Nitrate nitrogen is an important nitrogen source for tea plants’ growth and development. LBD transcription factors play important roles in response to the presence of nitrate in plants. The functional study of LBD transcription factors in tea plants remains limited. In this study, the LBD family gene CsLBD39 was isolated and characterized from tea plants. Sequence analysis indicated that CsLBD39 contained a highly conserved CX₂CX₆CX₃CX domain. The phylogenetic tree assay showed that CsLBD39 belonged to class II subfamily of the LBD family. CsLBD39 was highly expressed in flowers and root; we determined that its expression could be induced by nitrate treatment. The CsLBD39 protein was located in the nucleus and has transcriptional activation activity in yeast. Compared with the wild type, overexpression of CsLBD39 gene in Arabidopsis resulted in smaller rosettes, shorter main roots, reduced lateral roots and lower plant weights. The nitrate content and the expression levels of genes related to nitrate transport and regulation were decreased in transgenic Arabidopsis hosting CsLBD39 gene. Compared with the wild type, CsLBD39 overexpression in transgenic Arabidopsis had smaller cell structure of leaves, shorter diameter of stem cross section, and slender and compact cell of stem longitudinal section. Under KNO₃ treatment, the contents of nitrate, anthocyanins, and chlorophyll in leaves, and the content of nitrate in roots of Arabidopsis overexpressing CsLBD39 were reduced, the expression levels of nitrate transport and regulation related genes were decreased. The results revealed that CsLBD39 may be involved in nitrate signal transduction in tea plants as a negative regulator and laid the groundwork for future studies into the mechanism of nitrate response.

The circadian system of plants is a complex physiological mechanism, a biological process in which plants can adjust themselves according to the day and night cycle. To understand the effects of different photoperiods on the biological clock of tea plants, we analyzed the expression levels of core clock genes (CCA1, PRR9, TOC1, ELF4) and photosynthesis-related genes (Lhcb, RbcS, atpA) under normal light (light/dark = 12 h/12 h, 12L12D) and took the cost function defined by cycle and phase errors as the basic model parameter. In the continuous light environment (24 h light, 24L), the peak activity and cycle of key genes that control the biological clock and photosynthesis were delayed by 1–2 h. Under a skeleton photoperiod (6L6D, 3L3D), the expression profiles of clock genes and photosynthesis-related genes in tea plants were changed and stomatal opening showed a circadian rhythm. These observations suggest that a skeleton photoperiod may have an effect on the circadian rhythm, photosynthetic efficiency and stomatal regulation of tea plants. Our study and model analyzed the components of circadian rhythms under different photoperiodic pathways, and also revealed the underlying mechanisms of circadian regulation of photosynthesis in tea plants.

The circadian clock refers to the formation of a certain rule in the long-term evolution of an organism, which is an invisible ‘clock’ in the body of an organism. As one of the largest TF families in higher plants, the MYB transcription factor is involved in plant growth and development. MYB is also inextricably correlated with the circadian rhythm. In this study, the transcriptome data of the tea plant ‘Baiyeyihao’ were measured at a photoperiod interval of 4 h (24 h). A total of 25,306 unigenes were obtained, including 14,615 unigenes that were annotated across 20 functional categories within the GO classification. Additionally, 10,443 single-gene clusters were annotated to 11 sublevels of metabolic pathways using KEGG. Based on the results of gene annotation and differential gene transcript analysis, 22 genes encoding MYB transcription factors were identified. The G10 group in the phylogenetic tree had 13 members, of which 5 were related to the circadian rhythm, accounting for 39%. The G1, G2, G8, G9, G15, G16, G18, G19, G20, G21 and G23 groups had no members associated with the circadian rhythm. Among the 22 differentially expressed MYB transcription factors, 3 members of LHY, RVE1 and RVE8 were core circadian rhythm genes belonging to the G10, G12 and G10 groups, respectively. Real-time fluorescence quantitative PCR was used to detect and validate the expression of the gene transcripts encoding MYB transcription factors associated with the circadian rhythm.

Theanine, a unique non-protein amino acid, is specifically accumulated in tea plants during winter. This study explored the theanine accumulation patterns in ‘Longjing 43’ and ‘Huangjinya’ under different N supply conditions and analyzed the expression of genes involved in theanine biosynthesis during winter dormancy. We found that the two tea plant cultivars shared similar theanine accumulation patterns in winter. After 30 d of cultivation with various N forms and N deficiency, the theanine content in the tissues of both cultivars was highest in the control group, followed by NH₄⁺ treatment. Furthermore, we noted that root growth of tea plants was inhibited to varying degrees under different N sources and N-deficient conditions. Gene expression analysis revealed that both N forms can induce the transcription of key genes, including CsADC, CsALT, CsCuAO, CsGDH2, CsPAO, CsNiR, CsNR, and CsTS1 in ‘Longjing 43’ and ‘Huangjinya’. The expression of these genes was strongly correlated with theanine levels under the N treatments. The winter theanine accumulation was finely tuned by the interplay of multiple related genes, with expression levels varying across different cultivars and tissues.