Planta DOI 10.1007/s00425-014-2097-z
Original Article
Effects of temperature on leaf hydraulic architecture of tobacco plants Jing Hu · Qiu‑Yun Yang · Wei Huang · Shi‑Bao Zhang · Hong Hu
Received: 4 March 2014 / Accepted: 16 May 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Main Conclusion Modifications in leaf anatomy of tobacco plants induced greater leaf water transport capacity, meeting greater transpirational demands and acclimating to warmer temperatures with a higher vapor pressure deficit. Temperature is one of the most important environmental factors affecting photosynthesis and growth of plants. However, it is not clear how it may alter leaf hydraulic architecture. We grew plants of tobacco (Nicotiana tabacum) ‘k326’ in separate glasshouse rooms set to different day/ night temperature conditions: low (LT 24/18 °C), medium (MT 28/22 °C), or high (HT 32/26 °C). After 40 days of such treatment, their leaf anatomies, leaf hydraulics, photosynthetic rates, and instantaneous water-use efficiency (WUEi) were measured. Compared with those under LT, plants exposed to HT or MT conditions had significantly higher values for minor vein density (MVD), stomatal density (SD), leaf area, leaf hydraulic conductance (Kleaf), and light-saturated photosynthetic rate (Asat), but lower values for leaf water potential (ψl) and WUEi. However, those parameters did not differ significantly between HT and MT conditions. Correlation analyses demonstrated that SD and J. Hu · Q.-Y. Yang · W. Huang · S.-B. Zhang (*) · H. Hu (*) Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China e-mail: [email protected] H. Hu e-mail: [email protected] J. Hu University of Chinese Academy of Sciences, Beijing 100049, China
Kleaf increased in parallel with MVD. Moreover, greater SD and Kleaf were partially associated with accelerated stomatal conductance. And then stomatal conductance was positively correlated with Asat. Therefore, under well-watered, fertilized conditions, when relative humidity was optimal, changes in leaf anatomy seemed to facilitate the hydraulic acclimation to higher temperatures, meeting greater transpirational demands and contributing to the maintenance of great photosynthetic rates. Because transpiration rate increased more with temperature than photosynthetic rate, WUEi reduced under warmer temperatures. Our results indicate that the modifications of leaf hydraulic architecture are important anatomical and physiological strategies for tobacco plants acclimating to warmer temperatures under a higher vapor pressure deficit. Keywords Temperature · Leaf hydraulic conductance · Transpiration · Leaf anatomy · Nicotiana tabacum ‘k326’ Abbreviations MVD Minor vein density SD Stomatal density VPD Higher vapor pressure deficit LA Leaf area HT High temperature MT Medium temperature LT Low temperature Kleaf Leaf hydraulic conductance ψl Leaf water potential WUEi Instantaneous water-use efficiency gs Stomatal conductance Asat Light-saturated photosynthetic rate PPFD Photosynthetic photon flux density Aarea Area-based net CO2 assimilation rate E Transpiration rate
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Planta
Introduction Warmer temperatures can lead to a higher vapor pressure deficit (VPD), increased evaporative demand, and greater transpiration rates (Way et al. 2012), consequently altering the water status of leaves. Leaf hydraulic conductance (Kleaf) represents the capacity of the transport system to deliver water, allowing the stomata to remain open for photosynthesis (Scoffoni et al. 2011). Greater capacity for leaf water transport means that more water is available for meeting transpirational demands as well as maintaining wider stomata and optimal photosynthetic rates (Brodribb et al. 2005). Because of the close relationship between leaf water-related anatomy and Kleaf (Medek et al. 2011), anatomical features such as minor vein density (MVD) may also be influenced by temperature. However, whereas the impacts of elevated temperatures on plant growth and photosynthesis have been broadly studied (Berry and Bjorkman 1980; Wolf et al. 1990; Pastenes and Horton 1996), the influences of temperature on hydraulic architecture remain unclear. The effects of temperature on leaf water-related anatomy have been paid more attention recently (Brodribb et al. 2007; Sack et al. 2012; Sack and Scoffoni 2013). Plants grown at warmer temperatures and with a high VPD often have larger vessel diameters, which can contribute to greater Kleaf (Medek et al. 2011). A denser pattern of venation also helps plants supply more water to leaves to satisfy high transpirational demands in a warmer environment (Medek et al. 2011). These anatomical modifications may be a response to increased leaf-to-air VPD and transpiration rates (Rejšková et al. 2012; Way et al. 2012). They might also be accomplished by altered leaf development, such as cell expansion (Murphy et al. 2014). There is also strong evidence that vein and stomatal densities are closely related, i.e., water entry through the vessels is coordinated with water exit through the stomata (Brodribb and Jordan 2011; Murphy et al. 2012). Although the responses of Kleaf to growth temperature have been examined previously, no general consensus exists on this issue. For example, Way et al. (2012) have reported a trend of greater capacity for leaf water transport by seedlings grown in chambers at warmer temperatures with greater VPDs. However, Phillips et al. (2011) have shown that seedlings of Eucalyptus saligna exposed to ambient temperature have Kleaf values similar to those exposed to elevated temperatures with greater VPDs. Therefore, further study is needed to determine the effect of temperature on Kleaf. Structural traits and water-related leaf anatomy, e.g., MVD, are closely associated with leaf hydraulic conductance (Roth-Nebelsick et al. 2001; Sack and Frole 2006;
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Brodribb et al. 2007; Rodriguez-Gamira et al. 2010). For example, under a warmer regime, Poa foliosa plants have larger, denser vessels that allow for greater water transport (Medek et al. 2011). Higher vein density can correspond to a greater capacity for water supply because it can enlarge the surface area available for exchanging xylem water with surrounding mesophyll cells while also reducing the distance through which water must travel outside of the xylem (Sack and Holbrook 2006). As proven by the Hagen–Poiseuille equation (Hellemans et al. 1980), a small increase in conduit radius leads to a large increase in vascular hydraulic conductance, i.e., conductance through an ideal pipe is proportional to the fourth power of its radius. Leaf hydraulic conductance is also related to palisade mesophyll thickness and the ratio of palisade to spongy-mesophyll thicknesses (Sack et al. 2003). Because temperature has important effects on water-related leaf anatomy and, thus, leaf hydraulic efficiency, it is critical that researchers investigate how both leaf anatomy and leaf hydraulic conductance are influenced by temperature. Positive correlations have been reported between Kleaf and the light-saturated photosynthetic rate (Asat) (Hubbard et al. 2001; Santiago et al. 2004; Taylaran et al. 2011). Values for maximum stomatal conductance and Asat increased significantly with Kleaf in oak (Quercus robur) (Rust and Roloff 2002). A rise in temperature leads to a higher VPD and increased transpiration rate (Way et al. 2012). Greater water transport capacity is helpful for satisfying transpirational demands and keeping stomata wide-open, thereby ensuring continuous photosynthesis (Brodribb et al. 2002). Under different temperatures, if the transpiration rate increases more with temperature than does the maximum rate of photosynthesis, instantaneous water-use efficiency (WUEi) will decrease with increased temperatures (Allen et al. 2003). Furthermore, WUEi and Asat can be viewed as a trade-off between water loss and carbon gain (Cao et al. 2012). However, it remains unknown how plants optimize their photosynthetic rate and degree of water loss by modifying leaf hydraulic architecture under different temperatures. Here, we grew a single tobacco cultivar in three separate glasshouse rooms with different day/night temperatures. After 40 days, we evaluated their leaf anatomies, leaf hydraulics, WUEi, and leaf gas exchange. Our goal was to investigate how these plants alter their hydraulic architecture when acclimating to changes in temperature. We hypothesized that plants exposed to warmer conditions would have greater SD, MVD, and leaf water transport capacity to meet their higher transpirational demands. This would then reduce WUEi and contribute to the maintenance of a more ideal photosynthetic rate.
Planta
Materials and methods
Hydraulics
Experimental conditions Seeds of a tobacco cultivar (Nicotiana tabacum ‘k326’) were obtained from the Seed Center of Yunnan Academy of Tobacco (Yunnan, China). Seeds were sown and cultured within a germination cabinet at ambient temperature and CO2 concentration. At 30 days after planting, the seedlings were transferred to pots (50 cm diam. × 50 cm tall) filled with red soil and humus (3:1, v:v). The pots were placed in three adjacent glasshouse rooms (4.0 m long × 3.4 m wide × 3.5 m high). The normal range of daytime temperatures for this species is 24–32 °C (Haroon et al. 1972). Day/night temperature treatments were programmed for a low (LT 24/18 °C), medium (MT 28/22 °C), or high (HT 32/26 °C) regime, with the temperatures set to change at 8:00 a.m. and 8:00 p.m. The rooms received natural lighting, with a maximum photosynthetic photon flux density (PPFD) of 1,400 μmol m−2 s−1. Relative humidity in each room was maintained at a constant 70 ± 3 % over the growing season, such that the VPD covaried with temperature. These conditions were meant to represent a realistic scenario for future climates in which warming is expected to occur with a constant relative humidity and, thus, a higher VPD (Way et al. 2012). During the daytime, the VPD under LT, MT, and HT averaged 0.90, 1.13, and 1.43 kPa, respectively, based on the combined calculation of air temperature and relative humidity (http://autogrow.com/downloads/download-software-anddrivers). All plants were irrigated daily and provided with adequate fertilizers during the experimental period. Measurements were made after 40 days of glasshouse growth.
Leaf hydraulic conductance was determined from the same fresh leaves assessed for photosynthetic traits, using a high-pressure flow meter (HPFM; Dynamax Inc., Houston, TX, USA). After gas exchange measurements, the whole plants for measurements were moved from the glasshouse rooms to the laboratory for measuring Kleaf. The high-pressure flow meter measured resistance (the inverse of conductance) as the force required to push water through a sample for a given flow rate. Leaf hydraulic conductance was measured by the ‘transient’ method between 11:00 a.m. and 2:00 p.m., and was calculated on a leaf area basis (Sack et al. 2002; Tyree et al. 2005). Briefly, the petiole was recut under water and then connected to the flow meter. Water flowed into the leaf and the necessary applied pressure were recorded every 3 s while pressure was ramped, at a constant rate of 3–7 kPa s−1, from 0 to 550 kPa. Here, Kleaf was computed as the water flow rate per unit leaf area divided by the pressure drop (between 200 and 550 kPa for most measurements) driving the flow. Leaf area (LA) was determined with a LI-3100C leaf area meter (Li-Cor, Inc.). During this measurement period, the laboratory temperature was 19.5–21.5 °C and laboratory irradiance was 10– 30 μmol m−2 s−1. Leaf water potential was measured on one fully expanded, mature leaf per plant (ninth to tenth leaf from the stem base) for a total of five leaves per treatment. Samples were taken at the same time when photosynthesis of the same plant was measured. Leaf water potential was measured with WP4 Dewpoint Potentiometer (Decagon Devices, Inc. WA, USA).
Gas exchange
Leaf anatomy
The response of CO2 assimilation to light (incident PPFD of 0–2,000 μmol m−2 s−1) was measured in the laboratory on one fresh leaf per plant (ninth to tenth leaf from the stem base), for a total of five leaves per treatment. The net photosynthetic rate per unit leaf area, transpiration rate, and stomatal conductance (gs) were determined with an open gas exchange system that incorporated infrared CO2 and water vapor analyzers (Li-6400; Li-Cor, Inc., Lincoln, NE, USA). Each leaf was illuminated by red light-emitting diodes (656–680 nm; Li-6400-02, Li-Cor, Inc.). Maximum stomatal conductance was defined as the value when PPFD was between 800 and 1,000 μmol m−2 s−1. Instantaneous water-use efficiency was calculated as the net photosynthetic rate divided by the transpiration rate. Instantaneous water-use efficiency was estimated at PPFD of 1,400 μmol m−2 s−1. While the measurements were being recorded, the leaves were exposed to their specified treatment temperature, an relative humidity of approximately 70 %, and a CO2 concentration of 380 μmol mol−1.
After the parameters for photosynthesis and leaf hydraulics were assessed, we examined the anatomical features of those leaves in the laboratory. Leaves were boiled in 7 % NaOH for 3 min and then stained with safranin. The sections were then photographed under a light microscope at 4× magnification (Nikon Optiphot; Nikon, Tokyo, Japan). Values for MVD were expressed as the sum of the lengths of third- and higher-order veins per unit area (Zhu et al. 2012). Stomatal density (SD) was measured on abaxial cuticles (stomata were absent from the adaxial surfaces) sampled from the same leaves on which MVD was measured. SD was measured from digital photomicrographs of the cuticle preparation at 20× magnification. These preparations included one leaf per plant, for a total of five leaves per treatment. We used ImageJ software (US National Institutes of Health, Bethesda, MD, USA) to obtain both MVD and SD and examined at least five fields of view per section.
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Planta
Statistical analyses All measurements for each temperature treatment were made with three to five plants. Means and standard errors were estimated from those three to five replicates. Error bars indicated the standard error of the mean across replicates. One-way ANOVAs were applied to investigate the differences in leaf anatomy, leaf hydraulics, photosynthesis, and WUEi among the three treatments. Relationships between variables were examined by Pearson’s productmoment correlations. The results were accepted as significant at P
Original Article
Effects of temperature on leaf hydraulic architecture of tobacco plants Jing Hu · Qiu‑Yun Yang · Wei Huang · Shi‑Bao Zhang · Hong Hu
Received: 4 March 2014 / Accepted: 16 May 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Main Conclusion Modifications in leaf anatomy of tobacco plants induced greater leaf water transport capacity, meeting greater transpirational demands and acclimating to warmer temperatures with a higher vapor pressure deficit. Temperature is one of the most important environmental factors affecting photosynthesis and growth of plants. However, it is not clear how it may alter leaf hydraulic architecture. We grew plants of tobacco (Nicotiana tabacum) ‘k326’ in separate glasshouse rooms set to different day/ night temperature conditions: low (LT 24/18 °C), medium (MT 28/22 °C), or high (HT 32/26 °C). After 40 days of such treatment, their leaf anatomies, leaf hydraulics, photosynthetic rates, and instantaneous water-use efficiency (WUEi) were measured. Compared with those under LT, plants exposed to HT or MT conditions had significantly higher values for minor vein density (MVD), stomatal density (SD), leaf area, leaf hydraulic conductance (Kleaf), and light-saturated photosynthetic rate (Asat), but lower values for leaf water potential (ψl) and WUEi. However, those parameters did not differ significantly between HT and MT conditions. Correlation analyses demonstrated that SD and J. Hu · Q.-Y. Yang · W. Huang · S.-B. Zhang (*) · H. Hu (*) Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China e-mail: [email protected] H. Hu e-mail: [email protected] J. Hu University of Chinese Academy of Sciences, Beijing 100049, China
Kleaf increased in parallel with MVD. Moreover, greater SD and Kleaf were partially associated with accelerated stomatal conductance. And then stomatal conductance was positively correlated with Asat. Therefore, under well-watered, fertilized conditions, when relative humidity was optimal, changes in leaf anatomy seemed to facilitate the hydraulic acclimation to higher temperatures, meeting greater transpirational demands and contributing to the maintenance of great photosynthetic rates. Because transpiration rate increased more with temperature than photosynthetic rate, WUEi reduced under warmer temperatures. Our results indicate that the modifications of leaf hydraulic architecture are important anatomical and physiological strategies for tobacco plants acclimating to warmer temperatures under a higher vapor pressure deficit. Keywords Temperature · Leaf hydraulic conductance · Transpiration · Leaf anatomy · Nicotiana tabacum ‘k326’ Abbreviations MVD Minor vein density SD Stomatal density VPD Higher vapor pressure deficit LA Leaf area HT High temperature MT Medium temperature LT Low temperature Kleaf Leaf hydraulic conductance ψl Leaf water potential WUEi Instantaneous water-use efficiency gs Stomatal conductance Asat Light-saturated photosynthetic rate PPFD Photosynthetic photon flux density Aarea Area-based net CO2 assimilation rate E Transpiration rate
13
Planta
Introduction Warmer temperatures can lead to a higher vapor pressure deficit (VPD), increased evaporative demand, and greater transpiration rates (Way et al. 2012), consequently altering the water status of leaves. Leaf hydraulic conductance (Kleaf) represents the capacity of the transport system to deliver water, allowing the stomata to remain open for photosynthesis (Scoffoni et al. 2011). Greater capacity for leaf water transport means that more water is available for meeting transpirational demands as well as maintaining wider stomata and optimal photosynthetic rates (Brodribb et al. 2005). Because of the close relationship between leaf water-related anatomy and Kleaf (Medek et al. 2011), anatomical features such as minor vein density (MVD) may also be influenced by temperature. However, whereas the impacts of elevated temperatures on plant growth and photosynthesis have been broadly studied (Berry and Bjorkman 1980; Wolf et al. 1990; Pastenes and Horton 1996), the influences of temperature on hydraulic architecture remain unclear. The effects of temperature on leaf water-related anatomy have been paid more attention recently (Brodribb et al. 2007; Sack et al. 2012; Sack and Scoffoni 2013). Plants grown at warmer temperatures and with a high VPD often have larger vessel diameters, which can contribute to greater Kleaf (Medek et al. 2011). A denser pattern of venation also helps plants supply more water to leaves to satisfy high transpirational demands in a warmer environment (Medek et al. 2011). These anatomical modifications may be a response to increased leaf-to-air VPD and transpiration rates (Rejšková et al. 2012; Way et al. 2012). They might also be accomplished by altered leaf development, such as cell expansion (Murphy et al. 2014). There is also strong evidence that vein and stomatal densities are closely related, i.e., water entry through the vessels is coordinated with water exit through the stomata (Brodribb and Jordan 2011; Murphy et al. 2012). Although the responses of Kleaf to growth temperature have been examined previously, no general consensus exists on this issue. For example, Way et al. (2012) have reported a trend of greater capacity for leaf water transport by seedlings grown in chambers at warmer temperatures with greater VPDs. However, Phillips et al. (2011) have shown that seedlings of Eucalyptus saligna exposed to ambient temperature have Kleaf values similar to those exposed to elevated temperatures with greater VPDs. Therefore, further study is needed to determine the effect of temperature on Kleaf. Structural traits and water-related leaf anatomy, e.g., MVD, are closely associated with leaf hydraulic conductance (Roth-Nebelsick et al. 2001; Sack and Frole 2006;
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Brodribb et al. 2007; Rodriguez-Gamira et al. 2010). For example, under a warmer regime, Poa foliosa plants have larger, denser vessels that allow for greater water transport (Medek et al. 2011). Higher vein density can correspond to a greater capacity for water supply because it can enlarge the surface area available for exchanging xylem water with surrounding mesophyll cells while also reducing the distance through which water must travel outside of the xylem (Sack and Holbrook 2006). As proven by the Hagen–Poiseuille equation (Hellemans et al. 1980), a small increase in conduit radius leads to a large increase in vascular hydraulic conductance, i.e., conductance through an ideal pipe is proportional to the fourth power of its radius. Leaf hydraulic conductance is also related to palisade mesophyll thickness and the ratio of palisade to spongy-mesophyll thicknesses (Sack et al. 2003). Because temperature has important effects on water-related leaf anatomy and, thus, leaf hydraulic efficiency, it is critical that researchers investigate how both leaf anatomy and leaf hydraulic conductance are influenced by temperature. Positive correlations have been reported between Kleaf and the light-saturated photosynthetic rate (Asat) (Hubbard et al. 2001; Santiago et al. 2004; Taylaran et al. 2011). Values for maximum stomatal conductance and Asat increased significantly with Kleaf in oak (Quercus robur) (Rust and Roloff 2002). A rise in temperature leads to a higher VPD and increased transpiration rate (Way et al. 2012). Greater water transport capacity is helpful for satisfying transpirational demands and keeping stomata wide-open, thereby ensuring continuous photosynthesis (Brodribb et al. 2002). Under different temperatures, if the transpiration rate increases more with temperature than does the maximum rate of photosynthesis, instantaneous water-use efficiency (WUEi) will decrease with increased temperatures (Allen et al. 2003). Furthermore, WUEi and Asat can be viewed as a trade-off between water loss and carbon gain (Cao et al. 2012). However, it remains unknown how plants optimize their photosynthetic rate and degree of water loss by modifying leaf hydraulic architecture under different temperatures. Here, we grew a single tobacco cultivar in three separate glasshouse rooms with different day/night temperatures. After 40 days, we evaluated their leaf anatomies, leaf hydraulics, WUEi, and leaf gas exchange. Our goal was to investigate how these plants alter their hydraulic architecture when acclimating to changes in temperature. We hypothesized that plants exposed to warmer conditions would have greater SD, MVD, and leaf water transport capacity to meet their higher transpirational demands. This would then reduce WUEi and contribute to the maintenance of a more ideal photosynthetic rate.
Planta
Materials and methods
Hydraulics
Experimental conditions Seeds of a tobacco cultivar (Nicotiana tabacum ‘k326’) were obtained from the Seed Center of Yunnan Academy of Tobacco (Yunnan, China). Seeds were sown and cultured within a germination cabinet at ambient temperature and CO2 concentration. At 30 days after planting, the seedlings were transferred to pots (50 cm diam. × 50 cm tall) filled with red soil and humus (3:1, v:v). The pots were placed in three adjacent glasshouse rooms (4.0 m long × 3.4 m wide × 3.5 m high). The normal range of daytime temperatures for this species is 24–32 °C (Haroon et al. 1972). Day/night temperature treatments were programmed for a low (LT 24/18 °C), medium (MT 28/22 °C), or high (HT 32/26 °C) regime, with the temperatures set to change at 8:00 a.m. and 8:00 p.m. The rooms received natural lighting, with a maximum photosynthetic photon flux density (PPFD) of 1,400 μmol m−2 s−1. Relative humidity in each room was maintained at a constant 70 ± 3 % over the growing season, such that the VPD covaried with temperature. These conditions were meant to represent a realistic scenario for future climates in which warming is expected to occur with a constant relative humidity and, thus, a higher VPD (Way et al. 2012). During the daytime, the VPD under LT, MT, and HT averaged 0.90, 1.13, and 1.43 kPa, respectively, based on the combined calculation of air temperature and relative humidity (http://autogrow.com/downloads/download-software-anddrivers). All plants were irrigated daily and provided with adequate fertilizers during the experimental period. Measurements were made after 40 days of glasshouse growth.
Leaf hydraulic conductance was determined from the same fresh leaves assessed for photosynthetic traits, using a high-pressure flow meter (HPFM; Dynamax Inc., Houston, TX, USA). After gas exchange measurements, the whole plants for measurements were moved from the glasshouse rooms to the laboratory for measuring Kleaf. The high-pressure flow meter measured resistance (the inverse of conductance) as the force required to push water through a sample for a given flow rate. Leaf hydraulic conductance was measured by the ‘transient’ method between 11:00 a.m. and 2:00 p.m., and was calculated on a leaf area basis (Sack et al. 2002; Tyree et al. 2005). Briefly, the petiole was recut under water and then connected to the flow meter. Water flowed into the leaf and the necessary applied pressure were recorded every 3 s while pressure was ramped, at a constant rate of 3–7 kPa s−1, from 0 to 550 kPa. Here, Kleaf was computed as the water flow rate per unit leaf area divided by the pressure drop (between 200 and 550 kPa for most measurements) driving the flow. Leaf area (LA) was determined with a LI-3100C leaf area meter (Li-Cor, Inc.). During this measurement period, the laboratory temperature was 19.5–21.5 °C and laboratory irradiance was 10– 30 μmol m−2 s−1. Leaf water potential was measured on one fully expanded, mature leaf per plant (ninth to tenth leaf from the stem base) for a total of five leaves per treatment. Samples were taken at the same time when photosynthesis of the same plant was measured. Leaf water potential was measured with WP4 Dewpoint Potentiometer (Decagon Devices, Inc. WA, USA).
Gas exchange
Leaf anatomy
The response of CO2 assimilation to light (incident PPFD of 0–2,000 μmol m−2 s−1) was measured in the laboratory on one fresh leaf per plant (ninth to tenth leaf from the stem base), for a total of five leaves per treatment. The net photosynthetic rate per unit leaf area, transpiration rate, and stomatal conductance (gs) were determined with an open gas exchange system that incorporated infrared CO2 and water vapor analyzers (Li-6400; Li-Cor, Inc., Lincoln, NE, USA). Each leaf was illuminated by red light-emitting diodes (656–680 nm; Li-6400-02, Li-Cor, Inc.). Maximum stomatal conductance was defined as the value when PPFD was between 800 and 1,000 μmol m−2 s−1. Instantaneous water-use efficiency was calculated as the net photosynthetic rate divided by the transpiration rate. Instantaneous water-use efficiency was estimated at PPFD of 1,400 μmol m−2 s−1. While the measurements were being recorded, the leaves were exposed to their specified treatment temperature, an relative humidity of approximately 70 %, and a CO2 concentration of 380 μmol mol−1.
After the parameters for photosynthesis and leaf hydraulics were assessed, we examined the anatomical features of those leaves in the laboratory. Leaves were boiled in 7 % NaOH for 3 min and then stained with safranin. The sections were then photographed under a light microscope at 4× magnification (Nikon Optiphot; Nikon, Tokyo, Japan). Values for MVD were expressed as the sum of the lengths of third- and higher-order veins per unit area (Zhu et al. 2012). Stomatal density (SD) was measured on abaxial cuticles (stomata were absent from the adaxial surfaces) sampled from the same leaves on which MVD was measured. SD was measured from digital photomicrographs of the cuticle preparation at 20× magnification. These preparations included one leaf per plant, for a total of five leaves per treatment. We used ImageJ software (US National Institutes of Health, Bethesda, MD, USA) to obtain both MVD and SD and examined at least five fields of view per section.
13
Planta
Statistical analyses All measurements for each temperature treatment were made with three to five plants. Means and standard errors were estimated from those three to five replicates. Error bars indicated the standard error of the mean across replicates. One-way ANOVAs were applied to investigate the differences in leaf anatomy, leaf hydraulics, photosynthesis, and WUEi among the three treatments. Relationships between variables were examined by Pearson’s productmoment correlations. The results were accepted as significant at P
