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Yang C.,Huazhong Agricultural University | Yang C.,Key Laboratory of Horticultural Plant Biology | Yang C.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Liu T.,Huazhong Agricultural University | And 17 more authors.
Molecular Genetics and Genomics | Year: 2015

Corky split vein can develop under long-term boron deficient conditions in Citrus sinensis L. Osbeck cv. Newhall. This symptom only occurs in the upper rather than the lower epidermis of old leaves. Our previous study demonstrated that vascular hypertrophy was involved in the symptoms, and the 3rd developmental stage of corky split vein (BD3) was the critical stage for phenotype formation. Here, we performed an intensive study on the BD3 vein and its control sample (CK3 vein). A lignin test demonstrated that the lignin content in BD3 vein was approximately 1.7 times more than the CK3 vein. Anatomical investigation of the corky split vein indicated that the upper epidermis was destroyed by overgrowing vascular cells, and the increased lignin may contribute to vascular cell differentiation and wounding-induced lignification. In a subsequent small RNA sequencing of the BD3 and CK3 veins, 99 known miRNAs and 22 novel miRNAs were identified. Comparative profiling of these miRNAs demonstrated that the 57 known miRNAs and all novel miRNAs exhibited significant expression differences between the two small RNAs libraries of the BD3 and CK3 veins. Associated with our corresponding digital gene expression data, we propose that the decreased expression of two miRNAs, csi-miR156b and csi-miR164, which leads to the up-regulation of their target genes, SPLs (csi-miR156b-targeted) and CUC2 (csi-miR164-targeted), may promote vascular cell division and orderless stage transition in old leaves. © 2015, Springer-Verlag Berlin Heidelberg.

Islam M.Z.,Huazhong Agricultural University | Islam M.Z.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Jin L.-F.,Huazhong Agricultural University | Jin L.-F.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | And 6 more authors.
Tree Genetics and Genomes | Year: 2015

The sucrose transporter (SUT) plays a major role in the transport of sucrose apoplastically. An extensive mining of the data confirmed that at least three SUT genes (CitSUT1, CitSUT2, and CitSUT3) were found in the databases of the three currently available citrus genomes. The exon and intron sizes varied among the three CitSUT genes, and the similarities among the genes were 40–50 %. The phylogenetic analysis revealed that the CitSUT1, CitSUT2, and CitSUT3 were distributed into groups SUT I, SUT II, and SUT III, respectively. The analysis of the spatiotemporal expression of the genes showed that the CitSUT1, CitSUT2, and CitSUT3 were predominantly expressed in the stamens, young leaves, and fruits [juice sacs (JS) and/or segment membrane (SM)], respectively. Moreover, in addition to the significant increase in sucrose levels from 114 days after anthesis (DAA) to 160 DAA, the transcript levels of CitSUT1 and CitSUT3 increased significantly in the JS and decreased significantly in the SM, whereas the CitSUT2 transcript levels decreased significantly either in the JS or in the SM. Additionally, treatment with ABA increased the accumulations of sucrose and fructose in fruits, which were accompanied by the induction of the CitSUT2 transcripts in fruits (JS and SM) and the CitSUT3 transcripts in the SM and the reduction of the CitSUT1 and CitSUT3 transcript levels in the JS. Thus, the CitSUT1 and CitSUT3 might play important roles in the transport of sucrose into the fruit JS during normal fruit development; the transcript alterations of the CitSUT2 and CitSUT3 under the ABA treatment might contribute to the increased accumulation of sucrose. © 2015, Springer-Verlag Berlin Heidelberg.

Zhu F.,Huazhong Agricultural University | Zhu F.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Chen J.,Huazhong Agricultural University | Chen J.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | And 15 more authors.
Food Chemistry | Year: 2016

To comprehensively analyze the effects of salicylic acid (SA) on the storability of Satsuma mandarin (Citrus unshiu), fruits were treated with 2 mM SA. The disease incidence of control/SA-treated fruit at 50 d and 120 d after treatment was 23.3%/10% and 67.3%/23.3%, respectively, suggesting that SA treatment can significantly reduce the rot rate of postharvest citrus fruit. Fruit quality assays revealed that the treatment can maintain fruit firmness without affecting the inner quality. Furthermore, the contents of H2O2 and some defense-related metabolites, such as ornithine and threonine, in citrus pericarp, were significantly increased by SA treatment. Moreover, it was lipophilic polymethoxylated flavones, rather than flavanone glycosides, that accumulated in SA-treated fruits and these can directly inhibit pathogen development. These results suggest that the effects of SA on postharvest citrus fruit may be attributed to the accumulation of H2O2 and defense-related metabolites. © 2016 Published by Elsevier Ltd.

Liu X.,Huazhong Agricultural University | Guo L.-X.,Huazhong Agricultural University | Jin L.-F.,Huazhong Agricultural University | Liu Y.-Z.,Huazhong Agricultural University | And 4 more authors.
Molecular Biology Reports | Year: 2016

Growth-regulating factor (GRF) is an important protein in GA-mediated response, with key roles in plant growth and development. However, it is not known whether or how the GRF proteins in citrus to regulate organ size. In this study, nine citrus GRF genes (CsGRF1–9) were validated from the ‘Anliu’ sweet orange (AL, Citrus sinensis cv. Anliu) by PCR amplification. They all contain two conserved motifs (QLQ and WRC) and have 3–4 exons. The transcript levels of genes were detected by qRT-PCR. Transcript analysis showed that (1) CsGRF 1, 2, 5, 6, 7, and 9 expressed predominantly in young leaf, CsGRF 3 and 4 expressed predominantly in fruit immature juice sacs and CsGRF 8 expressed predominantly in root; (2) all citrus GRF genes had significantly higher expression in young leaves than mature leaf; (3) in juice sacs, the transcript levels of CsGRF1, 4, 5, 6, and 8 increased significantly while the transcript levels of CsGRF2, 3, 7, and 9 had no significant change from 80 DAF to 100 DAF. Besides, GA3 treatment did not affect the transcript levels of CsGRF5 and CsGRF6 but significantly increased the transcript levels of the other seven CsGRF genes in young leaves. These results suggested that all CsGRF genes involve in the leaf development, CsGRF1, 4, 5, 6, and 8 act developmentally whilst CsGRF2, 3, 7, and 9 play fundamental roles in fruit cell enlargement, which may be through GA pathway or GA-independent pathway. © 2016 Springer Science+Business Media Dordrecht

Zheng X.,Key Laboratory of Horticultural Plant Biology of Ministry of Education | Zheng X.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Zheng X.,Huazhong Agricultural University | Xie Z.,Key Laboratory of Horticultural Plant Biology of Ministry of Education | And 14 more authors.
Molecular Genetics and Genomics | Year: 2015

In plants, the carotenoid cleavage dioxygenase 4 (CCD4) could target on plastoglobules and cleave specific carotenoids, producing apocarotenoids and volatile compounds. These compounds are important for color and aroma formation in fruits and flowers. In this study, five CCD4 gene members (CCD4a, b, c, d, and e) were investigated in different citrus species including mandarin, pummelo, and sweet orange. Sequence analysis showed that the CCD4 genes from all the species examined exhibited extensive allelic variability (including SNPs and frame-shift mutations). Furthermore, the distribution of the CCD4 allelic mutation sites supported our previous hypothesis that the sweet orange originated from the hybridization of mandarin and pummelo. A derived cleaved amplified polymorphic sequence (dCAPs) marker was then successfully developed based on the allelic polymorphism of CCD4c, providing an ideal molecular marker for studying the genetic relationship between citrus species. Quantitative RT-PCR analysis identified differential expression patterns for the CCD4 genes in tissues/organs, and CCD4b was shown to have a high-level expression in citrus fruit flavedos (especially those with a deep orange-reddish color). HPLC-based detection of a key component (i.e., β-citraurin) for orange-reddish flavedo formation in different citrus revealed a positive correlation between CCD4b expression levels and the presence of β-citraurin, suggesting that CCD4b may be responsible for β-citraurin biosynthesis in flavedo. In summary, this study not only reinforced the anticipated roles of CCD4 genes in flavedo color formation in citrus, but also provided new information about gene expression patterns, allelic polymorphism characteristics, and sequence variability for this gene subfamily. © 2015, Springer-Verlag Berlin Heidelberg.

Fu L.,Huazhong Agricultural University | Fu L.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Chai L.,Huazhong Agricultural University | Chai L.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | And 5 more authors.
Journal of the American Society for Horticultural Science | Year: 2016

Iron (Fe) deficiency caused by calcareous soil is a serious problem in the cultivation of citrus (Citrus L.) trees. In this study, we report that ‘Zhique’ (Citrus wilsonii Tanaka) citrus rootstock from Chenggu county of Shaanxi province, China, shows tolerance to Fe deficiency under calcareous soil conditions. In the same calcareous field conditions, ‘Miyagawa Wase’ Satsuma mandarin (Citrus unshiu Marc.) grafted on trifoliate orange [Poncirus trifoliate (L.) Raf.] rootstock, the most commonly used rootstock, showed obvious interveinal chlorosis in young leaves, though some leaves or branches are asymptomatic, whereas no symptoms were found on those grafted on ‘Zhique’ rootstock. This was further evidenced by the fact that the chlorophyll concentration in chlorotic leaves of ‘Miyagawa Wase’ grafted on trifoliate orange was significantly lower than in those grafted on ‘Zhique’. In addition, transmission electron microscopy (TEM) analysis revealed a significant reduction of grana and stroma thylakoid of chloroplasts in chlorotic leaves. Measurement of Fe concentrations revealed that the total Fe and cell wall Fe showed no difference between ‘Zhique’ and trifoliate orange roots, whereas the ferrous Fe was significantly higher in ‘Zhique’ than trifoliate orange roots. Interestingly, both total Fe and ferrous Fe concentrations in chlorotic leaves were significantly lower than in green leaves of ‘Miyagawa Wase’ grafted on either ‘Zhique’ or trifoliate orange, whereas the cell wall Fe concentration of ‘Miyagawa Wase’ leaves only showed significant difference between the ‘Zhique’ and trifoliate orange samples. Further transcript assessment found that the Fe acquisition-related genes FIT, HA, FRO, and NRAMP were upregulated in roots of ‘Zhique’ compared with trifoliate orange, thus suggesting ‘Zhique’ might be more capable of Fe uptake under calcareous soil conditions. The novel citrus rootstock reported here could be used as an ideal material for Fe-uptake research, and as a Fe-deficiency-tolerant rootstock for citrus cultivation in calcareous soils. © 2016 American Society for Horticultural Science, All rights reserved.

Jin L.-F.,Huazhong Agricultural University | Jin L.-F.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Liu Y.-Z.,Huazhong Agricultural University | Liu Y.-Z.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | And 4 more authors.
Acta Physiologiae Plantarum | Year: 2016

Boron (B) toxicity reduces crop productivity and is a serious abiotic stress presenting in many parts of the world. MicroRNAs (miRNAs) play important roles in nutrient toxicity. In this study, we found that the B concentrations in roots and leaves of trifoliate orange (Poncirus trifoliata) were increased by 1.4- and 1.2-fold, respectively, after 10 days of excess B treatment (DAEBT). After 20 DAEBT, the B concentrations in roots and leaves increased by 2.8- and 2.0-fold, respectively. Transcript analysis showed that the miR397 relative transcript level decreased following the excess B treatment. Laccase7 (LAC7) was shown to be the target of miR397, and its transcription increased after the excess B treatment. In addition, the activity of laccase increased significantly following this treatment. Because LAC7 plays a role in lignin biosynthesis, we also measured the lignin concentrations in roots and leaves and found that they were increased following the excess B treatment. Our work demonstrates that decreased miR397 transcription plays a possible role in enhancing tolerance to B toxicity stress via negatively regulating LAC7 transcription and increasing the lignin concentration. © 2015, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.

Shi C.-Y.,Huazhong Agricultural University | Shi C.-Y.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | Song R.-Q.,Huazhong Agricultural University | Song R.-Q.,Key Laboratory of Horticultural Crop Biology and Genetic Improvement Central Region | And 8 more authors.
Frontiers in Plant Science | Year: 2015

PH5 is a petunia gene that encodes a plasma membrane H+-ATPase and determines the vacuolar pH. The citrate content of fruit cell vacuoles influences citrus organoleptic qualities. Although citrus could have PH5-like homologs that are involved in citrate accumulation, the details are still unknown. In this study, extensive data-mining with the PH5 sequence and PCR amplification confirmed that there are at least eight PH5-like genes (CsPH1-8) in the citrus genome. CsPHs have a molecular mass of approximately 100 kDa, and they have high similarity to PhPH5, AtAHA10 or AtAHA2 (from 64.6 to 80.9%). They contain 13–21 exons and 12–20 introns and were evenly distributed into four subgroups of the P3A-subfamily (CsPH1, CsPH2, and CsPH3 in Group I, CsPH4 and CsPH5 in Group II, CsPH6 in Group IV, and CsPH7 and CsPH8 in Group III together with PhPH5). A transcript analysis showed that CsPH1, 3, and 4 were predominantly expressed in mature leaves, whereas CsPH2 and 7 were predominantly expressed in roots, CsPH5 and 6 were predominantly expressed in flowers, and CsPH8 was predominantly expressed in fruit juice sacs (JS). Moreover, the CsPH transcript profiles differed between orange and pummelo, as well as between high-acid and low-acid cultivars. The low-acid orange “Honganliu” exhibits low transcript levels of CsPH3, CsPH4, CsPH5, and CsPH8, whereas the acid-free pummelo (AFP) has only a low transcript level of CsPH8. In addition, ABA injection increased the citrate content significantly, which was accompanied by the obvious induction of CsPH2, 6, 7, and 8 transcript levels. Taken together, we suggest that CsPH8 seems likely to regulate citrate accumulation in the citrus fruit vacuole. © 2015 Shi, Song, Hu, Liu, Jin and Liu.

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