Transcriptional Analysis Of Citrus Nucellar Embryo Initiation And Functional Characterization Of CsFUS3 Gene Preferentially Expressed During Somatic Embryogenesis

Citrus is one of the most widely grown and economically important fruit trees in the world. However, conventional breeding of citrus is hindered by some of its inherent characters like sterility of reproductive, long juvenility, nucellar polyembryony. Biotechnological approaches could be effective substitutes for citrus improvement and somatic embryogenesis(SE) is usually an inevitable step for successful plant regeneration. However, calluses of most genotypes tended to lose SE capability gradually as long term subculture. In addition, despite nucellar embryos(somatic embryos in vivo) enables to perpetuate agricultural traits of great value, little is known about its molecular mechanisms. Therefore, there is an urgent need to deeper understanding of the underlying molecular mechanisms of SE in citrus. Our project identified the reference genes that are most suitable for normalizing the gene expression data in citrus and revealed that CsFUS3, which was assigned to the B3 family, can enhance embryogenic competence of citrus callus. We have generated transcriptional profiles between two pairs of poly- and mono-embryonic cultivars using a combination of cellular observation and RNA-seq, which provides new insights into transcriptional regulation underlying nucellar embryo initiation. The results are as follows:1. Selection and validation of suitable reference genes for mRNA qRT-PCR analysis using somatic embryogenic cultures, floral and vegetative tissues in citrus. The expression stability of ten potential reference genes including CitACT7, CiteIF-1A, CiteIF4α, CitHistone H3, CitHistone H4, CitTUA3, CitTUB8, CitUBL5, CitUBQ1 and CitUBQ14 was assessed. Furthermore, this was validated by qRT-PCR in diverse sets of biological samples, including embryonic cultures at seven stages at three different temperatures(20°C, 25°C and 30°C), five distinct plant organs, four floral tissues and four stages of flower development. Three distinct statistical algorithms, geNorm, NormFinder and Bestkeeper, were used to evaluate the expression stability of the candidate reference genes. GeNorm was also used to determine both the optimal number and the best combination of reference genes for each experimental set. The expression of CitUBQ1 was the most stable one across the set of all samples, flower developmental stages and somatic embryogenesis process under two conditions i.e. different temperature treatment and normal temperature treatment. CitUBQ14 presented more stable expression in different plant organs and floral tissues. CitHistone H3 was the most unsuitable reference gene in many citrus sample sets. In addition, the relative gene expression profile of CitSERK1-like was conducted to confirm the validity of the reference genes in this study.2. Cloning and functional characterization of CsFUS3. The full-length sequence of CsFUS3 was obtained by RACE PCR. It was detected mainly in embryonic tissues and its abundance was consistent with SE capability of different citrus genotypes. However, the level of endogenous CsFUS3 protein was hard to detect. Overexpression of CsFUS3 in ‘Guoqing No.1’ Satsuma mandarin callus resulted in enhanced embryogenic competence. RNA-Seq transcriptome profiling revealed that a number of SE-promoting transcription factors were up-regulated in the overexpression(OE) line. In addition, the expression of pathway genes involved in biosynthesis, deactivation, and signaling of ABA and GA was altered. The pattern of ABA/GA ratio was similar to the expression pattern of CsFUS3 during SE and showed a positive correlation with embryogenic competence of various citrus varieties. The OE lines possessed higher ABA/GA ratio, and displayed stronger SE capability under ABA or GA inhibitor(paclobutrazol) treatments.3. Identification candidate transcripts related to nucellar embryo initiation. Histochemical observation showed that the time points for the appearance of nucellar embryo initial cells. RNA-seq was employed to compare transcriptomic profiling between two pairs of poly- and mono-embryonic cultivars ovules(Cocktail grapefruit/Huanong red pummelo; Ponkan ‘Huagan No.2’/Nour clementina). Using P-value≤0.05 and Abs [log(fold-change)] ≥1 as a selection threshold, a total of 71 differentially expressed transcripts were overlapped from pairwise comparisons. In ovules of the poly- or mono-embryonic cultivars, the over-represented transcripts were involved in cell wall modification and synthesis, hormone biosynthesis and response, signal transduction, transcriptional regulation and stress response. The expression of UGT1 that could activate callose synthesis process was up-regulated in the polyembryonic cultivars at NEI stage, suggesting that callose wall has specific function during nucellar embryo initiation.