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Somatic embryogenesis (SE) is a key regeneration pathway in various biotechnology approaches to crop improvement, especially for economically important perennial woody crops like citrus. However, maintenance of SE capability has long been a challenge and becomes a bottleneck in biotechnology-facilitated plant improvement. In the embryogenic callus (EC) of citrus, we identified two csi-miR171c targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 (CsSCL2/3), which exert positive feedback regulation on csi-miR171c expression. Suppression of CsSCL2 expression by RNA interference (RNAi) enhanced SE in citrus callus. A thioredoxin superfamily protein CsClot was identified as an interactive protein of CsSCL2/3. Overexpression of CsClot disturbed reactive oxygen species (ROS) homeostasis in EC and enhanced SE. ChIP-Seq and RNA-Seq identified 660 genes directly suppressed by CsSCL2 that were enriched in biological processes including development related processes, auxin signaling pathway and cell wall organization. CsSCL2/3 bound to the promoters of regeneration-related genes, such as WUSCHEL-RELATED HOMEOBOX 2 (CsWOX2), CsWOX13 and Lateral Organ Boundaries Domain 40 (LBD40), and repressed their expression. Overall, CsSCL2/3 modulate ROS homeostasis through the interactive protein CsClot and directly suppress the expression of regeneration-related genes, thus regulating SE in citrus. We uncovered a regulatory pathway of miR171c-targeted CsSCL2/3 in SE, which shed light on the mechanism of SE and regeneration capability maintenance in citrus.

MicroRNA390 (miR390) is involved in plant growth and development by down-regulating the expression of the downstream genes trans-acting short interfering RNA3 (TAS3) and AUXIN RESPONSE FACTORs (ARFs). There is a scarcity of research on the involvement of the miR390-TAS3-ARFs pathway in the stem development of Populus. Here, differentially expressed miRNAs during poplar stem development were screened by small RNA sequencing analysis, and a novel function of miR390b in stem development was revealed. Overexpression of miR390b (OE-miR390b) resulted in a large increase in the number of xylem fiber cells and a slight decrease in the cell length at the longitudinal axis. Overall increases in stem elongation and plant height were observed in the OE-miR390b plants. According to transcriptome sequencing results and transient co-expression analysis, TAS3.1 and TAS3.2 were identified as the target genes of miR390 in poplar and were negatively regulated by miR390 in the apex. The transcription levels of ARF3.2 and ARF4 were significantly repressed in OE-miR390b plants and strongly negatively correlated with the number of xylem fiber cells along the longitudinal axis. These findings indicate that the conserved miR390-TAS3-ARFs pathway in poplar is involved in stem elongation and plant height growth.

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