Wei Xin

In vitro regeneration of plants from tissues such as leaf or root explants typically starts with auxin-induced formation of a pluripotent cell mass termed callus. This cellular reprogramming is essential for the subsequent regeneration of shoots or roots. However, whether or how lipid metabolism is involved in callus formation and thus plant regeneration remains largely unclear. Here, we report that phospholipid remodeling is critical for auxin-induced callus formation during the regeneration of Arabidopsis thaliana. We show that phospholipid metabolism changes dynamically during auxin-induced pluripotent callus formation, and that interfering with the phospholipid metabolic pathway enhances or dampens callus formation induced by auxin. We further demonstrate that the phospholipid metabolite phosphatidic acid (PA) inhibits callus formation by binding to auxin signaling repressors of the INDOLE-3-ACETIC ACID INDUCIBLE (IAA) family and enhancing their stability, thereby decreasing auxin signaling and the callus-forming program. Our findings reveal the molecular interplay between phospholipid metabolism and auxin signaling during plant regeneration and identify developmental programs that may participate in this process.

Induction of the pluripotent cell mass termed callus from detached organs or tissues is an initial step in typical in vitro plant regeneration, during which auxin-induced ectopic activation of root stem cell factors are required for subsequent de novo shoot regeneration. While Arabidopsis (Arabidopsis thaliana) AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 and their downstream transcription factors LATERAL ORGAN BOUNDARIES DOMAIN (LBD) are known to play key roles in directing callus formation, the molecules responsible for activation of root stem cell factors and thus establishment of callus pluripotency are unclear. Here, we identified Arabidopsis WRKY23 and BASIC HELIX-LOOP-HELIX 041 (bHLH041) as a transcriptional activator and repressor, respectively, of root stem cell factors during establishment of auxin-induced callus pluripotency. We show that auxin-induced WRKY23 downstream of ARF7 and ARF19 directly activates the transcription of PLETHORA 3 (PLT3) and PLT7 and thus that of the downstream genes PLT1, PLT2 and WUSCHEL-RELATED HOMEOBOX 5 (WOX5), while LBD-induced removal of bHLH041 derepresses the transcription of PLT1, PLT2 and WOX5. We provide evidence that transcriptional activation by WRKY23 and loss of bHLH041-imposed repression act synergistically in conferring shoot-regenerating capability on callus cells. Our findings thus disclose a transcriptional mechanism underlying auxin-induced cellular reprogramming, which, together with previous studies, outlines the molecular framework of auxin-induced pluripotent callus formation for in vitro plant regeneration programs.

Epigenetic modifications within promoter sequences can act as regulators of gene expression. Shoot regeneration is influenced by both DNA methylation and histone methylation, but the mechanistic basis of this regulation is obscure. Here, we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli (RC) and non‐regenerable calli (NRC) in Arabidopsis thaliana. An analysis of the promoters of five of the differentially expressed genes (FWA, ACC1, TFL1, MAX3, and GRP3) pointed to an inverse relationship between cytosine methylation and transcription. The FWA promoter was demethylated and highly expressed in NRC, whereas it was methylated and expressed at low levels in RC. Explants of the hypomethylation mutants fwa‐1 and fwa‐2 showed strong levels of FWA expression and regenerated less readily than the wild type, suggesting that FWA inhibits direct in vitro shoot regeneration. WUSCHEL‐RELATED HOMEOBOX 9 (WOX9), which is required for shoot apical meristem formation, was directly repressed by FWA. Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa‐2 plants. These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration.