Analysis of Functional Single-Nucleotide Polymorphisms (SNPs) and Leaf Quality in Tea Collection under Nitrogen-Deficient Conditions.

This study discusses the genetic mutations that have a significant association with economically important traits that would benefit tea breeders. The purpose of this study was to analyze the leaf quality and SNPs in quality-related genes in the tea plant collection of 20 mutant genotypes growing without nitrogen fertilizers. Leaf N-content, catechins, L-theanine, and caffeine contents were analyzed in dry leaves via HPLC. Additionally, the photochemical yield, electron transport efficiency, and non-photochemical quenching were analyzed using PAM-fluorimetry. The next generation pooled amplicon-sequencing approach was used for SNPs-calling in 30 key genes related to N metabolism and leaf quality. The leaf N content varied significantly among genotypes (p ≤ 0.05) from 2.3 to 3.7% of dry mass. The caffeine content varied from 0.7 to 11.7 mg g-1, and the L-theanine content varied from 0.2 to 5.8 mg g-1 dry leaf mass. Significant positive correlations were detected between the nitrogen content and biochemical parameters such as theanine, caffeine, and most of the catechins. However, significant negative correlations were observed between the photosynthetic parameters (Y, ETR, Fv/Fm) and several biochemical compounds, including rutin, Quercetin-3-O-glucoside, Kaempferol-3-O-rutinoside, Kaempferol-3-O-glucoside, Theaflavin-3'-gallate, gallic acid. From our SNP-analysis, three SNPs in WRKY57 were detected in all genotypes with a low N content. Moreover, 29 SNPs with a high or moderate effect were specific for #316 (high N-content, high quality) or #507 (low N-content, low quality). The use of a linear regression model revealed 16 significant associations; theaflavin, L-theanine, and ECG were associated with several SNPs of the following genes: ANSa, DFRa, GDH2, 4CL, AlaAT1, MYB4, LHT1, F3'5'Hb, UFGTa. Among them, seven SNPs of moderate effect led to changes in the amino acid contents in the final proteins of the following genes: ANSa, GDH2, 4Cl, F3'5'Hb, UFGTa. These results will be useful for further evaluations of the important SNPs and will help to provide a better understanding of the mechanisms of nitrogen uptake efficiency in tree crops.

Long-term cold, freezing and drought: overlapping and specific regulatory mechanisms and signal transduction in tea plant (Camellia sinensis (L.) Kuntze).

Introduction: Low temperatures and drought are two main environmental constraints reducing the yield and geographical distribution of horticultural crops worldwide. Understanding the genetic crosstalk between stress responses has potential importance for crop improvement. Methods: In this study, Illumina RNA-seq and Pac-Bio genome resequencing were used to annotate genes and analyze transcriptome dynamics in tea plants under long-term cold, freezing, and drought. Results: The highest number of differentially expressed genes (DEGs) was identified under long-term cold (7,896) and freezing (7,915), with 3,532 and 3,780 upregulated genes, respectively. The lowest number of DEGs was observed under 3-day drought (47) and 9-day drought (220), with five and 112 genes upregulated, respectively. The recovery after the cold had 6.5 times greater DEG numbers as compared to the drought recovery. Only 17.9% of cold-induced genes were upregulated by drought. In total, 1,492 transcription factor genes related to 57 families were identified. However, only 20 transcription factor genes were commonly upregulated by cold, freezing, and drought. Among the 232 common upregulated DEGs, most were related to signal transduction, cell wall remodeling, and lipid metabolism. Co-expression analysis and network reconstruction showed 19 genes with the highest co-expression connectivity: seven genes are related to cell wall remodeling (GATL7, UXS4, PRP-F1, 4CL, UEL-1, UDP-Arap, and TBL32), four genes are related to calcium-signaling (PXL1, Strap, CRT, and CIPK6), three genes are related to photo-perception (GIL1, CHUP1, and DnaJ11), two genes are related to hormone signaling (TTL3 and GID1C-like), two genes are involved in ROS signaling (ERO1 and CXE11), and one gene is related to the phenylpropanoid pathway (GALT6). Discussion: Based on our results, several important overlapping mechanisms of long-term stress responses include cell wall remodeling through lignin biosynthesis, o-acetylation of polysaccharides, pectin biosynthesis and branching, and xyloglucan and arabinogalactan biosynthesis. This study provides new insight into long-term stress responses in woody crops, and a set of new target candidate genes were identified for molecular breeding aimed at tolerance to abiotic stresses.

InDel and SCoT Markers for Genetic Diversity Analysis in a Citrus Collection from the Western Caucasus.

Citrus collections from extreme growing regions can be an important source of tolerant germplasms for the breeding of cold-tolerant varieties. However, the efficient utilization of these germplasms requires their genetic background information. Thus, efficient marker systems are necessary for the characterization and identification of valuable accessions. In this study, the efficiency of 36 SCoT markers and 60 InDel markers were evaluated as part of the broad citrus collection of the Western Caucasus. The interspecific and intraspecific genetic diversity and genetic structures were analyzed for 172 accessions, including 31 species and sets of the locally derived cultivars. Single markers, such as SCoT18 (0.84), SCoT20 (0.93), SCoT23 (0.87), SCoT31 (0.88), SCoT36 (0.87) и LG 1-4 (0.94), LG 4-3 (0.86), LG 7-11 (0.98), and LG 8-10 (0.83), showed a high discriminating power, indicating the good applicability of these markers to assess intraspecific diversity of the genus Citrus. Overall, SCoT markers showed a higher level of polymorphism than InDel markers. According to analysis of population structure, SCoT and InDel markers showed K = 9 and K = 5 genetic clusters, respectively. The lowest levels of genetic admixtures and diversity were observed among the locally derived satsumas and lemons. The highest level of genetic admixtures was observed in the lime group. Phylogenetic relationships indicated a high level of interspecific genetic diversity but a low level of intraspecific diversity in locally derived satsumas and lemons. The results provide new insight into the origin of citrus germplasms and their distribution in colder regions. Furthermore, they are important for implementing conservation measures, controlling genetic erosion, developing breeding strategies, and improving breeding efficiency.

Evolutionary Landscape of Tea Circular RNAs and Its Contribution to Chilling Tolerance of Tea Plant.

Chilling stress threatens the yield and distribution pattern of global crops, including the tea plant (Camellia sinensis), one of the most important cash crops around the world. Circular RNA (circRNA) plays roles in regulating plant growth and biotic/abiotic stress responses. Understanding the evolutionary characteristics of circRNA and its feedbacks to chilling stress in the tea plant will help to elucidate the vital roles of circRNAs. In the current report, we systematically identified 2702 high-confidence circRNAs under chilling stress in the tea plant, and interestingly found that the generation of tea plant circRNAs was associated with the length of their flanking introns. Repetitive sequences annotation and DNA methylation analysis revealed that the longer flanking introns of circRNAs present more repetitive sequences and higher methylation levels, which suggested that repeat-elements-mediated DNA methylation might promote the circRNAs biogenesis in the tea plant. We further detected 250 differentially expressed circRNAs under chilling stress, which were functionally enriched in GO terms related to cold/stress responses. Constructing a circRNA-miRNA-mRNA interaction network discovered 139 differentially expressed circRNAs harboring potential miRNA binding sites, which further identified 14 circRNAs that might contribute to tea plant chilling responses. We further characterized a key circRNA, CSS-circFAB1, which was significantly induced under chilling stress. FISH and silencing experiments revealed that CSS-circFAB1 was potentially involved in chilling tolerance of the tea plant. Our study emphasizes the importance of circRNA and its preliminary role against low-temperature stress, providing new insights for tea plant cold tolerance breeding.

Effects of exogenous calcium on the drought response of the tea plant (Camellia sinensis (L.) Kuntze).

Background: Drought is one of the major factors reducing the yield of many crops worldwide, including the tea crop (Camellia sinensis (L.) Kuntze). Calcium participates in most of cellular signaling processes, and its important role in stress detection and triggering a response has been shown in many crops. The aim of this study was to evaluate possible effects of calcium on the tea plant response to drought. Methods: Experiments were conducted using 3-year-old potted tea plants of the best local cultivar Kolkhida. Application of ammonium nitrate (control treatment) or calcium nitrate (Ca treatment) to the soil was performed before drought induction. Next, a 7-day drought was induced in both groups of plants. The following physiological parameters were measured: relative electrical conductivity, pH of cell sap, and concentrations of cations, sugars, and amino acids. In addition, relative expression levels of 40 stress-related and crop quality-related genes were analyzed. Results: Under drought stress, leaf electrolyte leakage differed significantly, indicating greater damage to cell membranes in control plants than in Ca-treated plants. Calcium application resulted in greater pH of cell sap; higher accumulation of tyrosine, methionine, and valine; and a greater Mg2+ content as compared to control plants. Drought stress downregulated most of the quality-related genes in both groups of tea plants. By contrast, significant upregulation of some genes was observed, namely CRK45, NAC26, TPS11, LOX1, LOX6, Hydrolase22, DREB26, SWEET2, GS, ADC, DHN2, GOLS1, GOLS3, and RHL41. Among them, three genes (LOX1, RHL41, and GOLS1) showed 2-3 times greater expression in Ca-treated plants than in control plants. Based on these results, it can be speculated that calcium affects galactinol biosynthesis and participates in the regulation of stomatal aperture not only through activation of abscisic-acid signaling but also through jasmonic-acid pathway activation. These findings clarify calcium-mediated mechanisms of drought defense in tree crops. Thus, calcium improves the drought response in the tea tree.

Genetic Diversity and Genome Size Variability in the Russian Genebank Collection of Tea Plant [Camellia sinensis (L). O. Kuntze].

The tea collection of the FRC SSC RAS (Sochi, Maykop in Russia) represents one of the northernmost germplasm comprising a number of locally derived cultivars and ɣ-irradiation mutants. The latter are often characterized by larger genome size, which may lead to better adaptation to biotic and abiotic stress. Such genotypes may be a valuable genetic resource for better adaptability to extreme environmental conditions, which could enable tea cultivation outside global growing regions. Microsatellite markers are often the best choice for genetic diversity analysis in genebank collections. However, their use in polyploid species is questionable because simple sequence repeat (SSR) allele dosage cannot be readily determined. Therefore, the efficiency of SSR and start codon targeted (SCoT) markers was investigated using 43 selected cultivars from the Russian genebank collection derived from mutant breeding and clonal selection. Previously, the increase in genome size was confirmed in 18 mutants within this collection. Despite the presence of polyploid tea genotypes, our study revealed higher efficiency of SSR markers than SCoT markers. Subsequent SSR analysis of the 106 genotypes in the Russian genebank collection revealed three distinct genetic clusters after STRUCTURE analysis. Greater genetic variation was observed within genetic clusters than between clusters, indicating low genetic variation between collections. Nevertheless, the northernmost tea collection exhibited a greater genetic distance from the other two clusters than they did from each other. Close genetic relationships were found between many cultivars with particularly large leaves and mutant forms. Pearson's correlation analysis revealed a significant, moderate correlation between genome size and leaf area size. Our study shows that microsatellite fingerprinting is useful to estimate the genetic diversity and genetic background of tea germplasm in Russia despite polyploid tea accessions. Thus, the results of our study contribute to the development of future tea germplasm conservation strategies and modern tea breeding programs.

Biochemical and Genetic Responses of Tea (Camellia sinensis (L.) Kuntze) Microplants under Mannitol-Induced Osmotic Stress In Vitro.

Osmotic stress is a major factor reducing the growth and yield of many horticultural crops worldwide. To reveal reliable markers of tolerant genotypes, we need a comprehensive understanding of the responsive mechanisms in crops. In vitro stress induction can be an efficient tool to study the mechanisms of responses in plants to help gain a better understanding of the physiological and genetic responses of plant tissues against each stress factor. In the present study, the osmotic stress was induced by addition of mannitol into the culture media to reveal biochemical and genetic responses of tea microplants. The contents of proline, threonine, epigallocatechin, and epigallocatechin gallate were increased in leaves during mannitol treatment. The expression level of several genes, namely DHN2, LOX1, LOX6, BAM, SUS1, TPS11, RS1, RS2, and SnRK1.3, was elevated by 2-10 times under mannitol-induced osmotic stress, while the expression of many other stress-related genes was not changed significantly. Surprisingly, down-regulation of the following genes, viz. bHLH12, bHLH7, bHLH21, bHLH43, CBF1, WRKY2, SWEET1, SWEET2, SWEET3, INV5, and LOX7, was observed. During this study, two major groups of highly correlated genes were observed. The first group included seven genes, namely CBF1, DHN3, HXK2,SnRK1.1, SPS, SWEET3, and SWEET1. The second group comprised eight genes, viz. DHN2, SnRK1.3, HXK3, RS1, RS2,LOX6, SUS4, and BAM5. A high level of correlation indicates the high strength connection of the genes which can be co-expressed or can be linked to the joint regulons. The present study demonstrates that tea plants develop several adaptations to cope under osmotic stress in vitro; however, some important stress-related genes were silent or downregulated in microplants.

Physiological, biochemical and genetic responses of Caucasian tea (Camellia sinensis (L.) Kuntze) genotypes under cold and frost stress.

BACKGROUND: Cold and frost are two serious factors limiting the yield of many crops worldwide, including the tea plant (Camellia sinensis (L.) Kuntze). The acclimatization of tea plant from tropical to temperate climate regions resulted in unique germplasm in the North-Western Caucasus with extremely frost-tolerant genotypes. METHODS: The aim of the current research was to evaluate the physiological, biochemical and genetic responses of tolerant and sensitive tea cultivars exposed to cold (0 to +2 °C for 7 days) and frost (-6 to -8 °C for 5 days). Relative water content, cell membranes integrity, pH of the cell sap, water soluble protein, cations, sugars, amino acids were measured under cold and frost. Comparative expression of the following genes ICE1, CBF1, WRKY2, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, SnRK1.1, SnRK1.2, SnRK1.3, bHLH7, bHLH43, P5CS, LOX1, LOX6, LOX7 were analyzed. RESULTS: We found elevated protein (by 3-4 times) and cations (potassium, calcium and magnesium) contents in the leaves of both cultivars under cold and frost treatments. Meanwhile, Leu, Met, Val, Thr, Ser were increased under cold and frost, however tolerant cv. Gruzinskii7 showed earlier accumulation of these amino acids. Out of 18 studied genes, 11 were expressed at greater level in the frost- tolerant cultivar comparing with frost-sensitive one: ICE1, CBF1, WRKY2, DHN2, NAC17, NAC26, SnRK1.1, SnRK1.3, bHLH43, P5CS and LOX6. Positive correlations between certain amino acids namely, Met, Thr, Leu and Ser and studied genes were found. Taken together, the revealed cold responses in Caucasian tea cultivars help better understanding of tea tolerance to low temperature stress and role of revealed metabolites need to be further evaluated in different tea genotypes.

Comparative Expression Analysis of Stress-Inducible Candidate Genes in Response to Cold and Drought in Tea Plant [Camellia sinensis (L.) Kuntze].

Cold and drought are two of the most severe threats affecting the growth and productivity of the tea plant, limiting its global spread. Both stresses cause osmotic changes in the cells of the tea plant by decreasing their water potential. To develop cultivars that are tolerant to both stresses, it is essential to understand the genetic responses of tea plant to these two stresses, particularly in terms of the genes involved. In this study, we combined literature data with interspecific transcriptomic analyses (using Arabidopsis thaliana and Solanum lycopersicum) to choose genes related to cold tolerance. We identified 45 stress-inducible candidate genes associated with cold and drought responses in tea plants based on a comprehensive homologous detection method. Of these, nine were newly characterized by us, and 36 had previously been reported. The gene network analysis revealed upregulated expression in ICE1-related cluster of bHLH factors, HSP70/BAM5 connected genes (hexokinases, galactinol synthases, SnRK complex, etc.) indicating their possible co-expression. Using qRT-PCR we revealed that 10 genes were significantly upregulated in response to both cold and drought in tea plant: HSP70, GST, SUS1, DHN1, BMY5, bHLH102, GR-RBP3, ICE1, GOLS1, and GOLS3. SnRK1.2, HXK1/2, bHLH7/43/79/93 were specifically upregulated in cold, while RHL41, CAU1, Hydrolase22 were specifically upregulated in drought. Interestingly, the expression of CIP was higher in the recovery stage of both stresses, indicating its potentially important role in plant recovery after stress. In addition, some genes, such as DHN3, bHLH79, PEI54, SnRK1.2, SnRK1.3, and Hydrolase22, were significantly positively correlated between the cold and drought responses. CBF1, GOLS1, HXK2, and HXK3, by contrast, showed significantly negative correlations between the cold and drought responses. Our results provide valuable information and robust candidate genes for future functional analyses intended to improve the stress tolerance of the tea plant and other species.

Comprehensive co-expression analysis provides novel insights into temporal variation of flavonoids in fresh leaves of the tea plant (Camellia sinensis).

Flavonoids are the major class of characteristic secondary compounds in Camellia sinensis that affect quality of tea. However, the temporal variation and the underlying regulatory mechanism of flavonoid biosynthesis during different growth months require a further investigation. Here, we combined analyses of the metabolomics and transcriptomics to tea leaves freshly collected during five different months for a comprehensive understanding of flavonoid metabolism regulation in tea plants. Through loading plot analysis, significant changes in the contents of metabolites during growing months were discovered, and further co-expression and association analysis indicated that one flavone glycoside (naringenin-7-O-glucoside) and two flavonol glycosides (quercetin-3-O-galactoside and kaemferol-3-O-(6″-O-p-courmaroyl)-glucoside) were evaluated as growth markers, which may explain the high bitterness and astringency of August teas; additionally, the high levels of two flavan-3-ols (gallocatechin and catechin gallate) may contribute to the flavor formation of April tea. Meanwhile, multiple flavonoid-related structural genes, MYB and bHLH transcription factors exhibit specific expression patterns to modulate the biosynthesis of these key flavonoids. A co-expression regulatory sub-network was constructed based on profiles of differentially expressed genes; one CsbHLH and six transcription factors (three CsbHLHs and three CsMYBs) exhibited negative and positive roles in the regulation of flavonoid biosynthetic genes, respectively. Taken together, our results provide new insights into the regulation of principle flavonoids for unique flavor of tea regulated by many flavonoid-related structural genes and transcription factors during different growth months.

Duplication and transcriptional divergence of three Kunitz protease inhibitor genes that modulate insect and pathogen defenses in tea plant (Camellia sinensis).

Kunitz protease inhibitors (KPIs) are ubiquitous in plants and act as crucial compounds in defense responses against insect attack and pathogen infection. However, the influence of gene duplication on the postdivergence of the CsKPI genes involved in biotic stresses in tea plant is not well known. Here, we identified three CsKPI genes from tea plant (Camellia sinensis) and characterized their expression and evolutionary patterns among plant species. We found that CsKPI1, CsKPI2, and CsKPI3 diverged from their common ancestor 72.94 million years ago (MYA), and the tandem duplication of CsKPI2 and CsKPI3 occurred 26.78 MYA. An in vitro protein assay showed that the three CsKPI proteins were functional and inhibited the production of p-nitroanilide (PNA) from an artificial substrate. The three CsKPI-GFP fusion proteins localized to the cytoplasm. We showed that salicylic acid (SA) and transcripts of CsKPI2 and CsKPI3 significantly accumulated after infection with Glomerella cingulata. The application of exogenous SA stimulated the high expression of both CsKPI2 and CsKPI3 by activating cis-elements within their promoters. Under Ectropis oblique attack, CsKPI1 expression and jasmonic acid (JA) levels were more abundant in both insect-damaged leaf tissues and undamaged neighboring leaves. The application of jasmonic acid methyl ester elicited high expression levels of CsKPI1, suggesting that CsKPI1 accumulation requires JA production in tea plant. The overall findings suggest that the transcriptional divergence of KPI genes after duplication led to the specialized role of CsKPI1 in the physiological response to insect stress; the functional conservation between CsKPI2 and CsKPI3 confers resistance to pathogen infection in tea plant.