Limited-transpiration response to high vapor pressure deficit in crop species

Accepted Manuscript Title: Limited-transpiration response to high vapor pressure deficit in crop species Authors: Thomas R. Sinclair, Jyostna Devi, Avat Shekoofa, Sunita Choudhary, Walid Sadok, Vincent Vadez, Mandeep Riar, Thomas Rufty PII: DOI: Reference:

S0168-9452(16)30611-2 http://dx.doi.org/doi:10.1016/j.plantsci.2017.04.007 PSL 9589

To appear in:

Plant Science

Received date: Revised date: Accepted date:

11-11-2016 28-3-2017 7-4-2017

Please cite this article as: Thomas R.Sinclair, Jyostna Devi, Avat Shekoofa, Sunita Choudhary, Walid Sadok, Vincent Vadez, Mandeep Riar, Thomas Rufty, Limited-transpiration response to high vapor pressure deficit in crop species, Plant Sciencehttp://dx.doi.org/10.1016/j.plantsci.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Limited-transpiration response to high vapor pressure deficit in crop species Thomas R. Sinclaira, Jyostna Devia, Avat Shekoofaa, Sunita Choudharyb, Walid, Sadokc, Vincent Vadezb, Mandeep Riara, Thomas Ruftya

a b

Crop Science Department, North Carolina State University, Raleigh, NC 27695-7620, USA. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),

Patancheru 502324, Greater Hyderabad, Telangana, India. c

Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108-

6026, USA. Correspondence Thomas R Sinclair 1304 Williams Hall, Campus Box 7620, Department of Crop Science, North Carolina State University, Raleigh, NC 27695 E-mail: [email protected]

Highlights     

Limited transpiration at elevated vapor pressure deficit confers drought tolerance Trait associated with low hydraulic conductivity in plant Aquaporin expression related to hydraulic conductivity and limited transpiration Genotypic variability for limited transpiration identified in major crop species Success in basic physiological research being used in improved crops

ABSTRACT

Water deficit under nearly all field conditions is the major constraint on plant yields. Other than empirical observations, very little progress has been made in developing crop plants in which 1

specific physiological traits for drought are expressed. As a consequence, there was little known about under what conditions and to what extent drought impacts crop yield. However, there has been rapid progress in recent years in understanding and developing a limited-transpiration trait under elevated atmospheric vapor pressure deficit to increase plant growth and yield under water-deficit conditions. This review paper examines the physiological basis for the limitedtranspiration trait as result of low plant hydraulic conductivity, which appears to be related to aquaporin activity. Methodology was developed based on aquaporin involvement to identify candidate genotypes for drought tolerance of several major crop species. Cultivars of maize and soybean are now being marketed specifically for arid conditions. Understanding the mechanism of the limited-transpiration trait has allowed a geospatial analyses to define the environments in which increased yield responses can be expected. This review highlights the challenges and approaches to finally develop physiological traits contributing directly to plant improvement for water-limited environments.

Abbreviations: AQP, aquaporins; VPD, vapor pressure deficit

Key words: Aquaporins, Drought, Hydraulic conductivity, Transpiration, Vapor pressure deficit

1. Introduction

Changes in precipitation patterns as a result of climate change are expected to have significant impact on available water for human consumption, agriculture and energy generation (Confalonieri et al. 2007). Identification of fundamental drought-tolerant traits and their incorporation into productive crop genotypes is essential to sustain agriculture viability under current and future climates with declining amounts or frequency of precipitation. Nevertheless, only a few plant traits have actually been considered to improve drought resistance despite the many physiological traits involved in plant responses to drought (Pita et al. 2005). Various efforts have been made to enhance the efficiency of selection for drought-tolerant genotypes based on yield and specific physiological traits. Unfortunately, most of the efforts 2

have failed due to genotype x environment interactions causing yield variability and also, to the lack of precise screening techniques for trait evaluation (Branch and Hilderbrand, 1985; Cooper and Hammer, 1996; Mitra, 2001). Hence, the selection of a physiological trait to screen for water-deficit tolerance requires a comprehensive understanding of the nature of any putative trait, the responsiveness of the trait to the environment, and most importantly, its contribution to increasing crop yield (Ludlow and Muchow, 1990; Sheshshayee et al., 2003; Sinclair, 2011). Several adaptive strategies have evolved in plants to cope with drought stress especially in environments where water deficit frequently occurs (Maroco et al., 1997; Borrell et al., 2006; Araus et al. 2008). However, many of these traits relate to plant survival under drought, which are generally of little or no economic benefit in annual crop production. This is because severe drought conditions that threaten plant survival mean there is very little water for crop growth and yield formation, and yields necessarily remain extremely low. Therefore, traits to increase crop survival still result in severely threatened economic viability for growers. It is essential that any putative drought trait should result in monetarily relevant yield benefit and should have genetic variability within the target crop species (Sadok and Sinclair, 2011). The traits that appear to be particularly desirable to improve performance of crops under water-deficit conditions generally are associated with limited-water use early in the growing season so that water is conserved for use later in season to sustain plant productivity during seed fill (Richards and Passioura, 1989; Sinclair et al., 2005; Zaman-Allah et al., 2011a,b). One approach to achieve water conservation is for plants to have low leaf gas exchange during periods of high vapor pressure deficit (VPD), which usually occur during midday. While the limited-transpiration (TRlim) trait appears to be rare in commercial crop varieties, such a trait would have the dual benefit of (1) improving crop water use efficiency by limiting crop gas exchange during high VPD conditions, and (2) conserving soil water early in the growing season for use to sustain plant activity during late-season drought events. The TRlim response to high VPD in selected genotypes is examined in detail in this review. First, the nature of the TRlim trait is discussed. Second, evidence for the involvement of hydraulic conductivity in expression of TRlim is considered. Third, the involvement of aquaporins in hydraulic conductivity and in the TRlim trait is discussed. Fourth, approaches to screen among populations of genotypes for expression of the trait are presented. Fifth, reports of expression of the TRlim trait in various crop

3

species are reviewed. And finally, the impacts of other environmental factors, particularly high temperature, on TRlim are considered.

2. Vapor Pressure Deficit and Transpiration Rate

Vapor pressure deficit is the difference between the pressure of water vapor held in saturated air at a given temperature and the pressure of water vapor that exists in the ambient air.
From the perspective of plants, VPD is the difference between the vapor pressure inside the leaf compared to the vapor pressure of the air, and this is the driving force for transpiration rate (TR) of plants. Since the saturated vapor pressure is temperature sensitive, a challenge is to separate the impact of temperature on VPD and of temperature directly on plants (Sinclair et al., 2007a). Figure 1 illustrates the impact of the TRlim trait on TR as atmosphere VPD changes through the day. In this example, two values are shown for limited TR through the midday. The two cases of TRlim illustrate the potential decrease in TR as compared to the unrestricted TR, and the degree of water conservation. A key consequence of this behavior is that in the midday under high VPD there is conservation of soil water. Also, this trait would increase daily transpiration efficiency by decreasing the proportion of the transpiration that occurs during periods of high VPD (Sinclair et al., 2005). Of course, the negative impact of this trait would be an immediate decrease in CO2 assimilation rate due to the inherently close relationship between water vapor and CO2 exchange by leaves and crop canopies (Tanner and Sinclair, 1983). A key issue is whether the negative impact on CO2 assimilation rate is compensated during the latter part of the growing season by sustained physiological activity supported by soil water conserved earlier in the growing season. This issue was examined in a simulation analysis of the TRlim trait for soybean (Glycine max Merr. L.) based on 50 years of weather data across the USA (Sinclair et al., 2010). Based on the 50 years of simulations a probability of yield increase was calculated (Figure 2a). These results showed that there was soybean yield increase with the TRlim trait in most regions in more than 85% of the years. The change in absolute yield was also calculated by the introduction of TRlim. The yield changes were ranked at each location so that specific percentiles in the yield ranking could be presented across locations. Yield gains were simulated in most locations at percentile rankings of both 25% (dry years, Figure 2d) and 75% (wet years, Figure 2b). Of course, the greatest yield increases were simulated for the drier years. 4

The TRlim trait was similarly found in a simulation study across Africa to result in yield increases in more than 70% of the growing seasons in many locations (Sinclair et al., 2014). However, the simulation results for Africa showed a only small benefit in yield in the higher rainfall regions.

3. Water flow in leaves

An increase in VPD resulting in increased TR must be matched by water transport to leaves, or leaves will rapidly dehydrate and senesce since crop leaves have a very small water storage capacity. A critical option to sustain leaves is to have restricted TR under such high VPD conditions by partial stomatal closure so that TR is decreased to match water flux into the leaf. In those cases where such regulation exists, a VPD threshold can be identified at which further increases in VPD result in little or no further increase in TR (Bunce, 1981; Turner et al., 1984), which generally reflects the water transport capacity in the soil-plant system. Stomatal regulation of leaf water balance has been proposed to be controlled by active metabolic processes and/or by passive hydraulic process (McAdam and Brodribb, 2014). The active metabolic process is presumably triggered by low, bulk leaf water potential that cause synthesis of abscisic acid (McAdam and Brodribb, 2016). For the annual crop species considered in this review, however, there is no evidence that bulk water deficits develop in leaves to trigger active metabolic process at the onset in expression of the TRlim response. We measured leaf water status as VPD was increased experimentally. There was no decrease in leaf water potential or relative water content at the threshold of TRlim, which is shown by a vertical line in the two examples presented in Figure 3. In fact, these results showed these leaves are essentially in a well-watered state over the entire range of increase in VPD with a water potential of about -0.4 MPa in the soybean example and a relative water content of about 90% in the peanut (Arachis hypogaea L.) example. The insensitivity to any decreases in bulk leaf water status is consistent with the observation of McAdam and Brodribb (2016) that excised pea (Pisum sativum L.) leaves had to be exposed to 1.0 MPa of pressure to induce a sufficient loss in turgor to result in an increase in leaf abscisic acid concentration. McAdam and Brodribb (2014) concluded that the VPD response in conifer Metasequoia glyptostroboides was a result of passive control of stomatal conductance. The passive control was dominant in this species until leaf water potential reaches -1.1 MPa. Hence, it appears in the 5

studied crop species that there is passive control of transpiration rate in those genotypes expressing the limited-transpiration trait. Given that bulk leaf water status does not decline as VPD increases for genotypes expressing the TRlim trait (Figure 3), a possibility is that localized decrease in water status in the leaf results in the stomatal response to increasing VPD. The long-standing proposal of Meidner (1975) was that the site of much water evaporation within the leaf, hence loss in cell water status, was in the epidermis including the stomatal guard cells. He suggested that little water evaporation may occur from mesophyll cells so the bulk leaf water status might, in fact, be little changed with increasing VPD. This scheme for liquid water flow and evaporation is illustrated in Figure 4. That is, the bulk of water evaporation occurs from the epidermis, and particularly the guard cells, which makes these cells the terminal destination of much of the water flow in leaves. As a result, guard cells may be especially vulnerable to turgor loss when high evaporation exists, and, consequently, the observed stomatal closure at high VPD. Limited-transpiration based on a passive plant hydraulic scheme involving the guard cells would be an effective approach to soil water conservation.

In this scheme, hydraulic

conductivity of water to and in leaves would be critical variables in determining TR that can be supported by the plant water balance. A comparatively low hydraulic conductivity would result in partial stomata closure whenever water flux into leaves is insufficient to meet unrestricted TR. In these cases, low water flux to leaves would necessitate partial stomatal closure as VPD increases to restrict TR to match the water flux into the leaves (Bunce, 2006). In soybean genotype PI 416937, Sinclair et al. (2008) demonstrated that TRlim rate under high VPD is likely to be the result of low hydraulic conductivity located in the leaves of this genotype between the xylem and into the guard cells. Choudhary et al. (2013b) measured hydraulic limitation in two genotypes of sorghum (Sorghum bicolor L.). They found in genotype SC 15 that a low leaf hydraulic conductivity was associated with its TRlim response. In another study with maize hybrids (Choudhary et al., 2014), TRlim was correlated with low hydraulic conductivity in both leaves and roots indicating a common, underlying basis for limiting hydraulic conductivity in these two tissues. Ocheltree et al. (2014) also found in C3 and C4 grass species that decreased stomatal conductance at increasing VPD was related to low leaf hydraulic conductivity. They observed

6

that leaf hydraulic conductivity was correlated with the hydraulic conductivity of large longitudinal veins, but was not related to root hydraulic conductivity.

4. Aquaporins

It has been hypothesized (Heinen et al., 2009) that the hydraulic conductivity associated with the TRlim trait may be related to transmembrane transport of water via aquaporins (AQP). The involvement of AQP offers a basis for environmental sensitivity (Nardini and Salleo, 2005; Levin et al., 2007, Shatil-Cohen et al., 2011) including drought and varying VPD. One approach to examining the possible involvement of AQPs in the phenotype of TRlim at high VPD has been based on studies using AQP inhibitors. In the study by Sadok and Sinclair (2010a), de-rooted shoots of soybean genotypes with and without expression of TRlim rate were subjected to various metal AQP inhibitors while under constant VPD. Those genotypes that expressed TRlim and low leaf hydraulic conductivity tended to have TR relatively insensitive to feeding of silver ions (Sadok and Sinclair, 2010 a,b). This result was hypothesized to indicate that silver-sensitive AQPs were not present or their abundance was at low levels in the TRlim genotypes. Four QTLs were identified as putatively associated with the silver response under constant VPD (2.3 kPa) from a population of soybean recombinant inbred lines derived from the mating of silver-insensitive, slow-wilting PI 416937 and silver-sensitive, fast-wilting Benning (Carpentieri-Pipolo et al, 2012). Recently, a difference in AQP transcript abundance was identified studies in soybean following were studied following exposure to high and low VPD (Devi et al., 2016a). Genotype PI 416937 with TRlim showed 944 genes differentially expressed when subjected to high VPD as compared to low VPD. Most of these expressed genes were transcription factors and genes related to cell wall development and metabolism, and transport genes. The transport genes included mostly AQPs which were down regulated (Devi et al., 2015a), which supports the hypothesis that AQPs are involved in the TRlim trait to conserve water. The genotype PI 416937 also showed lower expression of expansin and extension genes under high VPD conditions which might co-relate to the low leaf expansion rate under high VPD as compared to low VPD conditions (Devi et al., 2015b). Genotype Hutcheson with increasing transpiration rate with

7

increasing VPD showed only one gene differentially expressed at high VPD compared to low VPD. De-rooted shoots of peanut genotypes with/without TRlim treated with AQP inhibitors showed differences among genotypes in sensitivity to silver as well as gold (Devi et al., 2012; Shekoofa et al., 2013). These genotypes also differed in their expression of the TRlim trait and AQP gene expression when subjected to different AQP inhibitors (Devi et al., 2016b). In addition, the tested AQP genes differed for their expression in the genotypes differing in the TRlim trait when exposed to high VPD compared to low VPD (Devi et al., 2016b). In maize (Zea mays L.) and sorghum, differences in sensitivity to AQP inhibitors were also found among genotypes that varied in expression of TRlim. A significant correlation between the VPD threshold for TRlim and transpiration response to silver was observed in maize (Choudhary et al., 2015). In sorghum, the genotype with TRlim trait and low hydraulic conductivity was linked to an apparent lower population of silver-sensitive AQPs (Choudhary et al., 2013). Differences in the response of TR to high VPD in sorghum cultivars were also related to

differences in the expression of AQP, specifically plasma membrane intrinsic proteins (unpublished, Vincent Vadez, 2016)

5. Physiological screening for limited transpiration

The challenge in exploiting nearly all physiological traits is an ability to phenotype the expression of the trait in a range of genetic backgrounds and environmental conditions (Ghanem et al., 2015). This is certainly true for the TRlim trait. The success in developing an understanding of the TRlim trait and applying it in conjunction with breeding programs has generally resulted from the use of top-down, multi-level phenotyping (Ghanem et al., 2015). Several options exist in phenotyping for the TRlim trait depending on the stage in the breeding process and the available resources. An indirect approach, which allows a large number of genotypes to be phenotype, depends on observations of plants in the field. A delay in leaf wilting following exposure to water deficit would be likely for those genotypes that expressed TRlim and had conserved soil water prior to imposition of the water deficit. This approach would allow hundreds of genotypes to be phenotyped at one time. The problem is that there are several

8

reasons for delayed wilting other than TRlim. Therefore, observations on delayed wilting in themselves cannot be considered definitive. A corollary method to the delayed-wilting approach is to remotely measure temperature images of a field location. In this case, high temperature for well-watered plots under high VPD conditions could indicate partial stomata closure as a consequence of the TRlim trait. This approach is demanding of fairly unique environmental conditions and like delayed wilting, temperature differences can results from several possibilities. Therefore, the temperature measurements do not offer definitive results. A more narrowly focused screen that can be applied to fewer lines has been developed based on the response of TR of de-rooted shoots to being fed silver ions. As described previously, there tends to be an insensitivity in TR when de-rooted shoots of TRlim lines are fed silver ions. However, the results among plants tend to be variable so a large number of replicates are needed to establish statistical differences. Nevetheless, this approach has successfully applied by Carpentieri-Pipolo et al. (2012) to phenotype about 100 genotypes in a RIL population. The most intense approach to phenotype for expression of TRlim involves direct measurements of water loss in response to variation in VPD. Individual plant chambers have been used to measure plant TR over an induced range of VPD (Fletcher et al., 2007). This approach requires a capability to regulate both temperature and the humidity in the chambers. An alternative is to measure stomatal conductance on plants growing in the field during the natural daily variations in VPD (Gilbert et al., 2011; Shekoofa et al., 2015). However, the field approach can be constrained by weather conditions when measurements are to be made and in the number of lines that can actually be measured repeatedly through the day for stomatal conductance. A solution to some of the limitations to the direct measurements of plant response to VPD has been developed based on plants grown in large pots in the field (Vadez et al., 2015). In this case, the pots are weighed frequently through the day to obtain estimates of TR under natural varying VPD conditions. To account for variation in leaf area among plants it is necessary to express plant TR on a leaf area basis. The LeasyScan platform allows expeditious measurements of pot weight and plant leaf area. The platform is equipped with load cells on which plants cultivated in a density similar to the field are constantly weighed, providing a transpiration 9

measurement every hour. Using 3-D laser scanning, leaf area is estimated. Proof of concept in observing the TRlim response has been demonstrated for lines of sorghum, pearl millet (Pennisetum glaucum L.), cowpea (Vigna unguiculata L.), and peanut. In Figure 5 are examples of TR measured in this system through the day for sorghum (Figure 5A) and pearl millet (Figure 5B) (Vadez et al., 2015).

6. Limited-Transpiration Trait in Various Crop Species

The response of transpiration rate with increasing VPD has now been studied in several crop species. While commonly the trait is not observed in most crop genotypes, genotypic variations within species have now been identified. Briefly reviewed here are reports of genotypes expressing limited transpiration under high VPD in chickpea (Cicer arietinum L.), cowpea, maize, peanut, pearl millet, sorghum, soybean, and wheat (Triticum aestivum L.).

Chickpea. Zaman-Allah et al. (2011a) studied the water use response to VPD of eight chickpea genotypes with comparable phenology in three years of field testing. Among these genotypes, they found two genotypes with TRlim above about 2.5 kPa VPD and the other six with linear increase in TR with increasing VPD. The VPD-sensitive entries clearly had high early growth vigor and leaf development, but ultimately they had lower seed set. Using a lysimeter system, those lines with the TRlim response during the vegetative stage along with smaller canopy size, contributed to soil water conservation during the vegetative stage and this trait was associated with increased seed yield (Zaman-Allah et al., 2011b).

Maize. Gholipoor et al. (2013) tested a total of 35 single-cross maize hybrids for their TR response to VPD. A two-segment transpiration response to VPD was observed in eleven of the tested hybrids in which a VPD threshold for TRlim was observed in the range of 1.7 to 2.5 kPa. The remaining hybrids showed a linear response in their TR to increases in VPD. The results of TR plotted against VPD are presented for two hybrids in Figure 6 a,b. The TRlim trait has already been incorporated into commercial AQUAmax® hybrids being sold by DuPont-Pioneer for more arid regions of maize production (Gaffney et al., 2015). In on-farm trails across a wide

10

area of water-limited environments over three years, the mean yield advantage of the AQUAmax hybrids as compared to non-AQUAmax hybrids was 36, 53, and 22 g m-2, respectively. To fully assess the possible importance of the TRlim trait across the US, a simulation study was done to examine yield changes as a result of the TRlim trait (Messina et al., 2015). It was found that the TRlim trait resulted in improved yield (~135 g m-2) in specific water-deficit environments while a small yield penalty (~33 g m-2) was simulated for very wet seasons when water was less limiting.

Peanut. Peanut genotypes were found to exhibit differences in expression of the TRlim traits under both controlled-environmental conditions and field (Devi et al., 2010; Devi and Sinclair, 2011; Shekoofa et al., 2013; Shekoofa et al., 2015). In these studies, the threshold for the TRlim trait in breeding, commercial, and parents of recombinant inbred lines of peanut was observed in the VPD range of 1.6 to 2.9 kPa. Examples of the change in TR with increasing VPD are presented for two peanut cultivars in Figures 6 c,d.

Pearl Millet. Drought-tolerant genotypes of pearl millet exhibited TRlim above VPD of 1.4 to 1.9 kPa (Kholova et al., 2010). Terminal-drought-tolerant cultivar PRLT-2/89-33, and near-isogenic lines introgressed with a terminal drought tolerance QTL from PRLT-2/89-33 in the background of terminal drought sensitive H77/833-2, were found to have TRlim at high VPD. The testing of these lines in a lysimeter system then confirmed that the water use at the vegetative stage for these VPD-sensitive lines was less than in VPD-insensitive lines, and this led to yield increases reaching 60 g m-2 (Vadez et al., 2013). A QTL was mapped for the TR sensitivity to VPD (Kholova et al., 2012), which co-mapped with the terminal drought tolerance QTL identified earlier from yield assessments.

Sorghum. Sorghum is a warm-season grass and mostly grown in dryland regions where it would clearly be beneficial to improve the crop performance under water-deficit conditions. Sensitivity of stomatal conductance to increase in VPD was observed by Bunce et al. (2003) under field conditions in sorghum. Genotypic variation for TRlim under high VPD conditions in sorghum genotypes has been identified (Gholipoor et al., 2010; Choudhary et al., 2013a). Out of the 26 sorghum genotypes tested by Gholipoor et al. (2010), 17 exhibited the TRlim trait and nine 11

genotypes had continually increasing TR with increase in VPD. The VPD at which TRlim was initially expressed ranged between 1.6 to 2.7 kPa. In another study with 12 sorghum genotypes, Choudhary et al. (2013a) found five genotypes had a linear increase in TR and seven had TRlim rate at VPD levels from 1.2 to 2.9 kPa. Shekoofa et al. (2014) compared expression of the TRlim trait under field and controlled environmental conditions in nine sorghum genotypes. They found several sorghum genotypes that exhibited TRlim rate under high VPD conditions both under field and controlled-environment conditions. The TRlim under high VPD was shown in a simulation study with sorghum (Sinclair et al., 2005) to be beneficial for production in Australia. In that study, 115 years were simulated at each of four locations in Australia. The TRlim trait resulted in yield increases in 75% or more of the years at each location. In the remaining years in which there was greater rainfall, there were only modest decreases in yields. Sinclair et al. (2005) concluded that the tradeoff in yields would be economically desirable for growers. Recently, Kholova et al. (2014) simulated sorghum in a range of post-rainy season environments and found the greatest positive benefit of alteration of any plant trait resulted from the TRlim trait.

Soybean. In soybean, Fletcher et al. (2007) found that genotype PI 416937, which was observed to have delayed wilting in the field, expressed the TRlim trait under high VPD conditions. Their results confirmed the earlier observation by Bunce (1984) that leaf conductance of some soybean cultivars decreased in response to increased leaf-to-air VPD difference. In contrast to PI 416937, another delayed-wilting cultivar, PI 471938, and fast-wilting cultivar A5959 showed a linear increase in TR with increasing VPD (Figure 6 e,f). Genetic variation for the TRlim trait in soybean was studied in 31 recombinant inbred lines from a mating between delayed-wilting PI 416937 and fast-wilting Benning (Sadok and Sinclair, 2009 a,b). Across these studies, those genotypes that expressed the TRlim trait had thresholds above which there was little or no further increase at VPD between 1.1 to 2.2 kPa. Gilbert et al. (2011) compared genotypes for stomatal conductance in the field and controlled-environment conditions. Genotypes varied from no stomatal response to VPD, to strong negative responses resulting in full stomata closure at ∼4 kPa. Overall, the response to VPD observed for individual genotypes in the field corresponded to their responses in the controlled-environment.

12

Genotype PI 416937 has been now been used as a parent to develop drought-tolerant, commercial cultivars. Advanced, elite public genotypes that are high-yielding for water-limited environments have been generated for the southern US (Devi et al., 2014). There is also evidence that some private, commercial cultivars also express the limited-transpiration trait (unpublished, T.R. Sinclair, June 2016).

Wheat. Schoppach and Sadok (2012) studied eight cultivars and found six displayed TRlim above VPD ranging from 2.4 to 3.9 kPa. In a follow-up investigation, Schoppach et al. (2017) examined the diversity in TR response curves to increasing VPD within a larger and diverse pool of 23 Australian wheat genotypes. Surprisingly, in this population all cultivars displayed a bi-linear response of TR to VPD. The VPD at which there was a change in slope varied among genotypes between 1.9 and 2.3 kPa. The consistent nature of the bi-linear responses indicated that over the 120 years of selection for dryland wheat production in Australia, breeders appeared to have “unconsciously” favored a water-conservation drought-tolerance trait Another intriguing finding of the study of the 23 Australian genotypes was that the slope in TR at VPD greater than the VPD threshold was correlated strongly and positively (P<0.0001; R2=0.72) with the year of release, suggesting that over 120 years of selection resulted in the progressive increase in canopy conductivity under high VPD conditions (Schoppach et al. 2017). This may seem counter-intuitive considering that the increase in the TR slope at high VPD should diminish the water-conserving capability of the more modern genotypes, but this might come with the positive trade-off of enhancing the opportunity for CO2 fixation that comes with increasing canopy conductance. Such a possibility is in line with the fact that the increase of the TR slope at high VPD over time tracks the historic yield increases that took place over the same time period.

Other species. Substantial genotypic differences were also recorded in cowpea with tolerant genotypes having TRlim above a VPD of about 2.2 kPa (Belko et al. 2012). Limited-transpiration was also observed in C₃ turfgrass (Wherley and Sinclair, 2009; Sermons et al., 2012) and coolseason grass Festuca arundinacea (Sinclair et al., 2007b). Increases in TR were limited at VPD above 1.4 kPa in creeping bentgrass (Agrostis palustris) and perennial rye grass (Lolium perenne). Yield advantage of alfalfa (Medicago sativa) , red clover (Trifolium pratense), cocks foot (Dactylis glomerata) and perennial rye grass expressing conservative water use under high VPD has also been observed (Hainaut et al., 2016). 13

7. Environment and Limited Transpiration

7.1. Temperature effects High temperature has been shown to alter expression of the TRlim trait in soybean. Seversike et al. (2013) found in the cultivar Hutcheson that while the TRlim trait was expressed when the plants were grown and measured at 30 C, the trait was no longer apparent when the VPD response was measured at 35 oC. In addition, Seversike et al. (2013) found in other soybean genotypes that growth temperature of 25 C vs. 30 oC influenced expression of the TRlim trait. Expression of the TRlim trait was found to be sensitive to temperature in some maize hybrids. Yang et al. (2012) observed the transpiration response to VPD of four maize hybrids in the temperature range of 25 to 30 oC. The threshold of VPD to TRlim at high temperature (30 oC), occurred at higher VPD (2.0 to 2.2 kPa) than at low temperature (25 oC) where the threshold was expressed at 1.7 to 1.9 kPa. Shekoofa et al. (2015) studied the influence of high temperature on the limited-transpiration response of 12 maize hybrids, which all expressed the limitedtranspiration trait at 32 0C. In the transition from 36 to 38 0C, five of the hybrids lost the TRlim characteristic. Recently, Shekoofa et al. (2015) compared the responses of peanut cultivars for the TRlim trait in growth chamber and under field conditions. In controlled environments, the trait was evaluated on whole plants. In the field, the trait was evaluated by measuring TR of leaves under a range of naturally occurring VPD. Expression of TRlim observed in the growth chamber was not found under field conditions apparently a result of the high temperature in the field. Based on these results, expression of the limited-transpiration trait in peanut appears to be especially sensitive to a shift in temperature between 31and 36°C. High temperature also modulated the VPD response in tall fescue (Festuca arundinacea Schreb.). Sermons et al. (2012), exposed tall fescue to an acclimation period with increased temperature and VPD for several days. They demonstrated that at lower temperature (21 C), water use was similar at VPDs of 1.2 and 1.8 kPa, indicating a restriction of TR at high VPD. At temperature 27°C, transpiration limitation at high VPD was weakened. The general observation that high temperature can result in an increase in the VPD at which the TRlim is expressed, or in some cases completely lost, corresponds to the observations that 14

plant hydraulic conductivity generally increases with temperature (Bolger et al., 1992; Cochard et al., 2000; Matzner and Comstock, 2001). That is, greater plant hydraulic conductivity allows water to be more readily replenished in leaves so partial stomatal closure moves to a higher TR, i.e. a higher VPD. Although, root hydraulic conductivity was found to decrease in tomato with high temperature shock (Morales et al., 2003), but no measure on response in TR was reported. The sensitivity of expression of the TRlim trait at high temperature for those genotypes that express the trait at lower temperature may be critical in crop breeding and genotype selection. The temperature environment in which the genotype is to be grown appears to be an important consideration in plant selection for the TRlim trait. Genotypes that retain TRlim at high temperature seems suitable for environments where there would be only brief periods of exposure to high temperature and restricted transpiration rate would not result in prolonged high leaf temperature causing serious damage. On the other hand, genotype selection for loss of the TRlim response may well be appropriate in high-temperature environments where plants are subject to prolonged exposure to high temperature so that evaporative cooling of leaves may be necessary to aid in the prevention of loss of physiological activity. Certainly, further study is required to fully understand the thermal and temporal dynamics in the expression and adaptation of the TRlim trait among genotypes and across environments.

7.2. Soil water content The transpiration response to increasing VPD can also be modified by long-term moderate water-deficit stress. In the study of Forseth and Ehleringer (1983), relatively well-watered plants of Lupinus arizonicus appeared to have an abrupt change in transpiration at a VPD threshold of 2.5 kPa. Not surprisingly, when the water potential of the leaves dropped to about -1.3 MPa, the slope of the linear response was substantially lower than that of the well-watered plants and TRlim appeared to be lost. In sunflower (Helianthus annuus), two features can be observed as a result of decreased soil water (Turner et al., 1983): the slope of the relationship between TR and VPD below the VPD threshold for TRlim is lower than when soil-water content is high, and the VPD threshold to limit the TR itself is shifted earlier in the curve, i.e. TRlim is initiated at a lower VPD. Since experimental results on the interaction of the TRlim trait with soil water content is so limited, this is a topic needing much investigation.

15

7.3. Chemical Induction One possibility for activating expression of the TRlim trait appears to be the external application of a chemical to plants. Shekoofa et al. (2016) recently demonstrated that application of Daconil-Action® fungicide in a series of repeated tests resulted in a TRlim response in bentgrass after chemical application. If this can be replicated in crop plants, the external application of a chemical can open new management options. Instead of relying on genetically developed genotypes that always express the TRlim trait, high-yielding genotypes without the TRlim trait might be used in production systems. When late-season water deficits are predicted, then chemical spray of the crop could be done to achieve the benefit of the TRlim trait prior to the development of the drought. Thus, the putative benefit of the TRlim trait could be obtained in water-deficit growing seasons with the chemical but the crop does not necessarily need to be penalized by expression of the trait in wet seasons. The extent of the benefit of a chemical treatment will depend to a great extent on the dynamics and nature of crop response to treatment of any specific chemical.

8. Conclusions

While the TRlim trait is not commonly expressed in crop lines bred for high yield under wellwater conditions, recent study have identified the trait in selected genotypes of many crop species. The empirical evidence indicates that the TRlim trait is desirable for yield increase in many production systems. The physiological basis for the trait appears to be a consequence of limited hydraulic conductivity in the plant, which may be related to aquaporin expression and abundance. However, expression of TRlim at high VPD is not static and is influenced by other environmental factors. Consequently, it may not be enough to treat this trait as qualitative but rather as a trait that is continually modulated by the plant environment. Nevertheless, variable response among genotypes offers an opportunity for plant breeding to be region specific and maximize the probability of increasing crop production.

16

Acknowledgment

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors for the preparation of this review paper.

17

References Araus JL, Slafer GA, Royo C, Serret MD. 2008. Breeding for yield potential and stress adaptation in cereals. Critical Reviews in Plant Sciences 27, 377-412. Belko N, Zaman-Allah M, Cisse N, Diop NN, Zombre G, Ehlers JD et al. 2012. Lower soil moisture threshold for transpiration decline under water deficit correlates with lower canopy conductance and higher transpiration efficiency in drought-tolerant cowpea. Functional Plant Biology 39, 306-322. Bolger P, Upchurch DR, McMichael BL. 1992. Temperature effects on cotton root hydraulic conductance. Environmental and Experimental Botany 32, 49-54. Borrell A, Jordan D, Mullet J, Henzell B, Hammer G. 2006. Drought adaptation in sorghum. In: Ribaut JM, editor. Drought Adaptation in Cereals. Binghamton, USA: The Haworth Press, p.335-399. Branch WD, Hilderbrand GL. 1985. Pod yield comparison of pure-line peanut selections simultaneously developed from Georgia and Zimbabwe breeding programs. Plant Breeding 102, 260-263. Bunce JA. 1981. Comparative responses of leaf conductance to humidity in single attached leaves. Journal of Experimental Botany 32, 629-634. Bunce JA. 1984. Identifying soybean lines differing in gas exchange sensitivity to humidity. Annals of Applied Biology 105, 313-318. Bunce JA. 2003. Effects of water vapor pressure difference on leaf gas exchange in potato and sorghum at ambient and elevated carbon dioxide under field conditions. Field Crops Research 82, 37-47. Bunce JA. 2006. How do leaf hydraulics limit stomatal conductance at high water vapour pressure deficits? Plant, Cell and Environment 29, 1644-1650. Carpentieri-Pipolo V, Pipolo AE, Abdel-Haleem H, Boerma HR, Sinclair TR. 2012. Identification of QTLs associated with limited leaf hydraulic conductance in soybean. Euphytica 186, 679-686. Choudhary S, Mutava RN, Shekoofa A, Sinclair TR, Prasad PV. 2013a. Is the stay-green trait in sorghum a result of transpiration sensitivity to either soil drying or vapor pressure deficit? Crop Science 53, 2129-2134. 18

Choudhary S, Sinclair TR, Prasad PV. 2013b. Hydraulic conductance of intact plants of two contrasting sorghum lines, SC15 and SC1205. Functional Plant Biology 40, 730-738. Choudhary S, Sinclair TR, Messina CD, Cooper M. 2014. Hydraulic conductance of maize hybrids differing in transpiration response to vapor pressure deficit. Crop Science 54, 11471152. Choudhary S, Sinclair TR, Messina CD, Cai W, Warner D, Cooper M. 2015. Inhibitor screen for limited-transpiration trait among maize hybrids. Environmental and Experimental Botany 109, 161-167. Cochard H, Martin R, Gross P, Bogeat-Triboulot. 2000. Temperature effects on hydraulic conductance and water relations of Quercus robur L. Journal of Experimental Botany 51, 1255-1259. Confalonieri U, Menne B, Akhtar R, Ebi KL, Hauengue M, Kovats RS, et al. 2007. Human health. Climate Change 2007: Impacts, Adaptation and Vulnerability. Contribution of Working Group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge, UK: Cambridge University Press; p. 391-431. Cooper MA, Hammer GL. 1996. Synthesis of strategies for crop improvement. In: Cooper MA, Hammer GL, editors. Plant adaptation and crop improvement: ICRISAT and IRRI. Wallingford, UK: CAB International: p. 591-623. Devi MJ, Sinclair TR, Vadez V. 2010. Genotypic variation in peanut for transpiration response to vapor pressure deficit. Crop Science 50, 191-196. Devi MJ, Sinclair TR. 2011. Diversity in drought traits among commercial southeastern US peanut cultivars. International Journal of Agronomy 22. Available from URL: http://dx.doi.org/10.1155/2011/754658 Devi MJ, Sadok W, Sinclair TR. 2012. Transpiration response of de-rooted peanut plants to aquaporin inhibitors. Environmental and Experimental Botany 78, 167-172. Devi JM, Sinclair TR, Chen P, Carter TE. 2014. Evaluation of elite southern maturity soybean breeding lines for drought-tolerant traits. Agronomy Journal 106, 1947-1954. Devi MJ, Sinclair TR, Taliercio E. 2015a. Comparisons of the effects of elevated vapor pressure deficit on gene expression in leaves among two fast-wilting and a slow-wilting soybean. PloS one 10 e0139134. doi: 10.1371/journal.pone.0139134.

19

Devi MJ, Taliercio EW, Sinclair TR. 2015b. Leaf expansion of soybean subjected to high and low atmospheric vapour pressure deficits. Journal of Experimental Botany 66, 1845-1850. Devi MJ, Sinclair TR, Taliercio E. 2016a. Silver and zinc inhibitors influence transpiration rate and aquaporin transcript abundance in intact soybean plants. Environmental and Experimental Botany 122, 168-175. Devi MJ, Sinclair TR, Jain M, Gallo M. 2016b. Leaf aquaporin transcript abundance in peanut genotypes diverging in expression of the limited‐transpiration trait when subjected to differing vapor pressure deficits and aquaporin inhibitors. Physiologia Plantarum 4, 387-396. Fletcher AL, Sinclair TR, Allen LH. 2007. Transpiration responses to vapor pressure deficit in well watered ‘slow-wilting’ and commercial soybean. Environmental and Experimental Botany 61, 145–151. Forseth IN, Ehleringer JR. 1983. Ecophysiology of two solar tracking desert winter annuals III. Gas exchange responses to light, CO2 and VPD in relation to long-term drought. Oecologia 57, 344-351. Gaffney J, Schussler J, Loffler C, Cai W, Paszkiewicz S, Messina C, Groeteke J, Keaschall J, Cooper M. 2015. Industry-scale evaluation of maize hybrids selected for increased yield in drought-stress conditions of the US corn belt. Crop Science 55, 1608-1618. Ghanem ME, Marrou H, Sinclair TR. 2015. Physiological phenotyping of plants for crop improvement. Trends in Plant Science 20, 139-144. Gholipoor M, Prasad PV, Mutava RN, Sinclair TR. 2010. Genetic variability of transpiration response to vapor pressure deficit among sorghum genotypes. Field Crops Research 119, 8590. Gholipoor M, Choudhary S, Sinclair TR, Messina CD, Cooper M. 2013. Transpiration response of maize hybrids to atmospheric vapour pressure deficit. Journal of Agronomy and Crop Science 199, 155-160. Gilbert ME, Holbrook NM, Zwieniecki, Sadok W, Sinclair TR. 2011. Field confirmation of genetic variation in soybean transpiration response to vapor pressure deficit and photosynthetic compensation. Field Crops Research 124, 85-92. Hainaut P, Remacle T, Decamps C, Lambert R, Sadok W. 2016. Higher forage yields under temperate drought explained by lower transpiration rates under increasing evaporative demand. European Journal of Agronomy 72, 91-98. 20

Heinen RB, Ye Q, Chaumont F. 2009. Role of aquaporins in leaf physiology. Journal of Experimental Botany 60, 2971-2985. Kholova J, Hash CT, Kakkera A, Kocova M, Vadez V. 2010. Constitutive water-conserving mechanisms are correlated with the terminal drought tolerance of pearl millet Pennisetum glaucum (L.) R. Br. Journal of Experimental Botany 61, 369–377. Kholová J, Nepolean T, Hash CT, Supriya A, Rajaram V, Senthilvel S, Kakkera A, Yadav R, Vadez V. 2012. Water saving traits co-map with a major terminal drought tolerance quantitative trait locus in pearl millet [Pennisetum glaucum (L.) R. Br.]. Molecular Breeding 30, 1337-1353. Kholová J, Murugesan T, Kaliamoorthy S, Malayee S, Baddam R, Hammer GL et al. 2014. Modelling the effect of plant water use traits on yield and stay-green expression in sorghum. Functional Plant Biology 41, 1019-1034. Levin M, Lemcoff JH, Cohen S, Kapulnik Y. 2007. Low air humidity increases leaf-specific hydraulic conductance of Arabidopsis thaliana (L.) Heynh (Brassicaceae). Journal of Experimental Botany 58, 3711-3718. Ludlow MM, Muchow RC. 1990. A critical evaluation of traits for improving crop yields in water-limited environments. Advances in Agronomy 43, 107–153. Maroco JP, Pereira JS, Chaves MM. 1997. Stomatal responses to leaf-to-air vapour pressure deficit in sahelian species. Australian Journal of Plant Physiology 24, 381–387. Matzner S, Comstock J. 2001. The temperature dependence of shoot hydraulic resistance: implications for stomatal behaviour and hydraulic limitation. Plant, Cell and Environment 24, 1299-1307. McAdam SAM, Brodribb, TJ. 2014. Separating active and passive influences on stomatal control of transpiration. Plant Physiology 164, 1578-1586. McAdam SAM, Brodribb, TJ. 2016. Linking turgor with ABA biosynthesis: Implications for stomatal responses to vapor pressure deficit across land plants. Plant Physiology 171, 20082016. Meidner H. 1975. Water supply, evaporation, and vapour diffusion in leaves. Journal of Experimental Botany 26, 666-673.

21

Messina CD, Sinclair TR, Hammer GL, Curan D, Thompson J, Oler Z et al. 2015. Limitedtranspiration trait may increase maize drought tolerance in the US Corn Belt. Agronomy Journal 107, 1978-1786. Mitra J. 2001. Genetics and genetic improvement of drought resistance in crop plants. Current Science 80, 758–763. Morales D, Rodríguez P, Dell'Amico J, Nicolás E, Torrecillas A, Sánchez-Blanco MJ. 2003. High-temperature preconditioning and thermal shock imposition affects water relations, gas exchange and root hydraulic conductivity in tomato. Biologia Plantarum 47, 203-208. Nardini A, Salleo S. 2005. Water stress-induced modifications of leaf hydraulic architecture in sunflower: co-ordination with gas exchange. Journal of Experimental Botany 56, 3083-3101. Ocheltree TW, Nippert JB, Prasad PV. 2014. Stomatal responses to changes in vapor pressure deficit reflect tissue‐specific differences in hydraulic conductance. Plant, Cell and Environment 37, 132-139. Pita P, Cañas I, Soria F, Ruiz F, Toval G. 2005. Use of physiological traits in tree breeding for improved yield in drought-prone environments. The case of Eucalyptus globulus. Forest Systems 14, 383-393. Richards RA, Passioura JB. 1989. A breeding program to reduce the diameter of the major xylem vessel in the seminal roots of wheat and its effect on grain-yield in rain-fed environments. Australian Journal of Agricultural Research 40, 943–950. Sadok W, Sinclair TR. 2009a. Genetic variability of transpiration response to vapor pressure deficit among soybean cultivars. Crop Science 49, 955-60. Sadok W, Sinclair TR. 2009b. Genetic variability of transpiration response to vapor pressure deficit among soybean (Glycine max [L.] Merr.) genotypes selected from a recombinant inbred line population. Field Crops Research 113, 156-160. Sadok W, Sinclair TR. 2010a. Transpiration response of ‘slow-wilting’and commercial soybean (Glycine max (L.) Merr.) genotypes to three aquaporin inhibitors. Journal of Experimental Botany 61, 821-829. Sadok W, Sinclair TR. 2010b. Genetic variability of transpiration response of soybean (Glycine max (L.) Merr.) shoots to leaf hydraulic conductance Inhibitor AgNO3. Crop Science 50, 1423-1430.

22

Sadok W, Sinclair TR. 2011. Crops yield increase under water-limited conditions: review of recent physiological advances for soybean genetic improvement. Advances in Agronomy 113, 313-337. Schoppach R, Sadok W. 2012. Differential sensitivities of transpiration to evaporative demand and soil water deficit among wheat elite cultivars indicate different strategies for drought tolerance. Environmental and Experimental Botany 84, 1–10. Sermons SM, Seversike TM, Sinclair TR, Fiscus EL, Rufty TW. 2012. Temperature influences the ability of tall fescue to control transpiration in response to atmospheric vapour pressure deficit. Functional Plant Biology 39, 979-986. Seversike TM, Sermons SM, Sinclair TR, Carter TE, Rufty TW. 2013. Temperature interactions with transpiration response to vapor pressure deficit among cultivated and wild soybean genotypes. Physiologia Plantarum 148, 62–73 Shatil-Cohen A, Attia Z, Moshelion M. 2011. Bundle-sheath cell regulation of xylemmesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA? Plant Journal 67, 72–80. Shekoofa A, Devi JM, Sinclair TR, Holbrook CC, Isleib TG. 2013. Divergence in droughtresistance traits among parents of recombinant peanut inbred lines. Crop Science 53, 25692576. Shekoofa, A., M. Balota, and T.R. Sinclair. 2014. Limited-transpiration trait evaluated in growth chamber and field for sorghum genotypes. Environmental and Experimental Botany 99, 175–179. Shekoofa A, Sinclair TR, Messina CD, Cooper M. 2015a. Variation among maize hybrids in response to high vapor pressure deficit at high temperatures. Crop Science, 55, 392-396. Shekoofa A, Rosas-Anderson P, Sinclair TR, Balota M, Isleib TG. 2015b. Measurement of limited-transpiration trait under high vapor pressure deficit for peanut in chambers and in field. Agronomy Journal 107, 1019-1024. Shekoofa A, Rosas-Anderson P, Carley DS, Sinclair TR, Rufty TW. 2016. Limited transpiration under high vapor pressure deficits of creeping bentgrass by application of Daconil-Action®. Planta 243, 421-427.

23

Sheshshayee MS, Bindumadhava H, Shankar AG, Prasad TG, Udayakumar M. 2003. Breeding strategies to exploit water use efficiency for crop improvement. Journal of Plant Biology 30, 253–268. Sinclair, TR. 2011. Challenges in breeding for yield increase for drought. Trends in Plant Science 16, 289-293. Sinclair TR, Hammer GL,van Oosterom EJ. 2005. Potential yield and water-use efficiency benefits in sorghum from limited maximum transpiration rate. Functional Plant Biology 32, 945-952. Sinclair TR, Salado-Navarro LR, Salas G, Purcell LC. 2007a. Soybean yields and soil water status in Argentina: simulation analysis. Agricultural Systems 94, 471–477. Sinclair T, Fiscus E, Wherley B, Durham M, Rufty T. 2007b. Atmospheric vapor pressure deficit is critical in predicting growth response of “cool-season” grass Festuca arundinacea to temperature change. Planta 227, 273-276. Sinclair TR, Zwieniecki MA, Holbrook NM. 2008. Low leaf hydraulic conductance associated with drought tolerance in soybean. Physiologia Plantarum 132, 446-451. Sinclair TR, Messina CD, Beatty A, Samples M. 2010. Assessment across the United States of the benefits of altered soybean drought traits. Agronomy Journal 102, 475-482. Sinclair TR, Marrou H, Soltani A, Vadez V, Chandolu KC. 2014. Soybean production potential in Africa. Global Food Security 3, 31-40. Sinclair TR, Devi JM, Carter Jr TE. 2016. Limited-transpiration trait for increased yield for water-limited soybean: From model to phenotype to genotype to cultivars. In: Yin X, Struik PC, editors. Crop Systems Biology. Springer International Publishing; p. 129-146. Tanner CB, Sinclair, TR. 1983. Efficient water in crop production: Research or re-search? In: Taylor, Jordan WR, Sinclair TR, editors. Limitations to Efficient Water Use in Crop Production. American Society of Agronomy, p. 1-27. Turner NC, Schulze ED, Gollan T. 1984. The responses of stomata and leaf gas exchange to vapour pressure deficits and soil water content. I. Species comparisons at high soil water contents. Oecologia 63, 338-342. Turner NC, Schulze ED, Gollan T. 1985. The responses of stomata and leaf gas exchange to vapour pressure deflcits and soil water content II. In the mesophytic herbaceous species Helianthus annuus. Oecologia 65, 348-355. 24

Vadez V, Kholova J, Yadav RS, Hash CT. 2013. Small temporal differences in water uptake among varieties of pearl millet (Pennisetum glaucum (L.) R. Br.) are critical for grain yield under terminal drought. Plant and Soil 371, 447-462. Vadez V, Kholova J, Medina S, Kakkera A, Anderberg H. 2014. Transpiration efficiency: new insights into an old story. Journal of Experimental Botany 65, 6141-6153. Vadez V, Kholová J, Hummel G, Zhokhavets U, Gupta SK, Hash CT. 2015. LeasyScan: a novel concept combining 3D imaging and lysimetry for high-throughput phenotyping of traits controlling plant water budget. Journal of Experimental Botany 61, 5581-5593. Wherley BG, Sinclair TR. 2009. Differential sensitivity of C 3 and C 4 turfgrass species to increasing atmospheric vapor pressure deficit. Environmental and Experimental Botany 67, 372-376. Yang Z, Sinclair TR, Zhu M, Messina CD, Cooper M, Hammer GL. 2012. Temperature effect on transpiration response of maize plants to vapor pressure deficit. Environmental and Experimental Botany 78, 157-162 Zaman-Allah M, Jenkinson DM, Vadez V. 2011a. Chickpea genotypes contrasting for seed yield under terminal drought stress in the field differ for traits related to the control of water use. Functional Plant Biology 38, 270-281. Zaman-Allah M, Jenkinson DM, Vadez V. 2011b. A conservative pattern of water use, rather than deep or profuse rooting, is critical for the terminal drought tolerance of chickpea. Journal of Experimental Botany 62, 4239–4252. Zwieniecki MA, Brodribb TJ, Holbrook NM. 2007. Hydraulic design of leaves: insights from rehydration kinetics. Plant, Cell and Environment 30, 910-921.

25

Figure Captions

Figure 1. Calculated daily cycle of the transpiration rate of two limited-transpiration phenotypes with maximum transpiration rated of 0.4 to 0.6 mm h-1 (dotted line with triangles) at high vapor pressure deficit (VPD) and standard phenotype (solid line with circles) with no limitation on transpiration rate at high VPD. The vapor pressure deficit through the daily cycle used in these calculations is included as a reference (Dotted line with diamonds). (Sinclair et al., 2005)

Figure 2. Results of simulated changes in soybean yields across the USA with inclusion of the limited-transpiration trait as compared to the absence of the trait (Sinclair et al., 2010). (a) Probability of yield increase with limited transpiration trait as compared to soybean without the trait. (b to d) Absolute yield difference between the simulations with the limited-transpiration trait and those without the trait standard simulation were calculated for each year. The yield differences were ranked to obtain for each grid location the (b) 75%, (c) median, (d) and 25% percentile ranking across years. (Sinclair et al., 2010)

Figure 3. Top panel: Leaf water potential measured over a range of VPD in the field for a soybean cultivar that expressed the limited-transpiration trait (Gilbert et al., 2011). Bottom panel: Relative water content measured over a range of VPD in the field for a peanut cultivar that expressed the limited-transpiration trait (Shekoofa et al., 2015b).

Figure 4. Schematic of water flow in leaves showing the major pathway of water flow from xylem to guard cells. The solid lines represent liquid flow and the dashed lines show vapor flow. The resistances to liquid water flow are indicated as rI in the pathway from the xylem to the epidermis and rII in the pathway to the mesophyll cells. (Zwieniecki et al., 2007). -2

-1

Figure 5. Transpiration rate (in mg cm min ) as a function of cumulative temperature units (calculated with base temperature of 10 ºC and optimal temperature of 25 and 35 ºC) in two genotypes of sorghum (VPD-insensitive R16 and VPD-sensitive S35) (a), and in two genotypes of pearl millet (VPD-insensitive H77/833-2 and VPD-sensitive PRLT-2/89-33) (b). The incept in each figure represents a close-up of a 3-days period between 191 and 227 cumulative 26

temperature units. Each data point is the mean (+/- SE) of 6 replicated sectors for each genotype (Vadez et al., 2015).

Figure 6. Transpiration rate of (a,b) maize (c,d) peanut and (e,f) soybean in response to increase in vapor pressure deficit (VPD). The regressions in panels a, c, and e are two segmental linear relationship where plants showed limited transpiration response above VPD threshold as shown in the figures. In panels b, d, and f, plants had linear increase in transpiration rate across all VPD. (Gholipoor et al., 2013; Devi et al., 2010; Flectcher et al., 2007, respectively).

Figure 1

27

Figure 2

28

Leaf Water Potentail ()

Figure 3

-0.0

Soybean (PI 416937)

-0.2 -0.4 -0.6 -0.8 1.0

Limited Transpiration Threshold

1.5

2.0

2.5

3.0

3.5

4.0

Rel. Water Content (%)

Vapor Pressure Deficit (kPa)

100 90 80

Peanut 70 1.0

Limited Transpiration Threshold

(ASL 177)

1.5

2.0

2.5

3.0

3.5

4.0

Vapor Pressure Deficit

29

Figure 4

30

Figure 5

31

Figure 6

32