Phloem parenchyma transfer cells in Arabidopsis - an experimental system to identify transcriptional regulators of wall ingrowth formation

Abstract

In species performing apoplasmic loading, phloem cells adjacent to sieve elements often develop into transfer cells (TCs) with wall ingrowths. The highly invaginated wall ingrowths serve to amplify plasma membrane surface area to achieve increased rates of apoplasmic transport, and may also serve as physical barriers to deter pathogen invasion. Wall ingrowth formation in TCs therefore plays an important role in phloem biology, however, the transcriptional switches regulating the deposition of this unique example of highly localized wall building remain unknown. Phloem parenchyma (PP) TCs in Arabidopsis veins provide an experimental system to identify such switches. The extent of ingrowth deposition responds to various abiotic and applied stresses, enabling bioinformatics to identify candidate regulatory genes. Furthermore, simple fluorescence staining of PP TCs in leaves enables phenotypic analysis of relevant mutants. Combining these approaches resulted in the identification of GIGANTEA as a regulatory component in the pathway controlling wall ingrowth development in PP TCs. Further utilization of this approach has identified two NAC (NAM, ATAF1/2 and CUC2)-domain and two MYB-related genes as putative transcriptional switches regulating wall ingrowth deposition in these cells.

Keywords: Arabidopsis; phloem parenchyma; transcription factors; transfer cells; wall ingrowths.

FIGURE 1

Imaging of PPTCs in Arabidopsis veins using fluorescence staining and scanning electron microscopy. Calcofluor White staining of cleared leaf tissue (A–C) showing presence of PP TCs in a terminating minor vein (arrow in A) and as more continuous linear strands of staining running along major veins (arrows in B). Higher magnification reveals a central band of mottled fluorescence (arrows in C, asterisks mark cell edges) in a PP TC which corresponds to the deposition pattern of reticulate wall ingrowths seen by scanning electron microscopy in these cells (arrows in D). Staining of PP TCs by aniline blue (E, F) shows the same patterns of staining as revealed by Calcofluor White, albeit with superior signal-to-noise properties (see F). Punctate staining indicating the non-continuous development of PP cells into PP TCs along a given length of vein is particularly evident in E. The images in A–D are reproduced from Edwards et al. (2010) and E and F are unpublished data. Staining with aniline blue was performed identically to that of Calcofluor White, except that 0.01 (w/v) aniline blue in 70 mM phosphate buffer, pH 8.5, was used to replace 0.05% (w/v) Calcofluor White. Scale bars: A, B, E = 100 μm; F = 200 μm; C = 5 μm; D = 2 μm.