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Background: G1 + 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. Results: A 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. Conclusion: Our 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. [ABSTRACT FROM AUTHOR]

Background: Polyploidy has often been considered to confer plants a better adaptation to environmental stresses. Tetraploid citrus rootstocks are expected to have stronger stress tolerance than diploid. Plenty of doubled diploid citrus plants were exploited from diploid species for citrus rootstock improvement. However, limited metabolic and molecular information related to tetraploidization is currently available at a systemic biological level. This study aimed to evaluate the occurrence and extent of metabolic and transcriptional changes induced by tetraploidization in Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka), which is a special citrus germplasm native to China and widely used as an iron deficiency tolerant citrus rootstock. Results: Doubled diploid Ziyang xiangcheng has typical morphological and anatomical features such as shorter plant height, larger and thicker leaves, bigger stomata and lower stomatal density, compared to its diploid parent. GC-MS (Gas chromatography coupled to mass spectrometry) analysis revealed that tetraploidization has an activation effect on the accumulation of primary metabolites in leaves; many stress-related metabolites such as sucrose, proline and ?-aminobutyric acid (GABA) was remarkably up-regulated in doubled diploid. However, LC-QTOF-MS (Liquid chromatography quadrupole time-of-flight mass spectrometry) analysis demonstrated that tetraploidization has an inhibition effect on the accumulation of secondary metabolites in leaves; all the 33 flavones were down-regulated while all the 6 flavanones were up-regulated in 4x. By RNA-seq analysis, only 212 genes (0.8% of detected genes) are found significantly differentially expressed between 2x and 4x leaves. Notably, those genes were highly related to stress-response functions, including responses to salt stress, water and abscisic acid. Interestingly, the transcriptional divergence could not explain the metabolic changes, probably due to post-transcriptional regulation. Conclusion: Taken together, tetraploidization induced considerable changes in leaf primary and secondary metabolite accumulation in Ziyang xiangcheng. However, the effect of tetraploidization on transcriptome is limited. Compared to diploid, higher expression level of stress related genes and higher content of stress related metabolites in doubled diploid could be beneficial for its stress tolerance. [ABSTRACT FROM AUTHOR]

Background: After several years in the juvenile phase, trees undergo flowering transition to become mature (florally competent) trees. This transition depends on the balanced expression of a complex network of genes that is regulated by both endogenous and environmental factors. However, relatively little is known about the molecular processes regulating flowering transition in woody plants compared with herbaceous plants. Results: Comparative transcript profiling of spring shoots after self-pruning was performed on a spontaneously early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) with a short juvenile phase and the wild-type (WT) tree by using massively parallel signature sequencing (MPSS). A total of 16,564,500 and 16,235,952 high quality reads were obtained for the WT and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed genes in the MT (31,468) was larger than in the WT (29,864), suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts revealed that 2735 genes had more than twofold expression difference in the MT compared with the WT. In addition, we identified 110 citrus flowering-time genes homologous with known elements of flowering-time pathways through sequencing and bioinformatics analysis. These genes are highly conserved in citrus and other species, suggesting that the functions of the related proteins in controlling reproductive development may be conserved as well. Conclusion: Our results provide a foundation for comparative gene expression studies between WT and precocious trifoliate orange. Additionally, a number of candidate genes required for the early flowering process of precocious trifoliate orange were identified. These results provide new insight into the molecular processes regulating flowering time in citrus. [ABSTRACT FROM AUTHOR]