Gene Expression Programs during Shoot, Root, and Callus Development in Arabidopsis Tissue Culture

Abstract

Shoots can be regenerated from Arabidopsis (Arabidopsisthaliana) root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium. Regenerating tissues can be directed along different developmental pathways leading to the formation of shoots, new roots, or callus by transferring to the appropriate organ induction medium. Using gene-profiling methods, we identified groups of genes that serve as molecular signatures of the different developmental processes, i.e. genes that were specifically up- or down-regulated on one developmental pathway, but not on others. One transcription factor gene that was up-regulated during early shoot development was RAP2.6L (At5g13330), a member of the ERF (ethylene response factor) subfamily B-4 of the ERF/APETALA2 transcription factor gene family. RAP2.6L functions in shoot regeneration because T-DNA knockdown mutations in the gene reduced the efficiency of shoot formation in tissue culture, but not normal embryo or seedling development. RAP2.6L promoter:β-glucuronidase fusions demonstrated that the up-regulation of the gene during shoot regeneration was, at least in part, transcriptionally controlled. The promoter:β-glucuronidase fusions also demonstrated that RAP2.6L expression was localized to the shoot and emerging leaves, but expression declined in the leaf lamina as leaves expanded. T-DNA knockdown mutations in RAP2.6L reduced the expression of many genes that are normally up-regulated during shoot development including CUP-SHAPED COTYLEDON2 that is involved in shoot meristem specification. Thus, RAP2.6L appears to be part of a network involved in regulating the expression of many other genes in shoot regeneration.

BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways

“…Dissecting the phenomenon of in vitro regeneration at the moleculargenetic level has been difficult due to the length of the regeneration process and the many developmental events that take place as explants dedifferentiate and then redifferentiate new tissues and organs. Research in Arabidopsis has greatly facilitated our knowledge on multiple aspects of the regeneration process, including how competent cells are formed and induced to regenerate (Che et al 2006(Che et al , 2007. Mutant screens and functional studies have been particularly helpful in identifying proteins that control developmental pathways leading to both direct and indirect regeneration, either by promoting spontaneous (hormonefree) regeneration or by stimulating regeneration under sub-optimal conditions (Chuck et al 1996;Gallois et al 2002;Banno et al 2001;Zuo et al 2002;Marsch-Martinez et al 2006).…”

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

“…The esr1-1/drn-2 loss-offunction mutant, referred to as esr1-1 hereafter, however, does not display strong defects in shoot regeneration when cultured on CIM and SIM (Matsuo et al, 2011). By contrast, loss-of-function mutations in ESR2 and RAP2.6L cause clear defects in in vitro shoot regeneration (Matsuo et al, 2011;Che et al, 2006), suggesting that they play more profound roles.…”

“…Our RT-qPCR analysis confirmed these results and further showed that the late activation of ESR2 expression is dependent on ESR1 ( Figure 7B). Similarly, the expression of key shoot regulators, such as CUC1, WUS, STM, and RAP2.6L, is also increased after transfer to SIM (Gordon et al, 2007;Che et al, 2006;Chatfield et al, 2013), and their expression requires (A) Callus formation at wound sites of wild-type, Pro WIND1 :WIND1-SRDX (WIND1-SRDX), esr1-D, and WIND1-SRDX esr1-D leaf explants. Leaf explants were cultured on phytohormone-free MS medium, and callus phenotypes were scored at 8 d after wounding.…”

“…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. Previous studies have also identified several other regulators, such as ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN (ESR1/DRN), ESR2/DRN-LIKE (DRNL), and RAP2.6L, that contribute to shoot regeneration in vitro (Banno et al, 2001;Kirch et al, 2003;Che et al, 2006). An early study showed that overexpression of ESR1 promotes shoot regeneration without or at low doses of exogenous cytokinin (Banno et al, 2001).…”

“…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.…”