Chongyi Xu

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.

Induction of a pluripotent cell mass, called callus, from detached organs is an initial step in in vitro plant regeneration, during which phytohormone auxin-induced ectopic activation of a root developmental program has been shown to be required for subsequent de novo regeneration of shoots and roots. However, whether other signals are involved in governing callus formation, and thus plant regeneration capability, remains largely unclear. Here, we report that the Arabidopsis calcium (Ca ²⁺ ) signaling module CALMODULIN IQ-MOTIF CONTAINING PROTEIN (CaM–IQM) interacts with auxin signaling to regulate callus and lateral root formation. We show that disruption of IQMs or CaMs retards auxin-induced callus and lateral root formation by dampening auxin responsiveness, and that CaM–IQM complexes physically interact with the auxin signaling repressors INDOLE-3-ACETIC ACID INDUCIBLE (IAA) proteins in a Ca ²⁺ -dependent manner. We further provide evidence that the physical interaction of CaM6 with IAA19 destabilizes the repressive interaction of IAA19 with AUXIN RESPONSE FACTOR 7 (ARF7), and thus regulates auxin-induced callus formation. These findings not only define a critical role of CaM–IQM-mediated Ca ²⁺ signaling in callus and lateral root formation, but also provide insight into the interplay of Ca ²⁺ signaling and auxin actions during plant regeneration and development.