(2) How Plants Manage Water Deficit and Why It Matters

Plant-Water Relations (2) How plants manage water deficit and why it matters

Photo credits: Mel Oliver; William M. Ciesla, Forest Health Management International; R.L. Croissant Bugwood.org

1. Water scarcity is a growing problem 2. Desiccation-tolerant plants and xerophytes 3. Plant responses to water deficit Perception and signaling Transcriptional responses Effects on photosynthesis Effects on growth and development 4. Towards water-optimized and drought-tolerant agriculture Optimizing irrigation Monitoring water stress 5. Breeding for drought-tolerance Classical approaches Candidate gene approaches

Desiccation: Extreme water loss, equilibration to the water potential of the air which is generally low Desiccation tolerance: The ability to withstand and recover from desiccation Dehydration: The process of water loss from a cell or system Water deficit: Sub-optimal water status from the perspective of plant function Drought: Lack of rainfall or water supply leading to water deficit Drought tolerance: The ability to withstand suboptimal water availability through adaptations or acclimations (not the same as desiccation tolerance) Xerophyte: A plant adapted to live in a low-water environment Mesophyte: A plant without specialized adaptions to low-water or high- water environments Adaptation: Long-term evolutionary process by which an organism becomes suited to its environment Acclimation: Short-term physiological process by which an organism adjusts to changes in its environment

In the past 40 years, Lake Chad has Severe droughts are increasing in shrunk by 95% because of irrigation frequency and forcing up food prices demands & climate change

In 2013, a major drought affected large parts of the United States

US National Drought Mitigation Center / NASA

Reprinted by permission from Macmillan Publishers Ltd. NATURE: Vorosmarty, C.J., McIntyre, P.B., Gessner, M.O., Dudgeon, D., Prusevich, A., Green, P., Glidden, S., Bunn, S.E., Sullivan, C.A., Liermann, C.R. and Davies, P.M. (2010). Global threats to human water security and river biodiversity. Nature. 467: 555-561, copyright 2010.

<1% of Earth’s water is 97% saltwater fresh and potentially available to plants

3% freshwater 68.7% snow and ice (including polar icecaps)

30.9% groundwater 0.4% lakes and rivers NASA: Earth Observatory

Rainwater and snowmelt are renewable

Oil is a Solar energy is a nonrenewable renewable resource resource, limited Aquifer-derived only by our ability water is not to capture and harness it

Almost one third of the groundwater used for irrigation in the United Aquifers around the world States comes from the are similarly running dry Ogallala aquifer (shown in color), but this water resource is running out

USGS

World Development Report 2010

Understanding how plants manage water deficit is essential for meeting the needs of the growing (and growing in affluence) human population

Source: FAO Water at a Glance

© 2014 American Society of Plant Biologists Some plants and plant tissues are desiccation-tolerant Seeds and pollen – usually desiccation tolerant Bryophytes (mosses, liverworts, hornworts) – many are desiccation tolerant

Some vascular plants have acquired vegetative desiccation tolerance Vegetative tissues of vascular plants – usually not desiccation tolerant Photo credit: Anne Wangalachi/CIMMYT; Mel Oliver; Dartmouth Electron Microscope Facility

Within families and species, a wide range of drought sensitivities are present Drought tolerant Drought sensitive

Many bryophytes

A small number of Succulents and vascular plants non-succulents

Photo credits: Mel Oliver ; Hoekstra, F.A., Golovina, E.A. and Buitink, J. (2001). Mechanisms of plant desiccation tolerance. Trends Plant Sci. 6: 431-438; Mwilliams; Halley; Lopes,

M.S., Araus, J.L., van Heerden, P.D.R., and Foyer, C.H. (2011). Enhancing drought tolerance in C4 crops. J. Exp. Bot. 62: 3135 – 3153; Paxton Payton

• Equilibrate their Cellular cellular water potential Use morphological, Adaptations protection and with the ambient water phenological and and acclimations repair potential biochemical strategies to to avoid mechanisms • Tolerate desiccation maintain constant through biochemical cellular water potential desiccation and structural and / or relative water

Ψ fluctuates adjustments content Ψ fairly constant

Death

Cellular water water Cellular potential

potential Cellular water water Cellular External water External water potential potential

Desiccation-tolerant Most plants – bryophytes under -3 to -4 MPa

lab lethal limit -

conditions MPa 250 All desiccation- (-12 MPa Larrea) -

tolerant plants 40MPa

Dehydration levels

10MPa

600 MPa 600 100 MPa 100

All resurrection plants - tends to be

the lowest end point

in natural environments Most bryophytes for brief periods

In a practical context: Plants and plant organs (e.g., seeds) equilibrated at water potentials Adapted from Oliver, M.J., Cushman, J.C. and Koster, K.L. (2010).Dehydration tolerance below -100 MPa are considered desiccated in plants. Methods in Molecular Biology (Clifton, N.J.) 639: 3-24.

Note that most seeds and pollen retain desiccation tolerance

Vegetative DT lost Desiccation tolerance (DT) A small number of is common in bryophytes tracheophytes (vascular plants) (Iiverworts, mosses) called “resurrection plants” have reacquired vegetative desiccation tolerance

Liverworts (~7000 Mosses (~10,000 – Hornworts (~200 species) – 8500 species) 17,000 species) Vascular plants

Green algae

Land plants

Adapted from Chang, Y. and Graham, S.W. (2011). Inferring the higher-order phylogeny of mosses (Bryophyta) and relatives using a large, multigene plastid data set. Am. J. Bot. 98: 839-849 and Ligrone, R., Duckett, J.G. and Renzaglia, K.S. (2012). Major transitions in the evolution of early land plants: a bryological perspective. Ann. Bot. 109: 851-871. Photo credits Tom Donald, Mary Williams and gjshepherd_br / Foter.com / CC BY-NC-SA

Wheat, corn, rice, beans and other human food staples produce “orthodox” seeds that desiccate and can be stored stably

Rhizophora mangle Cocos nucifera Some mangroves, Stenocereus alamosensis red mangrove coconut cacti and large- seeded plants such as coconut have “recalcitrant” seeds that do not desiccate or become quiescent

Viviparous seeds germinating on the tree or in the fruit

Reprinted with permission of the Botanical Society of America from Cota-Sánchez, J.H., Reyes-Olivas, Á. and Sánchez-Soto, B. (2007). Vivipary in coastal cacti: a potential reproductive strategy in halophytic environments. Am. J. Bot. 94: 1577-1581. Jaro Nemcok Vencel

Embryogenesis

seeds Orthodox Dry state, Water content Dispersal

Embryogenesis Germination

Seed reserve accumulation Recalcitrant seeds

disperse without the stability associated with

Dispersal desiccation

Recalcitrant Recalcitrant seeds Water content

Adapted from Franchi, G.G., Piotto, B., Nepi, M., Baskin, C.C., Baskin, J.M. and Pacini, E. (2011). Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. J. Exp. Bot. 62: 5267-5281.

Some plants make recalcitrant pollen that Most plants produce does not desiccate and cannot tolerate Some grasses desiccated, orthodox pollen prolonged exposure to dry air live in dense that can tolerates exposure groups so pollen to dry air during dispersal Some orchids package disperses very pollen inside a protective short distances membrane, shown here attached to the head of a pollinator

Squash (Cucrbita pepo) male flowers are each open for only a few hours

Firon, N., Nepi, M. and Pacini, E. (2012). Water status and associated processes mark critical stages in pollen development and functioning. Ann. Bot. 109: 1201-1214. Dartmouth Electron Microscope Facility H. Zell Spedona Rasbak David Midgley

A desiccation tolerant plant Selaginella showing a cycle of lepidophylla dehydration and rehydration

Oliver, M.J., Velten, J. and Mishler, B.D. (2005). Desiccation tolerance in bryophytes: A reflection of the primitive strategy for plant survival in dehydrating habitats? Integr. Comp. Biol. 45: 788-799 by permission of The Society for Integrative and Comparative Biology. Yobi, A., Wone, B.W.M., Xu, W., Alexander, D.C., Guo, L., Ryals, J.A., Oliver, M.J. and Cushman, J.C. (2013). Metabolomic Profiling in Selaginella lepidophylla at various hydration states provides new insights into the mechanistic basis of desiccation tolerance. Mol. Plant. 6: 369-385 by permission of Chinese Academy of Sciences and Chinese Society of Plant Biology

Xerophyta Myrothamnus Sporobolus stapfianus Selaginella humilis flabellifolia lepidophylla Mohria caffrorum (fern)

Angiosperms

Vegetative DT lost Craterostigma Tortula ruralis Bryophyte plantagineum (moss)

Reprinted from Farrant, J.M. and Moore, J.P. (2011). Programming desiccation-tolerance: from plants to seeds to resurrection plants. Curr. Opin. Plant Biol. 14: 340-345 with permission from Elsevier.

Craterostigma pumilum, an angiosperm desiccation-tolerant “resurrection plant”

Upon desiccation, • leaves curl in, • purple anthocyanin photoprotective sunlight- absorbing pigments accumulate, and • small molecules Selaginella lepidophylla accumulate that stabilize cell integrity

Reprinted from Hoekstra, F.A., Golovina, E.A. and Buitink, J. (2001). Mechanisms of plant desiccation tolerance. Trends Plant Sci. 6: 431-438 with permission from Elsevier; Kristian Peters

• Membranes and cell walls can break or adhere to each other • Proteins can aggregate or denature • Toxic reactive oxygen species can accumulate

Desiccation-tolerant cells must prevent CW=cell wall, C= chloroplast, T= thylakoids, V= vacuole, L=lipid droplet irreversible damage

Reprinted from Thomson, W.W. and Platt, K.A. (1997). Conservation of cell order in desiccated mesophyll of Selaginella lepidophylla ([Hook and Grev.] Spring). Ann. Bot. 79: 439-447 by permission of Oxford University Press.

Desiccation tolerant species Protein in native state (N)

Fully hydrated cell Intermediate dehydration Dry (glassy)

Denatured Desiccation (D) protein sensitive Small molecules and species proteins help maintain proteins in their native forms

Reprinted from Hoekstra, F.A., Golovina, E.A. and Buitink, J. (2001). Mechanisms of plant desiccation tolerance. Trends Plant Sci. 6: 431-438 with permission from Elsevier.

LEA proteins (grey coil) accumulate during dehydration and protect cell structures

Late Embryo Abundant (LEA) proteins were first characterized in seeds during embryo maturation, but accumulate in vegetative tissues during dehydration

Sun, X., Rikkerink, E.H.A., Jones, W.T. and Uversky, V.N. (2013). Multifarious roles of intrinsic disorder in proteins illustrate its broad impact on plant biology. Plant Cell. 25: 38-55.

Orderly folding helps membranes and walls, survive desiccation (bcw = bundle sheath cell wall, mcw = mesophyll cell wall)

Hydrated Dehydrated

Vander Willigen, C., Pammenter, N.W., Mundree, S.G. and Farrant, J.M. (2004). Mechanical stabilization of desiccated vegetative tissues of the resurrection grass Eragrostis nindensis: does a TIP 3;1 and/or compartmentalization of subcellular components and metabolites play a role? J. Exp. Bot. 55: 651-661 by permission of Oxford University Press. Reprinted with permission from Moore, J.P., Vicré-Gibouin, M., Farrant, J.M. and Driouich, A. (2008). Adaptations of higher plant cell walls to water loss: drought vs desiccation. Physiol. Plant. 134: 237-245.

Light energy absorbed by Dehydration stress interferes with photosynthesis, chlorophyll creates excited so the excess energy accumulates as reactive electrons for photosynthesis oxygen species

Photosynthesis Chl* e-

Photon Reactive oxygen species (ROS)

Chl

ROS image source: BioTek

These systems can be up-regulated under water stress From: Buchanan, B.B., Gruissem, W. and Jones, conditions R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

Synthesis of: Severe Cytoplasmic vitrification Moderate • Osmolytes water deficit Increase in antioxidant systems water deficit • LEA proteins (Desiccation- Photopigment protection (All plants) • Dehydrins tolerant Vacuolar fragmentation • ROS- Leaf shrinkage or folding detoxification plants)

Hydrated Dehydrated

Tortula ruralis is a highly desiccation- tolerant moss that has been used as a model system to understand desiccation tolerance

Adapted from Rascio, N. and La Rocca, N. (2005). Resurrection plants: The puzzle of surviving extreme vegetative desiccation. Crit.Rev. Plant Sci. 24: 209–225; Oliver, M.J., Tuba, Z., and Mishler, B.D. (2000). The evolution of vegetative desiccation tolerance in land plants. Plant Ecol. 151: 85 – 100.

Many bryophytes and a few vascular plants are desiccation tolerant. They use many strategies shared with non-desiccation tolerant plants, but take them to an extreme Selaginella lepidophylla

Yobi, A., Wone, B.W.M., Xu, W., Alexander, D.C., Guo, L., Ryals, J.A., Oliver, M.J. and Cushman, J.C. (2013). Metabolomic Profiling in Selaginella lepidophylla at various hydration states provides new insights into the mechanistic basis of desiccation tolerance. Mol. Plant. 6: 369-385 by permission of Chinese Academy of Sciences and Chinese Society of Plant Biology

Anatomical Thick cuticle, Phenological rolled leaves, Drought evaders: stomatal crypts Grow and reproduce after rain, otherwise remain quiescent

Morphological Biochemical Deep roots, high Crassulacean acid xylem transport and / metabolism or tissue storage (CAM): stomata capacity, and tiny or open at night to absent leaves avoid water loss

Photo credits:; Amrum; SAPS; James Henderson, Mary Williams

Euphorbia

Cacti

Agave, Yucca, Aloe

Anatomical traits frequently found in xerophytes: • Succulent (water storing) leaves or stems • Thick cuticle • Sunken stomata Agave Map of Life - "Succulent desert plants"

Responses vary with extent of water deficit, rate of dehydration, and by genotype

Lopes, M.S., Araus, J.L., van Heerden, P.D.R., and Foyer, C.H. (2011). Enhancing drought tolerance in C4 crops. J. Exp. Bot. 62: 3135 – 3153 by permission of Society of Experimental Biology; Oliver, M.J., Cushman, J.C. and Koster, K.L. (2010).Dehydration tolerance in plants. Methods in Molecular Biology (Clifton, N.J.) 639: 3-24 with kind permission from Springer Science and Business Media.

Photosynthesis There is little Transpiration response to a few days without water, but after a Critical water critical soil water potential threshold potential is Soil moisture reached, the plant responds

Marshall, A. et al., and De Smet, I. (2012). Tackling drought stress: RECEPTOR-LIKE KINASES present new approaches. Plant Cell. 24: 2262-2278; See also Boyer, J.S. (1970). Leaf enlargement and metabolic rates in corn, soybean, and sunflower at various leaf water potentials. Plant Physiol. 46: 233-235..

Slow rate of water loss Fast rate of water Slow drying allows plants time loss Sensitive species to respond and avoid injury Acclimation / Protection Injury / Repair

Resistant species

Acclimation / Drought-resistant species Protection suffer less damage with the Injury / Repair same extent of water deficit

TIME

Adapted from Bray, E.A. (1997). Plant responses to water deficit. Trends Plant Sci. 2: 48-54.

• Cell turgor Between cells: Short term: • Membrane pressure • ABA • Decrease in stomatal • Osmotic content • Ethylene conductance • Reactive oxygen • Hydraulic signals • Alterations in hydraulic • ? Other unknown • Water potential conductivity • Xylem pH • Osmotic adjustments • Other signals… Long term: Within cells: • Induction of drought- • ABA induced genes • Reactive oxygen • Changes in growth rate • Transcriptional including root architecture cascades • Other signals…

High evaporative demand

The site of perception can be cells in the leaf or root or both

Soil water depletion

Several plasma-membrane Similar proteins may be localized pressure sensors in involved in sensing water yeast have been identified deficit in plants

Reprinted from Christmann, A., Grill, E., and Huang, J. (2013). Hydraulic signals in long-distance signaling. Curr. Opin. Plant Biol. 16: 293–300 with permission from Elsevier.

Drought responses are mediated by ABA and Altered ion Stomatal closure hydraulic processes ABA channel and inhibition of biosynthetic ABA activity opening enzymes Transcription Cellular defenses Water Cellular Turgor factor and osmotic deficit dehydration sensor activation adjustment Transcription factor activation Altered hydraulic conductance Growth (cell Arrows indicate division and interactions, whether expansion) positive or negative

Adapted from Zhang, J., Jia, W., Yang, J. and Ismail, A.M. (2006). Role of ABA in integrating plant responses to drought and salt stresses. Field Crops Research. 97: 111-119.

© 2014 American Society of Plant Biologists Abscisic acid accumulation is a rapid response to water deficit Synthesis ABA levels are tightly controlled induced by by synthesis, conjugation and water deficit degradation

Reversible inactivation by conjugation Abscisic acid (ABA)

Irreversible hydrolysis and inactivation

Zeevaart, J.A.D. (1980). Changes in the levels of abscisic acid and its metabolites in excised leaf blades of Xanthium strumarium during and after water stress. Plant Physiol. 66: 672-678.

Nakashima, K., Ito, Y. and Yamaguchi-Shinozaki, K. (2009). Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol. 149: 88-95.

Hundreds of studies have revealed 1000s of genes involved in drought responses

Some are regulated by ABA, others are ABA-independent

Many different transcription factors are involved

Many of the targets are involved in cellular protection or conserving water

Fukao, T. and Xiong, L. (2013). Genetic mechanisms conferring adaptation to submergence and drought in rice: simple or complex? Curr. Opin. Plant Biol. 16: 196-204.

© 2014 American Society of Plant Biologists Water deficit induces stomatal closure & decreased photosynthesis In this study of maize growing in a pot, stomatal resistance and ABA levels increased after three days

without irrigation

Leaf waterLeaf

potential potential (bars)

) When a leaf is detached, the rate 2 of transpiration decreases over

time, indicating stomatal closure

(sec /cm) (sec ABA (ng level / cm ABA

Stomatal resistanceStomatal Rewatering

Beardsell, M.F. and Cohen, D. (1975). Relationships between leaf water status, abscisic acid levels, and stomatal resistance in maize and sorghum. Plant Physiol. 56: 207-212 Hopper, D.W., Ghan, R. and Cramer, G.R. (2014). A rapid dehydration leaf assay reveals stomatal response differences in grapevine genotypes. Hort. Res. 1: 2..

gs is a rapid response to water deficit

OPEN

gs↓ ABA Guard cells respond to ABA produced ABA ψleaf↓ locally and distally, ABA↑ ψ ↓ leaf and to a change in leaf water potential CLOSED ψxylem↓

ψroot↓

ψsoil↓

Ion flux and water ABA promotes flow into the guard stomatal cell causes stomata closure to open

June Kwak, University of Maryland See also Schroeder, J.I., Allen, G.J., Hugouvieux, V., Kwak, J.M. and Waner, D. (2001). Guard cell signal transduction. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 627-658.

ABA-deficient tomato

ABA-deficient Arabidopsis

ABA-insensitive Arabidopsis

Reprinted from Bauer, H., Ache, P., Lautner, S., Fromm, J., Hartung, W., Al-Rasheid, Khaled A.S., Sonnewald, S., Sonnewald, U., Kneitz, S., Lachmann, N., Mendel, Ralf R., Bittner, F., Hetherington, Alistair M. and Hedrich, R. (2013). The stomatal response to reduced relative humidity requires guard cell-autonomous ABA synthesis. Curr. Biol. 23: 53-57 with permission from Elsevier. Fujii, H., and Zhu, J.-K. (2009). Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc. Natl. Acad. Sci. USA 106: 8380-8385; SolGenomics.net

Ca is [CO2] g is stomatal conductance of ambient Ca s CO2 (from outside the leaf to inside the leaf air spaces)

Ci is [CO2] inside the C i gm is mesophyll conductance of CO2 leaf (from air spaces into chloroplasts)

Cc is [CO2] Cc inside the chloroplast

2 x 3-phosphogylcerate A is the rate of photosynthetic carbon assimilation + Rubisco A depends on the amounts of enzyme and substrates CO2 Ribulose-1,5- bisphosphate

Under ideal conditions, A

depends on [CO2] inside the chloroplast (where the enzyme is located)

© 2014 American Society of Plant Biologists Water deficit can but does not have to affect photosynthesis In a plant with wide-open stomata, the rate of transpiration may be more than necessary for the maximum photosynthetic rate

In a plant with tightly closed A stomata, CO2 might limit photosynthetic There is an ideal point at carbon assimilation which the exchange of CO and H O is optimal Ci 2 2

Water deficit often reduces crop growth and yield (shown here – wheat)

Water content: Percent of control 100% 16% 4% 2% Ψw (MPa) -0.03 -0.20 -0.81 -1.60

As water become scarce, the plant’s resources are preferentially allocated to the root Well watered Water deficit

Image source: USDA; Hsiao, T.C. and Xu, L.K. (2000). Sensitivity of growth of roots versus leaves to water stress: biophysical analysis and relation to water transport. J. Exp. Bot. 51: 1595- 1616. Sharp, R.E., Silk, W.K. and Hsiao, T.C. (1988). Growth of the maize primary root at low water potentials: I. Spatial distribution of expansive growth. Plant Physiol. 87: 50-57.

What limits shoot and leaf growth? • Decrease in photosynthetic rate? • ABA signaling? • Hydraulic effects on cell elongation?

How is root growth maintained relative to shoot growth under water deficit?

Zea mays Water withheld Well watered for 10 days

Reduced leaf Fully area, leaf expanded, margins green curled to conserve water, yellowing

Marshall, A. et al., and De Smet, I. (2012). Tackling drought stress: RECEPTOR-LIKE KINASES present new approaches. Plant Cell. 24: 2262-2278.

Leaf enlargement is more sensitive to mild water deficit than photosynthesis; therefore, factors other than availability of photosynthate must limit leaf growth

Boyer, J.S. (1970). Leaf enlargement and metabolic rates in corn, soybean, and sunflower at various leaf water potentials. Plant Physiol. 46: 233-235.

Water deficit’s effect on leaf expansion may be independent from those of photosynthesis (although smaller leaves means less area for photosynthesis)

Reprinted from Tardieu, F., Granier, C. and Muller, B. (2011). Water deficit and growth. Co-ordinating processes without an orchestrator? Curr. Opin. Plant Biol. 14: 283-289 with permission from Elsevier.

The developmental state of the leaf / cell affects its responses to water deficit Red = proliferating (dividing) tissues Green = expanding tissues White = mature tissues

Summary of tissue- Control specific responses to water deficit and signals involved “Acclimation” Stressed

ABA = abscisic acid GA = gibberellin ACC = 1-aminocyclopropane-1- Time affects carboxylic (ethylene precursor) AOX = alternative oxidase responses KRP & SIM = cell cycle regulators

Skirycz, A., et al and Inzé, D. (2010). Developmental stage specificity and the role of mitochondrial metabolism in the response of Arabidopsis leaves to prolonged mild osmotic stress. Plant Physiol. 152: 226-244. Skirycz, A. and Inzé, D. (2010). More from less: plant growth under limited water. Curr. Opin. Biotech. 21:. 197-203 with permmission from Elsevier; see also Muller, B., Pantin, F., Génard, M., Turc, O., Freixes, S., Piques, M. and Gibon, Y. (2011). Water deficits uncouple growth from photosynthesis, increase C content, and modify the relationships between C and growth in sink organs. J. Exp. Bot. 62: 1715-1729.

Claeys, H. and Inzé, D. (2013). The agony of choice: How plants balance growth and survival under water-limiting conditions. Plant Physiol. 162: 1768-1779.

Root hair elongation

Increase in number or diameter or xylem elements Production of short-lived, drought-induced Increase in hydraulic lateral roots conductance, through expression of Preferential aquaporins or changes primary root Preferential lateral root in exodermis and elongation proliferation in deep soil endodermis

Adapted from Wasson, A.P., Richards, R.A., Chatrath, R., Misra, S.C., Prasad, S.V.S., Rebetzke, G.J., Kirkegaard, J.A., Christopher, J. and Watt, M. (2012). Traits and selection strategies to improve root systems and water uptake in water-limited wheat crops. J. Exp. Bot. 63: 3485-3498.

Soil moisture in the upper soil zones is depleted during drought

A gene that promotes deeper rooting (enhanced gravitropic response confers drought protection)

Reprinted by permission from Macmillan Publishers Ltd from Uga, Y., Sugimoto, K., Ogawa, S., Rane, J., Ishitani, M., Hara, N., Kitomi, Y., Inukai, Y., Ono, K., Kanno, N., Inoue, H., Takehisa, H., Motoyama, R., Nagamura, Y., Wu, J., Matsumoto, T., Takai, T., Okuno, K. and Yano, M. (2013). Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions. Nat. Genet. 45: 1097-1102.

ABA may act by suppressing the Fluridone, an ABA synthesis accumulation of growth-inhibitory inhibitor, inhibits primary root reactive oxygen and ethylene elongation at low water potentials

Reprinted with permission of the Society of Experimental Biology from Sharp, R.E., Wu, Y., Voetberg, G.S., Saab, I.N. and LeNoble, M.E. (1994). Confirmation that abscisic acid accumulation is required for maize primary root elongation at low water potentials. J. Exp. Bot. 45: 1743-1751. Sharp, R.E., Poroyko, V., Hejlek, L.G., Spollen, W.G., Springer, G.K., Bohnert, H.J. and Nguyen, H.T. (2004). Root growth maintenance during water deficits: physiology to functional genomics. J. Exp. Bot. 55: 2343-2351.

The developmental stage of a plant experiencing water deficit affects the outcomes

In wheat and rice, seed (kernel) number is highly sensitive just before anthesis (pollen maturation)

Reprinted from Plants in Action, published by the Australian Society of Plant Scientists, http://plantsinaction.science.uq.edu.au/edition1 with permission Plants in Action

Wheat Maize Control Water deficit

Reprinted from McLaughlin, J.E. and Boyer, J.S. (2004). Sugar-responsive gene expression, invertase activity, and senescence in aborting maize ovaries at low water potentials. Ann. Bot.. 94: 675-689 by permission of Oxford University Press; Reprinted from Dolferus, R., Ji, X. and Richards, R.A. (2011). Abiotic stress and control of grain number in cereals. Plant Sci. 181: 331-341 with permission from Elsevier.

Photosynthesis Plant responses to Stomatal aperture water deficit are pleiotropic, complex Shoot meristem and context and leaf growth dependent, but typically include an increase in root growth, decrease in shoot growth, and decrease in Root growth transpiration and photosynthesis Solute accumulation (synthesis and uptake) Water uptake

Maggio, A., Zhu, J.-K., Hasegawa, P.M. and Bressan, R.A. (2006). Osmogenetics: Aristotle to Arabidopsis. Plant Cell. 18: 1542-1557.

New technologies are making it possible to prevent, diagnose and ultimately eliminate the harmful effects of water deficit

Prevent: Monitor the weather and soil moisture, use water wisely

Diagnose: Monitor plant stress responses

Breed tolerance: Genetic strategies to confer drought tolerance

Image source: USDA

Soil moisture sensors use a variety of technologies and can indicate moisture at deep as well as shallow depths

Radios and cell phones inform farmers about weather forecasts so they can decide when to plant or irrigate Satellites can monitor soil moisture and rainfall

Photo credit: Francesco Fiondella; NASA / JPL

A false-color thermal image of several fields. Blue and green colored fields are cooler due to evaporative transpiration, indicating greater stomatal aperture and less water stress

The VegDRI integrates satellite- based observations of vegetative conditions, climate data and other information to indicate drought level

US National Drought Mitigation Center; NASA Earth Observatory

Drip irrigation minimizes wasteful evaporation

Overhead watering allows wasteful evaporation from the spray and the bare soil

Photo credit: USDA; JISL

Sector-specific irrigation Grid-specific irrigation Sensor-based irrigation (static) (dynamic)

By adjusting the rotation By adjusting the rotation speed of the irrigation speed and flow from Soil or plant information can arm, different amounts of sprinklers, zones can be sent to the irrigation water can be delivered to each have a customized system for real-time different sectors water application irrigation optimization

Grisso, R., Alley, M., Thomason, W., Holshouser, D., and Roberson, G.T. (2011). Precision farming tools: Variable-rate application. Virginia Cooperative Extension

(PRD) PRD often results in equivalent harvests, even if the leaf size is smaller

Well PRD watered irrigation

In PRD irrigation, the plants are watered on alternating sides, so each half of the root system Hypothesis: The wet roots hydrate the shoot, alternates between wet and dry the dry roots send a signal that causes partial stomatal closing and water conservation

Image sources: CIP; Adapted from Davies, W.J., and Hartung, W. (2004). Has extrapolation from biochemistry to crop functioning worked to sustain plant production under water scarcity?. Proc. 4th Intl. Crop Sci. Congress, Brisbane, Australia, 2004.

© 2014 American Society of Plant Biologists F3.11 An ideal climate-smart agricultural landscape of the future would enable farmers to use new technologies and techniques to maximize yields and allow land managers to protect natural systems, with natural habitats integrated into agriculturally productive landscapes

World Development Report 2010

© 2014 American Society of Plant Biologists Diagnosing plant physiological activities and drought stress effects What would a plant “tricorder”* measure? •Plant water potential •Photosynthetic gas exchange

(CO2 assimilation, O2 evolution) •Chlorophyll fluorescence (indicates efficiency of photosystem II)

•Transpiration (H2O release, change in temperature)

•Water use efficiency (ratio of CO2 assimilated to H2O transpired) •Stress-responses: hormones, transcripts, metabolites •Growth rate

*In the TV series Star Trek, a tricorder is a •Growth direction and orientation handheld scanning and analysis diagnostic device

Image credit: fanboyfactor.com

Image credit: fanboyfactor.com and LI-COR

The light energy absorbed by It is possible to determine the photosynthetic chlorophyll has three fates: rate indirectly by measurements of fluorescence photosynthesis, dissipation as heat, or re-emission as longer wavelength light Easy to measure (fluorescence) Fluorescence

Photo- Hard to measure Chl* synthesis

Photon Can be accounted for with Heat appropriate Chl controls

1. Block photosynthesis and 2. Unblock photosynthesis and The difference, measure maximal measure fluorescence (Fʹ) normalized to Fmʹ, fluorescence (Fmʹ) indicates the photosynthetic operating efficiency Fluorescence Fluorescence Fmʹ Fʹ Fmʹ - Fʹ

Fmʹ Photo- Photo- Chl* synthesis Chl* synthesis

A X The energy not This value can be All of the energy emitted as used to calculate

is emitted as fluorescence is used A, the CO2 fluorescence for photosynthesis assimilation rate

Light Light Gas exchange Closed system in a leaf can be Open system measured in a Air in Air out closed or open (flow through) system

Light on Light off Light on Light off [CO2] is

measured by O2 infrared gas O2 analysis (IRGA) Ambient [O2] and [O ] by an

2 Ambient [CO2] in chamber in

CO oxygen air exiting in CO2 Concentration Concentration Concentration Concentration 2 electrode

Light Transpirational water loss cam Thermal imaging: transpiration be measured by weight loss requires heat energy and cools from a plant growing in a plants sealed pot Air Leaf chamber Air in out

Sealed pot Light on Light off

Light on Light off

H2O Top row: Wild-type Arabidopsis. in exiting air exiting in

Concentration Concentration

The concentration of water in a Bottom two rows: Mutants (ost1-1 sealed leaf chamber can be and ost1-2) defective in stomatal monitored in a flow-through or in pot closure. More transpiration = cooler

closed system plants Weight of Weight plant

Xin, Z., Franks, C., Payton, P. and Burke, J.J. (2008). A simple method to determine transpiration efficiency in sorghum. Field Crops Res. 107: 180-183; Mustilli, A.-C., et al., and. and Giraudat, J. (2002). Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell. 14: 3089-3099.

CO2 assimilated to H2O transpired

H2O ambient CO2 ambient WUE values are typically on the

order of 1 mol CO2 fixed to 100 - 400 mol H2O transpired (for C3 plants) H O inside 2 CO2 inside

Crop yield (kg)

Water consumption (kg) On a larger scale, WUE can be measured as yield relative to water consumed

Instantaneous WUE can be calculated from simultaneous measurements of chlorophyll fluorescence (CO2 assimilation) and temperature (transpiration)

Reprinted from McAusland, L., Davey, P.A., Kanwal, N., Baker, N.R. and Lawson, T. (2013). A novel system for spatial and temporal imaging of intrinsic plant water use efficiency. J. Exp. Bot.. 64: 4993-5007 by permission of the Society for Experimental Biology.

12C 12C Approximately 1.1% of the Earth’s 98.9% 13 carbon has an extra neutron, making C 1.1% 12C it larger and more massive. The 13C isotope is stable (non-radioactive) (unlike the radioactive 14C isotope)

The carbon-fixing enzyme 12C Rubisco preferentially uses 12 + C Rubisco 12 CO2 as a substrate

CO2 Ribulose-1,5- bisphosphate 2 x 3-phosphogylcerate

[CO2]inside high, [CO2]inside low, Mostly 12CO assimilated Relatively more 13CO 2 2 The isotope assimilated differential (δ 13C) reflects stomatal conductance 12 12 C C 12C 12 12 C C 12C 13C 12C 12C Rubisco 12 Rubisco 13 See Farquhar, G. D., Ehleringer, J.R., and Hubick, K.T. C C (1989). Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 503 – 537.

water use efficiency in C3 plants As the stomata are more closed,

30 C3 [CO2]i / [CO2]a decreases, relative 13 abundance of CO2 increases, C4 and the carbon isotope 20 discrimination decreases

10 Note that in C plants there is Discrimination % Discrimination 4 no additional isotope discrimination because

0 PEPCase (the CO2-fixing 0.2 0.4 0.6 0.8 1.0 enzyme in C plants) does not 13CO diffuses less 4 2 discriminate between stable effectively through the [CO2] inside / [CO2] atmospheric isotopes stomatal pores, so inside the leaf there is a 4.4% non-enzymatic isotope discrimination

Adapted from Farquhar, G. D., Ehleringer, J.R., and Hubick, K.T. (1989). Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 503 – 537.

Automated greenhouse

Donald Danforth Plant Science Center See video here

Imaging chamber

Educational resources here See a video here

Reflected light: Single wavelength NDVI is a Normalized 770 nm normalized difference 440 nm 550 nm 660 nm Near indicator of vegetation Blue Green Red infrared chlorophyll index (NDVI) absorption RNIR - RR

RNIR + RR R+G+B NIR+G+B Much Much reflected reflected

Much absorbed by chlorophyll

Reprinted from Fiorani, F., Rascher, U., Jahnke, S. and Schurr, U. (2012). Imaging plants dynamics in heterogenic environments. Curr. Opin. Biotechnol. 23: 227-235 with permission from Elsevier.

Plant: Shoot of a sugar beet Tree

Forest

Height – Leaf angle distance relative to sun from camera

Reprinted from Fiorani, F., Rascher, U., Jahnke, S. and Schurr, U. (2012). Imaging plants dynamics in heterogenic environments. Curr. Opin. Biotechnol. 23: 227-235 with permission from Elsevier. Reprinted from Omasa, K., Hosoi, F. and Konishi, A. (2007). 3D lidar imaging for detecting and understanding plant responses and canopy structure. J. Exp. Bot. 58: 881-898 with permission from Society for Experimental Biology. See also Biskup, B., Scharr, H., Schurr, U. and Rascher, U.W.E. (2007). A stereo imaging system for measuring structural parameters of plant canopies. Plant Cell Environ. 30: 1299-1308.

Successful breeding programs, whether using forward or reverse genetics, require accurate, preferably high- throughput, phenomics tools

Reprinted from Furbank, R.T. and Tester, M. (2011). Phenomics – technologies to relieve the phenotyping bottleneck. Trends Plant Sci. 16: 635-644 with permission from Elsevier.

The phenomobile carries equipment to measure: •leaf greenness and ground cover • canopy temperature • volume (biomass) of plants, plant height and plant density • crop chemical composition

See Araus, J.L. and Cairns, J.E. (2014). Field high-throughput phenotyping: the new crop breeding frontier. Trends Plant Sci. 19: 52–61.See also White, J.W., Andrade-Sanchez, P., Gore, M.A., Bronson, K.F., Coffelt, T.A., Conley, M.M., Feldmann, K.A., French, A.N., Heun, J.T., Hunsaker, D.J., Jenks, M.A., Kimball, B.A., Roth, R.L., Strand, R.J., Thorp, K.R., Wall, G.W. and Wang, G. (2012). Field-based phenomics for plant genetics research. Field Crops Research. 133: 101-112. Image source: Australian Plant Phenomics Facility and Phenomobile.

Reverse Genetics: Identify drought-tolerance genes and alter Two different expression levels in plants inbred maize varieties showing different responses to water deficit Forward Genetics: Classical approach, select for drought tolerance based on phenotype

Lopes, M.S., Araus, J.L., van Heerden, P.D.R., and Foyer, C.H. (2011). Enhancing drought tolerance in C4 crops. J. Exp. Bot. 62: 3135 – 3153 by permission of Society of Experimental Biology

If you find a solution that works in a model organism in a controlled environment, will it work in a vastly more complex environment?

The same problem occurs when translating lab mice studies to humans with all their diversity and complexity

Dave Clark, USDA. Charles Andrès; Nelissen, H., Moloney, M. and Inzé, D. (2014). Translational research: from pot to plot. Plant Biotech. J. 12: 277-285.

For example, these two cultivated types of peanut (Arachis hypogaea) are very different in their response to drought. The drought-tolerant variety produces plenty of peanuts, while the drought-sensitive variety produces almost none Drought tolerant Drought sensitive

Peanuts are legumes whose fruits grow underground

Photos by Paxton Payton and Franz Eugen Köhler, Köhler's Medizinal-Pflanzen see also NOAA.

© 2014 American Society of Plant Biologists For success, drought-tolerant traits have to be in the breeding population Genetic diversity of original, wild population Many ancestral traits are not represented in modern varieties, but can still be found in germplasm collections

Genetic diversity of modern cultivated varieties

Lopes, M.S., Araus, J.L., van Heerden, P.D.R., and Foyer, C.H. (2011). Enhancing drought tolerance in C4 crops. J. Exp. Bot. 62: 3135 – 3153 by permission of Society of Experimental Biology

© 2014 American Society of Plant Biologists Components of yield under drought stress: WU, WUE and HI Yield under drought is the product of water uptake (WU), water use efficiency (WUE), and harvest index (conversion of biomass to grain)

See Salekdeh, G.H., Reynolds, M., Bennett, J. and Boyer, J. (2009). Conceptual framework for drought phenotyping during molecular breeding. Trends Plant Science. 14: 488-496; Reprinted from Reynolds, M., and Tuberosa, R. (2008). Translational research impacting on crop productivity in drought-prone environments. Curr. Opin. Plant Biol. 11: 171 – 179 with permission from Elsevier.

© 2014 American Society of Plant Biologists Different “drought tolerance” traits have different consequences Extensive Decreased stomatal Cellular Early root system aperture / leaf area protectants reproduction

Benefits Decreases Avoids cell death under Avoids late season Increases water uptake transpiration severe drought water deficit Drawbacks Decreased Biomass sequestered Reduced growth rate, photosynthesis and Shortened in root not available for energy diverted to biomass growing season shoot / reproduction protection accumulation

Adapted from Tardieu, F. (2012). Any trait or trait-related allele can confer drought tolerance: just design the right drought scenario. J. Exp. Bot. 63: 25-31; Photo credits: Michael Thompson.

Scenario for maximum Scenario for maximum Trait positive effect negative effect

Extensive / deep Deep water available Low availability of water / root system (enhanced uptake) nutrients in deep layers

Decreased stomatal Severe drought (less Favorable year (loss of aperture / leaf area transpiration uses less water) potential biomass)

Could lead to reduced Cellular Very severe drought growth rate in non- protectants (avoid death) extreme conditions

Early Very dry year, late season Favorable year (loss of reproduction drought (ensure reproduction) potential biomass)

Adapted from Tardieu, F. (2012). Any trait or trait-related allele can confer drought tolerance: just design the right drought scenario. J. Exp. Bot. 63: 25-31.

Osmotic adjustment Based on our (mannitol, proline, Transcription understanding of glycine betaine etc.) factors plant responses to desiccation and water limitation, hundreds of Protective proteins different candidate (chaperones, dehydrins) genes have been Signaling molecules analyzed for and hormones, drought-tolerance hormone responses effects Transport proteins (e.g., vacuolar Na+ / K+ antiporter, aquaporins)

See Deikman, J., Petracek, M., and Heard, J.E. (2013). Drought tolerance through biotechnology: improving translation from the laboratory to farmers’ field. Curr. Opin. Biotech. 23: 243 – 250; Lawlor, D.W. (2013). Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. J. Exp. Bot. 64: 83-108. Yang, S., Vanderbeld, B., Wan, J. and Huang, Y. (2010). Narrowing down the targets: Towards successful genetic engineering of drought-tolerant crops. Mol. Plant. 3: 469-490; Chen, M.-X., Lung, S.-C., Du, Z.-Y. and Chye, M.-L. (2014). Engineering plants to tolerate abiotic stresses. Biocat. Agric. Biotech. 3: 81 – 87.

Engineered genes

Stress perception

Transcription networks Plant Trait Regulatory Stress engineering assessment approval Metabolic pathways Plants made using genetic engineering methods have additional regulatory Effectors hurdles to cross

Adapted from Chen, M.-X., Lung, S.-C., Du, Z.-Y. and Chye, M.-L. (2014). Engineering plants to tolerate abiotic stresses. Biocat. Agric. Biotech. 3: 81 – 87.

A gene encoding a bacterial RNA chaperone increases yields in plants that experience late-season drought

After extensive testing, the first plants carrying a transgene for drought tolerance were launched in the US in 2013 and have been approved for use in China

Castiglioni, P., Warner, D., Bensen, R.J., Anstrom, D.C., Harrison, J., Stoecker, M., Abad, M., Kumar, G., Salvador, S., D'Ordine, R., Navarro, S., Back, S., Fernandes, M., Targolli, J., Dasgupta, S., Bonin, C., Luethy, M.H. and Heard, J.E. (2008). Bacterial RNA chaperones confer abiotic stress tolerance in plants and improved grain yield in maize under water-limited conditions. Plant Physiol. 147: 446-455.

Pyrophosphate 2 x

H+

H+-PPase Hormone synthesis and response H+

The vacuolar Membrane Transcription H+- pyrophosphatase transporters factors (H+-PPase) hydrolyzes and pumps pyrophosphate to pump protons into the vacuole And many more….

Nelson, D.E., et al., and Heard, J.E. (2007). Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc. Natl. Acad. Sci. 104: 16450-16455 copyright National Academy of Sciences. Qin, H., et al., and Zhang, H. (2011). Regulated expression of an isopentenyltransferase gene (IPT) in peanut significantly improves drought tolerance and increases yield under field conditions. Plant Cell Physiol. 52: 1904-1914 with permission of the Japanese Society of Plant Physiologists; Gaxiola, R.A., Li, J., Undurraga, S., Dang, L.M., Allen, G.J., Alper, S.L. and Fink, G.R. (2001). Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc. Natl. Acad. Sci. 98: 11444-11449.

Models can test lots of scenarios “virtually” to identify those worth testing “in the real world”

Types of questions models can investigate: • Which genotype will work best for my environment? • Should I grow a cover or companion crop? • When should I plant? • Should I spend my money on water or fertilizers? • Which combinations of alleles will work best Diagram showing various simulated breeding paths for my needs? from low yield (blue) to • Which genes should I start introducing now in high yield (red) anticipation of future climate changes?

Reprinted from Hammer, G., Cooper, M., Tardieu, F., Welch, S., Walsh, B., van Eeuwijk, F., Chapman, S. and Podlich, D. (2006). Models for navigating biological complexity in breeding improved crop plants. Trends Plant Sci. 11: 587-593 with permission from Elsevier.

Testing Experimentation Modeling Selection

Reprinted from Messina, C.D., Podlich, D., Dong, Z., Samples, M. and Cooper, M. (2011). Yield–trait performance landscapes: from theory to application in breeding maize for drought tolerance. J. Exp. Bot. 62: 855-868 with permission of Society for Experimental Biology.

Historical data about QTL effects on leaf growth and water sensitivity were fed into a model that indicated the expected effects of each on yield during various drought-stress scenarios

Reprinted from Tardieu, F. and Tuberosa, R. (2010). Dissection and modelling of abiotic stress tolerance in plants. Curr. Opin. Plant Biol. 13: 206-212 with permission from Elsevier.

Research is key to advancing: • Irrigation management • Plant stress monitoring • Phenotyping methods • High throughput phenotyping platforms • –omics methods • Breeding strategies and tools • Modeling plant responses to water deficit • Modeling gene x environment interactions

Image source: USDA

Excellent model systems are available for learning how cells respond to desiccation and how plants respond to drought, but translating that understanding into drought- tolerant food production is not easy

Osakabe, Y., Osakabe, K., Shinozaki, K. and Tran, L.-S.P. (2014). Response of plants to water stress. Frontiers in Plant Science. 5: 86. R.L. Croissant, Bugwood.org

Source: FAO

At the time of this article’s publication, California was facing the prospect of its worst drought in 500 years. These photos show the snow accumulated in January 2013 vs January 2014.

How will governments, growers and consumers respond to rising food prices and food shortages as a consequence of this drought? NASA Earth Observatory