Abstract
BackgroundLeaf cellular architecture plays an important role in setting limits for carbon assimilation and, thus, photosynthetic performance. However, the low density, fine structure, and sensitivity to desiccation of plant tissue has presented challenges to its quantification. Classical methods of tissue fixation and embedding prior to 2D microscopy of sections is both laborious and susceptible to artefacts that can skew the values obtained. Here we report an image analysis pipeline that provides quantitative descriptors of plant leaf intercellular airspace using lab-based X-ray computed tomography (microCT). We demonstrate successful visualisation and quantification of differences in leaf intercellular airspace in 3D for a range of species (including both dicots and monocots) and provide a comparison with a standard 2D analysis of leaf sections.ResultsWe used the microCT image pipeline to obtain estimates of leaf porosity and mesophyll exposed surface area (Smes) for three dicot species (Arabidopsis, tomato and pea) and three monocot grasses (barley, oat and rice). The imaging pipeline consisted of (1) a masking operation to remove the background airspace surrounding the leaf, (2) segmentation by an automated threshold in ImageJ and then (3) quantification of the extracted pores using the ImageJ ‘Analyze Particles’ tool. Arabidopsis had the highest porosity and lowest Smes for the dicot species whereas barley had the highest porosity and the highest Smes for the grass species. Comparison of porosity and Smes estimates from 3D microCT analysis and 2D analysis of sections indicates that both methods provide a comparable estimate of porosity but the 2D method may underestimate Smes by almost 50%. A deeper study of porosity revealed similarities and differences in the asymmetric distribution of airspace between the species analysed.ConclusionsOur results demonstrate the utility of high resolution imaging of leaf intercellular airspace networks by lab-based microCT and provide quantitative data on descriptors of leaf cellular architecture. They indicate there is a range of porosity and Smes values in different species and that there is not a simple relationship between these parameters, suggesting the importance of cell size, shape and packing in the determination of cellular parameters proposed to influence leaf photosynthetic performance.
Highlights
Leaf cellular architecture plays an important role in setting limits for carbon assimilation and, photosynthetic performance
Significant advances have recently begin to be reported via engineering photosynthetic biochemistry [3,4,5], less progress has been made in the optimisation of internal leaf architecture which is thought to limit photosynthetic carbon assimilation [6]
The surface area of mesophyll cells exposed to intercellular airspaces (Smes) has been shown to be positively correlated with photosynthetic performance [7, 8] presumably by facilitating increased diffusional flux of C O2
Summary
Leaf cellular architecture plays an important role in setting limits for carbon assimilation and, photosynthetic performance. Measurements of lengths or areas from the cross-sectional images are transformed using correction factors to generate estimations of three-dimensional (3D) geometry [9,10,11]. These techniques are destructive, labour intensive, and in some cases the process of tissue preparation can alter the parameters being measured (e.g. poorly sectioned samples or sectioned at oblique angles), potentially leading to underestimation of values for Smes by as much as 30% [12]
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