It is essential for tea plants (Camellia sinensis) to undergo bud endodormancy in order to survive winter and grow again the following spring. Molecular pathways underlying the release and break of endodormancy, however, remain unclear. Accessible chromatin regions play a crucial role in precise gene expression regulation. Here, we profiled the chromatin accessibility and transcriptional features of transcription factors (TFs) using ATAC- and RNA-seq technologies. We revealed dynamic changes during bud dormancy break in tea plants and detected open chromatin regions and TF binding sites. Numerous transcripts associated with open chromatin regions were found to exhibit dormant-preferential expression among TF-target genes. We focused on several crucial TF motifs, including MADS-box, AP2EREBP, bZIP, MYB, bHLH, E2FDP, REMB3, TCP, and G2-like factors, that were enriched in open chromatin regions at different budbreak stages, and predicted networks of TF-target genes. Then, we used qRT-PCR to investigate the expression patterns of budbreak-related genes.We found a set of differentially regulated peaks of MADS-box TFs and related genes enriched in plant hormone signal transduction pathway, photosynthesis, secondary metabolism, plant-pathogen interaction functions. Correlation analysis with the phytohormones abscisic acid and indole-3-acetic acid also provided details of the regulation network. Moreover, we identified the core TFs (SVP, CO, AP1, SOC, ABI, CBF, FRUITFULL, and ULTRAPETALA) of several pathways related to budbreak, providing the guideposts for future study.

C-repeat binding factors/dehydration responsive element binding factors 1 (CBFs/DREB1s) are a small family of transcription factors that play important roles in plant resistance to various external stresses. However, functional characterization of tea plant (Camellia sinensis L.) CBF gene (CsCBF) was still seldom reported. Here, functional study of the cold-responsive CBF gene (CsCBF2) was done. Results showed that CsCBF2 had conserved AP2 DNA-binding domain and the typical PKK/RPAGRxKFxETRHP and DSAWR signature sequences of CBF/DREB1. Yeast one-hybrid and transcription activation assays revealed that the activation domain of CsCBF2 could activate the reporter gene expression, and the N terminal of CsCBF2 displayed an inhibitory effect. Although CsCBF2 was conserved to bind the C-repeat/dehydration-responsive element (CRT/DRE), intact CsCBF2 protein preferred the CRT cis element. Under normal growth conditions, CsCBF2-overexpressing tobacco plants (CsCBF2-OX) exhibited lighter green leaf color, growth retardation, and dwarfism. Smaller leaf of CsCBF2-OX was only seen in eight weeks after been sown in soil. Under cold, salinity, or drought stress, CsCBF2-OX displayed better growing with longer roots, heavier fresh weight, higher germination rate, and accumulated more proline and sugar contents, but lower electrolyte leakage. The results demonstrated that CsCBF2 enhanced plant tolerance to multiple abiotic stresses.

Tea plant (Camellia sinensis) is an important economic beverage crop. Drought stress seriously affects the growth and development of tea plant and the accumulation of metabolites, as well as the production, processing, yield and quality of tea. Therefore, it is necessary to understand the reaction mechanism of tea plant under drought conditions and find efficient control methods. Based on transcriptome sequencing technology, this study studied the difference of metabolic level between sexual and asexual tea plants under drought stress. In this study, there were multiple levels of up-regulation and down-regulation of differential genes related to cell composition, molecular function and biological processes. Transcriptomic data show that the metabolism of tea plants with different propagation modes of QC and ZZ is different under drought conditions. In the expression difference statistics, it can be seen that the differential genes of QC are significantly more than ZZ; GO enrichment analysis also found that although differential genes in biological process are mainly enriched in the three pathways of metabolic, single organism process and cellular process, cellular component is mainly enriched in cell, cell part, membrane, and molecular function, and binding, catalytic activity, and transporter activity; the enrichment order of differential genes in these pathways is different in QC and ZZ. This difference is caused by the way of reproduction. The further study of these differential genes will lay a foundation for the cultivation methods and biotechnology breeding to improve the quality of tea.

[目的]明确陕西省茶树种质资源的遗传多样性,为茶树育种提供依据.[方法]以陕西地方和野生种质资源、陕西临近省份以及西南茶区共8个省份的118份茶树资源为材料,利用43对高质量EST-SSR引物,用PROSize 2.0软件统计扩增条带,用Powermarker V3.25分析软件计算主等位基因频率(MAF)、基因型、等位基因数(Na)、观测杂合度(Ho)、Nei基因多样性指数(H)和多态信息含量(PIC),用MEGA 6软件进行UPGMA聚类分析,对陕西茶树资源的遗传多样性进行分析.[结果]从118份茶树材料中共检测到310个等位基因和832个基因型.主等位基因频率(MAF)、每个位点等位基因数(N.)、扩增基因型和多态信息含量(PIC)的平均值分别为0.3368,7.21个,19.35和0.7272.Nei基因多样性指数(H)在0.2323～0.9110,平均值为0.7600;观察到的杂合度(Ho)在0.1057～0.9918,平均值为0.8112.聚类分析结果将陕西省茶树种质资源分为3大类群,同一地区的陕西茶树资源多聚在一起;对不同地区茶树资源的聚类结果表明,陕西省茶树资源聚为一支,且与湖北的茶树资源关系较近.[结论]陕西省茶树资源具有丰富的遗传多样性和相对独立的遗传背景,且与湖北省茶树资源遗传关系较近.

[目的]探究不同氮素形态对茶树叶片品质及其氮代谢相关基因的影响,为茶树合理施肥提供依据.[方法]以一年生舒茶早(Camellia sinensis cv.Shuchazao)扦插苗为材料,采用水培法,对不同铵硝比(4:0,3∶1,2∶2,1∶3和0∶4)处理条件下,茶树叶片水浸出物、游离氨基酸、茶多酚、咖啡碱、酚氨比,以及茶树叶片氮代谢相关基因进行分析,探讨不同氮素形态对茶树叶片品质及其氮代谢相关基因表达的影响.[结果]铵硝比4∶0、3∶1和2∶2处理下茶树叶片游离氨基酸含量显著高于铵硝比0∶4和1∶3处理;茶多酚含量4∶0和3:1处理较高,显著高于其他处理组;各处理组水浸出物和咖啡碱含量无显著差异.与铵硝比4∶0、0∶4和1∶3处理相比,铵硝比3∶1和2∶2处理使GS1;1、GS1;3、GOGAT、AMT3;1和GDH基因的表达量升高;NRT1;2和NR基因表达量随着硝态氮比例的增加呈现上调趋势.[结论]铵硝配比施肥能增加茶树叶片中游离氨基酸和咖啡碱含量,提高茶树氮代谢相关基因的表达,其中铵硝比3∶1处理的效果较优.

Albino cultivars of Camellia sinensis are widely cultivated in China for producing a unique type of green teas with high abundance of free amino acid. However, the underlying mechanism of enhanced amino acids metabolism in albino leaves at different growth stages is still elusive. This study examined the profile changes of amino acids in the albino tea cultivar 'Huangshanbaicha' (HSBC), compared with the normal green cultivar 'Huangshanzhong' (HSZ). High-performance liquid chromatography (HPLC) was employed to assess the free amino acid content in the current shoots grown in spring from albinostic to regreening stages. Transcript levels of the key genes involved in amino acid metabolism, and transportation were also quantified by qRT-PCR. The results indicated that the contents of free amino acids in four developmental stages of the 'HOC' current shoots were in descending order: albinostic stage (S1) > bud stage (S0) > albescent stage (S2) > re-greening stage (S4). The total amino acid content at S1 stage reached to 7.10%, 1.8 and 2.6 times higher than the corresponding levels at S0 and S4, respectively. Moreover, from S0 to S4, the leaf composition of amino acids did not significantly change. Theanine, a characteristic amino acid of the tea plant, accounted for the largest proportion of 61.3%-70.2% relative to the total amount of all amino acids. The second most abundant component, glutamate, had a proportion far lower than that of theanine and varied between 6.3% and 15.7%. Expression levels of the genes involved in theanine synthesis were significantly higher in 'HSBC' than in 'HSZ', suggesting enhanced ability of theanine synthesis was appeared in 'HSBC' at the albinostic stage. Moreover, the transcript levels of amino acid transporter genes in 'HSBC' were significantly lower than those in 'HSZ'. Together, these findings demonstrated that the significantly increased amino acid content in the leaves of 'HSBC' during the albinostic stage, which might be partially resulted from enhanced ability of theanine synthesis and reduced capability amino acids transport in the leaves of 'HSBC' tea plants during the albinostic stage.

为了解干旱-低温交叉适应对茶树抗寒性的影响,以一年生无性系品种舒茶早茶苗为试验材料,用20％聚乙二醇6000 (PEG-6000)模拟干旱预处理,测定低温胁迫过程中茶树半致死温度、丙二醛、抗氧化酶活性、渗透物质和内源激素等部分生理指标的变化.结果 表明,经过PEG-6000预处理的茶苗半致死温度LT50明显下降.低温条件下,茶苗叶片丙二醛、抗氧化酶活性、渗透物质和激素含量整体呈上升趋势.低温胁迫第7天时,与对照相比,PEG-6000预处理的茶苗叶片丙二醛含量下降44.6％,SOD、POD酶活分别升高78％和25％,可溶性蛋白、溶性糖含量分别增加44.6％及20.0％;内源激素ABA与SA含量分别提高97.7％和122.0％.干旱可诱导茶树对低温胁迫的交叉适应性,这种交叉适应性与茶树的抗氧化酶活性、渗透物质和内源激素的调节能力有关.

基于表型性状和生化成分对陕西茶树种质资源遗传多样性进行研究,以期为优异茶树种质资源选育提供信息和科学依据,并加快利用和丰富陕西茶树种质资源种类.利用统计分析、主成分分析和聚类分析方法,对陕西茶树种质资源表型性状和生化成分遗传多样性进行研究,并评价其遗传多样性.陕西茶树种质资源表型性状变异较丰富,不同表型性状变异系数和遗传多样性指数差异较大,变异系数为19.88％～55.96％,平均变异系数31.84％,遗传多样性指数为0.6918～2.0030,平均遗传多样性指数1.1059;陕西茶树种质资源内含物质成分较丰富,基于生化成分的变异系数为10.89％～30.03％,平均变异系数21.78％,遗传多样性指数为1.7701～2.0492,平均遗传多样性指数1.8900;基于表型性状和生化成分的聚类分析结果不一致,表明聚类分析结果与茶树种质资源表型性状、生化成分、DNA分子的遗传多样性和地域分布关系密切,单一依据表型性状或生化成分进行茶树种质资源聚类分析具有一定的局限性.研究结果表明,陕西茶树种质资源遗传多样性较丰富,优异资源筛选潜力较大,具有独特地域性,但属于江北茶区的陕南茶树种质资源遗传多样性丰富程度稍低于南方茶区的四川、广西、贵州等地.基于生化成分初步筛选出高水浸出物(＞50％)茶树种质资源3份,高茶多酚(＞20％)茶树种质资源4份,高氨基酸(＞3％)茶树种质资源4份,低咖啡碱(＜1.6％)茶树种质资源4份.

BACKGROUND:Low temperature restricts the planting range of all crops, but cold acclimation induces adaption to cold stress in many plants. Camellia sinensis, a perennial evergreen tree that is the source of tea, is mainly grown in warm areas. Camellia sinensis var. sinensis (CSS) has greater cold tolerance than Camellia sinensis var. assamica (CSA). To gain deep insight into the molecular mechanisms underlying cold adaptation, we investigated the physiological responses and transcriptome profiles by RNA-Seq in two tea varieties, cold resistant SCZ (classified as CSS) and cold susceptible YH9 (classified as CSA), during cold acclimation. RESULTS:Under freezing stress, lower relative electrical conductivity and higher chlorophyll fluorescence (Fv/Fm) values were detected in SCZ than in YH9 when subjected to freezing acclimation. During cold treatment, 6072 and 7749 DEGs were observed for SCZ and YH9, respectively. A total of 978 DEGs were common for both SCZ and YH9 during the entire cold acclimation process. DEGs were enriched in pathways of photosynthesis, hormone signal transduction, and transcriptional regulation of plant-pathogen interactions. Further analyses indicated that decreased expression of Lhca2 and higher expression of SnRK2.8 are correlated with cold tolerance in SCZ. CONCLUSIONS:Compared with CSA, CSS was significantly more resistant to freezing after cold acclimation, and this increased resistance was associated with an earlier expression of cold-induced genes. Because the greater transcriptional differentiation during cold acclimation in SCZ may contribute to its greater cold tolerance, our studies identify specific genes involved in photoinhibition, ABA signal conduction, and plant immunity that should be studied for understanding the processes involved in cold tolerance. Marker-assisted breeding focused on the allelic variation at these loci provides an avenue for the possible generation of CSA cultivars that have CSS-level cold tolerance.

Fruit cracking in watermelon (Citrullus lanatus) causes a great economic loss. To understand the molecular mechanisms underlying watermelon fruit cracking, the differentially expressed genes (DEGs) between resistant and susceptible-cracking watermelon were analyzed using transcriptome sequencing. We selected the parent inbred W11 (resistant-cracking) and W13 (susceptible-cracking), and Near-Isogenic Lines W96 (resistant cracking) and W85 (susceptible-cracking) as materials. Differentially expressed genes (DEGs) analysis showed that 290 DEGs had detectable in "W11" VS "W13", while 165 DEGs had detectable in "W96" VS "W85". There were 56 DEGs between the four samples. Additionally, 14 DEGs related to fruit cracking were identified from the transcriptome. Only 8 DEGs among them, were involved in 14 KEGG pathway. The expression patterns of the 14 DEGs related to fruit cracking were analyzed by qRT-PCR to explore their putative functions. This transcriptome dataset will aid in understanding and carrying out future studies on the molecular basis of fruit cracking and contribute to watermelon breeding..

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future.

Table S8. Differentially expressed genes related to osmoprotectans in response to cold treatment of SCZ and YH9 leaves. Significant (Pâ

Apoplastic ascorbate oxidase (AO) plays a major role in cell growth. Although AO genes have been studied in depth, some articles have mistakenly identified AO homologues as AO genes. Overall, the divergence between AO genes and AO homologues has not been explored. Meanwhile, there is little information concerning AO and the AO homologue with respect to Camellia sinensis. In the present study, one CsAO homologue and three CsAOs were confirmed by RT-PCR amplification, cloning and sequencing. Multicopper oxidase type 1 (PF00394), type 2 (PF07731) or type 3 (PF07732) domains and one transmembrane helix were the key domains for each member of the cupredoxin family. The CsAOs with their counterparts from seven dicotyledonous plants and three monocotyledonous plants were used to build phylogenetic tree and compare the deduced polypeptides. CsAO may be strongly expressed in the stretch expanded tissues, including bud and root. The abiotic stress-induced expression pattern of the CsAO homologue (CsAO2) is similar to those of CsAO1, CsAO3 and CsAO4. A new and very large group of AO homologues, which may function as AO genes, was present in both dicotyledonous and monocotyledonous plants. Our study may help in identifying stress-responsive AO genes of plants.

Late embryogenesis abundant (LEA) proteins are widely known to be present in higher plants and are believed to play important functional roles in embryonic development and abiotic stress responses. However, there is a current lack of systematic analyses on the LEA protein gene family in tea plant. In this study, a total of 48 LEA genes were identified using Hidden Markov Model profiles in C. sinensis, and were classified into seven distinct groups based on their conserved domains and phylogenetic relationships. Genes in the CsLEA_2 group were found to be the most abundant. Gene expression analyses revealed that all the identified CsLEA genes were expressed in at least one tissue, and most had higher expression levels in the root or seed relative to other tested tissues. Nearly all the CsLEA genes were found to be involved in seed development, and thirty-nine might play an important role in tea seed maturation concurrent with dehydration. However, only sixteen CsLEA genes were involved in seed desiccation, and furthermore, most were suppressed. Additionally, forty-six CsLEA genes could be induced by at least one of the tested stress treatments, and they were especially sensitive to high temperature stress. Furthermore, it was found that eleven CsLEA genes were involved in tea plant in response to all tested abiotic stresses. Overall, this study provides new insights into the formation of CsLEA gene family members and improves our understanding on the potential roles of these genes in normal development processes and abiotic stress responses in tea plant, particularly during seed development and desiccation. These results are beneficial for future functional studies of CsLEA genes that will help preserve the recalcitrant tea seeds for a long time and genetically improve tea plant.

Tea plant (Camellia sinensis) is a typical plant that accumulates abundant aluminum (Al) and fluoride (F). Al and F play vital roles in the growth and development in tea plant. MYB (myeloblastosis) is one of the largest transcription factor families in plant, which plays an important role in the stress responses. As the largest and multifunctional subfamily of MYB, R2R3-MYB subfamily participates in multiple abiotic stresses in plant. However, there are few studies about R2R3-MYB in tea plant, especially in the accumulation of Al and F during its growth. Here, we identified 43 CsMYB genes from the transcriptome database of tea plant and analyzed the relative expression in Al and F treatments. According to the Arabidopsis thaliana classification, 43 CsMYB genes are divided into 18 subgroups via phylogenetic tree analysis. All 43 CsMYB proteins have the typical R2R3-MYB domain by MEME prediction. Moreover, 9 CsMYB genes (CsMYB11, CsMYB17, CsMYB29, CsMYB38 to CsMYB43) that related to abiotic stress were selected from 43 CsMYB genes for a further study, and the relative expressions showed that they are tissue-specifically expressed. In addition, the results of different concentrations of Al treatments showed that CsMYB11, -17, -29, -38 and -41 are stimulated by Al treatment. The results of different concentrations of F treatments showed that the CsMYB11, -17, -38, -39, -40, -41, -42 and -43 participate in F accumulation of C. sinensis roots. Our research establishes a solid foundation for further investigation into the molecular functions of R2R3-MYB transcription factors in C. sinensis.

Tea (Camellia sinensis) plants inhabit arbuscular mycorrhizal fungi (AMF) in rhizosphere, whereas it is not clear whether AMF improves leaf food quality of tea plants. A potted study was conducted to determine effects of Claroideoglomus etunicatum, Diversispora spurca, D. versiformis and a mixture of the three AMF species on leaf sugar, amino acid, soluble protein, tea polyphenol, catechuic acid, and flavonoid contents of Camellia sinensis ‘Fuding Dabaicha’ seedlings. After 12 weeks of AMF inoculation, mycorrhizal plants recorded significantly higher shoot biomass and total leaf area, whilst the effect was ranked as C. etunicatum > D. spurca > mixed-AMF > D. versiformis in the decreasing order. AMF treatments significantly increased leaf total amino acid concentrations, accompanied with up-regulation of amino acid synthetic enzymes genes glutamine synthetase (CsGS), glutamate synthase (CsGOGAT) and glutamate dehydrogenase (CsGDH). Leaf glucose, sucrose, total soluble protein, tea polyphenol, catechuic acid, and flavonoid contents were significantly higher in AMF- than in non-AMF-inoculated plants. In addition, mycorrhizal inoculation notably up-regulated the expression level of leaf 3-hydroxy-3-methylglutaryl coenzyme gene (CsHMGR), ascorbate peroxidase gene (CsAPX), and tea caffeine synthase 1 gene (CsTCS1). These results implied that AMF inoculation had positive effects on leaf food quality partly by means of up-regulation of relevant gene expression in ‘Fuding Dabaicha’ seedlings. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Background The tea plant is a crucial economic crop. The floral organ development consumes a large amount of nutrients, which affects the leaf yield. To understand the mechanism by which the tea plant produces sterile floral buds, we obtained a sterile tea plant by artificial hybridization. RNA-sequencing based transcriptome analysis was implemented in three samples to determine the differentially expressed genes (DEGs) related to flower development. Results In this study, a total of 1991 DEGs were identified; 1057 genes were up-regulated and 934 genes were down-regulated in sterile hybrid floral buds. These were mainly distributed in the regulation of biological and metabolic processes. Significantly, auxin biosynthesis genes YUCCA , AUX1 and PIN were dramatically down-regulated, and ARF gene was up-regulated in the sterile hybrid floral buds, and flower development-related genes AP1 , AP2 and SPL were changed. A total of 12 energy transfer-related genes were significantly decreased. Furthermore, the expression of 11 transcription factor genes was significantly different. Conclusion The transcriptome analysis suggested that the production of sterile floral buds is a complex bioprocess, and that low auxin-related gene levels result in the formation of sterile floral buds in the tea plant.

Significance A high-quality genome assembly of Camellia sinensis var. sinensis facilitates genomic, transcriptomic, and metabolomic analyses of the quality traits that make tea one of the world’s most-consumed beverages. The specific gene family members critical for biosynthesis of key tea metabolites, monomeric galloylated catechins and theanine, are indicated and found to have evolved specifically for these functions in the tea plant lineage. Two whole-genome duplications, critical to gene family evolution for these two metabolites, are identified and dated, but are shown to account for less amplification than subsequent paralogous duplications. These studies lay the foundation for future research to understand and utilize the genes that determine tea quality and its diversity within tea germplasm.

以一年生舒茶早(C.sinensis cv.‘Shuchazao’)扦插苗为材料研究不同氮素形态和氮素水平对茶树叶片光合作用、叶片含氮量和氮素利用相关基因的影响.结果表明,在氮素形态上,铵硝比例3∶1和2∶2处理茶树叶片光合作用能力显著高于其他处理组;铵硝比例3∶1组叶片含氮量显著高于其他组;铵硝比例2∶2的谷氨酰胺合成酶基因(GS)的表达量最高,全铵处理组表达量最低,只有铵硝比例2∶2处理组的66.67％.铵硝比例2∶2的谷氨酸合成酶基因(GOGAT)的表达量最高,铵硝比例1∶3表达量最低,只有铵硝比例2∶2组的20％.在氮素水平上,正常氮素营养液培养下,茶树叶片的光合作用和叶片含氮量显著高于不合氮和4倍氮处理组;茶树叶片的GS和GOGAT基因的表达量也最高,分别是4倍氮素处理组的1.3倍和2.1倍,是不含氮素处理组的5.2倍和5.0倍.综合来看,茶园施肥需要铵硝配比施肥并适当提高铵态氮含量,同时不宜过度施肥,不仅造成肥料浪费还可能不利于茶树生长.

The research and utilization of the ancient tea germplasm resources were the new topic in the reasearch of the tea germplasm resources in China. In order to identify and evaluate the phenotypic traits diversity index of the 38 ancient tea germplasm resources in Shaanxi,the phenotypic traits diversity index was researched and analyzed with the methods of the correlation analysis,the principal compoent analysis and the clustering analysis in the paper. The results showed that,the diversity and variation of the six main phenotypic traits(leaf size,leaf shape,leaf colour,leaf cross section,leaf upper surface and leaf attitude)of the 38 ancient tea germplasm resources in Shaanxi was abundant,the diversity index among the main phenotypic traits was different,the diversity index was between 0.7934-0.9969,and the average diversity index reaching 0.8811;the 38 ancient tea germplasm resources in Shaanxi could clustered into two large groups at the euclidean distance of ten,they were clustered accordance with the phenotypic traits diversity index,were not in strict accordance with the regional cluster. The variation of the ancient tea germplasm resources in Hanzhong and Ankang of Shaanxi was abundant,possessing the high genetic diversity and breeding potential of the excellent tea germplasm resources to a certain extent.