Molecular Mechanisms of Plant Regeneration from Differentiated Cells: Approaches from Historical Tissue Culture Systems.

Plants can exert remarkable capacity for cell reprogramming even from differentiated cells. This ability allows plants to regenerate tissues/organs and even individuals in nature and in vitro. In recent decades, Arabidopsis research has uncovered molecular mechanisms of plant regeneration; however, our understanding of how plant cells retain both differentiated status and developmental plasticity is still obscure. In this review, we first provide a brief outlook of the representative modes of plant regeneration and key factors revealed by Arabidopsis research. We then re-examine historical tissue culture systems that enable us to investigate the molecular details of cell reprogramming in differentiated cells and discuss the different approaches, specifically highlighting our recent progress in shoot regeneration from the epidermal cell of Torenia fournieri.

The SUMO E3 Ligase SIZ1 Negatively Regulates Shoot Regeneration.

Plants form calluses and regenerate new organs when incubated on phytohormone-containing media. While accumulating evidence suggests that these regenerative processes are governed by transcriptional networks orchestrating wound response and developmental transitions, it remains unknown if posttranslational regulatory mechanisms are involved in this process. In this study, we demonstrate that SAP AND MIZ1 DOMAIN- CONTAINING LIGASE1 (SIZ1), an E3 ligase-catalyzing attachment of the SMALL UBIQUITIN-LIKE MODIFIER (SUMO) to proteins, regulates wound-induced signal transduction and organ regeneration in Arabidopsis (Arabidopsis thaliana). We show that loss-of-function mutants for SIZ1 exhibit overproduction of shoot meristems under in vitro tissue culture conditions, while this defect is rescued in a complementation line expressing pSIZ1::SIZ1 RNA sequencing analysis revealed that siz1-2 mutants exhibit enhanced transcriptional responses to wound stress, resulting in the hyper-induction of over 400 genes immediately after wounding. Among them, we show that elevated levels of WOUND INDUCED DEDIFFERENTIATION1 (WIND1) and WIND2 contribute to the enhanced shoot regeneration observed in siz1 mutants, as expression of the dominant-negative chimeric protein WIND1-SRDX (SUPERMAN repression domain) in siz1-3 mutants partly rescues this phenotype. Although compromised SIZ1 function does not modify the transcription of genes implicated in auxin-induced callus formation and/or pluripotency acquisition, it does lead to enhanced induction of cytokinin-induced shoot meristem regulators such as WUSCHEL, promoting the formation of WUSCHEL-expressing foci in explants. This study thus suggests that SIZ1 negatively regulates shoot regeneration in part by repressing wound-induced developmental reprogramming.

Molecular Mechanisms of Plant Regeneration.

Plants reprogram somatic cells following injury and regenerate new tissues and organs. Upon perception of inductive cues, somatic cells often dedifferentiate, proliferate, and acquire new fates to repair damaged tissues or develop new organs from wound sites. Wound stress activates transcriptional cascades to promote cell fate reprogramming and initiate new developmental programs. Wounding also modulates endogenous hormonal responses by triggering their biosynthesis and/or directional transport. Auxin and cytokinin play pivotal roles in determining cell fates in regenerating tissues and organs. Exogenous application of these plant hormones enhances regenerative responses in vitro by facilitating the activation of specific developmental programs. Many reprogramming regulators are epigenetically silenced during normal development but are activated by wound stress and/or hormonal cues.

A Gene Regulatory Network for Cellular Reprogramming in Plant Regeneration.

Wounding triggers organ regeneration in many plant species, and application of plant hormones, such as auxin and cytokinin, enhances their regenerative capacities in tissue culture. Recent studies have identified several key players mediating wound- and/or plant hormone-induced cellular reprogramming, but the global architecture of gene regulatory relationships underlying plant cellular reprogramming is still far from clear. In this study, we uncovered a gene regulatory network (GRN) associated with plant cellular reprogramming by using an enhanced yeast one-hybrid (eY1H) screen systematically to identify regulatory relationships between 252 transcription factors (TFs) and 48 promoters. Our network analyses suggest that wound- and/or hormone-invoked signals exhibit extensive cross-talk and regulate many common reprogramming-associated genes via multilayered regulatory cascades. Our data suggest that PLETHORA 3 (PLT3), ENHANCER OF SHOOT REGENERATION 1 (ESR1) and HEAT SHOCK FACTOR B 1 (HSFB1) act as critical nodes that have many overlapping targets and potentially connect upstream stimuli to downstream developmental decisions. Interestingly, a set of wound-inducible APETALA 2/ETHYLENE RESPONSE FACTORs (AP2/ERFs) appear to regulate these key genes, which, in turn, form feed-forward cascades that control downstream targets associated with callus formation and organ regeneration. In addition, we found another regulatory pathway, mediated by LATERAL ORGAN BOUNDARY/ASYMMETRIC LEAVES 2 (LOB/AS2) TFs, which probably plays a distinct but partially overlapping role alongside the AP2/ERFs in the putative gene regulatory cascades. Taken together, our findings provide the first global picture of the GRN governing plant cell reprogramming, which will serve as a valuable resource for future studies.