Grafting, cutting, and pruning are important horticultural techniques widely used in the establishment of clonal forestry. After the application of these techniques, some properties of the plants change, however, the underlying molecular mechanisms are still unclear. In our previous study, 27 age-related transcripts were found to be expressed differentially between the juvenile vegetative (1- and 2-year-old) and adult reproductive (25- and 50-year-old) phases of Larix kaempferi. Here, we re-analyzed the 27 age-related transcripts, cloned their full-length cDNA sequences, and measured their responses to grafting, cutting, and pruning. After sequence analysis and cloning, 20 transcription factors were obtained and annotated, most of which were associated with reproductive development, and six (LaAGL2-1, LaAGL2-2, LaAGL2-3, LaSOC1-1, LaAGL11, and LaAP2-2) showed regular expression patterns with L. kaempferi aging. Based on the expression patterns of these transcription factors in L. kaempferi trees subjected to grafting, cutting, and pruning, we concluded that (1) cutting and pruning rejuvenate the plants and change their expression, and the effects of cutting on gene expression are detectable within 14 years, although the cutting seedlings are still maturing during these years; (2) within three months after grafting, the rootstock is more sensitive to grafting than the scion and readily becomes mature with the effect of the scion, while the scion is not readily rejuvenated by the effect of the rootstock; and (3) LaAGL2-2 and LaAGL2-3 are more sensitive to grafting, while LaAP2-2 is impervious to it. These findings not only provide potential molecular markers to assess the state of plants but also aid in studies of the molecular mechanisms of rejuvenation.

In animals and plants, organ shape is primarily determined during primordium development by carefully coordinated growth and cell division^1–3. Rare examples of post-primordial change in morphology (reshaping) exist that offer tractable systems for the study of mechanisms required for organ shape determination and diversification. One such example is morphogenesis in Capsella fruits whose heart-shaped appearance emerges by reshaping of the ovate spheroid gynoecium upon fertilization⁴. Here we use whole-organ live-cell imaging and single-cell RNA sequencing (scRNA-seq) analysis to show that Capsella fruit shape determination is based on dynamic changes in cell growth and cell division coupled with local maintenance of meristematic identity. At the molecular level, we reveal an auxin-induced mechanism that is required for morphological alteration and ultimately determined by a single cis-regulatory element. This element resides in the promoter of the Capsella rubella SHOOTMERISTEMLESS ⁵ (Cr STM) gene. The CrSTM meristem identity factor positively regulates its own expression through binding to this element, thereby providing a feed-forward loop at the position and time of protrusion emergence to form the heart. Independent evolution of the STM-binding element in STM promoters across Brassicaceae species correlates with those undergoing a gynoecium-to-fruit shape change. Accordingly, genetic and phenotypic studies show that the STM-binding element is required to facilitate the shape transition and suggest a conserved molecular mechanism for organ morphogenesis.

Key message: Expression analysis of Larix kaempferi mature miR171s and their primary transcripts and target gene LaSCL6 during somatic embryogenesis revealed the transcriptional and post-transcriptional regulation of the miR171-LaSCL6 module. Somatic embryogenesis provides a useful experimental system for studying the regulatory mechanisms of plant development. The level and activity of microRNA171 (miR171) fluctuate during somatic embryogenesis in Larix kaempferi, but the underlying mechanisms are still unclear. Here, in L. kaempferi we identified five members of the miR171 family, which cleave LaSCL6 mRNA at different sites. In addition, we improved the method of measuring miRNA activity in a more direct way. Furthermore, we measured the expression patterns of mature miR171s and their primary transcripts during somatic embryogenesis in L. kaempferi and found that their patterns differed, indicating that the transcription of MIR171 genes and the subsequent cleavage of their intermediate products are regulated. Taken together, our findings not only offer a means to study the regulation of miRNA activity, but also provide further insight into the regulation of L. kaempferi somatic embryogenesis by miR171-LaSCL6.

Fruits are a key feature defining angiosperms, yet how local growth is coordinated during development to generate diverse fruits remains unclear. Here, we demonstrate that the Capsella rubella C2H2-zinc finger transcription factor JAGGED (CrJAG) controls fruit shape determination by promoting both cell division and anisotropic growth. At the molecular level, CrJAG physically interacts with members of the Capsella rubella MULTICOPY SUPPRESSOR OF IRA1 (CrMSI) histone chaperone family, increasing the chromatin accessibility and thereby sustaining the expression of genes involved in fruit morphogenesis. The resulting closed chromatin state in Crjag fruits is characterized by a reduction of the active histone marker (H3K18ac) and an increase of the repressive marker (H3K27me3). Further expression and pharmacological treatment analyses indicate that the developmental defects in Crjag fruits are largely attributable to down-regulation of the key cell-cycle regulator CrAUR2. Collectively, our findings therefore suggest that fine-tuning the cell cycle via epigenetic modification represents an additional, essential layer of regulation critical to organ development and diversification.

Fruits that protect the seeds and aid their dispersal are a key feature distinguishing angiosperms from other land plants, and fruit shape varies widely between species. However, it remains largely unknown how local growth behaviors are coordinated during development to generate fruits with characteristic sizes and shapes. Members of the Capsella genus in the mustard family produce unique fruits with an evolutionarily derived heart shape, making it an exceptional system suited for addressing this question. In this study, we demonstrate that the Capsella rubella C2H2-zinc finger transcription factor JAGGED (CrJAG) controls fruit shape. Whole-mount live imaging analysis indicates that CrJAG regulates valve development by promoting both cell division and anisotropic growth. Ectopic expansion of the valve epidermal cells in Crjag mutants is accompanied by mis-patterned endoreduplication events. CrJAG physically interacts with the histone chaperones Capsella rubella MULTICOPY SUPPRESSOR OF IRA1 members, increasing the chromatin accessibility and thereby sustaining the expression of genes involved in fruit morphogenesis. The resulting closed chromatin state in Crjag fruits is characterized by a reduction of the active histone marker (H3K18ac) and an increase of the repressive marker (H3K27me3). Expression analyses and pharmacological treatments indicate that the developmental defects in Crjag fruits are largely attributable to down-regulation of the key cell-cycle regulator CrAUR2 . Collectively, our findings suggest that fine-tuning the cell cycle via epigenetic modification represents an additional, essential layer of regulation critical to organ development.