Photosynth Res DOI 10.1007/s11120-017-0436-1
ORIGINAL ARTICLE
Acclimation of Swedish and Italian ecotypes of Arabidopsis thaliana to light intensity Jared J. Stewart1 · Stephanie K. Polutchko1 · William W. Adams III1 · Barbara Demmig‑Adams1
Received: 14 February 2017 / Accepted: 22 August 2017 © Springer Science+Business Media B.V. 2017
Abstract This study addressed whether ecotypes of Arabidopsis thaliana from Sweden and Italy exhibited differences in foliar acclimation to high versus low growth light intensity, and compared CO2 uptake under growth conditions with light- and C O2-saturated intrinsic photosynthetic capacity and leaf morphological and vascular features. Differential responses between ecotypes occurred mainly at the scale of leaf architecture, with thicker leaves with higher intrinsic photosynthetic capacities and chlorophyll contents per leaf area, but no difference in photosynthetic capacity on a chlorophyll basis, in high light-grown leaves of the Swedish versus the Italian ecotype. Greater intrinsic photosynthetic capacity per leaf area in the Swedish ecotype was accompanied by a greater capacity of vascular infrastructure for sugar and water transport, but this was not associated with greater C O2 uptake rates under growth conditions. The Swedish ecotype with its thick leaves is thus constructed for high intrinsic photosynthetic and vascular flux capacity even under growth chamber conditions that may not permit full utilization of this potential. Conversely, the Swedish ecotype was less tolerant of low growth light intensity than the Italian ecotype, with smaller rosette areas and lesser aboveground biomass accumulation in low light-grown plants. Foliar vein density and stomatal density were both enhanced by high growth light intensity with no significant difference between ecotypes, and the ratio of water to sugar conduits was also similar between the two ecotypes during light acclimation. These findings add to the understanding of
* Barbara Demmig‑Adams barbara.demmig‑[email protected] 1
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309‑0334, USA
the foliar vasculature’s role in plant photosynthetic acclimation and adaptation. Keywords Arabidopsis · Light acclimation · Photosynthesis · Phloem · Xylem Abbreviations CC Companion cell HL High light LL Low light PC Phloem parenchyma cell PFD Photon flux density SE Sieve element TE Tracheary element
Introduction Plants in nature experience a wide range of different light conditions, both temporally over the course of seconds to seasons and spatially in different habitats (Demmig-Adams et al. 2012). Examples for the range of variation among locations include the extreme radiation of open arid habitats compared to deeply shaded forest understories as well as the greater range of photoperiods experienced by plants over seasons at higher versus lower latitudes. In addition, pronounced genetic differences exist among species in their adaptations and acclimation potential to variation in light environment. For example, while certain hemiepiphytic climbing vines are capable of acclimating equally well to extremely low light intensities and to full sunlight, annual species typically do not thrive in deep shade (DemmigAdams et al. 2006). Plant acclimatory adjustment to growth light environment takes place at multiple levels of organization, ranging from
13
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molecules, organelles, cells, and organs to the whole-plant level (Givnish 1988; Cookson and Granier 2006; Mishra et al. 2012). Growth light environment affects whole-plant architecture and development (Givnish 1988), leaf architecture (James and Bell 2000; Hanba et al. 2002), and chloroplast composition (e.g., the relative capacity for light absorption versus CO2 fixation; Walters and Horton 1994; Bailey et al. 2004; Cookson and Granier 2006). Evans and Poorter (2001) examined acclimation to growth light environment (200 vs. 1000 µmol photons m−2 s−1 in growth chambers) in ten C3 species, including both annuals and evergreens, and reported that high light-grown leaves typically exhibited thicker leaves and higher light-saturated rates of photosynthesis expressed per leaf area than low light-grown leaves, whereas the ratio of light-saturated rates of photosynthesis to leaf dry weight (both of which adjusted concomitantly) was unaffected by growth light intensity. With regard to the impact of growth light intensity on foliar vasculature, minor vein density was greater in leaves of some species grown in high light irrespective of the effect of growth light intensity on leaf size. High- versus low-lightgrown leaves had a higher minor vein density in species exhibiting smaller leaves in high light (Carins Murphy et al. 2012), larger leaves in high light (Amiard et al. 2005; Adams et al. 2007), or leaves of a similar size in high light (Stewart et al. 2017). The leaf’s major veins provide structural support of the leaf lamina and serve as conduits for the import of water and nutrients and the export of photosynthate (Sack et al. 2012). In addition, leaf minor veins serve in sugar loading and interchange of photosynthates and water between the major veins and the sites of photosynthesis (Sack and Scoffoni 2013; see Haritatos et al. 2000 for Arabidopsis thaliana). While major vein size scales with leaf area (Sack et al. 2012), adjustment of the minor foliar veins takes place in response to growth environment and can involve varying adjustment of different minor vein features dependent on plant species, ecotype, and physiological state (Adams et al. 2005, 2007, 2013, 2014, 2016; Amiard et al. 2005, 2007; Dumlao et al. 2012; Cohu et al. 2013a, b, 2014a; Muller et al. 2014a; Stewart et al. 2016, 2017). Ecotypes of A. thaliana from central Italy and central Sweden, which are separated by over 20° of latitude, have been investigated by a collaborative of multiple laboratories (Akiyama and Ågren 2012, 2014; Ågren and Schemske 2012; Ågren et al. 2013, 2017; Cohu et al. 2013a, b, 2014a, b; Grillo et al. 2013; Adams et al. 2014, 2016; Dittmar et al. 2014; Oakley et al. 2014, 2015; Gehan et al. 2015; Postma and Ågren 2015, 2016; Stewart et al. 2015, 2016; Mojica et al. 2016; Postma et al. 2016). For two other ecotypes of A. thaliana originating from sites separated by an even greater latitudinal distance (43° of latitude), Pons (2012) reported no significant ecotypic difference in photosynthetic acclimation to growth temperatures or light intensities. However,
13
Photosynth Res
the Swedish ecotype featured in the current study exhibited constitutively thicker leaves than the Italian ecotype under a relatively low light intensity of 200 µmol photons m−2 s−1 and a greater upregulation of intrinsic photosynthetic capacity in response to six evenly spaced 5-min periods of higher light (800 µmol photons m −2 s−1) during the photoperiod (Stewart et al. 2015). Moreover, there was differential acclimation of the Swedish and Italian ecotypes in response to contrasting growth temperatures (Cohu et al. 2013a, b; Adams et al. 2014, 2016; Stewart et al. 2016). The Swedish ecotype exhibited significantly greater upregulation of photosynthetic capacity, leaf thickness, and number of minor vein phloem cells per minor vein in response to growth under low (8 °C) compared to high (35 °C) air temperature (Adams et al. 2016; Stewart et al. 2016). The present study was undertaken to further evaluate the Swedish and Italian ecotypes of A. thaliana for differences in foliar morphological and vascular acclimation to two extremes in growth light intensity (100 and 1000 µmol photons m−2 s−1) and to compare CO2 uptake under ambient growth conditions with light- and CO2-saturated intrinsic photosynthetic capacity.
Materials and methods Plant material and growth conditions Following stratification at 4 °C for 4 days, seeds of Swedish and Italian ecotypes of A. thaliana (L.) Heynhold (for more details, see Stewart et al. 2015; Adams et al. 2016) were germinated in soil (Canadian Growing Mix 2; Conrad Fafard Inc., Agawam, MA, USA) in growth chambers (E15 and PGR15; Conviron, Winnipeg, Canada) set to 25 °C/20 °C (light/dark air temperature) under a 9-h photoperiod of either 100 (LL) or 1000 (HL) µmol photons m −2 s−1. Following germination, individual seedlings were transplanted (with 50 mL soil) into large pots (2.8 L of soil) and transferred to an intermediate temperature regime (20 °C/15 °C and 15 °C/15 °C [light/dark air temperature] for LL and HL plants, respectively) for 7 days before transfer to their final growth temperature regime. To maintain identical leaf temperatures under the different photon flux densities (PFDs), air temperature during the photoperiod was set to 20 °C for the LL regime or 12 °C for the HL regime and 12 °C during the night for both light regimes. Plants received daily watering with nutrients added every other day after emergence of the first leaves. Nutrients were added with a Dosatron D14MZ2 injector system (Dosatron International Inc., Clearwater, FL, USA) at an injection ratio of 1:100 from a concentrated solution of 10.0% (w/v) 15-5-15 Calcium + Magnesium LX and 1.5% (w/v) 10-0-0 Mag Trate LX (Jack’s Professional LX Water-Soluble Fertilizer, J.R. Peters, Inc., Allentown, PA, USA). Several fully expanded
Photosynth Res
leaves from non-flowering plants [approximately 6 (HL) or 8 (LL) weeks old] were used for the various parameters assessed, typically one leaf from each of 3–4 plants for a given parameter for each ecotype from each growth environment (i.e., 3–4 biological replicates). As reported by Stewart et al. (2017) for the Col-0 genotype of A. thaliana, leaf size was similar in low and high light-grown leaves of the Swedish and Italian ecotypes and minor vein density remained constant soon after leaf emergence in a given growth environment (see also Kang and Dengler 2004; Sack et al. 2012). Leaf and plant parameters Leaf disks were dried in an oven (at 70 °C) for 1 week, weighed using a balance (A-160; Denver Instrument Company, Denver, CO, USA), and the mass divided by the leaf area of each disk in order to calculate the leaf mass per area. Minor vein density was measured as total minor vein length within a 9.842-mm2 area from leaf segments that had been cleared by soaking in 70% (v/v) ethanol followed by soaking in 5% (w/v) NaOH (Stewart et al. 2017). Leaf thickness, palisade mesophyll thickness, spongy mesophyll thickness, number of palisade mesophyll cell layers, and minor vein cell numbers and cross-sectional areas were determined from leaf cross sections of 0.7–1.0 µm thickness that were cut with an ultramicrotome (UltraCut E; Reichert Technologies Life Sciences, Buffalo, NY, USA) and stained with a 0.1% (w/v) toluidine blue and 1% (w/v) sodium borate solution. Leaf segments used for these microscopic analyses were initially fixed in 2% (w/v) glutaraldehyde and 2% (w/v) paraformaldehyde in a 70-mM sodium cacodylate buffer (balanced to pH 6.9 with 6 M HCl), the process of which was facilitated through the application of a vacuum with a vacuum pump (Duo-Seal 1405; Sargent-Welch Scientific CO., Skokie, IL, USA) and a refrigerated vapor trap (RVT400; Savant Instruments, Inc., Farmingdale, NY, USA), dehydrated in an acetone series, and embedded in Spurr resin (Spurr 1969) as described by Dumlao et al. (2012). Four different types of minor vein cells were characterized. Tracheary elements (TEs) of the xylem are the conduits through which water is delivered to, and distributed throughout, leaves. Cross-sectional area of such conduits should affect the flux capacity of such pipelines, but it is the number of TEs per minor vein normalized for differences in vein density among species that showed the strongest relationship with photosynthetic capacity (Muller et al. 2014b; but see mixed results for A. thaliana in Cohu et al. 2013b). The same is true for the sieve elements (SEs) of the phloem as a proxy for flux capacity to move sugars out of the leaf. Photosynthetic capacity, again, exhibited a stronger relationship with the number of SEs per minor vein than with SE cross-sectional area per minor vein in A. thaliana, but both relationships were highly significant (Cohu et al. 2013b; see
also Adams et al. 2016). In A. thaliana—as a species that loads sugars into the phloem via membrane-bound transporters—all phloem cells in the minor veins are involved in facilitating this active loading: H +-sucrose symporters move sucrose across the cell membranes of SEs and companion cells (CCs), ATPases on the cell membranes of CCs and phloem parenchyma cells (PCs) pump protons into the cell wall space, and sucrose efflux channels permit sucrose to flow across PC membranes. As a proxy for the capacity to load sugars into the phloem, the numbers of all of these phloem cells per minor vein (normalized for vein density when differences in vein density existed) have been found to exhibit a significant relationship with photosynthetic capacity in A. thaliana and among other apoplastic phloem loaders (Adams et al. 2013, 2016; Cohu et al. 2013b; Muller et al. 2014a, b; Stewart et al. 2016, 2017). To quantify the number of stomata per unit of leaf surface area, impressions of the abaxial and adaxial epidermis were obtained by evenly applying a clear nail polish to small areas of the leaf surfaces while avoiding midrib and the leaf margin. After drying, the nail polish was gently removed using fine tweezers and mounted to a glass slide. Stomatal densities were determined by counting the number of stomata over a 1.476-mm2 leaf area. With the exception of leaf mass per area, all measurements of the previously described leaf and minor vein parameters were made using ImageJ (Rasband WS, National Institute of Health, Bethesda, MD, USA) from images obtained with an Axioskop 20 microscope (Carl Zeiss AG, Oberkochen, Germany) and an OptixCam OCView camera system (The Microscope Store, Roanoke, VA, USA). Sample size for parameters was four leaves (one leaf each from four different plants) for each ecotype from each growth environment (i.e., four biological replicates). Chlorophyll level was determined via high-performance liquid chromatography of leaf extracts as described in Stewart et al. (2015) using 0.30-cm2 leaf disks from one leaf from each of 3–4 different plants for each ecotype from each growth environment (i.e., 3–4 biological replicates). Aboveground biomass and rosette area were measured as described in Stewart et al. (2015) from non-flowering plants that were approximately 6 weeks (HL regime) or 8 weeks (LL regime) old. Foliar photosynthesis Measurements of photosynthetic capacity, which was assessed as light- [1500 [LL] or 2000 (HL) µmol photons m−2 s−1] and CO2- (50,000 ppm) saturated rate of photosynthetic oxygen evolution, were obtained from leaves maintained at 25 °C in a water-saturated atmosphere (Hansatech oxygen electrode systems, King’s Lynn, Norfolk, UK; Delieu and Walker 1981). Rates of C O2 uptake (using an
13
Photosynth Res
LCi Portable Photosynthesis System; ADC Bioscientific Ltd., Hoddesdon, Herts, England, UK) were determined as described in Stewart et al. (2015) under the respective PFDs experienced by the plants during the photoperiod (100 µmol photons m−2 s−1 for those that developed under LL and 1000 µmol photons m−2 s−1 for those that developed under HL). All rates of CO2 exchange were determined at a leaf temperature of 27.1 ± 1.0 °C (mean ± standard deviation; n = 16) in the presence of 411 ± 32 ppm CO2 (mean ± standard deviation; n = 16) and a vapor pressure deficit of 2.05 ± 0.19 kPa (mean ± standard deviation; n = 16). Sample size for photosynthetic rates was four leaves (one leaf each from four different plants) for each ecotype from each growth environment (i.e., four biological replicates). Statistical analyses Comparisons of two means were statistically evaluated via Student’s t tests, while the effects of ecotype and growth light intensity, as well as the ecotypic response to growth light intensity, were assessed via two-way analysis of variance. All statistical analyses were conducted using JMP software (Pro 13.0.0; SAS Institute Inc., Cary, NC, USA).
Results
Fig. 1 Photographic images of representative plants of the a, b Italian and c, d Swedish ecotypes of Arabidopsis thaliana grown under a 9-h photoperiod of 100 (LL; a, c) or 1000 (HL; b, d) µmol photons m−2 s−1 for eight or six weeks, respectively. Photographs were taken in the growth chamber during the photoperiod before characterization
Ecotypic comparison of morphology and photosynthesis under low and high growth PFD Rosette area (light-exposed leaf area per plant assessed from photographic images such as those depicted in Fig. 1) did not differ significantly between the two ecotypes grown under the high light intensity (PFD for photon flux density; Fig. 2a). In contrast, the Italian ecotype developed a 77% larger rosette area (Fig. 2a) and accumulated significantly more (64%) aboveground biomass per plant (Fig. 2b) compared to the Swedish ecotype when both ecotypes were grown under low PFD (Table 1). Conversely, the Swedish ecotype accumulated significantly (24%) more aboveground biomass per plant (Fig. 2b) compared to the Italian ecotype when grown under high PFD (see also Table 1). It should be noted that this percentage is similar to the rosette-area difference of 22%, although this latter difference was not significant (Fig. 2a). Rates of photosynthetic CO2 uptake (means ± standard deviations) by leaves of the Swedish ecotype (6.0 ± 0.5 µmol CO2 m−2 s−1) were slightly but significantly (P
ORIGINAL ARTICLE
Acclimation of Swedish and Italian ecotypes of Arabidopsis thaliana to light intensity Jared J. Stewart1 · Stephanie K. Polutchko1 · William W. Adams III1 · Barbara Demmig‑Adams1
Received: 14 February 2017 / Accepted: 22 August 2017 © Springer Science+Business Media B.V. 2017
Abstract This study addressed whether ecotypes of Arabidopsis thaliana from Sweden and Italy exhibited differences in foliar acclimation to high versus low growth light intensity, and compared CO2 uptake under growth conditions with light- and C O2-saturated intrinsic photosynthetic capacity and leaf morphological and vascular features. Differential responses between ecotypes occurred mainly at the scale of leaf architecture, with thicker leaves with higher intrinsic photosynthetic capacities and chlorophyll contents per leaf area, but no difference in photosynthetic capacity on a chlorophyll basis, in high light-grown leaves of the Swedish versus the Italian ecotype. Greater intrinsic photosynthetic capacity per leaf area in the Swedish ecotype was accompanied by a greater capacity of vascular infrastructure for sugar and water transport, but this was not associated with greater C O2 uptake rates under growth conditions. The Swedish ecotype with its thick leaves is thus constructed for high intrinsic photosynthetic and vascular flux capacity even under growth chamber conditions that may not permit full utilization of this potential. Conversely, the Swedish ecotype was less tolerant of low growth light intensity than the Italian ecotype, with smaller rosette areas and lesser aboveground biomass accumulation in low light-grown plants. Foliar vein density and stomatal density were both enhanced by high growth light intensity with no significant difference between ecotypes, and the ratio of water to sugar conduits was also similar between the two ecotypes during light acclimation. These findings add to the understanding of
* Barbara Demmig‑Adams barbara.demmig‑[email protected] 1
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309‑0334, USA
the foliar vasculature’s role in plant photosynthetic acclimation and adaptation. Keywords Arabidopsis · Light acclimation · Photosynthesis · Phloem · Xylem Abbreviations CC Companion cell HL High light LL Low light PC Phloem parenchyma cell PFD Photon flux density SE Sieve element TE Tracheary element
Introduction Plants in nature experience a wide range of different light conditions, both temporally over the course of seconds to seasons and spatially in different habitats (Demmig-Adams et al. 2012). Examples for the range of variation among locations include the extreme radiation of open arid habitats compared to deeply shaded forest understories as well as the greater range of photoperiods experienced by plants over seasons at higher versus lower latitudes. In addition, pronounced genetic differences exist among species in their adaptations and acclimation potential to variation in light environment. For example, while certain hemiepiphytic climbing vines are capable of acclimating equally well to extremely low light intensities and to full sunlight, annual species typically do not thrive in deep shade (DemmigAdams et al. 2006). Plant acclimatory adjustment to growth light environment takes place at multiple levels of organization, ranging from
13
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molecules, organelles, cells, and organs to the whole-plant level (Givnish 1988; Cookson and Granier 2006; Mishra et al. 2012). Growth light environment affects whole-plant architecture and development (Givnish 1988), leaf architecture (James and Bell 2000; Hanba et al. 2002), and chloroplast composition (e.g., the relative capacity for light absorption versus CO2 fixation; Walters and Horton 1994; Bailey et al. 2004; Cookson and Granier 2006). Evans and Poorter (2001) examined acclimation to growth light environment (200 vs. 1000 µmol photons m−2 s−1 in growth chambers) in ten C3 species, including both annuals and evergreens, and reported that high light-grown leaves typically exhibited thicker leaves and higher light-saturated rates of photosynthesis expressed per leaf area than low light-grown leaves, whereas the ratio of light-saturated rates of photosynthesis to leaf dry weight (both of which adjusted concomitantly) was unaffected by growth light intensity. With regard to the impact of growth light intensity on foliar vasculature, minor vein density was greater in leaves of some species grown in high light irrespective of the effect of growth light intensity on leaf size. High- versus low-lightgrown leaves had a higher minor vein density in species exhibiting smaller leaves in high light (Carins Murphy et al. 2012), larger leaves in high light (Amiard et al. 2005; Adams et al. 2007), or leaves of a similar size in high light (Stewart et al. 2017). The leaf’s major veins provide structural support of the leaf lamina and serve as conduits for the import of water and nutrients and the export of photosynthate (Sack et al. 2012). In addition, leaf minor veins serve in sugar loading and interchange of photosynthates and water between the major veins and the sites of photosynthesis (Sack and Scoffoni 2013; see Haritatos et al. 2000 for Arabidopsis thaliana). While major vein size scales with leaf area (Sack et al. 2012), adjustment of the minor foliar veins takes place in response to growth environment and can involve varying adjustment of different minor vein features dependent on plant species, ecotype, and physiological state (Adams et al. 2005, 2007, 2013, 2014, 2016; Amiard et al. 2005, 2007; Dumlao et al. 2012; Cohu et al. 2013a, b, 2014a; Muller et al. 2014a; Stewart et al. 2016, 2017). Ecotypes of A. thaliana from central Italy and central Sweden, which are separated by over 20° of latitude, have been investigated by a collaborative of multiple laboratories (Akiyama and Ågren 2012, 2014; Ågren and Schemske 2012; Ågren et al. 2013, 2017; Cohu et al. 2013a, b, 2014a, b; Grillo et al. 2013; Adams et al. 2014, 2016; Dittmar et al. 2014; Oakley et al. 2014, 2015; Gehan et al. 2015; Postma and Ågren 2015, 2016; Stewart et al. 2015, 2016; Mojica et al. 2016; Postma et al. 2016). For two other ecotypes of A. thaliana originating from sites separated by an even greater latitudinal distance (43° of latitude), Pons (2012) reported no significant ecotypic difference in photosynthetic acclimation to growth temperatures or light intensities. However,
13
Photosynth Res
the Swedish ecotype featured in the current study exhibited constitutively thicker leaves than the Italian ecotype under a relatively low light intensity of 200 µmol photons m−2 s−1 and a greater upregulation of intrinsic photosynthetic capacity in response to six evenly spaced 5-min periods of higher light (800 µmol photons m −2 s−1) during the photoperiod (Stewart et al. 2015). Moreover, there was differential acclimation of the Swedish and Italian ecotypes in response to contrasting growth temperatures (Cohu et al. 2013a, b; Adams et al. 2014, 2016; Stewart et al. 2016). The Swedish ecotype exhibited significantly greater upregulation of photosynthetic capacity, leaf thickness, and number of minor vein phloem cells per minor vein in response to growth under low (8 °C) compared to high (35 °C) air temperature (Adams et al. 2016; Stewart et al. 2016). The present study was undertaken to further evaluate the Swedish and Italian ecotypes of A. thaliana for differences in foliar morphological and vascular acclimation to two extremes in growth light intensity (100 and 1000 µmol photons m−2 s−1) and to compare CO2 uptake under ambient growth conditions with light- and CO2-saturated intrinsic photosynthetic capacity.
Materials and methods Plant material and growth conditions Following stratification at 4 °C for 4 days, seeds of Swedish and Italian ecotypes of A. thaliana (L.) Heynhold (for more details, see Stewart et al. 2015; Adams et al. 2016) were germinated in soil (Canadian Growing Mix 2; Conrad Fafard Inc., Agawam, MA, USA) in growth chambers (E15 and PGR15; Conviron, Winnipeg, Canada) set to 25 °C/20 °C (light/dark air temperature) under a 9-h photoperiod of either 100 (LL) or 1000 (HL) µmol photons m −2 s−1. Following germination, individual seedlings were transplanted (with 50 mL soil) into large pots (2.8 L of soil) and transferred to an intermediate temperature regime (20 °C/15 °C and 15 °C/15 °C [light/dark air temperature] for LL and HL plants, respectively) for 7 days before transfer to their final growth temperature regime. To maintain identical leaf temperatures under the different photon flux densities (PFDs), air temperature during the photoperiod was set to 20 °C for the LL regime or 12 °C for the HL regime and 12 °C during the night for both light regimes. Plants received daily watering with nutrients added every other day after emergence of the first leaves. Nutrients were added with a Dosatron D14MZ2 injector system (Dosatron International Inc., Clearwater, FL, USA) at an injection ratio of 1:100 from a concentrated solution of 10.0% (w/v) 15-5-15 Calcium + Magnesium LX and 1.5% (w/v) 10-0-0 Mag Trate LX (Jack’s Professional LX Water-Soluble Fertilizer, J.R. Peters, Inc., Allentown, PA, USA). Several fully expanded
Photosynth Res
leaves from non-flowering plants [approximately 6 (HL) or 8 (LL) weeks old] were used for the various parameters assessed, typically one leaf from each of 3–4 plants for a given parameter for each ecotype from each growth environment (i.e., 3–4 biological replicates). As reported by Stewart et al. (2017) for the Col-0 genotype of A. thaliana, leaf size was similar in low and high light-grown leaves of the Swedish and Italian ecotypes and minor vein density remained constant soon after leaf emergence in a given growth environment (see also Kang and Dengler 2004; Sack et al. 2012). Leaf and plant parameters Leaf disks were dried in an oven (at 70 °C) for 1 week, weighed using a balance (A-160; Denver Instrument Company, Denver, CO, USA), and the mass divided by the leaf area of each disk in order to calculate the leaf mass per area. Minor vein density was measured as total minor vein length within a 9.842-mm2 area from leaf segments that had been cleared by soaking in 70% (v/v) ethanol followed by soaking in 5% (w/v) NaOH (Stewart et al. 2017). Leaf thickness, palisade mesophyll thickness, spongy mesophyll thickness, number of palisade mesophyll cell layers, and minor vein cell numbers and cross-sectional areas were determined from leaf cross sections of 0.7–1.0 µm thickness that were cut with an ultramicrotome (UltraCut E; Reichert Technologies Life Sciences, Buffalo, NY, USA) and stained with a 0.1% (w/v) toluidine blue and 1% (w/v) sodium borate solution. Leaf segments used for these microscopic analyses were initially fixed in 2% (w/v) glutaraldehyde and 2% (w/v) paraformaldehyde in a 70-mM sodium cacodylate buffer (balanced to pH 6.9 with 6 M HCl), the process of which was facilitated through the application of a vacuum with a vacuum pump (Duo-Seal 1405; Sargent-Welch Scientific CO., Skokie, IL, USA) and a refrigerated vapor trap (RVT400; Savant Instruments, Inc., Farmingdale, NY, USA), dehydrated in an acetone series, and embedded in Spurr resin (Spurr 1969) as described by Dumlao et al. (2012). Four different types of minor vein cells were characterized. Tracheary elements (TEs) of the xylem are the conduits through which water is delivered to, and distributed throughout, leaves. Cross-sectional area of such conduits should affect the flux capacity of such pipelines, but it is the number of TEs per minor vein normalized for differences in vein density among species that showed the strongest relationship with photosynthetic capacity (Muller et al. 2014b; but see mixed results for A. thaliana in Cohu et al. 2013b). The same is true for the sieve elements (SEs) of the phloem as a proxy for flux capacity to move sugars out of the leaf. Photosynthetic capacity, again, exhibited a stronger relationship with the number of SEs per minor vein than with SE cross-sectional area per minor vein in A. thaliana, but both relationships were highly significant (Cohu et al. 2013b; see
also Adams et al. 2016). In A. thaliana—as a species that loads sugars into the phloem via membrane-bound transporters—all phloem cells in the minor veins are involved in facilitating this active loading: H +-sucrose symporters move sucrose across the cell membranes of SEs and companion cells (CCs), ATPases on the cell membranes of CCs and phloem parenchyma cells (PCs) pump protons into the cell wall space, and sucrose efflux channels permit sucrose to flow across PC membranes. As a proxy for the capacity to load sugars into the phloem, the numbers of all of these phloem cells per minor vein (normalized for vein density when differences in vein density existed) have been found to exhibit a significant relationship with photosynthetic capacity in A. thaliana and among other apoplastic phloem loaders (Adams et al. 2013, 2016; Cohu et al. 2013b; Muller et al. 2014a, b; Stewart et al. 2016, 2017). To quantify the number of stomata per unit of leaf surface area, impressions of the abaxial and adaxial epidermis were obtained by evenly applying a clear nail polish to small areas of the leaf surfaces while avoiding midrib and the leaf margin. After drying, the nail polish was gently removed using fine tweezers and mounted to a glass slide. Stomatal densities were determined by counting the number of stomata over a 1.476-mm2 leaf area. With the exception of leaf mass per area, all measurements of the previously described leaf and minor vein parameters were made using ImageJ (Rasband WS, National Institute of Health, Bethesda, MD, USA) from images obtained with an Axioskop 20 microscope (Carl Zeiss AG, Oberkochen, Germany) and an OptixCam OCView camera system (The Microscope Store, Roanoke, VA, USA). Sample size for parameters was four leaves (one leaf each from four different plants) for each ecotype from each growth environment (i.e., four biological replicates). Chlorophyll level was determined via high-performance liquid chromatography of leaf extracts as described in Stewart et al. (2015) using 0.30-cm2 leaf disks from one leaf from each of 3–4 different plants for each ecotype from each growth environment (i.e., 3–4 biological replicates). Aboveground biomass and rosette area were measured as described in Stewart et al. (2015) from non-flowering plants that were approximately 6 weeks (HL regime) or 8 weeks (LL regime) old. Foliar photosynthesis Measurements of photosynthetic capacity, which was assessed as light- [1500 [LL] or 2000 (HL) µmol photons m−2 s−1] and CO2- (50,000 ppm) saturated rate of photosynthetic oxygen evolution, were obtained from leaves maintained at 25 °C in a water-saturated atmosphere (Hansatech oxygen electrode systems, King’s Lynn, Norfolk, UK; Delieu and Walker 1981). Rates of C O2 uptake (using an
13
Photosynth Res
LCi Portable Photosynthesis System; ADC Bioscientific Ltd., Hoddesdon, Herts, England, UK) were determined as described in Stewart et al. (2015) under the respective PFDs experienced by the plants during the photoperiod (100 µmol photons m−2 s−1 for those that developed under LL and 1000 µmol photons m−2 s−1 for those that developed under HL). All rates of CO2 exchange were determined at a leaf temperature of 27.1 ± 1.0 °C (mean ± standard deviation; n = 16) in the presence of 411 ± 32 ppm CO2 (mean ± standard deviation; n = 16) and a vapor pressure deficit of 2.05 ± 0.19 kPa (mean ± standard deviation; n = 16). Sample size for photosynthetic rates was four leaves (one leaf each from four different plants) for each ecotype from each growth environment (i.e., four biological replicates). Statistical analyses Comparisons of two means were statistically evaluated via Student’s t tests, while the effects of ecotype and growth light intensity, as well as the ecotypic response to growth light intensity, were assessed via two-way analysis of variance. All statistical analyses were conducted using JMP software (Pro 13.0.0; SAS Institute Inc., Cary, NC, USA).
Results
Fig. 1 Photographic images of representative plants of the a, b Italian and c, d Swedish ecotypes of Arabidopsis thaliana grown under a 9-h photoperiod of 100 (LL; a, c) or 1000 (HL; b, d) µmol photons m−2 s−1 for eight or six weeks, respectively. Photographs were taken in the growth chamber during the photoperiod before characterization
Ecotypic comparison of morphology and photosynthesis under low and high growth PFD Rosette area (light-exposed leaf area per plant assessed from photographic images such as those depicted in Fig. 1) did not differ significantly between the two ecotypes grown under the high light intensity (PFD for photon flux density; Fig. 2a). In contrast, the Italian ecotype developed a 77% larger rosette area (Fig. 2a) and accumulated significantly more (64%) aboveground biomass per plant (Fig. 2b) compared to the Swedish ecotype when both ecotypes were grown under low PFD (Table 1). Conversely, the Swedish ecotype accumulated significantly (24%) more aboveground biomass per plant (Fig. 2b) compared to the Italian ecotype when grown under high PFD (see also Table 1). It should be noted that this percentage is similar to the rosette-area difference of 22%, although this latter difference was not significant (Fig. 2a). Rates of photosynthetic CO2 uptake (means ± standard deviations) by leaves of the Swedish ecotype (6.0 ± 0.5 µmol CO2 m−2 s−1) were slightly but significantly (P
