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Genetic variability of transpiration response to vapor pressure deficit among sorghum genotypes
Field Crops Research 119 (2010) 85–90
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Genetic variability of transpiration response to vapor pressure deficit among sorghum genotypes Manoochehr Gholipoor a , P.V. Vara Prasad b , Raymond N. Mutava b , Thomas R. Sinclair c,∗ a b c
Department of Agronomy and Plant Breeding, Shahrood University of Technology, P.O. Box 36155-316, Shahrood, Iran Department of Agronomy, Kansas State University, Manhattan, KS 66506-5501, USA Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA
a r t i c l e
i n f o
Article history: Received 20 April 2010 Received in revised form 24 June 2010 Accepted 28 June 2010 Keywords: Drought stress Genotypic variation Sorghum Transpiration Vapor pressure deficit
a b s t r a c t Simulation studies have demonstrated that limited maximum transpiration rate (TR) at high air vapor pressure deficit (VPD) in water-limited environments could result in significant increases in sorghum yield. However, such a restriction on TR at high VPD has not been documented in sorghum. The objective of this study was to search within sorghum germplasm for the possibility of restricted TR at high VPD. Twenty six genotypes were selected for measurement of VPD response based on field observations including yield, leaf temperature, and the stay-green phenotype. These genotypes were grown in a greenhouse for about 24-d growth, and then placed into individual chambers in which VPD was varied and TR measured. The results of this study showed marked variation among sorghum genotypes in TR response to VPD. Seventeen genotypes were identified as exhibiting a breakpoint in their VPD response in the range from 1.6 to 2.7 kPa, above which there was little or no further increase in TR. Therefore, these genotypes with a breakpoint have the possibility of soil water conservation when VPD during the midday cycle exceeds the breakpoint VPD. This trait would be desirable in less humid environments for increasing yields in water-deficit seasons. The observed range in the value of the BP among genotypes offers the possibility of developing genotypes with BP appropriate for specific environments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Water scarcity is a major constraint for increasing crop yields in many regions (Huang et al., 2002; Rosegrant and Cline, 2003). Projections for future climate changes indicate that water availability for crops in some regions may be decreased due to more infrequent rain events, lengthened intervals between rain events, and weather circulation patterns resulting in less rainfall during the crop growing season (Allen et al., 2010). Crops with traits that are generally associated with drought tolerance may become more important in such future climates. Hence, specific plant traits that enhance drought tolerance to water-deficits will be of interest to physiologists and plant breeders. Sorghum [Sorghum bicolor (L.) Moench], a C4 crop that diverged from maize (Zea mays L.) about 15 million years ago, is the fifth most important cereal crop grown worldwide (Doggett, 1988). It is known as a multifunctional crop and is especially important in the semiarid tropics because of its substantial tolerance of hot and dry environments. Sorghum has been identified as a key plant species as a source of beneficial genes for agriculture, particularly those
∗ Corresponding author. Tel.: +1 919 513 1620. E-mail addresses: [email protected], [email protected]fl.edu (T.R. Sinclair). 0378-4290/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2010.06.018
related to abiotic and biotic stress tolerance. Sorghum characteristics including relatively small genome [∼730 megabase] and the availability of its full sequence (Paterson et al., 2009), extraordinary diversity of germplasm (Djè et al., 2000; Kong et al., 2000) and incremental divergence from maize and rice (Oryza sativa L.; Doebley et al., 1990), make it ideally suited to aid in the identification of desired genes for dry conditions through comparative genomics. Conservation of soil water is one approach to enhance crop yields for late season water-deficit conditions (Passioura, 1972). Richards and Passioura (1989) approached this possibility by selecting wheat genotypes with smaller diameter xylem elements in the stem. While they found a yield increase with small-xylem germplasm under drought conditions, this approach was never pursued beyond the experimental stage. An alternate approach to water conservation proposed by Sinclair et al. (2005) is for plants to limit transpiration rate (TR) at a constant, maximum rate under high evaporative conditions. Their hypothesis was examined in a simulation study with sorghum to examine the putative benefits of restricted transpiration rate during high vapor pressure deficit (VPD) conditions during the midday. In the simulations, TR was limited to a maximum transpiration rate of 0.4 mm h−1 at four locations in Australia. This limitation resulted in average yield gains of about 5–7%, but more importantly yield gain was increased in
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65–78% of the seasons with the largest benefit coming in dry, lowyielding seasons. Sinclair et al. (2005) concluded that this trait would be economically desirable for sorghum growers in these locations. However, we were able to identify only one experimental study of sorghum gas exchange in response to VPD. Bunce (2003) measured stomata conductance of one sorghum genotype over several days on which VPD differed. His results showed little change in stomata conductance over the studied VPD range of 1.5–2.6 kPa. Recent evidence has shown that there are differences among genotypes in their VPD response such that at high VPD there was little or no further increases in TR with increasing VPD. This variability has been found within species comparing four soybean (Glycine max (L.) Merr.) genotypes (Fletcher et al., 2007), 17 peanut (Arachis hypogea L.) genotypes (Devi et al., 2010), and seven pearl millet (Pennisetum glaucum (L.) R.Br.) genotypes (Kholova et al., 2010). Fletcher et al. (2007) found the limited-TR trait in the soybean genotype PI 416927, which had been identified in the field as “slow-wilting”, at VPD > 2.1 kPa. Kholova et al. (2010) found that genotypes tolerant of terminal drought exhibited a decreased rate of TR increase at high VPD (>2.0 kPa). In the study with peanut (Devi et al., 2010), the threshold of VPD above which there was restricted TR varied from 2.0 to 2.6 kPa for nine genotypes. Sinclair et al. (2008) examined in greater detail the unique response of soybean genotype PI 416937 to increasing VPD. They found that the limitation on TR at higher VPD was linked to low hydraulic conductance at the leaf level, which was not observed in two genotypes that had no breakpoint (BP) in the TR response to VPD. Since the genetic background of this crop in which the twosegment TR response to VPD was limited to genotype PI 416937, Sadok and Sinclair (2009a) explored the existence of this trait in seven diverse genotypes: four lines that had PI 416937 in their pedigree and three lines did not. They found that there was a BP in two cultivars which were not derived from PI 416937, indicating more than one genetic source for this trait. In another study, Sadok and Sinclair (2009b) showed variation in BP among a recombinant inbred line population derived from PI 416937, and suggested that the variation in the BP could allow cultivars to be developed with specific BP for differing water-deficit environments. The objective of this study was to identify possible variation among sorghum genotypes for TR response to VPD. Specifically, the question was whether there are sorghum genotypes that exhibit a breakpoint in their response to increasing VPD and genotypes that do not. Twenty six genotypes were selected as initial candidates based on results from field screening. Transpiration rate of these selected lines was measured in controlled environments under a range of VPD.
2. Materials and methods 2.1. Field screen Sorghum genotypes were identified for measurement of the response of their transpiration rates to VPD based on field observations at Ashland Bottoms, Manhattan, KS (39◦ 13 N, 96◦ 62 W). In 2006, 297 genotypes were grown under rainfed conditions and the focus of the selection was on high-yielding genotypes under water-limited conditions. In addition, two stay-green genotypes were selected. All genotypes were sown in single rows 6-m long and spaced 0.75 m apart. There were two replications of each genotype. Standard crop management practices for rainfed conditions were adopted throughout the season. Grain yield was measured on all lines. At maturity, 2-m long rows were marked and the number of plants counted and panicles harvested. The panicles were dried at 40 ◦ C for 2 d and threshed to obtain seed yield. In 2007, the 297 genotypes were again grown in the same manner as 2006
except the field was irrigated to maintain well-watered conditions. In this second year, leaf temperature was measured using a handheld infrared temperature sensor (OS 534, Omega Engineering, Stamford, CT). The hypothesis was that those lines that had restricted transpiration rate under high VPD conditions at midday were likely to have higher leaf temperatures than other lines. Leaf temperature was measured at about 20 d intervals starting from booting through maturity on tagged plants. Data was collected on individual, top-most fully expanded leaves (mostly boot leaf). Measurements were made on a clear sunny day around noon when the VPD was approaching its daily maximum. 2.2. Controlled environment VPD response TR response to increasing VPD was measured using chambers that enclosed individual plants so that the VPD around the plants could be modified. The relative simple design of the measurement system allowed 12 chambers to be constructed so that 12 plants could be studied at one time. Since at least three replicate plants were studied at a time, three or four genotypes were included in each experiment. A total of seven experiments were performed to measure the TR response to VPD of all 26 sorghum genotypes identified in the field experiments. The plants were grown in pots constructed so that shoot chambers could be readily attached when VPD treatments were to be imposed. The pots were constructed from polyvinyl chloride pipe (0.1-m diameter and 0.32-m tall). They were filled with 1.8–2 kg of compost garden soil (Miracle-Gro Lawn Products, Inc., Marysville, OH) containing slow-release fertilizer (15% N–5% P–10% K). Five small holes were drilled in the center of the bottom end cap of the pot for drainage. A PVC toilet flange was glued to the top, open end of the pots to facilitate attachment of the VPD chamber. Four seeds were sown in each pot. The plants were grown in a greenhouse at Raleigh, NC (35◦ 47 N, 78◦ 39 W) with temperature regulated for a minimum temperature of 22 ◦ C and maximum temperature of 30 ◦ C. Pots were watered every 2–3 d. About 10 d after sowing, each pot was thinned to a single plant. Plants were grown for approximately 24 d until the projected canopy diameters of the plants were near the diameter of the VPD chambers for the TR measurements, i.e. 0.3 m. The day before TR measurements, 12 plants were placed in a walk-in growth chamber (31 ◦ C) and watered to dripping. Aluminum foil was placed over the soil and sealed around the stem of the plant to eliminate soil evaporation into the chamber. The foil combined with a positive air pressure on the foil minimized soil evaporation. The following morning the aerial parts of each plant were individually sealed into 0.38-m tall × 0.34-m diameter, 21-L clear plastic food containers (Cambro Manufacturing, Huntington Beach, CA). The containers were inverted over the plants and sealed to its lid which had been previously attached to the toilet flange on the pot. Each container was equipped with a 12 V, 76-mm diameter computer box fan (Northern Tool and Equipment, Burnsville, MN) to mix the air inside the chamber. A humidity/temperature sensor (Extech Instruments, League City, TX) was mounted through the side wall of each container. The photosynthetically active radiation in the chamber was constant at about 800 mole m−2 s−1 . Continuous flow of air through the VPD chamber ensured that no carbon dioxide deficit developed in th chambers. Three VPD ranges were targeted (0.5–1.5 kPa, 1.5–2.5 kPa, 2.5–3.5 kPa) for each set of plants on each day. Different VPD levels were established in the chambers by adjusting the air source and flow rate into the chambers. The lowest VPD in the chambers was established by varying input air flow rate between 0.4 and 0.8 L min−1 . The middle range of VPD was achieved by increasing the air flow rate to between 0.8 and 1.2 L min−1 . The highest VPD was obtained by first flowing air through a column of Drierite
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(W.A. Hammond Drierite Co., Xenia, OH) before the air entered the VPD chambers at 1.2–2.2 L min−1 . The VPD treatments were applied sequentially, from the lowest to the highest. Prior to data collection for each VPD treatment, the humidity was adjusted and the atmosphere in the containers was allowed to equilibrate for 25–30 min. The TR at each VPD was measured for 90 min. The measurement period for the low VPD treatment usually began at about 08:30 (standard time). Temperature and relative humidity were periodically recorded in each chamber to calculate the actual atmospheric VPD in each chamber during measurements. Since the chambers incorporated a fan to stir the air, previous tests showed the leaf temperature was approximately equal to air temperature (Sinclair et al., 2008), justifying the estimate of VPD based on air temperature. Once the atmosphere in the chambers was equilibrated, the entire unit of plant, container, and pot were weighed on a balance with a resolution of 0.1 g (Model SI-8001, Denver Instrument, Denver, CO). The loss in weight of each pot after a 90-min exposure was determined by again weighing the pots. The corresponding VPD values were calculated from the temperature and relative humidity data and averaged for the measurement period. On the second day, the entire sequence of measurements was repeated. Following TR measurements on the second day, leaves were removed from the plants and passed through an area meter (Model LI-3100, Licor, Lincoln). Transpiration rate for each plant was expressed per unit of leaf area. Rate of transpiration for each genotype was regressed against atmospheric VPD determined for each genotype. The data were first submitted to a two-segment linear regression analysis intersecting at a common value (Prism 2.01, GraphPad Software Inc., San Diego, CA). This method relied on an iterative procedure starting with initial conditions provided by the user. Initial values had to be given for slopes of the two linear segments and the breakpoint (BP) VPD between the two segments. The iteration tested whether the slopes of the two linear segments derived from linear regression were significantly different (P < 0.05). Convergence of the iterative algorithm, however, proved to be dependent on the scatter of the data in the region of the BP and on the initial BP value. Therefore, repeated segmented regression fittings were done on the data by providing initial BP values at 0.2 kPa increments over the range from 0.8 to 3 kPa. For a given genotype, the final regression parameters were those obtained as the same results from two to four analyses starting with successive initial BP values. Two linear regression equations for the successful regression fit to the two-segment model was the coefficients in the following two equations. A key output was the VPD value for BP where the two linear segments intersected. If VPD < BP,
TR = intercept1 + slope1 (VPD)
(1)
If VPD ≥ BP,
TR = intercept2 + slope2 (VPD)
(2)
Analysis of variance for leaf area was performed by using the SAS software (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Field screen A total of 13 genotypes were identified in the 2006 field study for intensive measurement of their TR response to VPD. Eight of these genotypes were selected based on a wide range in yield (Table 1). The yield among these genotypes ranged from 2741 kg ha−1 for SC532 to 5656 kg ha−1 for the cultivar RTX430. In addition to the eight selections based on yield, four genotypes were selected based on clear differences in their expression of the stay-green trait. Leaf temperature was measured in 2006 although these data were not used in selecting genotypes for the VPD test. Among the selected
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Table 1 Field observations on selected genotypes. Genotypes were selected under the rainfed conditions of 2006 based on high yield or on stay-green characteristics. Genotypes were selected under the irrigation conditions of 2007 based on measurements of leaf temperature. Air temperature during the canopy measurements was approximately 33 ◦ C. Genotype
Characteristic
Leaf temperature (◦ C)
Grain yield (kg ha−1 )
2006 (rainfed) SC532 SC489 SC630 SC299 SC982 BTXARG1 SC1345 RTX430 SC599 B35 TX3042 TX7078
High yield High yield High yield High yield High yield High yield High yield High yield Stay green rating Stay green rating Non-stay green Non-stay green
34.9 34.9 34.5 34.0 35.6 34.7 36.5 35.2 36.7
2741 2916 3675 3836 3903 4221 5189 5656 4447
2007 (irrigated) BTX378 BTX623 BTX2752 Macia BTX3197 SN149 SC1047 SC1019 SC1074 SC979 SC803 DK28 DK54
Low leaf temperature Low leaf temperature Low leaf temperature Low leaf temperature Low leaf temperature High leaf temperature High leaf temperature High leaf temperature High leaf temperature High leaf temperature High leaf temperature Hybrid Hybrid
32.8 32.8 32.9 33.3 33.4 35.0 36.8 36.8 36.9 37.1 37.1 32.9 33.8
5487 5993 5545 5516 5516 6393 5496 9065 5301 5189 5354 7040 7960
lines leaf temperature ranged from 33.4 to 36.7 ◦ C with the highest leaf temperature observed for the stay-green genotype SC599. The forage genotype Hegari was also selected because of its history of good performance under dryland conditions. The basis for selection of genotypes for the VPD test in 2007 was leaf temperature. Five genotypes were selected for exhibiting low leaf temperature, which is hypothetically indicative of fully open stomata (Table 1). Among these low-temperature lines, the observed average leaf temperatures were 32.8–33.4 ◦ C. Six genotypes were selected for high leaf temperatures which might result from partially closed stomata. Among these high-temperature genotypes, the average leaf temperature range was 35.0–37.1 ◦ C. In addition to the 11 genotypes selected based on leaf temperature, two high-yielding hybrids were identified for inclusion in the VPD test. 3.2. Controlled environment VPD response An effort was made to select uniform plants when thinning the pots, so that the leaf area of the plants was approximately constant within and across genotypes. An equivalent leaf was fairly well achieved within a genotype with a standard deviation for the three replicate plants being less than 22.2 cm2 plant−1 (Tables 2 and 3). Mean leaf area among the genotypes ranged from 257.7 cm2 plant−1 to 310.6 cm2 plant−1 (Tables 2 and 3). Across all experiments and genotypes, the average temperature of the VPD chambers was 30.5 ◦ C. Variation in VPD was induced in the chambers mainly by alteration of humidity levels as a result of differing air sources and flow rates. The desired levels of VPD were obtained for each genotype ranging from about 0.5 to about 3.9 kPa. There was no observable difference in the TR response to VPD between the 2 days of measurements of each genotype. As illustrated in Fig. 1, no significant differences were found within any genotype in the regression results for the TR response to VPD when
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Table 2 Leaf area (LA; cm2 plant−1 ) and regression results [breakpoint and slopes of two linear regression segments (mg H2 O m−2 s−1 kPa−1 ) and R2 ] for 17 genotypes that were found to fit the two-segment regression model. Genotype
LA
SC982 SC1074 BTX623 SC979 BTX2752 SC1019 SC630 SC599 SC1047 SC803 BTX3197 SN149 Macia BTX378 B35 TX3042 TX7078
Breakpoint
Slope1
R2
Slope2
Value
S.D.
value
S.E.
Value
S.E.
Value
S.E.
295 280 265 290 288 261 277 286 258 267 280 294 270 268 265 278 300
20.6 17.7 10.0 14.6 13.0 13.7 15.2 15.1 14.8 12.8 13.2 17.4 12.6 16.0 22.2 13.8 17.4
1.62 1.99 2.05 2.06 2.08 2.08 2.16 2.23 2.24 2.29 2.29 2.30 2.51 2.55 2.61 2.69 2.72
0.23 0.17 0.18 0.18 0.20 0.25 0.31 0.27 0.23 0.19 0.29 0.19 0.27 0.39 0.18 0.23 0.23
50.0 45.8 56.3 47.7 52.0 43.8 40.1 35.5 32.2 39.8 38.8 38.2 35.7 30.2 40.3 33.5 37.7
12.6 9.5 10.7 7.8 9.0 8.1 9.7 7.0 5.6 6.5 6.5 5.2 3.8 3.3 6.8 4.6 6.0
6.3 1.0 10.4 0 11 1.3 −1.5 3.4 1.1 −6.1 3.2 −1.8 4.0 11.3 2.4 −0.5 1.4
4.8 4.1 5.2 6.5 6.7 10.1 8.2 8.4 6.5 6.8 9.2 8.8 12.4 11.0 7.7 11.4 10.2
the data for the 2 days were analyzed independently. Hence, the data collected in the 2 days for each genotype were combined. In fact, this consistency in results allowed adjustment in the VPD treatments if needed on the second day to gain the desired distribution of VPD treatments. Fig. 1 shows that the TR response to VPD clearly diverged among genotypes. Among the 26 genotypes, 17 were found to exhibit a BP in the increase in transpiration rate as VPD was increased (Table 2). The value for the BP ranged from 1.6 to 2.7 kPa, with an average of 2.26 kPa for the 17 genotypes. The lowest BP was found in SC982. The genotypes TX7078, TX3042, B35, BTX378 and Macia had the highest BP. The R2 for the two-segment regression for each of these 17 genotypes ranged from 0.74 to 0.91 (Table 2). The slope of the linear regression below the BP, i.e. slope1, ranged from 30.2 to 56.3 mg H2 O m−2 s−1 kPa−1 with an average 41.0 mg H2 O m−2 s−1 kPa−1 for the 17 genotypes that exhibited a BP. There was significant (P < 0.01) inverse relation between BP and initial slope (Fig. 2), although there was considerable scatter in the data (R2 = 0.49). The slope of the TR response to VPD above the BP (i.e. slope2) was much less than slope1. The value of slope2 varied a great deal among genotypes from −6.1 to 11.3 mg H2 O m−2 s−1 kPa−1 . The average of slope2 for all 17 genotypes was 2.8 mg H2 O m−2 s−1 kPa−1 . The distribution between negative and positive slopes was four negative and 13 positive slopes. There was no association between the value of the BP and slope2. Data for nine genotypes were found not to fit the two-segment model, and hence, a single linear regression generally fit these data well with the R2 ranging from 0.81 to 0.90 (Table 3). The slope of these genotypes, i.e. with single-linear TR response,
0.79 0.84 0.9 0.84 0.89 0.81 0.74 0.8 0.84 0.84 0.85 0.87 0.91 0.91 0.88 0.89 0.88
ranged from 23.1 to 28.6 mg H2 O m−2 s−1 kPa−1 with an average 25.8 mg H2 O m−2 s−1 kPa−1 for these nine genotypes. Interestingly, the average slope of these nine genotypes is 63% of slope1 of the 17 genotypes exhibiting a BP.
Table 3 Leaf area (LA; cm2 plant−1 ) and regression results [slope (mg H2 O m−2 s−1 kPa−1 ) and R2 ] for 9 genotypes that were found to fit a linear regression model. Genotype
DK28 BTXARG-1 SC1345 Hegari DK54 RTX430 SC489 SC532 SC299
LA
R2
Slope
Value
S.D.
Value
S.E.
311 277 308 280 300 289 298 283 283
14.7 22.4 14.3 14.9 20.4 19.7 15.6 13.9 14.1
28.6 27.5 27.5 26.3 25.4 25.4 24.8 23.9 23.1
2.5 2.4 3.0 2.8 2.9 2.8 2.8 2.8 2.3
0.89 0.90 0.84 0.84 0.83 0.87 0.83 0.81 0.86
Fig. 1. Transpiration rate response of three genotypes to increasing vapor pressure deficit as three general patterns of response that was found for the tested sorghum genotypes. The two symbols indicate data recorded on 2 consecutive days of observations. The detail of regression information was presented in Tables 2 and 3.
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Fig. 2. The relation between BP and slope of regression line at low VPD before the BP.
4. Discussion In contrast to the usual model of transpiration rate increasing linearly with increasing VPD (e.g., Sinclair and Bennett, 1998), this study with 26 sorghum genotypes showed a number of genotypes that deviated for the linear response of TR to VPD. Only approximately one-third of the studied genotypes had continuously increasing transpiration rate with increasing VPD. Two-thirds of the genotypes had a clear BP in transpiration rate at VPD from 1.6 to 2.7 kPa. The BP observed in these 17 sorghum genotypes is similar to the VPD for the BP of genotypes in other species in which a BP has been reported (Comstock and Ehleringer, 1993; Fletcher et al., 2007; Sinclair et al., 2008; Sadok and Sinclair, 2009a,b). No clear value in identifying sorghum genotypes in the two field experiments that might exhibit the BP with VPD increase was found. Of the eight genotypes selected in 2006 for high yield under water-limited conditions, only two exhibited a BP although SC982 had the lowest BP of any studied genotype. These results indicate that yield itself does not appear to be a sufficient criterion for identifying limited transpiration rate at high VPD. Interesting, all four of the genotypes selected in 2006 for difference in the stay-green trait exhibited a BP. All 11 lines selected in 2007 based on leaf temperature measurements exhibited a BP. That is, the measured leaf temperature differences in the field did not result in a segregation of genotypes for response to VPD. Many plant factors can contribute to elevated leaf temperature such as background stomata conductance, leaf size, and canopy architecture. Single-row plots in the field screen may have also influenced the expression of leaf temperature differences. In any event, it appears the influence of these factors overwhelmed a clear leaf temperature difference as a result of VPD response under field conditions. Only the two hybrids with high yield in the irrigated conditions in 2007 exhibited no BP. Sinclair et al. (2008) interpreted the BP in soybean PI 416937 as a response to a limiting hydraulic conductance in the leaf that constrains the flow of water from the xylem into the guard cells. A recent study of Sadok and Sinclair (2010a) indicated that existence of BP in soybean may be linked to limited leaf hydraulic conductance associated with the apparent lack of an AgNO3 sensitive leaf aquaporin population. This trait accounted for 25–50% of the decrease in TR at high VPD (Sadok and Sinclair, 2010b). Their suggestion is that in the absence of the silver-sensitive population of aquaporins in soybean leaves water flow can be restricted causing the plant to have stable TR at high VDP, i.e. a BP (King et al., 2009). Parent et al. (2009) studied transgenic lines of maize that expressed antisense of one of the NCED genes resulting in low abscisic acid concentrations in the xylem sap. The transgenic lines with low abscisic acid had restricted hydraulic conductance in the roots and this resulted in lower plant transpiration rates. Low
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hydraulic conductance was associated with decreased gene expression and protein content measured for PIP aquaporin isoforms. It appeared that posttranscriptional mechanisms regulated the amount of PIP proteins that influenced root hydraulic conductance. It is unknown whether the mechanism for expression of a BP at high VPD identified in this study exists in the leaves or the roots. One interesting possibility is that in those genotypes with especially low BP, e.g. SC982, there might be limiting hydraulic conductance in both leaves and roots (Sadok and Sinclair, 2010b). Further, research with these lines is needed to quantify and to resolve the site of the hydraulic conductance limitation in these sorghum genotypes. Among these sorghum genotypes exhibiting a BP, there was a significant negative relationship between the value of the BP and the linear slope at low VPD (i.e. slope1) (Fig. 2). This is an interesting relationship in that it indicates that plants that can transport large rates of water in the plant at low VPD reach the maximum transpiration rate more rapidly as VPD increases, i.e. a low BP. Of course, this hypothesis based on differences in hydraulic conductance in the plants cannot be resolved without identifying and measuring hydraulic conductance in these genotypes and its possible limitation on water flux in the plant. The maximum TR of the genotypes, however, differs so that this relationship is not rigidly limited by the same maximum transpiration rate across all genotypes. These differences offer the opportunity to develop genotypes with the appropriate BP and maximum transpiration for each geographical location. The genotypic variability identified in this study offers candidate parental lines for breeding programs seeking to improve sorghum yields under water-deficit conditions. Those lines without a BP and low rate of increase in TR with VPD might be well suited for consistently dry environmental conditions. However, these genotypes would also have restricted CO2 assimilation under these conditions so that such genotypes are likely to be slow growing, and probably low yielding under well-watered conditions. Genotypes with a high initial slope and a low BP, e.g. SC982, may offer a better approach to developing drought tolerance. These lines maximize gas exchange under low VPD and then shift to water conservation at high VPD. This is the water-conservation strategy currently being pursued with pearl millet (Kholova et al., 2010) for terminal drought conditions. Selection of the BP could be matched with the likelihood of water-deficit conditions. A low BP would give the greatest water conservation when soil water is still available while a high BP imposes less-restrictive water conservation. The results of this study clearly indicate sorghum genotypes could be selected to pursue soil water conservation as a way to improve yields under water-deficit conditions. Acknowledgments The authors gratefully acknowledge the financial support of the Kansas Grain Sorghum Commission. Contribution no. 10-313-J from Kansas Agricultural Experiment Station. References Allen, C.D., Macalady, A.K., Chenchouni, H., et al., 2010. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. Forest Ecol. Manage. 259, 660–684. Bunce, J.A., 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 Res. 82, 37–47. Comstock, J., Ehleringer, J., 1993. Stomatal response to humidity in common bean (Phaseolus vulgaris): implications for maximum transpiration rate, water-use efficiency and productivity. Aust. J. Plant Physiol. 20, 669–691. Devi, M.J., Sinclair, T.R., Vadez, V., 2010. Genotypic variation in peanut for transpiration response to vapor pressure deficit. Crop Sci. 50, 191–196. Djè, Y., Heuertz, M., Lefèbvre, C., Vekemans, X., 2000. Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers. Theor. Appl. Genet. 100, 918–925.
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Paterson, A.H., Bowers, J.E., Bruggmann, R., Dubchak, I., Grimwood, J., et al., 2009. The Sorghum bicolor genome and the diversification of grasses. Nature 457, 551–556. Richards, R.A., Passioura, J.B., 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. Aust. J. Agric. Res. 40, 943–950. Rosegrant, M., Cline, S., 2003. Global food security: challenges and policies. Science 302, 1917–1919. Sadok, W., Sinclair, T.R., 2009a. Genetic variability of transpiration response to vapor pressure deficit among soybean cultivars. Crop Sci. 49, 955–960. Sadok, W., Sinclair, T.R., 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 Res. 113, 156–160. Sadok, W., Sinclair, T.R., 2010a. Transpiration response of ‘slow-wilting’ and commercial soybean (Glycine max (L.) Merr.) genotypes to three aquaporin inhibitors. J. Exp. Bot. 61, 821–829. Sadok, W., Sinclair, T.R., 2010b. Genetic variability of transpiration response to AgNO3 aquaporin inhibitor. Crop Sci. 50, 1423–1430. Sinclair, T.R., Bennett, J.M., 1998. Water. In: Sinclair, T.R., Gardner, F.P. (Eds.), Principles of Ecology in Plant Production. CAB Int., Wallingford, UK. Sinclair, T.R., Hammer, G.L., van Oosterom, E.J., 2005. Potential yield and water-use efficiency benefits in sorghum from limited maximum transpiration rate. Funct. Plant Biol. 32, 945–952. Sinclair, T.R., Zwieniecki, M.A., Holbrook, N.M., 2008. Low leaf hydraulic conductance associated with drought tolerance in soybean. Physiol. Plant. 132, 446–451.
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Field Crops Research journal homepage: www.elsevier.com/locate/fcr
Genetic variability of transpiration response to vapor pressure deficit among sorghum genotypes Manoochehr Gholipoor a , P.V. Vara Prasad b , Raymond N. Mutava b , Thomas R. Sinclair c,∗ a b c
Department of Agronomy and Plant Breeding, Shahrood University of Technology, P.O. Box 36155-316, Shahrood, Iran Department of Agronomy, Kansas State University, Manhattan, KS 66506-5501, USA Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA
a r t i c l e
i n f o
Article history: Received 20 April 2010 Received in revised form 24 June 2010 Accepted 28 June 2010 Keywords: Drought stress Genotypic variation Sorghum Transpiration Vapor pressure deficit
a b s t r a c t Simulation studies have demonstrated that limited maximum transpiration rate (TR) at high air vapor pressure deficit (VPD) in water-limited environments could result in significant increases in sorghum yield. However, such a restriction on TR at high VPD has not been documented in sorghum. The objective of this study was to search within sorghum germplasm for the possibility of restricted TR at high VPD. Twenty six genotypes were selected for measurement of VPD response based on field observations including yield, leaf temperature, and the stay-green phenotype. These genotypes were grown in a greenhouse for about 24-d growth, and then placed into individual chambers in which VPD was varied and TR measured. The results of this study showed marked variation among sorghum genotypes in TR response to VPD. Seventeen genotypes were identified as exhibiting a breakpoint in their VPD response in the range from 1.6 to 2.7 kPa, above which there was little or no further increase in TR. Therefore, these genotypes with a breakpoint have the possibility of soil water conservation when VPD during the midday cycle exceeds the breakpoint VPD. This trait would be desirable in less humid environments for increasing yields in water-deficit seasons. The observed range in the value of the BP among genotypes offers the possibility of developing genotypes with BP appropriate for specific environments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Water scarcity is a major constraint for increasing crop yields in many regions (Huang et al., 2002; Rosegrant and Cline, 2003). Projections for future climate changes indicate that water availability for crops in some regions may be decreased due to more infrequent rain events, lengthened intervals between rain events, and weather circulation patterns resulting in less rainfall during the crop growing season (Allen et al., 2010). Crops with traits that are generally associated with drought tolerance may become more important in such future climates. Hence, specific plant traits that enhance drought tolerance to water-deficits will be of interest to physiologists and plant breeders. Sorghum [Sorghum bicolor (L.) Moench], a C4 crop that diverged from maize (Zea mays L.) about 15 million years ago, is the fifth most important cereal crop grown worldwide (Doggett, 1988). It is known as a multifunctional crop and is especially important in the semiarid tropics because of its substantial tolerance of hot and dry environments. Sorghum has been identified as a key plant species as a source of beneficial genes for agriculture, particularly those
∗ Corresponding author. Tel.: +1 919 513 1620. E-mail addresses: [email protected], [email protected]fl.edu (T.R. Sinclair). 0378-4290/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2010.06.018
related to abiotic and biotic stress tolerance. Sorghum characteristics including relatively small genome [∼730 megabase] and the availability of its full sequence (Paterson et al., 2009), extraordinary diversity of germplasm (Djè et al., 2000; Kong et al., 2000) and incremental divergence from maize and rice (Oryza sativa L.; Doebley et al., 1990), make it ideally suited to aid in the identification of desired genes for dry conditions through comparative genomics. Conservation of soil water is one approach to enhance crop yields for late season water-deficit conditions (Passioura, 1972). Richards and Passioura (1989) approached this possibility by selecting wheat genotypes with smaller diameter xylem elements in the stem. While they found a yield increase with small-xylem germplasm under drought conditions, this approach was never pursued beyond the experimental stage. An alternate approach to water conservation proposed by Sinclair et al. (2005) is for plants to limit transpiration rate (TR) at a constant, maximum rate under high evaporative conditions. Their hypothesis was examined in a simulation study with sorghum to examine the putative benefits of restricted transpiration rate during high vapor pressure deficit (VPD) conditions during the midday. In the simulations, TR was limited to a maximum transpiration rate of 0.4 mm h−1 at four locations in Australia. This limitation resulted in average yield gains of about 5–7%, but more importantly yield gain was increased in
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65–78% of the seasons with the largest benefit coming in dry, lowyielding seasons. Sinclair et al. (2005) concluded that this trait would be economically desirable for sorghum growers in these locations. However, we were able to identify only one experimental study of sorghum gas exchange in response to VPD. Bunce (2003) measured stomata conductance of one sorghum genotype over several days on which VPD differed. His results showed little change in stomata conductance over the studied VPD range of 1.5–2.6 kPa. Recent evidence has shown that there are differences among genotypes in their VPD response such that at high VPD there was little or no further increases in TR with increasing VPD. This variability has been found within species comparing four soybean (Glycine max (L.) Merr.) genotypes (Fletcher et al., 2007), 17 peanut (Arachis hypogea L.) genotypes (Devi et al., 2010), and seven pearl millet (Pennisetum glaucum (L.) R.Br.) genotypes (Kholova et al., 2010). Fletcher et al. (2007) found the limited-TR trait in the soybean genotype PI 416927, which had been identified in the field as “slow-wilting”, at VPD > 2.1 kPa. Kholova et al. (2010) found that genotypes tolerant of terminal drought exhibited a decreased rate of TR increase at high VPD (>2.0 kPa). In the study with peanut (Devi et al., 2010), the threshold of VPD above which there was restricted TR varied from 2.0 to 2.6 kPa for nine genotypes. Sinclair et al. (2008) examined in greater detail the unique response of soybean genotype PI 416937 to increasing VPD. They found that the limitation on TR at higher VPD was linked to low hydraulic conductance at the leaf level, which was not observed in two genotypes that had no breakpoint (BP) in the TR response to VPD. Since the genetic background of this crop in which the twosegment TR response to VPD was limited to genotype PI 416937, Sadok and Sinclair (2009a) explored the existence of this trait in seven diverse genotypes: four lines that had PI 416937 in their pedigree and three lines did not. They found that there was a BP in two cultivars which were not derived from PI 416937, indicating more than one genetic source for this trait. In another study, Sadok and Sinclair (2009b) showed variation in BP among a recombinant inbred line population derived from PI 416937, and suggested that the variation in the BP could allow cultivars to be developed with specific BP for differing water-deficit environments. The objective of this study was to identify possible variation among sorghum genotypes for TR response to VPD. Specifically, the question was whether there are sorghum genotypes that exhibit a breakpoint in their response to increasing VPD and genotypes that do not. Twenty six genotypes were selected as initial candidates based on results from field screening. Transpiration rate of these selected lines was measured in controlled environments under a range of VPD.
2. Materials and methods 2.1. Field screen Sorghum genotypes were identified for measurement of the response of their transpiration rates to VPD based on field observations at Ashland Bottoms, Manhattan, KS (39◦ 13 N, 96◦ 62 W). In 2006, 297 genotypes were grown under rainfed conditions and the focus of the selection was on high-yielding genotypes under water-limited conditions. In addition, two stay-green genotypes were selected. All genotypes were sown in single rows 6-m long and spaced 0.75 m apart. There were two replications of each genotype. Standard crop management practices for rainfed conditions were adopted throughout the season. Grain yield was measured on all lines. At maturity, 2-m long rows were marked and the number of plants counted and panicles harvested. The panicles were dried at 40 ◦ C for 2 d and threshed to obtain seed yield. In 2007, the 297 genotypes were again grown in the same manner as 2006
except the field was irrigated to maintain well-watered conditions. In this second year, leaf temperature was measured using a handheld infrared temperature sensor (OS 534, Omega Engineering, Stamford, CT). The hypothesis was that those lines that had restricted transpiration rate under high VPD conditions at midday were likely to have higher leaf temperatures than other lines. Leaf temperature was measured at about 20 d intervals starting from booting through maturity on tagged plants. Data was collected on individual, top-most fully expanded leaves (mostly boot leaf). Measurements were made on a clear sunny day around noon when the VPD was approaching its daily maximum. 2.2. Controlled environment VPD response TR response to increasing VPD was measured using chambers that enclosed individual plants so that the VPD around the plants could be modified. The relative simple design of the measurement system allowed 12 chambers to be constructed so that 12 plants could be studied at one time. Since at least three replicate plants were studied at a time, three or four genotypes were included in each experiment. A total of seven experiments were performed to measure the TR response to VPD of all 26 sorghum genotypes identified in the field experiments. The plants were grown in pots constructed so that shoot chambers could be readily attached when VPD treatments were to be imposed. The pots were constructed from polyvinyl chloride pipe (0.1-m diameter and 0.32-m tall). They were filled with 1.8–2 kg of compost garden soil (Miracle-Gro Lawn Products, Inc., Marysville, OH) containing slow-release fertilizer (15% N–5% P–10% K). Five small holes were drilled in the center of the bottom end cap of the pot for drainage. A PVC toilet flange was glued to the top, open end of the pots to facilitate attachment of the VPD chamber. Four seeds were sown in each pot. The plants were grown in a greenhouse at Raleigh, NC (35◦ 47 N, 78◦ 39 W) with temperature regulated for a minimum temperature of 22 ◦ C and maximum temperature of 30 ◦ C. Pots were watered every 2–3 d. About 10 d after sowing, each pot was thinned to a single plant. Plants were grown for approximately 24 d until the projected canopy diameters of the plants were near the diameter of the VPD chambers for the TR measurements, i.e. 0.3 m. The day before TR measurements, 12 plants were placed in a walk-in growth chamber (31 ◦ C) and watered to dripping. Aluminum foil was placed over the soil and sealed around the stem of the plant to eliminate soil evaporation into the chamber. The foil combined with a positive air pressure on the foil minimized soil evaporation. The following morning the aerial parts of each plant were individually sealed into 0.38-m tall × 0.34-m diameter, 21-L clear plastic food containers (Cambro Manufacturing, Huntington Beach, CA). The containers were inverted over the plants and sealed to its lid which had been previously attached to the toilet flange on the pot. Each container was equipped with a 12 V, 76-mm diameter computer box fan (Northern Tool and Equipment, Burnsville, MN) to mix the air inside the chamber. A humidity/temperature sensor (Extech Instruments, League City, TX) was mounted through the side wall of each container. The photosynthetically active radiation in the chamber was constant at about 800 mole m−2 s−1 . Continuous flow of air through the VPD chamber ensured that no carbon dioxide deficit developed in th chambers. Three VPD ranges were targeted (0.5–1.5 kPa, 1.5–2.5 kPa, 2.5–3.5 kPa) for each set of plants on each day. Different VPD levels were established in the chambers by adjusting the air source and flow rate into the chambers. The lowest VPD in the chambers was established by varying input air flow rate between 0.4 and 0.8 L min−1 . The middle range of VPD was achieved by increasing the air flow rate to between 0.8 and 1.2 L min−1 . The highest VPD was obtained by first flowing air through a column of Drierite
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(W.A. Hammond Drierite Co., Xenia, OH) before the air entered the VPD chambers at 1.2–2.2 L min−1 . The VPD treatments were applied sequentially, from the lowest to the highest. Prior to data collection for each VPD treatment, the humidity was adjusted and the atmosphere in the containers was allowed to equilibrate for 25–30 min. The TR at each VPD was measured for 90 min. The measurement period for the low VPD treatment usually began at about 08:30 (standard time). Temperature and relative humidity were periodically recorded in each chamber to calculate the actual atmospheric VPD in each chamber during measurements. Since the chambers incorporated a fan to stir the air, previous tests showed the leaf temperature was approximately equal to air temperature (Sinclair et al., 2008), justifying the estimate of VPD based on air temperature. Once the atmosphere in the chambers was equilibrated, the entire unit of plant, container, and pot were weighed on a balance with a resolution of 0.1 g (Model SI-8001, Denver Instrument, Denver, CO). The loss in weight of each pot after a 90-min exposure was determined by again weighing the pots. The corresponding VPD values were calculated from the temperature and relative humidity data and averaged for the measurement period. On the second day, the entire sequence of measurements was repeated. Following TR measurements on the second day, leaves were removed from the plants and passed through an area meter (Model LI-3100, Licor, Lincoln). Transpiration rate for each plant was expressed per unit of leaf area. Rate of transpiration for each genotype was regressed against atmospheric VPD determined for each genotype. The data were first submitted to a two-segment linear regression analysis intersecting at a common value (Prism 2.01, GraphPad Software Inc., San Diego, CA). This method relied on an iterative procedure starting with initial conditions provided by the user. Initial values had to be given for slopes of the two linear segments and the breakpoint (BP) VPD between the two segments. The iteration tested whether the slopes of the two linear segments derived from linear regression were significantly different (P < 0.05). Convergence of the iterative algorithm, however, proved to be dependent on the scatter of the data in the region of the BP and on the initial BP value. Therefore, repeated segmented regression fittings were done on the data by providing initial BP values at 0.2 kPa increments over the range from 0.8 to 3 kPa. For a given genotype, the final regression parameters were those obtained as the same results from two to four analyses starting with successive initial BP values. Two linear regression equations for the successful regression fit to the two-segment model was the coefficients in the following two equations. A key output was the VPD value for BP where the two linear segments intersected. If VPD < BP,
TR = intercept1 + slope1 (VPD)
(1)
If VPD ≥ BP,
TR = intercept2 + slope2 (VPD)
(2)
Analysis of variance for leaf area was performed by using the SAS software (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Field screen A total of 13 genotypes were identified in the 2006 field study for intensive measurement of their TR response to VPD. Eight of these genotypes were selected based on a wide range in yield (Table 1). The yield among these genotypes ranged from 2741 kg ha−1 for SC532 to 5656 kg ha−1 for the cultivar RTX430. In addition to the eight selections based on yield, four genotypes were selected based on clear differences in their expression of the stay-green trait. Leaf temperature was measured in 2006 although these data were not used in selecting genotypes for the VPD test. Among the selected
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Table 1 Field observations on selected genotypes. Genotypes were selected under the rainfed conditions of 2006 based on high yield or on stay-green characteristics. Genotypes were selected under the irrigation conditions of 2007 based on measurements of leaf temperature. Air temperature during the canopy measurements was approximately 33 ◦ C. Genotype
Characteristic
Leaf temperature (◦ C)
Grain yield (kg ha−1 )
2006 (rainfed) SC532 SC489 SC630 SC299 SC982 BTXARG1 SC1345 RTX430 SC599 B35 TX3042 TX7078
High yield High yield High yield High yield High yield High yield High yield High yield Stay green rating Stay green rating Non-stay green Non-stay green
34.9 34.9 34.5 34.0 35.6 34.7 36.5 35.2 36.7
2741 2916 3675 3836 3903 4221 5189 5656 4447
2007 (irrigated) BTX378 BTX623 BTX2752 Macia BTX3197 SN149 SC1047 SC1019 SC1074 SC979 SC803 DK28 DK54
Low leaf temperature Low leaf temperature Low leaf temperature Low leaf temperature Low leaf temperature High leaf temperature High leaf temperature High leaf temperature High leaf temperature High leaf temperature High leaf temperature Hybrid Hybrid
32.8 32.8 32.9 33.3 33.4 35.0 36.8 36.8 36.9 37.1 37.1 32.9 33.8
5487 5993 5545 5516 5516 6393 5496 9065 5301 5189 5354 7040 7960
lines leaf temperature ranged from 33.4 to 36.7 ◦ C with the highest leaf temperature observed for the stay-green genotype SC599. The forage genotype Hegari was also selected because of its history of good performance under dryland conditions. The basis for selection of genotypes for the VPD test in 2007 was leaf temperature. Five genotypes were selected for exhibiting low leaf temperature, which is hypothetically indicative of fully open stomata (Table 1). Among these low-temperature lines, the observed average leaf temperatures were 32.8–33.4 ◦ C. Six genotypes were selected for high leaf temperatures which might result from partially closed stomata. Among these high-temperature genotypes, the average leaf temperature range was 35.0–37.1 ◦ C. In addition to the 11 genotypes selected based on leaf temperature, two high-yielding hybrids were identified for inclusion in the VPD test. 3.2. Controlled environment VPD response An effort was made to select uniform plants when thinning the pots, so that the leaf area of the plants was approximately constant within and across genotypes. An equivalent leaf was fairly well achieved within a genotype with a standard deviation for the three replicate plants being less than 22.2 cm2 plant−1 (Tables 2 and 3). Mean leaf area among the genotypes ranged from 257.7 cm2 plant−1 to 310.6 cm2 plant−1 (Tables 2 and 3). Across all experiments and genotypes, the average temperature of the VPD chambers was 30.5 ◦ C. Variation in VPD was induced in the chambers mainly by alteration of humidity levels as a result of differing air sources and flow rates. The desired levels of VPD were obtained for each genotype ranging from about 0.5 to about 3.9 kPa. There was no observable difference in the TR response to VPD between the 2 days of measurements of each genotype. As illustrated in Fig. 1, no significant differences were found within any genotype in the regression results for the TR response to VPD when
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Table 2 Leaf area (LA; cm2 plant−1 ) and regression results [breakpoint and slopes of two linear regression segments (mg H2 O m−2 s−1 kPa−1 ) and R2 ] for 17 genotypes that were found to fit the two-segment regression model. Genotype
LA
SC982 SC1074 BTX623 SC979 BTX2752 SC1019 SC630 SC599 SC1047 SC803 BTX3197 SN149 Macia BTX378 B35 TX3042 TX7078
Breakpoint
Slope1
R2
Slope2
Value
S.D.
value
S.E.
Value
S.E.
Value
S.E.
295 280 265 290 288 261 277 286 258 267 280 294 270 268 265 278 300
20.6 17.7 10.0 14.6 13.0 13.7 15.2 15.1 14.8 12.8 13.2 17.4 12.6 16.0 22.2 13.8 17.4
1.62 1.99 2.05 2.06 2.08 2.08 2.16 2.23 2.24 2.29 2.29 2.30 2.51 2.55 2.61 2.69 2.72
0.23 0.17 0.18 0.18 0.20 0.25 0.31 0.27 0.23 0.19 0.29 0.19 0.27 0.39 0.18 0.23 0.23
50.0 45.8 56.3 47.7 52.0 43.8 40.1 35.5 32.2 39.8 38.8 38.2 35.7 30.2 40.3 33.5 37.7
12.6 9.5 10.7 7.8 9.0 8.1 9.7 7.0 5.6 6.5 6.5 5.2 3.8 3.3 6.8 4.6 6.0
6.3 1.0 10.4 0 11 1.3 −1.5 3.4 1.1 −6.1 3.2 −1.8 4.0 11.3 2.4 −0.5 1.4
4.8 4.1 5.2 6.5 6.7 10.1 8.2 8.4 6.5 6.8 9.2 8.8 12.4 11.0 7.7 11.4 10.2
the data for the 2 days were analyzed independently. Hence, the data collected in the 2 days for each genotype were combined. In fact, this consistency in results allowed adjustment in the VPD treatments if needed on the second day to gain the desired distribution of VPD treatments. Fig. 1 shows that the TR response to VPD clearly diverged among genotypes. Among the 26 genotypes, 17 were found to exhibit a BP in the increase in transpiration rate as VPD was increased (Table 2). The value for the BP ranged from 1.6 to 2.7 kPa, with an average of 2.26 kPa for the 17 genotypes. The lowest BP was found in SC982. The genotypes TX7078, TX3042, B35, BTX378 and Macia had the highest BP. The R2 for the two-segment regression for each of these 17 genotypes ranged from 0.74 to 0.91 (Table 2). The slope of the linear regression below the BP, i.e. slope1, ranged from 30.2 to 56.3 mg H2 O m−2 s−1 kPa−1 with an average 41.0 mg H2 O m−2 s−1 kPa−1 for the 17 genotypes that exhibited a BP. There was significant (P < 0.01) inverse relation between BP and initial slope (Fig. 2), although there was considerable scatter in the data (R2 = 0.49). The slope of the TR response to VPD above the BP (i.e. slope2) was much less than slope1. The value of slope2 varied a great deal among genotypes from −6.1 to 11.3 mg H2 O m−2 s−1 kPa−1 . The average of slope2 for all 17 genotypes was 2.8 mg H2 O m−2 s−1 kPa−1 . The distribution between negative and positive slopes was four negative and 13 positive slopes. There was no association between the value of the BP and slope2. Data for nine genotypes were found not to fit the two-segment model, and hence, a single linear regression generally fit these data well with the R2 ranging from 0.81 to 0.90 (Table 3). The slope of these genotypes, i.e. with single-linear TR response,
0.79 0.84 0.9 0.84 0.89 0.81 0.74 0.8 0.84 0.84 0.85 0.87 0.91 0.91 0.88 0.89 0.88
ranged from 23.1 to 28.6 mg H2 O m−2 s−1 kPa−1 with an average 25.8 mg H2 O m−2 s−1 kPa−1 for these nine genotypes. Interestingly, the average slope of these nine genotypes is 63% of slope1 of the 17 genotypes exhibiting a BP.
Table 3 Leaf area (LA; cm2 plant−1 ) and regression results [slope (mg H2 O m−2 s−1 kPa−1 ) and R2 ] for 9 genotypes that were found to fit a linear regression model. Genotype
DK28 BTXARG-1 SC1345 Hegari DK54 RTX430 SC489 SC532 SC299
LA
R2
Slope
Value
S.D.
Value
S.E.
311 277 308 280 300 289 298 283 283
14.7 22.4 14.3 14.9 20.4 19.7 15.6 13.9 14.1
28.6 27.5 27.5 26.3 25.4 25.4 24.8 23.9 23.1
2.5 2.4 3.0 2.8 2.9 2.8 2.8 2.8 2.3
0.89 0.90 0.84 0.84 0.83 0.87 0.83 0.81 0.86
Fig. 1. Transpiration rate response of three genotypes to increasing vapor pressure deficit as three general patterns of response that was found for the tested sorghum genotypes. The two symbols indicate data recorded on 2 consecutive days of observations. The detail of regression information was presented in Tables 2 and 3.
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Fig. 2. The relation between BP and slope of regression line at low VPD before the BP.
4. Discussion In contrast to the usual model of transpiration rate increasing linearly with increasing VPD (e.g., Sinclair and Bennett, 1998), this study with 26 sorghum genotypes showed a number of genotypes that deviated for the linear response of TR to VPD. Only approximately one-third of the studied genotypes had continuously increasing transpiration rate with increasing VPD. Two-thirds of the genotypes had a clear BP in transpiration rate at VPD from 1.6 to 2.7 kPa. The BP observed in these 17 sorghum genotypes is similar to the VPD for the BP of genotypes in other species in which a BP has been reported (Comstock and Ehleringer, 1993; Fletcher et al., 2007; Sinclair et al., 2008; Sadok and Sinclair, 2009a,b). No clear value in identifying sorghum genotypes in the two field experiments that might exhibit the BP with VPD increase was found. Of the eight genotypes selected in 2006 for high yield under water-limited conditions, only two exhibited a BP although SC982 had the lowest BP of any studied genotype. These results indicate that yield itself does not appear to be a sufficient criterion for identifying limited transpiration rate at high VPD. Interesting, all four of the genotypes selected in 2006 for difference in the stay-green trait exhibited a BP. All 11 lines selected in 2007 based on leaf temperature measurements exhibited a BP. That is, the measured leaf temperature differences in the field did not result in a segregation of genotypes for response to VPD. Many plant factors can contribute to elevated leaf temperature such as background stomata conductance, leaf size, and canopy architecture. Single-row plots in the field screen may have also influenced the expression of leaf temperature differences. In any event, it appears the influence of these factors overwhelmed a clear leaf temperature difference as a result of VPD response under field conditions. Only the two hybrids with high yield in the irrigated conditions in 2007 exhibited no BP. Sinclair et al. (2008) interpreted the BP in soybean PI 416937 as a response to a limiting hydraulic conductance in the leaf that constrains the flow of water from the xylem into the guard cells. A recent study of Sadok and Sinclair (2010a) indicated that existence of BP in soybean may be linked to limited leaf hydraulic conductance associated with the apparent lack of an AgNO3 sensitive leaf aquaporin population. This trait accounted for 25–50% of the decrease in TR at high VPD (Sadok and Sinclair, 2010b). Their suggestion is that in the absence of the silver-sensitive population of aquaporins in soybean leaves water flow can be restricted causing the plant to have stable TR at high VDP, i.e. a BP (King et al., 2009). Parent et al. (2009) studied transgenic lines of maize that expressed antisense of one of the NCED genes resulting in low abscisic acid concentrations in the xylem sap. The transgenic lines with low abscisic acid had restricted hydraulic conductance in the roots and this resulted in lower plant transpiration rates. Low
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hydraulic conductance was associated with decreased gene expression and protein content measured for PIP aquaporin isoforms. It appeared that posttranscriptional mechanisms regulated the amount of PIP proteins that influenced root hydraulic conductance. It is unknown whether the mechanism for expression of a BP at high VPD identified in this study exists in the leaves or the roots. One interesting possibility is that in those genotypes with especially low BP, e.g. SC982, there might be limiting hydraulic conductance in both leaves and roots (Sadok and Sinclair, 2010b). Further, research with these lines is needed to quantify and to resolve the site of the hydraulic conductance limitation in these sorghum genotypes. Among these sorghum genotypes exhibiting a BP, there was a significant negative relationship between the value of the BP and the linear slope at low VPD (i.e. slope1) (Fig. 2). This is an interesting relationship in that it indicates that plants that can transport large rates of water in the plant at low VPD reach the maximum transpiration rate more rapidly as VPD increases, i.e. a low BP. Of course, this hypothesis based on differences in hydraulic conductance in the plants cannot be resolved without identifying and measuring hydraulic conductance in these genotypes and its possible limitation on water flux in the plant. The maximum TR of the genotypes, however, differs so that this relationship is not rigidly limited by the same maximum transpiration rate across all genotypes. These differences offer the opportunity to develop genotypes with the appropriate BP and maximum transpiration for each geographical location. The genotypic variability identified in this study offers candidate parental lines for breeding programs seeking to improve sorghum yields under water-deficit conditions. Those lines without a BP and low rate of increase in TR with VPD might be well suited for consistently dry environmental conditions. However, these genotypes would also have restricted CO2 assimilation under these conditions so that such genotypes are likely to be slow growing, and probably low yielding under well-watered conditions. Genotypes with a high initial slope and a low BP, e.g. SC982, may offer a better approach to developing drought tolerance. These lines maximize gas exchange under low VPD and then shift to water conservation at high VPD. This is the water-conservation strategy currently being pursued with pearl millet (Kholova et al., 2010) for terminal drought conditions. Selection of the BP could be matched with the likelihood of water-deficit conditions. A low BP would give the greatest water conservation when soil water is still available while a high BP imposes less-restrictive water conservation. The results of this study clearly indicate sorghum genotypes could be selected to pursue soil water conservation as a way to improve yields under water-deficit conditions. Acknowledgments The authors gratefully acknowledge the financial support of the Kansas Grain Sorghum Commission. Contribution no. 10-313-J from Kansas Agricultural Experiment Station. References Allen, C.D., Macalady, A.K., Chenchouni, H., et al., 2010. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests. Forest Ecol. Manage. 259, 660–684. Bunce, J.A., 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 Res. 82, 37–47. Comstock, J., Ehleringer, J., 1993. Stomatal response to humidity in common bean (Phaseolus vulgaris): implications for maximum transpiration rate, water-use efficiency and productivity. Aust. J. Plant Physiol. 20, 669–691. Devi, M.J., Sinclair, T.R., Vadez, V., 2010. Genotypic variation in peanut for transpiration response to vapor pressure deficit. Crop Sci. 50, 191–196. Djè, Y., Heuertz, M., Lefèbvre, C., Vekemans, X., 2000. Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers. Theor. Appl. Genet. 100, 918–925.
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