Global Transcriptional Analysis Reveals the Complex Relationship between Tea Quality, Leaf Senescence and the Responses to Cold-Drought Combined Stress in Camellia sinensis

Abstract

In field conditions, especially in arid and semi-arid areas, tea plants are often simultaneously exposed to various abiotic stresses such as cold and drought, which have profound effects on leaf senescence process and tea quality. However, most studies of gene expression in stress responses focus on a single inciting agent, and the confounding effect of multiple stresses on crop quality and leaf senescence remain unearthed. Here, global transcriptome profiles of tea leaves under separately cold and drought stress were compared with their combination using RNA-Seq technology. This revealed that tea plants shared a large overlap in unigenes displayed "similar" (26%) expression pattern and avoid antagonistic responses (lowest level of "prioritized" mode: 0%) to exhibit very congruent responses to co-occurring cold and drought stress; 31.5% differential expressed genes and 38% of the transcriptome changes in response to combined stresses were unpredictable from cold or drought single-case studies. We also identified 319 candidate genes for enhancing plant resistance to combined stress. We then investigated the combined effect of cold and drought on tea quality and leaf senescence. Our results showed that drought-induced leaf senescence were severely delayed by (i) modulation of a number of senescence-associated genes and cold responsive genes, (ii) enhancement of antioxidant capacity, (iii) attenuation of lipid degradation, (iv) maintenance of cell wall and photosynthetic system, (v) alteration of senescence-induced sugar effect/sensitivity, as well as (vi) regulation of secondary metabolism pathways that significantly influence the quality of tea during combined stress. Therefore, care should be taken when utilizing a set of stresses to try and maximize leaf longevity and tea quality.

Keywords: Camellia sinensis; RNA-Seq; cold-drought combined stress; leaf senescence; tea quality.

Figure 1

The phenotypes and physiological analyses of tea plants under CT, DT and CD. (A) The phenotypes of control (CK) and plants treated with gradually cold (CT) and drought (DT) stress individually or together (CD) after 15 days. (B) Leaf maximum photochemical quantum yield of PS II (Fv/Fm), (C) leaf relative electrolyte conductivity (REC), and (D) relative water content (RWC) were determined at six time-points (0, 3, 6, 9, 12 and 15 days) during CT, DT, and CD.

Figure 2

Relationships between transcriptome responses during CT, DT, and CD. (A) The histogram showing the number of common and specific DEGs during CT, DT, and CD (left), and the cumulative log-fold changes of 1000 most significantly CT- and DT-responding unigenes in CD (right), respectively. Venn diagram of genes (B) down-regulated or (C) up-regulated by each stress. The numbers of common and specific DEGs were shown in the overlapping and non-overlapping regions, respectively. The total numbers of up- and down-regulated DEGs were indicated in parentheses.

Figure 3

Clustering of unigenes to predefined expression profiles generating the transcriptional response modes. The unigene sets were created by the union of the 500 most significant unigenes for CT, DT, and CD. These unigenes were clustered to 20 predefined expression profiles, each categorizing a potential expression change that may occur when combined stresses are applied. The dotted line in the boxes represents unigene expression with no change compared with the control. The value and color in the right boxes represents the percentage of unigenes that correlate with the particular predefined expression profile (red is higher). Combinatorial, similar levels in the CT and DT but a different response to CD; canceled, unigene response to either CT or DT returned to control levels; prioritized, opposing responses to the CT and DT and one stress response prioritized in response to CD; independent, response to only CT or DT and a similar response to CD; similar, similar responses to CT, DT, and CD.

Figure 4

Heat map of selected differentially expressed SAGs with known effect. Heat map shown the expression level of SAGs with promote (A) or delay (B) effect in LSD. The selection was based on the clustering analysis results (i.e., mode 1, 2, 5, 6, 9, 10, 11, 12, 15, 16, 19, and 20 in Figure 3). Color indicates fold change of DEGs under CT, DT and CD, as shown in the top.

Figure 5

GO and KEGG pathway analysis of DEGs under CT, DT, and CD. (A) Multi-series chord graphs for the top 20 level-3 GO terms of each stress treatment. The size of the segments around the circumference of the circle indicate the number of DEGs in specific GO terms. The orange (up-regulated) or blue (down-regulated) lines within the circle indicate the DEGs involved in different biological processes. The numbers of up- and down-regulated DEGs were shown in the right bar graphs and parentheses. (B) Heat map showing the overall expression levels of 25 enriched pathways during CT, DT, and CD. The color represents the mean expression values of DEGs within the pathways. The total numbers of DEGs in the pathways were shown in the right bar graphs and parentheses.

Figure 6

Heat map of 319 co-up-regulated DEGs that showed enhanced expression under CD compared with CT and DT. Color indicates fold change of DEGs under CT, DT and CD, as shown in the top.

Figure 7

Flavonoid and theanine biosynthetic pathway in C. sinensis under CT, DT, and CD. Heat maps on the left show fold change of the DEGs involved in (A) flavonoid biosynthetic pathway and (B) theanine biosynthetic pathway under CT, DT, and CD. The numbers in the brackets following each gene name indicate the number of corresponding unigenes identified in transcriptome. The color in the ellipses represents the gene was regulated under particular stress treatment (blue indicates CT; yellow indicates DT; red indicates CD). Full names of enzymatic genes are expanded in Supplementary Tables 10, 13.

Figure 8

Model representing the complex relationship between tea quality, leaf senescence and the responses to combined CD stress in C. sinensis. Sharp and blunt arrow denote activation and suppression, respectively. Heat maps show fold change of DEGs among the model during CT, DT, and CD. Full names of various genes are expanded in Supplementary Tables 11, 13.