REVIEW published: 22 January 2020 doi: 10.3389/fpls.2019.01676 Drought Resistance by Engineering Plant Tissue-Specific Responses Damiano Martignago , Andrés Rico-Medina , David Blasco-Escámez , Juan B. Fontanet-Manzaneque and Ana I. Caño-Delgado * Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona, Spain Drought is the primary cause of agricultural loss globally, and represents a major threat to food security. Currently, plant biotechnology stands as one of the most promising fields when it comes to developing crops that are able to produce high yields in water-limited conditions. From studies of Arabidopsis thaliana whole plants, the main response mechanisms to drought stress have been uncovered, and multiple drought resistance genes have already been engineered into crops. So far, most plants with enhanced drought resistance have displayed reduced crop yield, meaning that there is still a need to search for novel approaches that can uncouple drought resistance from plant growth. Our Edited by: Oscar Lorenzo, laboratory has recently shown that the receptors of brassinosteroid (BR) hormones use University of Salamanca, tissue-specific pathways to mediate different developmental responses during root Spain growth. In Arabidopsis, we found that increasing BR receptors in the vascular plant Reviewed by: tissues confers resistance to drought without penalizing growth, opening up an Parvathi Madathil Sreekumar, Kerala Agricultural University, exceptional opportunity to investigate the mechanisms that confer drought resistance India with cellular specificity in plants. In this review, we provide an overview of the most Pablo Albertos, Technical University of Munich, promising phenotypical drought traits that could be improved biotechnologically to obtain Germany drought-tolerant cereals. In addition, we discuss how current genome editing *Correspondence: technologies could help to identify and manipulate novel genes that might grant Ana I. Caño-Delgado resistance to drought stress. In the upcoming years, we expect that sustainable ana.cano@cragenomica.es solutions for enhancing crop production in water-limited environments will be identified Specialty section: through joint efforts. This article was submitted to Plant Physiology, a section of the Keywords: drought, Arabidopsis, cereals, genome editing, cell-specific regulation journal Frontiers in Plant Science Received: 08 July 2019 Accepted: 28 November 2019 INTRODUCTION Published: 22 January 2020 Citation: Today, agriculture is facing an unprecedented challenge. Arable land is being reduced by soil Martignago D, Rico-Medina A, erosion and degradation, desertification, and salinization, destructive processes that are being Blasco-Escámez D, further accelerated by climate change. This could jeopardize global food production, which will need Fontanet-Manzaneque JB and to be maximized to cope with the world´s growing population and to match the food security goals Caño-Delgado AI (2020) Drought established by United Nations. More than ever, drought is a major threat to agriculture worldwide. Resistance by Engineering Plant Tissue-Specific Responses. The Food and Agriculture Organization (FAO) of the United Nations documented that between Front. Plant Sci. 10:1676. 2005 and 2015, drought caused USD 29 billion in direct losses to agriculture in the developing doi: 10.3389/fpls.2019.01676 world, with the 2008–2011 drought in Kenya alone accounting for USD 1.5 billion (FAO, 2018). In Frontiers in Plant Science | www.frontiersin.org 1 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses addition, more than 70% of the world´s available fresh water is identified physiological traits that result from drought responses being used in irrigation (Organization for Economic and contribute to the adaptation of plants in water-limited Cooperation and Development, 2017). To cope with these conditions. Understanding the molecular and physiological challenges, plant breeders will need to begin producing novel mechanisms behind these traits is essential for improving crop varieties that have increased yield, that are tolerant to crops through biotechnology. abiotic stresses, and that have improved water and nutrient In this review, we describe some of the drought resistance uptake efficiencies (Fita et al., 2015). traits of the model plant Arabidopsis that have the potential of In agronomy, drought can generally be defined as a prolonged being transferrable to crops, focusing on strategies that involve lack of water that affects plant growth and survival, ultimately the manipulation of cell- and tissue-specific responses. As these reducing crop yield. In plant science, the broadest definition of strategies open up opportunities to uncouple drought resistance drought stress coincides with the definition of water deficit, from the commonly associated growth and yield penalties, we which happens when the rate of transpiration exceeds water will discuss their biotechnological application in cereal species. uptake (Bray, 1997). This could be the result of a lack of water, but also of increased salinity or osmotic pressure. From a molecular biology perspective, the first event during drought MAJOR TRAITS CONTRIBUTING TO stress is the loss of water from the cell, or dehydration. DROUGHT RESISTANCE Dehydration usually triggers signals that are osmotic and hormone related, with abscisic acid (ABA) mainly involved in Early Flowering and Drought Escape the latter (Blum, 2015). These signals are followed by a response The molecular control of flowering time is complex, and has been that could be broadly categorized into three main strategies: i) highly studied in Arabidopsis (Michaels and Amasino, 1999; drought escape (DE), ii) dehydration avoidance, and iii) Simpson and Dean, 2002) as well as in many other plant species dehydration or desiccation tolerance (Kooyers, 2015; Blum and (Corbesier et al., 2007). During the developmental switch from Tuberosa, 2018). DE is the attempt of a plant to accelerate the vegetative to the reproductive stage, the photoperiodic light flowering time before drought conditions hinder its survival. signal from the environment is perceived by leaves, where the This response is common to annual plants including the model FLOWERING LOCUS T (FT) protein is synthesized. FT is species Arabidopsis thaliana (Arabidopsis), and is exploited by loaded into the phloem and transported to the shoot apical cereal plant breeders (Shavrukov et al., 2017). In dehydration meristem (SAM) where it initiates floral transition (Andrés and avoidance, the plant is able to maintain a high relative water Coupland, 2012). It is now known that in the SAM, FT forms a content (RWC% = [fresh mass − dry mass]/[water saturated complex with the bZIP protein FD in specific cells beneath the mass − dry mass] × 100) even during water scarcity. This is tunica layers in which FD is expressed, with these cells then achieved by physiological and morphological responses that originating the floral primordia (Abe et al., 2019). include the reduction of transpiration via ABA-mediated When Arabidopsis is exposed to drought conditions, it can stomatal closure, the deposition of cuticular waxes, and the activate the DE response. DE is one of the main defense slowing down the plant´s life cycle. Dehydration avoidance mechanisms against drought in Arabidopsis, and it integrates usually leads to survival through delaying plant growth, and the photoperiodic pathway with drought-related ABA signaling thus senescence and mortality. This strategy evolved as a (Conti, 2019). DE has mainly been studied in an evolutionary response to moderate, temporary drought stress in which the context in natural populations (McKay et al., 2003; Franks et al., plant undergoes a developmental stand-by until the next rainfall 2007), and the molecular mechanisms that regulate it have only (or irrigation). While effective in increasing plant survival rate, been unraveled recently. It is known that, to trigger DE, the key dehydration avoidance often comes with growth and yield photoperiodic gene GIGANTEA (GI) needs to be activated by penalties, which are, of course, major negative traits for crop ABA (Riboni et al., 2013; Riboni et al., 2016). A recent breeders (Skirycz and Inzé, 2010). On the other hand, in breakthrough was the discovery that the ABRE-BINDING dehydration tolerance, the plant is able to maintain its FACTORS (ABF) 3 and 4, which act on the master floral gene functions in a dehydrated state, usually by regulation of plant SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) m e t a b ol i s m t o i nc r e a s e t he p r o d u c t i o n o f s u g a r s , in response to drought, are involved in this process. The mutants osmoprotectants, antioxidants, and reactive oxygen species abf3 abf4 are insensitive to ABA-induced flowering and have a (ROS) scavengers (Hu and Xiong, 2014). These responses are reduced DE response (Hwang et al., 2019). However, the precise usually activated by gibberellic acid (GA) signaling through the molecular mechanisms that link ABA to GI and ultimately to DE modulation of the GA-signaling molecule DELLA, a pathway are still rather obscure, and different crop species might have that integrates multiple hormone- and stress-related pathways evolved unknown pathways that trigger DE in different (Vandenbussche et al., 2007; Navarro et al., 2008; Colebrook environments (Figure 1A). et al., 2014). From an agronomic perspective, DE and early flowering Ultimately, drought resistance is determined by how a plant varieties with faster life cycles are interesting because an efficiently and timely senses changing environmental conditions, anticipated switch to the reproductive stage might allow grain adopting and combining the aforementioned strategies in filling before the onset of seasonal terminal drought. response to diminished water availability. Plant breeders have Furthermore, a shorter crop season reduces the need for Frontiers in Plant Science | www.frontiersin.org 2 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses FIGURE 1 | Major traits contributing to drought resistance in Arabidopsis thaliana: For each of the traits, we highlight recent and comprehensive review papers, and prominent articles discussed in the main text. (A) Early flowering and drought escape: Conti, 2019—spotlight article about the latest discoveries in drought escape; Riboni et al., 2016— relationship between abscisic acid (ABA), GIGANTEA, and flowering time in drought escape; Hwang et al., 2019—molecular mechanisms that allow ABA-responsive element (ABRE)–binding factors (ABFs) to bind to the promoter of the floral master regulator SOC1 by interacting with a nuclear factor Y subunit C (NF-YC). (B) Leaf traits, including senescence, the stay-green trait, and leaf area: Abdelrahman et al., 2017—review about stay-green traits: in Arabidopsis, the SGR gene family regulates chlorophyll metabolism during senescence; Distelfeld et al., 2014—review about senescence in cereals. (C) Stomatal-mediated drought responses: Bertolino et al., 2019—review about stomatal manipulation toward drought tolerant plants; Papanatsiou et al., 2019—expression of a synthetic K+ channel in guard cells improved stomata kinetics and drought avoidance in Arabidopsis. (D) Cuticular wax production: Lee and Suh, 2015; Xue et al., 2017—reviews about cuticular wax evolution, chemical composition, biosynthesis, and drought responses; Lee et al., 2014—Camelina sativa plants overexpressing Arabidopsis MYB96 have increased wax biosynthesis and accumulation, and an improved survival rate; Zhou et al., 2014—rice plants that constitutively express the transcription factor OsWR2 have increased cuticular wax deposition and improved tolerance, but the yield was negatively affected; Bi et al., 2018—wheat plants constitutively expressing TaSHN1 have altered wax composition, reduced stomatal density, and an improved drought survival rate. (E) Carbon allocation: Paul et al., 2018—research update about trehalose 6-phosphate (T6P) and T6P-related approaches to improving crop yield and stress resilience; Griffiths et al., 2016—chemical supply of synthetic analogs of T6P improves dehydration tolerance in Arabidopsis and wheat; Fàbregas et al., 2018—overexpression of the Arabidopsis brassinosteroid receptor BRL3 improved survival rate by altering carbohydrate distribution in vascular tissues. (F) Architecture and response of roots: Rogers and Benfey, 2015—review about root system architecture regulation and possible biotechnological applications in crop improvement; Fàbregas et al., 2018—root vascular expression of AtBRL3 enhanced hydrotropic response of the Arabidopsis root. agricultural inputs (e.g., fertilizers, pesticides) and might stress and GA, and when overexpressed in transgenic rice plants facilitate double cropping (i.e., the farming of two different confers early flowering and improves drought tolerance (Fang crops in the same field within the same year). On the other et al., 2019). However, as these transgenic rice lines were not hand, crops that switch too early to flowering will have their yield tested in a field trial, it is unknown whether engineering FTL reduced. Despite DE being an emerging research field in crop genes could deliver cereal varieties with superior drought science, there are not any biotechnologically improved crops that performances and good yield in both dry and well-watered exploit DE as a drought resistance trait. Still, it has been proposed conditions. Nonetheless, the manipulation of the DE pathway that DE can be used to obtain quick-growing, early-flowering could be an innovative and valid strategy especially in the context cereal varieties, which would be especially useful in temperate of highly variable water availability. As DE involves specific regions like the Mediterranean area where terminal drought is tissues (leaf, phloem) and cell types (phloem companion cells, expected to affect plants toward the end of the crop season FD-expressing SAM cells), it might be possible to devise (Shavrukov et al., 2017). Furthermore, it has been recently shown strategies aimed at developing drought-resistant plants via that OsFTL10, one of the 13 FLOWERING LOCUS T-LIKE (FTL) manipulation of these plant components, adjusting DE to the genes annotated in the rice genome, is induced by both drought different environmental conditions. Frontiers in Plant Science | www.frontiersin.org 3 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses Leaf Traits: Senescence, Stay-Green, jasmonic acid and ethylene pathways (Dezar et al., 2005; and Leaf Area Manavella et al., 2008). Interestingly, HB4-expressing soybean Senescence is a developmental stage of plant leaves that leads to has increased yield in both water-limited and well-watered the arrest of photosynthesis, the degradation of chloroplasts and conditions. As shown in extensive field trials, this same gene proteins, and the mobilization of nitrogen, carbon, and other confers similar drought tolerance properties without yield nutrient resources from the leaves to other organs. As most penalties when transferred to bread wheat (Gonzalez et al., cereals are monocarpic annual species, these resources are 2019), with the transgenic wheat having an unaltered quality directed to developing seeds, and senescence therefore plays a and nutritional content when compared with its parental non- relevant role in crop yield. Environmental stresses like transgenic variety Cadenza (Ayala et al., 2019, Figure 2C). As temperature, lack of nutrients, and drought might initiate such, it is likely that the HB4 cassette could confer drought senescence prematurely, affecting seed nutritional composition resistance to other cereals. It is worth pointing out that the and crop yield (Buchanan-Wollaston, 1997; Distelfeld et al., success of HB4 is due to the exploitation of drought-responsive 2014). In crops threatened by terminal drought, the ability to promoters rather than of constitutive strong promoters. sustain photosynthetic activity longer by delaying or slowing Using a rather different approach, Monsanto expressed the down senescence could be an effective strategy to avoid yield bacterial cold shock protein B (CSPB) under the control of the losses. As such, leaf senescence has been extensively studied in constitutive rice ACTIN1 promoter. The expressed CSPB protein crops (Figure 1B). bears RNA-binding motifs named cold shock domains (CSDs) Plant breeders commonly refer to the trait that confers that act as RNA chaperones and regulate translational activity. In extended photosynthetic activity as stay-green, also defined as the analyzed transgenic plants, chlorophyll content and green leaf area at maturity (GLAM). This trait is well studied in photosynthetic rates were improved (Castiglioni et al., 2008). sorghum [Sorghum bicolor (L.) Moench], a dry climate-adapted These transgenic plants were tested in 3-year field trials in two cereal in which a number of stay-green quantitative trait loci different locations, and yields were on average 6% higher than for (QTLs) have been identified (Vadez et al., 2011). However, the the control plants in water-limited conditions (Figure 2B). genes underlying these QTLs have not yet been identified Although the molecular mechanisms are not fully understood, (Harris-Shultz et al., 2019). Stay-greenness in sorghum is a improved performances in water-limited conditions have been complex trait, and it is also connected with the perennial linked to a transient reduction in leaf area that leads to reduced tendencies of some varieties (Thomas and Howarth, 2000). water use and improved overall water use efficiency (WUE). This Other plant species achieve stay-green characteristics via temporary dehydration avoidance does not negatively affect yield substantially different pathways that include disabling due to an improved ear partitioning, which is probably also a chlorophyll catabolism (like in the case of Gregor Mendel’s consequence of reduced stress exposure during vegetative growth green peas, Armstead et al., 2007), and altering the responses (Nemali et al., 2015). This work led to the first biotechnologically to plant hormones. Indeed, some stay-green genes have also been improved crop for drought tolerance, called Genuity™ identified in Arabidopsis and rice (Hörtensteiner, 2009), notably DroughtGard™ by Monsanto (Figure 2, event code MON- the Stay-Green Rice (SGR) genes and their homologs in 87460-4, ISAAA, 2019). Even though this result was achieved Arabidopsis SGR1, SGR2, and SGR-like (SGRL). The respective by the constitutive expression of a bacterial TF, we speculate that molecular pathways have been elucidated, with the leaf-specific or meristem-specific genes expressed in specific phytohormones ethylene, ABA, cytokinin (CK), and developmental stages could lead to similar results. strigolactone (SL) having a prominent role in stress-induced leaf senescence (Abdelrahman et al., 2017). The connection Stomatal-Mediated Drought Responses between ethylene and leaf senescence is long known (Bleecker Stomata, which are openings on the surface of the aerial portion et al., 1988; Grbić and Bleecker, 1995), and numerous attempts to of plants, are enclosed by two specialized guard cells that can improve photosynthetic activity and drought performance by open and close the pore by changing their turgor pressure. manipulating ethylene biosynthesis have been published in Stomata are vital for CO2 uptake in photosynthetic organs and dicots (John et al., 1995) and cereal plants (Young et al., 2004). are finely regulated by a molecular pathway that allows plants to The first biotechnologically produced plant ever to reach the acquire CO2 while minimizing water loss. Manipulating stomatal market with improved drought resistance due to reduced number, size, and regulation was one of the earliest strategies ethylene sensitivity and delayed senescence was produced by adopted by scientists in attempt to produce drought-resistant Verdeca and named HB4 ® Drought Tolerance Soybeans plants, and recent advances in Arabidopsis and crops to this effect (Bergau, 2019 ). HB4 is a modified version of the are thoroughly reviewed in Bertolino et al., 2019 (Figure 1C). homeodomain-leucine zipper (HD-zip) transcription factor The main hormone signal that triggers stomatal closure in (TF) HaHB4 from sunflower (Helianthus annuus). It is water-limited conditions is ABA (Sussmilch and McAdam, expressed under the control of the native soybean HaHB4 2017). In Arabidopsis, expression of the CLAVATA3/ promoter, which is stress inducible (Waltz, 2015). Although EMBRYO-SURROUNDING REGION-RELATED 25 (CLE25) HaHB4 does not have conserved homologs in Arabidopsis, gene is upregulated in the root vascular tissues upon drought upon ectopically expressing HaHB4 in this model species, it stress. The CLE25 peptide is translocated to the leaves where it was discovered that the TF acts at the intersection between the binds to BARELY ANY MERISTEM (BAM) receptors, which, in Frontiers in Plant Science | www.frontiersin.org 4 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses FIGURE 2 | Drought tolerance genes that have been discovered or tested in model species and translated successfully into crop species. All of these genes have been expressed in engineered cereal crops and have been tested in field trials. Major agronomical traits, including yield, have been assessed, and conditions and drought performances have been successfully improved without negatively affecting plant growth or crop yield. (A) Hahb4: The sunflower transcription factor Hahb4 was expressed in soybean under the control of the native stress-inducible promoter of a homologous gene. Transgenic plants have reduced ethylene sensitivity, delayed senescence, increased osmoprotectant content, and an increased yield in the presence or absence of drought stress (Waltz, 2015). These plants are currently on the market as Verdeca Drought Tolerance Soybeans HB4®. The same Hahb4 has also been transferred to bread wheat under the control of the constitutive promoter of maize ubiquitin 1 with similar promising results (Gonzalez et al., 2019; Ayala et al., 2019). (B) CspA, CspB: Maize plants overexpressing Escherichia coli CspB have high chlorophyll content, an improved photosynthetic rate, and reduced leaf area during vegetative growth. The best performing lines were commercialized as Genuity® DroughtGard™ by Monsanto (now Bayer) in 2010 (Castiglioni et al., 2008; Nemali et al., 2015). (C) NF-YB1, NF-YB2: Maize plants overexpressing ZmNF-YB2 have higher stomatal conductance and chlorophyll content, and delayed senescence. These lines were not assessed in the field for performance under well-watered conditions and were never introduced to the market (Nelson et al., 2007). (D) TPS/TPP, TsVP: Carbon allocation, root/shoot ratio. In maize, floral-specific expression of T6P phosphatase (TPP) altered carbon allocation and improved yield in both well-watered and water-limited field trials (Nuccio et al., 2015). Also, in maize, the constitutive expression of the TsVP gene from the halophyte Thellungiella halophila under the control of the endogenous ubiquitin promoter increased total soluble sugars and proline under osmotic stress. Improvements in dehydration tolerance were assessed in a small-scale field trial (Li et al., 2008). (E) OsNACs, OsERF71, HVA1, DRO1. In rice, expression of the transcription factor OsNAC5 under the control of the root-specific promoter RCc3 improved drought and high salinity resistance by enlarging the root diameter. Yield improvements in normal and stress conditions were assessed in a 3-year field trial in three different locations (Jeong et al., 2013). Similar results were obtained with OsNAC9 and OsNAC10 (Jeong et al., 2010; Redillas et al., 2012). Root-specific expression. Also, in rice, the expression of the barley HVA1 under the control of a synthetic ABA-inducible promoter enhanced root growth, leading to better water use efficiency and abiotic stress tolerance. as confirmed by a small-scale field trial (Chen et al., 2015). The DRO1 allele from deep-rooting rice cultivars increases gravitropic response and root depth, increasing rice yield in both drought and normal conditions (Uga et al., 2013; Arai-Sanoh et al., 2014). (F) AtOSR1, ARGOS8: Arabidopsis ORGAN SIZE RELATED1 (AtOSR1) 1 and its maize homolog ZmARGOS1 improve dehydration avoidance in both plant species by reducing ethylene sensitivity (Shi et al., 2015); Moderate constitutive expression of ARGOS8, which was obtained by promoter swapping using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) homology-directed recombination, improved drought tolerance in a field trial under stress conditions without affecting yield in well-watered control experiments (Shi et al., 2017). Commercialization of these lines is under evaluation by the developer Corteva Agriscience™ (former DuPont Pioneer). Frontiers in Plant Science | www.frontiersin.org 5 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses turn, induce ABA accumulation in leaves leading to stomatal established. Overall, engineering the physiological behavior of closure (Takahashi et al., 2018). The manipulation of ABA stomata represents a remarkable innovation in Arabidopsis that sensitivity to increase stomatal responses in response to has yet to be applied to crops. drought could help plants to survive. However, diminished photosynthetic activity due to limited CO2 uptake is usually Cuticular Wax Production detrimental to carbon assimilation and negatively impacts crop Aerial plant organs have an external cuticle layer of which waxes yield. In addition, water evaporation through stomatal openings are a major component. This hydrophobic barrier physically prevents plants from overheating. As drought in a natural protects the epidermis against a plethora of external factors environment is likely to be accompanied by warm including UV light, cold temperatures, fungal pathogens, and temperatures, reducing stomata capacity might not be a insects, and also regulates permeability and water loss. However, sustainable approach to enhance drought resistance while despite the fact that a number of studies in Arabidopsis and crops securing yield and biomass production. For instance, a series of have shown a connection between drought stress and changes in rice mutants of the ABA receptors pyrabactin resistance 1-like 1 cuticular wax content, composition, and morphology, many of (pyl1), pyl4, and pyl6 have improved yield but are more sensitive the key genes involved in wax metabolism, regulation, and to drought (Miao et al., 2018), a result that resonates with the transport still need to be characterized (Xue et al., 2017; improved drought resistance but reduced yield of the transgenic Patwari et al., 2019). Cuticular wax composition has been plants that overexpress PYL5 (Kim et al., 2014). studied both in Arabidopsis and crop species; wax composition In an early attempt to produce drought-resistant plants, it was not only varies between plant species, but also between specific observed that the constitutive expression of AtNF-YB1 in tissues or organs within the same plant. In the most well-studied Arabidopsis improved the survival rate of the transgenic plants model, the biosynthesis of cuticular waxes occurs in epidermal (Nelson et al., 2007). NUCLEAR FACTOR Y (NF-Y) are cells where de novo synthesized C16–C18 fatty acids produced in heterotrimeric TFs that regulate multiple developmental plastids are exported by acyl–acyl carrier proteins (acyl–ACP). pathways (Zhao et al., 2017), including stomatal responses via These proteins are subsequently hydrolyzed by the fatty acyl– modulation of the ABA signaling pathway (Bi et al., 2017), with ACP thioesterase B (FATB) and the C16–C18 fatty acids are conserved functions in Arabidopsis and cereals during both imported into the endoplasmic reticulum following activation by flowering (Siriwardana et al., 2016; Goretti et al., 2017) and DE long-chain acyl–coenzyme A (acyl-CoA) synthetases, which are (Hwang et al., 2019). One maize homolog of AtNF-YB1, ZmNF- encoded by the long-chain acyl-CoA synthetase genes LCAS1 YB2, was constitutively expressed under the control of the rice and LCAS2. The carbon chains are then elongated with C2 units actin 1 promoter. Maize transgenic plants showed an improved from the malonyl-CoA by the fatty acids elongase complex. This survival rate in a greenhouse experiment, confirming the complex biosynthesizes C20–C34 very-long-chain fatty acids functional conservation between Arabidopsis and maize NF- (VLCFA) that are modified via two different pathways, namely YBs. In field trials, the transgenic plants were also drought the alcohol-forming pathway and the alkane-forming pathway. resistant due to a combination of higher stomatal conductance, These pathways produce the aliphatic compounds of cuticular cooler leaf temperatures, higher chlorophyll content, and delayed waxes. While the alcohol-forming pathway produces very-long- onset of senescence (Nelson et al., 2007). Nevertheless, even if chain (VLC) primary alcohols and wax esters, the alkane- these transgenic lines show promising results in field trials, with forming pathway produces VLC aldehydes, VLC alkanes, the best performing line having a 50% increase in yield relative to secondary alcohols, and ketones (Yeats and Rose, 2013). controls under severe drought conditions, these lines were never Besides these ubiquitous wax compounds that are common to introduced to the market, maybe because the yield in well- almost all plant species, there are a plethora of specialty wax watered conditions was negatively affected (Figure 2A). compounds that vary in carbon number, terminal carbon The trade-off between stomatal conductance and drought oxidation state, and the presence and oxidation state of resistance could be avoided by manipulating stomatal kinetics, secondary functional groups, with about 125 different or more precisely, by improving the speed of stomatal responses compounds identified in over 100 plant species whose (McAusland et al., 2016). Recently, enhanced plant stomatal biosynthetic pathways are not yet fully described (Busta and kinetics was achieved by expressing a synthetic, blue light– Jetter, 2018). All wax components are synthesized in the induced K+ channel 1 (BLINK1) under the control of the endoplasmic reticulum and need to be exported to the plasma strong guard cell–specific promoter pMYB60 (Cominelli et al., membrane and then secreted from the cell wall of the epidermal 2011). This effectively accelerated stomatal responses, producing cells where they constitute the cuticle (Fernández et al., 2016). plants that responded faster to changing light conditions. The secretion of wax molecules from the plasma membrane to Arabidopsis WUE (i.e., the biomass per transpired water) was the extracellular matrix in Arabidopsis is known to be mediated improved without reducing carbon fixation rates, resulting in a by the ATP binding cassette (ABC) transporters, CER5 (from 2.2-fold increase in total biomass in the transgenic plants grown eceriferum, waxless mutants) and WBC11 (Pighin et al., 2004; in water-deficit conditions when compared with the control Bird et al., 2007). On the other hand, the intracellular trafficking plants (Papanatsiou et al., 2019). Whether this approach would that governs the transport of wax constituents is not fully be efficient in crops in an open field, or whether the increased understood, but involves more than a single mechanism. biomass would correspond to a better yield, is yet to be Gnom-like 1-1 and echidna mutants (gnl1-1 and ech), which Frontiers in Plant Science | www.frontiersin.org 6 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses are defective in vesicle trafficking, show a decrease in surface cuticular deposition and drought tolerance when ectopically waxes, thereby indicating that endomembrane vesicle trafficking expressed in Arabidopsis, but also leads to detrimental effects is required in wax transport (McFarlane et al., 2014). In addition, on plant growth and development. However, when expressed membrane-localized lipid transfer proteins (LTPs) may be under the control of the stress-inducible Arabidopsis RD29 involved in wax delivery to the cuticle through the hydrophilic promoter, EsWAX1 is able to improve the rate of drought cell wall. In fact, Arabidopsis LTPG1 and LTPG2 genes have been survival without causing any major negative pleiotropic effect characterized (DeBono et al., 2009; Kim et al., 2012). Novel (Zhu et al., 2014). Even if seed number or yield was not assessed, proteins with yet unknown molecular functions involved in the use of drought-responsive promoters helps overcome the extracellular wax transport are also being discovered in undesirable effects of ectopic overexpression. Another well- monocots through the characterization of mutants. One such known TF controlling wax biosynthesis is the Arabidopsis example is maize GL6 (Li et al., 2019). A comprehensive coverage ABA-responsive R2R3-type MYB TF, MYB96. MYB96 is highly of cuticular wax biosynthesis and deposition can be found in the expressed in stem epidermal cells, is activated by drought, and review articles by Bernard and Joubès (2013), and Lee and binds directly to the promoter of multiple wax biosynthetic genes Suh (2015). to upregulate their transcripts and increase wax production (Seo Cuticular waxes can be regulated post-translationally, post- et al., 2011). Overexpressing Arabidopsis MYB96 in the close transcriptionally, and transcriptionally. In terms of post- relative Brassicaceae biofuel crop Camelina sativa led to an translational regulation, the CER9 gene, which encodes a increase in wax biosynthesis and deposition, and also putative E3 ubiquitin ligase, plays a role in the homeostasis of improved drought survival of the transgenic camelina plants cuticular wax biosynthetic enzymes through ubiquitination and (Lee et al., 2014). Taken together, these results show relevant degradation of proteins in the endoplasmic reticulum. advances in the quest to obtaining drought-resistant plants by Arabidopsis cer9 mutants showed an increase in lipid manipulating cuticular wax biosynthesis. Important differences deposition and drought tolerance, suggesting that it has a between Arabidopsis and crop species in terms of wax negative role with regards to the regulation of cuticular wax composition, localization, and quantity need to be considered biosynthesis (Lü et al, 2012). CER7, on the other hand, which when attempting to transfer drought resistance traits. encodes an exosomal exoribonuclease that was proposed to play Furthermore, an excessive wax production might have negative a role in the degradation of small RNA species that negatively effects on plants because of the high amount of carbon resources regulate the CER3 transcript (an enzyme involved in wax that need to be redirected from seeds to leaves, and because of the biosynthesis), is part of the post-transcriptional mechanisms of reduced CO2 permeability of the wax-covered leaves. As such, it regulation. However, transcriptional mechanisms are considered is essential that the any biotechnologically induced increase in to be the main regulator of wax biosynthesis (Yeats and Rose, wax production occurs in specific cell types and in response to 2013). Accordingly, most of the biotechnological approaches that dehydration rather than constitutively. have attempted to improve drought performance by manipulating cuticular wax levels focus on TFs that control the Carbon Allocation overall process rather than on overexpressing multiple Plants are photosynthetic organisms able to fix atmospheric components of the biosynthetic pathways. In Arabidopsis, carbon into macromolecules essential for growth and survival. overexpression of the TF WAX INDUCER1/SHINE1 (WIN1/ Thus, it is evident that carbon metabolism and allocation are SHN1) was found to activate wax biosynthesis, increase wax highly regulated and this regulation has a vital role in plant deposition, and confer drought resistance in a survival rate resilience to stresses and crop yield. In cereals, carbon is the main experiment (Aharoni et al., 2004; Broun et al., 2004). In studies determinant of crop yield, and carbohydrates from cereals are the performed in apple and mulberry, WIN1/SHN1 homologs have primary source of calories in the human diet (Lafiandra et al., been shown to have conserved functions, and therefore might 2014). One of the main pathways that regulates carbon allocation similarly increase drought tolerance (Sajeevan et al., 2017; Zhang in plants is the trehalose 6-phosphate (T6P)/SNF1-related/ et al., 2019). In rice, overexpression of the WIN1/SHN1 homolog AMPK protein kinases (SnRK1) pathway. T6P is a OsWR1 improves drought tolerance at the seedling stage (Wang nonreducing disaccharide present in trace quantities in plants, et al., 2012). While constitutive expression of OsWR2 and it acts as a signal for sucrose levels. The T6P/SnRK1 pathway dramatically increased cuticular wax deposition (48.6% in has been unraveled through studies in Arabidopsis that led to the leaves) and improved dehydration avoidance, yield was identification and characterization of the TREHALOSE negatively affected, with a reduction of 30% in seed number PHOSPHATE SYNTHASE ( TPS) a n d T RE HA L OS E per panicle (Zhou et al., 2014). On the other hand, in wheat, the PHOSPHATE PHOSPHATASE (TPP) genes. This pathway overexpression of the OsWR1 ortholog TaSHN1—cloned from has also been linked to auxin and ABA signaling (Paul et al., the drought-tolerant genotype RAC875—was able to improve 2018). T6P is known to act as a signaling molecule during drought tolerance in a survival experiment without an evident flowering, and Arabidopsis tps1 mutants are extremely late to loss in yield under controlled conditions. These transgenic plants flower (Wahl et al., 2013). Increasing the intracellular content of have an altered wax composition and a lower stomatal density the disaccharide T6P is a well-known strategy for improving (Bi et al., 2018, Figure 1D). EsWAX1, a novel TF that was drought tolerance in plants (Romero et al., 1997). While T6P is isolated from the halophyte Eutrema salsugineum, improves present in trace amounts in most of temperate plants, it Frontiers in Plant Science | www.frontiersin.org 7 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses accumulates in resurrection plants (Wingler, 2002). However, 2016). Notably, seed-specific manipulation of T6P might manipulating T6P levels through the expression of T6P increase drought tolerance as well as resistance to flooding regulatory or biosynthetic genes under the control of strong (Kretzschmar et al., 2015). As most of the plant sugar constitutive promoters significantly alters plant growth and trafficking happens through the phloem, shoot and root development, and might negatively affect crop yield (Guan and vascular tissues are also candidate targets for T6P Koch, 2015). In rice, the overexpression of Escherichia coli T6P manipulation (Griffiths et al., 2016; Fàbregas et al., 2018). biosynthetic genes under the control of an artificial ABA- inducible promoter derived from the rbcS (RuBisCO) leaf- Root Traits specific promoter, avoided the negative effect of ectopic T6P Roots are the main plant organ dedicated to the uptake of water, biosynthetic gene overexpression. In a laboratory-scale and are the first place where a lack of water is perceived. As such, experiment, rice transgenic plants were drought tolerant, with an abundance of studies have examined root responses to improved photosynthetic activity and reduced photo-oxidative dehydration. The most relevant root traits capable of damage under drought conditions (Garg et al., 2002). In maize, improving drought tolerance and their biotechnological the catabolic enzyme T6P phosphatase (TPP) has been applications have recently been reviewed by Koevoets et al. specifically expressed in female floral components using the (2016) and by Rogers and Benfey (2015), respectively. Here, we rice floral promoter gene Mads6 (MCM1, AGAMOUS, will focus on the solutions offered by manipulation of the BR DEFICIENS, and serum response factor). This reduced the pathway, and will provide a brief overview on the most concentration of T6P in female reproductive tissues, increased promising biotechnological strategies aimed at improving the sucrose content in the whole developing spikelet, and affected drought resistance through manipulating root-related traits. the T6P/SnRK1 regulatory pathway. Effects on drought BRs are a class of plant hormones that are widely involved in resistance were assessed in extensive field trials, and the yield plant growth and development, as well as in stress responses. was consistently improved in well-watered, mild, and severe Along with other plant hormones, BRs play a key role in root drought conditions, with no obvious impact on plant or ear growth. As BR levels are finely regulated to permit proper root morphology (Nuccio et al., 2015). Crop yield and stress resilience development, BR metabolism and signaling are clear targets for has also been increased using chemical treatments that stimulate the manipulation of root responses (Singh and Savaldi- T6P production in Arabidopsis and wheat. As plants are Goldstein, 2015; Planas-Riverola et al., 2019). Indeed, impermeable to exogenous T6P, synthetic precursors were exogenous application of BRs has been extensively tested on a produced and used as treatments that triggered a light- variety of crops with variable outcomes (Khripach et al., 2000). inducible endogenous production of T6P (Griffiths et al., However, from a genetic perspective, the only BR-related mutant 2016). Trehalose was also shown to accumulate in the roots of widely used in agriculture is the barley uzu mutant, which carries plants with augmented brassinosteroid (BR) signaling, together a single amino acid substitution in the BR receptor HvBRI1, with other osmoprotective sugars like sucrose and raffinose. homolog of the Arabidopsis BR receptor Brassinosteroid Furthermore, T6P-related gene expression was specifically insensitive-1 (BRI1) and displays a semi-dwarf phenotype upregulated in the root phloem cells (Fàbregas et al., 2018, (Chono et al., 2003). Recently, the triple mutant of wrky46, Figure 1E). In turn, by mediating BR signaling, sugars act as wrky54, and wrky70—positive regulators of BR signaling signaling molecules in Arabidopsis to control different aspects of Arabidopsis group III WRKY TFs—was shown to be drought root system architecture, such as primary root elongation, lateral resistant. Due to a significant upregulation and downregulation root development, and root directional response (Gupta et al., of dehydration-induced and dehydration-repressed genes, 2015; Zhang and He, 2015). Notably, manipulation of BR respectively, these TFs operate as negative regulators of signaling results in an increase in osmoprotectant metabolites drought tolerance (Chen et al., 2017). BR biosynthetic dwarf including proline, an amino acid long known for conferring and semi-dwarf mutants were also shown to be drought tolerant drought and salinity tolerance (Kishor et al., 1995). Thus, (Beste et al., 2011). Somehow, contrasting with these results, it accumulation of sugars and proline might also be a valid has recently been demonstrated that the overexpression of strategy to achieve dehydration tolerance in cereals, as shown vascular-specific BR receptor BRI1-LIKE 3 (BRL3) increases by transgenic maize plants that constitutively express the the survival rate of Arabidopsis plants exposed to severe vacuolar H+-pyrophosphatase (V-H+-PPase) gene (TsVP) from drought stress. Interestingly, these transgenic plants do not the halophyte Thellungiella halophila. In a small-scale field show reduced growth, which is typically associated with experiment, these transgenic plants showed a higher yield drought-resistant BR mutants, and retain the same RWC as under drought conditions than the control plants (Li et al., wild-type plants. As previously mentioned, these transgenic 2008, Figure 2E). plants displayed an osmoprotectant signature (proline, Altering sugar distribution via the T6P pathway is a trehalose, sucrose, and raffinose) in response to drought, with promising biotechnological approach for producing drought- the corresponding biosynthetic and metabolic genes upregulated tolerant plants, with the best results being obtained when in the root phloem. This might suggest that BRs are involved in manipulation is directed to specific tissues like developing dehydration tolerance as well as in dehydration avoidance reproductive structures (Nuccio et al., 2015; Oszvald et al., (Fàbregas et al., 2018, Figure 1F). BRs are also involved in 2018) and seeds (Kretzschmar et al., 2015; Griffiths et al., hydrotropism, with the receptors BRI1-LIKE 1 (BRL1) and BRL3 Frontiers in Plant Science | www.frontiersin.org 8 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses having a prominent role that is independent of the pathway. the overexpression of AP2/ERF genes increases stress tolerance Interestingly, BRL3 is structurally and functionally very similar in Arabidopsis (Haake et al., 2002). The Arabidopsis HARDY to BRI1, but its expression is confined to the root stem cell niche (HRD) gene, a AP2/ERF TF, was identified as a dominant while that of BRI1 is ubiquitously found in the root. This suggest mutant with increased root density, and its ectopic expression that the BR-related drought responses in roots could be led by BR was able to improve the survival rate of both Arabidopsis and rice receptors in specific cells, such as the root meristematic region (Karaba et al., 2007). Ectopic HRD expression also alters leaf and vascular tissues (Fàbregas et al., 2018). In crops, increments morphology, with thicker deep-green leaves in Arabidopsis and in the BR biosynthetic pathway were shown to improve both increased shoot biomass in rice contributing to an improved stress tolerance—including dehydration and heat stress—and WUE of the transgenic plants. However, the increased WUE was seed yield in the oil crop Brassica napus (Sahni et al., 2016). measured as an increase in biomass and no data regarding seed Furthermore, in wheat, the overexpression of the BES/BZR production and yield were reported. Similar promising results family TF gene TaBZR2, a positive regulator of BR signaling, were obtained in the fodder dicot Trifolium alexandrinum by enhanced the expression of wheat glutathione S-transferase 1, constitutively expressing Arabidopsis HRD. This transgenic plant TaGST1. These transgenic plants showed an increase in ROS had a larger biomass in drought and salt stress conditions as scavenging and a drought-resistant phenotype without being tested in a controlled environment and in field trials dwarf (Cui et al., 2019). The seemingly opposed behavior of BR- (Abogadallah et al., 2011). In bread wheat, the expression of engineered plants could be partly explained by the drought stress the Arabidopsis AP2/ERF TF DREB1A gene under the control of experimental setup. Most BR biosynthetic and signaling mutants the stress-inducible RD29A promoter delayed leaf wilting in a exhibit an evident dwarf phenotype. However, in drought water withholding experiment in a controlled environment survival experiments, dwarf plants often show a passive (Pellegrineschi et al., 2004). In rice, a root-specific drought- drought resistance phenotype, and it is challenging to dissect responsive AP2/ERF TF OsERF71 was cloned and expressed whether a phenotype is due to a direct genetic effect on drought- either in the whole plant using the rice GOS2 promoter (de Pater related gene expression, or if it is a dehydration avoidance et al., 1992), or specifically in the roots using the RCc3 promoter. mechanism due to limited water consumption. Still, the Both transgenic lines proved to be drought resistant. In addition, manipulation of BR pathways retains its full potential with the root-specific expression was able to improve grain yield in respect to the development of stress-tolerant varieties, drought conditions. OsERF71 can bind to the promoter of the particularly if directed to specific cell types to avoid key lignin biosynthesis gene OsCINNAMOYL-COENZYME A unnecessary ectopic expression, and because of the REDUCTASE1, and it was proposed that changes in cell wall and involvement of these pathways in many agriculturally relevant root structure were the basis of the drought-resistant phenotype traits such as grain shape and size, cell elongation and plant (Lee et al., 2016). However, OsERF71 overexpression has a much height, leaf angle, and root development (Espinosa-Ruiz et al., wider impact on plant transcriptional regulation; it induces the 2017; Martins et al., 2017; Tong and Chu, 2018). Unfortunately, oxidative response and DNA replication, and reduces translating root responses from Arabidopsis to cereals is photosynthesis, thereby diverting more resources toward particularly challenging as the root system and architecture survival-related mechanisms (Ahn et al., 2017). Native differ greatly among plant species. Nevertheless, interesting OsERF71 expression is induced by ABA, and in turn regulates results were obtained in cereals by engineering root responses. the expression of ABA-related and proline biosynthesis genes in In rice, expressing the TF OsNAC5 under the control of the root- drought stress conditions (Li et al, 2018b). Another set of studies specific promoter RCc3 (Xu et al., 1995) improved drought aimed at improving abiotic stress tolerance in rice found that the resistance by increasing root diameter. Specifically, enlarged expression of barley late embryogenesis abundant (LEA) protein metaxylematic vascular tissues permitted the transgenic plants HVA1 under the control of a synthetic ABA-inducible promoter to have a better water flux. The use of a tissue-specific promoter increased the root system expansion. LEA proteins are encoded was paramount for the success of this experimental approach. by stress-responsive genes, and barley HVA1 and its rice Indeed, when expressed under a constitutively strong promoter, homolog LEA3 are well known for being regulated in roots in the same OsNAC5 was not able to increase yield under drought response to ABA, salt, and abiotic stresses. LEA proteins could because of a reduction in the grain filling rate (Jeong et al., 2013, work as osmoprotectants by maintaining cell functionality and Figure 2F). Another example is the rice NAC family, which is conferring dehydration tolerance. In this rice study, the synthetic well known for its effect on root architecture and stress promoter 3xABRC321, which carries a series of ABA-responsive responses. Several genes of this family have been overexpressed elements, drove the expression of HVA1 in response to abiotic (OsNAC9, Redillas et al., 2012) or expressed under the control of stress specifically in the root apical meristem and lateral root the root-specific promoter RCc3 (OsNAC10, Jeong et al., 2010), primordia. In turn, both primary and secondary root growth was with similar effects on drought and stress tolerance. Another significantly promoted through an auxin-dependent process. superfamily of stress-related TFs, the APETALA2/ethylene These transgenic rice plants showed a better WUE and abiotic responsive element binding factors (AP2/ERF), has been stress tolerance in a small-scale field trial (Chen et al., 2015). In extensively studied in attempt to enhance root traits and rice, QTL DEEPER ROOTING 1 (DRO1), which controls root achieve improved drought tolerance. AP2/ERF TFs participate angle, was studied using shallow- and deep-rooting cultivars, and in drought and cold stress responses (Shinozaki et al., 2003) and was identified by developing a near-isogenic line homozygous for Frontiers in Plant Science | www.frontiersin.org 9 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses the allele conferring the deep-root trait. DRO1 is expressed in the tracrRNA were fused together in a unique fragment, the root meristematic region, is controlled by auxins, and regulates gRNA. Simply modifying the 20 nucleotides corresponding to the gravitropic response. The DRO1 deep-root allele from the the spacer is sufficient to target the Cas9 to a sequence of choice cultivar Kinandang Patong (DRO1-kp) contains a 1-bp deletion (Cong et al., 2013). that results in a premature stop codon, shortening the C- The implementation of CRISPR-based genome editing terminal domain of the protein that it encodes. DRO-kp lines technologies in plant science opened up a wealth of have an enhanced gravitropic response that leads to deeper roots opportunities to plant scientists and plant breeders alike (Li and drought avoidance, and ultimately improves rice yield under et al., 2013; Shan et al., 2013). The most straightforward drought conditions (Uga et al., 2013). Furthermore, as shown in application of CRISPR/Cas9 is the production of out-of-frame paddy field trials, the yield of DRO-kp lines is also improved in loss-of-function mutants. Interestingly, loss-of-function normal growth conditions (Arai-Sanoh et al., 2014). Although mutations are the most frequent kind of genomic modification DRO1 does not have a clear homolog in Arabidopsis, it has that happened during the domestication of crops. In fact, from a homologs in other monocots like maize. The C-terminal position genetic perspective, crop domestication was achieved by stacking of the stop codon in the DRO1-kp alleles makes DRO1 an ideal loss-of-function mutants in key genes controlling traits like seed target for CRISPR-based gene targeting. shattering, flowering time, seed color, or size (Meyer and Purugganan, 2013). By targeting these genes, scientists have been able to swiftly retrace thousands of years of crop CHALLENGES AND improvement in a process known as de novo domestication FUTURE PERSPECTIVES (Zsögön et al., 2017; Li et al., 2018c). Ideally, this approach could assist with the rapid improvement of highly resilient, Genome Editing for Drought-Resistant locally adapted species to obtain new commercially relevant Crops crops that still retain the unaltered stress resistance During the past 10 years, genome editing technologies like zinc characteristics of their wild-type relatives. For some crops, de fingers nucleases (ZFNs), transcription activator–like effectors novo domestication could be more efficient than breeding into nucleases (TALENs), and homing meganucleases (also known as modern commercial varieties since stress resistance traits, often meganucleases) have enabled scientists to produce targeted controlled by multiple, sometimes unknown, genes, have been genetic modifications in organisms of choice (Arnould et al., lost during crop domestication. 2011; Bogdanove and Voytas, 2011; Carroll, 2011). Innovative Genome editing technologies might also help speed up cloning approaches like the Golden Gate system made the molecular breeding and crop improvement for so-called assembly of these tools more straightforward (Cermak et al., orphan crops, plants that are critical to local food security but 2011); however, genome editing protocols were still relatively are less relevant on a global scale (e.g., sweet potato, chickpea, or time consuming and labor intensive. With the advent of the sorghum) (Lemmon et al., 2018; Li et al., 2018a). The rationale engineered clustered regularly interspaced short palindromic behind these approaches is similar to that of the de novo repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system to domestication strategy: improving a resilient, locally adapted, perform targeted mutagenesis, genome editing became accessible and highly specialized crop might provide better results than to most research laboratories (Gasiunas et al., 2012; Jinek et al., attempting to restore stress tolerance in currently used elite 2012). In CRISPR-based genome editing, specificity to the target varieties in which complex multigenic traits were lost over the sequence is conferred by a programmable short fragment of RNA domestication process (Khan et al., 2019). One of the main limits called guide RNA (gRNA), and the Cas9 protein itself does not to genome editing in crops is plant transformation efficiency, require any structural modification to change target recognition. which hampers the delivery of genome editing material into the This is similar to what happens in the case of ZFNs and TALENs target cells. For most wild or orphan species, genetic (Cong et al., 2013). CRISPR/Cas9 is derived from the bacterial transformation has never been attempted, and in the immune system against viral infections. It was first observed by remaining cases, current protocols are only efficient for a small sequencing the DNA of E. coli, where a short series of DNA subset of the lab amenable varieties. Nonetheless, the high repeats are separated by spacer sequences (Ishino et al., 1987). potential of genome editing in plant sciences is driving the The spacer sequences are DNA from viral invaders that the development of more efficient crop transformation methods bacteria store as a sort of immune memory (Mojica et al., 2000; (Figure 3A). Mojica et al., 2005). These CRISPR sequences are transcribed and DuPont Pioneer scientists have successfully used CRISPR/ processed into short CRISPR RNAs (crRNAs), which are Cas9 to engineer drought tolerance by swapping the native composed of a variable spacer portion and a conserved promoter of the ARGOS8 gene for the promoter of maize protospacer repeat, and subsequently associated with a trans- GOS2. The maize GOS2 promoter was identified from the rice activating CRISPR RNA (tracrRNA). The ribonucleoprotein homolog GOS2 (de Pater et al., 1992), and in this case conferred complex composed by Cas9, crRNA, and tracrRNA are finally a moderate ubiquitous expression to ARGOS8. In field trials, directed toward the invading DNA complementary to the spacer these cis-genic lines showed increased yield under drought (Jansen et al., 2002; Bolotin et al., 2005; Pourcel et al., 2005). This conditions (Shi et al., 2017). ARGOS were previously studied system was engineered in such a way that the crRNA and as negative regulators of ethylene signaling in both Arabidopsis Frontiers in Plant Science | www.frontiersin.org 10 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses FIGURE 3 | A general frame for translating research in Arabidopsis thaliana to crops to improve drought performance in cereals. (A) Translation of promising genes/ traits in crops: In recent years, the improvement of genome editing technologies has enabled targeted genetic modifications of organisms of choice, and has opened up a wealth of opportunities to plant scientists and plant breeders alike. Genome editing technologies might also help drive the development of more efficient crop transformation methods. (B) Development of cell-specific stress response promoters for monocots: It has been shown that the use of moderate constitutive, tissue- specific, and drought-responsive promoters could limit unintended pleiotropic effects in terms of growth or yield penalty while maintaining the improved trait (Waltz, 2015; Fàbregas et al., 2018; Papanatsiou et al., 2019). (C) Analysis of cell-specific drought responses: As of today, cell-specific promoters available for cereals are limited. By performing transcriptomic studies coupled to FACS (fluorescence-activated cell sorting) in the crops of interest, it could be possible to identify novel cell- specific promoters. Subsequently, these promoters could be exploited and introduced into crops. Drought stress research will greatly benefit from tissue-specific -omics. (D) Correct experimental design and phenotyping in Arabidopsis: Many Arabidopsis drought-stress experiments are performed without recording traits such as yield, plant biomass, relative water content (RWC), etc. Moreover, near-lethal conditions do not reflect in crop performances in open fields. Future studies should consider all these aspects. Recording more detailed data, plus validating these promising results in crops, should be a priority for any research group working in Arabidopsis. (E) High-throughput plant phenotyping (HTPP) to corroborate Arabidopsis results and test novel crop genes: HTPP for drought has been implemented in Arabidopsis, and it is in development for many crops. HTTP will help increase and improve the reproducibility and quality of data from drought adaptation studies. The widespread adoption of HTTP platforms could represent a valid intermediate step between laboratory conditions and open, large-scale field trials. Frontiers in Plant Science | www.frontiersin.org 11 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses and maize. The constitutive overexpression of ZmARGOS1, revealed that the overexpression of the vascular AtBRL3 receptor ZmARGOS8, and Arabidopsis ARGOS were shown to decrease confers drought tolerance without any evidence of growth ethylene sensitivity in transgenic Arabidopsis plants (Shi et al., penalty (Fàbregas et al., 2018), and thereby opens up new and 2015). ARGOS genes and their molecular mechanisms are exciting possibilities to address the societal demand for conserved in both model and crop species. However, producing “more crop per drop” and to ensure global food constitutively high expression had a negative effect in cool and security goals in the upcoming years. It would be interesting to high humidity conditions (Guo et al., 2014). The use of the maize assess what the effect would be of expressing the drought GOS2 promoter enabled a moderate constitutive expression that resistance genes that have been isolated over the years under delivered drought resistance without affecting yield in normal or the control of a promoter that is both stress-inducible and cell- humid conditions (Shi et al., 2017). This work shows that specific, similarly to what was demonstrated in rice by expressing combining genome editing with promoters of tailored activity the barley LEA protein HVA1 under the control of a synthetic levels can provide the basis for successfully producing drought- promoter (Chen et al., 2015). resistant crops (Figure 2D). The main drawback of this approach is the limited availability of crop promoters that allow such specific gene expression. This Tissue-Specific Promoters to Drive hurdle could be overcome by performing transcriptomics in Drought Tolerance crops under normal and stress conditions that accurately Basic plant science research, as well as most traditional breeding differentiate between tissues. For example, in a study and biotechnological approaches, are based on loss-of-function or performed in rice, metabolomic and transcriptomic profiling gain-of-function mutants, or on the constitutive expression of a was performed using samples representing developed leaves and gene conferring a certain trait. As an example, mutations in the the SAM region exposed to progressively harsher drought MILDEW RESISTANCE LOCUS O (Mlo) genes confer broad- conditions. Different responses from the plant were recorded. spectrum resistance against fungal pathogens to a large number Mild stress induced stomatal responses, decreased auxin and CK of plant species including major cereals like wheat and barley levels, and thus plant growth, while more severe stress resulted in (Kusch and Panstruga, 2017). Similarly, Bt crops constitutively the production of ABA and the remobilization of sugars (Todaka expressing bacterial toxins from Bacillus thuringiensis are used et al., 2017). Differentially regulated genes identified by this and worldwide to protect crops from pathogens (ISAAA Brief, 2017). similar studies will hopefully lead to the isolation of tissue- However, improving resistance to abiotic stresses does not seems specific, drought-responsive promoters. Species-specific to follow the same pattern (Todaka et al., 2015). Dehydration responses were also observed in this study; in contrast to what avoidance based on a reduction in size or density of stomata previously reported in Arabidopsis, moderate drought stress did comes with a growth or yield penalty (Bertolino et al., 2019). not activate ethylene-responsive genes in rice (Skirycz et al., Manipulating hormone signaling pathways by means of ectopic 2011). Thus, as drought response pathways might differ overexpression of its components, or alternatively, by knocking significantly from those of Arabidopsis, it will be important to them out, often has an undesired pleiotropic negative effect on perform transcriptomic studies directly on the crops of interest. overall plant growth and development. In contrast, it has already This might also help identify novel, species-specific components been shown in maize that the use of moderate constitutive of the drought response. promoters instead of strong promoters could limit this Once a sufficient number of promoters are identified and undesired effect while maintaining the improved trait (Shi tested in crops, a virtuous circle might be triggered in which et al., 2017) (Figure 2B). transgenic cereals expressing tissue-specific markers would The use of tissue-specific promoters to drive gene expression enable tissue-specific transcriptomics. This in turn could lead in particular cells upon drought stress stands as a promising to the discovery of novel, cell type– and response-specific solution to break the deadlock between drought resistance and promoters that might provide innovative solutions to plant yield penalties. Several emerging studies show that when a tissue- biotechnologists. Using fluorescence-activated cell sorting specific promoter is used, it is possible to reap the benefits of the (FACS), a large number of plant seedlings expressing cell type– expressed genes while avoiding any major alteration to overall specific fluorescent markers could be grown in the desired plant phenotype. This is the case for the guard cell–specific experimental conditions, and then protoplasts prepared and promoter pMYB60, which was used to express the synthetic sorted by flow cytometry to collect cells for -omics studies protein BLINK1 in stomata (Papanatsiou et al., 2019), and the (Birnbaum et al., 2005). In Arabidopsis, methods to perform rbcS leaf-specific promoter that was used to express T6P RNAseq transcriptomics with as few as 40 cells isolated using biosynthetic genes in leaves (Garg et al., 2002). Similarly, the FACS have been developed (Clark et al., 2018). In fact, efficient use of stress-inducible promoters has proven to be an effective protoplast preparation protocols that enable quick preparation strategy to improve drought performances without penalizing and sorting of protoplasts—avoiding major transcriptional yield in soybean and wheat (Waltz, 2015; Gonzalez et al., 2019). changes—are already available for rice (Zhang et al., 2011), Importantly, these transgenic plants were tested extensively in and FACS has already been used to study stress responses in field trials, and the transgenic HB4 soybean is one of the very few this crop (Evrard et al., 2012). Similarly, a protocol to prepare biotechnologically improved drought-resistant plants ever to be protoplasts for FACS has already been developed for maize introduced on the market (ISAAA, 2019). Our recent findings (Ortiz-Ramirez et al., 2018). An alternative approach for Frontiers in Plant Science | www.frontiersin.org 12 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses single-cell isolation is INTACT (isolation of nuclei tagged in stimulating genes in plant transformation (Ebinuma et al., specific cell types), where cell type–specific nuclei are isolated by 1997). Co-delivery of BBM and WUS, either as proteins or affinity purifying a transgenic label targeted to the cell nucleus coding sequences, together with the target sequences seems to (Deal and Henikoff, 2011). INTACT does not require specialized considerably improve transformation efficiency in a number of instruments for cell sorting and might be preferred for chromatin notoriously recalcitrant species like sorghum and sugarcane, as studies (Deal and Henikoff, 2010). However, so far it has been well as in elite varieties of maize (Lowe et al., 2016; Mookkan tested exclusively on the model plant Arabidopsis. Both methods et al., 2017). need transgenic plants expressing a fluorescent marker or a Scientists at the University of California, Berkeley (USA), nuclear protein label. In cereals, depending on the species, plant developed an interesting approach to plant transformation, transformation might still be challenging and a protoplast which is distinct from both Agrobacterium- and biolistic-based preparation protocol might be tedious, time consuming, and systems. In this novel approach, a DNA delivery system makes may significantly alter the transcriptional responses. In fact, so use of carbon nanotubes (Demirer et al., 2019). While far most of the tissue-specific studies performed in monocots have transformation was achieved only transiently in leaves or used laser capture microdissection to isolate tissues. However, it is protoplasts of the target plants, this method has the notable expected that cereal-adapted protocols will soon be developed to advantage of being genotype independent. The lack of transgene enable advanced transcriptomics. Regardless of the method of integration could actually represent a critical advantage when choice, drought stress research in cereals will greatly benefit from CRISPR/Cas9 genome editing is involved. Indeed, it has been tissue-specific -omics, especially considering how just a few proven that transient expression of Cas9 and gRNAs in the target relevant cell types appear to control major responses (Efroni cells is sufficient to produce stable and heritable edits (Zhang and Birnbaum, 2016) (Figure 3C). In Arabidopsis, this field of et al., 2016). The advantage of this approach is that the first research is quickly developing and has led to even more exciting generation of mutants after transformation can carry the desired approaches that allow high-throughput single-cell RNA modifications, with no need to segregate the CRISPR material. sequencing (scRNA-seq). In scRNA-seq, protoplasted cells are This makes it attractive, especially for use in crops that are encapsulated in individual droplets and each cell transcriptome is difficult or impossible to cross. Even though this approach is still individually analyzed. Cells are then bioinformatically organized in its proof-of-concept stage, it represents an innovative and into tissues and cell types based on the presence of marker genes, potentially groundbreaking technology. which in the case of Arabidopsis, are well established for each cell type. In turn, novel, highly specific marker genes can be identified. High-Throughput Plant Phenotyping for This approach is called Drop-seq, and while it was initially Drought Traits developed for animal cell studies (Macosko et al., 2015), it has Despite the vast amount of information that has been reported to been adapted for Arabidopsis root cells to study development date regarding drought in Arabidopsis, Bayer’s (then Monsanto) (Denyer et al., 2019; Ryu et al., 2019) and responses to treatments DroughtGard® maize, Verdeca’s HB4 soybean and wheat, and (Shulse et al., 2019). Indonesian Perkebunan Nusantara’s NXI-4T sugarcane are the only biotechnologically improved drought-resistant crops ever Cereal Transformation introduced onto the market (Nuccio et al., 2018). This gap can With the notable exceptions of rice and maize, for which only partially be explained by societal and market opposition to transformation efficiencies can reach up to 100% and 70%, genetically modified (GM) crops. The main hurdle in translating respectively, plant transformation is notoriously challenging in Arabidopsis-developed drought-resistant traits into crops is the cereal crops and involves time-consuming protocols that often fact that most of the laboratory-scale, Arabidopsis-based drought need to be performed by highly skilled technicians (Singh and studies have limited data collection and phenotyping (Blum, Prasad, 2016). Even when efficient protocols have been 2014). As a general example, most of the Arabidopsis drought- developed for the species of interest, high transformation stress experiments are performed by suspending irrigation for an efficiency is usually confined to just a few laboratory varieties extended time (12–21 days) followed by re-watering. The (Harwood, 2012). In addition, as these varieties are often survival rate (live/dead plants) is then measured a few days obsolete, an introgression program into current elite varieties (2–7) after. In this set of experiments, data regarding soil must follow, thereby further hampering applicability of plant moisture, plant biomass, RWC, and seed yield are often not research in plant breeding. recorded. These dehydration survival experiments in near lethal The advent of genome editing is rapidly altering this scenario. conditions do not often reflect in crop performances in open The wealth of opportunities that are opening up as a result of the fields, and do not relate to improved yield under drought or rapidly advancing CRISPR-based technologies are driving a new normal conditions (Figure 3D). Furthermore, plants have wave of technological development in plant transformation evolved survival traits to maximize fitness when growth (Kausch et al., 2019). A recent breakthrough by DuPont conditions are not ideal, often by decreasing total seed number Pioneer (now Corteva) scientists was achieved by applying to ensure the full viability of a limited number of seeds. However, morphogenic regulator genes like BABYBOOM (BBM) and the ultimate goal of plant breeding is to increase or secure plant WUSCHEL (WUS) as transformation adjuvants (Lowe et al., yield and production, not plant survival (Skirycz et al., 2011; 2016). This ultimately optimized the idea of using growth- Blum and Tuberosa, 2018). Future experimental planning with Frontiers in Plant Science | www.frontiersin.org 13 January 2020 | Volume 10 | Article 1676 Martignago et al. Drought Resistance by Tissue-Specific Responses more thorough data collection beyond mere survival rate and On the other hand, traits like cuticular waxes, senescence, and that includes yield evaluations might overcome this limitation stay-green might have significant differences that would need to (Zhou et al., 2016). Validating the most promising results in a be carefully assessed using a species-by-species approach. crop model should become the priority for any research group Nonetheless, Arabidopsis could still provide a useful that works in Arabidopsis. Alternatively, collaborations between heterologous system to test novel genes discovered in cereal Arabidopsis and crop scientists and/or plant breeders should be species and their relative molecular responses. established to streamline the translation of innovative As a general frame to help translate research in Arabidopsis biotechnological approaches for use in agricultural science. into crops, and with the ultimate goal of improving drought Furthermore, industrial partnerships with plant breeding performance in cereals, we suggest the following measures to be companies or seed companies could provide both the means adopted: a) use an accurate experimental design in Arabidopsis; and the expertise to test engineered plants in extensive field trials, b) timely translate promising genes/traits in model crops (i.e., tests which would otherwise prove unpractical or financially rice); c) include HTPP to corroborate Arabidopsis results and to unsustainable in most research laboratories. test novel crop genotypes; d) investigate tissue- and cell type– In parallel, the advent of high-throughput plant specific drought responses; and e) clone tissue- and cell type– phenotyping (HTPP) platforms and the establishment of specific, stress-responsive promoters for monocots and make research infrastructure networks like the EPPN2020 (https:// available them to the entire scientific community. eppn2020.plant-phenotyping.eu/) will definitively help to It is crucial to strengthen the bridges between Arabidopsis and increase and improve the reproducibility and quality and crop scientists. Moreover, the coordination of research groups quantity of data from drought adaptation studies. HTPP for and institutes working with Arabidopsis and crop species at the drought responses has been implemented for Arabidopsis same time will be important in facilitating this process. In (Granier et al., 2006; Jansen et al., 2009; Skirycz et al., 2011; addition, academia–industry partnerships could prove Fujita et al., 2018) and applied to drought research (Rosa et al., instrumental not only for rapidly scaling up promising results, 2019). Outdoor or greenhouse HTPP facilities to study drought but also for designing potential drought-resistant strategies that performances are being developed for crops (Fahlgren et al., might have a high impact on global agriculture. 2015). These facilities are usually capable of capturing multispectral images of the plants, weighing the pots as a gravimetric, indirect measure of soil moisture, and differentially irrigating the plants to allow drought-exposed AUTHOR CONTRIBUTIONS and control plants to be placed in the same environment. DM and AC-D outlined and wrote the manuscript together with Generally, these systems are based either on a robotized AR-M (designed the figures), JF-M, and DB-E. All authors apparatus that moves around the plants performing reviewed and edited the manuscript. measurements and irrigation (Gosseau et al., 2019), or exploits a mobile device that scans plants for images while the pots stand on scales (Vadez et al., 2015). Alternatively, the pots are arranged on a conveyor belt system that transports the FUNDING plants to watering or imaging stations, like in the APPP systems at IPK Gatersleben, Germany (Junker et al., 2014). AIC-D is a recipient of a BIO2016-78150-P grant funded by the As reproducibility of results (Editorial, 2016) might deter plant Spanish Ministry of Economy and Competitiveness and Agencia breeders and investors, a widespread adoption of HTPP could Estatal de Investigación (MINECO/AEI) and Fondo Europeo de not only support the validity of experimental results, but also Desarrollo Regional (FEDER), and a European Research represent a valid intermediate step before large-scale field Council, ERC Consolidator Grant (ERC-2015-CoG – 683163). trials, especially when a series of different genotypes with JBF-M is supported by the grant 2017SGR718 from Secretaria comparable drought performances are involved (Figure 3E). d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and by the ERC- 2015-CoG – 683163 granted to the AIC-D laboratory. AR-M is a SUMMARY predoctoral fellow from Fundación Tatiana Pérez de Guzmán el Bueno. DB-E and DM are funded by the ERC-2015-CoG – In this review, we highlight that many physiological mechanisms 683163 granted to the AIC-D laboratory. This project has underlying drought-resistance traits are conserved between received funding from the European Research Council (ERC) Arabidopsis and crops. DE, control of flowering time, stomatal under the European Union’s Horizon 2020 research and responses, T6P pathways, and some root traits are highly innovation programme (Grant Agreement No 683163). This conserved among plants. Therefore, Arabidopsis is an excellent work was supported by the CERCA Programme from the model to test drought responsive strategies. Still, when studies Generalitat de Catalunya. 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Bot. tion in other forums is permitted, provided the original author(s) and the copyright 58 (15-16), 4269–4281. doi: 10.1093/jxb/erm288 owner(s) are credited and that the original publication in this journal is cited, in Wahl, V., Ponnu, J., Schlereth, A., Arrivault, S., Langenecker, T., Franke, A., et al. accordance with accepted academic practice. No use, distribution or reproduction is (2013). Regulation of flowering by trehalose-6-phosphate signaling in permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 19 January 2020 | Volume 10 | Article 1676