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

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.

Heteroblastic Development of Transfer Cells Is Controlled by the microRNA miR156/SPL Module

“…We recently reported semiquantitative assessment of wall ingrowth deposition in PP TCs of Arabidopsis veins by confocal imaging of leaves stained by a modified pseudo-Schiff propidium iodide staining technique (Nguyen and McCurdy, 2015). This article described four classes of PP TC development depending on abundance of wall ingrowth deposition, ranging from Class I with no wall ingrowths to Class IV with abundant deposition (Nguyen and McCurdy, 2015).…”

“…This article described four classes of PP TC development depending on abundance of wall ingrowth deposition, ranging from Class I with no wall ingrowths to Class IV with abundant deposition (Nguyen and McCurdy, 2015). By assigning an arbitrary value of 0, 2, 4, and 6 for Classes I, II, III, and IV, respectively, a semiquantitative assessment of wall ingrowth deposition in PP TCs across single leaves of developing and developed shoots could be generated (Nguyen and McCurdy, 2015).…”

“…Haritatos et al, 2000;Amiard et al, 2007;Maeda et al, 2006Maeda et al, , 2008. Recently, we used a modified pseudo-Schiff-propidium iodide (mPS-PI) procedure for staining cell walls (Truernit et al, 2008;Wuyts et al, 2010) to image wall ingrowth deposition in PP TCs by confocal microscopy (Nguyen and McCurdy, 2015). This procedure enables more extensive and semiquantitative assessment of PP TC development at the whole leaf level and across rosette development compared to using TEM, and was used to assess, among other criteria, the extent of wall ingrowth deposition in response to defoliation (Nguyen and McCurdy, 2015).…”

“…Recently, we used a modified pseudo-Schiff-propidium iodide (mPS-PI) procedure for staining cell walls (Truernit et al, 2008;Wuyts et al, 2010) to image wall ingrowth deposition in PP TCs by confocal microscopy (Nguyen and McCurdy, 2015). This procedure enables more extensive and semiquantitative assessment of PP TC development at the whole leaf level and across rosette development compared to using TEM, and was used to assess, among other criteria, the extent of wall ingrowth deposition in response to defoliation (Nguyen and McCurdy, 2015). In surveying PP TCs across individual leaf and rosette development, we identified that wall ingrowth deposition was highly abundant in early emerged organs such as cotyledons (Nguyen and McCurdy, 2015) and juvenile leaves, and was dramatically less so in later-emerged adult leaves.…”

“…This procedure enables more extensive and semiquantitative assessment of PP TC development at the whole leaf level and across rosette development compared to using TEM, and was used to assess, among other criteria, the extent of wall ingrowth deposition in response to defoliation (Nguyen and McCurdy, 2015). In surveying PP TCs across individual leaf and rosette development, we identified that wall ingrowth deposition was highly abundant in early emerged organs such as cotyledons (Nguyen and McCurdy, 2015) and juvenile leaves, and was dramatically less so in later-emerged adult leaves. These observations led us to speculate that PP TC development, as assessed by levels of wall ingrowth deposition, is specific to leaf identity.…”