Intraspecific variation in thermal acclimation of photosynthesis across a range of temperatures in a perennial crop.

Interest in the thermal acclimation of photosynthesis has been stimulated by the increasing relevance of climate change. However, little is known about intra-specific variations in thermal acclimation and its potential for breeding. In this article, we examined the difference in thermal acclimation between alfalfa (Medicago sativa) cultivars originating from contrasting origins, and sought to analyze the mechanisms in play. A series of experiments was carried out at seven growth temperatures between 5 and 35 °C using four cultivars from temperate and Mediterranean origin. Leaf traits, the photosynthetic rate at 25 °C (A400 (25)), the photosynthetic rate at optimal temperature (A400 (opt)), the thermal optimum of photosynthesis (Topt), and the photosynthetic parameters from the Farqhuar model were determined. Irrespective of cultivar origin, a clear shift in the temperature responses of photosynthesis was observed as a function of growth temperature, affecting thermal optimum of photosynthesis, photosynthetic rate at optimal temperature and photosynthetic rate at 25 °C. For both cultivars, Topt values increased linearly in leaves grown between 5 and 35 °C. Relative homeostasis of A400 (25) and A400 (opt) was found between 10 °C and 30 °C growth temperatures, but sharp declines were recorded at 5 and 35 °C. This homeostasis was achieved in part through modifications to leaf nitrogen content, which increased at extreme temperatures. Significant changes were also recorded regarding nitrogen partitioning in the photosynthetic apparatus and in the temperature dependence of photosynthetic parameters. The cultivars differed only in terms of the temperature response of photosynthetic parameters, with Mediterranean genotypes displaying a greater sensitivity of the maximum rate of Rubisco carboxylation to elevated temperatures. It was concluded that intra-specific variations in the temperature acclimation of photosynthesis exist among alfalfa cultivars, but that Mediterranean genotypes presented no evidence of superior performance at high temperatures. Published by Oxford University Press on behalf of the Annals of Botany Company.

Introduction

Because plants cannot move, adaptation of their photosynthetic characteristics is essential to maximize performance at their growth temperature (Tgrowth) (Berry and Bjorkman 1980; Sage and Kubien 2007; Way and Yamori 2014; Yamori ). In addition to short-term responses of photosynthesis, the thermal acclimation of the photosynthetic system has long been acknowledged as a phenomenon (Berry and Bjorkman 1980; Mooney ). It is now seen as a critical process to predict impacts of climate change (IPCC 2013 and infer species adaptation (Gunderson ; Kattge and Knorr 2007).

Berry and Bjorkman (1980) defined photosynthetic acclimation as ‘environmentally induced changes in photosynthetic characteristics that result in an improved performance under the new growth regime’. The thermal acclimation of photosynthesis has been associated with modifications to several photosynthetic variables (Way and Yamori 2014) which include: (i) shifts in the thermal optimum (Topt) of photosynthesis toward a new Tgrowth, (ii) a relative homeostasis of the maximum photosynthetic rate between Tgrowth (Cowling and Sage 1998; Gunderson ) and (iii) altered photosynthetic characteristics measured at 25 °C, such as the ratio between the maximum electron transport rate (Jmax) and the maximum rate of Rubisco carboxylation (Vcmax) (Berry and Bjorkman 1980; Leuning 1997, 2002; Medlyn ). Growth temperature is also known to affect leaf expansion (Louarn ) and the structure of mature leaves (Bula 1972; Hanson ; Ku and Hunt 1973). Ultimately, growth effects alter leaf traits, such as the final leaf area (Larea), the specific leaf area (SLA) and the amount of nitrogen per unit area (Na) (Field and Mooney 1986; Garnier ; Hikosaka 2004; Reich ), which are tightly related to photosynthetic capacity (Evans 1989; Yamori ). These growth responses may in part be adaptive, and contribute to the thermal acclimation of photosynthesis (Onoda ; Sage and Kubien 2007).

Inter-specific differences in the thermal acclimation of photosynthesis have been studied extensively within each of the different photosynthetic pathways (Sage and Kubien 2007; Yamori ). Among C3 species, significant inter-specific differences have been reported relative to the magnitude of responses (Bunce 2000; Hikosaka ; Mooney 1980; Yamasaki ; Yamori , 2014). These differences were to some extent linked to the ecological thermal niches of species. For instance, cold-tolerant herbaceous species displayed a more marked degree of maximal temperature homeostasis and a greater ability to shift Topt than cold-sensitive ones (Yamori ). Among trees, species adapted to habitats with broader seasonal or diel temperature variations have also been reported to have greater capacities for acclimation (Cunningham and Read 2002). The mechanisms underpinning these inter-specific differences have not been fully elucidated. However, differences in the capacity to balance nitrogen partitioning in the photosynthetic apparatus with growth temperature were associated with differences in thermal acclimation among herbaceous species (Hikosaka ; Onoda ; Yamori ). Differences in the ability to adjust the temperature dependencies of photosynthetic reactions limiting CO2 assimilation, namely the rates of ribulose-1,5-biphospate (RuBP) carboxylation and regeneration, were also identified (Bunce 2000). Finally, differences have been reported in the heat stability of Rubisco activase, resulting in the de-activation of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) at lower temperatures in species adapted to cold environments (Hikosaka ; Salvucci and Crafts-Brandner 2004; Sage ).

By comparison, the study of intra-specific variations in thermal acclimation has received far less attention. A couple of studies showed that differences exist among ecotypes (Bjorkman ; Ishikawa ; Mooney 1980; Pearcy 1977). However, little is known about the range of plasticity existing within a species compared to the inter-specific range, in part because most previous studies were limited to comparing two Tgrowth only. Furthermore, positive relationships were found between the acclimation potential and the altitude or thermal regime of ecotypes in some (Ishikawa ), but not all species (Gunderson ; Teskey and Will 1999). Nevertheless, it is still necessary to determine how intra-specific diversity in terms of photosynthesis acclimation might be related to the ability of a species to occupy a broad range of habitats and achieve this acclimation.

The objectives of this study were to characterize the differences in thermal acclimation between alfalfa (Medicago sativa) cultivars originating from contrasting temperate and Mediterranean areas, and to analyse the mechanisms in play. Alfalfa is a temperate perennial forage legume with a broad geographic distribution that ranges from Northern Europe and Canada to North Africa and Florida (Michaud ). It can generally cope with cold winters, as well as hot dry summers, and could potentially express a broad thermal acclimation of photosynthesis (Yamori ). The species has been shown to present significant genetic diversity for heat and cold tolerance (McKenzie ) as well as maximal photosynthesis at 25 °C (Delaney and Dobrenz 1974; Heichel ). We compared the thermal acclimation of two cultivars and two clones propagated from cuttings at seven growth temperatures between 5 and 35 °C. Thermal acclimation was analysed in terms of leaf traits, thermal optimum, maximum photosynthesis and photosynthetic parameters (Vcmax, Jmax and their temperature dependencies) at each Tgrowth. The Farquhar model (Farquhar ) was used to infer the relative importance of the different photosynthetic parameters to the thermal acclimation observed.

Methods

Plant materials and growing conditions

A series of experiments was performed in a 8.1 m2 growth chamber (model 97132/7NU, Froids et Mesures, Beaucouzé, France) at the INRA Lusignan research station, France. Independent experiments were carried out successively at seven growth temperatures (Tgrowth), ranging from 5 to 35 °C with 5 °C increments. During each experiment, two alfalfa (M. sativa) commercial cultivars (Harpe and Barmed from temperate and Mediterranean origin, respectively) and two clones propagated from stem cuttings (G3 and 7_7 clones isolated from temperate and Mediterranean cultivars Orca and Demnate, respectively; Maamouri et al. 2015) were used. Because alfalfa cultivars are synthetic varieties (i.e. populations of half-sibs containing significant genetic diversity; Julier ), clones were selected in order to replicate identical genotypes in the different experiments. One clone and eight seedlings of each cultivar were replicated four times in a random block design.

The cuttings were produced in a greenhouse about 3 months before each experiment. Seeds of the synthetic varieties were pre-germinated in the dark at 25 °C for 96 h before each experiment. Seedlings and clone cuttings were transplanted individually into 1.5 L pots (10 × 20 cm cylindrical pots) filled with fine quartz (0.8–1.4 mm mesh). The pots were ferti-irrigated with a complete nutrient solution at intervals ranging from three times (5 °C) to eight times a day (35 °C). At the 15 °C Tgrowth, a problem encountered during the propagation of the cuttings prevented their study.

Two phases were distinguished during each experiment. First, a conditioning period at 25 °C and 70 % relative humidity was applied for 3 weeks. At the end of this period, plant development had achieved five leaves on the main stems of seedlings, and 8–11 leaves on cuttings. The plants were then placed at the studied Tgrowth for a period corresponding to five phyllochrones. The air temperature was adjusted to ensure a daily average leaf temperature of Tgrowth. Day/night temperatures were thus 5/3, 10/8, 15/13, 20/18, 25/23, 30/28 and 35/33 °C at Tgrowth of 5, 10, 15, 20, 25, 30 and 35 °C, respectively. The vapour pressure deficit (VPD) was maintained below 1.5 kPa at all Tgrowth by adjusting the relative humidity. Lights were set on a 14-/10-h (light/dark) photoperiod (POWERSTAR, HQI-BT 400WD lamps, OSRAM, Munich, Germany). The photosynthetic photon flux density (PPFD) measured at the pot level ranged from 400 to 450 µmol photon m−2 s−1.

Gas exchange measurements and determination of photosynthetic parameters

At the end of each experiment, gas exchange measurements were performed using a portable photosynthesis system (LI-6400, Li-Cor, Lincoln, NE, USA) with a 6 cm2 (2*3 cm) cuvette. The youngest mature leaf on the main stem (i.e. node ranks 8–9 for seedlings and 12–16 for cuttings) were used for each plant [.

Light saturated net photosynthesis at ambient CO2 (400 ppm, PPFD of 1500 µmol photon m−2 s−1) and dark respiration were measured at 25 °C (A40025) and T (A400growth) on four (clones) or eight (seedlings) plants per cultivar. In addition, the responses of A to internal CO2 concentration (Ci) at the substomatal level (A–Ci curves) were determined on the cuttings leaves. Different levels of Ci were obtained by modifying the ambient CO2 concentration (Ca) in the leaf measurement chamber. The A-Ci curves were compiled as proposed by Long and Bernacchi (2003). First, the value of A at the actual Ca level was recorded, and then Ca was gradually reduced to five different levels below the ambient concentration. Thereafter, Ca was returned to the initial value and increased to seven different levels up to 1500 µmol.mol−1. Each Ca step was maintained for 5 minutes in order to record stable values. All curves were compiled at 1500 µmol m−2 s−2 PPFD, the leaf temperature was controlled at 25 °C and the VPD between the leaf and the air was kept at 1 ± 0.5 kPa. The photosynthetic parameters (Vcmax and Jmax) were estimated simultaneously by fitting the biochemical model developed by Farquhar to the whole A–Ci curve, according to the procedure proposed by Sharkey . Overall, A–Ci curves and photosynthetic parameters were determined at five different leaf temperatures (Tgrowth, 10, 25, 35 and 42 °C) for each clone (G3 and 7_7) and each growth temperature.

Leaf traits

Immediately after the gas exchange measurements, the leaves were scanned (Konica Minolta C352/C300, Konica Minolta Sensing, Osaka, Japan). The leaf area (Larea) was determined by image analysis (ImageJ software, http://rsbweb.nih.gov/ij/, last accessed 31 May 2016). The leaves were then dried at 60 °C for 2 days, weighed to determine their dry mass and ground in a vibrating ball mill (MM400, Retsch GmbH and Co, Haan, Germany). Leaf samples were analyzed with an elemental analyser (model EA 1108, Carlo Erba Instruments, Milan, Italy) to determine their N concentration. The SLA (m2 g−1) and leaf nitrogen content per unit of area (Na, g N m−2) were then calculated.

Thermal optimum and determination of the temperature dependencies of photosynthetic parameters

The response curves were fitted to temperature using the nls procedure under R software (R Development Core Team 2005). The thermal optimum and response to temperature of light saturated net photosynthesis at 400 ppm CO2 were determined using a beta function (Equationn 1, Yan and Hunt 1999):

The temperature dependencies of Vcmax and Jmax were fitted using the Arrhenius model if there was an exponential increase with temperature (Equation 2), or using a modified Arrhenius function if a significant decline was measured at high leaf temperatures (Equation 3.): where r is the rate normalized by the parameter value at 25 °C, C is equal to 1 + exp[(ΔS.T −ΔHd)/(R.T0)] (no dimension), T1 is the leaf temperature.

Determination of the limiting steps of photosynthesis

The model developed by Farquhar was used to infer the limiting steps of photosynthesis in the response of A to temperature under the different combinations of genotypes and growth temperatures studied. The version implemented on the OpenAlea Modelling Platform was used (Prieto ) with the default parameters for alfalfa (Louarn ). All the variables, parameters and symbols used are detailed in Appendix 1. The Farquhar model assumes that the net photosynthetic rate is limited by either the activation state, quantity and kinetic properties of Rubisco (Ac) or RuBP regeneration in the Calvin cycle (Ar):

It was assumed that the kinetic properties of Rubisco (Kc, Ko, and Γ* which depend on the specificity factor of the Rubisco for CO2 and O2) were constant between genotypes and growth temperatures (Bernacchi ; Harley ; Sharkey ).

Ci was calculated from measured Ca and leaf temperature by coupling the semi-empirical stomatal conductance model proposed by Ball et al. (1987) to the Farquhar model. Rd was calculated from measured values of dark respiration at 25 °C (Rd25) and an Arrhenius function accounting for its temperature dependence. The set of gas exchange parameters estimated by Louarn et al. (2015) was used.

For each genotype and Tgrowth, the photosynthetic parameters and their temperature dependencies were used to examine the relationship between changes in the A400 response to temperature and changes in parameter values. First of all, simulations were performed using the parameters actually measured in each situation. Then, three series of simulations were conducted to assess the sensitivity of Topt and the overall temperature response shape to photosynthetic parameters: (i) assuming a constant ratio between Jmax25 and Vcmax25, (ii) assuming unchanged temperature dependencies for Vcmax and Jmax and (iii) assuming both a constant ratio and unchanged temperature dependencies.

Statistical analyses

Statistical analyses were performed using R software (R Development Core Team 2005). Analyses of variance (ANOVA, aov procedure) were used to test for significant differences between means and assess effects of temperature treatments and genotype on the values of leaf traits (Larea, SLA and N), assimilation rates (A40025) and photosynthetic parameters (Vcmax and Jmax). Multiple comparison tests were performed using the LSD procedure. Linear regression lines were fitted to the data using the lm procedure. Linear relationships were tested between observed and predicted A400 and Topt values and were compared with the theoretical 1:1 line. In addition, the model error (RMSE) and bias (Bias) were calculated as follow:

Results

Impact of growth temperature on leaf growth and leaf nitrogen content

The impacts of growth temperature (Tgrowth) on final leaf size (Larea), SLA and leaf nitrogen content (Na) are presented in Figure 1. Growth temperature significantly affected the three leaf traits studied (ANOVA, F6,42 > 5.9, P < 10−3) in both seedling (Fig. 1) and cutting (not shown) plants. The final leaf size was highest at intermediate Tgrowth (20 and 25 °C) and was smaller at both ends of the temperature range tested (5 and 35 °C). SLA was also maximum at a moderate Tgrowth (20–30 °C). The reduction in SLA due to low Tgrowth (5 and 10 °C) appeared to be greater than that observed at high temperatures (35 °C) within the range tested. Concerning the nitrogen content, Na patterns mirrored the pattern observed for SLA. Na was lowest at a moderate Tgrowth (15–30 °C) and maximum at extreme Tgrowth values (5, 10 and 35 °C). ANOVA analyses did not demonstrate any significant differences regarding the origins of the plants in terms of Larea and SLA (ANOVA, F1,85 < 29.0, P > 0.2), but were significant for Na (ANOVA, F1,85 = 17.7, P < 0.05).

Figure 1.

Impact of growth temperature on the final leaf size, SLA and leaf nitrogen concentration of two alfalfa cultivars of temperate (open circles, Harpe cv) and Mediterranean (filled circles, Barmed cv) origins. Leaves were all sampled on the main stem between node ranks 8 and 9. The letters indicate homogeneous groups of means between the different growth temperatures (Fisher’s LSD test). No significant cultivar effect was found.

At a Tgrowth of 35 °C, symptoms of thermal stress became apparent in the form of heat-bleached leaves on some plants from the Mediterranean cultivars, but never on plants from temperate cultivars (see [). These symptoms occurred at a frequency of about 30% in the Barmed cv. and were observed on the 7_7 clone.

Impact of growth temperature on the net assimilation rate at 25 °C

The impact of growth temperature on the net rate of light saturated photosynthesis measured under standard conditions (i.e. at a leaf temperature of 25 °C, A40025) is presented in Figure 2. A significant effect of Tgrowth on A40025 was observed in both seedlings and cuttings from temperate and Mediterranean origins (ANOVA, F6,42 > 6.1, P < 10−3). A40025 remained relatively constant over a broad range of Tgrowth between 10 and 30 °C. However, a drop at extreme Tgrowth values (5 and 35 °C) was observed for both alfalfa cultivars and cuttings. The different plant materials displayed similar response patterns, but with slight differences between seedlings and cuttings. In either plant materials, Mediterranean and temperate origins did not affect A40025 between 5 and 30 °C (ANOVA, F1,69 < 1.5, P > 0.21). Mediterranean plants displayed a decreased photosynthetic capacity at the 35 °C growth temperature (P < 0.02).

Figure 2.

Impact of growth temperature on the net rate of light-saturated photosynthesis measured at 25 °C and 400 ppm CO2 (A40025) on seedlings (a) and cuttings (b) of alfalfa of Mediterranean (filled symbols) and temperate (open symbols) origins. Measurements were performed on the youngest mature leaf of the main stem at the end of the experiment (i.e. node ranks 8–9 for seedlings and 12–16 for clones). The letters indicate homogeneous groups of means between the different growth temperatures (Fisher’s LSD test).

Acclimation of net assimilation to growth temperature

The responses of A400 to leaf temperature are presented at the different growth temperatures in Figure 3. A clear shift in the temperature responses of photosynthesis was observed as a function of Tgrowth, affecting both the optimal temperature (Topt) and the net assimilation rate at T (A400opt). Over the whole range of growth temperatures, Topt (as determined by fitting a beta function, Equation 1) rose regularly from about 18 °C (for leaves grown at 5 °C) to 35 °C (for leaves grown at 35 °C). An acclimation of the response curve thus occurred in the two genotypes studied, which tended to maximize photosynthetic rates within a temperature range close to the growth temperature. However, A400opt did not remain constant in response to temperature (ANOVA, F5,13 > 7.3, P < 10−2). The maximum rate peaked for leaves grown at 25 °C and was significantly reduced with Tgrowth below 15 °C and above 30 °C. A marked difference in the maximum rates of the two genotypes was observed at a Tgrowth of 35 °C, but not with the other Tgrowth. Significant differences were also observed between the two genotypes relative to photosynthetic rates at high Tleaf (42 °C), the Mediterranean genotype displaying an accentuated decrease at 10, 25 and 30 °C Tgrowh (ANOVA, F1,10 > 4.2, P < 0.05). This resulted in slightly altered shapes of the response curves.

Figure 3.

Temperature responses of the net assimilation rate at ambient CO2 (A400) for growth temperatures of 5 °C (a), 10 °C (b), 20 °C (c), 25 °C (d), 30 °C (e) and 35 °C (f). Data are presented for two alfalfa cuttings of Mediterranean (filled triangles, 7_7 cutting) and temperate (open triangles, G3 cutting) origins. Lines represent fits of Equation (1). Each point is the average of measurements on three to four mature leaves (node ranks 12–16).

Impact of growth temperature on photosynthetic parameters and their responses to leaf temperature

The photosynthetic parameters determined under standard conditions are presented in Figure 4 at the different growth temperatures. The Vcmax25 and Jmax25 parameters were significantly affected by Tgrowth (ANOVA, F5,12 > 15.9, P < 10−3). Vcmax25 values remained constant between 10 and 30 °C, but were lower at 5 and 35 °C. The values of Jmax25, on the other hand, decreased between 10 and 30 °C and presented a relatively more limited drop at 5 °C. Overall, the Jmax25/Vcmax 25 ratio fell regularly for leaves grown at between 5 and 35 °C (Fig. 4c). No significant differences between genotypes from temperate and Mediterranean origins were observed (ANOVA, F =1.4, P > 0.29), except at 35 °C where the declines in both Vcmax25 and Jmax25 were more pronounced in the Mediterranean genotype (heat-bleached leaves).

Figure 4.

Impact of growth temperature on the maximum catalytic rate of Rubisco (Vcmax25, a), the maximum electron transport rate (Jmax25, b) and their ratio (c, Jmax25/Vcmax25) for two alfalfa genotypes of Mediterranean (filled triangles, 7_7) and temperate (open triangles, G3) origins. The letters indicate homogeneous groups of means between the different growth temperatures (Fisher’s LSD test).

At each Tgrowth, the dependencies of photosynthetic parameters on leaf temperature were also determined. The parameters of these response curves are summarized in [ Figure 5 presents three examples of these curves at contrasting growth temperatures for the two cuttings studied. The responses of Vcmax and Jmax to leaf temperature were modified by growth temperature in both genotypes. The magnitude of the normalized responses increased with the rise in growth temperature for both Vcmax and Jmax. In addition, changes to the response curves differed between the temperate and Mediterranean genotypes. Vcmax displayed a typical increasing Arrhenius response curve (Equation 2) irrespective of Tgrowth in the temperate genotype, whereas a shift toward an optimum curve (best fitted by a Johnson function, Equation 3) was observed in the Mediterranean genotype grown at 25 and 30 °C. Similarly, Jmax responses to leaf temperature appeared to be flatter in the Mediterranean genotype at the highest Tgrowth (25 and 30 °C). The rates did not exceed 1.7 at these temperatures, while they reached 2 in the temperate genotype.

Figure 5.

Temperature dependencies of the maximum rate of Rubisco carboxylation (Vcmax, a and b) and maximum electron transport rate (Jmax, c and d) of alfalfa leaves grown at different temperatures. Data are given for two genotypes of Mediterranean (b–d, 7_7) and temperate (a–c, G3) origins at growth temperatures of 5 °C (open circles), 25 °C (grey circles) and 30 °C (filled circles). Normalizations by the rates at 25 °C were performed. The lines represent fits of either Equations (2) or (3), the parameters of which are given in [

Impact of growth temperature on the limiting step of photosynthesis

The photosynthetic parameters and their responses to leaf temperature [ were used as inputs for the Farquhar model in order to investigate their respective roles in the acclimation of photosynthesis to Tgrowth. When the whole set of measured parameters was applied, the model proved able to account for the changes in A400 observed both within (i.e. different Tleaf) and between Tgrowth (RMSE < 2 µmol m−2 s−1, no significant bias; [). In the two genotypes, the relationship between observed and predicted A400 values did not differ from the 1:1 line (t-value < 1.07, P > 0.3 for the intercept; t-value > 32.0, P < 10−3 for a slope equal to 0.95 ± 0.05). The limiting steps of photosynthesis associated with these simulations are presented in Figure 6. As expected from the response curves of the Vcmax and Jmax parameters, the temperature dependencies of the assimilation rates limited by RuBP carboxylation (Ac) and RuBP regeneration (Ar) differed at all growth temperatures. In both temperate and Mediterranean genotypes, the optimal temperature predicted by the model (arrows, Fig. 6) increased with rising Tgrowth and was closely related to observed Topt [. Except at 5 °C, Topt was determined by the intersection of A400,c and A400,r in all the situations studied. At Topt, photosynthesis was thus generally co-limited by RuBP carboxylation and RuBP regeneration. At 5 °C however, A400,r was higher than A400,c irrespective of leaf temperature (about 1.5-fold), and RuBP carboxylation systematically appeared as the limiting step. Outside the optimal temperature range, the limiting step of photosynthesis also changed as a function of Tgrowth. Under cold growing conditions (i.e. below 10 °C), A400,c fully explained the temperature dependency. In contrast, at warmer Tgrowth, A400,c generally appeared to be the limiting step above Topt, whereas A400,r limited assimilation below Topt.

Figure 6.

Predicted responses to leaf temperature of the RuBP carboxylation limited (full line, Ac) and the RuBP regeneration limited (dotted line, Ar) assimilation rates at 400 ppm CO2 and growth temperatures of 5 °C (a–g), 10 °C (b–h), 20 °C (c–i), 25 °C (d–j), 30 °C (e–k) and 35 °C (f–l). Data are for two alfalfa genotypes of temperate (a–l, G3) and Mediterranean (g–l, 7_7) origins. Arrows indicate the predicted thermal optimum Heat-bleached leaves (l) were unsuitable to derive Vcmax and Jmax parameters from gas exchange measurements, and therefore no Ac and Ar curves were simulated.

Furthermore, a sensitivity analysis was carried out to assess the role of different parameters in acclimation of the A400 response to temperature. The respective impacts of the plasticity of the Jmax25/Vcmax 25 ratio and of changes to the temperature dependencies of Jmax and Vcmax were examined [. Simulations which did not account for modulation of the Jmax25/Vcmax25 ratio (only considering the acclimation of temperature dependencies) showed a much narrower variation in Topt between growth temperatures (ranging from 25 to 32 °C) than that actually observed. In contrast, considering changes in the Jmax25/Vcmax25 ratio only (unchanged temperature dependencies) led to an accurate prediction of the acclimation of Topt (ranging from 20 to 32 °C), but to more inaccurate simulations of the photosynthetic rates at extreme temperatures (as reflected by the significant increase in RMSE). Under these assumptions, photosynthesis did not decrease as much as expected above 35 °C leaf temperatures for Tgrowth such as 25 or 30 °C. Accounting for both processes was necessary to minimize model errors over the broad range of situations studied.

Discussion

Acclimation of the thermal optimum of net photosynthesis occurred over a broad range of growing temperatures irrespective of temperate and Mediterranean origins

Shifts in the optimal temperature of light saturated photosynthesis (Topt) as a function of growth temperature have been reported in a number of C3 species (e.g. Bunce 2000; Mooney 1980; Yamasaki ; Yamori ) and are central to the thermal acclimation of CO2 assimilation (Yamori ). A significant plasticity of Topt was also characterized for alfalfa during our experiments, increasing from about 18 °C for leaves grown at 5–32 °C for leaves grown at 35 °C. Acclimation to low and high temperatures were both covered by this range of conditions. Remarkably, the plasticity of Topt observed in alfalfa alone matched the range of Topt shifts reported by Yamori across a set of contrasting C3 species (Fig. 7). The average shift in Topt was about 0.48 °C for each 1 °C increase in Tgrowth in both temperate and Mediterranean alfalfa cultivars (no significant genotype effect; y = 0.48*x + 17.4, r2 = 0.85, for the common regression line), as compared with 0.49 °C−1 on average across C3 species (Yamori ). Whether this high degree of Topt acclimation is related to the broad geographic distribution of alfalfa and its perennial growth, or whether it is a more general feature of temperate C3 species, still needs to be tested. However, during this study, the same range of Topt variations was observed for both of the genotypes studied, irrespective of their origin. No significant difference was found in the Topt–Tgrowth relationship, suggesting that the ability of a genotype to shift Topt towards actual growth conditions did not necessarily depend upon the environment in which it was selected. Similarly, Pearcy (1977) and Mooney (1980) found identical Topt variations in clones of Atriplex lentiformis and Heliotropum carassivicum collected from contrasting cool coastal and desert habitats. However, these findings differed from those of several other studies which reported differences in the acclimation potential of populations occupying ecological niches with dissimilar thermal regimes (Berry and Bjorkman 1980; Ishikawa ).

Figure 7.

Impact of growth temperature on the optimal temperature of net assimilation rate measured at 400 ppm for two alfalfa genotypes of Mediterranean (filled triangles, 7_7 cutting) and temperate (open triangles, G3 cutting) origins. A linear relationship was fitted for each clone and is represented by a dashed line for Mediterranean and a full line for temperate materials. No significant difference was observed between clones. The regression line found across C3 species by Yamori et al. (2014) is also represented (dotted line).

The reasons for variations in Topt as a function of growth temperature had previously been analyzed using the biochemical model developed by Farquhar , which assumes that the photosynthetic rate may be limited by either RuBP carboxylation by the Rubisco (Ac) or by RuBP regeneration (Ar). Inter-specific differences have been shown in the limiting step of net assimilation at Topt. Yamori suggested that C3 plant species could belong to one of three categories: net assimilation limited at Topt by Ac (i), by Ar (ii) or co-limited and determined by the intersection of Ac and Ar (iii). Our results showed that alfalfa belonged to the co-limitation group in all cases, except at 5 °C Tgrowth (Ac group). This behaviour was consistent with other cold tolerant temperate species such as wheat, rye or faba bean (Yamori ). In the case where Topt is determined by the co-limitation of Ac and Ar, the optimum temperature of net assimilation can shift through changes in the temperature dependencies of each partial reaction, and also simply because of changes to the balance between Ac and Ar, even if their temperature response does not change (Farquhar and Von Caemmerer 1982). In our study, both contributed to the observed shifts in alfalfa. The temperature dependencies of Vcmax and Jmax were modified in the two genotypes studied as a function of growth temperature, resulting in shifts of Ac and Ar. Similarly, the Jmax25/Vcmax25 ratio decreased regularly in leaves grown at between 5 and 35 °C. However, sensitivity analysis revealed that the Jmax25/Vcmax25 ratio had the strongest impact in determining Topt [. Changes to the Jmax25/Vcmax25 ratio have also been found to be the variable which correlates best to shifts in Topt among different species (Onoda ; Yamori ).

The increase in this ratio at low temperatures had previously been associated with elevated concentrations of enzymes involved in the capacity of the Calvin cycle and thylakoid reactions to regenerate RuBP (Antolin ; Berry and Bjorkman 1980) or decreased investment in Calvin Cycle capacity. For instance, Ishikawa reported an increased ratio between fructose-1,6-biphosphatase and Rubisco contents in Plantago asiatica genotypes able to adapt their Topt at low temperatures. In our study, the Jmax25/Vcmax25 ratio in the two genotypes displayed the same response to temperature. A significant plasticity of nitrogen partitioning in the photosynthetic apparatus was thus observed irrespective of the origin of genotypes, which might explain why the same range of Topt variations was observed.

Homeostasis of light-saturated photosynthesis occurred within a narrower range of growing temperatures

Changes to Tgrowth not only modified nitrogen partitioning in the photosynthetic apparatus but also dramatically altered leaf growth, leaf structure (as reflected by SLA) and the leaf nitrogen content. Despite these growth modifications, an apparent homeostasis of light saturated net photosynthesis (A40025) was observed within a broad range of temperatures from 10 to 30 °C, irrespective of the origin of the cultivars. This temperature homeostasis of photosynthesis may play an important role in the ability of alfalfa to grow successfully in habitats with contrasting temperature regimes, and to adapt to changes in temperatures over its extended growing season (Berry and Bjorkman 1980). Yamori indeed indicated that perennial herbaceous species generally display greater temperature homeostasis of photosynthesis than deciduous woody and annual herbaceous species.

The calculation of photosynthetic nitrogen use efficiency (PNUE) at each growth temperature suggested that these growth modifications could be adaptive. Indeed, PNUE values peaked at intermediate temperatures (25 °C), falling steadily on either side of the curve [. The photosynthetic capacity per unit of leaf nitrogen was thus not constant and was reduced under high and low growth temperatures. Modifications to CO2 diffusion, nitrogen allocation between photosynthetic and non-photosynthetic nitrogenous compounds, and the kinetics of photosynthetic enzymes could be three possible reasons for these reductions in PNUE (Field and Mooney 1986; Hikosaka 2004; Reich ). By modifying the structure of leaves, and increasing their nitrogen content at high and low growth temperatures, alfalfa developed responses which contributed to maintaining high rates of net assimilation per unit of leaf area. However, at the most extreme temperatures (5 and 35 °C), these structural acclimations were not enough to compensate for the important drop in PNUE values, causing significant reductions in A40025. The same decrease was seen to affect A400Opt (Fig. 3) [. This suggests that, at least in alfalfa, adaptive acclimation may occur within a much narrower range of growth temperatures (10–30 °C) than that within which Topt may shift (5–35 °C in this study).

Thermal stresses irreversibly disrupting the integrity of the photosynthetic apparatus most likely reduced maximal photosynthesis at the two extreme growth temperatures (Berry and Bjorkman 1980). Although not characterized, the reduction in net assimilation seen below 10 °C might be related to photo-inhibition (Jones and Kok 1966a,b; Louarn et al. 2008). This hypothesis is consistent with the high Jmax25/Vcmax25 ratio observed at these temperatures, where Ac was shown to be the limiting factor of CO2 assimilation. Over-investment in the electron transport chain may indeed provide protection against photo-oxidative damage (Hikosaka 2005). No difference in response was found between the two genotypes with respect to this stress. On the other hand, at high growth temperatures, a drop in the net assimilation rate was seen in both genotypes, but was dramatically more pronounced in the Mediterranean cuttings. Symptoms of thermal stress were apparent in the form of heat-bleached leaves in the Mediterranean material [ (Feierabend and Mikus 1977; Smillie ). The stress only affected new leaves that emerged after the transfer at 35 °C, and resulted in leaves lacking chlorophylls (as assessed by chlorophyll chromatography, not shown). All the 7_7 cuttings and about 30% of the plants in the Barmed cultivar were affected. McKenzie had previously noticed that a genetic variability existed in alfalfa concerning heat-induced reductions in maximal photosynthesis.

Temperate and Mediterranean genotypes differed in their tolerance to high temperatures

Except at the stressful growth temperature of 35 °C, the temperate and Mediterranean genotypes did not differ in terms of their A40025 or A400Opt. However, at high leaf temperatures (42 °C), the photosynthetic rate was almost always lower in the Mediterranean genotype (10, 25 and 30 °C Tgrowth). Leaves showing no apparent signs of thermal stress, and with similar maximal photosynthesis, thus displayed different temperature responses in terms of net photosynthesis. These differences were linked to those in the acclimation of temperature dependencies of the photosynthetic parameters Vcmax and Jmax as a function of Tgrowth (Fig. 5). They were modified in both genotypes, but these changes differed in nature. In the temperate genotype, Vcmax conserved a typical, rising Arrhenius response curve irrespective of growth temperature, while responses decreasing at high leaf temperatures were observed in the Mediterranean genotype. Such decreases in Vcmax at high leaf temperatures had previously been reported in several species (Sage ; Yamori , 2012). They were associated with reversible changes in the activation state of the Rubisco induced by high leaf temperatures, with gradual de-activation occurring under moderate heat stresses (Salvucci and Crafts-Brandner 2004; Yamori ). This Vcmax reduction resulted primarily from a reduction in the activity of Rubisco activase (Kumar ; Kurek ; Sage ), rather than from a reduction of Rubisco catalytic activity (Galmés , 2015). The difference between the two genotypes might also have resulted from their varying abilities to produce heat-shock proteins, affording better protection of the various components of the photosynthetic apparatus in temperate genotype (Burke 2001). Overall, these biochemical responses resulted in net assimilation rates that were more limited at high leaf temperatures in the Mediterranean genotype, because of depreciated Ac values.

Given the thermal differences in their original habitats, the greater sensitivity of the Mediterranean material to high temperatures had not necessarily been anticipated. Whether this genetic variability was circumstantial (sampling effect), or whether it had some ecological significance and was indicative of adaptation to local conditions, now needs to be confirmed. However, the evidence of a greater sensitivity of Mediterranean plants was consistent in the two types of plant material tested (seedlings from the Barmed cv. and a clone selected from the Demnate cv.). Observations in the same genotype of a greater thermal sensitivity of the Rubisco in response to short-term heat exposure, and of more severe damage in response to prolonged heat stress (heat bleaching), were also coherent with a niche differentiation. One possible explanation for this counter-intuitive result is that many species from the Mediterranean basin, even herbaceous perennials, actually develop stress avoidance rather than stress tolerance strategies, and are dormant or non-productive during periods of high temperatures in the summer (Volaire , 2014). Typical Mediterranean plants are thus adapted to growing during the cooler seasons rather than to maintaining active photosynthesis during heat waves (MacColl and Cooper 1967).

Conclusions

To conclude, a significant intra-specific variability was found in terms of the thermal acclimation of photosynthesis in alfalfa. Within the non-stressful range of growth temperatures, this was ascribed to changes in the temperature dependence of the maximum rate of Rubisco carboxylation rather than to differences in nitrogen partitioning within the photosynthetic apparatus, as reported previously (Hikosaka 2005; Ishikawa ). This resulted in differentiated responses of net photosynthesis at high leaf temperatures, but not in different maximal photosynthetic rates, rates at 25 °C or shifts of optimal temperature. The response of the Mediterranean genotypes tested was consistent with a strategy of thermal stress avoidance, so they do not seem suitable to be used as a genetic resource for breeding to improve the thermal tolerance of photosynthesis. On the other hand, room for improvement exists to increase the summer photosynthesis of southern varieties.

Sources of Funding

This study received support from the CLIMAGIE project of INRA’s ACCAF metaprogramme, the REFORMA project funded by the ARIMNet call of the ERA-CAPS 7th EU Framework Programme and the MODEXTREME project funded by the FP7. Serge Zaka’s PhD grant was financed by the Poitou-Charentes Regional Council (CPER).

Contributions by the Authors

S.Z., G.L. and E.F. designed the experiments and conducted measurements. S.Z. and G.L. contributed to model development and ran simulations. S.Z. performed data analyses. B.J. and G.L rose funding for these research. All of the authors contributed to writing the manuscript.

Conflict of Interest Statement

None declared.

Table A1.

Abbreviations of all the variables, parameters and symbols used

A	µmol CO2 m−2 s−1	Net photosynthetic rate
A400	µmol CO2 m−2 s−1	Net photosynthetic rate at 400 ppm CO2
A40025	µmol CO2 m−2 s−1	Net photosynthetic rate at 400 ppm CO2 and 25 °C leaf temperature
A400growth	µmol CO2 m−2 s−1	Net photosynthetic rate at 400 ppm CO2 and leaf at the growth temperature
A400opt	µmol CO2 m−2 s−1	Net photosynthetic rate at 400 ppm CO2 and thermal optimum
Ac	µmol CO2 m−2 s−1	Rubisco-limited rate of carboxylation
Ar	µmol CO2 m−2 s−1	Electron transport-limited rate of carboxylation
Ca	ppm	Ambient CO2 partial pressure
Ci	ppm	Intercellular CO2 partial pressure
ΔHa	J mol−1	Enthalpy of activation
ΔHd	J mol−1	Enthalpy of de-activation
Jmax	µmol m−2 s−1	Maximum electron transport rate
Kc	µmol mol−1	Michaelis-Menten constant for CO2
Ko	mmol mol−1	Michaelis-Menten constant for O2
Larea	cm2	Final leaf area
Na	g m−2	Amount of nitrogen per unit leaf area
O	KPa	Oxygen partial pressure
P25	µmol m−2 s−1	Value of Vcmax or Jmax at 25 °C
PNUE	µmol CO2 g N−1 s−1	Photosynthetic nitrogen use efficiency
PPFD	µmol photon m−2 s−1	Photosynthetic photon flux density
Γ*	µmol mol−1	CO2 compensation point in the absence of dark respiration
R	KJ mol−1 K−1	Universal gas constant for perfect gases
Rd	µmol m−2 s−1	Mitochondrial respiration
RuBP	–	Ribulose-1,5-biphosphate
ΔS	J mol−1 K−1	Entropy term
SLA	cm2 g	Specific leaf area
Tgrowth	°C	Growth temperature
Tleaf	°C	Leaf temperature
Tmax	°C	Maximum temperature of a process
Tmin	°C	Minimum temperature of a process
Topt	°C	Thermal optimum of photosynthesis
Vcmax	µmol m−2 s−1	Maximum rate of Rubisco carboxylation
VPD	kPa	Vapour pressure deficit