Javier Fernando Palatnik

Summary: Plant organ size depends on the coordination of cell proliferation and expansion, a process regulated by numerous factors, including transcription factors and small RNAs. However, how different small‐RNA pathways are integrated with growth‐regulating transcriptional networks remains only partly understood. Here, we studied how the Arabidopsis TRANS‐ACTING siRNA3A ( TAS3A )‐derived trans ‐acting small RNA pathway controls organ size in Arabidopsis thaliana . We found that TAS3A overexpression increased leaf and floral organ size, whereas tas3a mutants showed reduced organ growth. We show that TAS3A acts by modulating AUXIN‐RESPONSE‐FACTOR2 (ARF2) activity and that plants expressing a trans ‐acting siRNA (tasiRNA)‐resistant ARF2 allele showed reduced organ growth. In turn, ARF2 repressed several GROWTH‐REGULATING FACTOR ( GRF ) genes, including GRF3 , which is a direct ARF2 target and is also controlled by microRNA miR396. Furthermore, uncoupling GRF activity from ARF2 repression was sufficient to increase floral organ size. Interestingly, whereas the TAS3A‐ARF2‐GRF module regulates leaf and petal growth predominantly through changes in cell proliferation, gynoecium growth involves a more complex balance between cell proliferation and elongation. Together, our results identify ARF2 as a central molecular node connecting tasiRNA‐ and miRNA‐dependent pathways and establish TAS3A as a key regulator of Arabidopsis aerial organ growth.

Plants display a remarkable regeneration capacity, which allows them to replace damaged or lost cells, tissues and organs, and thus recover from a broad spectrum of injuries^1,2. Even lost stem cells can be regenerated from non-stem cells after competence acquisition, highlighting the enormous plasticity of plant cells. However, the molecular mechanisms underlying this process are still poorly understood. In the root, the highly conserved microRNA miR396 and its targets, the GROWTH-REGULATING FACTORs (GRFs), control the transition from stem cells to proliferative cells. miR396 promotes stem cell activity by repressing and excluding the GRFs from the stem cell area. In turn, the GRFs promote cell division in the proliferation zone³. Here we show that the miR396–GRF regulatory module guides stem cell reconstitution after root tip excision, playing a dual role: while miR396 promotes competence, the GRFs control regeneration speed. Moreover, plants with ectopic miR396 expression have defined stem cell niches before the excision but do not reconstitute them afterwards, remaining in an open state despite continuing to grow. We propose that this phenomenon is caused by dispersed stem cell activity, which supports growth after root tip excision without reconstituting the organized and spatially restricted stem cell niche typical of Arabidopsis roots.

MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.

Plants rely on complex regulatory mechanisms to ensure proper growth and development. As plants are sessile organisms, these mechanisms must be flexible enough to adapt to changes in the environment. GROWTH-REGULATING FACTORS (GRFs) are plant-specific transcription factors that act as a central hub controlling plant growth and development, which offer promising biotechnological applications to enhance plant performance. Here, we analyze the complex molecular mechanisms that regulate GRFs activity, and how their natural and synthetic variants can impact on plant growth and development. We describe the biological roles of the GRFs and examine how they regulate gene expression and contribute to the control of organ growth and plant responses to a changing environment. This review focuses on the premise that unlocking the full biotechnological potential of GRFs requires a thorough understanding of the various regulatory layers governing GRF activity, the functional divergence among GRF family members, and the gene networks that they regulate.

The growth-regulating factor (GRF) family of transcriptional factors are involved in the control of leaf size and senescence, inflorescence and root growth, grain size, and plant regeneration. However, there is limited information about the genes regulated by these transcriptional factors, which are in turn responsible for their functions. Using a meta-analysis approach, we identified genes encoding Arabidopsis (Arabidopsis thaliana) zinc-finger homeodomain (ZF-HD) transcriptional factors, as potential targets of the GRFs. We further showed that GRF3 binds to the promoter of one of the members of the ZF-HD family, HOMEOBOX PROTEIN 33 (HB33), and activates its transcription. Increased levels of HB33 led to different modifications in leaf cell number and size that were dependent on its expression levels. Furthermore, we found that expression of HB33 for an extended period during leaf development increased leaf longevity. To cope with the functional redundancy among ZF-HD family members, we generated a dominant repressor version of HB33, HB33-SRDX. Expression of HB33-SRDX from HB33 regulatory regions was seedling-lethal, revealing the importance of the ZF-HD family in plant development. Misexpression of HB33-SRDX in early leaf development caused a reduction in both cell size and number. Interestingly, the loss-of-function of HB33 in lines carrying a GRF3 allele insensitive to miR396 reverted the delay in leaf senescence characteristic of these plants. Our results revealed functions for ZF-HDs in leaf development and linked them to the GRF pathway.

Key message Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. Abstract The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.

Gene expression is reconfigured rapidly during the cell cycle to execute the cellular functions specific to each phase. Studies conducted with synchronized plant cell suspension cultures have identified hundreds of genes with periodic expression patterns across the phases of the cell cycle, but these results may differ from expression occurring in the context of intact organs. Here, we describe the use of fluorescence-activated cell sorting to analyze the gene expression profile of G2/M cells in the growing root. To this end, we isolated cells expressing the early mitosis cell cycle marker CYCLINB1;1-GFP from Arabidopsis root tips. Transcriptome analysis of these cells allowed identification of hundreds of genes whose expression is reduced or enriched in G2/M cells, including many not previously reported from cell suspension cultures. From this dataset, we identified SCL28, a transcription factor belonging to the GRAS family, whose messenger RNA accumulates to the highest levels in G2/M and is regulated by MYB3R transcription factors. Functional analysis indicates that SCL28 promotes progression through G2/M and modulates the selection of cell division planes. Significance The mitotic cell cycle (MCC) sustains plant organ growth by producing new cells in the meristems. Multiple mechanisms of gene expression regulation determine the dynamics of gene expression across the successive phases of the cell cycle. At the transcriptional level, only E2F and MYB3R transcription factors (TFs) have been identified as controlling gene expression dynamics in the MCC. In this work, we analyze the transcriptome of cells in the G2/M phases of the cell cycle and identify SCL28, a previously uncharacterized GRAS TF, as a regulator of the MCC in plants.

Members of the GROWTH REGULATING FACTOR (GRF) family of transcription factors play key roles in the promotion of plant growth and development. Many GRFs are post-transcriptionally repressed by microRNA (miRNA) miR396, an evolutionarily conserved small RNA, which restricts their expression to proliferative tissue. We performed a comprehensive analysis of the GRF family in eudicot plants and found that in many species all the GRFs have a miR396-binding site. Yet, we also identified GRFs with mutations in the sequence recognized by miR396, suggesting a partial or complete release of their post-transcriptional repression. Interestingly, Brassicaceae species share a group of GRFs that lack miR396 regulation, including Arabidopsis GRF5 and GRF6. We show that instead of miR396-mediated post-transcriptional regulation, the spatiotemporal control of GRF5 is achieved through evolutionarily conserved promoter sequences, and that AUXIN RESPONSE FACTOR 2 (ARF2) binds to such conserved sequences to repress GRF5 expression. Furthermore, we demonstrate that the unchecked expression of GRF5 in arf2 mutants is responsible for the increased cell number of arf2 leaves. The results describe a switch in the repression mechanisms that control the expression of GRFs and mechanistically link the control of leaf growth by miR396, GRFs, and ARF2 transcription factors.

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4–GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4–GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4–GIF1 with CRISPR–Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF–GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.