Lysine crotonylation, as a novel evolutionarily conserved type of post-translational modifications, is ubiquitous and essential in cell biology. However, its functions in tea plants are largely unknown, and the full functions of lysine crotonylated proteins of tea plants in nitrogen absorption and assimilation remains unclear. Our study attempts to describe the global profiling of nonhistone lysine crotonylation in tea leaves and to explore how ammonium (NH4+) triggers the response mechanism of lysine crotonylome in tea plants. Here, we performed the global analysis of crotonylome in tea leaves under NH4+ deficiency/resupply using high-resolution LC-MS/MS coupled with highly sensitive immune-antibody. A total of 2288 lysine crotonylation sites on 971 proteins were identified, of which contained in 15 types of crotonylated motifs. Most of crotonylated proteins were located in chloroplast (37%) and cytoplasm (33%). Compared with NH4+ deficiency, 120 and 151 crotonylated proteins were significantly changed at 3 h and 3 days of NH4+ resupply, respectively. Bioinformatics analysis showed that differentially expressed crotonylated proteins participated in diverse biological processes such as photosynthesis (PsbO, PsbP, PsbQ, Pbs27, PsaN, PsaF, FNR and ATPase), carbon fixation (rbcs, rbcl, TK, ALDO, PGK and PRK) and amino acid metabolism (SGAT, GGAT2, SHMT4 and GDC), suggesting that lysine crotonylation played important roles in these processes. Moreover, the protein-protein interaction analysis revealed that the interactions of identified crotonylated proteins diversely involved in photosynthesis, carbon fixation and amino acid metabolism. Interestingly, a large number of enzymes were crotonylated, such as Rubisco, TK, SGAT and GGAT, and their activities and crotonylation levels changed significantly by sensing ammonium, indicating a potential function of crotonylation in the regulation of enzyme activities. The results indicated that the crotonylated proteins had a profound influence on metabolic process of tea leaves in response to NH4+ deficiency/resupply, which mainly involved in diverse aspects of primary metabolic processes by sensing NH4+, especially in photosynthesis, carbon fixation and amino acid metabolism. The data might serve as important resources for exploring the roles of lysine crotonylation in N metabolism of tea plants. Data were available via ProteomeXchange with identifier PXD011610.

Vacuolar invertases (VINs) have been reported to regulate plant growth and development and respond to abiotic stresses such as drought and cold. With our best knowledge, the functions of VIN genes little have been reported in tea plant (Camellia sinensis L.). Therefore, it is necessary to develop research in this field. Here, we identified a VIN gene, CsINV5, which was induced by cold acclimation and sugar treatments in the tea plant. Histochemical assays results showed that the 1154 bp 5′-flanking sequence of CsINV5 drove β-glucuronidase (GUS) gene expression in roots, stems, leaves, flowers and siliques of transgenic Arabidopsis during different developmental stages. Moreover, promoter deletion analysis results revealed that an LTRE-related motif (CCGAAA) and a WBOXHVISO1 motif (TGACT) within the promoter region of CsINV5 were the core cis-elements in response to low temperature and sugar signaling, respectively. In addition, overexpression of CsINV5 in Arabidopsis promoted taproot and lateral root elongation through glucose-mediated effects on auxin signaling. Based on physiological and RNA-seq analysis, we found that overexpression of CsINV5 improved cold tolerance in transgenic Arabidopsis mainly by increasing the contents of glucose and fructose, the corresponding ratio of hexose to sucrose, and the transcription of osmotic-stress-related genes (P5CS1, P5CS2, AtLEA3, COR413-PM1 and COR15B) to adjust its osmotic potential. Comprehensive experimental results suggest that overexpression of CsINV5 may enhance the cold tolerance of plant through the modification of cellular sugar compounds contents and osmotic regulation related pathways.

Light is an important environmental factor which affects plant growth, through changes of intensity and quality. In this study, monochromatic white (control), red (660 nm), and blue (430 nm) light-emitting diodes (LEDs) were used to treat tea short cuttings. The results showed the most adventitious roots in blue light treated tea cuttings, but the lowest roots in that treated by red light. In order to explore the molecular mechanism of light quality affecting adventitious root formation, we performed full-length transcriptome and metabolome analyses of mature leaves under three light qualities, and then conducted weighted gene co-expression network analysis (WGCNA). Phytohormone analysis showed that Indole-3-carboxylic acid (ICA), Abscisic acid (ABA), ABA-glucosyl ester (ABA-GE), trans-Zeatin (tZ), and Jasmonic acid (JA) contents in mature leaves under blue light were significantly higher than those under white and red light. A crosstalk regulatory network comprising 23 co-expression modules was successfully constructed. Among them, the “MEblue” module which had a highly positive correlation with ICA (R = 0.92, P = 4e-04). KEGG analysis showed that related genes were significantly enriched in the “Plant hormone signal transduction (ko04075)” pathway. YUC (a flavin-containing monooxygenase), AUX1, AUX/IAA, and ARF were identified as hub genes, and gene expression analysis showed that the expression levels of these hub genes under blue light were higher than those under white and red light. In addition, we also identified 6 auxin transport-related genes, including PIN1, PIN3, PIN4, PILS5, PILS6, and PILS7. Except PILS5, all of these genes showed the highest expression level under blue light. In conclusion, this study elucidated the molecular mechanism of light quality regulating adventitious root formation of tea short cutting through WGCNA analysis, which provided an innovation for “rapid seedling” of tea plants.

The type of adventitious root formation of tea softwood cuttings is different from that of single node cuttings. In addition to the callus at the base of cuttings, the adventitious roots are also formed in the upper cortex of softwood cuttings. In order to find out the similarities and differences between the above two types of adventitious roots of softwood cuttings and the influencing factors for the differences, an integrated analysis of plant hormones, untargeted metabolomics, and transcriptomics of the softwood cutting stems at different positions is performed. The phytohormone results show that IAA plays a leading role, and a high ratio of in vivo auxin to GA3 or ABA facilitated root formation. The ratios of IAA/GA3 and IAA/ABA in the upper and base rooting parts of cuttings are both higher than the middle non-rooting transition zone. Differences in metabolites indicate that 73 metabolites are involved in the formation of adventitious roots in cuttings. Compared with the middle non-rooting transition zone, most saccharides are downregulated in the upper and base rooting parts of softwood cuttings, and the saccharides in the base rooting parts of cuttings are more consumed than that in the upper rooting parts. Most organic acids in the callus at the base of cuttings show an upward trend, while those in the upper rooting parts show a downward trend. Furthermore, coniferyl alcohol is the key metabolite for adventitious root formation in the upper and base rooting parts of cuttings. Transcriptome results show 1099 differentially expressed genes (DEGs) are obtained, and KEGG enrichment analysis show that these DEGs are significantly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, and flavonoid biosynthesis pathways. Based on weighted gene co-expression network analysis (WGCNA), two key modules which have a highly positive correlation with IAA are identified. In summary, maintaining the balance of endogenous hormones and sufficient nutritional elements is very important for adventitious root formation in tea plants. Clarifying the material basis of softwood cutting rooting of tea plant is of great significance to improve the cutting survival rate of tea plant (especially difficult rooting varieties) and shorten the breeding cycle.

Background: As a type of calmodulin binding protein, CAMTAs are widely involved in vegetative and reproductive processes as well as various hormonal and stress responses in plants. To study the functions of CAMTA genes in tea plants, we investigated bioinformatics analysis and performed qRT-PCR analysis of the CAMTA gene family by using the genomes of ‘ShuChaZao’ tea plant cultivar. Results: In this study, 6 CsCAMTAs were identified from tea plant genome. Bioinformatics analysis results showed that all CsCAMTAs contained six highly conserved functional domains. Tissue-specific analysis results found that CsCAMTAs played great roles in mediating tea plant aging and flowering periods. Under hormone and abiotic stress conditions, most CsCAMTAs were upregulated at different time points under different treatment conditions. In addition, the expression levels of CsCAMTA1/3/4/6 were higher in cold-resistant cultivar ‘LongJing43’ than in the cold-susceptible cultivar ‘DaMianBai’ at cold acclimation stage, while CsCAMTA2/5 showed higher expression levels in ‘DaMianBai’ than in ‘LongJing43’ during entire cold acclimation periods. Conclusions: In brief, the present results revealed that CsCAMTAs played great roles in tea plant growth, development and stress responses, which laid the foundation for deeply exploring their molecular regulation mechanisms.

Pectin methylesterase inhibitor (PMEI) inhibits pectin methylesterase (PME) activity at post-translation level, which plays core roles in vegetative and reproductive processes and various stress responses of plants. However, the roles of PMEIs in tea plant are still undiscovered. Herein, a total of 51 CsPMEIs genes were identified from tea plant genome. CsPMEI1-4 transcripts were varied in different tea plant tissues and regulated by various treatments, including biotic and abiotic stresses, sugar treatments, cold acclimation and bud dormancy. Overexpression of CsPMEI4 slightly decreased cold tolerance of transgenic Arabidopsis associated with lower electrolyte leakage, soluble sugars contents and transcripts of many cold-induced genes as compared to wild type plants. Under long-day and short-day conditions, CsPMEI2/4 promoted early flowering phenotypes in transgenic Arabidopsis along with higher expression levels of many flowering-related genes. Moreover, overexpression of CsPMEI2/4 decreased PME activity, but increased sugars contents (sucrose, glucose, and fructose) in transgenic Arabidopsis as compared with wild type plants under short-day condition. These results indicate that CsPMEIs are widely involved in tea plant vegetative and reproductive processes, and also in various stress responses. Moreover, CsPMEI4 negatively regulated cold response, meanwhile, CsPMEI2/4 promoted early flowering of transgenic Arabidopsis via the autonomous pathway. Collectively, these results open new perspectives on the roles of PMEIs in tea plant.