High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

Suong T T Nguyen,David W Mccurdy

doi:10.1186/s12870-015-0483-8

Abstract

BackgroundTransfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy.Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level.ResultsThe simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation.ConclusionsUse of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0483-8) contains supplementary material, which is available to authorized users.

Highlights

Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces
To develop a procedure for confocal imaging of wall ingrowths in phloem parenchyma (PP) TCs in Arabidopsis leaves we used the technique of Wuyts et al [17], modified by first peeling away the abaxial epidermis of rosette leaves immediately prior to fixation
The highly localized deposition of wall ingrowths in PP TCs is evidenced by their occurrence only along the wall shared with a cell of the Sieve element (SE)/Companion cell (CC) complex (Figure 1A,B)

Summary

Introduction

Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. The formation of nucellar projection and endosperm TCs in barley grains involves differential expression of at least 815 genes [7], while the development of epidermal TCs in Vicia faba cotyledons is predicted to involve up to 650 genes [8] These and other observations have led to the proposition that wall ingrowth deposition in TCs involves hierarchical regulation of cascades of gene expression, presumably controlled by key transcription factors [9], a model based on the genetic regulation of secondary wall deposition in xylem tissue [10,11]. The identification of such factors putatively regulating wall ingrowth deposition in TCs is best undertaken in a genetic model such as Arabidopsis thaliana (Arabidopsis)

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