Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 (CsS40) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in ‘Fuding Dabai’ developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of ‘Fuding Dabai’. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar ‘Fuding Dabai’. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

The tea plant (Camellia sinensis) is an important economic crop, which is becoming increasingly popular worldwide, and is now planted in more than 50 countries. Tea green leafhopper is one of the major pests in tea plantations, which can significantly reduce the yield and quality of tea during the growth of plant. In this study, we report a genome assembly for DuyunMaojian tea plants using a combination of Oxford Nanopore Technology PromethION^™ with high-throughput chromosome conformation capture technology and used multi-omics to study how the tea plant responds to infestation with tea green leafhoppers. The final genome was 3.08 Gb. A total of 2.97 Gb of the genome was mapped to 15 pseudo-chromosomes, and 2.79 Gb of them could confirm the order and direction. The contig N50, scaffold N50 and GC content were 723.7 kb, 207.72 Mb and 38.54%, respectively. There were 2.67 Gb (86.77%) repetitive sequences, 34,896 protein-coding genes, 104 miRNAs, 261 rRNA, 669 tRNA, and 6,502 pseudogenes. A comparative genomics analysis showed that DuyunMaojian was the most closely related to Shuchazao and Yunkang 10, followed by DASZ and tea-oil tree. The multi-omics results indicated that phenylpropanoid biosynthesis, α-linolenic acid metabolism, flavonoid biosynthesis and 50 differentially expressed genes, particularly peroxidase, played important roles in response to infestation with tea green leafhoppers (Empoasca vitis Göthe). This study on the tea tree is highly significant for its role in illustrating the evolution of its genome and discovering how the tea plant responds to infestation with tea green leafhoppers will contribute to a theoretical foundation to breed tea plants resistant to insects that will ultimately result in an increase in the yield and quality of tea.

Duyun Maojian Tea mainly cultivated in Duyun City is one of the ten famous teas in China. A brown leaf spot was discovered in March 2023 in Qiannan Normal University for Nationalities' Maojian Tea experimental field (26.29°N, 107.52°E). The incidence of the disease is about 40% surveyed on about 300 m2 of tea plants. Infected leaves showed light brown, round or irregular lesions. With disease progression, they enlarged and merged, creating dark brown necrotic zones along leaf edges. To identify the responsible pathogen, three symptomatic leaves from the tea nursery base were collected. The samples were cut 3 mm x 3 mm tissues with 75% ethanol for 30 s and 5% sodium hypochlorite for 60 s, rinsing three times with sterile water. Tissues were taken to potato dextrose agar (PDA) medium and incubated dark at 28 °C for 3 days. Three isolates (TF1, TF3 and TF5) with similar morphology were obtained from three samples, respectively. The colony showed white, with short, villous hyphae expanding radially. Notably, mycelium near the disc was erect and sparse 2 cm away. After 30 days, conidia were observed. Alpha conidia (5.9 to 8.6 μm × 2.0 to 2.8 μm (n = 50)) were colorless and transparent, no septum, ellipsoid to spindle-shaped. Beta conidia (11.1 to 25.3 μm × 1.0 to 1.8 μm (n = 50)) were transparent, no septum, filiform, hooklike, tapering towards the ends. The isolates were identified as Diaporthe pescicola based on the morphology (Zhang et al. 2022). Genomic DNA was extracted, the internal transcribed spacer region (ITS), β-tubulin (tub2), and translation elongation factor 1-alpha (TEF) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively, and sequenced. The ITS (PQ163865, PQ685064 and PQ685065), tub2 (PQ197032, PV055438 and PV055439), and TEF (PQ197029, PV055440 and PV055441) sequences of isolates (TF1, TF3 and TF5) were deposited in GenBank, respectively. BLAST searches showed >97% nucleotide identity to sequences of D. pescicola MFLUCC 16-0105 (ITS, 98.40% to KU557555.1; tub2, 97.40% to KU557579.1; and TEF, 97.63% to KU557623.1). The phylogenetic tree of ITS, tub2, and TEF gene sequences was constructed using MEGA 6 software with the neighbour-joining method (NJ), and the bootstrap method was employed for 1,000 repeated tests. Isolates TF1, TF3, TF5, and D. pescicola DT 1-1 were grouped together in a node with a bootstrap value of 99. Isolates were identified as D. pescicola. Since large quantities of conidia are difficult to produce in cultures, pathogenicity on the wounded leaves of 5-year-old tea plants of Duyun Maojian Tea local population was conducted by inoculating 6 mm mycelium discs of TF1. The control group and the treatment group were inoculated with 3 tea plants, repeated 2 times. All tea plants were incubated at 25 ± 1 °C, 70% humidity, and natural daylight. The onset of symptoms appeared 7 days later. Lesions were brown, similar to field symptoms. In contrast, the control were healthy. A strain exhibiting the same morphology as TF1 was re-isolated from the diseased portion. ITS, tub2, and TEF gene sequences were consistent with TF1, completing Koch's postulates. D. pescicola as a pathogen has caused leaf spot on Kerria japonica (Zhang et al. 2022), dieback on Prunus persica (Dissanayake et al. 2017) and Vitis vinifera (Manawasinghe et al. 2019), and shoot canker on Pyrus plants (Guo et al. 2020). To our knowledge, this is the first report of tea leaf spot caused by D. pescicola in China and worldwide. It is imperative to develop and implement control measures to manage the disease.