CsAHL25 regulates citrus somatic embryogenesis by affecting the expression of CsHB1 and LEC1/B3 genes

Abstract: 【Objective】Somatic embryogenesis (SE) is widely used in the conservation and utilization of plant germplasm resources. However, there is significant variation in the somatic embryogenesis (SE) capacity of calls derived from different citrus varieties. Furthermore, their SE capacity gradually diminishes during culture, posing a significant hindrance to the conservation and utilization of citrus germplasm resources. CsHB1, an HD-ZIP II gene associated with enhancing SE, was isolated from a citrus variety exhibiting robust SE capabilities. In this study, we harnessed the promoter of CsHB1 (pCsHB1) to search for upstream transcription factors to provide reliable candidate genes for the study on plant somatic embryogenesis.【Methods】To identify the upstream transcription factors of CsHB1, we cloned pCsHB1 (-1018 to -558 bp) into pAbAi and utilized a yeast one-hybrid (Y1H) assay to obtain the candidate transcription factor CsAHL25 from a yeast library. Using SMART, candidate genes were analyzed for domains and named based on annotations in the Citrus Pangenome Breeding Database. The expression pattern of this gene was measured by qRT-PCR in various somatic embryo developmental stages of citrus, aiming to deduce the function of CsAHL25. The gene was cloned and inserted into pRI121, trans-ferred into GV3101 and Marker mixed annotated Nicotiana benthamiana. After 2 d, the localization in the cells was observed using the laser scanning confocal microscopy. CsAHL25 was cloned and inserted into pGADT7 and transfected into Y1HGold with pCsHB1-AbAi for the Y1H assay. A Y1H assay was performed to determine whether the two were complementary or not based on the growth of the yeast cells in the screening medium. The gene was cloned and inserted into the overexpression vector pCMBAI1300-35S, and pCsHB1 (-2377-0 bp) was cloned and inserted into pGreenII 0800-LUC. The two vectors were then separately transferred into GV3101 and mixed to transiently transform N. benthamiana, with empty vector used as a control. After 2 d, the fluorescence of LUC was observed using an in vivo Plant Fluorescent Imaging System, and the LUC/REN ratio was calculated. This was followed by a comparison with the control to determine the role of this gene in the downstream gene regulation of pCsHB1. To explore the function of this gene, we transiently expressed the gene in the callus of Citrus sinensis‘Next’, and qRT- PCR was used to detect the expression of somatic embryogenesis- related genes.【Results】A candidate transcription factor, named CsAHL25, which is involved in the regulation of CsHB1 expression, was identified from the results of Y1H screening. Sequence analysis revealed that CsAHL25 possesses a typeⅠ AT-HOOK domain and a type A PPC domain and belongs to the AHL15- 29 subfamily of AT-HOOK. Subcellular localization analysis demonstrated that, similar to other AHL transcription factors, CsAHL25 is a nucleus-localized transcription factor. CsAHL25 exhibited high expression levels at 60 d and 120 d of somatic cell embryo induction. The expression pattern of CsAHL25 suggested that this gene may play a role in SE. The Y1H results showed that yeast cells containing CsAHL25 and pCsHB1 were able to grow well in SD-Leu/ABA200, indicating that CsAHL25 was bound to pCsHB1. The results of plant fluorescent imaging indicated that 1300+pCsHB1-LUC exhibited higher LUC values than CsAHL25-1300+pCsHB1-LUC. The LUC/REN results were consistent with the Plant Fluorescent Imaging outcomes, with the strongest LUC- related activity observed in 1300 + pCsHB1-LUC. These results showed that CsAHL25 was bound to the integral pCsHB1 and repressed its transcription. To further investigate the function of CsAHL25 during SE, we performed transient transfection of CsAHL25 in the callus of C. sinensis‘Anliucheng’, and then analyzed gene expression by using qRT-PCR. The results showed that the expression of CsHB1 was significantly downregulated, while the LEC1/B3 genes promoting somatic embryogenesis, such as CsLEC1, CsL1L, CsFUS3 and CsABI3, were significantly upregulated.【Conclusion】The results of this study indicated that CsAHL25 was an upstream transcription factor of CsHB1, which can inhibit CsHB1 expression, and transient expression of CsAHL25 can cause upregulation of the expression of LEC1/B3 genes. Based on the expression pattern of CsAHL25, we studied the functions of HD-ZIP, LEC1/B3 and AHL25 in relation to citrus somatic embryogenesis. Finally, we hypothesized that CsAHL25 regulated citrus somatic embryogenesis.