The non-protein amino acid γ-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish endogenous GABA as a bona fide plant signal, acting via a mechanism not found in animals. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to reduce stomatal opening and transpirational water loss, which improves water use efficiency and drought tolerance, via negative regulation of a stomatal guard cell tonoplast-localised anion transporter. We find GABA modulation of stomata occurs in multiple plants, including dicot and monocot crops. This study highlights a role for GABA metabolism in fine tuning physiology and opens alternative avenues for improving plant stress resilience.

MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted. Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response. We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in ‘Yingshuang’, and 46 up-regulated miRNA and 45 down-regulated miRNAs in `Baiye 1’ in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5′RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

Purple-leaf tea plants, as anthocyanin-rich cultivars, are valuable materials for manufacturing teas with unique colors or flavors. In this study, a new purple-leaf cultivar “Zixin” (“ZX”) was examined, and its biochemical variation and mechanism of leaf color change were elucidated. The metabolomes of leaves of “ZX” at completely purple, intermediately purple, and completely green stages were analyzed using ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS). Metabolites in the flavonoid biosynthetic pathway remained at high levels in purple leaves, whereas intermediates of porphyrin and chlorophyll metabolism and carotenoid biosynthesis exhibited high levels in green leaves. In addition, fatty acid metabolism was more active in purple leaves, and steroids maintained higher levels in green leaves. Saponin, alcohol, organic acid, and terpenoid-related metabolites also changed significantly during the leaf color change process. Furthermore, the substance changes between “ZX” and “Zijuan” (a thoroughly studied purple-leaf cultivar) were also compared. The leaf color change in “Zijuan” was mainly caused by a decrease in flavonoids/anthocyanins. However, a decrease in flavonoids/anthocyanins, an enhancement of porphyrin, chlorophyll metabolism, carotenoid biosynthesis, and steroids, and a decrease in fatty acids synergistically caused the leaf color change in “ZX”. These findings will facilitate comprehensive research on the regulatory mechanisms of leaf color change in purple-leaf tea cultivars.

Internal γ-Aminobutyric Acid (GABA) interacting with stress response substances may be involved in the regulation of differentially abundant proteins (DAPs) associated with optimum temperature and cold stress in tea plants (Camellia sinensis (L.) O. Kuntze). Tea plants supplied with or without 5.0 mM GABA were subjected to optimum or cold temperatures in this study. The increased GABA level induced by exogenous GABA altered levels of stress response substances – such as glutamate, polyamines and anthocyanins – in association with improved cold tolerance. Isobaric tags for relative and absolute quantification (iTRAQ) – based DAPs were found for protein metabolism and nucleotide metabolism, energy, amino acid transport and metabolism other biological processes, inorganic ion transport and metabolism, lipid metabolism, carbohydrate transport and metabolism, biosynthesis of secondary metabolites, antioxidant and stress defense. The iTRAQ analysis could explain the GABA-induced physiological effects associated with cold tolerance in tea plants. Analysis of functional protein–protein networks further showed that alteration of endogenous GABA and stress response substances induced interactions among photosynthesis, amino acid biosynthesis, and carbon and nitrogen metabolism, and the corresponding differences could contribute to improved cold tolerance of tea plants.

Intercropping, a green and sustainable planting pattern, has demonstrated positive effects on plant growth and the soil environment. However, there is currently little research on the influence of intercropping leguminous plants and using them as green manure on the soil environment and tea quality. During the profuse flowering period of Chinese milkvetch, the contents of tea amino acids and soluble sugar in intercropping tea plants with soybean increased by 6.89 and 54.58%. Moreover, there was 27.42% increase in soil ammonium nitrogen and 21.63% increase in available nitrogen. When Chinese milkvetch was returned to soil for 1 month during its profuse flowering period, the soybean and Chinese milkvetch as green manure enhanced tea amino acids and soluble sugar by 9.11 and 33.96%, and soil ammonium nitrogen, nitrate nitrogen and available nitrogen increased by 25.04, 77.84, and 48.90%. Intercropping systems also have positive effects on tea quality components, soil fertility, and soil microbial communities during the profuse flowering period of soybeans and when soybeans with this period were returned to the field for 1 month. Furthermore, the soil fertility index was significantly increased, especially in the intercropping system of tea–soybean–Chinese milkvetch. The soil bacterial community complexity and fungal community interactions were significantly increased. Soil pH, nitrate nitrogen, and available phosphorus were found to be crucial influencing factors on soil microbial communities, specifically bacterial communities. These results highlight the significance of optimizing intercropping systems to improve the soil environment and tea quality components. They also provide a theoretical foundation for promoting the sustainable development of tea plantations.