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.

Nε-Acetylation of lysine residues, a frequently occurring post-translational modification, plays important functions in regulating physiology and metabolism. However, the information of global overview of protein acetylome under nitrogen-starvation/resupply in tea (Camellia sinensis) leaves was limited. And the full function of lysine acetylated proteins of tea plants in nitrogen absorption and assimilation remains unclear. Here, we performed the global review of lysine acetylome in tea leaves under nitrogen (N)-starvation/resupply, using peptide prefractionation, immunoaffinity enrichment, and coupling with high sensitive LC-MS/MS combined with affinity purification analysis. Altogether, 2229 lysine acetylation sites on 1286 proteins were identified, of which 16 conserved motifs in E*KacK, Kac*K, Kac*R, Kac*HK, Kac*N, Kac*S, Kac*T, Kac*D, were extracted from 2180 acetylated peptides. Approximately, 36.76% of the acetylated lysines were located in the regions of ordered secondary structures. The most of the identified lysine acetylation proteins were located in the chloroplast (39%) and cytoplasm (29%). The largest group of acetylated proteins consisted of many enzymes, such as ATP synthase, ribosomal proteins and malate dehydrogenase [NADP], which were related to metabolism (38%) in the biological process. These acetylated proteins were mainly enriched in three primary protein complexes of photosynthesis: photosystem I, photosystem II and the cytochrome b6/f complex. And some acetylated proteins related to glycolysis and secondary metabolite biosynthesis were increased/decreased under N-resupply. Moreover, the PPI (protein-protein interaction) analysis revealed that the diverse interactions of identified acetylated proteins mainly involved in photosynthesis and ribosome. The results suggested that lysine acetylated proteins might play regulating roles in metabolic process in tea leaves. The critical regulatory roles mainly involved in diverse aspects of metabolic processes, especially in photosynthesis, glycolysis and secondary metabolism. A lot of proteins related to the photosynthesis and glycolysis were found to be acetylated, including LHCA1, LHCA3, LHCB6, psaE, psaD, psaN, GAPDH, PEPC, ENL and petC. And some proteins related to flavonoids were also found to be acetylated, including PAL, DFR, naringenin 3-dioxygenase and CHI. The provided data may serve as important resources for exploring the physiological, biochemical, and genetic role of lysine acetylation in tea plants. Data are available via ProteomeXchange with identifier PXD008931.

The positive aspects of the tea plant/legume intercropping system draw attention to the Chinese tea industry for its benefit for soil fertility improvement with low fertilizer input. However, limited information exists as to the roles of intercropped legumes in the rhizosphere microbiome and tea quality. Hereby, soybean was selected as the intercropped plant to investigate its effect on bacterial communities, nutrient competition, tea plant development, and tea quality. Our data showed that intercropped soybean boosted the uptake of nitrogen in tea plants and enhanced the growth of young tea shoots. Nutrient competition for phosphorus and potassium in soil existed between soybeans and tea plants. Moreover, tea/soybean intercropping improved tea quality, manifested by a significantly increased content of non-ester type catechins (C, EGC, EC), total catechins and theanine, and decreased content of ester type catechins (EGCG). Significant differences in rhizobacterial composition were also observed under different systems. At the genus level, the relative abundance of beneficial bacteria, such as Bradyrhizobium, Saccharimonadales and Mycobacterium, was significantly increased with the intercropping system, while the relative abundance of denitrifying bacteria, Pseudogulbenkiania, was markedly decreased. Correlation analysis showed that Pseudogulbenkiania, SBR1031, and Burkholderiaceae clustered together showing a similar correlation with soil physicochemical and tea quality characteristics; however, other differential bacteria showed the opposite pattern. In conclusion, tea/soybean intercropping improves tea quality and nutrition uptake by increasing the relative abundance of beneficial rhizosphere bacteria and decreasing denitrifying bacteria. This study strengthens our understanding of how intercropping system regulate the soil bacterial community to maintain the health of soils in tea plantations and provides the basis for replacing chemical fertilizers and improving the ecosystem in tea plantations.

Freezing damage has been a common natural disaster for tea plantations. Quantitative detection of low temperature stress is significant for evaluating the degree of freezing injury to tea plants. Traditionally, the determination of physicochemical parameters of tea leaves and the investigation of freezing damage phenotype are the main approaches to detect the low temperature stress. However, these methods are time-consuming and laborious. In this study, different low temperature treatments were carried out on tea plants. The low temperature response index (LTRI) was established by measuring seven low temperature-induced components of tea leaves. The hyperspectral data of tea leaves was obtained by hyperspectral imaging and the feature bands were screened by successive projections algorithm (SPA), competitive adaptive reweighted sampling (CARS) and uninformative variable elimination (UVE). The LTRI and seven indexes of tea plant were modeled by partial least squares (PLS), support vector machine (SVM), random forests (RF), back propagation (BP) machine learning methods and convolutional neural networks (CNN), long short-term memory (LSTM) deep learning methods. The results indicated that: (1) the best prediction model for the seven indicators was LTRI-UVE-CNN (R^{2 =} 0.890, RMSEP=0.325, RPD=2.904); (2) the feature bands screened by UVE algorithm were more abundant, and the later modeling effect was better than CARS and SPA algorithm; (3) comparing the effects of the six modeling algorithms, the overall modeling effect of the CNN model was better than other models. It can be concluded that out of all the combined models in this paper, the LTRI-UVE-CNN was a promising model for predicting the degree of low temperature stress in tea plants.

Traditionally, the supplement of organic manure in tea plantations has been a common approach to improving soil fertility and promoting terroir compounds, as manifested by the coordinated increase in yield and quality for the resulting teas. However, information regarding the effect of organic manure in the metabolome of tea plants is still inadequate. The metabolite profiles of tea shoots applied with cow manure, urea or no fertilizer were studied using gas chromatography–mass spectrometry (GC–MS). In total, 73 metabolites were detected, and the modulated metabolites included mainly amino acids, organic acids and fatty acids. In particular, glutamine, quinic acid and proline accumulated more in tea shoots in soils treated with cow manure, but octadecanoic acid, hexadecanoic acid and eicosanoic acid were drastically reduced. Pearson correlation analysis indicated that organic acids and amino acids in tea shoots were the two major metabolite groups among the three treatments. The analysis of metabolic pathways demonstrated that the cow manure treatment significantly changed the enrichment of pathways related to amino acids, sugars and fatty acids. Sensory evaluation showed that the quality of green teas was higher when the plants used to make the tea were grown in soil treated with cow manure rather than urea during spring and late summer. The results indicated that the application of cow manure in soils changed the metabolic characteristics of tea shoots and improved the qualities of the resulting teas.

Aroma substances are the most crucial criteria for the sensory evaluation of tea quality, and also key attractors influencing consumers to make the decision for purchasing tea. Understanding the aromatic properties of tea infusion during different brewing time is crucial to control the tea aromatic quality. Here, headspace and direct immersion solid-phase microextraction (HS-SPME and DI-SPME), coupled with GC-MS, were employed to investigate the impact of brewing time on the changes of the volatile features of green tea infusion. Esters, aldehydes, alcohols, fatty acids, and alkaloids were the predominant volatile groups from tea infusions. Two to three minutes was identified as the best duration for the tea brewing that can maximize the abundance of aromatic chemicals in the headspace emitted from the tea infusions. The variation of the key aromatic contributors between the tea infusion and the headspace over the infusion tended to equilibrate during the tea brewing process. This study provides a theory-based reference method by analyzing the real-time aromatic characteristics in green tea. The optimal time was determined for aromatic quality control, and the complementary relationship between the volatiles in the headspace and its counterpart, tea infusion, was primarily elucidated.