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Recent characterization of genes and core regulatory networks has revolutionised our understanding of how tissues develop. However, the development of individual tissues is only one requirement for building complex organisms. Another, less understood process is how the development of spatially discrete but functionally interdependent tissues is coordinated. One possible mechanism for such coordination is colocation of primordial tissues. Thus, lymphatic and blood-carrying vessels of mammals develop from a common embryonic vascular system, and the xylem and phloem of plants derive from a shared cambium. However, complex organisms also depend on the coordinated development of many tissues with different origins (Cavalier-Smith, 2005); for example, lung capacity, vascular volume and muscle mass are necessarily coordinated (Rubner, 1883). Similarly, developmental coordination is essential for plants because their primordial tissues have indeterminate growth. Thus, plants can show great plasticity in response to the environment, but this plasticity is only effective if the diverse tissues involved remain functionally coordinated.
One important example of coordination between discrete tissues is found between the veins and stomata in the leaves of land plants. Branching density in the leaf vein network determines water transport efficiency of the lamina (leaf hydraulic conductance), which is closely linked to maximum rates of photosynthesis (Brodribb et al., 2005) and transpiration (Boyce et al., 2009). Because leaf vascular networks replace water lost by evaporation during the uptake of CO2 for photosynthesis (Sack & Holbrook, 2006), plants with higher rates of photosynthesis per unit leaf area lose more water (Cowan & Farquhar, 1977) and thus demand greater investment in leaf veins (McKown et al., 2010). This investment comes largely as increased branching of leaf minor veins, because a greater density of minor veins delivers water closer to sites of evaporation in the leaf (Brodribb et al., 2007), leading to increased transport efficiency (Sack & Frole, 2006). However, these veins are expensive to synthesize, and plants are likely to coordinate the production of photosynthetic and water supply tissues to maximize returns on investments in the water transport system (Brodribb & Jordan, 2011). Furthermore, while vein density determines water supply in the leaf, the density of stomata determines maximum rates of water loss and photosynthesis, and thus maintaining a balance between these traits during adaptation to the environment should be of high functional and adaptive importance. Such coordination has been demonstrated both within trees during plastic adaptation to light (Murphy et al., 2012) and between species (Edwards, 2006; Dunbar-Co et al., 2009; Zhang et al., 2012).
However, little is known about how this critically important link between vascular and stomatal tissues is maintained. A recent study of a tree species showed that plasticity in epidermal cell size changed vein and stomatal density in concert during light acclimation. Hence, larger epidermal cells in the shade result in larger leaves that have lower densities of veins and stomata than sun leaves (Murphy et al., 2012). This coordinating role of cell size during plastic adaptation of leaves to different evaporative and photosynthetic conditions of sun and shade raises the prospect that changing cell size could also be an important mechanism for evolutionary adaptation in plants.
A correlation between cell volume and genome size has been long recognized as a fundamental feature of eukaryotic organisms (Mirsky & Ris, 1951; Cavalier-Smith, 1985); however, the evolutionary significance of variation in cell size, and associated genome size in plants and animals, has been hotly debated (Cavalier-Smith, 1978, 2005; Petrov, 2001; Hodgson et al., 2010). In animals, transitions in cell and genome size are implicated in several important evolutionary transitions (such as the evolution of birds from dinosaurs; Organ et al., 2007), but in plants the adaptive significance of cell size variation remains obscure. Attempts to account for the enormous range in genome and cell size in plants have recently focused on variation in stomatal size as a potentially important functional consequence of variable cellular and nuclear volume (Beaulieu et al., 2008). Theory and observation suggest that large stomata are associated with low rates of gas exchange as a result of limits on the packing density of guard cells (if stomata become larger, then fewer can fit on the leaf surface), and diminishing benefits in terms of maximum diffusive conductance of larger, deeper pores (Franks & Beerling, 2009). Other potentially important tissues that share size-constrained functional properties include leaf veins, which have analogous associations between the size of cells and the density (Field & Brodribb, 2013) and conductivity (Sack & Frole, 2006; Brodribb et al., 2007) of the vascular system. Epidermal cell size also appears to be a primary determinant of the final size of leaves, as well as influencing the thickness of the photosynthetic mesophyll (Pérez-Pérez et al., 2011). Here we examine how these interconnected systems in the leaf respond to family-wide variation in cell size.
Considering the diversity of influences that cell size has on leaf physiology, we investigate how key functional attributes of leaves remain coordinated if cell size changes. This is of particular significance considering that cell size and genome size appear not only to be rather labile within angiosperms, but also to exhibit some long-term adaptive patterns (Masterson, 1994; Franks et al., 2012b). In this study, we examine the relationship between cell size and functional anatomy in the leaves of a morphologically and ecologically diverse sample of Proteaceae trees and shrubs. The primary question we address in this study is whether the cell sizes in functionally linked tissues are coordinated in such a way as to preserve integrated function. Specifically we hypothesize that greater vein density and stomatal density should be associated with smaller cell size, leading to coordination in tissues related to water supply and water loss in the leaf.
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As leaf anatomy and morphology evolve and adapt in response to environmental change, there is the potential for functionally interdependent tissues to change independently of one another. Without some coordinating mechanism, this would lead to inefficiency or plant death. Our results show that coordinated changes in cell size can function as a linkage between the vascular system and epidermis during adaptive variation in leaves of Proteaceae. Over an extreme range of leaf size, morphology and habitat, our sample of 48 species of shrubs and trees showed strongly correlated changes in the sizes of distinct cell types (stomatal, epidermal, xylem and palisade cells) in the leaf. This resulted in a unified relationship between the density of stomata on the leaf surface and the density of vein branching in the lamina. As these tissues are responsible for regulating interlocked processes of water delivery and water loss in leaves, modification of cell size therefore provides a rapid way for plants to adapt and evolve to the prevailing conditions without compromising the coordination of component tissues necessary for the whole leaf to function effectively (Fig. 7).
Figure 7. Schematic representation of the linkages between variation in cell size and species adaptation, explaining why cell size should be coordinated and why variation in cell size is a useful adaptive tool. As long as mutations in cell size produce parallel changes in the size of all cell lines in the leaf, the critical link between water supply and transpiration/photosynthesis will be maintained. Selection can then act upon the functional outputs of cell size variation in the leaf, which are photosynthetic performance and leaf thickness, both of which are highly adaptive under different conditions. The selective advantage of high rates of photosynthesis is relatively straightforward, while leaf thickness is involved in a suite of associated functional characters, such as leaf mass per unit area (LMA), stomatal positioning and leaf water content (see the Discussion section).
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Variation in the sizes of cells and genomes appears to be closely related across biological kingdoms (Cavalier-Smith, 2005), but the selective processes driving this variation has been the subject of considerable controversy. Based upon a linear correlation (P < 0.01; data not shown) between the stomatal size data presented here for Proteaceae and genome size published by Stace et al. (1998), we can say that Proteaceae provide an exemplary demonstration of this so called ‘C-size paradox’. A commonly cited explanation for cell and genome size variation suggests that different cell sizes may be suited to different ecological strategies, and in particular whether a species is the product of r- or K-type selection (Cavalier-Smith, 1978). Owing to an apparent negative correlation between cell size and rates of cell division (Van't Hof & Sparrow, 1963), large cells/genomes are proposed to result from K-selection, yielding slow-growing species, while small cells are suggested as being associated with r-selected species, with rapid turnover. This idea has received some support from studies in vertebrates (Vinogradov, 1995), and has been tested in seed plants on the premise that K-selected species are typically those with high LMA and hence that LMA should be positively correlated with genome size. Although early studies provided some support for this hypothesis (Knight et al., 2005), a larger study found nonsignificant associations within a phylogenetically independent framework (Beaulieu et al., 2007), and similarly within the Proteaceae we found no correlations between cell size and LMA (Table 2).
Rather than expecting cell size to affect leaf economics in a predictable way, we argue here that by coordinating cell sizes in the leaf, plants are free to adapt the water transport and photosynthetic gas-exchange properties of leaves while still maintaining an optimal balance between water transport and water loss tissues. Coordination in stomatal and hydraulic physiology provides important functional advantages in terms of maintaining optimal investment and safety (Brodribb, 2009) and this pressure may be the adaptive driver that has led to cell size-dependent coordination between xylem and stomatal tissues in the leaf. Along with other studies showing correlations between cell size and the density of stomata and veins (Murphy et al., 2012; Zhang et al., 2012), the data here from such a diverse family as the Proteaceae provide strong evidence of the primacy of the stomata–vein linkage. Size correlations between other leaf cells, such as epidermal cells and palisade cells (Fig. 3), may have functional significance as well, but it is also possible that the correlated size of these cells is merely a consequence of a common genetic control of cell sizes, perhaps via genome size.
Other studies have indicated the importance of cell size in regulating the density of stomata on the leaf, because constraints on packing density mean that fewer large stomata can fit on the leaf than smaller stomata (Beaulieu et al., 2008; Franks & Beerling, 2009). Our data show that this relationship is particularly strong among Proteaceae (Fig. 3, Table 2). Owing to the connection between size and density in stomata, a critical implication of reduced stomatal cell size is a dramatic increase in the amount of water and CO2 that can be exchanged over the leaf epidermis, because stomatal density is allowed to increase and pore depth is reduced. The net result of this is that a leaf epidermis built from smaller cells will support a much higher capacity for gas exchange, in terms of both photosynthetic CO2 and transpirational water vapour. However, without a parallel increase in the density of minor veins in the leaf, the increased capacity for photosynthesis cannot be realized because stomata will not be sufficiently irrigated to open fully (Brodribb, 2009). Hence the discovery here, that vein density is also sensitive to cell size in such a way as to change in proportion with stomatal density, is highly significant because the result is a match between water supply and water loss from the leaf. This match is most clearly demonstrated by the proportionality between modelled maximum stomatal conductance and vein density across all species (Fig. 5), indicating that the hydraulic efficiency of the vein network (as determined by vein density; Brodribb et al., 2007) remains in proportion to the water-loss capacity of the leaf. Interestingly, we found that vein density was most strongly correlated with the size of stomatal and epidermal cells, and whilst these cells are the key determinants of stomatal density, they are separated from the xylem and mesophyll cells that make up the vein network and surrounding tissue.
One pathway to evolving enhanced photosynthetic rate in leaves can be easily visualized as a process of selection for small cell size, leading to high densities of stomata and veins in leaves (Fig. 7). Such an evolutionary trajectory appears to have been significant during the evolution of the Proteaceae. Thus, one major clade (the Grevilleoideae) is dominated by taxa with small stomata and high vein densities. This pattern makes ecological sense because cellular miniaturization in this group is associated with species that occupy tropical rainforests, where rapid gas exchange and growth tend to be selected for (Körner, 1994). Interestingly, however, several clades, including two large and ecologically successful groups (Persoonioideae and Protea, each with c. 100 species), have very large stomata. These cases are of particular interest because they imply that apparently less productive, large cell size must also confer selective advantages to plants under some conditions. In the Proteaceae, very large stomata occur almost exclusively in clades of open habitats (Fig. 6), and almost exclusively in species with thick and mostly amphistomous leaves. This association also fits the evidence that selection under high light conditions favours thick leaves and amphistomy (Smith et al., 1998; Cooper & Cass, 2003). Amphistomous leaves are highly efficient in terms of photosynthesis, water use and hydraulic supply (Mott et al., 1982), and the ability to spread stomata more sparsely on both leaf surfaces will greatly ameliorate stomatal crowding constraints, probably allowing stomatal size to increase. It is uncertain whether the primary selective force in open conditions is for thick leaves and hence large cells, ultimately allowing amphistomy to develop, or whether amphistomy is selected for initially, leading to a relaxation in spatial constraints in the leaf and allowing cell size and leaf thickness to increase.
Evidence suggests that amphistomy is strongly associated with high light environments and selected against in shady environments (Carpenter, 1994; Smith et al., 1998). While amphistomy provides for a more efficient use of water (Mott & O'Leary, 1984), mesophyll tissue (Parkhurst, 1978) and vein tissue (because veins can deliver water simultaneously to both surfaces of the leaf), these benefits require substantial illumination of both leaf surfaces, and under low light the significant internal self-shading makes this a highly inefficient architecture. Alternatively, the more common configuration of stomata confined to the lower leaf surface seems to provide plants with a more efficient light-harvesting configuration under shadier conditions. However, the restriction of stomata to the lower leaf surface requires that stomata become densely concentrated and hence vein density must be very high to sustain rapid water transport to this highly evaporative surface. The combination of hypostomy and high rates of gas exchange would therefore produce substantial pressure for simultaneous miniaturization of both stomata and veins (Field & Brodribb, 2013). However, as long as the size of these cells declines in concert, leaves will remain balanced in terms of hydraulic supply and evaporative load. Our data for Proteaceae indicate that this cell size-dependent coordination does operate, and we suggest that this is possibly mediated by the size of the species genome.
Recently, it has been suggested that changes in stomatal and genome size in land plants may be a response to Phanerozoic megacycles of CO2 in the atmosphere, with periods of low CO2 favouring smaller stomata (Franks et al., 2012a). The common link between stomatal and vein density, demonstrated here through cell size, provides an important connection between the evolutionary patterns described for stomatal density and similar patterns reported for vein density (Boyce et al., 2009). Studies of leaf veins across the phylogeny of vascular plants show that, although vein densities range from < 1 to > 25 mm mm−2, only angiosperms are able to produce leaves with vein densities in the upper part of this range (> 6 mm mm−2). This unique capacity in angiosperms has been discussed as an important factor contributing to their success over competing plant groups (Brodribb & Feild, 2010), and, similar to the stomatal trend, the rise in vein density requires a decrease in cell size (Field & Brodribb, 2013) and is likely to respond to changes in atmospheric CO2 (Brodribb & Feild, 2010). It seems reasonable to suggest that the transition from ferns and gymnosperms characterized by low densities of veins and stomata to angiosperms with high densities of veins and stomata might be mediated by a decrease in genome size, a proposition supported by the fact that ferns and gymnosperms tend to have large genome sizes relative to angiosperms (Leitch et al., 2005). It remains unresolved, however, whether changing genome size is a driver or a product of macroevolutionary changes in vascular plants. In the context of this question, it is relevant to note that the evolution of high vein density in angiosperms actually occurred after the divergence of early angiosperm lineages, and that the ancestral state for angiosperm vein density is low, despite the fact that the early angiosperms are thought to have small genomes (Masterson, 1994) and therefore potentially small cells. A focus on the evolutionary trajectories of genome size and cell size at the base of the angiosperms will provide important insights into the drivers of this critical evolutionary event, and whether a linkage among genome size, cell size and water use is universal among vascular plant species.