SummarySomatic embryogenesis (SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of miRNAs/siRNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing (HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel miRNAs, 130 miniature inverted-repeat transposable elements (MITEs) derived, 94 other and 235 phased small interfering RNAs (siRNAs), as well as 203 target genes. The majority of the abundantly expressed miRNAs/siRNAs exhibit lower expression levels in embryogenic callus (EC) or during SE process than in nonembryogenic callus (NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors (TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, miRNA-and siRNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved miRNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE.

Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1. Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation. Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP) with the aim to clarify their potential roles in anther development and male sterility. On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed differentially expressed profiles in one or more stages. Proteins up- or down-regulated in G1+HBP were mainly involved in carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase), nucleotide binding (RNA-binding proteins), protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits). Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.

HighlightIntegrative mRNA and small RNA analysis between poly- and monoembryonic ovules reveals involvement of an oxidative stress response and a novel miRNA-target regulatory module in initiation of citrus apomixis.

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BackgroundG1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from “Guoqing No.1” (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development.ResultsA total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5’ RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression.ConclusionOur study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2882-0) contains supplementary material, which is available to authorized users.