Xujun Zhu

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.

Exogenous abscisic acid significantly affects proteome in tea plant (Camellia sinensis) exposed to drought stress.

Tea [Camellia sinensis (L.) O. Kuntze] is an important economic crop, and drought is the most important abiotic stress affecting yield and quality. Abscisic acid (ABA) is an important phytohormone responsible for activating drought resistance. Increased understanding of ABA effects on tea plant under drought stress is essential to develop drought-tolerant tea genotypes, along with crop management practices that can mitigate drought stress. The objective of the present investigation is evaluation of effects of exogenous ABA on the leaf proteome in tea plant exposed to drought stress. Leaf protein patterns of tea plants under simulated drought stress [(polyethylene glycol (PEG)-treated] and exogenous ABA treatment were analyzed in a time-course experiment using two-dimensional electrophoresis (2-DE), followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Among the 72 protein spots identified by MALDI-TOF MS, 16 proteins were downregulated and two were upregulated by exogenous ABA. The upregulated proteins have roles in glycolysis and photosystem II stabilization. Twenty-one protein spots were responsive to drought stress and most participate in carbohydrate and nitrogen metabolism, control of reactive oxygen species (ROS), defense, signaling or nucleic acid metabolism. The combined treatments of exogenous ABA and drought showed upregulation of 10 protein spots at 12 h and upregulation of 11 proteins at 72 h after initiation of drought stress. The results support the importance of the role that ABA plays in the tea plant during drought stress, by improving protein transport, carbon metabolism and expression of resistance proteins.

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.

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.