This is the accepted version of the article: Gupta, Aditi; Rico Medina, Andrés; Caño Delgado, Ana I. «The physiology of plant responses to drought». Science, Vol. 368, no. 6488 (April 2020), p. 266-269. DOI 10.1126/science.aaz7614 This version is avaible at https://ddd.uab.cat/record/221568 under the terms of the license 1 MANUSCRIPT TYPE: Review article 2 TITLE: The Physiology of Plant Responses to Drought 3 4 AUTHORS: Aditi Gupta, Andrés Rico-Medina, and Ana I. Caño-Delgado*, 5 ADDRESS: Department of Molecular Genetics, Centre for Research in Agricultural Genomics 6 (CRAG) CSIC-IRTA-UAB-UB, Campus UAB (Cerdanyola del Vallès), 08193 Barcelona, Spain. 7 *Corresponding author email: ana.cano@cragenomica.es 8 9 KEYWORDS 10 Drought, abiotic stress, signaling, root, hydrotropism, stomata, water use efficiency, climate 11 change, food security, Arabidopsis, cereals, sorghum, crops. 12 13 ABSTRACT 14 Drought alone causes more yield loss annually than all pathogens combined (1). To adapt to 15 moisture gradients in soil, plants alter their physiology, modify root growth and architecture, and 16 close stomata on the above-ground plant. These tissue-specific responses modify the flux of signals 17 along the plant, resulting in early flowering or stunted plant growth that often reduce yield. 18 Physiological and molecular analyses of the model plant Arabidopsis thaliana (Arabidopsis) have 19 identified phytohormone signaling as key for regulating response to drought or water insufficiency. 20 Here, we discuss how engineering hormone signaling in specific cells and cellular domains 21 promises to improve plant responses to drought. We explore current knowledge and future 22 questions in the quest to produce high-yield, drought-resistant crops. 23 24 1 25 INTRODUCTION 26 Drought is a misfortune for agriculture, humanity and livestock alike. Climate change is leading 27 us towards a hotter, more parched world (2). There is an urgent need to produce high-yielding 28 plants that are more efficient at using water (Fig. 1a). In the last decade, global losses in crop 29 production due to drought estimated around 30 billion dollars. Global population raised from 5 30 billion inhabitants in the 1990 to over 7.5 billion presently and is predicted to raise to 9.7-10 billion 31 by 2050 (3), half of which (i.e. 5 billion people) is estimated to be living in water-scarce areas of 32 the planet (Fig. 1b) (4). Despite the moderate increase is global arable land, an additional 1 million 33 Ha will be needed to ensure food security (Fig. 1c) (5). In addition, water demand for agriculture 34 need to duplicate by 2050, whereas the fresh water availability is predicted to drop by half cause 35 of climate change (Fig. 1d) (6). Certainly, plant biotechnology holds one of the promises to meet 36 the societal demand to duplicate crop production in the planet. 37 38 Water is crucial for plant survival and water deficit limits plant growth. However, plants have 39 strategies to prevent water loss, to balance optimal water supply to all vital organs, to maintain the 40 cellular water content, and to persevere through periods of drought. The ability of a plant to sense 41 the water deficiency signal and initiate the coping strategies in response to drought signal is defined 42 as drought resistance. Drought resistance is a complex trait that impinges on several mechanisms 43 referred as: i) escape (acceleration of plant reproductive phase before stress that could hinder its 44 survival), ii) avoidance (endurance with increased internal water content and preventing tissue 45 damage), and iii) tolerance (endurance with low internal water content, whereas sustaining growth 46 over the drought period (7). The percentage of viable plants after a period of drought upon re- 47 watering is referred as drought survival. From molecular biology perspective, cellular water loss 2 48 marks the first event of drought stress. At the cellular level, drought signals promote production 49 of stress-protectant metabolites such as proline and trehalose; trigger antioxidant system to 50 maintain redox homeostasis and prevent acute cellular damage and membrane integrity by 51 peroxidase enzymes. Factors such as extent of water stress and the plant organ in which the stress 52 is sensed also trigger specific signaling responses, including but not limited to; abscisic acid, 53 brassinosteroids, and ethylene phytohormone pathways (8-11). 54 55 The impact that drought has on agriculture depends on the degree and duration of the reduced 56 precipitation and soil water gradients as well as on the species and developmental stage of the 57 plants (8). However, for most instances, crops experience moderate droughts caused by prolonged 58 precipitation deficits, reduced ground water levels, and/or limitation in accessing water supplies 59 leading to substantial losses in overall yield. Therefore, investigating the mechanisms of how a 60 plant sustains its growth during moderate drought and devising new strategies to improve plant 61 health during such moderate drought periods can provide us solution for future food security. 62 Understanding the response of cellular signaling to water shortage is a priority to answer the above- 63 mentioned questions for modern agriculture (12). Here we explore how water availability cues cell 64 and tissue growth patterns and how these growth patterns are coordinated in the whole plant to 65 improve drought resistance without loss of yield. Overexpression of drought-responsive genes 66 often results in growth deficits and yield loss. Tissue- or time-specific expression of drought- 67 response traits may improve drought response without depressing yield. A combination of 68 strategies may improve agricultural yields despite increased water insecurity. 69 70 Traits for improving drought resistance 3 71 During drought spells, plant systems work actively to maintain the physiological water balance. 72 This is achieved by: i) increased root water uptake from the soil, ii) reduced water loss by closing 73 stomata, and iii) osmotic adjustment within plant tissues (13). Activated stress response pathways 74 include phytohormone signaling, antioxidant and metabolite production and mobilization (11). 75 76 Roots respond to changes in soil moisture both at the cellular scale and at the entire root system 77 architecture. The root stem cell niche, meristem, and vasculature each coordinate responses for 78 drought (Fig. 2a, b). During water scarcity, the root system architecture undergoes morphological 79 changes to enhance its ability to absorb both water and nutrients (9,10). These modifications can 80 be traced to coordinated cell division, elongation and differentiation events in the root apex. Root 81 systems grow differentially to adapt their architecture to be either deep or shallow, in search of 82 moisture (Fig. 2c). Longer and deeper roots with reduced branching angles can efficiently capture 83 water from soil that may be dry at the surface but have deep layers retaining moisture. In contrast, 84 shallower root architectures are more beneficial in regions of low precipitations to maximize water 85 capture from the soil surface (9). Roots that encounter a soil environment with non-homogeneous 86 water distribution display hydro-patterning by favoring lateral root emergence towards soil patches 87 with higher water content, a process that is also mediated by auxin signaling (9, 14). Another 88 adaptive response to non-homogeneous distribution of moisture through soil is hydrotropism (Fig. 89 2d), where root tips grow towards zones with higher water content to optimize the root system 90 architecture for water acquisition (15). 91 92 Stomatal closure is a more rapid defense against dehydration (Fig. 2d, e). Stomatal pores, on leaf 93 surfaces, open or close according to turgidity of the surrounding guard cells. The turgor-driven 4 94 shape changes of guard cells are affected by cell wall structure, the plasma membrane, the 95 tonoplast properties, and cytoskeletal dynamics (16). Plant vascular tissues, xylem and phloem, 96 transmit water availability signals from roots to shoots and transmit photoassimilates from shoots 97 to roots respectively (17). Development of these inner vasculature tissues also affects drought 98 resistance. Crop yield becomes most vulnerable if the drought occurs during reproductive phase 99 of plant. In Arabidopsis, early flowering associated to drought escape is linked with the phloem 100 loading and transport of photoperiod-dependent protein FLOWERING LOCUS T (FT) from 101 leaves to the shoot apical meristem. (18) 102 103 Phytohormones to combat drought 104 The hormone abscisic acid (ABA) regulates plant responses to dehydration and optimizes water 105 use. Dehydration signals stimulate local production of ABA in different plant organs. However, 106 the ABA production is found to be most efficient in the leaf mesophyll cells compared to the root 107 tissues (19). The accumulated ABA then activates downstream signaling components (20). ABA 108 executes its function during stress by signal cross-talk with other pathways (Fig. 3) (21). Many 109 existing schemes to improve water use efficiency and drought resistance engage the ABA pathway. 110 111 Genetic engineering to improve Pyrabactin Resistance 1/PYR1-Like/Regulatory Component of 112 ABA Receptors (PYR/PYL/RCAR) and SnRK2s functions and repress the negative regulators 113 clade A type 2C protein phosphatases (PP2Cs) have resulted in improved water use efficiency in 114 plants such as Arabidopsis and wheat under controlled laboratory growth conditions and 115 glasshouses (22-25). A regulatory network of ABA pathway genes, a hierarchy of ABA-related 116 transcription factors, and signaling feedback were identified among ABA-mediated stress 5 117 responses to drought (26). Engineering the ABA receptor PYR1 for heightened sensitivity towards 118 the pre-existing agrochemical mandipropamid resulted in improved drought resistance in 119 Arabidopsis and tomato (22). Virtual screening for ABA receptor agonists led to the identification 120 of a bioactive ABA mimic called opabactin. This small molecule can enhance ABA receptor 121 activation and downstream signaling to improve water use efficiency and drought resistance in 122 Arabidopsis, tomato and wheat (27). Thus computational design combined with experimental 123 biology led to identify a small molecule that can mitigate the effects of drought on crop yields. 124 125 Brassinosteroid hormones also regulate drought response through signaling components linked to 126 the ABA response pathway (Fig. 3) (28, 29). Brassinosteroid signaling negative regulator 127 BRASSINOSTEROID-INSENSITIVE 2 (BIN2) is dephosphorylated by ABA INSENSITIVE1 128 (ABI1) and ABI2. ABA activates BIN2 by inhibiting ABI1/ABI2 activity (30). BIN2 129 phosphorylates SnRK2s and activates the downstream pathway (31). ABA signals can also 130 converge with the brassinosteroid pathway at the level of downstream transcription factors (Fig. 131 3). BRI1-EMS-SUPPRESSOR 1 (BES1) inhibited ABA induction of a drought-related 132 transcription factor RESPONSIVE TO DESICCATION 26 (RD26) (32). RD26 shows reciprocal 133 antagonism with brassinosteroid by modulating BES1-regulated transcription and inhibiting 134 brassinosteroid -regulated growth (33). WRKY46, 54, and 70 belong to another class of 135 transcription factors that interact with BES1 to promote plant growth while repressing drought 136 responses (34). BIN2 can phosphorylate and destabilize WRKY54 to negatively regulate its effect 137 on the BES1-mediated brassinosteroid response (35). BIN2 phosphorylates and activates the 138 ubiquitin receptor protein DSK2, which leads to BES1 degradation via autophagy and coordinates 139 plant growth and survival under drought conditions. (36). An AP2/ERF transcription factor called 6 140 TINY is another candidate that balances brassinosteroid-mediated stress adaptation with growth. 141 TINY interacts with BES1 and antagonizes brassinosteroid-regulated growth. BIN2 on the other 142 hand phosphorylates and stabilizes TINY to promote ABA-induced stomatal closure and drought 143 resistance (37). Thus, brassinosteroid as well as ABA aids drought resistance. 144 145 Tissue-specific responses for drought resistance 146 Stomatal closure preserves water in the plant. ABA content in leaves regulates stomatal movement 147 in response to water availability (25) (Fig. 3). Since stomatal movements control CO2 influx and 148 transpiration, efforts to reduce water loss via stomatal closure occur at the cost of photosynthesis, 149 growth, and yield (13). Therefore, most strategies to improve water efficiency and drought 150 resistance in plants focus on fine-tuning stomatal conductance and manipulating ABA signaling 151 via stomata-specific promoters (38). With optogenetics, scientists have improved the 152 responsiveness of the stomata and overcome the coupling of CO2 uptake with water vapor loss. 153 Upon introducing BLINK1, a light-activated synthetic K+ ion channel, into guard cells, stomata 154 became more synchronized with fluctuating light conditions (39). This manipulation improved the 155 performance of the stomata and, as a consequence, growth and productivity of the plant. Thus, 156 water use efficiency was improved by engineering stomata to maximize the amount of carbon 157 fixed per unit of water lost. 158 159 Improving water acquisition by roots can also improve plant performance upon drought. In 160 Arabidopsis, an auxin pathway modulator EXOCYST SUBUNIT EXO70 FAMILY PROTEIN A3 161 (EXO70A3) that regulates depth of the root system was identified using a genome-wide 162 association (GWA) mapping (40). EXO70A3, a component of the exocytosis system, is expressed 7 163 in root tips. EXO70A3 regulates local auxin transport by affecting the homeostasis of auxin efflux 164 carrier PIN-FORMED 4 in root columella cells (Fig. 3). Natural variation in EXO70A3 was 165 correlated with seasonal precipitation and conferred different adaptive root system architecture 166 configurations under different rainfall patterns. In areas with high temperatures and irrigated soils, 167 deeper root architectures proved better for drought adaptation. In rice, an auxin inducible gene 168 DEEPER ROOTING1 provides drought resistance by promoting a more vertical and deeper root 169 system architecture (41). Although auxin modulates root architecture under stress (40, 41), 170 hydrotropic root responses are fairly auxin-independent and involve ABA signaling in root 171 elongation zones. Coordinated activity of the ABA inducible MIZU-KUSSEI1 (MIZ1) and SNF1- 172 RELATED KINASE 2 (SnRK2.2), in root elongation zone cortical cells interprets water potential 173 gradients in soil environments (15, 42). 174 175 Brassinosteroid receptors regulate root hydrotropic responses (Fig. 3). Overexpression of a 176 vascular-enriched brassinosteroid receptor BRI1-Like3 (BRL3) promoted root hydrotropic 177 bending. The brl1brl3bak1 triple mutant of BRL3 signalosome showed reduced hydrotropic 178 response, suggesting a role for the vascular BRL3 receptor complex in regulating hydrotropic 179 responses (43) (Fig. 3). Activation of the BRL3 pathway in vasculature triggered accumulation of 180 osmoprotectant metabolites such as proline, trehalose, and raffinose family oligosaccharides in 181 plant roots in response to water withdrawal, which improved drought resistance without penalizing 182 growth (43) (Fig. 3). Phloem-specific localization of BRL3 is likely to be the determining factor 183 for promoting drought resistance without impairing yield (29, 43). 184 8 185 In drought, roots sense water scarcity from soil. The above-ground plant responds by closing 186 stomata in leaves, thus implicating a systemic communication system. Upon drought, the CLE25 187 peptide is produced in the roots and moves through the vasculature to plant leaves to drive ABA 188 production by activating the biosynthetic enzyme NCED3. This burst of ABA synthesis leads to 189 stomatal closure and improved water balance, thereby promoting drought survival (44) (Fig. 3). 190 This insight into small-peptide signaling in Arabidopsis may help with identification of similar 191 mechanisms in crop plants for root-to-shoot mobilization of stress signals. 192 193 A view to the future 194 Genetic traits that sustain crop plant growth under moderate drought may come from multiple 195 sources, including natural genetic variation in wild relatives or bioengineering. Traditional 196 breeding has been the main strategy for exploiting the genetic diversity of adaptive traits in natural 197 alleles. The advent of genomic technologies and gene mapping tools such as GWAS and precision 198 genome editing with the CRISPR/Cas9 system became instrumental for the generation of new 199 alleles that can improve plant yield and performance under various stresses Molecular studies 200 using tissue/cell-specific promoters coupled with live microscopy techniques for real-time 201 visualization of cellular processes pave the way for analysis of drought responses network that can 202 be used as targets for various biotechnological approaches (Fig. 4) . Small molecules, such as 203 peptides or hormone agonists, may be useful for fine-tuning drought-response pathways while 204 preserving yield in agriculture (. Together, uncovering the physiology of plant responses to drought 205 in model system and translating that to crops will deliver new strategies to combat water scarcity. 206 Agriculture is a thirsty enterprise. Discovering ways to ameliorate agriculture’s thirst will ease 207 competition for freshwater resources from increasing population growth. 9 208 209 FIGURE LEGENDS 210 Figure 1: The power of biotechnology to solve the problem of drought-induced food 211 insecurity. 212 a) Most scenarios predict that the scarcity of water will increase in years to come. With the world´s 213 population continuously raising, crop production must be duplicated to fulfill civilization´s basic 214 needs. For this purpose, the efficiency of water usage in plants will need to be improved. b) 215 Estimated world population for the 1990-2050-time period. Arrow indicates estimated number of 216 people potentially living in water-scarce areas of the planet. c) Global arable land for agriculture 217 for the 1990-2050-time period. Arrow indicate predicted demand of arable land to assure food 218 security, given current rates of crop production per hectare. d) Global fresh water demand for 219 agriculture for the 1990-2050-time period. Arrow indicates predicted decline in fresh water 220 availability for agriculture in the current tendency of climate change and precipitations. 221 222 Figure 2: Root and shoot traits that account for drought resistance. 223 a-b) Plants first sense drought through their roots, where particular cell types such as stem cells, 224 cells in the cortex and vasculature mediate adaptive responses towards water limitations. Roots 225 can modulate their system architecture to c) maximize access to superficial humidity or delve into 226 deep humid soil layers, and d) bend towards more humid soil zones (hydrotropism). e-f) In 227 aboveground plant organs such as leaves and stem stomata work actively against dehydration. In 228 water limiting conditions, stomata remain closed to reduce water loss. 229 230 Figure 3: Hormone signaling events underpinning drought. 10 231 Schematic representation of hormone signaling modules controlling drought adaptation. Plants 232 work against dehydration in all their organs such as leaves, vasculature and roots ABA, through 233 SnRK2, activates a variety of genes that cause stomata closure and improve water balance. When 234 drought is sensed in the roots, CLE25 peptide moves through the vasculature to leaves where 235 locally controls ABA biosynthesis and stomata closure. Brassinosteroids also play roles in 236 regulating plant drought response. Brassinosteroid pathway converge with ABA by activating 237 SnRK2 through downstream pathway component, BIN2 and vice versa. Independently of ABA, 238 brassinosteroid receptors (BRI1, BRL1, BRL3) modulate hydrotropic responses in the roots. 239 Vascular BRL3 receptor coordinates plant growth and survival under drought stress by promoting 240 the accumulation of osmoprotectant metabolites in the root tissues. Furthermore, non-canonical 241 auxin responses via EXO70A3 and PIN4 can modulate root architecture patterning and depth, to 242 improve water absorption from the soil improving drought tolerance. 243 244 Figure 4: The promise of overcoming drought in agriculture. 245 Various strategies can provide solutions to counteract drought and can be used to develop drought- 246 smart crops. Natural allelic variations found in plants can be selected to improve drought resistance 247 and yield. Traditional plant breeding approaches have successfully employed such useful traits to 248 obtain more resistant crop varieties in fields. 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A.R.M. is funded by a PhD fellowship (2018-2022) from the Fundación Tatiana Pérez 425 de Guzmán el Bueno. 426 427 AUTHOR CONTRIBUTIONS: 428 A.G. and A.I.C.-D wrote the manuscript. A.G. and A.R.-M. designed the figures. All authors 429 reviewed the manuscript. 430 431 COMPETING INTERESTS: 432 The authors declare no competing or financial interests. 16 a Year 2020 Year 2050 Duplicate Crop Production b c d 10 2,5 5 8 2 4 billions of cubic meters billions of hectares billions of people 6 1,5 3 4 1 2 2 0,5 1 0 0 0 1990 2020 2050 1990 2020 2050 1990 2020 2050 a c e 50 um 100 um b d f ia ed m ic ot m os gh hi 50 um 1 cm 20 um ABA PYR/PYL RCAR Stomata closure SnRK2 ABA-responding genes CL E2 BI 5 2N BRASSINOSTEROIDS BIN2 BRL3 Hydrotropism, osmoprotectant metabolites accumulation BES1 WRKYs/ RD26 TINY AUXIN 5 E2 CL EXO70A3 IAA Root system architecture CLE25 PIN4 Drought sensing Natural variation/ Genetic GWAS editing Traditional breeding Bioengineering Optogenetics Chemicals/ hormone mimetics Small peptides Biostimulants