What if a tea plant’s aging program also controls how much caffeine its leaves contain? This study identifies CsS40, a senescence-related transcription factor linked to lower TCS1 expression and reduced caffeine accumulation. Alkaloids are significant secondary metabolites in plants, with biological activities in medicines and defensive roles against herbivores and pathogens.

Caffeine is one of these alkaloids and is used as a neural stimulant. Caffeine accounts for two to five percent of tea dry weight and one to two point seven percent of coffee dry weight. It also protects plants against fungal and bacterial pathogens or herbivores and can serve as a signaling molecule.

Caffeine is synthesized in young plant tissues, predominantly in flowers, young leaves, and seeds. In tea, the major biosynthetic route is indicated to come from adenine. In the common caffeine-specific pathway, xanthosine becomes seven-methylxanthosine, seven-methylxanthine, theobromine, and finally caffeine.

N-methyltransferases facilitate methyl-group transfer to xanthine backbones. Tea caffeine synthase one, or TCS1, is a key enzyme that catalyzes both theobromine synthase and caffeine synthase reactions. Silencing TCS1 drastically reduced theobromine and caffeine contents.

These results indicate that TCS1 is crucial for caffeine biosynthesis, and manipulating TCS1 may help create tea plant variants with differing caffeine contents. Caffeine biosynthesis is tightly regulated at transcriptional and posttranscriptional levels in a spatiotemporal manner, and environmental and hormonal cues are involved through transcription factors.

Although several transcription factor families had been proposed to target TCS1, only one NAC and one MYB factor had been characterized in tea plants. More transcription factors therefore needed to be identified. To identify transcription factors controlling TCS1, a tea leaf protein expression library was constructed in the pGADT7 yeast expression library system for a yeast-one-hybrid screen of TCS1 promoter activators.

The library’s calculated capacity was four point four four times ten to the fourth colony-forming units. The TCS1 promoter fragment from the Fudingdabai genome was cloned into pHis2 as the bait construct. The bait did not display self-activation activity in the presence of three-amino-one, two, four-triazole.

The screening medium therefore contained fifty millimolar three-amino-one, two, four-triazole. Figure two documents the yeast-one-hybrid screening setup for finding transcription factors that activate the CsTCS1 promoter. Panel a shows library screening counts of seven hundred ninety-eight, two hundred eighty-two, and forty-seven colonies across tenfold, one-hundredfold, and one-thousandfold dilutions, supporting a calculated library capacity of four point four four times ten to the fourth colony-forming units.

Panel b tests promoter self-activation with zero, fifty, and one hundred nanomolar 3-AT, while panel c identifies seventeen candidate colonies for follow-up under selection conditions. The positive clones were sequenced and compared against the NCBI and GenBank databases.

Nine positive clones represented genes encoding nine different proteins, and XP underscore zero two eight zero seven five four eight six point one was a putative TCS1-promoter transcription factor. XP underscore zero two eight zero seven five four eight six point one was one thousand one hundred fifty-eight base pairs long and encoded a two-hundred-twelve-amino-acid polypeptide with a molecular weight of twenty-three point thirty-seven kilodaltons.

The protein had a conserved Senescence-reg S40 domain and was annotated as an uncharacterized Camellia sinensis protein. Because no related tea-plant studies were known, the gene was named CsS40. To verify CsS40’s activation function, the CsS40 coding sequence was inserted into pGADT7 and introduced into Y187 yeast together with the pHis2-pro-TCS1 bait.

Positive clones were grown on medium supplemented with three-amino-one, two, four-triazole at fifty, one hundred, one hundred fifty, two hundred, and two hundred fifty millimolar. Most clones grew well, indicating that CsS40 can bind and activate the TCS1 promoter.

Figure three validates CsS40 as a transcriptional regulator of TCS1. In yeast, CsS40 supported growth with the TCS1 promoter across three-AT selection levels, while the negative control did not; split-luciferase imaging and quantification likewise show signal when Pro-TCS1 and CsS40 are co-expressed.

The promoter contains multiple cis-acting elements, and EMSA testing across fragments P1 through P7 further examines direct binding, linking CsS40 to regulation of TCS1 expression. In the split-luciferase assay, the TCS1 promoter was fused to the amino-terminal luciferase fragment and CsS40 to the carboxy-terminal fragment, then both constructs were expressed in Nicotiana benthamiana leaves.

Luciferase activity appeared when Pro-TCS1-nLUC and CsS40 were co-expressed, but not with the negative-control pGreenII sixty-two-SK. These results indicate a regulatory effect on TCS1-promoter activation. For EMSA, purified CsS40 protein was incubated with biotin-labeled TCS1-promoter fragments.

Protein-bound fragments migrated more slowly, and binding was observed for fragments P1, P2, P4, P6, and P7. Figure four first localizes CsS40: the CsS40–GFP fusion appears in the nucleus and cell membrane, unlike the more diffuse GFP control. In tobacco, RT-qPCR confirms CsS40 expression in the overexpression lines, while HPLC measures altered xanthine and hypoxanthine accumulation.

The lower panels extend the test through VIGS: CsS40 transcript levels are reduced in transformed seedlings, alongside measurements of caffeine content, linking CsS40 activity to caffeine-related metabolism. Tobacco leaf disks were infected with Agrobacterium carrying either the CsS40 overexpression plasmid or an empty-vector control, and resistant positive plants were selected using kanamycin resistance.

The CsS40-overexpressing tobacco lines were named NtpSH, while the empty-vector lines were called CK. CsS40 expression in NtpSH was two point six six times higher than in CK plants. HPLC detected no caffeine in transgenic tobacco leaves, but it measured xanthine and hypoxanthine.

NtpSH plants contained one hundred fifty-one point zero one micrograms per gram of xanthine and fourteen point six seven micrograms per gram of hypoxanthine. To test CsS40 in tea, CsS40 transcription was repressed in Fuding Dabaicha leaf tissues using VIGS.

CsS40 expression was assessed by RT-qPCR, and caffeine content was measured by HPLC. Compared with wild-type plants and empty-vector controls, CsS40 expression in PTRV2-CsS40 leaves decreased to thirty-six point one nine percent of the control level, while caffeine content became one point two nine times higher.

These data demonstrated that silencing CsS40 repressed caffeine formation in Fuding Dabaicha, supporting CsS40 as a negative regulator of caffeine biosynthesis in tea plants. Figure five tests CsS40 function by comparing tea calli with CsS40 overexpressed or silenced, alongside GUS staining to verify transformation.

RT-qPCR shows that overexpression raises CsS40 expression to an average of four point seven one times the wild-type level, while TCS1 falls to fifty-four point three nine percent; the caffeine measurements in panel c extend this comparison to metabolite accumulation.

Together, the figure links CsS40 regulation with TCS1 expression and caffeine content in tea calli. In CsS40-overexpressing CspSH calli, CsS40 expression was four point seven one times higher than in wild-type calli, while TCS1 expression was fifty-four point three nine percent of wild type.

Overexpression therefore repressed TCS1 expression. In CsS40-silenced CspBWA calli, TCS1 expression was two point four six times higher and CsS40 expression was forty-one point two two percent of wild type. Silencing increased CsTCS1 expression.

CspSH calli contained one thousand four hundred fifty-three micrograms per gram of caffeine, compared with two thousand two hundred eighty-five micrograms per gram in wild type. CspBWA calli reached five thousand one hundred twenty-three micrograms per gram.

The callus results therefore show that CsS40 overexpression inhibited TCS1 expression and caffeine accumulation, while CsS40 silencing promoted TCS1 expression and increased caffeine accumulation. The S40 gene family has been shown to play a role in plant senescence. Fuding Dabaicha leaves were grouped as young, mature, and senescent, then analyzed by HPLC for caffeine accumulation.

As tea-leaf senescence increased, caffeine content gradually decreased. CsS40 expression levels in young, mature, and senescent leaves were twenty-three point eight two, forty-nine point three five, and one hundred twenty-four point one three.

The increasing CsS40 expression level was clearly correlated with tea-leaf aging. The resulting interpretation was that CsS40 may regulate caffeine content in close relation to tea-plant senescence. Figure six links CsS40 expression with leaf senescence in tea and tobacco.

In ‘Fuding Dabaicha’, CsS40 expression rises from young to senescent leaves, while caffeine content declines; in tobacco, CsS40-overexpressing leaves show visible yellowing after incubation. Following ethrel treatment, the figure compares chlorophyll, SOD-like activity, malondialdehyde, and LOX activity between transgenic and control leaves, providing physiological measurements of the senescence response.

CsS40 transcription was up-regulated by jasmonic acid, abscisic acid, gibberellin, and ethylene. CsS40 overexpression made tobacco leaves more susceptible to aging under darkness or ethylene-generator treatments. CsS40-overexpression tobacco plants also had higher xanthine and hypoxanthine levels than controls, indicating that CsS40 may also be involved in nitrogen metabolism.

Caffeine content in tea leaves gradually decreased during maturation, while CsS40 expression increased significantly. These patterns were consistent with CsS40 negatively regulating TCS1 expression in tea callus and caffeine accumulation in tea leaves. The proposed interpretation is that CsS40 may regulate tea-leaf maturation by regulating caffeine accumulation.

Yeast one-hybrid screening identified an unknown transcription factor that bound the TCS1 promoter and was identified as a S40 family protein in tea plants. CsS40 was further characterized through expression analysis, promoter-binding assays, VIGS in tea leaves, and overexpression and silencing in transgenic tea leaf calli.

In transgenic tobacco plants overexpressing CsS40, CsS40 was also confirmed to be involved in leaf senescence. Tea-leaf expression patterns connected CsS40 with TCS1 expression, caffeine accumulation, and leaf maturation. The study provides new insights into the molecular mechanisms underlying caffeine accumulation and its role related to leaf maturation and responses to abiotic stress in tea plants.

CsS40 is connected to leaf senescence and negatively regulates TCS1 and caffeine accumulation in tea. That links caffeine biosynthesis to leaf maturation, hormone signaling, and senescence.