Limited-transpiration trait evaluated in growth chamber and field for sorghum genotypes

Environmental and Experimental Botany 99 (2014) 175–179

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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Limited-transpiration trait evaluated in growth chamber and field for sorghum genotypes A. Shekoofa a , M. Balota b , T.R. Sinclair a,∗ a b

Department of Crop Science, North Carolina State University, Box 7620, Raleigh, NC 27695-7620, USA Virginia Tech Tidewater AREC, 6321 Holland Road Suffolk, VA 23437, USA

a r t i c l e

i n f o

Article history: Received 28 July 2013 Received in revised form 12 November 2013 Accepted 26 November 2013 Keywords: Drought stress Sorghum Stomatal conductance Vapor pressure deficit

a b s t r a c t Sorghum [Sorghum bicolor (L.) Moench] is commonly grown in water-limited environments throughout the world. Plant traits could be useful allowing for early-season water conservation so that more water is available for use later in the season when drought is most likely to develop. One trait that might result in early-season water conservation is the expression of a limited-transpiration trait defined as a limitation on further increases in transpiration rate (TR) under high vapor pressure deficit (VPD) conditions. The objective of this study was to compare the expression of the limited-TR trait measured for nine sorghum genotypes under both controlled chamber and field conditions. In the growth chamber, plant TR was measured over a range of imposed VPD to provide a direct measure of plant transpiration under high VPD. In the field, stomatal conductance (gs ) was measured over the daily cycle, which resulted in a range of ambient VPD. A decrease in gs under high VPD was evidence of the limited-TR trait. This study identified three sorghum genotypes (DKS 36-06, DKS 44-20, and DKS 54-00) that did not show any limitation on water loss at high VPD in either the greenhouse or field. On the other hand, four genotypes (BTX 2752, SC 599, SC 982, and B 35) exhibited the limited-TR trait in the growth chamber with breakpoints in response to VPD at values of 2.33 kPa and above. These four genotypes also expressed a breakpoint in gs in response to increasing VPD in the field. Two genotypes (TX ARG 1, TX 436) that differed between the growth chamber and field showed consistency in response on close examination of the field results. The overall general correspondence within genotypes between the controlled chamber and the field in expression or lack of expression of a breakpoint in response to increasing VPD demonstrated the possibility of selecting genotypes for the TRlim trait under differing environmental conditions. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Sorghum [Sorghum bicolor (L.) Moench] is the fifth most economically important cereal crop grown worldwide and adapted to a wide range of climatic conditions. World sorghum production has risen slightly from 60 million metric tons (2.4 billion bushels) to 65 million metric tons (2.6 billion bushels) over the past decade (Mutava, 2012). With predictions of less water available for crop production as a result of climate change, the adaptability of sorghum to dry environments may result in sorghum playing a larger role in global food security (Ciacci et al., 2007). Therefore, traits of sorghum that enhance its drought tolerance are likely to become of increasing importance.

Abbreviations: gs , stomatal conductance; TR, transpiration rate; VPD, vapor pressure deficit. ∗ Corresponding author. Tel.: +1 919 513 1620. E-mail address: [email protected] (T.R. Sinclair). 0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2013.11.018

Crop gas exchange between leaves and the atmosphere changes with the daily cycle and seasonally due to such factors as daylength, temperature and atmospheric vapor pressure (Allen et al., 1994). Atmospheric vapor pressured deficit (VPD) and transpiration rate (TR) follow a diurnal pattern with both commonly being lowest at sunrise and increasing to a maximum around 15:00 (Hirasawa and Hsiao, 1999). There is, however, an exception to the close link between atmospheric VPD and TR in cases where plants are unable to transport internally sufficient water to match the transpiration demand resulting from high VPD. In this case, plants exhibit a limited-TR associated with a decrease in stomatal conductance so that transpiration corresponds to the hydraulic conductance capacity in the plant (Zwieniecki et al., 2007). Since this phenomenon is expressed under high midday VPD, it is commonly observed as midday stomata closure. Two benefits of this trait are that it decreases the effective VPD under which water is transpired in the daily cycle resulting in improved water use efficiency, and it results in soil water conservation. Soil water conservation can be especially beneficial because it allows water to be retained in the soil to

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support plant growth later in the season in the event that drought conditions develop (Sinclair et al., 2005). Genotypic variation in the expression of the limited-TR trait has been identified in a number of crop species including sorghum (Gholipoor et al., 2010). Gholipoor et al. (2010) found variation between genotypes that expressed the limited-TR trait and those that did not. There was also variation in the VPD at which the break in TR with increasing VPD occurred among those genotypes expressing the trait. However, the study by Gholipoor et al. (2010) was done only on young plants (approximately 4–6 leaves) in a growth chamber. An unanswered question is whether the results obtained in the growth chamber translate to field-grown plants. Therefore, the objective of this study was to compare expression of transpiration response to VPD under growth chamber and field conditions. Transpiration measurements were made on whole, young plants contained in chambers as described by Gholipoor et al. (2010) and field measurements were based on leaf measurements of stomatal conductance under a range of naturally occurring VPD. Nine sorghum genotypes were compared under both growth conditions. 2. Materials and methods Nine sorghum genotypes were included in this comparison of growth chamber and field measurements (Table 1). The expression of the limited-TR trait was reported for six of the genotypes by Gholipoor et al. (2010). Additional data of the possible existence of the limited-TR trait were obtained on three genotypes: DKS 3606, DKS 44-20, and DKS 54-00. These genotypes represent cultivars adapted to the mid-Atlantic region of the US in which the field test was conducted. 2.1. Controlled chamber test The technique for measuring plant transpiration rates under a range of VPD was described by Gholipoor et al. (2010). Briefly, plants were grown in pots constructed for attachment of VPD chambers in which transpiration measurements in response to different VPD levels were made. Plants were grown under well-watered conditions in a greenhouse (North Carolina State University Phytotron, Raleigh, NC) regulated to temperatures of 31 ◦ C day/26 ◦ C night. Once approximately four leaves had fully expanded on the plants, the plants were moved into a walk-in growth chamber (31 ◦ C) and fully watered to dripping. The photosynthetic photon flux density in the growth chamber was about 650 ␮mol m−2 s−1 . The following morning the plants were enclosed individually in 21 L clear chambers for the measurement of transpiration rate under a range of VPD. Each container, i.e., VPD chamber, was fitted with a 12 V, 76 mm diameter computer box fan (Northern Tool and Equipment, Burnsville, MN) to continuously stir the air inside the chamber. In addition, a humidity/temperature data logger (Lascar Electronics, Erie, PA) was mounted through the side wall of each container to measure the chamber environment. Twelve VPD chambers were available for measurements, so that four genotypes with three replications were measured at one time. Transpiration responses were measured on two consecutive days at three VPD ranges on each day by starting with the lowest VPD (0.5–1.5 kPa), then the medium VPD (1.5–2.5 kPa), and finally the highest VPD (2.5–3.5). This sequence was used to avoid any recovery that might be needed if stomatal closure was induced by exposure to the high VPD treatment. The VPD in the chambers was allowed to stabilize for 30 min after introduction of each of the three humidity levels. Following this stabilization period, the initial weight of the pot was measured on a balance with a resolution of 0.1 g. The plants were exposed to each VPD treatment for 1 h and

then weighed. The VPD was averaged during the treatment period based on the measurements of temperature and relative humidity in the chamber. After completing measurements on the second day, the plants were dissected and the total area of the leaves on each plant was measured using a leaf area meter (LI-1300, Licor, Lincoln, NE). Transpiration rate was expressed on a plant leaf area basis. The 18 observations collected for each genotype (3 VPD × 2 d × 3 reps) were combined for a two-segment linear regression analysis (Prism 5.0, GraphPad, Software Inc., San Diego, CA) of transpiration rate vs. chamber VPD. When the slopes were not significantly different between the two segments, a simple linear regression was applied to all the data. For those genotypes found to be represented by the two segments, the regression analysis generated the VPD breakpoint between the two linear segments. 2.2. Field test The field experiment was done at the Tidewater Agricultural Research and Extension Center (TAREC) near Holland, VA (36◦ 68 N, 76◦ 77 W, 25 m elevation). The soil at this location is a Kenansville loamy sand (Arenic Hapludults, loamy, siliceous, thermic). The seeds of the nine sorghum genotypes were sown at a depth of 2.5 cm in 7 m2 plots with 20 plants m−2 on June 8, 2012. Genotypes were arranged in two-row plots in a randomized complete block (RCB) design with three replicates. Before sowing, 68 kg N ha−1 fertilizer was applied as urea on May 30. On 27 June, an additional 68 kg N ha−1 fertilizer was applied. During the experiment, weed and occasional insect-control was maintained to ensure optimal crop growing conditions. Stomatal conductance (gs ) of leaves was measured using a LI6400 (LICOR, Inc., Lincoln, Nebraska). To keep the leaf chamber within ±0.5 ◦ C of air temperature at the time of measurement, the constant block-temperature feature of the equipment was used. Relative humidity inside the sample chamber was maintained between 48% and 50%. During measurements, PPFD was maintained constant at 2000 ␮mol m−2 s−1 with a 6400-02B light source (LI-COR Biosciences Inc., Lincoln, NE). A 6400-01 CO2 mixer (LICOR Biosciences Inc., Lincoln, NE) was used to inject and maintain a constant concentration of 400 ␮mol CO2 mol−1 air during measurements. A 6 cm2 area of a leaf lamina was enclosed in the instrument chamber and allowed to equilibrate within the chamber for 60 s before recording measurements. When the sorghum plants entered the bloom stage, measurement of gs began (8 August 2012). Fully expanded leaves that had been previously exposed to full sunlight were used for the measurements (Balota et al., 2008). All measurements were taken between 1000 and 1500 EST. The sampling procedure for stomatal conductance consisted of measuring middle parts of the uppermost fully developed leaf of 3 plants per plot. The block replications were measured in rotation three times. Hence, 18 measurements (3 leaves × 3 reps × 2 times) were made on each genotype on each day. Measurements were made on 8 August (bloom stage), 15 August (half bloom), 21 August (soft dough), 31 August (hard dough), and 5 September (physiological maturity). Therefore, a total of 90 observations were obtained for each genotype. As a result of the measurement scheme, measurements for each genotype were made under the natural range in VPD to which the plants were subjected on each day. Ambient VPD was the basis for analyzing gs since gs would have been in response to ambient VPD conditions with little change during the 60 s in which the leaf segment was introduced to the measurement chamber. Ambient air temperature and relative humidity were continuously monitored with a humidity/temperature data logger (Lascar Electronics, Erie, PA) attached to the LI-6400 chamber. The ambient VPD for each measurement was calculated from the saturation vapor pressure (Buck, 1981) and mean relative humidity.

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Table 1 Controlled-chamber regression results [breakpoint and slopes of two linear regression segments (mol H2 O m−2 s−1 kPa−1 ) and R2] among nine genotypes tested for response of transpiration rate to increasing vapor pressure deficit. Genotypes

Date of experiment

Breakpoint ± S.E.

Slope1 ± S.E.

SC 982 BTX 2752 SC 599 B 35 BTX ARG 1 TX 436 DKS 36-06 DKS 44-20 DKS 54-00

Gholipoor et al. (2010) Gholipoor et al. (2010) Gholipoor et al. (2010) Gholipoor et al. (2010) Gholipoor et al. (2010) Choudhary et al. (2013) 26–27 February 2013 26–27 February 2013 26–27 February 2013

1.62 ± 0.23 2.08 ± 0.20 2.23 ± 0.27 2.61 ± 0.18 Linear Linear Linear Linear Linear

50.0 52.0 35.5 40.3 27.5 6.62 10.10 9.40 8.35

± ± ± ± ± ± ± ± ±

12.6 9.0 7.0 6.8 2.4 0.56 0.80 1.02 1.03

Slope2 ± S.E.

R2

6.3 ± 11 ± 3.4 ± 2.4 ± – – – – –

0.79 0.89 0.80 0.88 0.90 0.91 0.71 0.61 0.75

4.8 6.7 8.4 7.7

Fig. 1. Plot of rainfall during each day over the period of measurement of stomatal conductance and the day on which measurements were made.

Since no trend in gs was observed across the five measurement dates, all data were combined (90 observations) in the statistical analysis of each genotype. The analyses were done using the statistical software JMP 10.01 (SAS Institute, Cary, NC) and also GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). This software determined the breakpoint and the confidence interval for the breakpoint of the two-segment linear regression model of stomatal conductance vs. ambient VPD.

3. Results 3.1. Controlled chamber test The results of Gholipoor et al. (2010) and the measurements of the mid-Atlantic cultivars were combined to characterize the transpiration responses to increasing VPD under growth chamber conditions. The nine genotypes expressed significant differences in TR response with increasing VPD (Table 1). In four genotypes, TR response to increasing VPD showed two linear segments with a clear breakpoint between two segments. The value of the breakpoint ranged from 1.62 kPa for SC 982 to 2.61 kPa for B 35. The R2 for the two segments regression for the four genotypes ranged from 0.79 to 0.89. The remaining five genotypes, including all three of the mid-Atlantic cultivars exhibited a linear response in TR over the measured VPD range (Table 1). However, the slope of the linear slope of these five genotypes was less than the initial slope of the four genotypes expressing a breakpoint. The R2 of these five genotypes ranged from 0.61 to 0.90.

3.2. Field test Frequent rains (Fig. 1) assured that the plants in the field tests were under well-watered conditions. In particular, large rains occurred no more than 4 d prior to each set of stomatal conductance measurements. No stress symptoms were ever observed on these plants. The natural range of VPD in the field allowed measurements to be under VPD up to 5.0 kPa (Table 2). This was primarily a consequence of increasing midday temperature resulting in progressively higher saturated vapor pressure through the day. The mid-Atlantic cultivars exhibited no decrease in gs at high VPD indicating the lack of the limited-TR trait in these genotypes (Table 3). As illustrated in Fig. 2a by cultivar DKS 54-00, gs was nearly constant with changing ambient VPD. The slight upward trend in this and other cultivars with VPD may reflect a slight increase in gs as a result of the association between VPD and PPFD. In contrast to the lack of a breakpoint response of the three midAtlantic cultivars, the six genotypes originally studied by Gholipoor

Table 2 Maximum air temperature, photosynthetically active radiation (PAR), and ambient vapor pressure deficit (VPD) observed on each day during field measurement. Day of measurement

Max temperature (◦ C)

Max PAR (␮mol m−2 s−1 )

Max VPD (kPa)

8 August 2012 15 August 2012 21 August 2012 31 August 2012 5 September 2012

36.0 35.0 34.6 34.5 34.5

1813 1893 1100 1500 1500

3.8 3.5 4.4 5.0 3.9

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Table 3 Field regression results [breakpoint and slopes of two linear regression segments (mol H2 O m−2 s−1 kPa−1 ) and R2 ] among nine genotypes tested for response of stomatal conductance to increasing vapor pressure deficit. The significance level of the fit is indicated in the last column (ns = non-significance, *0.05, ***0.001). Genotypes

Breakpoint (X0 ) ±S.E.

Slope1 ± S.E.

TX 436 BTX 2752 SC 982 B 35 BTX ARG 1 SC 599 DKS 36-06 DKS 44-20 DKS 54-00

2.33 ± 2.10 3.28 ± 0.22 3.50 ± 0.15 3.45± 0.02 3.74 ± 0.09 3.87 ± 0.08 Linear Linear Linear

0.08 0.07 0.15 0.11 0.09 0.09 0.04 0.03 0.02

et al. (2010) exhibited breakpoints in gs from 3.3 to 3.9 kPa, as illustrated with cultivar B35 in Fig. 2b. Generally, the pattern of gs changes in the field was a small increase of gs with increasing VPD below the breakpoint (slope1) and a decrease of gs above the breakpoint (slope2). The exception to the common pattern of decreasing gs above the breakpoint was exhibited by TX 436. For TX 436, gs was essentially unchanged with increasing the VPD above the breakpoint (Table 3). The cultivar BTX ARG 1, which exhibited a linear response in TR with increasing VPD in the study of Gholipoor et al. (2010), was found to have a breakpoint in gs with increasing VPD in the field. The difference between the two measurements of response of TR to VPD seems likely to be a result of the range of VPD to which the plants were subjected under the two conditions. In the study of Gholipoor et al. (2010), the maximum VPD under which the plants were measured was 3.5 kPa while in the field study gs was measured under VPD of up to 5.0 kPa. Therefore, the high range of VPD in the field resulted in the identification of a breakpoint at a high VPD while in the growth chamber the range was insufficient to detect the breakpoint.

Fig. 2. Plot of field measurements of stomatal conductance vs. ambient vapor pressure deficit for cultivars (a) DKS 54-00 and (b) B 35.

± ± ± ± ± ± ± ± ±

0.12 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.02

Slope2 ± S.E.

R2

0.02 ± −0.13 ± −0.42 ± −0.06 ± −0.45 ± −0.84 ± – – –

0.20*** 0.23* 0.36*** 0.29*** 0.34*** 0.46*** 0.10* 0.02ns 0.05*

0.01 0.07 0.19 0.06 0.16 0.36

4. Discussion The results of whole-plant TR response to increasing VPD in the chambers highlighted two distinct patterns among the nine genotypes. Four genotypes exhibited a breakpoint with increasing VPD and five did not. Interestingly, no breakpoint was detected for the three genotypes that were adapted to the relative humid mid-Atlantic environment of the U.S. However, the slope of the whole-plant transpiration rate versus VPD of the genotypes with the no-breakpoint response was much less than those genotypes expressing a breakpoint. Hence, the no breakpoint cultivars would achieve soil water conservation by restricting transpiration under all conditions in contrast to the breakpoint genotypes in which water conservation occurred only under high VPD. The objective of this study was to compare the expression of TR response to increasing VPD measured in a growth chamber and in the field. In fact, there was correspondence in the determination of a breakpoint in TR in the growth chamber and a breakpoint in gs in the field for seven of the nine cultivars studied. Breakpoints were not detected for the three mid-Atlantic cultivars, DKS 36-06, DKS 44-20, and DKS 54-00, under either test condition. Four genotypes, SC 982, BTX 2752, SC 599, and B 35, expressed a breakpoint under both chamber and field conditions. However, no consistency in the value of the VPD at the breakpoint for those genotypes expressing the breakpoint was found between the chamber and field measurements. But, a comparison of the breakpoint value based on only four genotypes should not be considered conclusive. Two genotypes, TX 436 and BTX ARG1, had a linear response in plant TR measured in chambers but a breakpoint in gs response in the field. While a breakpoint was found in TX 436 in the field, the slope above the breakpoint was positive indicating a continuing increase in gs with increasing VPD, although at a slower rate. Therefore, TR for this genotype in the field was not severely restricted, similar to the observation of a linear response in the chamber measurements. The identification of the breakpoint in BTX ARG1 in the field but not in the chamber could result from the range of VPD that was tested under each condition. The VPD for the breakpoint in gs in the field for BTX ARG1 was 3.74 kPa, which was at the high end of the VPD range tested by Gholipoor et al. (2010). Hence, the failure to identify a breakpoint in the chamber study may simply have been a result of insufficient data at high VPD in the regression to establish a breakpoint. One distinction among those cultivars exhibiting a breakpoint both in the chamber and field was that the VPD at the breakpoints in the field were substantially greater than in the chamber study. There are at least two possibilities to explain this difference between measurements. One reason may be that the plants were at different stages of development. Young plants with only four expanded leaves were studied in the chamber measurements, while in the field the plants had completed vegetative development. The possibility exists that the breakpoint moves to a higher VPD for leaves that are produced late in vegetative development.

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A second reason to explain the difference in breakpoints between the chamber and field measurements was a difference between the two environments in which the plants developed and the measurements were made. The plants used in the chamber study were subjected to a day temperature of 31 ◦ C and measured at this same temperature. In the field, temperature was variable and temperature on each measurement day exceeded 34.5 ◦ C (Table 2). Yang et al. (2012) reported in each of four maize (Zea mays L.) hybrids that the VPD breakpoint was greater when measured at 30 ◦ C as compared to measurement at 25 ◦ C. They attributed this difference to changes in temperature sensitivity of hydraulic conductance in the plants, which could be a result of aquaporin adjustments to temperature. Sermons et al. (2012) reported a similar increase in breakpoint in the cool-season grass tall fescue (Festuca arundinacea Schreb.) when temperature was increased from 21 ◦ C to 29 ◦ C. Hence, a strong possibility is that the higher temperatures in the field could be important in the observed greater values of VPD at the breakpoint. Overall, this study showed a general correspondence between the expression or lack of expression of a breakpoint in TR with increasing VPD in chamber and field measurements. This is encouraging that this trait is expressed similarly under both conditions. While plant stage and/or temperature at measurement may influence the value of the breakpoint, the expression of the trait was generally characterized similarly in both situations. Consequently, the results of this study showing stability in the expression of the limited-transpiration trait supports the possibility of comparing germplasm for this trait under a range of conditions.

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