PI(4,5)P2 has trigger stress memory in primed plants also places the spot- been known for a long time to fulfill a crucial role as the light on identifying the role of the clock in providing a long- substrate for two major signaling pathways: the phosphoino- term memory for the stress response. To obtain meaningful sitide (PI)–phospholipase C (PLC) pathway that generates results, the stress-related experiments should be performed inositol-1,4,5-trisphosphate (IP3) and Ca2 + release, as well as under growing conditions that mimic as close as possible diacylglycerol (DAG) to activate protein kinase C (PKC); and the natural growth environment (Panter et al., 2019). the PI 3-kinase pathway that generates PI(3,4,5)P3, a crucial Likewise, analyzing combinatorial stresses that usually appear lipid second messenger in regulating cell proliferation and simultaneously or sequentially in nature (e.g. heat and many other physiologically important processes (Balla, 2013; drought) might provide much more reliable and relevant in- Dickson and Hille, 2019). formation to use for enhancing crop tolerance to abiotic Yet, higher plants clearly have a different agenda. They do stress (Rivero et al., 2022). Studies could also switch the fo- have the PI–PLC pathway to generate the above-mentioned cus from whole plants to single cells in order to get mean- signaling molecules, but they have no recognizable PKC or ingful conclusions about stress perception, signaling, and IP3 receptors, and no PI 3-kinases of Type I, the group that responses in specific cell types. Also, looking back to plant uses PI(4,5)P2 as a substrate (Munnik and Testerink, 2009). ancestors and comparisons of domesticated crops and their Yet Arabidopsis has 11 Type I PIP 5-kinases (PIP5Ks), nearly wild relatives (Markham and Greenham, 2021; Figure 6) will four times as many as us animals! And why despite this do surely provide novel avenues to understand how the circa- dian gating by the clock can be exploited for adaptation to plants maintain PI(4,5)P2 levels 1–2 orders of magnitude environmental stresses, thus providing novel opportunities lower than metazoans? The only logical answer to these for targeted approaches for improved tolerance to abiotic questions is that PI(4,5)P2 has an even more central role as stress (Bhatnagar-Mathur et al., 2008). a signaling entity in its own right than it does in animal biol- ogy. In plants, there seems to be a proliferation of PI(4,5)P2 How do lipid-derived second messengers functions in conjunction with an increasing understanding translate abiotic stress information into of PI(4,5)P2 compartmentalization (Munnik, 2014; Doumane et al., 2022). Together, these new insights are revolutionizing cellular responses for stress acclimation? how we think about this ancient and original PPI. (By Teun Munnik) Knockout of some Arabidopsis PIP5Ks (PIP5K1–PIP5K6) Phospholipids are crucial building blocks for membrane clearly indicate a role for PI(4,5)P2 in cell polarity, in particu- function. They create the bilayered, liquid structure that sur- lar during cell division and polar growth of root hairs and rounds every cell, organelle, and endosomal compartment, pollen tubes (van Leeuwen et al., 2007; Kusano et al., 2008; and hosts thousands of integral and peripheral membrane Ischebeck et al., 2010; Tejos et al., 2014). In contrast, no or 82 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 6 Circadian gating by the clock differentially regulates plant responses to stress depending on the time of day. Figurative examples of open questions related to the circadian gating via changes in chromatin, posttranscriptional, translational, and posttranslational mechanisms of regula- tion that ultimately define gene expression and protein function under stress conditions. Single-cell approaches with plants grown under condi- tions that closely follow the natural environment, including combined stresses, and comparisons of domesticated crops and their wild relatives are circadian research areas of increasing interest. Understanding the coupling among the different gating mechanisms will pave new ways for using the circadian clock to tackle the effects of abiotic stress on plants. Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 83 only subtle developmental phenotypes are observed for Another challenge remaining is to understand PI–PLC PIP5K7–PIP5K9 mutants as these PIP5Ks are instead in- signaling in plants (D’Ambrosio et al., 2017; van Wijk et al., volved in salinity stress and response to polyamines (Zarza 2018; Zhang et al., 2018c, 2018d). In vitro, the enzyme et al., 2020; Kuroda et al., 2021). Genetically encoded- equally likes PI4P and PI(4,5)P2 as substrate, but the gen- PI(4,5)P2 biosensors (van Leeuwen et al., 2007; Simon et al., eral absence of PI(4,5)P2 in plant plasma membranes, while 2014) revealed that PI(4,5)P2 typically accumulates at the PI4P is relatively abundant, makes it more likely that IP2 is plasma membrane, except during heat stress where addi- generated rather than IP3 in vivo. This would also explain tional punctates appeared in cytosol and near the nuclear the lack of IP3 receptors (Munnik and Vermeer, 2010; envelope (Mishkind et al., 2009). What these punctate com- Munnik, 2014). For DAG, it makes no difference to be con- partments are, and which PIP5K generates them, is still un- verted into PA. IP2 can be step-wise phosphorylated into known. Genetic manipulation of PI(4,5)P2 by inducible various inositolpolyphosphates (IPPs) that are emerging as production or depletion revealed crucial roles for PI(4,5)P2 signaling molecules, including IP5 and IP6, but also IPP- in endocytosis and regulating the actin- and microtubule cy- pyrophosphates IP7 and IP8 (Lorenzo-Orts et al., 2020). IP- Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 toskeleton, with dramatic consequences for development related signaling functions in plants include intracellular (Gujas et al., 2017; Doumane et al., 2021). Ca2 + release (ABA), acting as co-factor for auxin and But how is PI(4,5)P2 managing all this? In metazoan sys- jasmonate signaling (via TIR1 and COI1), as well as func- tems, several protein targets have been characterized, includ- tions in RNA transport and Pi sensing (Figure 7; Munnik, ing PI(4,5)P2 specific-binding domains, for example, PH, 2014; Lorenzo-Orts et al., 2020). Linking all these cellular Tubby, and SEC14 (De Jong and Munnik, 2021). While sev- phenotypes to PI–PLC signaling remains challenging eral plant proteins contain such domains, their PI(4,5)P2- (Zhang et al., 2018c, 2018d). binding specificity and functionality has remained largely While the above mostly deals with questions downstream unexplored (De Jong and Munnik, 2021). Since plant cells of abiotic stress-triggered lipid signaling, we are equally in contain much lower PI(4,5)P2 levels than animals, and since the dark on how lipid kinases, phosphatases, and phospholi- it is no problem to stably express PI(4,5)P2-biosensors (based pases are actually activated upstream. The next decade will on exogenous PH and Tubby domains) in Arabidopsis with- be an exciting venture into a much deeper understanding of out causing any phenotype (van Leeuwen et al., 2007; Simon how plants use signaling lipids and inositol phosphates to et al., 2014), plants are likely to contain distinct PI(4,5)P2- control their physiology and respond to stress. It will not binding domains with a much higher affinity. Such domains, just widen our basic knowledge of plant biology, but very however, remain to be identified. In animals, the gating of likely also our understanding of stress responses that will most K + channels is regulated by PI(4,5)P2, and there are most benefit efforts to improve plant resilience. indications this occurs in plants too (Zarza et al., 2020). How can laboratory stress research be Phosphatidic acid (PA) is another important plant lipid second messenger that is typically triggered upon abiotic applied to continuously stressed stress, including heat, cold, drought, and salinity stress, but plants in the field? also in response to pathogens and wounding (Munnik, 2001; (By Hilde Nelissen) Kim and Wang, 2020). In general, PA responses are fast One of the outstanding questions in plant abiotic stress bi- (min) and generated through hydrolysis of structural phos- ology is why the vast amount of knowledge on physiological pholipids by phospholipase D (PLD) and/or through phos- and molecular responses to abiotic stresses has resulted in phorylation of DAG by DAG kinase (DGK) (Munnik and so few climate resilient crop varieties that are currently on Testerink, 2009). Arabidopsis contains 12 PLDs and 7 DGKs, the market (Sanchez, 2013; Blum, 2014; Nuccio et al., 2018). and 9 PI–PLCs that produce DAG by hydrolyzing PI4P or, if Among the possible reasons that hamper the translation of generated, PI(4,5)P2. How PA is involved in the different molecular knowledge toward application, the gap between stress responses, via which PLD, DGK, and PLC, and at which environmentally controlled conditions versus more variable cell or compartment this takes place, has been a central field conditions was identified as one of the bottlenecks theme of the last decade, and will still be in the next. (Nuccio et al., 2018). Controlled conditions, here used as a Complementary approaches include KO and OE mutants on collective name for greenhouse and growth chamber experi- PA production, isolation, and characterization of PA targets, ments, allow higher throughput, increased speed of innova- and the construction of genetically encoded-PA biosensors tion and reduced cost compared with field trials (Simmons to monitor PA in living cells (Munnik and Testerink, 2009; et al., 2021). In addition, more detailed mechanistic insights Platre et al., 2018; Kim and Wang, 2020; Scholz et al., 2022). and mode of action studies often require controlled condi- While these tools helped to establish PA as a lipid second tions, where responses to changes in the environment messenger that rivals the importance of Ca2 + , we still know can be monitored with respect to time, space, amplitude, very little of either PA versus Ca2 + specificity in responding and other factors related to both the stress applied and to different stresses or their potential collaboration. the response output (Verslues et al., 2006; Bao et al., 2014; Monitoring both molecules simultaneously with ratiometric Alejo-Jacuinde et al., 2022; Hu et al., 2022). On the other biosensors would certainly help clarifying this. hand, field trials provide valuable information about the 84 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 7 Possible signaling mechanisms of PI(4,5)P2, PA, and PLC in plant abiotic-stress signaling. PI(4,5)P2 concentrations in plants are extremely low but synthesis is rapidly triggered by activation of one or more of the 11 PIP5Ks of Arabidopsis via unknown receptors or activation mecha- nisms (indicated by “?”). As such, PI(4,5)P2 is triggered by heat, salinity, osmotic stress, and polyamines, but also during polar growth, predomi- nantly accumulating at the plasma membrane. This likely causes recruitment of target proteins to the membrane or (in)activation of targets that are already present at the membrane. Putative plant targets are based on the mammalian literature and correlations with plant literature. Plant PLCs likely hydrolyse PI4P and generate membrane-bound DAG and the water-soluble headgroup, IP2. Again, receptors and activation mecha- nisms remain unknown (indicated by “?”). The newly formed DAG is rapidly phosphorylated by DGK to form the signaling lipid PA for which sev- eral protein targets have been identified. IP2 can be stepwise phosphorylated by IPP multi-kinases to produce IP5 and IP6, and IPP-pyrophosphates IP7 and IP8, for which several signaling functions and targets are emerging. Protein targets are indicated in purple [PI(4,5)P2] or dark red [PA]. Lipid (-derived) signals are indicated in blue. Solid lines indicate metabolic conversions; dashed lines represent “activation” or “cause.” Effects are indicated in green. potential of a stress-tolerant product for marketable yield As the name implies, controlled conditions aim to maxi- stability without yield drag. However, field evaluations are mally standardize the growing conditions to reassure repro- costly and labor-intensive because of the experimental de- ducibility and to facilitate detailed studies on the effects of a sign and replication needed to detect a desired effect, man- limited perturbation. In controlled condition stress experi- agement logistics, and weather hazards. Thus, given the ments, typically temperature, humidity, irrigation, or light is throughput and the costs involved, the logic pipeline for altered or a substance is added to mimic an abiotic stress plant improvement is a funnel-like screening in which many response. Ideally, the other environmental parameters are lines are screened in controlled conditions, of which the kept as standardized and stable as possible, so that only one most promising leads subsequently undergo field evaluations or a combination of a few abiotic stresses occur simulta- (Simmons et al., 2021). However, the realization is growing neously. However, to fully understand the impact of the ap- that the inability of typical laboratory- or greenhouse- plied stress(es), it might be necessary to monitor additional controlled conditions to properly model agronomic environ- parameters, such as soil water content in drought experi- ments hampers the translation of knowledge on abiotic ments. The stress(es) can be maintained for a substantial stresses to climate resilient crops (Sanchez, 2013; Blum, time of the plant’s development, sometimes followed by 2014; Nuccio et al., 2018; Simmons et al., 2021). stress alleviation, but more frequently stress treatments in Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 85 greenhouse and growth chamber conditions are short and soil, so that individual plant irrigation schemes or nutrient severe to evoke molecular changes. The setup of such an applications can be monitored. The pots are arranged to op- “ideal” laboratory stress assay is often more dependent on timize greenhouse space, irrespective of whether the plants the greenhouse and growth chamber facilities and the assay under study are typically grown as high-density row crops robustness than on reflecting the actual field conditions. or subjected to other management regimes. In this way, dif- Anyone who has performed field trials or has a private ferences in inter-plant population dynamics, interactions garden realizes that there is no typical growth season that with other organisms, or effects of crop rotation or inter- could be represented by standardized, control conditions. cropping are not accounted for when screening in con- Even when temperature and precipitation are close to the trolled abiotic stress conditions. By individualizing every multi-year average, there will be periods of extreme weather plant, field-born local differences that cause molecular conditions. Besides the conspicuous extreme weather condi- responses (Cruz et al., 2020) or trigger differences in devel- tions, there are also less obvious consequences of climate opmental programming (Robbins and Dinneny, 2015) can change that can have detrimental effects on crop yield. For be overlooked, along with the effects of soil differences re- Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 example, the global rising temperature is not only experi- lated to recent cropping history or biotic interactions enced during the daytime. Increased night temperature is (Beirinckx et al., 2020). also a problem for plants in that it enhances night respira- The fact that abiotic stress-related genes, identified in con- tion that increases utilization of photo-assimilates and trolled abiotic stress experiments, are transcriptionally regu- thereby reduces the amount of carbon available for grain fill- lated in the field, confirms their potential usefulness in ing (Desai et al., 2021; Xu et al., 2021). In the field, some improving plant resilience. However, this also urges plant sci- stresses are continuously present, like heavy metals in the entists to gain a better insight into the molecular “state of soil, but other stresses build up more gradually, like drought, mind” of field-grown plants in order to come up with more and can occur during any developmental stage with distinct clever ways to modulate these genes and the corresponding impacts on yield (Verbraeken et al., 2021). In field condi- networks to improve plant performance. To achieve this, tions, stresses rarely occur alone or in a coordinated way, the fields of breeding and molecular biology need to work and temperature, humidity, irrigation, light, and several addi- together more closely to grow and molecularly profile new tional factors fluctuate continuously rendering the plants varieties with improved traits in both field and controlled constantly stressed. conditions, irrespective of whether the lines were generated The fact that field-grown plants are continuously in a by traditional breeding, genome editing, or transgenic stressed state is reflected by the transcriptomic changes be- approaches. This would enable a virtuous cycle of each new tween plants of the same genotypes, grown in controlled variety to be field phenotyped and molecularly profiled, pro- conditions and multiyear field trials. Several stress-related viding information for further improvements (Figure 8). genes that confer abiotic stress tolerance in over-expression However, for such efforts to have a maximal impact on agri- lines exposed to controlled stress conditions were massively culture, there is also a need to ease and harmonize regula- upregulated in field-grown versus laboratory-grown plants, tions that govern the use of genome-edited or transgenic even under relatively normal field conditions (Nelissen et al., crops for sustainable agriculture and food production. 2020). Even neighboring plants of the same genotype grown Where is the plant most hydraulically in one field displayed transcriptional differences in stress- related genes (Cruz et al., 2020). Studies that also take into vulnerable? account the weather conditions when analyzing field (By Lawren Sack, Craig Brodersen, and Thomas N. transcriptomics (Nagano et al., 2012) or that incorporate Buckley) measurements of plant water status show that individual When storms threaten the power grids distributing electric- field-grown plants sense a micro-environment and react ity across a continent, we need to know their weakest links molecularly to local changes in environmental changes by to better prepare for and quickly remediate calamitous fail- altering stress-related genes, some of which were already ures (Weiss and Weiss, 2019). Equally, we need to ask, as in- identified in controlled conditions. creasingly frequent droughts face plants throughout our We showed that not only known stress-related genes globally important and vulnerable ecosystems (Hammond were differentially expressed between the controlled condi- et al., 2022), where are their weakest points? That is, where tions and the field (Nelissen et al., 2020). Genes involved in within the plant does the impact of water stress trigger the processes such as shade avoidance were also differentially most severe reductions in leaf gas exchange and whole plant expressed between the laboratory and the field, which, in productivity, the most irrecoverable damage, and the great- turn, may interact with stress responses (Hayes et al., 2019), est risk of mortality—especially with aggravating stresses complicating the situation even more. In addition, shade such as insect outbreaks and fire. Addressing this question is avoidance is a response that breeding attenuated to achieve critical to predicting the impacts of climate change on the higher planting density, an agronomic practice that is often local and global distribution of ecosystems, the future of ag- overlooked in laboratory or greenhouse experiments. In such ricultural and forest gas fluxes, and even the behavior of the experiments, plants are grown in pots filled with potting atmosphere and climate system. Equally, answering this 86 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 8 Schematic representation of the differences in management, light, and irrigation schemes between plants grown in the field (left) and in controlled conditions. Iterative cycles of field testing and molecular profiling of all lines with putatively improved traits will be necessary to in- crease our understanding how the stresses that plants experience in the field relate to the controlled abiotic stress conditions. Created with BioRender.com. question will inform the breeding of drought-resistant crops components yields a wealth of predictions and mechanistic for food security. representations by considering analogies of tissues, plants, Sensational or not, the analogy of plants as power grids is and ecosystems as including fixed resistors, variable resistors in fact a well-established quantitative approach. Much un- (potentiometers), capacitors, and diodes. Studies using this derstanding of plant hydraulics arose from the classical com- approach to analyze the distribution of hydraulic resistance parison of the soil–plant–atmosphere continuum to an within the plant network suggest that the extremities of the electrical circuit, where flow paths are resistors and water plant represent key bottlenecks to water flow, with the potential gradients are voltages, subject to the application of leaves and roots accounting for 475% of total resistance the analogy to Ohm’s Law (Van den Honert, 1948; Tyree and stems 525%, in a wide variety of growth forms (Tsuda and Zimmermann, 2002): and Tyree, 2000; Sack et al., 2003; Domec et al., 2009). Moreover, these resistances are dynamic, subject to internal DV ¼ IR; and environmental control. Most famously, in every compo- where V is the voltage, I the current, and R the electrical nent of the system, hydraulic conductance (K), the inverse resistance, of resistance, declines precipitously at lower ww, a “hydraulic By analogy, vulnerability” arising from a multiplicity of processes (Figure 9, A–F). In soils, air replaces water and thus removes Dww ¼ ER; flow paths for water to be sucked into the plant. In xylem where ww is the water potential, E the transpiration rate, throughout the plant, under strong dehydration, cavitation and R the hydraulic resistance. Thus, the stronger the flow occurs, which is the formation of air- or vapor-filled con- rate, and the resistance to water movement, the stronger duits that block water flow (Figure 9, E and F; Tyree and the drop in ww (or pressure) across the organ, or whole Zimmermann, 2002), However, in leaves and roots, water plant, between the soil and the atmosphere; conversely, the flows not only through xylem but also through living tissues stronger the ww gradient driving force, the stronger the flow (on the way from the soil to the root xylem, and from the rate through the system or any component. This electrical leaf xylem to the stomata). Before embolism forms in the analogy stands in for more detailed theory of fluid mechan- xylem, in dehydrating roots, cortical lacunae may form that ics and irreversible thermodynamics (Edlefsen, 1941; Slatyer break the hydraulic connection between the root xylem and and Taylor, 1960; Gibbs, 1961; Granger, 1995), and can have the soil (Figure 9, A and B; Cuneo et al., 2021), and in dehy- drawbacks (namely, when factors other than ww, such as drating stems and leaves, tissues shrink, which may influence thermal gradients drive water movement; Rockwell et al., flow pathways around and within cells, as the water channel 2014; Buckley et al., 2015). On the other hand, the analysis proteins that traverse cell membranes, known as aquaporins of the plant and its environment as a network of electronic (AQPs), can be gated even under mild dehydration (Scoffoni Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 87 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 9 The effects of drought on tissues across plant organs. Representative transverse X-ray micro-computed tomography images show the in vivo functional status of the vascular system and the surrounding tissues under well-watered (A, C, E) and drought (B, D, F) conditions in roots, stems, and leaves. In (A) and (B), the roots of grapevine (Vitis vinifera) the xylem network (XY) remains functional under a range of water poten- tials (ww) but mild drought (–1.5 MPa) leads to massive cellular damage and formation of cortical lacunae (CL in B) that physically decouple the vascular system from the surrounding soil (Cuneo et al., 2021). In (C) and (D), the vascular systems of the immature stems of conifer seedlings (Pseudotsuga menziesii) are highly drought tolerant (XY), but deformation of the cortex under strong drought in these resembles that in roots (Miller et al., 2020). In (E) and (F), leaves of ivy (Hedera canariensis), subjected to very strong dehydration (–4.0 MPa) show xylem cavitation in the midrib (XY) and the strong mesophyll tissue deformation that occurs during drought exceeding the turgor loss point (Scoffoni et al., 2017). Bars = 200 mm in (A) and (B); 100 mm in (C) and (D); 250 mm in (E) and (F). Values in the lower left corner of each panel indicate the ww of the plant during the experiment. et al., 2014; Figure 9, C–F). Given their extra-xylem pathways, challenge faced by plant organs will shift as drought pro- and despite similarity of their xylem in resistance to embo- ceeds, along with the distribution of different ww within the lism (Zhu et al., 2016a, 2016b; Klepsch et al., 2018; Li et al., plant. In drought mild enough that stomata remain open, 2020; Smith-Martin et al., 2020), leaves and roots tend to be transpiration causes ww to be lowest in the leaves, and given more hydraulically vulnerable than stems (Scoffoni et al., that the leaf is a bottleneck and highly vulnerable, K loss in 2017; Albuquerque et al., 2020; Cuneo et al., 2021). leaves can be drastic (Hernandez-Santana et al., 2016; Does the combination of the greater bottlenecks and vul- Scoffoni and Sack, 2017; Albuquerque et al., 2020). Leaf K nerabilities within leaves and roots than stems make these loss may, however, act as a brake on water loss by amplify- extremities the plant’s most sensitive fuses under strong ing stomatal closure (Scoffoni and Sack, 2017). In severe drought? Indeed, many have concluded that these organs droughts, where turgor is lost and stomata are fully closed, should be more vulnerable, to protect stem xylem from ten- the plant will be close to equilibrium with the soil, with all sions that would drive irreparable cavitation, as the stem is organs experiencing similar ww. As the soil and plant dehy- longer-lived and more costly to replace, ideas known as the drate further, exacerbated by the low “minimum con- “hydraulic-segmentation” and “vulnerability-segmentation” ductance” from incompletely closed stomata and/or across hypotheses (Tsuda and Tyree, 1997; Tyree and the cuticle (Martin-StPaul et al., 2017), the leaf xylem may Zimmermann, 2002; Pivovaroff et al., 2014). Yet, the suffer embolism, starting with major veins and proceeding 88 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. to minor veins, and this embolism may trigger leaf death 2021). In deciduous species, the leaves die first, and then the (Brodribb et al., 2021). When substantial cavitation occurs in buds on the stem, but in some species, roots apparently die the stem xylem, it usually kills the stem, because cavitation early on and spell the death of the plant (Sack, 2004). Plants tends to beget more cavitation, in a vicious cycle of with multiple stems and/or sectoriality among stems and “catastrophic embolism”—with gas bubbles spreading, roots may better survive drought due to redundancy and/or uncontrolled, among conduits (Tyree and Zimmermann, resprouting after mortality of stems and roots (Schenk et al., 2002). As for the roots, the vulnerability of their xylem and 2008; Zeppel et al., 2015; McElrone et al., 2021). Answering outside-xylem pathways may also be strong (Brunner et al., the critical question of the location of hydraulic bottlenecks, 2015; Rodriguez-Dominguez and Brodribb, 2020). Across spe- vulnerabilities, and triggers of mortality within plants, and of cies, this sequence of hydraulic decline—leaves before stems the traits with predominant influence across diverse plants, and roots—tends to be typical, and species’ thresholds for will open the door to the prediction of plant mortality and declines of K in organs and the effects of drought-induced ecosystem shifts, and the design of drought-hardy crop vari- damage are correlated (Bartlett et al., 2016; Dayer et al., eties. These imperatives are as urgent as readying our power Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 2020). grids for the storms of climate change. This overall simple scenario, however, has been tested in a few hundred plant species at best, and not in sufficient de- How does the continuing rise in CO2 affect tail to fully parameterize the hydraulic network for the bulk the regulation of stomatal apertures and of plants. Particularly little is known of the hydraulic vulner- water-use efficiency of plants? ability of roots, due to technical difficulties; recent studies (By Julian I. Schroeder and Po-Kai Hsu) on potted plants of a few species have proposed that the The atmospheric CO2 concentration is continuing to rise decline of hydraulic conductance in the root and/or root– and is now 50% larger than before the industrial revolu- soil interface can be strong enough even in relatively moist tion. This is resulting in increased absorption of infrared ra- soil to contribute to stomatal closure (Abdalla et al., 2021; diation by CO2, which in turn is causing temperatures to Bourbia et al., 2021; Duddek et al., 2022). Further, the poten- rise on Earth. Plants remove CO2 from the atmosphere via tial role of the root sheath and mycorrhizae in modulating photosynthesis. CO2 can be viewed as an abundant atmo- or perhaps protecting root and root interface hydraulic con- spheric fertilizer that contributes to plant growth, if ductance has yet to be fully clarified (e.g. Boomsma and nutrients and water are sufficiently available and depending Vyn, 2008; Brunner et al., 2015). Indeed, the fine roots are on the species and conditions (De Kauwe et al., 2021). more vulnerable than older roots, and their vulnerability Indeed, satellite monitoring of photosynthesis and plant needs separate quantification, especially if fine root death growth has shown global “greening” (Zhu et al., 2016a, and turnover occur even under mild soil moisture deficit 2016b; Chen et al., 2022). However, warming temperatures (Cuneo et al., 2021). globally can increase vapor pressure deficit, which arguably Given these numerous unknowns, the location of hydrau- may tend to counteract this greening trend (Zhu et al., lic triggers for declines in gas exchange and death is a critical 2016a, 2016b; Wang et al., 2020a, 2020b), with this hypothe- avenue for research. When enough hydraulic conductance is sis being a matter of debate and further studies being lost, water cannot be transported into and throughout the warranted. plant, and a spiral of mortality begins, potentially exacer- CO2 enters leaves for photosynthesis via stomatal pores. bated by carbon starvation and other biotic and abiotic Typically, plants lose 150 to 4500 water molecules via stresses (Choat et al., 2018; Hammond et al., 2022; stomatal transpiration for every CO2 molecule that is taken McDowell et al., 2022). Yet, water storage can buffer given in and assimilated by photosynthesis. The CO2 concentra- organs from loss of hydraulic conductance, and protect the tion itself is a regulator of the rapid stomatal closing and plant from dehydration, especially when the plant retracts opening responses. Elevated CO2 in the intercellular spaces its roots to prevent water loss to the soil. The general mag- of leaves, [CO2]i, occurs at night in C3 and C4 plants due to nitude and role of water storage “capacitors” is another, re- respiration, triggering stomatal closing. During light periods, lated critical unanswered question (McCulloh et al., 2014; photosynthesis reduces [CO2]i, which mediates stomatal Knipfer et al., 2019). Least of all is known about the triggers opening, together with a light-triggered signal transduction for death, and their timing and general order during dehy- network. The atmospheric [CO2] rise is adding to these diur- dration, for cells within tissues, tissues within organs, and nal changes in [CO2]i, thereby causing a narrowing of sto- organs throughout the plant. Indeed, new concepts are matal pores globally (Medlyn et al., 2001; Franks et al., 2013). needed—there is no generally agreed definition for the time This CO2 response can be beneficial to plants. Reduction in of death of an organ, tissue, or whole plant—and plants stomatal apertures resulting from elevated atmospheric may be incredibly diverse in this death pattern (Hammond [CO2] can enable plants to maintain photosynthetic CO2 as- and Adams, 2019). In many species, leaf cells are apparently similation rates, while losing substantially less water, thereby damaged or killed by dehydration below the turgor loss improving water use efficiency (WUE). However, there are point, but in resurrection plants, cells can recover limitations, including that many plants have a “weak” sto- completely (Stuart, 1968; Alpert, 2000; Prats and Brodersen, matal CO2 response (Raschke, 1975), thereby showing less Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 89 or no improvement in WUE. C4 plants show saturation of assimilation at relatively low [CO2]i levels and reduction of stomatal conductance may improve WUE depending on the species and conditions (De Kauwe et al., 2021). Moreover, this is also relevant for C3 plants (Doheny-Adams et al., 2012), including in forestry, where reduction in transpiration from trees could slow depletion of soil water content. On the other hand, in agricultural regions with sufficient rainfall, soil nutrients, and favorable growth conditions, the high CO2-induced reduction in stomatal apertures could limit photosynthesis. This applies particularly to C3 plants for which photosynthetic CO2 assimilation is not yet satu- rated at typical [CO2]i levels. C3 plants represent 85% of Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 plant species globally. Research is also needed to examine the impact of CO2-induced stomatal closing during heat stress, given that the CO2 response appears to be weaker at high temperatures and heat itself is a signal that promotes stomatal opening (Raschke, 1975). A better understanding of the molecular mechanisms that enable CO2 control of stomatal movements could aid in fu- ture molecular enhanced breeding-, engineering-, and/or gene editing-driven improvement of stomatal WUE traits that are better adapted to diverse environments in a high CO2 and climate change-challenged world. In recent years, advances have been made toward understanding the molec- ular mechanisms that cause CO2-regulated stomatal move- ments, with some key questions remaining to be resolved as highlighted here. Figure 10 Simplified model for CO2 sensing and signaling in stomatal Studies have shown CO2 sensing by guard cells, but also a closure. Schematic diagram of signaling components and pathways in role for the mesophyll (Mott et al., 2008), in sensing or am- CO2-induced stomatal closure. Black arrows indicate the directions of transport or enzymatic reaction in response to elevated CO2. Blue plifying the stomatal CO2 response. Forward genetic screens arrows and red blocks represent positive and negative regulation of have thus far identified guard cell localized molecular mech- high CO2-triggered stomatal closure, respectively. PIP2 AQPs (PIPs) anisms that function in CO2 control of stomatal move- and b-carbonic anhydrases (CA1/CA4) could facilitate CO2 influx and ments. Thus, the rapid mesophyll-derived signal remains one HCO 3 and proton production in guard cells. Protein kinases including of the open questions. We discuss guard cell CO2 signaling MPK4/MPK12, HT1, and CBC1/CBC2 are known early signaling com- mechanisms and open questions in the following ponents involved in regulating ion transporters. However, many de- (Figure 10). In brief, CO2 entry into guard cells could be fa- tailed mechanisms in CO2 sensing and signal transduction remain to cilitated by the CO2-permeable PIP2 aquaporins (Mori et al., be investigated as indicated by dash arrows and red dashed blocks. Basal ABA signaling and basal OST1/SnRK2 kinase activities facilitate 2014; for review Zhang et al., 2018a). Carbonic anhydrases in high CO2-induced stomatal closure. A receptor-like pseudokinase guard cells accelerate the stomatal response to CO2 shifts in GHR1 is required for SLAC1 activation under CO2 elevation and sev- Arabidopsis, rice, and maize (Hu et al., 2010; Chen et al., eral other environmental stimuli. 2017; Kolbe et al., 2018). Carbonic anhydrases mediate re- versible catalysis of CO2 in guard cells to bicarbonate and protons. The bCA4 isoform of carbonic anhydrases is lo- A forward genetic CO2 response screen identified recessive cated at the plasma membrane of guard cells and interacts high temperature 1 (ht1) mutant alleles in a RAF-like protein directly with the PIP2;1 aquaporin (for review, Zhang et al., kinase that cause a strong insensitivity to low CO2-induced 2018a). Studies have suggested that intracellular bicarbonate stomatal opening, while showing responsiveness to blue light (HCO 3 ) plays an important role as a second messenger in and ABA (Hashimoto et al., 2006). Mapping of natural var- transducing the CO2 signal in guard cells (e.g. Hu et al., iants in WUE and in stomatal regulation have independently 2010). However, the primary bicarbonate/CO2 sensor in identified the mitogen-activated protein kinase MPK12 as a guard cells that controls stomatal movements has remained rate-limiting genetic locus (Des Marais et al., 2014; Jakobson elusive (Note that a secondary sensor has been identified, as et al., 2016). Mechanistic research has shown that in discussed later). Identification of the primary HCO 3 /CO2 Arabidopsis double mutants of mpk12 together with the sensor that controls stomatal movements will be a key to close homolog mpk4 and in tobacco silencing of the close modifying dynamic CO2-dependent WUE. homolog NtMPK4 disrupt high CO2-induced stomatal 90 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. closing, while ABA-induced stomatal closing remains intact even though CO2 increases did not rapidly change their ac- (Marten et al., 2008; T~oldsepp et al., 2018). Neither MPK12 tivities (Hsu et al., 2018; Zhang et al., 2020). Thus, the link nor MPK4 protein kinase activities were found to be regu- from early CO2 signaling mechanisms to downstream targets lated by CO2/HCO 3 directly in vitro under several examined that mediate stomatal closure remains to be discovered. conditions (T~ oldsepp et al., 2018). There is still much to learn about how CO2 regulates sto- Two other RAF-like protein kinases were discovered, matal apertures, from CO2/HCO 3 sensors to a biochemical CONVERGENCE OF BLUE LIGHT AND CO2 (CBC1 and and physiological understanding of the signaling network, CBC2), for which double mutants show an impairment in which could drive future improvements in WUE of plants low CO2-induced stomatal opening (Hiyama et al., 2017). In including trees, depending on the species, with a need for contrast to recessive ht1 mutant alleles (Hashimoto et al., future quantitative field research (De Kauwe et al., 2021). 2006), cbc1/cbc2 double mutants disrupt blue light-induced Furthermore, leaf-level stomatal conductance models are a stomatal opening as well (Hiyama et al., 2017). Therefore, crucial part of Earth system climate models, and molecular CBC1 and CBC2 are proposed to represent a convergence insights could improve the accuracy of these models. Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 point of low CO2 and blue light-mediated stomatal opening (Hiyama et al., 2017). HT1 can phosphorylate CBC1 in vitro How can one aquaporin have so many roles (Hiyama et al., 2017), but the physiological relevance of this in a plant? reaction for CO2-mediated stomatal regulation is unknown. (By Stephen D. Tyerman) The carbonic anhydrases (bCAs), HT1, and MPK12/MPK4 Much research on plant AQPs has assessed their impact on proteins function in the early guard cell specific CO2 re- water transport across membranes in response to drought sponse pathway as positive regulators (bCAs, MPK12/MPK4) and salinity stress, but low temperature, anoxia, and nutrient and negative regulators (HT1) (Figure 10). Downstream of stress and combinations also feature (Kapilan et al., 2018). early CO2 signaling, a network of guard cell ion channels, Many studies show that overexpression of an AQP can con- pumps, transporters, and regulators in the plasma mem- fer tolerance to these stresses, sometimes multiple stresses, brane and vacuolar membrane as well as metabolic but it is by no means clear how such stress tolerance occurs responses (e.g. Flütsch et al., 2020) mediate CO2-regulated in the strict context of water transport. Some AQPs, origi- turgor driven stomatal movements. Elevated CO2 activates nally designated as true water channels, have been shown to both slow “S-type” and rapid “R-type” anion channels in transport multiple substrates besides water, including signal- guard cells (Raschke et al., 2003). Elevated CO2 can be pre- ing molecules (e.g. H2O2), neutral substrates for synthesis dicted to inhibit plasma membrane proton pumps that (CO2, O2, and NH3), and even ions (Na + , K + , and NO3–) drive stomatal opening, although direct evidence is needed. (Tyerman et al., 2021) that can all feature in responses to Furthermore, elevated bicarbonate enhances the activity of abiotic stress or photosynthesis (Ermakova et al., 2021). the S-type anion channel SLAC1 in heterologous cells and in AQPs are also under the control of many hormones (Maurel guard cells. Residues in SLAC1 that are required for this re- et al., 2021) and are important for regulating growth (Wang sponse have been identified and the respective SLAC1 et al., 2020a, 2020b). However, genetic evidence indicating mutants show impaired CO2 regulation, but intact ABA reg- AQP involvement in stress resistance, for example, the loca- ulation, of stomatal closing in intact plants, leading to the tion of AQP genes under stress-related QTLs, is uncommon model that SLAC1 can function as a secondary CO2/HCO 3 (Hostetler et al., 2021a), perhaps due to their complex regu- sensor in guard cells (Zhang et al., 2018b). Since SLAC1 acti- lation (Grondin et al., 2016) and multifunctionality. vation is known to require phosphorylation, the upstream The plant AQPs can be divided into several subfamilies primary CO2/HCO 3 sensor remains, however, unknown (Figure 11A) and not all of them are good water channels. (Zhang et al., 2018b). Within the NIPs (mainly), as well as some PIPs and XIPs, An important question remains on how early CO2 signal- transported substrates include metaloids, protonated or- ing mechanisms control these mediators of stomatal move- ganic acids, or metal complexes (Tyerman et al., 2021). ments. Research suggested that the elevated CO2 response Water can also be transported but not always. In each case, is mediated by the ABA receptor signaling pathway (Dittrich the transport is passive (down-hill) in response to the free et al., 2019). However, CO2-regulated stomatal conductance energy gradient for water, concentration gradients for the findings in ABA receptor mutants (Dittrich et al., 2019) solute, or electrochemical gradients for those shown to also could not be confirmed using several approaches and pass ions (Tomkins et al., 2021; Tyerman et al., 2021). showed CO2 responsiveness (e.g. Hsu et al., 2018; Zhang Multiple substrate transport through the same protein at et al., 2020). Moreover, recent research has led to the unex- the same time (e.g. ions and water, CO2 and water, and pected findings that CO2 triggers stomatal closing without H2O2 and water) could lead to interactions (Tyerman et al., further activating SNF1-related protein kinase2 (SnRK2s), in- 2021). It remains to be seen how some substrates permeate cluding OST1 (Hsu et al., 2018; Zhang et al., 2020), that are and the fifth pore through the center of the tetramer is a activated by ABA. Moreover, basal ABA levels and a basal candidate for ions and CO2 (Figure 11, B–E). activity of SnRK2/OST1 protein kinases were found in guard What may still occur, even for passive transport, is rectifi- cells, and these are required for amplifying the CO2 response cation (i.e. a greater flow or diffusion in one direction Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 91 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 11 How an aquaporin (AQP) can be multifunctional. A, AQP family with those indicated to transport ions electrogenically (under each subfamily, ? =maybe, Loc= membrane location for specific isoform, note many NIPs are located on the plasma membrane) (Tyerman et al., 2021). B, Features of PIP2;1 showing transmembrane helices (TM1-6), loops (A–E) and half helices forming the central selectivity NPA filter. Red dots = phosphorylation sites. C, Folded monomer structure (SoPIP2;1 PDB 1z98) (Törnroth-Horsefield et al., 2006). D, Homotetramer as the functional unit with four monomeric pores that transport water (blue arrows, dots) and probably H2O2, also NH3 in TIP2;1 (Kirscht et al., 2016) and the cen- tral pore (red arrow, dot) implicated in ion transport for mammalian AQP1 (Henderson et al., 2022). AQPs also likely form heterotetramers (e.g. ZmPIP1;2 + ZmPIP2;1) (Chaumont and Tyerman, 2014). E, View normal to the membrane plane on the cytoplasmic face. F, Hypothesis for multi- functionality of PIP2;1. Phosphorylation by specific kinases occurs at one or more of the sites on cytoplasmic loops like a digital dipswitch that de- termine protein interactions and/or substrate selectivity, for example, ion water reciprocity (Qiu et al., 2020) or H2O2 permeation (Rodrigues et al., 2017), or protein interactions (Prado et al., 2019). Selectivity is fixed to a certain degree in the monomeric pores by the structure of the ar/R selec- tivity filter and other residues. The hydrophobic central pore may still allow ion permeation and is proposed to be gated at a low probability de- pendent on monomer gating (Tyerman et al., 2021). There are likely feedbacks (small blue arrows) due to some substrates and protein interactions also affecting the signaling that determines the kinase/phosphatase activity. compared with the other depending on the direction and studied for plant AQPs at the molecular level though it was magnitude of the gradient) as is well known for some ion well studied in the past for water transport across plant cell channels. The aromatic/Arginine (ar/R) selectivity filter pre- membranes. This could occur for water to be trapped within sent on the lumen side of the monomeric pore may give the root xylem (Pascut et al., 2021) but reverse flow through rise to voltage and ion dependence (Mom et al., 2021) that a root with reversed gradients would argue against this. Ion could lead to rectification. Rectification has not been well flow through AtPIP2;1 and AtPIP2;2 can show rectification 92 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. with certain divalent cations (Ca2 + and Mg2 + ) present signaling for ABA responses due to H2O2 permeation (Ding (Kourghi et al., 2017). et al., 2021) but also for its water permeation in the root Some PIP AQPs can account for significant portions of (Ding et al., 2020). shoot and root hydraulic conductivity (Lpr) (Prado et al., Many introductions to papers on AQPs describe them as 2019; Ding et al., 2020; Domec et al., 2021). To determine exclusively important for water transport, implying that the contribution of AQPs to Lpr requires a combination of there is only water transport. But the situation now emerg- sophisticated models and measurements of water transport ing is far more complex and the PIPs seem to be implicated (Ding et al., 2021; Knipfer et al., 2021). The regulation of in various aspects of plant water relations that are more root AQPs in response to abiotic stress such as salinity and than just water transport across a membrane. This makes anoxia via gating or removal from the membrane results in interpretation of phenotypes in both reverse and forward rapid changes in Lpr (Tournaire-Roux et al., 2003; Boursiac genetics rather challenging. The question as to how a single et al., 2008). Shoot signals are also implicated in regulation AQP can function in such a broad range of cell types with of root AQPs (Chaumont and Tyerman, 2014). Changes in different transported substrates or signaling roles is Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 the Lpr can have large effects on stomatal conductance, addressed by the hypothesis in Figure 11F. The answer may shoot water relations, and growth (Ding et al., 2020; Knipfer lie in a digital-like switch through posttranslational modifica- et al., 2021) and perhaps ion content of the shoot related to tions and signaling pathways combined with many and var- the reflection coefficient of the root (Knipfer et al., 2021) or ied protein interactions. the capacity of some AQPs to transport ions. Ultimately, the amount of water relative to the amount of ions transported How do plants balance growth and abiotic to the xylem determines the xylem ion concentration for stress responses? delivery to the shoot. A common signal element that links (By Taishi Umezawa) plant Na + /K + ratios under salinity with control of AQPs in Plant growth is affected by many environmental factors that the root is the production and transport of H2O2 (Ma et al., alter the balance of energy use (Crepin and Rolland, 2019). 2011; Martiniere et al., 2019). Under favorable environmental conditions, plants are able Taking the PIP subfamily (with two clades; PIP1 and PIP2) to assign the energy obtained from photosynthesis to as the best studied example of multifunctionality, one PIP2 growth, especially during the vegetative growth phase. isoform can be permeable to water, cations, CO2, and the Conversely, under adverse environmental conditions, plants signaling molecule H2O2. For the cases of cations, water, and must redirect their energy to stress responses to overcome H2O2, phosphorylation on certain residues appears to be key the challenge and ensure individual survival. However, when (Figure 11) though there are some variations in the litera- stress conditions are prolonged, it is not always a suitable ture regarding the impact on water flow. Arabidopsis strategy to inhibit growth indefinitely. Under such condi- AtPIP2;1 has been shown to be required for circadian varia- tions, it may be advantageous for plants to maintain growth tion in rosette Lp with 14-3-3 proteins depending on to some extent, or to switch to their reproductive stage to C-term phosphorylation (Prado et al., 2019), for auxin regu- preserve the next generation. It is likely that plants have lation of lateral root outgrowth (Peret et al., 2012), CO2 up- evolved to adjust their energy balance precisely in response take into guard cells with a carbonic anhydrase (Wang et al., to abiotic stresses, because they are often forced to make 2016), H2O2 signaling in guard cells dependent on a Loop B such decisions throughout their life cycles. The question is phosphorylation (Rodrigues et al., 2017), and univalent cat- how plants tilt the balance toward stress responses or to- ion transport dependent on C-term phosphorylation (Qiu ward growth regulation (Figure 12). et al., 2020). The very similar AtPIP2;2, also permeable to In the case of drought, plant responses to this stress have cations but with increased sensitivity to Ca2 + compared been divided into three alternative strategies: drought with AtPIP2;1 (Kourghi et al., 2017), contributes to Lpr de- tolerance, drought avoidance, and drought escape (Kooyers, rived from root exudation (Lprex) but not Lpr derived from 2015). For short-term drought, drought avoidance or toler- pressure gradients (Lprpres) (Javot et al., 2003). Root exuda- ance can be effective. For example, it is well known that tion is important for xylem water continuity and may be plants can quickly close their stomata to prevent water loss more complicated than ion pumping into the xylem with from the leaf surface. Similarly, plant cells can rapidly adjust the subsequent osmotic gradient driving flow (Schenk et al., their osmotic potential to maintain water status (Zhu, 2002; 2021). Interestingly, AtPIP1;2 when knocked out reduces Yamaguchi-Shinozaki and Shinozaki, 2006). Molecular mech- Lprpres but not Lprex pointing to an interesting division of la- anisms that turn on such stress responses have been well bor that may depend on different locations of the two studied, especially responses induced by the phytohormone AQPs (Postaire et al., 2010). Another PIP2 from rose is impli- ABA. The major ABA signaling pathway consists of three cated in a drought signaling hub releasing a membrane core components: ABA receptors (PYL/RCAR), Clade A of bound transcription factor depending on phosphorylation the type 2C protein phosphatases (PP2C), and SnRK2 kin- status and environmental signals to control growth under ases (Cutler et al., 2010; Umezawa et al., 2010). Under nor- water stress (Zhang et al., 2019). The well-studied maize mal conditions, PP2Cs directly dephosphorylate and PIP2;5 is also multifunctional being implicated in guard cell inactivate SnRK2s (Umezawa et al., 2009; Vlad et al., 2009). Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 93 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 12 SnRK2 kinases regulate growth and stress response under drought. A, Under normal conditions, plants use energy to grow, and TORC or Raf36 are involved in this process. B, Under drought conditions, SnRK2s are activated and phosphorylate substrates, for example, bZIP transcrip- tion factors or slow anion channels (SLAC), to induce stress responses. SnRK2s also phosphorylates TORC or Raf36 to inhibit plant growth. In response to ABA, this inhibition is abated and active In nature, sudden and severe drought stress on plants is SnRK2s can phosphorylate various protein substrates to in- not likely to occur. In most cases, drought stress gradually duce ABA-associated responses including stomatal closure increases in stages. When drought stress is mild and pro- and large-scale changes in gene expression. Since SnRK2s are longed, it is likely not beneficial for plants to spend energy central players in drought responses, many studies have only on stress responses, and under such conditions, plants used SnRK2s as a starting point to identify signaling proteins may continue to grow as part of their drought avoidance or involved in ABA or osmotic stress signaling (Wang et al., escape strategies (Kooyers, 2015). For example, root growth 2018a; Kamiyama et al., 2021). often increases during mild drought as a means to increase In addition to their central role in ABA signaling, SnRK2s access to available water. In rice, a root angle QTL, DRO1, also function to regulate plant growth under drought stress was shown to be effective for selection of drought tolerance conditions. Recently, SnRK2s were shown to directly phos- in rice, demonstrating that drought avoidance is one of the phorylate Raptor, a component of the TOR complex promising breeding targets for drought resistance (Uga et al., (TORC) that regulates plant growth (Figure 12). Under stress 2013). conditions, phosphorylation of Raptor by SnRK2 resulted in In the drought escape response, plants accelerate floral de- velopment and the transition to the next generation dissociation of TORC and inhibition of plant growth (Wang (Kooyers, 2015). ABA is involved in early flowering as a et al., 2018a, 2018b). Separately, a recent study demon- drought escape response, and multiple pathways are pro- strated that Raf36, a group C Raf-like protein kinase, pro- posed to link ABA and flowering (Martignago et al., 2020). motes growth under normal conditions, and is degraded in For instance, several bZIP transcription factors, AREB/ABFs, response to ABA by SnRK2-dependent phosphorylation are phosphorylated by SnRK2s and involved in drought es- (Kamiyama et al., 2021). These two examples highlight an cape (Hwang et al., 2019). In addition, previous studies pro- important role for SnRK2s not only for ABA-dependent posed that the photoperiodic flowering pathway, consisting stress responses, but also for mediating growth inhibition of GIGANTEA (GI), CONSTANS, and FT, is essential for early under short-term and severe drought stress. In addition to flowering (Riboni et al., 2013, 2016). However, the connec- the SnRK2 pathway, Clade E Growth-Regulating (EGR) phos- tion between ABA and GI-dependent FT pathway is still un- phatases and Microtubule-Associated Stress Protein 1 are in- der investigation. volved in growth regulation during drought stress Drought stress is not constant and stress intensity fluctu- (Longkumer et al., 2022). It is expected that identifying and ates over time in nature. Once drought stress reaches a cer- functionally characterizing additional SnRK2 or EGR sub- tain level, plants cross a threshold and change the balance strates will aid our understanding of the mechanisms of between growth and stress response. It will be important to growth inhibition under drought stress. identify the molecular switch involved in such a stage-gate 94 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. of drought stress. Furthermore, if the intensity of drought to the pentose-phosphate pathway in a redox cycle (Hare stress changes frequently, plants can acquire stress memory. and Cress, 1997). However, this proposal has been little It is known that some epigenetic modifications could be in- tested. volved in stress memory (Sharma et al., 2022) and may regu- ProDH1 and P5CDH are also scavengers in that plants can late intra- or inter-generational responses to stress use proline as an alternative respiratory substrate during se- conditions. This is another topic to be clarified. nescence and dark-induced starvation (Zhang and Becker, As discussed in this section, plant growth regulation is 2015). This is facilitated by the fact that ProDH transfers re- complex, and current knowledge of plant growth regulation ductant directly to ubiquinone via its FADH cofactor under stress conditions is just beginning to scratch the sur- (Zheng et al., 2021). During stress recovery (after restoration face. Further studies will be required to understand how of water supply), when proline levels rapidly decline, or in plants balance stress response and growth regulation, and in response to exogenous proline, ProDH1 and P5CDH expres- depth understanding of such mechanisms could facilitate sion is induced (Figure 13) and proline catabolism can feed molecular breeding for yield and quality of agricultural pro- so much reductant into mitochondrial electron transport Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 duction under drought conditions. that some of it needs to be vented off by alternative oxidase Proline metabolism: protector, scavenger, or to prevent oxidative stress (Oh et al., 2022). The rapid catab- olism of proline after re-watering may be a way to channel executioner? the nitrogen from proline to other amino acids needed dur- (By Paul E. Verslues) ing the resumption of growth. Proline is highly soluble and zwitterionic, hallmarks of com- Interestingly, p5cs1 mutants, which have greatly reduced patible solutes that accumulate to reduce cellular osmotic proline accumulation, and prodh1 mutants, which have in- potential while also protecting protein and membrane struc- creased proline accumulation, have similar low ww-sensitive ture from dehydration-induced damage. However, making phenotypes (Sharma et al., 2011; Bhaskara et al., 2015). Low proline is not the only impact of proline metabolism and ww stress leads to ProDH1 downregulation in most of the the protective role of proline itself is not the only purpose plant tissues. However, meristematic and growing cells have of stress-induced proline accumulation (Bhaskara et al., steady or increased ProDH1 and P5CDH expression during 2015; Alvarez et al., 2022). The proline cycle (Figure 13) con- low ww stress (Sharma et al., 2011). This indicates that the sists of synthesis from glutamate by D1-pyrroline-5-carboxyl- proline cycle, with the synthesis versus catabolism sides of ate (P5C) synthetase (P5CS) and P5C reductase (P5CR) the cycle spatially separated, can also facilitate the move- while proline catabolism back to glutamate is catalyzed by ment of reducing potential, stored in the form of proline, proline dehydrogenase (ProDH) and P5C dehydrogenase away from photosynthetic tissue where it is in excess, to (P5CDH). In Arabidopsis, P5CS1 and ProDH1 are the most root and meristem tissue where proline is used for energy stress-responsive of the proline cycle genes and the proteins metabolism or osmotic adjustment (or in other words: to they encode catalyze the rate-limiting steps of proline syn- be a good protector, it is important to know when to also thesis and catabolism, respectively. One protective function be a scavenger). of the proline cycle is to regenerate NADP + to help ensure Proline metabolism shows its executioner side during the the supply of a safe electron acceptors during stress and hypersensitive response (HR) to incompatible pathogen in- when leaf CO2 becomes limited (Hebbelmann et al., 2011; fection (Figure 13). Proline accumulation mediated by P5CS2 Sharma et al., 2011; Signorelli, 2016). Both P5CS1 and P5CR and proline catabolism by ProDH1 and ProDH2 is required prefer NADPH over NADH as an electron donor (Giberti for HR-induced cell death and associated ROS burst (Fabro et al., 2014; Forlani et al., 2015; Sabbioni et al., 2021) and et al., 2004, 2016; Cecchini et al., 2011; Senthil-Kumar and p5cs1 mutants have a reduced NADP + /NADPH ratio during low water potential (ww) stress (Sharma et al., 2011). p5cs1– Mysore, 2012). During infection, cells marked for death have 4 also has substantial changes in photosynthesis-related up-regulated expression of P5CS2, P5CR, and ProDH1 but gene expression (Shinde et al., 2016). How such a proline P5CDH, leading to partial proline catabolism that is associ- synthesis-photosynthesis redox link could work depends on ated with cell death (Alvarez et al., 2022; Figure 13). the subcellular localization of P5CS1 and P5CR and how it is Whether the cell death is caused by proline-dependent ROS affected by stress. P5CS1 is likely to be localized in the cyto- production or a yet unknown signaling function of P5C (or plasm; however, this is ambiguous as both cytoplasmic a combination of the two) is unclear. P5C is the common (Funck et al., 2020) and chloroplast, or chloroplast- intermediate of both proline synthesis and catabolism and it associated, localization (Székely et al., 2008) have been has also been proposed that P5C may be exported from the reported. Similarly, fluorescently tagged P5CR was localized mitochondria and used for proline synthesis in the cyto- mainly in the cytoplasm (Funck et al., 2012) while biochemi- plasm, thus forming a proline–P5C cycle which could am- cal or immunoblot assays indicated that it could also be plify ProDH-dependent ROS production in the present in plastids (Szoke et al., 1992; Murahama et al., mitochondria (Miller et al., 2009). However, evidence sup- 2001). The preference of P5CS1 and P5CR for NADPH is also porting such a P5C cycle in plants is circumstantial and a consistent with the proposal that proline synthesis is linked mitochondrial P5C translocator has not been identified. Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 95 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 13 The protector, scavenger and executioner modes of proline metabolism and the conditions or tissue in which they are observed. Up and down arrows (red, brown, or gray) indicate changes in gene expression compared with unstressed control. In most cases where data are avail- able, protein levels of the corresponding enzyme change in the same direction. Thickness of the red or purple arrows represents predicted relative metabolic flux through different steps in proline metabolism. Abiotic stress refers to drought, freezing or high salt treatments where sustained ac- cumulation of free proline is observed in many plant species. Whether proline metabolism operates in protector, scav- excess reducing potential, including alternative oxidases and enger, or executioner mode depends on unknown environ- uncoupling proteins. Posttranslational modifications of mental and metabolic signals. Thus, proline metabolism is P5CS1 and ProDH1 (Alvarez et al., 2022) or interactions not only interesting in its own right in terms of how it pro- with regulatory proteins (Ren et al., 2018) are likely to affect tects or kills plant cells, it is also a useful model to discover their enzymatic properties but the roles of such factors in new aspects of stress signaling. For example, what sensing controlling the protector–scavenger–executioner modes of and signaling events occur during drought stress to allow proline metabolism are unknown. For P5CS1 and P5CR, high levels of proline to accumulate without inducing knowledge of their subcellular localization is also strikingly ProDH1 and without having proline metabolism switch into limited. Surprisingly, Savoure and co-workers have reported executioner mode to promote cell death (Miller et al., that a prodh1prodh2 double mutant, in which the only two 2005)? This is especially interesting as the sensing and up- ProDH genes in the Arabidopsis genome are knocked out, is stream signaling mechanism(s) plants use to detect and re- viable despite having no known way to catabolize proline spond to reduced water availability during drought stress (Alvarez et al., 2022). Is there a metabolic work around that remain unknown. For proline metabolism, the relative fluxes allows these plants to metabolize proline? And, Figure 13 through different parts of the proline cycle (indicated by dif- depicts a mitochondrial proline–glutamate exchanger and ferent arrow thicknesses in Figure 13) are inferred from gene such an activity, along with that of mitochondrial proline expression or protein levels of proline metabolism enzymes importer(s), has been biochemically observed (Di Martino but there is little information on actual metabolic flux rates et al., 2006); however, the genes encoding these activities re- through the proline cycle under different conditions. This is main unknown. important information for determining the conditions where Perhaps one of the most striking paradoxes of proline and proline catabolism is rapid enough to either significantly stress, given all the evidence of the importance of proline to contribute to respiration (scavenger mode) or significantly stress resistance, is that some plants apparently do without increase ROS levels (executioner mode) and how this is co- it. For example, most Arabidopsis accessions accumulate ordinated with mitochondrial mechanisms to dissipate high levels of proline during low ww stress; but a few have 96 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. very low levels of P5CS1 and greatly reduced levels of pro- different temperatures. From a population perspective, it is line accumulation similar to p5cs1 knockout mutants (Kesari possible to harness the power of genetics to identify geno- et al., 2012). Are these accessions more sensitive to drought mic regions and loci that show signatures of natural selec- (or salt) stress, or have they found a substitute for the tion in populations from different locations (Hancock et al., stress-protective (and executioner) effects of proline metab- 2011). Another strategy is to identify the underlying temper- olism? Also, some plants adapted to chronically dry condi- ature sensors in genetic screens or using a candidate gene tions have relatively low levels of free proline accumulation approach. This method is complicated by the often pleiotro- but instead convert proline to proline betaine or hydroxy- pic and redundant nature of temperature signaling path- proline betaine as these may be more effective osmoprotec- ways, reflecting their central role in many essential tants (Hanson et al., 1994). However, the implications of this responses. The use of carefully designed and controlled tem- conversion for the proline cycle are unknown and these perature screens can, however, overcome some of these compounds are likely to be more difficult to catabolize, thus issues. Sensors may then be investigated for natural variation impeding redeployment of nitrogen and reducing potential in the context of their mode of action. This approach has Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 when the stress subsides. the advantage that it is targeted and enables a test of func- Is proline metabolism a protector, scavenger, or execu- tionality, as well as identification of variation which directly tioner? It depends. Depends on what is the real question; a impacts temperature signaling. We will discuss some well- question whose answer will reveal much about the sensing, studied temperature responsive networks that suggest path- signaling, and metabolic mechanisms that plants use to ways to adaptation. cope with abiotic stresses that are of increasing concern for a warming and changing world. Vernalization Vernalization, the response of plants to prolonged cold, is a Temperature sensing: how do plants adapt to classical temperature response in Arabidopsis. Extensive nat- different climates? ural variation exists at the level of the major regulators FLC and FRI (Shindo et al., 2005; Figure 14). The complex nature (By Philip A. Wigge) of FLC silencing in response to cold via a proposed antisense A remarkable feature of plants is their ability to adapt to a RNA also lends itself to natural variation and modulation in wide range of climates, occupying almost every niche from terms of the extent and duration of cold requirement the tropics and hot springs to polar regions. To do this, (Duncan et al., 2015). plants have evolved an array of responses to temperature, over multiple scales, from minutes to months, which enable Thermomorphogenesis a suite of developmental and cell biological responses to Accelerated growth in response to warm temperature and maximize survival. Understanding how plants are able to flowering in Arabidopsis is termed thermomorphogenesis. adapt to different climates is a major open question, and of This process is dependent on enhanced activity of PIF4 particular relevance during a period of unprecedented rapid (Quint et al., 2016). PIF4 is regulated posttranslationally by global heating (Battisti and Naylor, 2009). the thermosensor phytochrome B (phyB). phyB measures Broadly, we can consider active and passive responses to temperature through its dark reversion rate (Jung et al., temperature. Passive responses refer to adaptations such as 2016a; Legris et al., 2016). Different rates of dark reversion membrane fluidity and protein stability. Proteins at high can evolve orthogonally to light sensing and cause a corre- temperature tend to denature and unfold. Thermophiles sponding change in temperature sensitivity. This could en- therefore have proteins with increased ionic interactions able thermorphogenesis to be tuned to the local and a larger stable hydrophobic core. At low temperature, environment. In addition to the identification of phyB in a there is reduced molecular motion due to low entropy and natural variation screen for thermal responsiveness, the gene enthalpy, and psychrophilic organisms adapt by having pro- EARLY FLOWERING3 (ELF3) was also identified (Box et al., teins with fewer salt bridges and hydrogen bonds to facili- 2014; Figure 14). ELF3 contains a thermoresponsive pre- tate flexibility (Brininger et al., 2018). dicted prion domain (Jung et al., 2020). This has been shown A major strategy of plants has been to evolve active tem- to have extensive natural variation in the length of a polyQ perature sensing and response pathways. These enable the repeat (Undurraga et al., 2012), and variation both anticipation of both seasonal temperature shifts as well as within Arabidopsis as well as with other plants such as shorter term temperature stresses. Temperature measure- Brachypodium distachyon directly changes temperature re- ments over the year, in concert with photoperiod, enable sponsive behavior. The polyQ region is encoded by a short plants to avoid unfavorable seasons in a dormant stage, and tandem repeat (STR), which can expand or contract during grow and flower during suitable months. While there is replication through DNA polymerase slippage, enabling the enormous diversity in the habitats and climates that plants generation of functional variation in ELF3 (Kashi and King, have adapted to, conservation of major signaling compo- 2006). Since the thermal responsiveness of the protein is nents appears to be a common theme. proportional to the size of the repeat, this may allow for ad- Broadly, two major approaches have been followed to aptation to different temperature ranges. It will be interest- identify the genes and mechanisms underlying adaptation to ing to see if this STR-based mechanism occurs in other Fifteen unknowns in abiotic stress THE PLANT CELL 2023: 35; 67–108 | 97 Downloaded from https://academic.oup.com/plcell/article/35/1/67/6677234 by guest on 13 April 2023 Figure 14 Temperature response mechanisms. Temperature controls many key adaptive traits in plants such as freezing tolerance, growth, flower- ing, and heat stress protection. Vernalization results in the stable repression of expression of the floral repressor FLC. Thermomorphogenesis inte- grates temperature information from at least three distinct biophysical responses: protein phase separation of ELF3, thermal reversion of phyB (Pfr to Pr) and enhanced translation of PIF7 in warm conditions. Heat stress also induces the induction of protective heat shock proteins via the activa- tion of HSFs via an unknown mechanism. temperature-responsive proteins, as has been suggested for independent sensors, while the heat stress is similarly inde- Drosophila (Sawyer et al., 1997). An additional thermosen- pendent from thermomorphogenesis. Nevertheless, it is sory mechanism is displayed by the RNA secondary struc- plausible to propose as few as 10–20 distinct temperature ture in the 50 UTR of PIF7, which facilitates enhanced sensors may account for most of the transcriptional translation at higher temperatures (Chung et al., 2020). responses to warm and cold temperatures observed in Since 50 UTR sequences can evolve independently of protein Arabidopsis. Thermomorphogenesis is perhaps the most function, this represents a mechanism to alter the levels of well-studied system, and in this case, it is interesting that protein rapidly in response to temperature. It is not known multiple discrete sensors act at different scales and levels if this is a widespread mechanism in plants. (transcriptional, translation, and posttranslation). Such re- dundancy may represent a mechanism to filter the inherent Temperature stress noise from temperature signals. How temperature informa- Survival of freezing stress is mediated by the CBF transcrip- tion is remembered and integrated over many months is tion factors in Arabidopsis, which are activated by cold per- also not understood. Heat stress in the field occurs in a ception (Jaglo-Ottosen et al., 1998). Heat stress responses complex environmental context, often with drought stress, are activated by the conserved heat shock factors (HSFs), and therefore how these various stresses are integrated is which activate protective heat shock proteins. In plants, important. Analysis of natural variation and field studies of a HSFs have undergone a remarkable expansion from 1 to 3 broader range of plants will also be critical for understanding family members in yeast and mammals to 21 members in mechanisms by which sequence variation can achieve rapid Arabidopsis (Baniwal et al., 2004). The basis for this is not changes in the temperature response range of thermosen- clear, but suggests an important role for this family of TFs in sors. This knowledge will enable the rational editing of crops mediating plant survival (Figure 14). to enhance thermal resilience. Critical to understanding adaptation to climate will be de- termining how many temperature sensors are present in Acknowledgments plants. The very distinct genetic and physiological responses The authors apologize to those researchers whose important to cold during vernalization and cold stress suggest work was not cited due to space limitations. J.I.S. and P.-K.H. 98 | THE PLANT CELL 2023: 35; 67–108 Verslues et al. dedicate their section to the memory of Klaus Raschke References (1928–2022) and his many contributions to stomatal biology 1001 Genomes Consortium (2016) 1,135 Genomes reveal the global and thank Peter Franks (University of Sydney) and pattern of polymorphism in Arabidopsis thaliana. Cell 166: Nattiwong Pankasem (UC San Diego) for helpful comments. 481–491 S.D.T. thanks Dr Caitlyn Byrt, Dr Jiaen Qiu, Dr Samantha Abdalla M, Carminati A, Cai GC, Javaux M, Ahmed MA (2021) Stomatal closure of tomato under drought is driven by an increase McGaughey, Nicholas Booth, Prof. David Day, and Prof. in soil–root hydraulic resistance. Plant Cell Environ 44: 425–431 Andrea Yool for inspiring discussions. 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