Incipient stem cell niche conversion in tissue culture: using a systems approach to probe early events in WUSCHEL‐dependent conversion of lateral root primordia into shoot meristems

Abstract

SUMMARYAdventitious shoot organogenesis contributes to the fitness of diverse plant species, and control of this process is a vital step in plant transformation and in vitro propagation. New shoot meristems (SMs) can be induced by the conversion of lateral root primorida/meristems (LRP/LRMs) or callus expressing markers for this identity. To study this important and fascinating process we developed a high-throughput methodology for the synchronous initiation of LRP by auxin, and subsequent cytokinin-induced conversion of these LRP to SMs. Cytokinin treatment induces the expression of the shoot meristematic gene WUSCHEL (WUS) in converting LRP (cLRP) within 24-30 h, and WUS is required for LRP ? SM conversion. Subsequently, a transcriptional reporter for CLAVATA3 (CLV3) appeared 32-48 h after transfer to cytokinin, marking presumptive shoot stem cells at the apex of cLRP. Thus the spatial expression of these two components (WUS and CLV3) of a regulatory network maintaining SM stem cells already resembles that seen in a vegetative shoot apical meristem (SAM), suggesting the very rapid initiation and establishment of the new SMs. Our high-throughput methodology enabled us to successfully apply a systems approach to the study of plant regeneration. Herein we characterize transcriptional reporter expression and global gene expression changes during LRP ? SM conversion, elaborate the role of WUS and WUS-responsive genes in the conversion process, identify and test putative functional targets, perform a comparative analysis of domainspecific expression in cLRP and SM tissue, and develop a bioinformatic tool for examining gene expression in diverse regeneration systems.

PLETHORA Genes Control Regeneration by a Two-Step Mechanism

“…Taken together, our data demonstrate that PLT3, PLT5 and PLT7 genes are necessary for de novo shoot regeneration, but not for callus formation. The regeneration phenotypes of plt mutants remained invariant in different culture conditions reported in the literature [3,4,19] (Fig. 2 E, S2 G).…”

“…As reported earlier de novo shoots were formed from wild-type callus after the ectopic overexpression of estradiolinducible WUS (pG10-90::WUS:3AT) or ESR2 (pG10-90::ESR2:3AT) on hormone-free medium supplemented with β-estradiol (Fig. 4 M-O) [19,23]. On the contrary, there was no sign of direct or callus mediated shoot regeneration in plt3; plt5-2; plt7 following overexpression of WUS or ESR2 (Fig.…”

“…S5). WUS has been shown to be required for the conversion of LRP into shoots [19]. Though the mutant LRP expresses WUS upon exposure to high cytokinin, it was unable to produce shoots.…”

A transcriptional view on somatic embryogenesis

“…Overexpression of WUS in arabidopsis is sufficient to induce organogenesis and SE in the shoot and root tip (Chatfield et al., 2013; Gallois, Nora, Mizukami, & Sablowski, 2004). WUS was also identified as PLANT GROWTH ACTIVATOR 6 ( PGA6 ) in an activation tagging screen for genes that induce somatic embryo formation from root callus (Zuo, Niu, Frugis, & Chua, 2002).…”

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis

“…By contrast, shoot regeneration on SIM ought to be more complex as it requires the conversion of root meristem fate into shoot meristem fate. What is central for the shoot meristem initiation is the activation of the key shoot stem cell regulator WUSCHEL (WUS) by cytokinin, which facilitates the partitioning of CIMinduced callus into WUS-expressing domains and CUC2-expressing domains (Che et al, 2006;Gordon et al, 2007;Chatfield et al, 2013). A cluster of CUC2-expressing cells continues to proliferate to form promeristems, in which polarized expression of the auxin transporter PIN-FORMED1 and another meristem regulator SHOOT MERISTEMLESS (STM) further organizes the formation of functional shoot meristem.…”