Unified Changes in Cell Size Permit Coordinated Leaf Evolution
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Research Unified changes in cell size permit coordinated leaf evolution Tim J. Brodribb1, Greg J. Jordan1 and Raymond J. Carpenter2 1School of Plant Science, University of Tasmania, Hobart, Tasmania 7001, Australia; 2School of Earth and Environmental Sciences, University of Adelaide, Adelaide, SA 5005, Australia Summary Author for correspondence: The processes by which the functions of interdependent tissues are coordinated as lineages Tim Brodribb diversify are poorly understood. Tel: + 61 362261707 Here, we examine evolutionary coordination of vascular, epidermal and cortical leaf tissues Email: [email protected] in the anatomically, ecologically and morphologically diverse woody plant family Proteaceae. Received: 11 February 2013 We found that, across the phylogenetic range of Proteaceae, the sizes of guard, epidermal, Accepted: 27 March 2013 palisade and xylem cells were positively correlated with each other but negatively associated with vein and stomatal densities. The link between venation and stomata resulted in a highly New Phytologist (2013) efficient match between potential maximum water loss (determined by stomatal conduc- doi: 10.1111/nph.12300 tance) and the leaf vascular system’s capacity to replace that water. This important linkage is likely to be driven by stomatal size, because spatial limits in the packing of stomata onto the Key words: adaptation, cell size, genome leaf surface apparently constrain the maximum size and density of stomata. size, leaf thickness, stomatal density, We conclude that unified evolutionary changes in cell sizes of independent tissues, possibly stomatal size, vein density. mediated by changes in genome size, provide a means of substantially modifying leaf function while maintaining important functional links between leaf tissues. Our data also imply the presence of alternative evolutionary strategies involving cellular miniaturization during radiation into closed forest, and cell size increase in open habitats. Introduction during the uptake of CO2 for photosynthesis (Sack & Holbrook, 2006), plants with higher rates of photosynthesis per unit leaf Recent characterization of genes and core regulatory networks area lose more water (Cowan & Farquhar, 1977) and thus has revolutionised our understanding of how tissues develop. demand greater investment in leaf veins (McKown et al., 2010). However, the development of individual tissues is only one This investment comes largely as increased branching of leaf requirement for building complex organisms. Another, less minor veins, because a greater density of minor veins delivers understood process is how the development of spatially discrete water closer to sites of evaporation in the leaf (Brodribb et al., but functionally interdependent tissues is coordinated. One pos- 2007), leading to increased transport efficiency (Sack & Frole, sible mechanism for such coordination is colocation of primor- 2006). However, these veins are expensive to synthesize, and dial tissues. Thus, lymphatic and blood-carrying vessels of plants are likely to coordinate the production of photosynthetic mammals develop from a common embryonic vascular system, and water supply tissues to maximize returns on investments in and the xylem and phloem of plants derive from a shared the water transport system (Brodribb & Jordan, 2011). Further- cambium. However, complex organisms also depend on the more, while vein density determines water supply in the leaf, the coordinated development of many tissues with different origins density of stomata determines maximum rates of water loss and (Cavalier-Smith, 2005); for example, lung capacity, vascular vol- photosynthesis, and thus maintaining a balance between these ume and muscle mass are necessarily coordinated (Rubner, traits during adaptation to the environment should be of high 1883). Similarly, developmental coordination is essential for functional and adaptive importance. Such coordination has been plants because their primordial tissues have indeterminate demonstrated both within trees during plastic adaptation to light growth. Thus, plants can show great plasticity in response to the (Murphy et al., 2012) and between species (Edwards, 2006; environment, but this plasticity is only effective if the diverse Dunbar-Co et al., 2009; Zhang et al., 2012). tissues involved remain functionally coordinated. However, little is known about how this critically important One important example of coordination between discrete tis- link between vascular and stomatal tissues is maintained. A recent sues is found between the veins and stomata in the leaves of land study of a tree species showed that plasticity in epidermal cell size plants. Branching density in the leaf vein network determines changed vein and stomatal density in concert during light accli- water transport efficiency of the lamina (leaf hydraulic conduc- mation. Hence, larger epidermal cells in the shade result in larger tance), which is closely linked to maximum rates of photosynthe- leaves that have lower densities of veins and stomata than sun sis (Brodribb et al., 2005) and transpiration (Boyce et al., 2009). leaves (Murphy et al., 2012). This coordinating role of cell size Because leaf vascular networks replace water lost by evaporation during plastic adaptation of leaves to different evaporative and Ó 2013 The Authors New Phytologist (2013) 1 New Phytologist Ó 2013 New Phytologist Trust www.newphytologist.com New 2 Research Phytologist photosynthetic conditions of sun and shade raises the prospect and it is an ecologically important group in the southern hemi- that changing cell size could also be an important mechanism for sphere where species range from trees in tropical rainforest to evolutionary adaptation in plants. shrubs in the arid zone. We sampled cell size and densities from A correlation between cell volume and genome size has been 48 species and stomatal size from 417 species of Proteaceae from long recognized as a fundamental feature of eukaryotic organisms all major branches of the phylogeny. (Mirsky & Ris, 1951; Cavalier-Smith, 1985); however, the evolu- Species were categorized as being from open vegetation or tionary significance of variation in cell size, and associated closed forest according to descriptions from regional floras. genome size in plants and animals, has been hotly debated Closed canopies are typically > 70% canopy cover, which is gen- (Cavalier-Smith, 1978, 2005; Petrov, 2001; Hodgson et al., erally only achieved in rainforest communities. Proteaceae species 2010). In animals, transitions in cell and genome size are impli- are typically canopy species, so regardless of habitat type, all cated in several important evolutionary transitions (such as the leaves were collected in the field from sun-exposed branches. In evolution of birds from dinosaurs; Organ et al., 2007), but in most cases, leaves were sampled from three trees and immediately plants the adaptive significance of cell size variation remains fixed in FAA (50% ethanol, 5% (v/v) acetic acid and 3.7% (v/v) obscure. Attempts to account for the enormous range in genome formaldehyde). Leaves were returned to the laboratory where they and cell size in plants have recently focused on variation in sto- were soaked in water in preparation for anatomical sectioning. matal size as a potentially important functional consequence of The leaf area and mass of at least 10 leaves per species were variable cellular and nuclear volume (Beaulieu et al., 2008). The- measured to yield leaf mass per unit area (LMA). ory and observation suggest that large stomata are associated with low rates of gas exchange as a result of limits on the packing Stomata and vein density density of guard cells (if stomata become larger, then fewer can fit on the leaf surface), and diminishing benefits in terms of maxi- Paradermal sections of leaves were made using a handheld razor mum diffusive conductance of larger, deeper pores (Franks & blade to remove the adaxial epidermis and palisade, exposing the Beerling, 2009). Other potentially important tissues that share minor veins. Sections were then bleached in commercial house- À À size-constrained functional properties include leaf veins, which hold bleach (50 g l 1 sodium hypochlorite and 13 g l 1 sodium have analogous associations between the size of cells and the den- hydroxide) until clear. Bleach was removed by washing, and sec- sity (Field & Brodribb, 2013) and conductivity (Sack & Frole, tions stained in 1% toluidine blue for 30 s to colour the lignin- 2006; Brodribb et al., 2007) of the vascular system. Epidermal rich veins. Finally sections were mounted in phenol glycerine jelly cell size also appears to be a primary determinant of the final size and photographed with a Nikon Digital Sight DS-L1 camera of leaves, as well as influencing the thickness of the photosyn- (Melville, NY, USA) mounted on a Leica DM 1000 microscope thetic mesophyll (Perez-Perez et al., 2011). Here we examine (Nussloch, Germany) with a 910 objective. ImageJ (http:// how these interconnected systems in the leaf respond to family- rsbweb.nih.gov/ij/index.html) was used to measure the total wide variation in cell size. length of venation in five fields of view that were aligned midway Considering the diversity of influences that cell size has on leaf between the midrib and the margin. Wire frames of the veins physiology, we investigate how key functional attributes of leaves were drawn manually and their total length counted. remain coordinated if cell size changes. This is of particular