© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Physiologia Plantarum 2014
Leaf anatomical and photosynthetic acclimation to cool temperature and high light in two winter versus two summer annuals Christopher M. Cohu† , Onno Muller‡ , William W. Adams III∗ and Barbara Demmig-Adams Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309-0334, USA
Correspondence *Corresponding author, e-mail: [email protected] Received 6 September 2013; revised 5 December 2013 doi:10.1111/ppl.12154
Acclimation of foliar features to cool temperature and high light was characterized in winter (Spinacia oleracea L. cv. Giant Nobel; Arabidopsis thaliana (L.) Heynhold Col-0 and ecotypes from Sweden and Italy) versus summer (Helianthus annuus L. cv. Soraya; Cucurbita pepo L. cv. Italian Zucchini Romanesco) annuals. Significant relationships existed among leaf dry mass per area, photosynthesis, leaf thickness and palisade mesophyll thickness. While the acclimatory response of the summer annuals to cool temperature and/or high light levels was limited, the winter annuals increased the number of palisade cell layers, ranging from two layers under moderate light and warm temperature to between four and five layers under cool temperature and high light. A significant relationship was also found between palisade tissue thickness and either cross-sectional area or number of phloem cells (each normalized by vein density) in minor veins among all four species and growth regimes. The two winter annuals, but not the summer annuals, thus exhibited acclimatory adjustments of minor vein phloem to cool temperature and/or high light, with more numerous and larger phloem cells and a higher maximal photosynthesis rate. The upregulation of photosynthesis in winter annuals in response to low growth temperature may thus depend on not only (1) a greater volume of photosynthesizing palisade tissue but also (2) leaf veins containing additional phloem cells and presumably capable of exporting a greater volume of sugars from the leaves to the rest of the plant.
Introduction Overwintering annual and biennial plant species have the remarkable ability to increase their intrinsic photosynthetic capacity, and remain photosynthetically active, under cool growth temperatures that strongly inhibit photosynthesis in summer-active species (Holaday et al. 1992, Adams et al. 1995, 2002, Martindale
and Leegood 1997, Strand et al. 1997, 1999, Dumlao et al. 2012, Cohu et al. 2013b). In this study, multiple leaf morphological and anatomical features as potential correlates of, and predictors for, photosynthetic capacity have been characterized in two winter-active and two summer-active annuals. We report highly significant correlations between the numbers and total
Abbreviations – CC, companion cell; LMA, leaf dry mass per leaf area; PC, phloem parenchyma cell; PFD, photon flux density; SE, sieve element. † Present
address: Dow AgroSciences, Portland, OR, USA ¨ ¨ Present address: Institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Julich GmbH, 52425 Julich, Germany
‡
Physiol. Plant. 2014
cross-sectional area of sugar-loading and transporting cells of the leaf’s minor veins and (1) several other leaf morphological features as well as (2) the rates of light- and CO2 -saturated photosynthesis. These results improve the understanding of what leaf features contribute to the ability of leaves to maximize photosynthesis rates in response to the environment and how plant species adapted to contrasting climatic conditions may differ in their ability to acclimate to cool temperatures. In previous studies (Boese and Huner 1990, Gorsuch et al. 2010, Dumlao et al. 2012), the leaves of winter-active species (the winter annuals spinach and Arabidopsis thaliana and the biennial Verbascum phoeniceum) exhibited greater thickness associated with development of additional layers of chloroplastpacked palisade mesophyll cells in response to growth under lower temperature compared to warmer temperature. The thicker leaves of plants grown under cool temperature furthermore exhibited rates of photosynthesis more than twice as high as those of leaves from plants grown under a moderate temperature (Dumlao et al. 2012). In contrast, no difference in palisade layer number, leaf thickness or foliar photosynthesis rate was seen in two summer annuals grown under two contrasting temperature regimes (Dumlao et al. 2012). In subsequent studies focusing on foliar minor veins (that function in the loading of sugars into the sugartransporting phloem), Cohu et al. (2013a, 2013b) found that both the numbers and total cross-sectional area of phloem cells per vein were greater in leaves of A. thaliana plants grown under higher light and lower temperature compared to those grown under lower light and higher temperature. This included both the sieve elements (SEs) serving as the conduits for sugar transport from the leaves to the rest of the plant as well as the companion cells (CCs) and phloem parenchyma cells (PCs) found in close association with the SE. The CCs, as well as possibly PCs, facilitate the active loading of sucrose as this sugar moves from the leaf’s photosynthetically active mesophyll cells to the SEs (Haritatos et al. 2000). Furthermore, there was a strong correlation between the latter foliar phloem metrics and photosynthesis, as well as weaker associations between minor vein tracheid number and cross-sectional area versus photosynthesis (Cohu et al. 2013b). In the present investigation, a full characterization of leaf morphology [leaf dry mass per leaf area (LMA), leaf thickness, number of cell layers comprising the palisade mesophyll tissue and thickness of the palisade mesophyll tissue], leaf vascular features (foliar vein density and numbers and total cross-sectional area per vein of SEs, CCs, PCs, xylem cells and tracheid
cells of the minor veins), as well as light- and CO2 saturated rates of photosynthesis at 25 and 12.5◦ C was undertaken from fully-expanded leaves of two winter annuals and two summer annuals grown under several different controlled conditions of temperature and photon flux density (PFD). This analysis revealed strong associations among many of the metrics, and considerable acclimation to temperature and light in the two winter annuals but not the summer annuals.
Materials and methods Plant species Four different annual plant species were chosen with well-characterized phloem loading mechanisms and different life histories. Three of the species [A. thaliana (L.) Heynhold, Helianthus annuus L. cv. Soraya (sunflower) and Spinacia oleracea L. cv. Giant Nobel (spinach)] utilize an apoplastic route for sugar loading into the phloem (Gamalei 1989, Lohaus et al. 1995, Haritatos et al. 2000, Turgeon et al. 2001). The other species [Cucurbita pepo L. cv. Italian Zucchini Romanesco (squash)] is from a well-characterized group of cucurbits that use a symplastic route for phloem loading (Volk et al. 1996). Two of these species typically germinate in the spring and complete their life cycle into the fall (summer annuals H. annuus and C. pepo), whereas the winter annuals A. thaliana and S. oleracea germinate in the fall, overwinter and complete their life cycle before the onset of summer. In addition, three ecotypes of A. thaliana (Swedish, Italian and Col-0, ˚ see Agren and Schemske 2012 and Cohu et al. 2013a, 2013b) were characterized. Growth conditions Plants were grown from seed under four (six for spinach) different controlled environmental conditions (for details on growth conditions, see the legend of Fig. 1 and Cohu et al. 2013a). For spinach, two additional growth conditions were utilized (9 h photoperiod at 400 μmol photons m−2 s−1 and daytime/nighttime leaf temperatures of 16–19/15◦ C and 34–36/25◦ C resulting from daytime/nighttime air temperatures of 15/15◦ C and 35/25◦ C, respectively). Fully expanded and mature leaves of non-flowering plants (6–8 weeks old) were characterized. Photosynthesis, leaf thickness and vein measurements Measurements of photosynthetic capacity (light- and CO2 -saturated rates of oxygen evolution at 12.5 and Physiol. Plant. 2014
Fig. 1. The light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 12.5◦ C (A, B), leaf dry mass per area (C, D), thickness of the foliar palisade mesophyll tissue (E, F) and the number of cell layers comprising the leaf mesophyll palisade tissue (G, H) from fully expanded leaves of Arabidopsis thaliana (Col-0; dark gray columns), spinach (black columns), squash (open columns) and sunflower (light gray columns). Plants were grown at a daytime leaf temperature of 25◦ C under 400 or 1000 μmol photons m−2 s−1 or at a daytime leaf temperature of 14◦ C under 400 or 1000 μmol photons m−2 s−1 . PFD, photon flux density in μmol photons m−2 s−1 and Temp., daytime leaf temperature during growth. Means ± standard deviation (n = 4 leaves, one from each of four plants) shown; significant differences among the mean values within a species (P < 0.05) are indicated by different lower case letters; n.s., not significantly different.
25◦ C), leaf vein density (length of vein per unit leaf area), LMA, leaf and palisade mesophyll tissue thickness and number of palisade mesophyll tissue cell layers, as well as leaf tissue embedding in Spurr resin, were conducted as previously described (Amiard et al. 2005, Dumlao et al. 2012). Leaf minor veins were determined and measured as described in Cohu et al. (2013a; also refer Figs 2 and 3 for details for each species) using IMAGE-J (Rasband W.S., ImageJ, U.S. National Institute of Health, Bethesda, MD, http://imagej.nih.gov/ij/, 1997–2012). In all species, the smallest class of veins (sixth or seventh order for squash and sunflower, fourth or fifth order for Physiol. Plant. 2014
Fig. 2. Relationship between the number of cell layers comprising the leaf mesophyll palisade tissue and (A) leaf dry mass per area (R2 = 0.88 for the winter annuals) and (B) the thickness of the leaf mesophyll palisade tissue (R2 = 0.77 for the winter annuals) from fully-expanded leaves. Species included the winter annuals Arabidopsis thaliana (three ecotypes, squares) and spinach (circles) and the summer annuals sunflower (triangles) and squash (diamonds). Plants were grown at a daytime leaf temperature of 25◦ C under 400 (open symbols) or 1000 (light gray symbols) μmol photons m−2 s−1 or at a daytime leaf temperature of 14◦ C under 400 (dark gray symbols) or 1000 (black symbols) μmol photons m−2 s−1 (see also the Key to Symbols in Figure 3), and spinach was also grown under 400 μmol photons m−2 s−1 at daytime leaf temperatures of 19◦ C (additional light gray circle) or 35◦ C (additional open circle). Means ± standard deviation (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001, with all points included in the lines except for the summer annual species (within the ovals).
spinach or third or fourth order for A. thaliana), which also contained the greatest proportion of tissue allocated to phloem cells relative to xylem cells (see Cohu et al. 2013a), was analyzed. Phloem and xylem parameters were quantified from 7–10 vein cross-sections per plant through characterization of vascular cells bound within the bundle sheath of these minor veins (Cohu et al. 2013a). Correlation coefficients and level of significance (ANOVA) were determined using JMP STATISTICAL software (SAS Institute, Cary, NC).
Results The two winter annual species exhibited higher light- and CO2 -saturated rates of oxygen evolution (determined at 12.5◦ C; Fig. 1A), higher LMA (Fig. 1C), greater thickness of the foliar mesophyll palisade tissue (Fig. 1E) and a greater number of palisade cell layers comprising that tissue (Fig. 1G) in response to growth under higher PFD and lower temperature. The summer annuals,
Fig. 3. Key to the symbols in Figs 2, 6–9 and 10. Spinach was also grown under 400 μmol photons m−2 s−1 at daytime leaf temperatures of 19◦ C (plotted as an additional light gray circle) and 35◦ C (plotted as an additional open circle).
on the other hand, showed little consistent response: photosynthesis was highest for sunflower grown at a high PFD under the warm temperature and lowest for squash grown at the high PFD under cool temperature (Fig. 1B); sunflower exhibited a significantly elevated LMA when grown under high PFD at cool temperature (Fig. 1D) and a significantly lower palisade thickness when grown at low PFD at warm temperature (Fig. 1F), whereas squash exhibited no significant differences in these metrics in response to the different growth conditions (Fig. 1D, F). The number of layers of palisade cells was approximately two for both sunflower and squash under all growth regimes (Fig. 1H). A strong and highly significant (P < 0.001) linear correlation between LMA and (1) the light- and CO2 saturated photosynthetic capacity [determined at both 12.5 and 25◦ C (not shown)], (2) total leaf thickness and (3) thickness of the palisade mesophyll tissue was observed among the leaves of all four species grown under all light and temperature regimes (data not shown). There was also a highly significant linear correlation between palisade layer number and (1) LMA (Fig. 2A) and palisade tissue thickness (Fig. 2B) for the winter annuals grown under the different temperature and light regimes, but not for the summer annuals. Representative sketches of minor vein cross-sections from leaves that developed under the moderate PFD of 400 μmol photons m−2 s−1 at two different temperatures are depicted in Fig. 4. There was no significant impact of growth temperature on the numbers or arrangement of phloem and xylem cells in the summer annuals squash and sunflower. On the other hand, development at cool temperature resulted in a greater number of phloem and xylem cells in the minor veins of the two winter annuals A. thaliana and spinach, with the total crosssectional area and numbers of SEs per vein being linearly related to the cross-sectional area and number of all CCs + PCs per vein, respectively, across all species and growth conditions (P < 0.001 for both; data not shown). The differences in the responsiveness of these phloem
Fig. 4. Representative sketches of minor veins from fully-expanded leaves of the winter annual apoplastic loaders Arabidopsis thaliana Col-0 and spinach, the summer annual symplastic loader squash and the summer annual apoplastic loader sunflower grown under a 9-h photoperiod of 400 μmol photons m−2 s−1 at an average day/night leaf temperature of 25/20◦ C (warm) or 14/12.5◦ C (cool). Filled dark gray cells are companion cells (CC), filled light gray cells are phloem parenchyma cells (PC), small white cells within the gray cells are sieve elements (SE), cells with light gray adjacent to the cell walls are tracheids (T) and large white cells are xylem parenchyma cells (XP). Arabidopsis thaliana images modified from Adams et al. (2013).
parameters to growth conditions are illustrated further in Fig. 5, where total cross-sectional area per minor vein (Fig. 5A, B), total cross-sectional area of the SEs per minor vein (Fig. 5C, D) and total cross-sectional area of the CCs + PCs (Fig. 5E, F) was significantly greater in leaves of winter annuals that developed under higher PFD and lower temperature, but not in the leaves of the summer annuals. The cross-sectional area of the xylem tissue in the minor veins did not vary significantly with growth conditions (data not shown). Consequently, the ratio of the cross-sectional area of the phloem to the crosssectional area of the xylem per minor vein increased with growth at higher PFD and lower temperature in the winter annuals (Fig. 5G) but did not vary significantly across growth regimes for the summer annuals (Fig. 5H). There was, however, a highly significant positive linear relationship between total cross-sectional area of foliar minor veins and the thickness of leaf palisade mesophyll tissue of all species grown under all light and temperature regimes (data not shown). Palisade mesophyll tissue thickness and either total crosssectional area of SEs per vein (Fig. 6A) or total crosssectional area of CCs and PCs per vein (Fig. 6B) were Physiol. Plant. 2014
Fig. 6. Relationship between foliar mesophyll palisade tissue thickness and (A) cross-sectional area of sieve elements (SE) per minor vein (R2 = 0.86) and (B) cross-sectional area of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.86) in fully-expanded leaves of two winter annuals and two summer annuals. For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Means ± standard error (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001.
Fig. 5. Total cross-sectional area per minor vein (A, B), total crosssectional sieve element (SE) area per minor vein (C, D), total crosssectional area of companion cells (CC) plus phloem parenchyma cells (PC) per minor vein (E, F) and the ratio of the cross-sectional area of the phloem to the cross-sectional area of the xylem in minor veins (G, H) of fully expanded leaves of two winter annuals and two summer annuals acclimated to different conditions. See the legend of Fig. 1 for additional details. Means ± standard deviation (n = 4 leaves, one from each of four plants) shown; significant differences among the mean values within a species (P < 0.05) are indicated by different lower case letters; n.s., not significantly different.
also significantly and positively correlated. Although there was a trend for the total cross-sectional area of the xylem tissue, as well as cross-sectional tracheid area, per vein to also increase with increasing palisade mesophyll tissue thickness, these positive relationships were not significant (data not shown). As a consequence of the strong response of the phloem and the less pronounced response of the xylem, the ratio of total phloem crosssectional area to total xylem cross-sectional area in the minor veins also increased significantly with increasing palisade mesophyll tissue thickness (data not shown). In contrast, the mean cross-sectional area of individual tracheids, individual SEs or individual CCs and PCs exhibited no relationship with palisade tissue thickness (data not shown). Physiol. Plant. 2014
As palisade mesophyll tissue thickness correlated well with total cross-sectional area of the vascular tissues of minor veins across all species and growth conditions, the former parameter was used as a metric against which to evaluate the numbers of cells in minor veins. The number of SEs per vein, the number of CCs and PCs per vein and the number of all (phloem and xylem) cells per vein exhibited two separate but highly significant positive linear relationships with palisade mesophyll tissue thickness (Fig. 7A–C). For a given palisade mesophyll tissue thickness, the veins of A. thaliana contained more cells compared to veins of the summer annuals or spinach (Fig. 7A–C). However, when normalized by multiplying the number of cells per vein by the density of veins per leaf area, the data for all four species converged onto a single positive linear relationship that was highly significant for all three parameters (Fig. 7D–F). While there were also positive trends for xylem and tracheid cell numbers versus palisade mesophyll tissue thickness, none of these were significant (data not shown). The numbers of cells per minor vein exhibited a highly significant negative correlation with foliar vein density, which was true for the total number of vascular cells (Fig. 8A), the number of SEs (Fig. 8B), the number of CCs and PCs (Fig. 8C) and the number of xylem cells per minor vein (Fig. 8D). Furthermore, cell numbers
Fig. 7. Relationship between foliar mesophyll palisade tissue thickness and (A) the number of sieve elements (SE) per minor vein (R2 = 0.85 for Arabidopsis thaliana and R2 = 0.93 for spinach and summer annuals), (B) the number of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.91 for A. thaliana and R2 = 0.95 for spinach and summer annuals), (C) the number of vascular (phloem + xylem) cells per minor vein (R2 = 0.83 for A. thaliana and R2 = 0.88 for spinach and summer annuals), (D) the product of the number of sieve elements per minor vein and vein density (VD = mm vein length mm−2 leaf area) (R2 = 0.81), (E) the product of the number of companion + phloem parenchyma cells (CC+PC) per minor vein and vein density (R2 = 0.77) and (F) the product of the number of vascular (phloem + xylem) cells per minor vein and vein density (R2 = 0.80) in fully-expanded leaves of two winter annuals and two summer annuals. For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Means ± standard error (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001.
per minor vein and vein density fell into three clusters (shown as three circles) representing (1) the minor veins of A. thaliana with the most cells and lowest vein density, (2) those of spinach with an intermediate number of cells and vein density and (3) the summer annuals with the fewest number of cells per minor vein but highest vein density (Fig. 8; see also Fig. 4). Photosynthetic capacity, on the other hand, exhibited no relationship with vein density for any of the species as growth conditions were varied (Fig. 8E). Figs 9 and 10 evaluate the relationship between numbers and total cross-sectional areas of phloem cells in minor veins and light- and CO2 -saturated rate of photosynthetic oxygen evolution determined at 12.5◦ C (the relationships were also significant for the rates determined at 25◦ C; data not shown). For spinach and A. thaliana, there were separate but highly significant positive linear relationships between photosynthesis and the number of SEs (Fig. 9A) and the number of CCs + PCs per vein (Fig. 9C), whereas neither anatomical metric exhibited a relationship with photosynthesis for the two summer annuals (Fig. 9A, C). Multiplying these anatomical metrics by vein density resulted in significant linear relationships between photosynthesis and the product of SE number per vein and vein density for spinach and A. thaliana that were still separate (Fig. 9B),
while the product of CC + PC number per vein and vein density converged onto a single highly significant linear relationship for the two winter annuals (Fig. 9D). No significant relationship between the product of these anatomical metrics and photosynthesis existed among the summer annuals grown under different temperature and PFD regimes (Fig. 9B, D). Remarkably, even without taking vein density into consideration, photosynthesis was significantly correlated with total cross-sectional SE area per vein (Fig. 10A) as well as total cross-sectional area of the CCs + PCs per vein (Fig. 10B) for the leaves of spinach and A. thaliana plants grown under various conditions. In contrast, the two summer annuals exhibited no relationship between photosynthesis and total phloem cross-sectional areas of minor veins (Fig. 10A, B).
Discussion Multiple leaf morphological and minor vein anatomical metrics, as well as light- and CO2 -saturated photosynthetic capacity, were evaluated in two winter annuals and two summer annuals acclimated to several light and temperature regimes. While many of the vein anatomical features (numbers and cross-sectional areas of phloem cells, either alone or normalized by vein density) and leaf Physiol. Plant. 2014
Fig. 9. Relationship between the light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 12.5◦ C and (A) the number of sieve elements (SE) per minor vein (R2 = 0.99 for spinach and R2 = 0.96 for Arabidopsis thaliana), (B) the product of the number of sieve elements per minor vein and vein density (VD = mm vein length mm−2 leaf area) (R2 = 0.92 for spinach and R2 = 0.85 for A. thaliana), (C) the number of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.99 for spinach and R2 = 0.96 for A. thaliana) and (D) the product of the number of companion + phloem parenchyma cells (CC+PC) per minor vein and vein density (R2 = 0.93 for spinach and A. thaliana) in fully-expanded leaves of two winter annuals and two summer annuals (in ovals). For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Data for A. thaliana in A and C re-plotted from Cohu et al. (2013b). Means ± standard deviation for photosynthesis and means ± standard error for vein metrics (n = 4 leaves, one from each of four plants) shown; regression lines for winter annuals significant at P < 0.001.
Fig. 8. Relationship between foliar vein density and (A) the number of vascular (phloem + xylem) cells per minor vein (R2 = 0.87), (B) the number of sieve elements (SE) per minor vein (R2 = 0.70), (C) the number of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.85), (D) the number of xylem cells per minor vein (R2 = 0.86) and (E) the light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 25◦ C in fully-expanded leaves of two winter annuals and two summer annuals. Ovals surround A. thaliana (squares), spinach (circles) and summer annuals (diamonds and triangles). For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Means ± standard deviation for photosynthesis and vein density, and means ± standard error for vein metrics (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001.
morphological features (leaf and palisade tissue thickness, leaf dry mass per unit leaf area) scaled across all four species and developmental growth regimes, only the two winter annuals exhibited strong acclimatory adjustments in palisade mesophyll cell layers, the numbers Physiol. Plant. 2014
of phloem cells in minor veins and photosynthesis in response to the growth light and temperature conditions examined here. In particular, total cross-sectional area per vein of either SEs or of CCs + PCs, as well as the number of CCs + PCs per minor vein normalized by vein density, were strong predictors of photosynthetic capacity in the leaves of the winter annuals. These results suggest that acclimatory modification of the phloem tissue in the foliar minor veins may be a necessary prerequisite to accommodate an increase in sugar loading and export to accompany the upregulation of photosynthesis that occurs in winter annuals. However, such adjustments to the minor veins should be viewed in the context of a suite of changes in response to growth at low temperature, including an increase in the amount of chloroplast-laden, highly photosynthetically active mesophyll tissue. Consistent with previous studies (Givnish 1988, Boese and Huner 1990, Terashima et al. 2001, Poorter et al.
Fig. 10. Relationship between the light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 12.5◦ C and (A) the cross-sectional area of sieve elements (SE) per minor vein (R2 = 0.81 for spinach and Arabidopsis thaliana) and (B) the cross-sectional area of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.84 for spinach and A. thaliana) in fully-expanded leaves of two winter annuals and two summer annuals (in ovals). For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Data for A. thaliana re-plotted from Cohu et al. (2013b). Means ± standard deviation for photosynthesis and means ± standard error for vein metrics (n = 4 leaves, one from each of four plants) shown; regression lines (excluding the data for the summer annuals in ovals) significant at P < 0.001.
2009, Gorsuch et al. 2010, Dumlao et al. 2012), leaf and palisade mesophyll tissue thickness and leaf dry mass per area were greater in leaves grown at higher PFD and/or lower temperature and exhibiting higher rates of photosynthesis. Leaf and palisade mesophyll tissue thickness were greater due to substantial increases in the number of palisade cell layers in the two winter annuals in response to higher PFD and/or lower temperature. However, in the two summer annuals, both leaf and palisade mesophyll tissue thickness were unresponsive to higher growth PFD and/or lower growth temperature – due to the number of palisade cell layers being limited to two under the growth conditions utilized in this study. Although the palisade tissue consisted of only a single cell layer in summer annuals grown in low light (9 h photoperiod of 100 μmol photons m−2 s−1 ; Amiard et al. 2005), the moderate PFD (9 h photoperiod of 400 μmol photons m−2 s−1 ) employed in this study was already high enough to elicit the maximal number of (two, or rarely three) palisade cell layers sunflower and squash are apparently capable of producing. Interestingly, overexpression of C-repeat binding factor (CBF) transcription factors resulted in thicker leaves, higher chlorophyll content, increased levels of photosynthetic enzymes and higher rates of photosynthesis in Arabidopsis and two other species (Gilmour et al. 2004, Savitch et al. 2005, Pino et al. 2008, Thomashow 2010), suggesting that the CBF regulon may be involved in orchestrating leaf anatomical and physiological acclimation to low temperature. It will
be interesting to determine whether CBF transcription factors also influence leaf vasculature. For species with dorsiventral leaves (as is the case for all species examined here), the majority of leaf photosynthetic activity presumably arises from the palisade mesophyll tissue packed with chloroplasts. To accommodate the export of sugars produced by photosynthesis, one might, in fact, predict the foliar phloem to scale with the volume of the palisade mesophyll tissue. Such a relationship was indeed suggested by the total cross-sectional area of phloem tissue per vein as well as the product of phloem cell number per vein and vein density, each of which exhibited a good correlation with palisade mesophyll tissue thickness (all species) and photosynthesis (only for winter annuals). On the other hand, cell number and total cross-sectional area per vein of the xylem tissue did not correlate with palisade mesophyll tissue thickness or photosynthesis among the four species studied here. However, number and total cross-sectional area of tracheids per vein did show a positive relationship with photosynthesis in A. thaliana (two ecotypes from Sweden and Italy) grown under different temperatures and PFDs (Cohu et al. 2013b). Moreover, total crosssectional xylem area and tracheid number per minor vein, both normalized for foliar vein density, exhibited strong, positive relationships with photosynthesis among eight summer annual species grown under common conditions of high light and warm temperature (Muller et al. 2014). It is possible that foliar vein density, which was highest in the summer annuals that would naturally be under the greatest pressure to supply water to foliar mesophyll tissue, plays a more important role in the distribution of water throughout the leaves than tracheid number or the cross-sectional xylem area per vein. Although investigations of the role of leaf hydraulics in influencing photosynthesis have drawn attention to a relationship between vein density and photosynthetic CO2 exchange (Brodribb et al. 2007, Brodribb and Feild 2010, Brodribb and Jordan 2011, Sack and Scoffoni 2013), no such relationship for intrinsic photosynthetic capacity (lightand CO2 -saturated oxygen evolution) was apparent among the species and growth conditions examined in this study. However, as shown in the accompanying paper (Muller et al. 2014), vein density (coupled with features of the xylem or phloem cells) was related to photosynthetic capacity among eight summer annual species grown under moderate conditions. Whereas vein density is fairly constant (and relatively low) in fully-expanded leaves of winter annuals regardless of growth conditions (Amiard et al. 2005, Cohu et al. 2013b; Fig. 8), vein density is typically high in summer Physiol. Plant. 2014
annuals grown in high light and lower in leaves grown in low light (Amiard et al. 2005, Adams et al. 2007). The moderately high growth PFD of 400 μmol photons m−2 s−1 used in this study was apparently high enough to result in high vein densities, as well as the maximal number of palisade tissue cell layers mentioned above, in the summer annuals. Among the four species examined here, there appeared to be a trade-off between either high vein density or high cell numbers per vein, with a continuum ranging from leaves of the two summer annuals (with the greatest vein densities and lowest cell numbers per vein) to spinach (with intermediate vein density and intermediate cell numbers per vein) to A. thaliana (with the lowest vein density and greatest cell number per vein). As mentioned previously, the two winter annuals, spinach and A. thaliana, exhibited an increased number and cross-sectional area of phloem cells per foliar minor vein in response to development under low temperature and/or high light that were also excellent predictors of photosynthesis (also see Cohu et al. 2013b). These anatomical adjustments presumably provide for a greater driving force for the loading of sugars into the phloem through increased CC and PC numbers that should thus be able to accommodate a greater number of transport proteins for the active movement of sucrose from the mesophyll tissue into the phloem in these apoplastic loaders (Lohaus et al. 1995, Haritatos et al. 2000, Amiard et al. 2007). Moreover, as was pointed out by Cohu et al. (2013a), there is a substantial increase in the viscosity of the phloem sap as temperature declines, and the increased cross-sectional area afforded by the greater number of SEs in the veins of winter annual leaves that develop under lower temperature may be critical to facilitating continued sucrose export and the maintenance of high rates of photosynthesis in the winter. Summer annuals were represented by a single apoplastic loader (sunflower) and a single symplastic loader (squash) in this study, and there was little evidence for a different response of these two species to growth environment. In order to further address summer annuals, the following study (Muller et al. 2014) examines four apoplastic and four symplastic summer annuals in greater detail under a single (warm) growth condition, revealing (1) both common relationships among leaf vascular features and photosynthesis across all species and (2) some contrasting relationships between certain aspects of foliar vein anatomy and photosynthesis for apoplastic versus symplastic phloem loaders. Acknowledgements – This work was supported by the National Science Foundation (Award Numbers IOS0841546 and DEB-1022236 to B. D-A and W. W. A.) and
Physiol. Plant. 2014
the University of Colorado at Boulder. We thank our ˚ colleagues Profs. Douglas Schemske and Jon Agren for providing seeds of the Arabidopsis thaliana ecotypes from Sweden and Italy, Tyler Dowd and Jared Stewart for assistance with vascular characterization, Dr Anza Darehshouri for embedding and cutting of some tissue samples and Dr Jennifer Mathias for one set of photosynthesis measurements.
References Adams WW III, Hoehn A, Demmig-Adams B (1995) Chilling temperatures and the xanthophyll cycle. A comparison of warm-grown and overwintering spinach. Aust J Plant Physiol 22: 75–85 Adams WW III, Demmig-Adams B, Rosenstiel TN, Brightwell AK, Ebbert V (2002) Photosynthesis and photoprotection in overwintering plants. Plant Biol 4: 545–557 Adams WW III, Watson AM, Mueh KE, Amiard V, Turgeon R, Ebbert V, Logan BA, Combs AF, Demmig-Adams B (2007) Photosynthetic acclimation in the context of structural constraints to carbon export from leaves. Photosynth Res 94: 455–466 Adams WW III, Cohu CM, Muller O, Demmig-Adams B (2013) Foliar phloem infrastructure in support of photosynthesis. Front Plant Sci 4: 194 ˚ Agren J, Schemske DW (2012) Reciprocal transplants demonstrate strong adaptive differentiation of the model organism Arabidopsis thaliana in its native range. New Phytol 194: 1112–1122 Amiard V, Mueh KE, Demmig-Adams B, Ebbert V, Turgeon R, Adams WW III (2005) Anatomical and photosynthetic acclimation to the light environment in species with differing mechanisms of phloem loading. Proc Natl Acad Sci USA 102: 12968–12973 Amiard V, Demmig-Adams B, Mueh KE, Turgeon R, Combs AF, Adams WW III (2007) Role of light and jasmonic acid signaling in regulating foliar phloem cell wall ingrowth development. New Phytol 173: 722–731 Boese SR, Huner HPA (1990) Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis. Plant Physiol 94: 1830–1836 Brodribb TJ, Feild TS (2010) Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification. Ecol Lett 13: 175–183 Brodribb TJ, Jordan GJ (2011) Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees. New Phytol 192: 437–448 Brodribb TJ, Feild TS, Jordan GJ (2007) Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiol 144: 1890–1898 Cohu CM, Muller O, Demmig-Adams B, Adams WW III (2013a) Minor loading vein acclimation for three Arabidopsis thaliana ecotypes in response to growth
under different temperature and light regimes. Front Plant Sci 4: 240 Cohu CM, Muller O, Stewart JJ, Demmig-Adams B, Adams WW III (2013b) Association between minor loading vein architecture and light- and CO2 -saturated rates of photosynthetic oxygen evolution among Arabidopsis thaliana ecotypes from different latitudes. Front Plant Sci 4: 264 Dumlao MR, Darehshouri A, Cohu CM, Muller O, Mathias J, Adams WW III, Demmig-Adams B (2012) Low temperature acclimation of photosynthetic capacity and leaf morphology in the context of phloem loading type. Photosynth Res 113: 181–189 Gamalei YV (1989) Structure and function of leaf minor veins in trees and herbs. A taxonomical review. Trees 3: 96–110 Gilmour SJ, Fowler SG, Thomashow MF (2004) Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Mol Biol 54: 767–781 Givnish TJ (1988) Adaptation to sun and shade: a whole-plant perspective. Aust J Plant Physiol 15: 63–92 Gorsuch PA, Pandey S, Atkin OK (2010) Temporal heterogeneity of cold acclimation phenotypes in Arabidopsis leaves. Plant Cell Environ 33: 244–258 Haritatos E, Medville R, Turgeon R (2000) Minor vein structure and sugar transport in Arabidopsis thaliana. Planta 211: 105–111 Holaday AS, Martindale W, Alred R, Brooks AL, Leegood RC (1992) Changes in activities of enzymes of carbon metabolism in leaves during exposure of plants to low temperature. Plant Physiol 98: 1105–1114 Lohaus G, Winter H, Heldt HW (1995) Further studies of the phloem loading process in leaves of barley and spinach: the comparison of metabolite concentrations in the apoplastic compartment with those in the cytosolic compartment and in the sieve tubes. Bot Acta 108: 270–275 Martindale W, Leegood RC (1997) Acclimation of photosynthesis to low temperature in Spinacia oleracea L. 1. Effects of acclimation on CO2 assimilation and carbon partitioning. J Exp Bot 48: 1865–1872 Muller O, Cohu CM, Stewart JJ, Protheroe JA, Demmig-Adams B, Adams WW III (2014) Association between photosynthesis and contrasting features of minor veins in leaves of summer annuals loading phloem via symplastic versus apoplastic routes. Physiol Plant. doi: #10.111/ppl.12155
Pino MR, Skinner JS, Jeknic Z, Hayes PM, Soeldner AH, Thomashow MF, Chen THH (2008) Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modifications in potato. Plant Cell Environ 31: 393–406 ¨ Poorter L, Wright IJ, Villar R Poorter H, Niinemets U, (2009) Causes and consequences of variation in leaf mass per area (LMA): a meta-analysis. New Phytol 182: 565–588 Sack L, Scoffoni C (2013) Leaf venation: structure, function, development, evolution, ecology and applications in the past, present and future. New Phytol 198: 983–1000 Savitch LV, Allard G, Seki M, Robert LS, Tinker NA, Huner NPA, Shinozaki K, Singh J (2005) The effect of overexpression of two Brassica CBF /DREB1-like transcription factors on photosynthetic capacity and freezing tolerance in Brassica napus. Plant Cell Physiol 46: 1525–1539 ˚ Hurry V, Gustafsson P, Gardestrom ¨ P (1997) Strand A, Development of Arabidopsis thaliana leaves at low temperature releases the suppression of photosynthesis and photosynthetic gene expression despite the accumulation of soluble carbohydrates. Plant J 12: 605–614 ˚ Hurry VM, Henkes S, Huner NPA, Gustafsson P, Strand A, ¨ P, Stitt M (1999) Acclimation of Arabidopsis Gardestrom leaves developing at low temperature: increasing cytoplasmic volume accompanies increased activities of enzymes in the Calvin cycle and in the sucrose-biosynthesis pathway. Plant Physiol 119: 1387–1397 Terashima I, Miyazawa SI, Hanba YT (2001) Why are sun leaves thicker than shade leaves? Consideration based on analyses of CO2 diffusion in the leaf. J Plant Res 114: 93–105 Thomashow MF (2010) Molecular basis of plant cold acclimation: insights gained from studying the CBF cold response pathway. Plant Physiol 154: 571–577 Turgeon R, Medville R, Nixon KC (2001) The evolution of minor vein phloem and phloem loading. Am J Bot 88: 1331–1339 Volk GM, Turgeon R, Beebe DU (1996) Secondary plasmodesmata formation in the minor-vein phloem of Cucumis melo L. and Cucurbita pepo L. Planta 199: 425–432
Edited by E. Scarpella
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Physiologia Plantarum 2014
Leaf anatomical and photosynthetic acclimation to cool temperature and high light in two winter versus two summer annuals Christopher M. Cohu† , Onno Muller‡ , William W. Adams III∗ and Barbara Demmig-Adams Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309-0334, USA
Correspondence *Corresponding author, e-mail: [email protected] Received 6 September 2013; revised 5 December 2013 doi:10.1111/ppl.12154
Acclimation of foliar features to cool temperature and high light was characterized in winter (Spinacia oleracea L. cv. Giant Nobel; Arabidopsis thaliana (L.) Heynhold Col-0 and ecotypes from Sweden and Italy) versus summer (Helianthus annuus L. cv. Soraya; Cucurbita pepo L. cv. Italian Zucchini Romanesco) annuals. Significant relationships existed among leaf dry mass per area, photosynthesis, leaf thickness and palisade mesophyll thickness. While the acclimatory response of the summer annuals to cool temperature and/or high light levels was limited, the winter annuals increased the number of palisade cell layers, ranging from two layers under moderate light and warm temperature to between four and five layers under cool temperature and high light. A significant relationship was also found between palisade tissue thickness and either cross-sectional area or number of phloem cells (each normalized by vein density) in minor veins among all four species and growth regimes. The two winter annuals, but not the summer annuals, thus exhibited acclimatory adjustments of minor vein phloem to cool temperature and/or high light, with more numerous and larger phloem cells and a higher maximal photosynthesis rate. The upregulation of photosynthesis in winter annuals in response to low growth temperature may thus depend on not only (1) a greater volume of photosynthesizing palisade tissue but also (2) leaf veins containing additional phloem cells and presumably capable of exporting a greater volume of sugars from the leaves to the rest of the plant.
Introduction Overwintering annual and biennial plant species have the remarkable ability to increase their intrinsic photosynthetic capacity, and remain photosynthetically active, under cool growth temperatures that strongly inhibit photosynthesis in summer-active species (Holaday et al. 1992, Adams et al. 1995, 2002, Martindale
and Leegood 1997, Strand et al. 1997, 1999, Dumlao et al. 2012, Cohu et al. 2013b). In this study, multiple leaf morphological and anatomical features as potential correlates of, and predictors for, photosynthetic capacity have been characterized in two winter-active and two summer-active annuals. We report highly significant correlations between the numbers and total
Abbreviations – CC, companion cell; LMA, leaf dry mass per leaf area; PC, phloem parenchyma cell; PFD, photon flux density; SE, sieve element. † Present
address: Dow AgroSciences, Portland, OR, USA ¨ ¨ Present address: Institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Julich GmbH, 52425 Julich, Germany
‡
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cross-sectional area of sugar-loading and transporting cells of the leaf’s minor veins and (1) several other leaf morphological features as well as (2) the rates of light- and CO2 -saturated photosynthesis. These results improve the understanding of what leaf features contribute to the ability of leaves to maximize photosynthesis rates in response to the environment and how plant species adapted to contrasting climatic conditions may differ in their ability to acclimate to cool temperatures. In previous studies (Boese and Huner 1990, Gorsuch et al. 2010, Dumlao et al. 2012), the leaves of winter-active species (the winter annuals spinach and Arabidopsis thaliana and the biennial Verbascum phoeniceum) exhibited greater thickness associated with development of additional layers of chloroplastpacked palisade mesophyll cells in response to growth under lower temperature compared to warmer temperature. The thicker leaves of plants grown under cool temperature furthermore exhibited rates of photosynthesis more than twice as high as those of leaves from plants grown under a moderate temperature (Dumlao et al. 2012). In contrast, no difference in palisade layer number, leaf thickness or foliar photosynthesis rate was seen in two summer annuals grown under two contrasting temperature regimes (Dumlao et al. 2012). In subsequent studies focusing on foliar minor veins (that function in the loading of sugars into the sugartransporting phloem), Cohu et al. (2013a, 2013b) found that both the numbers and total cross-sectional area of phloem cells per vein were greater in leaves of A. thaliana plants grown under higher light and lower temperature compared to those grown under lower light and higher temperature. This included both the sieve elements (SEs) serving as the conduits for sugar transport from the leaves to the rest of the plant as well as the companion cells (CCs) and phloem parenchyma cells (PCs) found in close association with the SE. The CCs, as well as possibly PCs, facilitate the active loading of sucrose as this sugar moves from the leaf’s photosynthetically active mesophyll cells to the SEs (Haritatos et al. 2000). Furthermore, there was a strong correlation between the latter foliar phloem metrics and photosynthesis, as well as weaker associations between minor vein tracheid number and cross-sectional area versus photosynthesis (Cohu et al. 2013b). In the present investigation, a full characterization of leaf morphology [leaf dry mass per leaf area (LMA), leaf thickness, number of cell layers comprising the palisade mesophyll tissue and thickness of the palisade mesophyll tissue], leaf vascular features (foliar vein density and numbers and total cross-sectional area per vein of SEs, CCs, PCs, xylem cells and tracheid
cells of the minor veins), as well as light- and CO2 saturated rates of photosynthesis at 25 and 12.5◦ C was undertaken from fully-expanded leaves of two winter annuals and two summer annuals grown under several different controlled conditions of temperature and photon flux density (PFD). This analysis revealed strong associations among many of the metrics, and considerable acclimation to temperature and light in the two winter annuals but not the summer annuals.
Materials and methods Plant species Four different annual plant species were chosen with well-characterized phloem loading mechanisms and different life histories. Three of the species [A. thaliana (L.) Heynhold, Helianthus annuus L. cv. Soraya (sunflower) and Spinacia oleracea L. cv. Giant Nobel (spinach)] utilize an apoplastic route for sugar loading into the phloem (Gamalei 1989, Lohaus et al. 1995, Haritatos et al. 2000, Turgeon et al. 2001). The other species [Cucurbita pepo L. cv. Italian Zucchini Romanesco (squash)] is from a well-characterized group of cucurbits that use a symplastic route for phloem loading (Volk et al. 1996). Two of these species typically germinate in the spring and complete their life cycle into the fall (summer annuals H. annuus and C. pepo), whereas the winter annuals A. thaliana and S. oleracea germinate in the fall, overwinter and complete their life cycle before the onset of summer. In addition, three ecotypes of A. thaliana (Swedish, Italian and Col-0, ˚ see Agren and Schemske 2012 and Cohu et al. 2013a, 2013b) were characterized. Growth conditions Plants were grown from seed under four (six for spinach) different controlled environmental conditions (for details on growth conditions, see the legend of Fig. 1 and Cohu et al. 2013a). For spinach, two additional growth conditions were utilized (9 h photoperiod at 400 μmol photons m−2 s−1 and daytime/nighttime leaf temperatures of 16–19/15◦ C and 34–36/25◦ C resulting from daytime/nighttime air temperatures of 15/15◦ C and 35/25◦ C, respectively). Fully expanded and mature leaves of non-flowering plants (6–8 weeks old) were characterized. Photosynthesis, leaf thickness and vein measurements Measurements of photosynthetic capacity (light- and CO2 -saturated rates of oxygen evolution at 12.5 and Physiol. Plant. 2014
Fig. 1. The light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 12.5◦ C (A, B), leaf dry mass per area (C, D), thickness of the foliar palisade mesophyll tissue (E, F) and the number of cell layers comprising the leaf mesophyll palisade tissue (G, H) from fully expanded leaves of Arabidopsis thaliana (Col-0; dark gray columns), spinach (black columns), squash (open columns) and sunflower (light gray columns). Plants were grown at a daytime leaf temperature of 25◦ C under 400 or 1000 μmol photons m−2 s−1 or at a daytime leaf temperature of 14◦ C under 400 or 1000 μmol photons m−2 s−1 . PFD, photon flux density in μmol photons m−2 s−1 and Temp., daytime leaf temperature during growth. Means ± standard deviation (n = 4 leaves, one from each of four plants) shown; significant differences among the mean values within a species (P < 0.05) are indicated by different lower case letters; n.s., not significantly different.
25◦ C), leaf vein density (length of vein per unit leaf area), LMA, leaf and palisade mesophyll tissue thickness and number of palisade mesophyll tissue cell layers, as well as leaf tissue embedding in Spurr resin, were conducted as previously described (Amiard et al. 2005, Dumlao et al. 2012). Leaf minor veins were determined and measured as described in Cohu et al. (2013a; also refer Figs 2 and 3 for details for each species) using IMAGE-J (Rasband W.S., ImageJ, U.S. National Institute of Health, Bethesda, MD, http://imagej.nih.gov/ij/, 1997–2012). In all species, the smallest class of veins (sixth or seventh order for squash and sunflower, fourth or fifth order for Physiol. Plant. 2014
Fig. 2. Relationship between the number of cell layers comprising the leaf mesophyll palisade tissue and (A) leaf dry mass per area (R2 = 0.88 for the winter annuals) and (B) the thickness of the leaf mesophyll palisade tissue (R2 = 0.77 for the winter annuals) from fully-expanded leaves. Species included the winter annuals Arabidopsis thaliana (three ecotypes, squares) and spinach (circles) and the summer annuals sunflower (triangles) and squash (diamonds). Plants were grown at a daytime leaf temperature of 25◦ C under 400 (open symbols) or 1000 (light gray symbols) μmol photons m−2 s−1 or at a daytime leaf temperature of 14◦ C under 400 (dark gray symbols) or 1000 (black symbols) μmol photons m−2 s−1 (see also the Key to Symbols in Figure 3), and spinach was also grown under 400 μmol photons m−2 s−1 at daytime leaf temperatures of 19◦ C (additional light gray circle) or 35◦ C (additional open circle). Means ± standard deviation (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001, with all points included in the lines except for the summer annual species (within the ovals).
spinach or third or fourth order for A. thaliana), which also contained the greatest proportion of tissue allocated to phloem cells relative to xylem cells (see Cohu et al. 2013a), was analyzed. Phloem and xylem parameters were quantified from 7–10 vein cross-sections per plant through characterization of vascular cells bound within the bundle sheath of these minor veins (Cohu et al. 2013a). Correlation coefficients and level of significance (ANOVA) were determined using JMP STATISTICAL software (SAS Institute, Cary, NC).
Results The two winter annual species exhibited higher light- and CO2 -saturated rates of oxygen evolution (determined at 12.5◦ C; Fig. 1A), higher LMA (Fig. 1C), greater thickness of the foliar mesophyll palisade tissue (Fig. 1E) and a greater number of palisade cell layers comprising that tissue (Fig. 1G) in response to growth under higher PFD and lower temperature. The summer annuals,
Fig. 3. Key to the symbols in Figs 2, 6–9 and 10. Spinach was also grown under 400 μmol photons m−2 s−1 at daytime leaf temperatures of 19◦ C (plotted as an additional light gray circle) and 35◦ C (plotted as an additional open circle).
on the other hand, showed little consistent response: photosynthesis was highest for sunflower grown at a high PFD under the warm temperature and lowest for squash grown at the high PFD under cool temperature (Fig. 1B); sunflower exhibited a significantly elevated LMA when grown under high PFD at cool temperature (Fig. 1D) and a significantly lower palisade thickness when grown at low PFD at warm temperature (Fig. 1F), whereas squash exhibited no significant differences in these metrics in response to the different growth conditions (Fig. 1D, F). The number of layers of palisade cells was approximately two for both sunflower and squash under all growth regimes (Fig. 1H). A strong and highly significant (P < 0.001) linear correlation between LMA and (1) the light- and CO2 saturated photosynthetic capacity [determined at both 12.5 and 25◦ C (not shown)], (2) total leaf thickness and (3) thickness of the palisade mesophyll tissue was observed among the leaves of all four species grown under all light and temperature regimes (data not shown). There was also a highly significant linear correlation between palisade layer number and (1) LMA (Fig. 2A) and palisade tissue thickness (Fig. 2B) for the winter annuals grown under the different temperature and light regimes, but not for the summer annuals. Representative sketches of minor vein cross-sections from leaves that developed under the moderate PFD of 400 μmol photons m−2 s−1 at two different temperatures are depicted in Fig. 4. There was no significant impact of growth temperature on the numbers or arrangement of phloem and xylem cells in the summer annuals squash and sunflower. On the other hand, development at cool temperature resulted in a greater number of phloem and xylem cells in the minor veins of the two winter annuals A. thaliana and spinach, with the total crosssectional area and numbers of SEs per vein being linearly related to the cross-sectional area and number of all CCs + PCs per vein, respectively, across all species and growth conditions (P < 0.001 for both; data not shown). The differences in the responsiveness of these phloem
Fig. 4. Representative sketches of minor veins from fully-expanded leaves of the winter annual apoplastic loaders Arabidopsis thaliana Col-0 and spinach, the summer annual symplastic loader squash and the summer annual apoplastic loader sunflower grown under a 9-h photoperiod of 400 μmol photons m−2 s−1 at an average day/night leaf temperature of 25/20◦ C (warm) or 14/12.5◦ C (cool). Filled dark gray cells are companion cells (CC), filled light gray cells are phloem parenchyma cells (PC), small white cells within the gray cells are sieve elements (SE), cells with light gray adjacent to the cell walls are tracheids (T) and large white cells are xylem parenchyma cells (XP). Arabidopsis thaliana images modified from Adams et al. (2013).
parameters to growth conditions are illustrated further in Fig. 5, where total cross-sectional area per minor vein (Fig. 5A, B), total cross-sectional area of the SEs per minor vein (Fig. 5C, D) and total cross-sectional area of the CCs + PCs (Fig. 5E, F) was significantly greater in leaves of winter annuals that developed under higher PFD and lower temperature, but not in the leaves of the summer annuals. The cross-sectional area of the xylem tissue in the minor veins did not vary significantly with growth conditions (data not shown). Consequently, the ratio of the cross-sectional area of the phloem to the crosssectional area of the xylem per minor vein increased with growth at higher PFD and lower temperature in the winter annuals (Fig. 5G) but did not vary significantly across growth regimes for the summer annuals (Fig. 5H). There was, however, a highly significant positive linear relationship between total cross-sectional area of foliar minor veins and the thickness of leaf palisade mesophyll tissue of all species grown under all light and temperature regimes (data not shown). Palisade mesophyll tissue thickness and either total crosssectional area of SEs per vein (Fig. 6A) or total crosssectional area of CCs and PCs per vein (Fig. 6B) were Physiol. Plant. 2014
Fig. 6. Relationship between foliar mesophyll palisade tissue thickness and (A) cross-sectional area of sieve elements (SE) per minor vein (R2 = 0.86) and (B) cross-sectional area of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.86) in fully-expanded leaves of two winter annuals and two summer annuals. For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Means ± standard error (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001.
Fig. 5. Total cross-sectional area per minor vein (A, B), total crosssectional sieve element (SE) area per minor vein (C, D), total crosssectional area of companion cells (CC) plus phloem parenchyma cells (PC) per minor vein (E, F) and the ratio of the cross-sectional area of the phloem to the cross-sectional area of the xylem in minor veins (G, H) of fully expanded leaves of two winter annuals and two summer annuals acclimated to different conditions. See the legend of Fig. 1 for additional details. Means ± standard deviation (n = 4 leaves, one from each of four plants) shown; significant differences among the mean values within a species (P < 0.05) are indicated by different lower case letters; n.s., not significantly different.
also significantly and positively correlated. Although there was a trend for the total cross-sectional area of the xylem tissue, as well as cross-sectional tracheid area, per vein to also increase with increasing palisade mesophyll tissue thickness, these positive relationships were not significant (data not shown). As a consequence of the strong response of the phloem and the less pronounced response of the xylem, the ratio of total phloem crosssectional area to total xylem cross-sectional area in the minor veins also increased significantly with increasing palisade mesophyll tissue thickness (data not shown). In contrast, the mean cross-sectional area of individual tracheids, individual SEs or individual CCs and PCs exhibited no relationship with palisade tissue thickness (data not shown). Physiol. Plant. 2014
As palisade mesophyll tissue thickness correlated well with total cross-sectional area of the vascular tissues of minor veins across all species and growth conditions, the former parameter was used as a metric against which to evaluate the numbers of cells in minor veins. The number of SEs per vein, the number of CCs and PCs per vein and the number of all (phloem and xylem) cells per vein exhibited two separate but highly significant positive linear relationships with palisade mesophyll tissue thickness (Fig. 7A–C). For a given palisade mesophyll tissue thickness, the veins of A. thaliana contained more cells compared to veins of the summer annuals or spinach (Fig. 7A–C). However, when normalized by multiplying the number of cells per vein by the density of veins per leaf area, the data for all four species converged onto a single positive linear relationship that was highly significant for all three parameters (Fig. 7D–F). While there were also positive trends for xylem and tracheid cell numbers versus palisade mesophyll tissue thickness, none of these were significant (data not shown). The numbers of cells per minor vein exhibited a highly significant negative correlation with foliar vein density, which was true for the total number of vascular cells (Fig. 8A), the number of SEs (Fig. 8B), the number of CCs and PCs (Fig. 8C) and the number of xylem cells per minor vein (Fig. 8D). Furthermore, cell numbers
Fig. 7. Relationship between foliar mesophyll palisade tissue thickness and (A) the number of sieve elements (SE) per minor vein (R2 = 0.85 for Arabidopsis thaliana and R2 = 0.93 for spinach and summer annuals), (B) the number of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.91 for A. thaliana and R2 = 0.95 for spinach and summer annuals), (C) the number of vascular (phloem + xylem) cells per minor vein (R2 = 0.83 for A. thaliana and R2 = 0.88 for spinach and summer annuals), (D) the product of the number of sieve elements per minor vein and vein density (VD = mm vein length mm−2 leaf area) (R2 = 0.81), (E) the product of the number of companion + phloem parenchyma cells (CC+PC) per minor vein and vein density (R2 = 0.77) and (F) the product of the number of vascular (phloem + xylem) cells per minor vein and vein density (R2 = 0.80) in fully-expanded leaves of two winter annuals and two summer annuals. For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Means ± standard error (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001.
per minor vein and vein density fell into three clusters (shown as three circles) representing (1) the minor veins of A. thaliana with the most cells and lowest vein density, (2) those of spinach with an intermediate number of cells and vein density and (3) the summer annuals with the fewest number of cells per minor vein but highest vein density (Fig. 8; see also Fig. 4). Photosynthetic capacity, on the other hand, exhibited no relationship with vein density for any of the species as growth conditions were varied (Fig. 8E). Figs 9 and 10 evaluate the relationship between numbers and total cross-sectional areas of phloem cells in minor veins and light- and CO2 -saturated rate of photosynthetic oxygen evolution determined at 12.5◦ C (the relationships were also significant for the rates determined at 25◦ C; data not shown). For spinach and A. thaliana, there were separate but highly significant positive linear relationships between photosynthesis and the number of SEs (Fig. 9A) and the number of CCs + PCs per vein (Fig. 9C), whereas neither anatomical metric exhibited a relationship with photosynthesis for the two summer annuals (Fig. 9A, C). Multiplying these anatomical metrics by vein density resulted in significant linear relationships between photosynthesis and the product of SE number per vein and vein density for spinach and A. thaliana that were still separate (Fig. 9B),
while the product of CC + PC number per vein and vein density converged onto a single highly significant linear relationship for the two winter annuals (Fig. 9D). No significant relationship between the product of these anatomical metrics and photosynthesis existed among the summer annuals grown under different temperature and PFD regimes (Fig. 9B, D). Remarkably, even without taking vein density into consideration, photosynthesis was significantly correlated with total cross-sectional SE area per vein (Fig. 10A) as well as total cross-sectional area of the CCs + PCs per vein (Fig. 10B) for the leaves of spinach and A. thaliana plants grown under various conditions. In contrast, the two summer annuals exhibited no relationship between photosynthesis and total phloem cross-sectional areas of minor veins (Fig. 10A, B).
Discussion Multiple leaf morphological and minor vein anatomical metrics, as well as light- and CO2 -saturated photosynthetic capacity, were evaluated in two winter annuals and two summer annuals acclimated to several light and temperature regimes. While many of the vein anatomical features (numbers and cross-sectional areas of phloem cells, either alone or normalized by vein density) and leaf Physiol. Plant. 2014
Fig. 9. Relationship between the light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 12.5◦ C and (A) the number of sieve elements (SE) per minor vein (R2 = 0.99 for spinach and R2 = 0.96 for Arabidopsis thaliana), (B) the product of the number of sieve elements per minor vein and vein density (VD = mm vein length mm−2 leaf area) (R2 = 0.92 for spinach and R2 = 0.85 for A. thaliana), (C) the number of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.99 for spinach and R2 = 0.96 for A. thaliana) and (D) the product of the number of companion + phloem parenchyma cells (CC+PC) per minor vein and vein density (R2 = 0.93 for spinach and A. thaliana) in fully-expanded leaves of two winter annuals and two summer annuals (in ovals). For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Data for A. thaliana in A and C re-plotted from Cohu et al. (2013b). Means ± standard deviation for photosynthesis and means ± standard error for vein metrics (n = 4 leaves, one from each of four plants) shown; regression lines for winter annuals significant at P < 0.001.
Fig. 8. Relationship between foliar vein density and (A) the number of vascular (phloem + xylem) cells per minor vein (R2 = 0.87), (B) the number of sieve elements (SE) per minor vein (R2 = 0.70), (C) the number of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.85), (D) the number of xylem cells per minor vein (R2 = 0.86) and (E) the light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 25◦ C in fully-expanded leaves of two winter annuals and two summer annuals. Ovals surround A. thaliana (squares), spinach (circles) and summer annuals (diamonds and triangles). For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Means ± standard deviation for photosynthesis and vein density, and means ± standard error for vein metrics (n = 4 leaves, one from each of four plants) shown; regression lines significant at P < 0.001.
morphological features (leaf and palisade tissue thickness, leaf dry mass per unit leaf area) scaled across all four species and developmental growth regimes, only the two winter annuals exhibited strong acclimatory adjustments in palisade mesophyll cell layers, the numbers Physiol. Plant. 2014
of phloem cells in minor veins and photosynthesis in response to the growth light and temperature conditions examined here. In particular, total cross-sectional area per vein of either SEs or of CCs + PCs, as well as the number of CCs + PCs per minor vein normalized by vein density, were strong predictors of photosynthetic capacity in the leaves of the winter annuals. These results suggest that acclimatory modification of the phloem tissue in the foliar minor veins may be a necessary prerequisite to accommodate an increase in sugar loading and export to accompany the upregulation of photosynthesis that occurs in winter annuals. However, such adjustments to the minor veins should be viewed in the context of a suite of changes in response to growth at low temperature, including an increase in the amount of chloroplast-laden, highly photosynthetically active mesophyll tissue. Consistent with previous studies (Givnish 1988, Boese and Huner 1990, Terashima et al. 2001, Poorter et al.
Fig. 10. Relationship between the light- and CO2 -saturated rate of oxygen evolution determined at a leaf temperature of 12.5◦ C and (A) the cross-sectional area of sieve elements (SE) per minor vein (R2 = 0.81 for spinach and Arabidopsis thaliana) and (B) the cross-sectional area of companion cells (CC) and phloem parenchyma cells (PC) per minor vein (R2 = 0.84 for spinach and A. thaliana) in fully-expanded leaves of two winter annuals and two summer annuals (in ovals). For detail on species and growth conditions, see the legend of Fig. 1 and the key in Fig. 3. Data for A. thaliana re-plotted from Cohu et al. (2013b). Means ± standard deviation for photosynthesis and means ± standard error for vein metrics (n = 4 leaves, one from each of four plants) shown; regression lines (excluding the data for the summer annuals in ovals) significant at P < 0.001.
2009, Gorsuch et al. 2010, Dumlao et al. 2012), leaf and palisade mesophyll tissue thickness and leaf dry mass per area were greater in leaves grown at higher PFD and/or lower temperature and exhibiting higher rates of photosynthesis. Leaf and palisade mesophyll tissue thickness were greater due to substantial increases in the number of palisade cell layers in the two winter annuals in response to higher PFD and/or lower temperature. However, in the two summer annuals, both leaf and palisade mesophyll tissue thickness were unresponsive to higher growth PFD and/or lower growth temperature – due to the number of palisade cell layers being limited to two under the growth conditions utilized in this study. Although the palisade tissue consisted of only a single cell layer in summer annuals grown in low light (9 h photoperiod of 100 μmol photons m−2 s−1 ; Amiard et al. 2005), the moderate PFD (9 h photoperiod of 400 μmol photons m−2 s−1 ) employed in this study was already high enough to elicit the maximal number of (two, or rarely three) palisade cell layers sunflower and squash are apparently capable of producing. Interestingly, overexpression of C-repeat binding factor (CBF) transcription factors resulted in thicker leaves, higher chlorophyll content, increased levels of photosynthetic enzymes and higher rates of photosynthesis in Arabidopsis and two other species (Gilmour et al. 2004, Savitch et al. 2005, Pino et al. 2008, Thomashow 2010), suggesting that the CBF regulon may be involved in orchestrating leaf anatomical and physiological acclimation to low temperature. It will
be interesting to determine whether CBF transcription factors also influence leaf vasculature. For species with dorsiventral leaves (as is the case for all species examined here), the majority of leaf photosynthetic activity presumably arises from the palisade mesophyll tissue packed with chloroplasts. To accommodate the export of sugars produced by photosynthesis, one might, in fact, predict the foliar phloem to scale with the volume of the palisade mesophyll tissue. Such a relationship was indeed suggested by the total cross-sectional area of phloem tissue per vein as well as the product of phloem cell number per vein and vein density, each of which exhibited a good correlation with palisade mesophyll tissue thickness (all species) and photosynthesis (only for winter annuals). On the other hand, cell number and total cross-sectional area per vein of the xylem tissue did not correlate with palisade mesophyll tissue thickness or photosynthesis among the four species studied here. However, number and total cross-sectional area of tracheids per vein did show a positive relationship with photosynthesis in A. thaliana (two ecotypes from Sweden and Italy) grown under different temperatures and PFDs (Cohu et al. 2013b). Moreover, total crosssectional xylem area and tracheid number per minor vein, both normalized for foliar vein density, exhibited strong, positive relationships with photosynthesis among eight summer annual species grown under common conditions of high light and warm temperature (Muller et al. 2014). It is possible that foliar vein density, which was highest in the summer annuals that would naturally be under the greatest pressure to supply water to foliar mesophyll tissue, plays a more important role in the distribution of water throughout the leaves than tracheid number or the cross-sectional xylem area per vein. Although investigations of the role of leaf hydraulics in influencing photosynthesis have drawn attention to a relationship between vein density and photosynthetic CO2 exchange (Brodribb et al. 2007, Brodribb and Feild 2010, Brodribb and Jordan 2011, Sack and Scoffoni 2013), no such relationship for intrinsic photosynthetic capacity (lightand CO2 -saturated oxygen evolution) was apparent among the species and growth conditions examined in this study. However, as shown in the accompanying paper (Muller et al. 2014), vein density (coupled with features of the xylem or phloem cells) was related to photosynthetic capacity among eight summer annual species grown under moderate conditions. Whereas vein density is fairly constant (and relatively low) in fully-expanded leaves of winter annuals regardless of growth conditions (Amiard et al. 2005, Cohu et al. 2013b; Fig. 8), vein density is typically high in summer Physiol. Plant. 2014
annuals grown in high light and lower in leaves grown in low light (Amiard et al. 2005, Adams et al. 2007). The moderately high growth PFD of 400 μmol photons m−2 s−1 used in this study was apparently high enough to result in high vein densities, as well as the maximal number of palisade tissue cell layers mentioned above, in the summer annuals. Among the four species examined here, there appeared to be a trade-off between either high vein density or high cell numbers per vein, with a continuum ranging from leaves of the two summer annuals (with the greatest vein densities and lowest cell numbers per vein) to spinach (with intermediate vein density and intermediate cell numbers per vein) to A. thaliana (with the lowest vein density and greatest cell number per vein). As mentioned previously, the two winter annuals, spinach and A. thaliana, exhibited an increased number and cross-sectional area of phloem cells per foliar minor vein in response to development under low temperature and/or high light that were also excellent predictors of photosynthesis (also see Cohu et al. 2013b). These anatomical adjustments presumably provide for a greater driving force for the loading of sugars into the phloem through increased CC and PC numbers that should thus be able to accommodate a greater number of transport proteins for the active movement of sucrose from the mesophyll tissue into the phloem in these apoplastic loaders (Lohaus et al. 1995, Haritatos et al. 2000, Amiard et al. 2007). Moreover, as was pointed out by Cohu et al. (2013a), there is a substantial increase in the viscosity of the phloem sap as temperature declines, and the increased cross-sectional area afforded by the greater number of SEs in the veins of winter annual leaves that develop under lower temperature may be critical to facilitating continued sucrose export and the maintenance of high rates of photosynthesis in the winter. Summer annuals were represented by a single apoplastic loader (sunflower) and a single symplastic loader (squash) in this study, and there was little evidence for a different response of these two species to growth environment. In order to further address summer annuals, the following study (Muller et al. 2014) examines four apoplastic and four symplastic summer annuals in greater detail under a single (warm) growth condition, revealing (1) both common relationships among leaf vascular features and photosynthesis across all species and (2) some contrasting relationships between certain aspects of foliar vein anatomy and photosynthesis for apoplastic versus symplastic phloem loaders. Acknowledgements – This work was supported by the National Science Foundation (Award Numbers IOS0841546 and DEB-1022236 to B. D-A and W. W. A.) and
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the University of Colorado at Boulder. We thank our ˚ colleagues Profs. Douglas Schemske and Jon Agren for providing seeds of the Arabidopsis thaliana ecotypes from Sweden and Italy, Tyler Dowd and Jared Stewart for assistance with vascular characterization, Dr Anza Darehshouri for embedding and cutting of some tissue samples and Dr Jennifer Mathias for one set of photosynthesis measurements.
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