Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus.

Cytoplasmic male sterility (CMS) has long been used to produce seedless fruits in perennial woody crops like citrus. A male-sterile somatic cybrid citrus (G1 + HBP) was generated by protoplast fusion between a CMS callus parent 'Guoqing No. 1' Satsuma mandarin (Citrus unshiu, G1) and a fertile mesophyll parent Hirado Buntan pummelo (Citrus grandis, HBP). To uncover the male-sterile mechanism of G1 + HBP, we compared the transcriptome profiles of stamen organ and cell types at five stages between G1 + HBP and HBP, including the initial stamen primordia, enlarged stamen primordia, pollen mother cells, tetrads, and microspores captured by laser microdissection. The stamen organ and cell types showed distinct gene expression profiles. A majority of genes involved in stamen development were differentially expressed, especially CgAP3.2, which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1 + HBP compared with HBP. Jasmonic acid- and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia, and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1 + HBP. In contrast, the content of auxin biosynthesis metabolites was lower in G1 + HBP. The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia, meiocytes, and microspores, indicating the dysfunction of mitochondria in stamen organ and cell types of G1 + HBP. Taken together, the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development, and thus lead to male sterility in the citrus cybrid.

miR171-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 regulate somatic embryogenesis in citrus.

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 2 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. Chromatin immunoprecipitation sequencing (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.

miR156 regulates somatic embryogenesis by modulating starch accumulation in citrus.

Somatic embryogenesis (SE) is a major regeneration approach for in vitro cultured tissues of plants, including citrus. However, SE capability is difficult to maintain, and recalcitrance to SE has become a major obstacle to plant biotechnology. We previously reported that miR156-SPL modules regulate SE in citrus callus. However, the downstream regulatory pathway of the miR156-SPL module in SE remains unclear. In this study, we found that transcription factors CsAGL15 and CsFUS3 bind to the CsMIR156A promoter and activate its expression. Suppression of csi-miR156a function leads to up-regulation of four target genes, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (CsSPL) genes, and reduction of SE efficiency. In the short tandem target mimic (STTM)-miR156a overexpression callus (MIM156), the number of amyloplasts and starch content were significantly reduced, and genes involved in starch synthesis and transport were down-regulated. csi-miR172d was down-regulated, whereas the target genes, CsTOE1.1 and CsTOE1.2, which inhibit the expression of starch biosynthesis genes, were up-regulated. In our working model, CsAGL15 and CsFUS3 activate csi-miR156a, which represses CsSPLs and further regulates csi-miR172d and CsTOEs, thus altering starch accumulation in callus cells and regulating SE in citrus. This study elucidates the pathway of miR156-SPLs and miR172-TOEs-mediated regulation of SE, and provides new insights into enhancing SE capability in citrus.

miR171 modulates induction of somatic embryogenesis in citrus callus.

KEY MESSAGE: Overexpression of miR171 restored SE competence in the recalcitrant citrus callus, and inhibition of miR171 function weakened SE competence in the strongly embryogenic citrus callus. Somatic embryogenesis (SE) is an important way of in vitro regeneration for plants. For perennial woody crops such as citrus, embryogenic callus is usually induced from unfertilized aborted ovules and widely used in biotechnology aided breeding. However, SE capacity always declines in callus during subculture, which makes regeneration difficult and hinders the application of biotechnology. We previously found that miR171 may be a regulator of SE in citrus, based on the abundant expression of csi-miR171c in the embryogenic callus and during SE of citrus. Here, we report that miR171 promotes SE and is required for SE in citrus. Overexpression of miR171 restored SE competence in the recalcitrant callus of 'Guoqing No.1' Satsuma mandarin (G1), whereas inhibition of miR171 function by Short Tandem Target Mimic (STTM) weakened SE competence in the strongly embryogenic callus of 'Valencia' sweet orange (V). The comparative transcriptomic analysis in miR171 overexpressed callus line (OE) and the wild type callus (WT) indicated that overexpression of miR171 decreased the expression level of its SCARECROW-LIKE (CsSCL) targets, and activated stress response related biological processes and metabolic processes that are required for cell differentiation. However, CsSCLs were up-regulated in the OE callus during SE induction process, which activated the cell division and developmental processes that are required for embryogenesis progress. Our results validate the function of miR171 in regulation of SE and reveal the biological responses provoked by miR171 in citrus that may promote SE.

miR156-SPL modules regulate induction of somatic embryogenesis in citrus callus.

miR156 is a highly conserved plant miRNA and has been extensively studied because of its versatile roles in plant development. Here, we report a novel role of miR156 in regulating somatic embryogenesis (SE) in citrus, one of the most widely cultivated fruit crops in the world. SE is an important means of in vitro regeneration, but over the course of long-term sub-culturing there is always a decline in the SE potential of the preserved citrus embryogenic callus, and this represents a key obstacle for citrus biotechnology. In this study, the SE competence of citrus callus of wild kumquat (Fortunella hindsii) was significantly enhanced by either overexpression of csi-miR156a or by individual knock-down of the two target genes, CsSPL3 and CsSPL14, indicating that the effect of miR156-SPL modules was established during the initial phases of SE induction. Biological processes that might promote SE in response to miR156 overexpression were explored using RNA-seq, and mainly included hormone signaling pathways, stress responses, DNA methylation, and the cell cycle. CsAKIN10 was identified as interacting protein of CsSPL14. Our results provide insights into the regulatory pathway through which miR156-SPL modules enhance the SE potential of citrus callus, and provide a theoretical basis for improvement of plant SE competence.