CsAAP7.2 is involved in the uptake of amino acids from soil and the long-distance transport of theanine in tea plants (Camellia sinensis L.).

Tea plant roots can uptake both inorganic nitrogen (NH4+ and NO3-) and organic nitrogen (amino acids) from the soil. These amino acids are subsequently assimilated into theanine and transported to young shoots through the xylem. Our previous study showed that CsLHT1 and CsLHT6 transporters take up amino acids from the soil, and CsAAPs participate in the transport of theanine. However, whether other amino acid transporters are involved in this process remains unknown. In this study, we identified two new CsAAPs homologous to CsAAP7, named CsAAP7.1 and CsAAP7.2. Heterologous expression of CsAAP7.1 and CsAAP7.2 in the yeast mutant 22Δ10α showed that CsAAP7.2 had the capacity to transport theanine and other amino acids, whereas CsAAP7.1 had no transport activity. Transient expression of the CsAAP7.2-GFP fusion protein in tobacco leaf epidermal cells confirmed its localization to the endoplasmic reticulum. Tissue-specific analysis showed that CsAAP7.2 was highly expressed in roots and stems. In addition, CsAAP7.2 overexpression lines were more sensitive to high concentrations of theanine due to the high accumulation of theanine in seedlings. Taken together, these findings suggested that CsAAP7.2 plays an important role in the uptake of amino acids from soil and the long-distance transport of theanine. These results provide valuable tools for nitrogen nutrition studies and enrich our understanding of theanine transport in tea plants.

Distribution of trace metals in a soil-tea leaves-tea infusion system: characteristics, translocation and health risk assessment.

The effects of metal pollution on tea are of great concern to consumers. We apply Geographic information systems technology to study the distribution of heavy metal elements in tea plantation ecosystems in Jiangsu Province, explore the relationships among metals in the soil, tea leaves and tea infusions, and assess the human safety risks of metals. The concentrations of nine metals in a soil-tea leaves-tea infusion system were studied at 100 randomly selected tea plantations in Jiangsu Province, China. Concentrations of selected metals, zinc (Zn), nickel (Ni), manganese (Mn), chromium (Cr) and copper (Cu), were quantified using an inductively coupled plasma-optical emission spectrometer (ICP-OES), and cadmium (Cd), arsenic (As), plumbum (Pb) and mercury (Hg) were quantified using inductively coupled plasma-mass spectrometry (ICP-MS). Arc-Map 10.3 was used for the spatial analysis of metals in soil, tea leaves and tea infusions. We found that the contents of Mn, Ni and Zn are high level in soil, tea leaves and tea infusions. The Mn level showed a spatial distribution pattern with greater concentrations at the junction of Nanjing and Yangzhou, southwest of Changzhou and west of Suzhou. The hazard index (HI) values in north-central Nanjing, southern Suzhou, southwestern Changzhou and northern Lianyungang were relatively greater. The Zn, Ni, Mn, Cr and Cu levels in the soil-tea infusion system were 17.3, 45.5, 54.5, 1.5 and 14.3%, respectively. The order of the leaching rates of the elements was Ni > Cr > Zn > Mn > Cu. The relative contribution ratios of HI were in the order of Mn > Ni > Cu > Zn > Cr > Pb > Cd > As > Hg. In tea infusions, the Mn level has the greatest potential health risks to consumers. Moreover, using Csoil it was inferred that the safety thresholds of Zn, Ni, Mn, Cr and Cu in soil were 27,700, 50, 1230, 493,000 and 16,800 mg L-1, respectively. The content of heavy metals in soil and tea varies greatly in different regions of Jiangsu Province, 92% of the soil has heavy metal content that meets the requirements of pollution-free tea gardens, 91% of tea samples met the requirements of green food tea. The thresholds for Ni (50 mg L-1) and Mn (1230 mg L-1) can be used as maximum limits in tea plantation soils. The consumption of tea infusions did not pose metal-related risks to human health.

Tea plantation intercropping green manure enhances soil functional microbial abundance and multifunctionality resistance to drying-rewetting cycles.

Climate change leads to more serious drying-rewetting alternation disturbance, which furtherly affects soil ecosystem function and agriculture production. Intercropping green manure, as an ancient agricultural practice, can improve the physical, chemical, and biological fertility of soil in tea plantation. However, the effects of intercropping green manure on soil multifunctional resistance to drying-rewetting disturbance in tea plantation has not been reported. In this study, the effects of different green manure practices over four years (tea plant monoculture, tea plant and soybean intercropping, tea plant and soybean + milk vetch intercropping) on soil multifunctionality resistance to drying-rewetting cycles, and the pivotal influencing factors were investigated. We used quantitative PCR array and analysis of multiple enzyme activities to characterize the abundance of functional genes and ecosystem multifunctionality, respectively. Compared with tea plantation monoculture, tea plant intercropping soybean and soybean + milk vetch significantly increased multifunctionality resistance by 12.07% and 25.86%, respectively. Random forest analysis indicated that rather than the diversity, the abundance of functional genes was the major drive of multifunctionality resistance. The structure equation model further proved that tea plantation intercropping green manure could improve the abundance of C cycling related functional genes mediated by soil properties, and ultimately increased multifunctionality resistance to drying-rewetting disturbance. Therefore, tea plantation intercropping green manure is an effective approach to maintain the multifunctionality resistance, which is conducive to maintain the soil nutrient supply capacity and tea production under the disturbance of drying-rewetting alternation.

The effects of tea plants-soybean intercropping on the secondary metabolites of tea plants by metabolomics analysis.

BACKGROUND: Intercropping, especially with legumes, as a productive and sustainable system, can promote plants growth and improves the soil quality than the sole crop, is an essential cultivation pattern in modern agricultural systems. However, the metabolic changes of secondary metabolites and the growth in tea plants during the processing of intercropping with soybean have not been fully analyzed. RESULTS: The secondary metabolomic of the tea plants were significant influence with intercropping soybean during the different growth stages. Especially in the profuse flowering stage of intercropping soybean, the biosynthesis of amino acids was significantly impacted, and the flavonoid biosynthesis, the flavone and flavonol biosynthesis also were changed. And the expression of metabolites associated with amino acids metabolism, particularly glutamate, glutamine, lysine and arginine were up-regulated, while the expression of the sucrose and D-Glucose-6P were down-regulated. Furthermore, the chlorophyll photosynthetic parameters and the photosynthetic activity of tea plants were higher in the tea plants-soybean intercropping system. CONCLUSIONS: These results strengthen our understanding of the metabolic mechanisms in tea plant's secondary metabolites under the tea plants-soybean intercropping system and demonstrate that the intercropping system of leguminous crops is greatly potential to improve tea quality. These may provide the basis for reducing the application of nitrogen fertilizer and improve the ecosystem in tea plantations.

GABA shunt contribution to flavonoid biosynthesis and metabolism in tea plants (Camellia sinensis).

γ-Aminobutyric acid (GABA), a signal molecule, is regarded as the intersection node of carbon and nitrogen metabolism, and its contributions to flavonoid metabolism in tea plant growth and development remain unclear. The correlation between the GABA shunt and flavonoid metabolism in tea plants is worth to explore. Secondary metabolites and their correlations with the taste of tea soup made from tea plants (Camellia sinensis) during different seasons were investigated. Related secondary metabolites and transcript profiles of genes encoding enzymes in the GABA shunt, flavonoid pathway and polyamine biosynthesis were measured throughout the tea plant growth seasons and after exogenous GABA applications. In addition, the abundance of differentially expressed proteins was quantified after treatments with or without exogenous GABA. The tea leaves showed the highest metabolite concentrations in spring season. CsGAD, CsGABAT, CsSPMS, CsODC, CsF3H and CsCHS were found to be important genes in the GABA and anthocyanin biosynthesis pathways. GABA and anthocyanin concentrations showed a positive correlation, to some extent, CsF3H and CsCHS played important roles in the GABA and anthocyanin biosynthesis.

Metabolomic Analyses Provide New Insights into Signaling Mechanisms for Nutrient Uptake by Lateral Roots of Pruned Tea Plant (Camellia sinensis).

Pruning is an important plant management practice in tea cultivation. However, the mechanism underlying the dynamics of nutrient uptake by roots of pruned tea is unknown. This study investigated the metabolic alterations in lateral roots of pruned tea to unveil the mechanism of nutrient uptake. Elemental analysis revealed that pruning significantly increases the uptake of nutrients by lateral roots. Metabolic profiling showed significant metabolic variations in lateral roots of pruned tea. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that flavonoid biosynthesis, phenylpropanoid biosynthesis, and amino acid metabolism were differentially regulated in lateral roots. Caffeine metabolism was significantly hindered, while ethylene signaling was significantly induced in lateral roots of pruned plants. In addition, intermediates in the tricarboxylic acid (TCA) cycle were upregulated, indicating high rates of the TCA cycle. Therefore, pathways related to phenylpropanoid biosynthesis, TCA cycle, ethylene biosynthesis, and metabolism of amino acids contribute to higher nutrient uptake by lateral roots of the tea plant.

A novel insight into nitrogen and auxin signaling in lateral root formation in tea plant [Camellia sinensis (L.) O. Kuntze].

BACKGROUND: Tea plant (Camellia sinensis) is one of the most popular non-alcoholic beverages worldwide. In tea, lateral roots (LRs) are the main organ responsible for the absorption of moisture and mineral nutrients from the soil. Lateral roots formation and development are regulated by the nitrogen and auxin signaling pathways. In order to understand the role of auxin and nitrogen signaling in LRs formation and development, transcriptome analysis was employed to investigate the differentially expressed genes involved in lateral roots of tea plants treated with indole-3-butyric acid (IBA), N-1-naphthylphthalamic acid (NPA), low and high concentrations of nitrogen. RESULTS: A total of 296 common differentially expressed genes were identified and annotated to four signaling pathways, including nitrogen metabolism, plant hormone signal transduction, glutathione metabolism and transcription factors. RNA-sequencing results revealed that majority of differentially expressed genes play important roles in nitrogen metabolism and hormonal signal transduction. Low nitrogen condition induced the biosynthesis of auxin and accumulation of transcripts, thereby, regulating lateral roots formation. Furthermore, metabolism of cytokinin and ethylene biosynthesis were also involved in lateral roots development. Transcription factors like MYB genes also contributed to lateral roots formation of tea plants through secondary cell wall biosynthesis. Reversed phase ultra performance liquid chromatography (RP-UPLC) results showed that the auxin concentration increased with the decreased nitrogen level in lateral roots. Thus, tea plant lateral roots formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation. CONCLUSION: This study provided insights into the mechanisms associated with nitrogen and auxin signaling pathways in LRs formation and provides information on the efficient utilization of nitrogen in tea plant at the genetic level.

Genome-wide identification, characterization and expression analysis of the amino acid permease gene family in tea plants (Camellia sinensis).

Amino acid permeases (AAPs) are involved in transporting a broad spectrum of amino acids and regulating physiological processes in plants. In this study, 19 AAP genes were identified from the tea plants genome database and named CsAAP1-19. Based on phylogenetic analysis, the CsAAP genes were classified into three groups, having significantly different structures and conserved motifs. In addition, an expression analysis revealed that most of CsAAP genes were specifically expressed in different tissues, especially CsAAP19 was expressed only in root. These genes also were significantly expressed in the Baiye 1 and Huangjinya cultivars. Nitrogen treatments indicated that the CsAAPs were obviously expressed in root. CsAAP2, -6, -12, -13 and - 16 were significantly expressed at 6 d after the glutamate treatment, while the expression trend at 24 h after contained the ammonium. These results improve our understanding of the CsAAP genes and their functions in nitrogen utilization in tea plants.

Genome-Wide Analysis Reveals Stress and Hormone Responsive Patterns of JAZ Family Genes in Camellia Sinensis.

JAZ (Jasmonate ZIM-domain) proteins play pervasive roles in plant development and defense reaction. However, limited information is known about the JAZ family in Camellia sinensis. In this study, 12 non-redundant JAZ genes were identified from the tea plant genome database. Phylogenetic analysis showed that the 12 JAZ proteins belong to three groups. The cis-elements in promoters of CsJAZ genes and CsJAZ proteins interaction networks were also analyzed. Quantitative RT-PCR analysis showed that 7 CsJAZ genes were preferentially expressed in roots. Furthermore, the CsJAZ expressions were differentially induced by cold, heat, polyethylene glycol (PEG), methyl jasmonate (MeJA), and gibberellin (GA) stimuli. The Pearson correlations analysis based on expression levels showed that the CsJAZ gene pairs were differentially expressed under different stresses, indicating that CsJAZs might exhibit synergistic effects in response to various stresses. Subcellular localization assay demonstrated that CsJAZ3, CsJAZ10, and CsJAZ11 fused proteins were localized in the cell nucleus. Additionally, the overexpression of CsJAZ3, CsJAZ10, and CsJAZ11 in E. coli enhanced the growth of recombinant cells under abiotic stresses. In summary, this study will facilitate the understanding of the CsJAZ family in Camellia sinensis and provide new insights into the molecular mechanism of tea plant response to abiotic stresses and hormonal stimuli.

Transcriptomic analysis between self- and cross-pollinated pistils of tea plants (Camellia sinensis).

BACKGROUND: Self-incompatibility (SI) is a major barrier that obstructs the breeding process in most horticultural plants including tea plants (Camellia sinensis). The aim of this study was to elucidate the molecular mechanism of SI in tea plants through a high throughput transcriptome analysis. RESULTS: In this study, the transcriptomes of self- and cross-pollinated pistils of two tea cultivars 'Fudingdabai' and 'Yulv' were compared to elucidate the SI mechanism of tea plants. In addition, the ion components and pollen tube growth in self- and cross-pollinated pistils were investigated. Our results revealed that both cultivars had similar pollen activities and cross-pollination could promote the pollen tube growth. In tea pistils, the highest ion content was potassium (K+), followed by calcium (Ca2+), magnesium (Mg2+) and phosphorus (P5+). Ca2+ content increased after self-pollination but decreased after cross-pollination, while K+ showed reverse trend with Ca2+. A total of 990 and 3 common differentially expressed genes (DEGs) were identified in un-pollinated vs. pollinated pistils and self- vs. cross-pollinated groups after 48 h, respectively. Function annotation indicated that three genes encoding UDP-glycosyltransferase 74B1 (UGT74B1), Mitochondrial calcium uniporter protein 2 (MCU2) and G-type lectin S-receptor-like serine/threonine-protein kinase (G-type RLK) might play important roles during SI process in tea plants. CONCLUSION: Ca2+ and K+ are important signal for SI in tea plants, and three genes including UGT74B1, MCU2 and G-type RLK play essential roles during SI signal transduction.

The CsHSP17.2 molecular chaperone is essential for thermotolerance in Camellia sinensis.

Small heat shock proteins (sHSPs) play important roles in responses to heat stress. However, the functions of sHSPs in tea plants (Camellia sinensis) remain uncharacterized. A novel sHSP gene, designated CsHSP17.2, was isolated from tea plants. Subcellular localization analyses indicated that the CsHSP17.2 protein was present in the cytosol and the nucleus. CsHSP17.2 expression was significantly up-regulated by heat stress but was unaffected by low temperature. The CsHSP17.2 transcript levels increased following salt and polyethylene glycol 6000 treatments but decreased in the presence of abscisic acid. The molecular chaperone activity of CsHSP17.2 was demonstrated in vitro. Transgenic Escherichia coli and Pichia pastoris expressing CsHSP17.2 exhibited enhanced thermotolerance. The transgenic Arabidopsis thaliana exhibited higher maximum photochemical efficiencies, greater soluble protein proline contents, higher germination rates and higher hypocotyl elongation length than the wild-type controls. The expression levels of several HS-responsive genes increased in transgenic A. thaliana plants. Additionally, the CsHSP17.2 promoter is highly responsive to high-temperature stress in A. thaliana. Our results suggest that CsHSP17.2 may act as a molecular chaperone to mediate heat tolerance by maintaining maximum photochemical efficiency and protein synthesis, enhancing the scavenging of reactive oxygen species and inducing the expression of HS-responsive genes.

Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana.

KEY MESSAGE: CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.

Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis.

BACKGROUND: 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. RESULT: 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. CONCLUSIONS: 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.