Wanping Fang

Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis

BackgroundMicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted.ResultSolexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response.ConclusionsWe discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in ‘Yingshuang’, and 46 up-regulated miRNA and 45 down-regulated miRNAs in `Baiye 1’ in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5′RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

Nitric Oxide Participates in Cold-Inhibited Camellia sinensis Pollen Germination and Tube Growth Partly via cGMP In Vitro

Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.

Seasonal and Habitat Dependent Variations in Culturable Endophytes of Camellia sinensis

Seasonal and habitat dependent variations in the endophytes of Camellia sinensis are investigated in this study. Stems and leaves of C. sinensis from naked and under-forestry tea garden have been collected for isolating endophytic fungi in January, April and October, respectively, as different seasons. Twenty-one endophytic species including a new species, are observed in this study. It is confirmed that species in under-forestry tea garden are more related to the naked tea garden. However, both habitats have similar evenness indices and Shannon-Wiener indices. Shannon-Wiener indices in spring are highest in all compared seasons. The diversity of species in stem is higher than in leaf. In all seasons, the evenness indices are slightly higher in leaf than stem. The number of endophytic fungi in autumn is the smallest, while the largest in winter. Furthermore, the frequencies of Neurospora crassa, Phomopsis sp4., Trichoderma viride, Phomopsis sp2., Pleosporales sp., Pestalotiopsis microspora, Glomerella sp., Colletotrichum gloeosporioides, Botryosphaeria sp., Penicillium sclerotiorum and Rosellinia sp. vary significantly in different habitats. The composition and diversity of endophytic fungi are different between leaves and stems. Guignardia mangiferae, T. viride, P. sclerotiorum, Pleosporales sp., Phomopsis sp4., C. gloeosporioides, P. clavispora, Glomerella sp. and N. crassa show remarkable organizational preference in tea plants.

Late-acting self-incompatibility in tea plant ( Camellia sinensis )

The self-incompatibility of tea plant (Camellia sinensis (L.) O. Kuntze) was studied with the methods of aniline blue fluorescence assay and paraffin sections. The characteristics of pollen tube elongation after hand pollination was analyzed in 4 tea cultivars, including ‘Keemenzhong’, ‘Longjing-changye’, ‘Fuding-dabaicha’ and ‘Yabukita’, under self-pollination and cross-pollination, respectively. Although there were some difference among cultivars, pollen tubes elongated through the style and reach the ovary successfully at 48 h after pollination for both cross- and self-pollen tubes in all the four cultivars of tea. Pollen tubes entered into the ovule micropyles, however, only for cross-pollination, but not for self-pollination. Pollen tubes of selfing plants, failed in fertilizing, seemed have some difficulties to enter the ovule. All of which indicated that the self-incompatibility of tea plant is a late-acting self-incompatibility system (LSI) or an ovarian sterility (OS), in which the self incompatibility was due to none self pollen tube penetrating into the ovule and no fertilization.

Functional analysis of CsCBF3 transcription factor in tea plant (Camellia sinensis) under cold stress

C-repeat binding factors (CBFs) are involved in multiple pathways of plant growth, development and stress responses. In this study, CsCBF3, a gene encoding a polypeptide of 274 amino acids and containing the structural feature of AP2 domain in CBF protein family, was characterized from tea plant [Camellia sinensis (L.) O. Kuntze]. CsCBF3 was located in cell nucleus and cytoplasm by subcellular localization analysis. Expression analysis revealed that CsCBF3 was induced by low temperature stress, abscisic acid and drought treatment in tea leaves. CsCBF3 overexpressed Arabidopsis displayed higher tolerance to cold stress and improved photosynthesis ability with less damage under cold condition compared to wild type plants. Furthermore, it was observed that the CsCBF3 gene regulates the expressions of downstream genes of cold responsive pathway, such as AtCOR15a and AtCOR78. These results indicate that CsCBF3 plays an important role in responding to cold stress and provide potential applications in molecular breeding to enhance cold tolerance in tea plant.

Zn, Ni, Mn, Cr, Pb and Cu in soil-tea ecosystem: The concentrations, spatial relationship and potential control

Excessive accumulation of heavy metals in tea has certain health risk to the consumers. To discuss the spatial relationship between heavy metals in tea leaves and soil in a county scale, 74 samples of tea leaves and corresponding subsoil (15-30 cm) were collected in the tea plantations of Lishui, Nanjing, China. The total Zn, Ni, Mn, Cr, Pb and Cu in both tea leaves and soil samples, as well as pH value in soil were analyzed. The results of regression showed that the Mn and Cr in tea leaves were increased when the Mn and Cr in soil were at high levels, and higher pH decreased Zn, Ni, Mn and Cu contents in tea leaves. Moreover, distance to village and metal industry had negative impacts on the Mn level in soil. It is necessary to control the pH of soil in tea plantations which are close to villages or metal industries, as well as, to search more appropriate areas, where are far away from villages or metal industries, to plant tea should be taken into consideration. In addition, 17.57% of the samples have higher concentration of Pb than the maximum limits was found in tea plantation of Lishui. Heavy metals (Cr) standards regarding leafy vegetables is used for reference in this study, as there are no maximum levels of contaminants (MLC) in tea. It is significant to construct a targeted standard of heavy metals contents that could applicable throughout the world for reduce the health risks to the consumers.