Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells

Abstract

Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL. Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells.

Phosphosite charge rather than shootward localization determines OCTOPUS activity in root protophloem

“…These attempts failed, presumably because OPS is expressed at comparatively low levels in relatively few cells (15). However, a recently established transdifferentiation assay (29) opened up the possibility of performing proteomics analyses of the transgenic OPS-GFP fusion protein when expressed under control of the native OPS promoter. Briefly, in this vascular cell induction culture system using Arabidopsis leaves (VISUAL) assay, cotyledon or leaf mesophyll cells are reprogrammed by hormonal treatments to differentiate into an ∼50:50 mix of xylem vessels and sieve elements.…”

“…Briefly, in this vascular cell induction culture system using Arabidopsis leaves (VISUAL) assay, cotyledon or leaf mesophyll cells are reprogrammed by hormonal treatments to differentiate into an ∼50:50 mix of xylem vessels and sieve elements. OPS transcription is induced in the future sieve elements during the early stages of this process (29) (Fig. S3A) protein could be detected after immunoprecipitation from an extract of 300-400 cultured cotyledons (Fig.…”

“…So far the VISUAL assay was the only system in which we were able to isolate sufficient OPS protein for proteomic analyses, but even then abundance of individual OPS-derived peptides was low, precluding quantitative evaluation of a differential OPS phosphorylation state. In addition, the VISUAL transdifferentiation process, including OPS induction, is strictly dependent on the presence of bikinin (29,32), a GSK3/BIN2 inhibitor (33). Therefore, it is at present not possible to create differential BIN2 activity states in this system that would then allow quantitative assessment of OPS phosphorylation status.…”

Auxin Contributes to the Intraorgan Regulation of Gene Expression in Response to Shade

“…Phloem marker genes, such as SUC2, SEOR1 (AT3G01680), and APL (AT1G79430) (Kondo et al, 2016;Yamaguchi et al, 2017), were expressed at much higher levels in VS and almost not at all in MN ( Fig. 2; Supplemental Fig.…”

Thinking Outside the Plant: Exploring Phloem Development Using VISUAL

“…However, unlike xylem cells, which undergo marked changesduringdevelopment,invitro-produced phloem cells are difficult to distinguish from other cell types without the use of phloemspecific markers. Kondo et al (2016) developed a powerful system for analyzing phloem differentiation called VISUAL (Vascular Cell Induction Culture System Using Arabidopsis Leaves). In this system, mesophyll cells from Arabidopsis thaliana leaf discs or cotyledons are induced to form procambial cells, which then develop into xylem (Kondo et al, 2015) or phloem cells.…”