Xiao-Xia Ge

Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange

Somatic embryogenesis (SE) is a remarkable process of plant somatic cells developing into an embryo capable of forming a complete plant. MiRNAs play important roles in plant development by regulating expression of their target genes, but its function in SE has rarely been studied. Herein, ten conserved miRNAs with critical functions in plant development are detected by stem-loop qRT-PCR in the SE system of Valencia sweet orange. Sixteen unigenes from citrus are predicted to be targeted by six of the miRNAs. Cleavage sites on 15 of these target mRNAs are mapped by 5′RACE, of which ten are reported in this study. Transcript abundances of the 16 target unigenes are detected by qRT-PCR during SE process. Stage and tissue-specific expressions of miRNAs and their targets suggest their possible modulation on SE of citrus callus: miR156, 168 and 171 exert regulatory function during somatic embryo induction process; miR159, 164, 390 and 397 are related to globular-shaped embryo formation; miR166, 167 and 398 are required for cotyledon-shaped embryo morphogenesis; in addition, target genes of miR164, 166 and 397 are associated with SE disability of nonembryogenic callus. Exploration of miRNA-mediated modulation on SE is expected to provide new insights into plant cell totipotency, as well as how to maintain and enhance the embryogenic capacity of somatic cells.

Comparative Transcript Profiling of a Male Sterile Cybrid Pummelo and Its Fertile Type Revealed Altered Gene Expression Related to Flower Development

Male sterile and seedless characters are highly desired for citrus cultivar improvement. In our breeding program, a male sterile cybrid pummelo, which could be considered as a variant of male fertile pummelo, was produced by protoplast fusion. Herein, ecotopic stamen primordia initiation and development were detected in this male sterile cybrid pummelo. Histological studies revealed that the cybrid showed reduced petal development in size and width, and retarded stamen primordia development. Additionally, disorganized cell proliferation was also detected in stamen-like structures (fused to petals and/or carpel). To gain new insight into the underlying mechanism, we compared, by RNA-Seq analysis, the nuclear gene expression profiles of floral buds of the cybrid with that of fertile pummelo. Gene expression profiles which identified a large number of differentially expressed genes (DEGs) between the two lines were captured at both petal primordia and stamen primordia distinguishable stages. For example, nuclear genes involved in nucleic acid binding and response to hormone synthesis and metabolism, genes required for floral bud identification and expressed in particular floral whorls. Furthermore, in accordance with flower morphology of the cybrid, expression of PISTILLATA (PI) was reduced in stamen-like structures, even though it was restricted to correct floral whorls. Down-regulated expression of APETALA3 (AP3) coincided with that of PI. These finding indicated that, due to their whorl specific effects in flower development, citrus class-B MADS-box genes likely constituted ‘perfect targets’ for CMS retrograde signaling, and that dysfunctional mitochondria seemed to cause male sterile phenotype in the cybrid pummelo.

Transcriptional profiling of genes involved in embryogenic, non-embryogenic calluses and somatic embryogenesis of Valencia sweet orange by SSH-based microarray

Somatic embryogenesis (SE) is a most promising technology that is used for in vitro germplasm conservation and genetic improvement via biotechnological approaches in citrus. 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 in citrus. A total of 880 unisequences were identified by microarray screening based on these three SSH libraries. Gene ontology 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. Transcription factors might also play an important role. LEC1/B3 domain regulatory network genes (LEC1, L1L, FUS3, ABI3, and ABI5) were isolated in citrus SE. Some new transcription factors associated with citrus SE, like a B3 domain containing gene and HB4, were identified. To understand the influence of these isolated genes on SE competence, their expression profiles were compared among callus lines of seven citrus cultivars with different SE competence. The expression dynamics suggested that these genes could be necessary for the SE initiation and might play a role in embryogenic competence maintenance in different cultivars. On the basis of gene expression profiles, an overview of major physiological and biosynthesis processes at different developmental stages during citrus SE is presented. For the first time, these data provide a global resource for transcriptional events important for SE in citrus, and the specific genes offer new information for further investigation on citrus SE maintenance and development.

Selection and validation of suitable reference genes for mRNA qRT-PCR analysis using somatic embryogenic cultures, floral and vegetative tissues in citrus

Accuracy of the quantitative real-time reverse transcription-PCR (qRT-PCR) depends on the stability of the reference gene(s), i.e. housekeeping gene(s) used for data normalization. Recent studies have shown that the expression of common reference genes can vary considerably in certain experimental conditions. However, reference genes of qRT-PCR in fruit trees have not been well identified. In this study, stability of expression of ten potential reference genes in citrus, 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 callus at seven stages, embryos maintained at three different temperatures, 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. The three outputs were merged by means of a non-weighted unsupervised rank aggregation method. 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 citrus receptor-like kinase gene CitSERK1-like was conducted to confirm the validity of the reference genes in this study. Taken together, this study identified the reference genes that are most suitable for normalizing the gene expression data in citrus. These results provide guidelines for the selection of reference gene(s) under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of samples in citrus.

Self-sterility in the mutant ‘Zigui shatian’ pummelo (Citrus grandis Osbeck) is due to abnormal post-zygotic embryo development and not self-incompatibility

Abstract‘Shatian’ pummelo (Citrus grandis Osbeck), one of the main citrus cultivars in China, is self-incompatible, and its pollen tubes are believed to be arrested in style after self-pollination.We have characterized one ‘Shatian’ pummelo mutant, named ‘Zigui shatian’ pummelo. The mutant pummelo had identical DNA ploidy level, morphology (leaf shape, stoma size and density, pollen shape and size) and developmental progress of pistil and male organs to that of the common ‘Shatian’ pummelo. However, unlike the common ‘Shatian’ pummelo, ‘Zigui shatian’ is self-compatible since its pollen tubes can self-pollinate allowing for successful fertilization. Histological analyses of ‘Shatian’ pummelo further verified abnormal post-zygotic development which led to seed abortion. Simple sequence repeats (SSR) analysis revealed polymorphism in 1 of the 120 primers screened showing that ‘Zigui shatian’ and ‘Shatian’ pummelo are different at the DNA level. Taken together, these data suggested mutant ‘Zigui shatian’ pummelo might be derived from ‘Shatian’ pummelo with self-sterility by self-incongruity after self-fertilization.

Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray

BackgroundSeedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored.ResultsAn integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated.ConclusionOur research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.

Genome-wide identification, classification and analysis of HD-ZIP gene family in citrus, and its potential roles in somatic embryogenesis regulation.

The homeodomain-leucine zipper (HD-Zip) transcription factors, which belong to a class of Homeobox proteins, has been reported to be involved in different biological processes of plants, including growth and development, photomorphogenesis, flowering, fruit ripening and adaptation responses to environmental stresses. In this study, 27 HD-Zip genes (CsHBs) were identified in Citrus. Based on the phylogenetic analysis and characteristics of individual gene or protein, the HD-Zip gene family in Citrus can be classified into 4 subfamilies, i.e. HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV containing 16, 2, 4, and 5 members respectively. The digital expression patterns of 27 HD-Zip genes were analyzed in the callus, flower, leaf and fruit of Citrus sinensis. The qRT-PCR and RT-PCR analyses of six selected HD-Zip genes were performed in six citrus cultivars with different embryogenic competence and in the embryo induction stages, which revealed that these genes were differentially expressed and might be involved in citrus somatic embryogenesis (SE). The results exhibited that the expression of CsHB1 was up-regulated in somatic embryo induction process, and its expression was higher in citrus cultivars with high embryogenic capacity than in cultivars recalcitrant to form somatic embryos. Moreover, a microsatellite site of three nucleotide repeats was found in CsHB1 gene among eighteen citrus genotypes, indicating the possible association of CsHB1 gene to the capacity of callus induction.

Overexpression of the CsFUS3 gene encoding a B3 transcription factor promotes somatic embryogenesis in Citrus

In citrus, genetic improvement via biotechnology is challenging due to insufficient understanding of molecular barriers that prevent regeneration by somatic embryogenesis (SE). Our previous study indicated that LEC genes were involved in SE in citrus, but their regulatory roles remain to be elucidated. Here, we cloned one of the LEC genes, CsFUS3, and show that it is preferentially expressed during SE and in the embryogenic callus (EC) derived from citrus varieties with strong embryogenic competence. The overexpression of CsFUS3 in recalcitrant citrus callus restored embryogenic competence. Complementation of the loss-of-function Arabidopsis fus3 mutant with the CsFUS3 gene restored normal late embryogenesis, which is consistent with the CsFUS3 and AtFUS3 proteins contributing to the same regulatory network in Arabidopsis. Transcriptome profiling revealed that the expression of particular TFs that promote SE was up-regulated in the citrus overexpression (OE) line. The 104 differentially expressed genes associated with hormone biosynthesis, catabolism, and signaling are particularly noteworthy. The dynamic change in the ratio of ABA to GA during SE in wild-type callus mirrored the expression pattern of CsFUS3. In contrast, in the OE line, the ratio of ABA to GA was higher and the capacity for SE was greater when the OE line was separately treated with ABA and GA biosynthesis inhibitors. Taken together, our results demonstrate that the overexpression of CsFUS3 appears to establish a cellular environment favorable to SE, at least in part by promoting a high ABA to GA ratio and by regulating the expression of TFs that promote SE.