Transcriptional Profiling Of Genes Involved In Embrogenic Calluses And Somatic Embryogenesis Of Valencia Sweet Orange By SSH Based Microarray

For citrus, callus is the most important explant for in vitro germplasm conservation and genetic improvement via biotechnological approaches such as transformation, protoplast fusion and somatic mutation breeding, and the somatic embryogenesis (SE) process is critical for plant regeneration from callus. Therefore, the maintainence of SE capacity becomes the key factor leading to successful germplasm conservation and improvement. However, varied citrus cultivars have different embryogenesis capacity, and long term subculture is generally accompanied by gradual loss of embryogenesis capacity. In an attempt to understand the mechanism of SE competence maintenance and embryo development in citrus, we used the embryogenic callus line of Citrus sinensis cv. ’Valencia’ sweet orange as experimental material, which still maintains strong SE capacity after preservation in vitro for over 26 years. A transcriptome study was carried out among non-embryogenic calluses, embryogenic calluses and somatic-embryo-induced calluses by applying suppression subtractive hybridization (SSH) followed by cDNA microarray analysis. In addition, genes specifically expressed in the early SE process were isolated and analyzed to understand the inherent mechanism of SE in citrus. The main results are as follow:1. The characteristic changes of EC, NEC and SE process were observed by cellular observation. We found that EC was unlike NEC not only in the SE capacity, but also in cell features, such as cell shape and size, callus colour and texture. Under the ultrastructural observation, the EC showed that small cells containing bigger nucleus with obvious nucleolus and amyloplasts. During IEC2 stage, cells of EC contained bigger nucleolus and rich nucleoplasm with high electron density. At IEC4 stage, cells of EC became mature with a big vacuole in the center of cell. Moreover, more protein bodies were found in EC than in NEC or IEC2, IEC4. The accumulation of protein body in EC and the dynamic changes of amyloplasts during SE, make it possible for the cells to access embryogenic differentiation to convert to the development of embryos and plant; the plasmodesma built the physiological organic connections between embryogenic cells facilitated the interchange of materials. Meanwhile, physiological isolation is the precondition of embryogenic capability.2. Herein, three suppression subtractive hybridization (SSH) libraries were constructed using calluses of Citrus sinensis cv.’Valencia’ to explore the molecular mechanisms that underlie the SE phenomenon in citrus. A total of 988 unisequences were identified by microarray screening based on our three libraries. Functional analysis of the differentially expressed genes indicated that nucleolus associated regulation and biogenesis processes, hormone signal transduction, and stress factors might be involved in SE process, and the transcription factors play important roles. LEC1/B3 domain regulatory network genes (LEC1, LIL, FUS3, ABI3, and ABI5) were first isolated in citrus SE, meanwhile some new transcription factors associated with citrus SE, e.g. a B3 domain containing gene, HB4, basic leucine zipper transcription factor、MYB protein, IAA9 and tubby like protein were identified.To understand the influence of these isolated genes(LEC1, FUS3, LIL, ABI3, ABI5, B3 domain containing gene, HB4 and TLP) on the somatic embryogenic capacity, their expression profiles were compared among callus lines of seven citrus cultivars with different responses to embryogenesis. It showed that the expression of most genes in cultivars with high embryogenic capacity was higher than that in cultivars recalcitrant to form somatic embryos (significantly low in C. unshui cv. Guoqing No.1) except that high expression of LIL gene was also detected in calluses of BT and O which maintain low SE capacity. All these results suggested that LEC1, FUS3, B3 domain containing gene, HB4 and TLP could be a central requisite for the SE initiation and might play a role in determining the embryogenic capacity in different cultivars. These data will offer new molecular information for citrus cultivars with different embryogenesis capacity.3. Full-length cDNA sequences of CsLECl and CsHB4 were cloned by RACE method, the full-length cDNA of CsLECl was 976 bp encoding 251 amino acid; while the full-length cDNA of CsHB4 was 1291 bp encoding 287 amino acid. By qRT-PCR, the up-regulated expression of CsLECl was detected in EC and IEC2, then the expression of CsLECl was down-regulated, but rare in NEC and other tissues, therefore we inferred that CsLEC1 gene play an important role in SE initiation stage, other than the rest of plant growth and development process. The highest expression of CsHB4 was detected in EC, then its transcription level decreased during IEC2 to IEC4 stages, and up-regulated in globular and cotyledon embryos at subsequent embryo-transition phases, while low expression level was detected in ovary, leaf, stem, style, young flowers. So it was suggested that CsHB4 played an essential roles in SE, also it would take part in the other plant development processes.4. A somatic embryogenesis receptor-like kinase gene was isolated, named CitSERKl-like from ’Valencia’. The predicted extracellular domain of CitSERK1-like protein shared all characteristic features of plant SERK proteins, including a putative leucine zipper (ZIP), a proline-rich region (SPP) and a C-terminal extension (C), there was fifteen different amino acids from CitSERK1 (C. unshui) in the leucine zipper domain. Our study in citrus revealed that the expression of CitSERK1-like was both present in EC and in NEC with high cell proliferation, these results were further confirmed by in situ hybridization analysis. However the spatial expression of CitSERK1-like in EC was uniform, rather than asymmetrically located in NEC cells. Consequently, the expression of CitSERK1-like in citrus might not be considered as the marker of SE competent cells. However, the higher expression in embryogenic callus and up-regulated CitSERK1-like expression during the induction stage might be related to the embryogenesis competence of callus. The expression of CitSERK1-like was detected in both the SE process and plant tissues. Its broader expression in various tissues indicated that the role of this gene was not restricted to the process of SE. It might have a broader role in plant development. Our study onunderstanding the precise expression of the SERK gene in citrus provided novel evidences on its roles in somatic embryogenesis and plant development.