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The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes
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The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes Margaret M. Barbour ∗ , Brent N. Kaiser Centre for Carbon, Water and Food, The University of Sydney, 380 Werombi Road, Brownlow Hill, NSW 2570, Australia
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Article history: Received 24 December 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online xxx Keywords: Water-use efficiency Mesophyll conductance Carbon isotope discrimination Drought Nitrogen availability Triticum aestivum
a b s t r a c t Increased mesophyll conductance (gm ) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction Increasing mesophyll conductance (gm ) has been suggested to provide an opportunity to improve water-use efficiency of crops, because a higher gm will result in higher chloroplastic CO2 partial pressures, Cc [1,2]. The increase in Cc will allow higher photosynthetic rates, all else being equal, without an increase in transpiration rate, resulting in an increase in leaf intrinsic water-use efficiency (the ratio of photosynthetic rate to stomatal conductance A/gs ). There is growing evidence of genotypic variation in gm among our important crop species [1,3,4]. The first hints of genetic control of gm were recently presented for
Abbreviations: A, photosynthetic rate; A/gs , leaf intrinsic water-use efficiency; CA, carbonic anhydrase; Cc , chloroplastic CO2 partial pressure; Cc /Ca , the ratio of chloroplastic to ambient CO2 partial pressure; Ci /Ca , the ratio of intercellular to ambient CO2 partial pressure; gm , mesophyll conductance; gs , stomatal conductance to water vapour; Jmax , electron transport rate; Sc , surface area of chloroplasts exposed to the intercellular air space per unit leaf surface area; Vcmax , maximum carboxylation rate; VPd, leaf-to-air vapour pressure difference; I , predicted photosynthetic carbon isotope discrimination assuming infinite mesophyll conductance; obs , measured photosynthetic carbon isotope discrimination; L , leaf water potential. ∗ Corresponding author. E-mail addresses: [email protected] (M.M. Barbour), [email protected] (B.N. Kaiser).
common wheat, with a region of genetic control of gm on chromosome 2A [5], raising the possibility of selecting for high gm to increase A/gs . Mesophyll conductance has been shown to respond to environmental conditions, both in terms of long-term, growth conditions, and more recently in response to dynamic changes in environment. Long-term exposure to low light resulted in decreased gm in walnut [6], maple [7], birch, linden, and the perennial herb goldenrod [8] and in beech [9]. The short-term response of gm to light is not as clear, with a positive relationship between the two in some species and experiments [10,11] but not others [12]. Similarly, the shortterm response of gm to temperature is variable, with some species showing limited response, and others showing a strong sensitivity of gm to temperature [13,14]. Both water and nitrogen availability strongly limit photosynthetic rate, the first through reductions in stomatal conductance and the second through lowered photosynthetic capacity. Reduced nitrogen availability has been shown to lower gm in spinach [15], Pinus radiata [16], and rice [17,18]. Moreover, positive correlations were found between leaf N content and gm in rice [19,20], a range of wheat cultivars [21], and four crop species (rice, wheat, spinach and tobacco) grown under differing N availabilities [22]. In contrast, the response of gm to limited N availability was small in Eucalyptus globulus [23]. In a review of published experiments, Flexas et al. [24] found that water stress often reduced gm (18 out of 20
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studies), but more recent work has suggested that reduction in gm under drought may be transient [25,26]. Decreased gm in response to reduced water and nitrogen availability may relate to changes in leaf anatomy [20], changes in membrane permeability due to aquaporin expression or activity [25], or differences in chloroplast size or location [17]. Given the observed changes in gm in response to growth conditions, any breeding program aiming to increase water-use efficiency through gm must ensure a full understanding of the ranking of genotypes for gm and A/gs under a wide range of growth conditions. Here we quantify variation in gm between wheat genotypes grown under conditions of varying nitrogen and water. We also investigate the stomatal and mesophyll limitations to photosynthesis under the differing growth environments, and assess the relative importance of stomatal and mesophyll conductance on leaf intrinsic water-use efficiency.
2. Materials and methods 2.1. Plant material and growth conditions Wheat (Triticum aestivum L.) plants of the cultivars ‘Dart’, Gregory’, ‘Livingston’, Spitfire’, ‘Sunguard’ and the pre-release line LPB10-0018 from LongReach Breeding Company were germinated in 6L pots filled with washed river sand, 3 plants per pot. Plants were grown in a controlled environment room at 25 ◦ C and 75% relative humidity during the 14-day light period (photosynthetically active radiation at the upper leaf surface was 800 mol m−2 s−1 ), and 17 ◦ C and 75% humidity during the 10-h dark period. The CO2 concentration was controlled at 400 ppm, but frequently increased to 600 ppm during the dark period due to respiration. However, the CO2 concentration and ␦13 C in the dark have little influence on photosynthetic assimilation. The ␦13 C of CO2 inside the room during the light period was measured using a stable isotope cavity ring down laser (G11101-i, Picarro CA, USA) and found to be −10.0‰ on average during the week prior to measurements and during the week that measurements were made. This value was used in the calculation of gm . All pots were supplied with 5 ml of either full nutrient solution (including 10 mM NH4 NO3 ; high N) or nutrient solution with adequate N but full supply of all other macro- and micro-nutrients (including 4.5 mM NH4 NO3 ; adequate N) three days a week, and well watered four days a week for four weeks. At this point, limited water availability was applied to half the pots by withholding water on the days that nutrient solution was not applied until 50% of plants had reached temporary wilting point, assessed visually. This occurred after 8 days for high N pots and after 10 days for adequate N plants. The weight of each pot was recorded at this point and designated as the target weight for the pot. Thereafter, the water content of droughted pots was maintained gravimetrically by adding nutrient solution (three days a week) and/or water (every day) to bring the pot to the target weight. This created four growth conditions, namely high N and well-watered, high N and droughted, adequate N and well-watered and adequate N and droughted. Five replicate plants were grown for each genotype in each of the four growth conditions. Prior to the start of the water availability treatment, plants were thinned to one per pot. One of extra plants was separated into the first leaf (leaf one), the fourth leaf (leaf 4) of the main tiller and the rest of the plant. Leaf gas exchange and water potential measurements occurred on the sixth and seventh weeks, and all plants were destructively sampled at the end of the measurements (eight weeks after planting seeds). At the time of harvest, all plants were pre-anthesis but all had at least some emerged heads. Plants were separated into emerging heads, leaves, stems and roots. Leaves 1 and 4 from the main tiller, plus the youngest fully expanded leaf
were sampled separately. All samples from both harvests were dried at 65 ◦ C for a minimum of 48 h before weighing for biomass. The individual leaves sampled prior to the water treatment and at the final harvest were ground to a fine powder and analysed for total N content (%N) and carbon isotope composition on a stable isotope ratio mass spectrometer (Delta V, Thermo Finnigan). 2.2. Leaf gas exchange and water potential measurements Photosynthetic CO2 response curves were measured using a portable gas exchange system (LI-6400xt, LiCor, Lincoln, NE, USA) for three of the six genotypes for all four growth conditions; Gregory, Livingston and Spitfire. The Li6400 was fitted with the standard 2 by 3 cm and red-blue light source. The light source was set to provide 1250 mol m−2 s−1 PAR, leaf temperature was controlled at 25 ◦ C and the leaf-to-air vapour pressure difference (VPd) between 0.75 and 1.0 kPa. The youngest fully expanded leaves from each plant was used for measurements. Maximum carboxylation rate (Vcmax ) and electron transport rate (Jmax ) were fitted using the spreadsheet from Sharkey et al. [27] but using estimated gm for each individual leaf. We also used the Sharkey et al. [27] spreadsheet in its original form to fit all parameters, including gm , for comparison with the fitted parameters using known gm . CO2 response curves and estimated gm values were used to calculate stomatal (Ls ) and mesophyll (Lm ) limitations to photosynthesis at an ambient CO2 concentration of 400 ppm using the method described by Warren et al. [28]. A Scholander-style pressure chamber (115, Soil Moisture Equipment, Santa Barbara, CA, USA) was used to measure the water potential (L ) at midday for all leaves immediately after gas exchange measurements. Leaves were wrapped in plastic film and cut just above the ligule with a razor blade prior to sealing in the pressure chamber for measurement. 2.3. Mesophyll conductance measurements Mesophyll conductance was estimated using a coupled leaf photosynthesis system (LI-6400xt, as above) and stable carbon isotope tunable diode laser (TGA100A, Campbell Scientific, Logan UT, USA) as described by Barbour et al. [1]. The Li-6400 was fitted with a 2 by 6 cm narrow leaf chamber (Li6400-11) and irradiance provided by a red-green-blue light source (Li6400-18) set to mimic the red-blue light source at an irradiance of 1250 mol m−2 s−1 PAR. Leaf temperature was controlled at 25 ◦ C and VPd between 0.8 and 1.6 kPa. Two or three of the youngest fully expanded leaves were placed side by side in the leaf chamber. gm was calculated from the difference between predicted discrimination assuming infinite mesophyll conductance (i ) and measured discrimination (obs ), using equations developed by Evans et al. [29] and Barbour et al. [1], and including a ternary effect as described by Farquhar and Cernusak [30]. We assume values of fractionation factors; carboxylation fractionation was assumed to be 29‰, fractionation during dissolution and diffusion through water was assumed to be 1.8‰, the fractionation associated with photorespiration was assumed to be 16.2‰, the fractionation occurring during diffusion through the leaf boundary layer was assumed to be 2.9‰ and the rate of day respiration was taken to be 1.5 mol m−2 s−1 (from Jahan et al. [4]). 2.4. Statistical analysis Differences between the various physiological measures were assessed using general analysis of variance, as implemented by GenStat 14th edition, and means were compared using Bonferroni significant difference test. Differences were considered statistically
Please cite this article in press as: M.M. Barbour, B.N. Kaiser, The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes, Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.2016.03.012
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3. Results 3.1. Plant growth As expected, above-ground growth was generally higher under high N compared to adequate N, and reduced under drought (Fig. 1). However, there were also significant interactive effects. For head biomass pre-anthesis, there was no significant nitrogen effect but there was a significant genotype by water availability interaction (P = 0.050) with a strong drought effect for LPB10-0018 but non-significant effects for other genotypes. There were significant differences between genotypes for leaf biomass (P > 0.001) with Gregory having the highest biomass under well-watered conditions, followed by LPB10-0018, Sunguard and Livingston with similar leaf biomass, then Spitfire and finally Dart with the lowest leaf biomass. There were also significant genotype by water (P < 0.001) and nitrogen by water (P = 0.018) interactions. Gregory, LPB10-0018 and Sunguard had greater reductions in leaf biomass under water limitation than did Dart and Spitfire. Across all genotypes the reduction in leaf biomass due to water limitation was greater at high N availability than at adequate N. There were also significant differences between genotypes for stem biomass (P < 0.001) with LBP10-0018 having the highest biomass under well-watered conditions, followed by Sunguard, Spitfire, Gregory and Dart with similar stem biomass, then Livingston with the
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Fig. 1. Biomass of six wheat genotypes grown under four conditions of variable nitrogen and water availability. Values are mean, ± standard error, n = 5.
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lowest stem biomass. There were also significant nitrogen by water availability (P < 0.001) interactive effects. Below ground biomass was also significantly affected by water and nitrogen availability. There was a significant genotype by water availability interaction (P = 0.016), with greater root biomass for droughted Spitfire plants compared to well-watered plants, while all other genotypes drought did not significantly affect root biomass. There was also a significant nitrogen by water availability interaction (P < 0.001) for root biomass. In general, under wellwatered conditions root biomass was higher for high N compared to adequate N, while under drought conditions root biomass was considerably higher under adequate N compared to high N for Spitfire (P, 0.05) but differences were statistically insignificant for other genotypes. These biomass effects resulted in significant differences in biomass allocation under the differing growth conditions. There was a significant genotype by water by nitrogen availability interaction (P < 0.001) for root to shoot ratio (Fig. 2). Dart was the least responsive to water and nitrogen limitation in terms of biomass allocation, with no significant differences in root/shoot across the treatments. Conversely, Spitfire allocated more than twice its biomass below ground under the combined drought and adequate N treatment (root/shoot is 2.66), but about half its biomass below ground under well-watered and high N conditions (root/shoot is 0.58). Significant genotype by water by nitrogen availability (P < 0.001) interactive effects were also found for the ratio of leaf to stem biomass (Fig. 2). Gregory allocated more biomass to leaves compared to stems than all other genotypes, and Dart allocated the least. In general, leaf/stem was higher for droughted compared to well-watered plants, except for Gregory and Sunguard which had higher leaf/stem for well-watered plants grown at adequate N compared to droughted plants at adequate N.
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B
C
Fig. 3. Growth environment effects on leaf water potential (A) and leaf gas exchange for six wheat genotypes. Values are mean, ± standard error, n = 5.
Using above ground biomass sampled prior to the start of the water availability treatment and the final harvest from the same pots, estimates of above-ground relative growth rate were made (Fig. 2C). Significant differences were found between cultivars (P < 0.001), Gregory, LPB10-0018 and Sunguard had the highest growth rates, and Dart and Spitfire had the lowest growth rates. There was also a significant water by nitrogen interaction (P = 0.003). Adequate nitrogen significantly reduced growth rate compared to high N under well-watered conditions (for Gregory, LPB10-0018, Sptifire and Sunguard) but not under drought conditions (except for LBP10-0018).
3.2. Leaf gas exchange As expected, water stress lowered leaf water potential (L Fig. 3). There were significant differences between cultivars (P < 0.001), Dart had the lowest L and Livingston the highest (other genotypes did not differ significantly from these two genotypes). There was also a significant nitrogen by water availability interactive effect (P < 0.001), where droughted plants at high N had lower L than droughted plants at adequate N. Genotypes had significantly different photosynthetic rates (A, P < 0.001), with Gregory having the highest and Spitfire the lowest overall average A, although genotypes did not differ significantly for A at any given growth treatment. There were significant nitrogen by water availability treatment effects for A (P < 0.0001) and stomatal conductance (gs , P < 0.001). The reduction in gs due to drought was stastically significant for Dart, Gregory, Livingston, Spitfire and Sunguard at high N, but the drought effect was not statistically
Fig. 4. Relationships between mesophyll conductance (gm) and other leaf gas exchange values for six wheat genotypes grown under four conditions of water and nitrogen availability. Values are mean, ± standard error, n = 5. Lines are least squares linear regression, with correlation coefficient and significance level provided.
significant for any genotype at adequate N, matching the effects of growth environment on L . We found a significant interactive effect of genotype by nitrogen by water availability (P < 0.001) for mesophyll conductance (gm ). Except for the genotypes Dart and Spitfire at adequate N, drought reduced gm (although it was statistically significant for Gergory at adequate N only). N availability did not have clear effects on gm at a given water availability for any genotypes except Gregory, for which gm at adequate N was higher than at high N under wellwatered conditions. The cultivars responded to drought differently in terms of leaf intrinsic water-use efficiency (A/gs , P = 0.004). There was a large increase in A/gs under drought and high N for Spitfire and Sunguard while Dart, Livingston and LPB10-0018 were less sensitive. There was a significant water by nitrogen availability interaction (P < 0.001) for A/gs ; the increase in A/gs in response to drought was greater for high N plants than for plants grown at adequate N. 3.3. Correlations between mesophyll conductance, leaf properties and gas exchange parameters When genotype and treatment averages were calculated, mesophyll conductance was positively related to A (P = 0.002, Fig. 4A) and gs (P = 0.003, Fig. 4B) and negatively related to A/gs (P = 0.0018, Fig. 4C). gm was also positively related to L (P = 0.001, Fig. 5A) and leaf N content (P = 0.008, Fig. 5B), but unrelated to leaf mass per unit
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area (Fig. 5C). So, high gm was found together with high A, gs , L and leaf N content. In this experiment, which included a moderate drought, A/gs was more strongly driven by gs than by A, resulting in a negative relationship between gm and A/gs .
A
3.4. Photosynthetic parameters and limitations Photosynthetic response curves to CO2 concentration were measured for three genotypes and, when combined with estimates of gm , allowed for the fitting of maximum carboxylation rate (Vcmax ), maximum electron transport rate (Jmax ), and stomatal (Ls ) and mesophyll (Lm ) conductance limitations to photosynthesis. We chose to calculate Ls and Lm at an ambient CO2 concentration of 400 mol mol−1 , to match the growth conditions. There were significant genotype differences in Vcmax (P = 0.017; Gregory had higher Vcmax than Livingston and Spitfire, Table 1) but no significant water or nitrogen availability effects. There were also significant genotype differences for Jmax (P < 0.001; Livingston had higher Jmax than Gregory or Spitfire), and significant nitrogen by water limitation interactions (P = 0.044). There was a nitrogen effect on Jmax for droughted plants only, in which Jmax was higher for adequate N than for high N. Mesophyll conductance was fitted from the CO2 response curves using the function developed by Sharkey et al. [27], with fitted values restricted to within a given range. The default range in fitted values is 0–3 mol m−2 s−1 bar−1 , and was used in the current study. Fitted gm varied between the upper limit and 0.26 mol m−2 s−1 bar−1 , but was unrelated to gm measured using the carbon isotope discrimination method (Fig. 6). A comparison of fitted Vcmax and Jmax using known gm , with values for which gm was also fitted revealed differences between the estimates. The average difference between the two estimates of Vcmax was only 1 mol m−2 s−1 , but this difference was significantly affected by water availability (P = 0.006), with droughted plants having an underestimate of Vcmax with fitted gm (on average, an underestimate of 8 mol m−2 s−1 , P = 0.034), and well-watered plants having an overestimate of Vcmax when gm was fitted (on average 10 mol m−2 s−1 , P = 0.015). There was also considerable
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Fig. 5. Relationships between mesophyll conductance (gm) and leaf traits for six wheat genotypes grown under four conditions of water and nitrogen availability. Values are mean, ± standard error, n = 5. Lines are least squares linear regression, with correlation coefficient and significance level provided.
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Table 1 Fitted values for maximum carboxylation rate (Vcmax , in mol m−2 s−1 ), electron transport rate (Jmax, in mol m−2 s−1 ), and stomatal (Ls , dimensionless) and mesophyll (Lm , dimensionless) limitations to photosynthesis for three wheat genotypes grown under four environments of water and nitrogen availability, using measured mesophyll conductance for individual plants. Values are mean, ± standard error, n = 5. Growth treatment
Parameter Gregory
High nitrogen, well-watered
Vcmax
131 ± 3
131 ± 5
151 ± 10
Jmax Ls Lm
199 ± 6 0.17 ± 0.01 0.06 ± 0.01
194 ± 7 0.17 ± 0.01 0.06 ± 0.01
184 ± 10 0.13 ± 0.01 0.08 ± 0.01
Vcmax
118 ± 14
152 ± 7
155 ± 22
Jmax Ls Lm
163 ± 21 0.30 ± 0.02 0.09 ± 0.01
209 ± 6 0.28 ± 0.02 0.14 ± 0.03
181 ± 19 0.36 ± 0.05 0.21 ± 0.06
Vcmax
110 ± 6
146 ± 9
131 ± 5
Jmax Ls Lm
170 ± 10 210 ± 11 170 ± 5 0.13 ± 0.02 0.18 ± 0.01 0.14 ± 0.01 0.03 ± 0.004 0.06 ± 0.004 0.07 ± 0.01
Vcmax
147 ± 9
156 ± 10
144 ± 9
Jmax Ls Lm
193 ± 10 0.22 ± 0.03 0.09 ± 0.01
227 ± 11 0.24 ± 0.01 0.09 ± 0.01
189 ± 7 0.30 ± 0.06 0.16 ± 0.05
High nitrogen, droughted
Adequate nitrogen, well-watered
Adequate nitrogen, droughted
Livingston
Spitfire
scatter in the relationship between the two estimates of Vcmax (Fig. 7), suggesting that fitting gm resulted in large under- and over-estimates for Vcmax of some individual leaves. In contrast, there was not wide scatter in the relationship between the two estimates of Jmax (Fig. 7). But, like Vcmax , water availability significantly (P = 0.045) affected the difference between the two estimates. Under droughted conditions fitting gm resulted in a 4.6 mol m−2 s−1 under estimate of Jmax compared to Jmax with known gm (P = 0.007), while under well-watered conditions there was no difference between the two estimates. The genotypes responded differently to water stress in terms of stomatal limitation to photosynthesis (Ls ); there was a stronger increase in Ls due to drought for Spitfire than either Gregory or Livingston (Table 1). There was also a significant nitrogen effect on Ls , with higher limitation at high N than at adequate N (P = 0.023). The mesophyll limitation to photosynthesis (Lm ) was stronger for droughted compared to well-watered plants (P < 0.001). There was also a significant difference (P = 0.006, Table 1) in Lm between genotypes, with Spitfire having the highest Lm and Gregory the lowest. 3.5. 13 C of whole leaf tissue, and relationships with leaf gas exchange The carbon isotope composition of the youngest fully expanded leaf at the final harvest varied between cultivars and was affected by both nitrogen and water availability. Decreased nitrogen availability increased 13 C, while drought reduced 13 C. There was a significant nitrogen by water availability interactive effect (P = 0.005), with a stronger effect of nitrogen availability under drought. The cultivars also responded differently to water availability (P = 0.018); Sunguard responded the most to drought with a 3.1‰ decrease from well-watered to droughted plants, while Gregory decreased by 2.0‰, and Livingston and LPB10-0018 were the least sensitive with a decrease of just 0.7 and 0.8‰, respectively. Across all growth environments, genotype average 13 C was negatively related to genotype averages from gas exchange measurements: A/gs , the ratio of intercellular to ambient CO2 partial pressure (Ci /Ca ) and ratio of chloroplastic to ambient CO2 partial
Fig. 7. The relationships between photosynthetic parameters (maximum carboxylation rate, Vcmax and electron transport rate, Jmax ) when fitted with known values of mesophyll conductance (gm ) and when gm was also fitted for three wheat genotypes grown under four growth environments. In both cases the procedure described by Sharkey et al. [27] was used, and there are five replicate plants for each genotype and growth environment. Values are for individual plants.
pressure (Cc /Ca ; Fig. 8). The closest correlation was between 13 C and A/gs , with an r2 of 0.71. 4. Discussion 4.1. Genotypes differ in their response to N availability, drought and the combined stresses The interactive effects of N and water availability on growth observed here confirm many previous studies (reviewed in Sadras [31] and Gonzalez-Dugo et al. [32]). Here, above ground relative growth rate was reduced by limited water availability more at high N availability than at adequate N. Also expected were differences between genotypes for growth rate and biomass allocation, photosynthesis and stomatal conductance and leaf intrinsic water-use efficiency under the four growth environments. The new information provided by this experiment is the interactive effect of N and water availability on mesophyll conductance, and the observation that the genotypes differ in their response to the growth environments. Overall, drought reduced gm and this response has been seen in many, but not all, previous experiments (see Flexas et al. [24] for a review). However, no genotypes grown at adequate N showed significantly reduced gm under drought. The genotypes also responded differently to N availability. Reduced N availability from high to adequate N generally did not alter gm , but the genotype
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Fig. 8. Relationships between leaf gas exchange (A, leaf intrinsic water-use efficiency; B, the ratio of intercellular to ambient CO2 partial pressures; C, the ratio of chloroplastic to ambient CO2 partial pressures) and the carbon isotope discrimination in leaf tissue. Values are mean, ± standard error, n = 5.
Gregory had higher gm at adequate N than at high N under droughted conditions. The positive relationship between leaf N content and gm found in the current study supports earlier observations for a range of crop species [17–20]. If leaves are treated individually, leaf N content explained 32% of measured variability in A but only 11% of measured variability in gm , while (again for individual leaves) variation in gm explained 33% of variation in A. With three-quarters of leaf N associated with photosynthesis (mostly Rubisco and chlorophyll, [33]) the relationship between N and gm may simply reflect the relatipnship between A and gm . However, the changes in gm in response to N availability and drought may have been due to changes in leaf anatomy [19,20]. The surface area of chloroplasts exposed to the intercellular air space (Sc ) has been shown to be positively related to gm across different species, while cell wall thickness was negatively related [34]. In Populus tremula, water stress reduced Sc and increased cell wall thickness, and these changes combined to reduce gm [35]. The reduced gm in response to drought in combination with limiting nitrate availability in rice was related to reductions in chloroplast size [17,18], which presumably also reduced Sc . High nitrogen availability increased chloroplast surface area per unit leaf area and reduced cell wall thickness in rice [20], and may have altered these traits here in wheat. Future studies should quantify genotypic variability in leaf anatomy in response to growth conditions in order to understand changes in genotype ranking of gm . Changes in gm under nutrient- or water-limited conditions may also result from changes in enzyme activity. Two enzymes have been implicated in regulation of gm , namely carbonic anhydrase and membrane aquaporins. Carbonic anhydrase (CA) catalyses the reversible conversion of CO2 to HCO3 − and is found in multiple forms and high abundance in the chloroplast, cytosol, mitochondria and plasma membrane of C3 plants [36]. While the importance of CA to gm has not been widely studied, the diffusivities of CO2
and HCO3 − do differ, so CA activity may play a role in gm [37]. The potential role for aquaporins in regulating gm is much clearer. gm in faba decreased significantly when leaves were fed with the aquaporin specific inhibitor HgCl2 [38]. Aquaporin over- and underexpressing lines display higher and lower gm , respectively, than their parental wildtypes in rice [39] and tobacco [40]. Some aquaporins have been shown to conduct CO2 [41]. It has been suggested that the composition of aquaporin tetramers in the membrane may facilitate differences in conductance of CO2 versus H2 O, compared to those with single isoform combinations [42]. While it is clear that water transport across membranes responds to water availability due to changes in aquaporin expression and activity [43,44], changes in the CO2 diffusivity of membranes in response to environmental conditions is less clear [37]. However, aquaporins remain a likely candidate for part regulation of gm . The interactive effects of N and drought observed for many plant traits have been shown here to be observed for mesophyll conductance in wheat, with genotype ranking depending on the growth environment. This suggests that (1) if gm is to be included as a trait in breeding programs [1,4], care must be taken to match the selection environment to the ultimate growth environment and (2) the underlying mechanism or mechanisms responsible for the observed genotypic variability in gm in response to growth environment should be determined. As suggested above, both leaf anatomy and aquaporin expression/activity/location are likely candidates to explain differences between genotypes. 4.2. Relative limitations on photosynthesis by stomata and mesophyll conductances depend on growth environment In wheat the limitation on photosynthesis imposed by stomatal conductance was, on average, slightly more than double the limitation imposed by mesophyll conductance. This is similar to that reported by Jahan et al. [4] under non-limiting conditions. When
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plants were droughted, the stomatal and mesophyll imitations both increased, but the degree of increase was greater for Ls than for Lm . Interestingly, there were significant differences between genotypes in the sensitivity of Ls to water stress; Spitfire was more sensitive to water stress than Gregory or Livingston. Consistent with limited change in actual values of gm in response to N availability, there were no significant N effects on Lm . However, Ls was reduced by lowered N availability. Photosynthesis was most limited by mesophyll diffusion of CO2 for Spitfire and least limited for Gregory, consistent with genotype rankings for photosynthetic rate itself. The lack of genotype differences in Ls reflects the lack of genotype differences in stomatal conductance. Considering diffusional photosynthetic limitations and selection for high water-use efficiency, it is logical that the combination of relatively high Ls and relatively low Lm should produce a plant with high WUE if photosynthetic capacity is constant. However, when the ratio of Ls to Lm from A/Ci curve analysis was compared to A/gs from instantaneous gas exchange measurements, there was no relationship between Ls /Lm and A/gs , even when leaves were grouped into similar Vcmax (data not shown). It seems when water availability is the key driver of variation in A/gs for wheat, stomatal limitations are much more important in determining water-use efficiency than mesophyll limitations [2], and indeed variation in Ls explained 39% of variation in A/gs while variation in Lm explained just 17% (data not shown). 4.3. Photosynthetic parameters are underestimated under drought when mesophyll conductance is unknown The photosynthetic parameters Vcmax and Jmax are central to models of photosynthesis such as the widely-used model of Farquhar et al. [45]. It has been recognized for some time that failure to account for gm will introduce significant errors in parameter estimation [46–48]. The parameter fitting procedure introduced by Sharkey et al. [36] attempted to overcome this problem by fitting gm at the same time as Vcmax and Jmax . However, the current study shows that Vcmax and Jmax are significantly underestimated under drought conditions (7% and 3% for Vcmax and Jmax respectively) and that Vcmax is overestimated (7%) under well-watered conditions when gm is unknown and fitted along with Vcmax and Jmax , as compared to using known gm values when fitting Vcmax and Jmax . Further, fitted gm was unrelated to measured gm , and fitted gm was often unrealistically high. Measurements of gm are recommended for accurate estimation of photosynthetic parameters, particularly under water limited conditions. One aspect that has not been addressed here is whether gm changes with CO2 concentration. A number of studies have found that gm declines as leaf internal CO2 partial pressure increases above 200 mol mol−1 [11,49,50]. However, Tazoe et al. [51] have shown that gm does not respond to CO2 in wheat. We have assumed that gm is constant throughout the response curve measurements, but this assumption has not been tested. Flexas et al. [24] assessed the effect of variable gm on CO2 response curves, and concluded that the effect may be substantial when gm is very low such as under severe water stress. Further work is warranted. 4.4. Mesophyll conductance and water-use efficiency When considering selection of genotypes for water-limited conditions, the combination of high carboxylation rate, relatively low gs but high gm , and high relative growth rate would provide the highest leaf-intrinsic water-use efficiency while still allowing for high productivity [1]. Of the genotypes studied here, Gregory has the highest Vcmax , A and gm , among the highest pre-anthesis relative growth rate, and the lowest Lm . Dart and Spitfire sit at the other end of the trait spectrum. The strong relationship between
gm and A suggests that mesophyll conductance can contribute to selection for high photosynthetic rate, however this study demonstrates that under water-limited conditions stomatal conductance has a stronger effect on leaf intrinsic water-use efficiency than does mesophyll conductance. Given the technical challenges of mesophyll conductance measurement, particularly in the field, a more effective selection strategy may be sampling leaf tissue for carbon isotope analysis via mass spectrometry combined with either photosynthetic rate or leaf N content as a proxy for photosynthetic rate. Use of 13 Cl as a selection trait has been particularly successful in wheat, providing the basis for the release of two commercial cultivars [51]. There are problems with interpreting variation in 13 Cl when gm is unknown, as discussed by Barbour et al. [1], but the strong relationship between A and gm found here in wheat, and earlier in a range of species [2], suggests that these concerns may be minor. A highly water-use efficient genotype, but one that is still productive, would be one with low 13 Cl combined with high leaf N content (or high A), and this would indirectly select for high gm . Based on this logic, we expect ‘Drysdale’ (the high water-use efficiency cultivar released using 13 Cl selection [52]) to have high gm , high leaf N content and high photosynthetic rate; a hypothesis to be tested in the future. Harvest index is a further complicating factor; genotypes that have desireable leaf traits during the pre-anthesis growth period in controlled conditions may not end up with high grain yield per unit water used at harvest if allocation to grain development and grain filling is not high. Field testing through to harvest is a necessity. In conclusion, wheat genotypes differ in their response to limited water and nitrogen availability in terms of leaf gas exchange, including mesophyll conductance. In this study, leaf intrinsic water-use efficiency was more closely related to stomatal than to mesophyll conductance. Mesophyll limitation to photosynthesis differed between genotypes and increased when plants were water-limited, while stomatal limitation to photosynthesis increased more in response to drought in the genotype Spitfire than in other genotypes. Selection for increased water-use efficiency through high mesophyll conductance should include screening under a range of growth conditions.
Acknowledgements This work was supported by the Grains Research and Development Corporation (contract US00056). Erin Lockhart, William Lin and Svetlana Ryazanova are thanked for technical assistance and Dr. Claudia Keitel for mass spectrometric analysis.
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The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes Margaret M. Barbour ∗ , Brent N. Kaiser Centre for Carbon, Water and Food, The University of Sydney, 380 Werombi Road, Brownlow Hill, NSW 2570, Australia
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Article history: Received 24 December 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online xxx Keywords: Water-use efficiency Mesophyll conductance Carbon isotope discrimination Drought Nitrogen availability Triticum aestivum
a b s t r a c t Increased mesophyll conductance (gm ) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction Increasing mesophyll conductance (gm ) has been suggested to provide an opportunity to improve water-use efficiency of crops, because a higher gm will result in higher chloroplastic CO2 partial pressures, Cc [1,2]. The increase in Cc will allow higher photosynthetic rates, all else being equal, without an increase in transpiration rate, resulting in an increase in leaf intrinsic water-use efficiency (the ratio of photosynthetic rate to stomatal conductance A/gs ). There is growing evidence of genotypic variation in gm among our important crop species [1,3,4]. The first hints of genetic control of gm were recently presented for
Abbreviations: A, photosynthetic rate; A/gs , leaf intrinsic water-use efficiency; CA, carbonic anhydrase; Cc , chloroplastic CO2 partial pressure; Cc /Ca , the ratio of chloroplastic to ambient CO2 partial pressure; Ci /Ca , the ratio of intercellular to ambient CO2 partial pressure; gm , mesophyll conductance; gs , stomatal conductance to water vapour; Jmax , electron transport rate; Sc , surface area of chloroplasts exposed to the intercellular air space per unit leaf surface area; Vcmax , maximum carboxylation rate; VPd, leaf-to-air vapour pressure difference; I , predicted photosynthetic carbon isotope discrimination assuming infinite mesophyll conductance; obs , measured photosynthetic carbon isotope discrimination; L , leaf water potential. ∗ Corresponding author. E-mail addresses: [email protected] (M.M. Barbour), [email protected] (B.N. Kaiser).
common wheat, with a region of genetic control of gm on chromosome 2A [5], raising the possibility of selecting for high gm to increase A/gs . Mesophyll conductance has been shown to respond to environmental conditions, both in terms of long-term, growth conditions, and more recently in response to dynamic changes in environment. Long-term exposure to low light resulted in decreased gm in walnut [6], maple [7], birch, linden, and the perennial herb goldenrod [8] and in beech [9]. The short-term response of gm to light is not as clear, with a positive relationship between the two in some species and experiments [10,11] but not others [12]. Similarly, the shortterm response of gm to temperature is variable, with some species showing limited response, and others showing a strong sensitivity of gm to temperature [13,14]. Both water and nitrogen availability strongly limit photosynthetic rate, the first through reductions in stomatal conductance and the second through lowered photosynthetic capacity. Reduced nitrogen availability has been shown to lower gm in spinach [15], Pinus radiata [16], and rice [17,18]. Moreover, positive correlations were found between leaf N content and gm in rice [19,20], a range of wheat cultivars [21], and four crop species (rice, wheat, spinach and tobacco) grown under differing N availabilities [22]. In contrast, the response of gm to limited N availability was small in Eucalyptus globulus [23]. In a review of published experiments, Flexas et al. [24] found that water stress often reduced gm (18 out of 20
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studies), but more recent work has suggested that reduction in gm under drought may be transient [25,26]. Decreased gm in response to reduced water and nitrogen availability may relate to changes in leaf anatomy [20], changes in membrane permeability due to aquaporin expression or activity [25], or differences in chloroplast size or location [17]. Given the observed changes in gm in response to growth conditions, any breeding program aiming to increase water-use efficiency through gm must ensure a full understanding of the ranking of genotypes for gm and A/gs under a wide range of growth conditions. Here we quantify variation in gm between wheat genotypes grown under conditions of varying nitrogen and water. We also investigate the stomatal and mesophyll limitations to photosynthesis under the differing growth environments, and assess the relative importance of stomatal and mesophyll conductance on leaf intrinsic water-use efficiency.
2. Materials and methods 2.1. Plant material and growth conditions Wheat (Triticum aestivum L.) plants of the cultivars ‘Dart’, Gregory’, ‘Livingston’, Spitfire’, ‘Sunguard’ and the pre-release line LPB10-0018 from LongReach Breeding Company were germinated in 6L pots filled with washed river sand, 3 plants per pot. Plants were grown in a controlled environment room at 25 ◦ C and 75% relative humidity during the 14-day light period (photosynthetically active radiation at the upper leaf surface was 800 mol m−2 s−1 ), and 17 ◦ C and 75% humidity during the 10-h dark period. The CO2 concentration was controlled at 400 ppm, but frequently increased to 600 ppm during the dark period due to respiration. However, the CO2 concentration and ␦13 C in the dark have little influence on photosynthetic assimilation. The ␦13 C of CO2 inside the room during the light period was measured using a stable isotope cavity ring down laser (G11101-i, Picarro CA, USA) and found to be −10.0‰ on average during the week prior to measurements and during the week that measurements were made. This value was used in the calculation of gm . All pots were supplied with 5 ml of either full nutrient solution (including 10 mM NH4 NO3 ; high N) or nutrient solution with adequate N but full supply of all other macro- and micro-nutrients (including 4.5 mM NH4 NO3 ; adequate N) three days a week, and well watered four days a week for four weeks. At this point, limited water availability was applied to half the pots by withholding water on the days that nutrient solution was not applied until 50% of plants had reached temporary wilting point, assessed visually. This occurred after 8 days for high N pots and after 10 days for adequate N plants. The weight of each pot was recorded at this point and designated as the target weight for the pot. Thereafter, the water content of droughted pots was maintained gravimetrically by adding nutrient solution (three days a week) and/or water (every day) to bring the pot to the target weight. This created four growth conditions, namely high N and well-watered, high N and droughted, adequate N and well-watered and adequate N and droughted. Five replicate plants were grown for each genotype in each of the four growth conditions. Prior to the start of the water availability treatment, plants were thinned to one per pot. One of extra plants was separated into the first leaf (leaf one), the fourth leaf (leaf 4) of the main tiller and the rest of the plant. Leaf gas exchange and water potential measurements occurred on the sixth and seventh weeks, and all plants were destructively sampled at the end of the measurements (eight weeks after planting seeds). At the time of harvest, all plants were pre-anthesis but all had at least some emerged heads. Plants were separated into emerging heads, leaves, stems and roots. Leaves 1 and 4 from the main tiller, plus the youngest fully expanded leaf
were sampled separately. All samples from both harvests were dried at 65 ◦ C for a minimum of 48 h before weighing for biomass. The individual leaves sampled prior to the water treatment and at the final harvest were ground to a fine powder and analysed for total N content (%N) and carbon isotope composition on a stable isotope ratio mass spectrometer (Delta V, Thermo Finnigan). 2.2. Leaf gas exchange and water potential measurements Photosynthetic CO2 response curves were measured using a portable gas exchange system (LI-6400xt, LiCor, Lincoln, NE, USA) for three of the six genotypes for all four growth conditions; Gregory, Livingston and Spitfire. The Li6400 was fitted with the standard 2 by 3 cm and red-blue light source. The light source was set to provide 1250 mol m−2 s−1 PAR, leaf temperature was controlled at 25 ◦ C and the leaf-to-air vapour pressure difference (VPd) between 0.75 and 1.0 kPa. The youngest fully expanded leaves from each plant was used for measurements. Maximum carboxylation rate (Vcmax ) and electron transport rate (Jmax ) were fitted using the spreadsheet from Sharkey et al. [27] but using estimated gm for each individual leaf. We also used the Sharkey et al. [27] spreadsheet in its original form to fit all parameters, including gm , for comparison with the fitted parameters using known gm . CO2 response curves and estimated gm values were used to calculate stomatal (Ls ) and mesophyll (Lm ) limitations to photosynthesis at an ambient CO2 concentration of 400 ppm using the method described by Warren et al. [28]. A Scholander-style pressure chamber (115, Soil Moisture Equipment, Santa Barbara, CA, USA) was used to measure the water potential (L ) at midday for all leaves immediately after gas exchange measurements. Leaves were wrapped in plastic film and cut just above the ligule with a razor blade prior to sealing in the pressure chamber for measurement. 2.3. Mesophyll conductance measurements Mesophyll conductance was estimated using a coupled leaf photosynthesis system (LI-6400xt, as above) and stable carbon isotope tunable diode laser (TGA100A, Campbell Scientific, Logan UT, USA) as described by Barbour et al. [1]. The Li-6400 was fitted with a 2 by 6 cm narrow leaf chamber (Li6400-11) and irradiance provided by a red-green-blue light source (Li6400-18) set to mimic the red-blue light source at an irradiance of 1250 mol m−2 s−1 PAR. Leaf temperature was controlled at 25 ◦ C and VPd between 0.8 and 1.6 kPa. Two or three of the youngest fully expanded leaves were placed side by side in the leaf chamber. gm was calculated from the difference between predicted discrimination assuming infinite mesophyll conductance (i ) and measured discrimination (obs ), using equations developed by Evans et al. [29] and Barbour et al. [1], and including a ternary effect as described by Farquhar and Cernusak [30]. We assume values of fractionation factors; carboxylation fractionation was assumed to be 29‰, fractionation during dissolution and diffusion through water was assumed to be 1.8‰, the fractionation associated with photorespiration was assumed to be 16.2‰, the fractionation occurring during diffusion through the leaf boundary layer was assumed to be 2.9‰ and the rate of day respiration was taken to be 1.5 mol m−2 s−1 (from Jahan et al. [4]). 2.4. Statistical analysis Differences between the various physiological measures were assessed using general analysis of variance, as implemented by GenStat 14th edition, and means were compared using Bonferroni significant difference test. Differences were considered statistically
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3. Results 3.1. Plant growth As expected, above-ground growth was generally higher under high N compared to adequate N, and reduced under drought (Fig. 1). However, there were also significant interactive effects. For head biomass pre-anthesis, there was no significant nitrogen effect but there was a significant genotype by water availability interaction (P = 0.050) with a strong drought effect for LPB10-0018 but non-significant effects for other genotypes. There were significant differences between genotypes for leaf biomass (P > 0.001) with Gregory having the highest biomass under well-watered conditions, followed by LPB10-0018, Sunguard and Livingston with similar leaf biomass, then Spitfire and finally Dart with the lowest leaf biomass. There were also significant genotype by water (P < 0.001) and nitrogen by water (P = 0.018) interactions. Gregory, LPB10-0018 and Sunguard had greater reductions in leaf biomass under water limitation than did Dart and Spitfire. Across all genotypes the reduction in leaf biomass due to water limitation was greater at high N availability than at adequate N. There were also significant differences between genotypes for stem biomass (P < 0.001) with LBP10-0018 having the highest biomass under well-watered conditions, followed by Sunguard, Spitfire, Gregory and Dart with similar stem biomass, then Livingston with the
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lowest stem biomass. There were also significant nitrogen by water availability (P < 0.001) interactive effects. Below ground biomass was also significantly affected by water and nitrogen availability. There was a significant genotype by water availability interaction (P = 0.016), with greater root biomass for droughted Spitfire plants compared to well-watered plants, while all other genotypes drought did not significantly affect root biomass. There was also a significant nitrogen by water availability interaction (P < 0.001) for root biomass. In general, under wellwatered conditions root biomass was higher for high N compared to adequate N, while under drought conditions root biomass was considerably higher under adequate N compared to high N for Spitfire (P, 0.05) but differences were statistically insignificant for other genotypes. These biomass effects resulted in significant differences in biomass allocation under the differing growth conditions. There was a significant genotype by water by nitrogen availability interaction (P < 0.001) for root to shoot ratio (Fig. 2). Dart was the least responsive to water and nitrogen limitation in terms of biomass allocation, with no significant differences in root/shoot across the treatments. Conversely, Spitfire allocated more than twice its biomass below ground under the combined drought and adequate N treatment (root/shoot is 2.66), but about half its biomass below ground under well-watered and high N conditions (root/shoot is 0.58). Significant genotype by water by nitrogen availability (P < 0.001) interactive effects were also found for the ratio of leaf to stem biomass (Fig. 2). Gregory allocated more biomass to leaves compared to stems than all other genotypes, and Dart allocated the least. In general, leaf/stem was higher for droughted compared to well-watered plants, except for Gregory and Sunguard which had higher leaf/stem for well-watered plants grown at adequate N compared to droughted plants at adequate N.
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A
B
C
Fig. 3. Growth environment effects on leaf water potential (A) and leaf gas exchange for six wheat genotypes. Values are mean, ± standard error, n = 5.
Using above ground biomass sampled prior to the start of the water availability treatment and the final harvest from the same pots, estimates of above-ground relative growth rate were made (Fig. 2C). Significant differences were found between cultivars (P < 0.001), Gregory, LPB10-0018 and Sunguard had the highest growth rates, and Dart and Spitfire had the lowest growth rates. There was also a significant water by nitrogen interaction (P = 0.003). Adequate nitrogen significantly reduced growth rate compared to high N under well-watered conditions (for Gregory, LPB10-0018, Sptifire and Sunguard) but not under drought conditions (except for LBP10-0018).
3.2. Leaf gas exchange As expected, water stress lowered leaf water potential (L Fig. 3). There were significant differences between cultivars (P < 0.001), Dart had the lowest L and Livingston the highest (other genotypes did not differ significantly from these two genotypes). There was also a significant nitrogen by water availability interactive effect (P < 0.001), where droughted plants at high N had lower L than droughted plants at adequate N. Genotypes had significantly different photosynthetic rates (A, P < 0.001), with Gregory having the highest and Spitfire the lowest overall average A, although genotypes did not differ significantly for A at any given growth treatment. There were significant nitrogen by water availability treatment effects for A (P < 0.0001) and stomatal conductance (gs , P < 0.001). The reduction in gs due to drought was stastically significant for Dart, Gregory, Livingston, Spitfire and Sunguard at high N, but the drought effect was not statistically
Fig. 4. Relationships between mesophyll conductance (gm) and other leaf gas exchange values for six wheat genotypes grown under four conditions of water and nitrogen availability. Values are mean, ± standard error, n = 5. Lines are least squares linear regression, with correlation coefficient and significance level provided.
significant for any genotype at adequate N, matching the effects of growth environment on L . We found a significant interactive effect of genotype by nitrogen by water availability (P < 0.001) for mesophyll conductance (gm ). Except for the genotypes Dart and Spitfire at adequate N, drought reduced gm (although it was statistically significant for Gergory at adequate N only). N availability did not have clear effects on gm at a given water availability for any genotypes except Gregory, for which gm at adequate N was higher than at high N under wellwatered conditions. The cultivars responded to drought differently in terms of leaf intrinsic water-use efficiency (A/gs , P = 0.004). There was a large increase in A/gs under drought and high N for Spitfire and Sunguard while Dart, Livingston and LPB10-0018 were less sensitive. There was a significant water by nitrogen availability interaction (P < 0.001) for A/gs ; the increase in A/gs in response to drought was greater for high N plants than for plants grown at adequate N. 3.3. Correlations between mesophyll conductance, leaf properties and gas exchange parameters When genotype and treatment averages were calculated, mesophyll conductance was positively related to A (P = 0.002, Fig. 4A) and gs (P = 0.003, Fig. 4B) and negatively related to A/gs (P = 0.0018, Fig. 4C). gm was also positively related to L (P = 0.001, Fig. 5A) and leaf N content (P = 0.008, Fig. 5B), but unrelated to leaf mass per unit
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area (Fig. 5C). So, high gm was found together with high A, gs , L and leaf N content. In this experiment, which included a moderate drought, A/gs was more strongly driven by gs than by A, resulting in a negative relationship between gm and A/gs .
A
3.4. Photosynthetic parameters and limitations Photosynthetic response curves to CO2 concentration were measured for three genotypes and, when combined with estimates of gm , allowed for the fitting of maximum carboxylation rate (Vcmax ), maximum electron transport rate (Jmax ), and stomatal (Ls ) and mesophyll (Lm ) conductance limitations to photosynthesis. We chose to calculate Ls and Lm at an ambient CO2 concentration of 400 mol mol−1 , to match the growth conditions. There were significant genotype differences in Vcmax (P = 0.017; Gregory had higher Vcmax than Livingston and Spitfire, Table 1) but no significant water or nitrogen availability effects. There were also significant genotype differences for Jmax (P < 0.001; Livingston had higher Jmax than Gregory or Spitfire), and significant nitrogen by water limitation interactions (P = 0.044). There was a nitrogen effect on Jmax for droughted plants only, in which Jmax was higher for adequate N than for high N. Mesophyll conductance was fitted from the CO2 response curves using the function developed by Sharkey et al. [27], with fitted values restricted to within a given range. The default range in fitted values is 0–3 mol m−2 s−1 bar−1 , and was used in the current study. Fitted gm varied between the upper limit and 0.26 mol m−2 s−1 bar−1 , but was unrelated to gm measured using the carbon isotope discrimination method (Fig. 6). A comparison of fitted Vcmax and Jmax using known gm , with values for which gm was also fitted revealed differences between the estimates. The average difference between the two estimates of Vcmax was only 1 mol m−2 s−1 , but this difference was significantly affected by water availability (P = 0.006), with droughted plants having an underestimate of Vcmax with fitted gm (on average, an underestimate of 8 mol m−2 s−1 , P = 0.034), and well-watered plants having an overestimate of Vcmax when gm was fitted (on average 10 mol m−2 s−1 , P = 0.015). There was also considerable
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Fig. 5. Relationships between mesophyll conductance (gm) and leaf traits for six wheat genotypes grown under four conditions of water and nitrogen availability. Values are mean, ± standard error, n = 5. Lines are least squares linear regression, with correlation coefficient and significance level provided.
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Table 1 Fitted values for maximum carboxylation rate (Vcmax , in mol m−2 s−1 ), electron transport rate (Jmax, in mol m−2 s−1 ), and stomatal (Ls , dimensionless) and mesophyll (Lm , dimensionless) limitations to photosynthesis for three wheat genotypes grown under four environments of water and nitrogen availability, using measured mesophyll conductance for individual plants. Values are mean, ± standard error, n = 5. Growth treatment
Parameter Gregory
High nitrogen, well-watered
Vcmax
131 ± 3
131 ± 5
151 ± 10
Jmax Ls Lm
199 ± 6 0.17 ± 0.01 0.06 ± 0.01
194 ± 7 0.17 ± 0.01 0.06 ± 0.01
184 ± 10 0.13 ± 0.01 0.08 ± 0.01
Vcmax
118 ± 14
152 ± 7
155 ± 22
Jmax Ls Lm
163 ± 21 0.30 ± 0.02 0.09 ± 0.01
209 ± 6 0.28 ± 0.02 0.14 ± 0.03
181 ± 19 0.36 ± 0.05 0.21 ± 0.06
Vcmax
110 ± 6
146 ± 9
131 ± 5
Jmax Ls Lm
170 ± 10 210 ± 11 170 ± 5 0.13 ± 0.02 0.18 ± 0.01 0.14 ± 0.01 0.03 ± 0.004 0.06 ± 0.004 0.07 ± 0.01
Vcmax
147 ± 9
156 ± 10
144 ± 9
Jmax Ls Lm
193 ± 10 0.22 ± 0.03 0.09 ± 0.01
227 ± 11 0.24 ± 0.01 0.09 ± 0.01
189 ± 7 0.30 ± 0.06 0.16 ± 0.05
High nitrogen, droughted
Adequate nitrogen, well-watered
Adequate nitrogen, droughted
Livingston
Spitfire
scatter in the relationship between the two estimates of Vcmax (Fig. 7), suggesting that fitting gm resulted in large under- and over-estimates for Vcmax of some individual leaves. In contrast, there was not wide scatter in the relationship between the two estimates of Jmax (Fig. 7). But, like Vcmax , water availability significantly (P = 0.045) affected the difference between the two estimates. Under droughted conditions fitting gm resulted in a 4.6 mol m−2 s−1 under estimate of Jmax compared to Jmax with known gm (P = 0.007), while under well-watered conditions there was no difference between the two estimates. The genotypes responded differently to water stress in terms of stomatal limitation to photosynthesis (Ls ); there was a stronger increase in Ls due to drought for Spitfire than either Gregory or Livingston (Table 1). There was also a significant nitrogen effect on Ls , with higher limitation at high N than at adequate N (P = 0.023). The mesophyll limitation to photosynthesis (Lm ) was stronger for droughted compared to well-watered plants (P < 0.001). There was also a significant difference (P = 0.006, Table 1) in Lm between genotypes, with Spitfire having the highest Lm and Gregory the lowest. 3.5. 13 C of whole leaf tissue, and relationships with leaf gas exchange The carbon isotope composition of the youngest fully expanded leaf at the final harvest varied between cultivars and was affected by both nitrogen and water availability. Decreased nitrogen availability increased 13 C, while drought reduced 13 C. There was a significant nitrogen by water availability interactive effect (P = 0.005), with a stronger effect of nitrogen availability under drought. The cultivars also responded differently to water availability (P = 0.018); Sunguard responded the most to drought with a 3.1‰ decrease from well-watered to droughted plants, while Gregory decreased by 2.0‰, and Livingston and LPB10-0018 were the least sensitive with a decrease of just 0.7 and 0.8‰, respectively. Across all growth environments, genotype average 13 C was negatively related to genotype averages from gas exchange measurements: A/gs , the ratio of intercellular to ambient CO2 partial pressure (Ci /Ca ) and ratio of chloroplastic to ambient CO2 partial
Fig. 7. The relationships between photosynthetic parameters (maximum carboxylation rate, Vcmax and electron transport rate, Jmax ) when fitted with known values of mesophyll conductance (gm ) and when gm was also fitted for three wheat genotypes grown under four growth environments. In both cases the procedure described by Sharkey et al. [27] was used, and there are five replicate plants for each genotype and growth environment. Values are for individual plants.
pressure (Cc /Ca ; Fig. 8). The closest correlation was between 13 C and A/gs , with an r2 of 0.71. 4. Discussion 4.1. Genotypes differ in their response to N availability, drought and the combined stresses The interactive effects of N and water availability on growth observed here confirm many previous studies (reviewed in Sadras [31] and Gonzalez-Dugo et al. [32]). Here, above ground relative growth rate was reduced by limited water availability more at high N availability than at adequate N. Also expected were differences between genotypes for growth rate and biomass allocation, photosynthesis and stomatal conductance and leaf intrinsic water-use efficiency under the four growth environments. The new information provided by this experiment is the interactive effect of N and water availability on mesophyll conductance, and the observation that the genotypes differ in their response to the growth environments. Overall, drought reduced gm and this response has been seen in many, but not all, previous experiments (see Flexas et al. [24] for a review). However, no genotypes grown at adequate N showed significantly reduced gm under drought. The genotypes also responded differently to N availability. Reduced N availability from high to adequate N generally did not alter gm , but the genotype
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Fig. 8. Relationships between leaf gas exchange (A, leaf intrinsic water-use efficiency; B, the ratio of intercellular to ambient CO2 partial pressures; C, the ratio of chloroplastic to ambient CO2 partial pressures) and the carbon isotope discrimination in leaf tissue. Values are mean, ± standard error, n = 5.
Gregory had higher gm at adequate N than at high N under droughted conditions. The positive relationship between leaf N content and gm found in the current study supports earlier observations for a range of crop species [17–20]. If leaves are treated individually, leaf N content explained 32% of measured variability in A but only 11% of measured variability in gm , while (again for individual leaves) variation in gm explained 33% of variation in A. With three-quarters of leaf N associated with photosynthesis (mostly Rubisco and chlorophyll, [33]) the relationship between N and gm may simply reflect the relatipnship between A and gm . However, the changes in gm in response to N availability and drought may have been due to changes in leaf anatomy [19,20]. The surface area of chloroplasts exposed to the intercellular air space (Sc ) has been shown to be positively related to gm across different species, while cell wall thickness was negatively related [34]. In Populus tremula, water stress reduced Sc and increased cell wall thickness, and these changes combined to reduce gm [35]. The reduced gm in response to drought in combination with limiting nitrate availability in rice was related to reductions in chloroplast size [17,18], which presumably also reduced Sc . High nitrogen availability increased chloroplast surface area per unit leaf area and reduced cell wall thickness in rice [20], and may have altered these traits here in wheat. Future studies should quantify genotypic variability in leaf anatomy in response to growth conditions in order to understand changes in genotype ranking of gm . Changes in gm under nutrient- or water-limited conditions may also result from changes in enzyme activity. Two enzymes have been implicated in regulation of gm , namely carbonic anhydrase and membrane aquaporins. Carbonic anhydrase (CA) catalyses the reversible conversion of CO2 to HCO3 − and is found in multiple forms and high abundance in the chloroplast, cytosol, mitochondria and plasma membrane of C3 plants [36]. While the importance of CA to gm has not been widely studied, the diffusivities of CO2
and HCO3 − do differ, so CA activity may play a role in gm [37]. The potential role for aquaporins in regulating gm is much clearer. gm in faba decreased significantly when leaves were fed with the aquaporin specific inhibitor HgCl2 [38]. Aquaporin over- and underexpressing lines display higher and lower gm , respectively, than their parental wildtypes in rice [39] and tobacco [40]. Some aquaporins have been shown to conduct CO2 [41]. It has been suggested that the composition of aquaporin tetramers in the membrane may facilitate differences in conductance of CO2 versus H2 O, compared to those with single isoform combinations [42]. While it is clear that water transport across membranes responds to water availability due to changes in aquaporin expression and activity [43,44], changes in the CO2 diffusivity of membranes in response to environmental conditions is less clear [37]. However, aquaporins remain a likely candidate for part regulation of gm . The interactive effects of N and drought observed for many plant traits have been shown here to be observed for mesophyll conductance in wheat, with genotype ranking depending on the growth environment. This suggests that (1) if gm is to be included as a trait in breeding programs [1,4], care must be taken to match the selection environment to the ultimate growth environment and (2) the underlying mechanism or mechanisms responsible for the observed genotypic variability in gm in response to growth environment should be determined. As suggested above, both leaf anatomy and aquaporin expression/activity/location are likely candidates to explain differences between genotypes. 4.2. Relative limitations on photosynthesis by stomata and mesophyll conductances depend on growth environment In wheat the limitation on photosynthesis imposed by stomatal conductance was, on average, slightly more than double the limitation imposed by mesophyll conductance. This is similar to that reported by Jahan et al. [4] under non-limiting conditions. When
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plants were droughted, the stomatal and mesophyll imitations both increased, but the degree of increase was greater for Ls than for Lm . Interestingly, there were significant differences between genotypes in the sensitivity of Ls to water stress; Spitfire was more sensitive to water stress than Gregory or Livingston. Consistent with limited change in actual values of gm in response to N availability, there were no significant N effects on Lm . However, Ls was reduced by lowered N availability. Photosynthesis was most limited by mesophyll diffusion of CO2 for Spitfire and least limited for Gregory, consistent with genotype rankings for photosynthetic rate itself. The lack of genotype differences in Ls reflects the lack of genotype differences in stomatal conductance. Considering diffusional photosynthetic limitations and selection for high water-use efficiency, it is logical that the combination of relatively high Ls and relatively low Lm should produce a plant with high WUE if photosynthetic capacity is constant. However, when the ratio of Ls to Lm from A/Ci curve analysis was compared to A/gs from instantaneous gas exchange measurements, there was no relationship between Ls /Lm and A/gs , even when leaves were grouped into similar Vcmax (data not shown). It seems when water availability is the key driver of variation in A/gs for wheat, stomatal limitations are much more important in determining water-use efficiency than mesophyll limitations [2], and indeed variation in Ls explained 39% of variation in A/gs while variation in Lm explained just 17% (data not shown). 4.3. Photosynthetic parameters are underestimated under drought when mesophyll conductance is unknown The photosynthetic parameters Vcmax and Jmax are central to models of photosynthesis such as the widely-used model of Farquhar et al. [45]. It has been recognized for some time that failure to account for gm will introduce significant errors in parameter estimation [46–48]. The parameter fitting procedure introduced by Sharkey et al. [36] attempted to overcome this problem by fitting gm at the same time as Vcmax and Jmax . However, the current study shows that Vcmax and Jmax are significantly underestimated under drought conditions (7% and 3% for Vcmax and Jmax respectively) and that Vcmax is overestimated (7%) under well-watered conditions when gm is unknown and fitted along with Vcmax and Jmax , as compared to using known gm values when fitting Vcmax and Jmax . Further, fitted gm was unrelated to measured gm , and fitted gm was often unrealistically high. Measurements of gm are recommended for accurate estimation of photosynthetic parameters, particularly under water limited conditions. One aspect that has not been addressed here is whether gm changes with CO2 concentration. A number of studies have found that gm declines as leaf internal CO2 partial pressure increases above 200 mol mol−1 [11,49,50]. However, Tazoe et al. [51] have shown that gm does not respond to CO2 in wheat. We have assumed that gm is constant throughout the response curve measurements, but this assumption has not been tested. Flexas et al. [24] assessed the effect of variable gm on CO2 response curves, and concluded that the effect may be substantial when gm is very low such as under severe water stress. Further work is warranted. 4.4. Mesophyll conductance and water-use efficiency When considering selection of genotypes for water-limited conditions, the combination of high carboxylation rate, relatively low gs but high gm , and high relative growth rate would provide the highest leaf-intrinsic water-use efficiency while still allowing for high productivity [1]. Of the genotypes studied here, Gregory has the highest Vcmax , A and gm , among the highest pre-anthesis relative growth rate, and the lowest Lm . Dart and Spitfire sit at the other end of the trait spectrum. The strong relationship between
gm and A suggests that mesophyll conductance can contribute to selection for high photosynthetic rate, however this study demonstrates that under water-limited conditions stomatal conductance has a stronger effect on leaf intrinsic water-use efficiency than does mesophyll conductance. Given the technical challenges of mesophyll conductance measurement, particularly in the field, a more effective selection strategy may be sampling leaf tissue for carbon isotope analysis via mass spectrometry combined with either photosynthetic rate or leaf N content as a proxy for photosynthetic rate. Use of 13 Cl as a selection trait has been particularly successful in wheat, providing the basis for the release of two commercial cultivars [51]. There are problems with interpreting variation in 13 Cl when gm is unknown, as discussed by Barbour et al. [1], but the strong relationship between A and gm found here in wheat, and earlier in a range of species [2], suggests that these concerns may be minor. A highly water-use efficient genotype, but one that is still productive, would be one with low 13 Cl combined with high leaf N content (or high A), and this would indirectly select for high gm . Based on this logic, we expect ‘Drysdale’ (the high water-use efficiency cultivar released using 13 Cl selection [52]) to have high gm , high leaf N content and high photosynthetic rate; a hypothesis to be tested in the future. Harvest index is a further complicating factor; genotypes that have desireable leaf traits during the pre-anthesis growth period in controlled conditions may not end up with high grain yield per unit water used at harvest if allocation to grain development and grain filling is not high. Field testing through to harvest is a necessity. In conclusion, wheat genotypes differ in their response to limited water and nitrogen availability in terms of leaf gas exchange, including mesophyll conductance. In this study, leaf intrinsic water-use efficiency was more closely related to stomatal than to mesophyll conductance. Mesophyll limitation to photosynthesis differed between genotypes and increased when plants were water-limited, while stomatal limitation to photosynthesis increased more in response to drought in the genotype Spitfire than in other genotypes. Selection for increased water-use efficiency through high mesophyll conductance should include screening under a range of growth conditions.
Acknowledgements This work was supported by the Grains Research and Development Corporation (contract US00056). Erin Lockhart, William Lin and Svetlana Ryazanova are thanked for technical assistance and Dr. Claudia Keitel for mass spectrometric analysis.
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Please cite this article in press as: M.M. Barbour, B.N. Kaiser, The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes, Plant Sci. (2016), http://dx.doi.org/10.1016/j.plantsci.2016.03.012