A model worker: Multifaceted modulation of AUXIN RESPONSE FACTOR3 orchestrates plant reproductive phases.

The key phytohormone auxin is involved in practically every aspect of plant growth and development. Auxin regulates these processes by controlling gene expression through functionally distinct AUXIN RESPONSE FACTORs (ARFs). As a noncanonical ARF, ARF3/ETTIN (ETT) mediates auxin responses to orchestrate multiple developmental processes during the reproductive phase. The arf3 mutation has pleiotropic effects on reproductive development, causing abnormalities in meristem homeostasis, floral determinacy, phyllotaxy, floral organ patterning, gynoecium morphogenesis, ovule development, and self-incompatibility. The importance of ARF3 is also reflected in its precise regulation at the transcriptional, posttranscriptional, translational, and epigenetic levels. Recent studies have shown that ARF3 controls dynamic shoot apical meristem (SAM) maintenance in a non-cell autonomous manner. Here, we summarize the hierarchical regulatory mechanisms by which ARF3 is regulated and the diverse roles of ARF3 regulating developmental processes during the reproductive phase.

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis.

BACKGROUND: Cotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton. RESULTS: We established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 µg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein-protein interaction studies. CONCLUSIONS: The protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

Cell- and noncell-autonomous AUXIN RESPONSE FACTOR3 controls meristem proliferation and phyllotactic patterns.

In cell-cell communication, noncell-autonomous transcription factors play vital roles in controlling plant stem cell fate. We previously reported that AUXIN RESPONSE FACTOR3 (ARF3), a member of the ARF family with critical roles in floral meristem maintenance and determinacy, has a distinct accumulation pattern that differs from the expression domain of its encoding gene in the shoot apical meristem (SAM). However, the biological meaning of this difference is obscure. Here, we demonstrate that ARF3 expression in Arabidopsis (Arabidopsis thaliana) is mainly activated at the periphery of the SAM by auxin where ARF3 cell autonomously regulates the expression of meristem-organ boundary-specific genes, such as CUP-SHAPED COTYLEDON1-3 (CUC1-3), BLADE ON PETIOLE1-2 (BOP1-2), and TARGETS UNDER ETTIN CONTROL3 (TEC3) to regulate the arrangement of organs in regular pattern, a phenomenon referred to as phyllotaxis. We also show that ARF3 is translocated into the organizing center where it represses cytokinin activity and WUSCHEL expression to regulate meristem activity noncell-autonomously. Therefore, ARF3 acts as a molecular link that mediates the interaction of auxin and cytokinin signaling in the SAM while coordinating the balance between meristem maintenance and organogenesis. Our findings reveal an ARF3-mediated coordination mechanism through cell-cell communication in dynamic SAM maintenance.