Plant growth-promoting rhizobacteria (PGPR) improve the growth and quality of several crops.

Plant growth-promoting rhizobacteria (PGPR) are known to have the effect of promoting plant growth. In this paper, three PGPR strains were selected from the previous work, which had plant growth-promoting activities such as phosphate solubilization, nitrogen fixation, phosphorus mobilization, etc. These strains named FJS-3(Burkholderia pyromania), FJS-7(Pseudomonas rhodesiae), and FJS-16(Pseudomonas baetica), respectively, were prepared into solid biological agents. Three widely planted commercial crops (tea plant, tobacco, and chili pepper) were selected for PGPR growth promotion verification. The results showed that the new shoots of tea seedlings under PGPR treatment were much more than the control. We also used tobacco, another important crop in Guizhou, to test the growth-promoting effect of individual bacteria, and the results showed that each of them could promote the growth of tobacco plants, and FJS-3(Burkholderia pyrrocinia) had the best effect. In addition, we carried out experiments on tobacco and pepper using multi-strain PGPR, the tobacco plants' height, fresh, and root weight increased by 30.15 %, 37.36 %, and 54.5 %, respectively, and the pepper plants' increased by 30.10 %, 56.38 % and 43.18 %, respectively, which both showed significantly better effects than that of a single strain. To further test the field performance, field trials were carried out in a mature Longjing43 tea plantation in Guizhou. There were four treatments: no fertilization (T1), combined application of PGPR biological agent and compound fertilizer (T2), only application of PGPR (T3), and only application of compound fertilizer (T4). In terms of yield, grouped with or without PGPR, there was a 15.38 % (T2:T4) and 92.31 % (T3:T1) increase between them, respectively. The tea's yield and tea flavor substances such as tea polyphenols, caffeine, and theanine were detected, and the T2 showed the most significant positive effect on both sides. Especially, an important indicator of Matcha green tea is the color, chlorophyll content was then tested, and PGPR application increased it and improved the appearance. All these results demonstrated that the PGPR we screened could significantly promote plant growth and quality improvement, and had good application potential in crop planting, which could contribute to environmental protection and economic growth.

CsMOF1-guided regulation of drought-induced theanine biosynthesis in Camellia sinensis.

The distinctive flavor and numerous health benefits of tea are attributed to the presence of theanine, a special amino acid found in tea plants. Nitrogen metabolite is greatly impacted by drought; however, the molecular mechanism underlying the synthesis of theanine in drought-stricken tea plants is still not clear. Through the drought transcriptome data of tea plants, we have identified a gene CsMOF1 that appears to play a role in theanine biosynthesis under drought stress, presenting a significantly negative correlation with both theanine content and the expression of CsGS1. Further found that CsMOF1 is a transcription factor containing a MYB binding domain, localized in the nucleus. Upon silencing CsMOF1, there was a prominent increase in the level of the theanine and glutamine, as well as the expression of CsGS1, while glutamic acid content decreased significantly. Conversely, overexpression of CsMOF1 yielded opposite effects. Dual luciferase reporter assay and electromobility shift assays demonstrated that CsMOF1 binds to the promoter of CsGS1, thereby inhibiting its activity. These results indicate that CsMOF1 plays a crucial role in theanine biosynthesis in tea plants under drought stress, acting as a transcriptional repressor related to theanine biosynthesis. This study provides new insights into the tissue-specific regulation of theanine biosynthesis and aids with the cultivation of new varieties of tea plants.

The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis.

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea, cacao, and coffee, but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear. Here, we report a novel transcription factor Camellia sinensis Senescnece 40 (CsS40) as a caffeine biosynthesis regulator, which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase (TCS1) promoter. Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays, a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter. Consistently, immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells. The expression pattern of CsS40 in 'Fuding Dabai' developing leaves was opposite to that of TCS1; and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels, respectively. The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of 'Fuding Dabai'. Furthermore, overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants, meanwhile, increased their susceptibility to aging. CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors. Taken together, we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar 'Fuding Dabai'. The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

Transcriptome Analysis Reveals Differentially Expressed Genes Involved in Aluminum, Copper and Cadmium Accumulation in Tea 'Qianmei 419' and 'Qianfu 4'.

Tea, as a global nonalcoholic beverage, is widely consumed due to its economic, health and cultural importance. Polyploids have the ability to solve the problems of low yield, cold resistance and insect resistance in tea tree varieties. However, the response mechanism to aluminum and heavy metal remains unclear. In this study, the content of Al, Cu and Cd were measured in the leaves and roots of 'Qianmei 419' and 'Qianfu 4', respectively. The content of Al, Cd and Cu in the roots of the 'Qianmei 419' tea variety were significantly higher than in 'Qianfu 4' roots. Only the content of Cu in the leaves of the 'Qianmei 419' tea variety was significantly higher than that in the roots of the 'Qianfu 4' tea variety. Moreover, we found that the content of Al, Cu and Cd in the soil around the root of 'Qianfu 4' were higher than in the soil around the root of 'Qianmei 419'. RNA-seq was performed to identify the DEGs involved in the accumulation of Al, Cu and Cd between 'Qianmei 419' and 'Qianfu 4'. A total of 23,813 DEGs were identified in the triploid tea variety, including 16,459 upregulated DEGs and 7354 downregulated DEGs. Among them, by analyzing the expression levels of some metal transporter genes, it was found that most of the metal transporter genes were downregulated in the triploid tea plants. In short, through the analysis of transcriptome data and metal content, it was found that changes in metal transporter gene expression affect the accumulation of metals in tea plants. These results provide candidate genes to enhance multi-metal tolerance through genetic engineering technology.

Genome-Wide Investigation and Functional Analysis Reveal That CsKCS3 and CsKCS18 Are Required for Tea Cuticle Wax Formation.

Cuticular wax is a complex mixture of very long-chain fatty acids (VLCFAs) and their derivatives that constitute a natural barrier against biotic and abiotic stresses on the aerial surface of terrestrial plants. In tea plants, leaf cuticular wax also contributes to the unique flavor and quality of tea products. However, the mechanism of wax formation in tea cuticles is still unclear. The cuticular wax content of 108 germplasms (Niaowang species) was investigated in this study. The transcriptome analysis of germplasms with high, medium, and low cuticular wax content revealed that the expression levels of CsKCS3 and CsKCS18 were strongly associated with the high content of cuticular wax in leaves. Hence, silencing CsKCS3 and CsKCS18 using virus-induced gene silencing (VIGS) inhibited the synthesis of cuticular wax and caffeine in tea leaves, indicating that expression of these genes is necessary for the synthesis of cuticular wax in tea leaves. The findings contribute to a better understanding of the molecular mechanism of cuticular wax formation in tea leaves. The study also revealed new candidate target genes for further improving tea quality and flavor and cultivating high-stress-resistant tea germplasms.

The Overexpression of Oryza sativa L. CYP85A1 Promotes Growth and Biomass Production in Transgenic Trees.

Brassinosteroids (BRs) are important hormones that play crucial roles in plant growth, reproduction, and responses to abiotic and biotic stresses. CYP85A1 is a castasterone (CS) synthase that catalyzes C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to CS, after which CS is converted into brassinolide (BL) in a reaction catalyzed by CYP85A2. Here, we report the functional characteristics of rice (Oryza sativa L.) OsCYP85A1. Constitutive expression of OsCYP85A1 driven by the cauliflower mosaic virus 35S promoter increased endogenous BR levels and significantly promoted growth and biomass production in three groups of transgenic Populus tomentosa lines. The plant height and stem diameter of the transgenic poplar plants were increased by 17.6% and 33.6%, respectively, in comparison with control plants. Simultaneously, we showed that expression of OsCYP85A1 enhanced xylem formation in transgenic poplar without affecting cell wall thickness or the composition of cellulose. Our findings suggest that OsCYP85A1 represents a potential target candidate gene for engineering fast-growing trees with improved wood production.

Comparative transcriptomic and proteomic analysis of nutritional quality-related molecular mechanisms of 'Qianmei 419' and 'Qianfu 4' varieties of Camellia sinensis.

In this study, the content of main nutrients in 'QianFu No. 4' were significantly higher than 'QianMei 419.'Transcriptome and proteome were combined to provide new insight of the molecular mechanisms linked to nutritional quality of 'QianFu No. 4' and 'QianMei 419' by leaf function analysis, RNA sequencing and isobaric tags for relative and absolute quantification techniques.A total of 23,813 genes and 361 proteins exhibited differential expression level in 'QianMei 419' when compared with 'QianFu No. 4'. These genes and proteins revealed that the pathway of flavonoids biosynthesis, caffeine metabolism, theanine biosynthesis and amino acid metabolism were linked to nutritional quality of tea. Our results provided transcriptomics and proteomics information with respect to the molecular mechanisms of nutritional changes of tea, identified key genes and proteins that associated with the metabolism and accumulation of nutrients, and helped clarify the molecular mechanisms of nutrient differences.

Preparation of Zn-Gly and Se-Gly and Their Effects on the Nutritional Quality of Tea (Camellia sinensis).

BACKGROUND: Micronutrient malnutrition affects millions of people due to a lack of Zn and Se. METHODS: The process conditions for the manufacture of glycine-chelated sodium selenite (Se-Gly) and zinc sulfate heptahydrate (Zn-Gly) were studied. The effects of ligand concentration, pH, reaction ratio, reaction temperature, and reaction time on fertilizer stability were assessed. The effects of Zn-Gly and Se-Gly on tea plants were determined. RESULTS: Orthogonal experiments showed that the optimal preparation conditions for Zn-Gly (75.80 % Zn chelation rate) were pH 6.0, ligand concentration 4 %, reaction ratio 1:2, reaction time 120 min, reaction temperature 70 ℃. The optimal preparation conditions for Se-Gly (56.75 % Se chelation rate) were pH 6.0, ligand concentration 10%, reaction ratio 2:1, reaction time 40 min, temperature 50 ℃. Each chelate was completely soluble in water and verified by infrared spectroscopy and ultraviolet spectroscopy. CONCLUSIONS: Zn-Gly and Se-Gly increased the Zn and Se content in tea plants, and foliar application was more effective than soil application. Combined application of Zn-Gly and Se-Gly was more effective than Zn-Gly or Se-Gly alone. Our findings suggest that Zn-Gly and Se-Gly provide a convenient method of addressing human Zn and Se deficiency.

Genome-Wide Investigation and Functional Analysis Reveal That CsGeBP4 Is Required for Tea Plant Trichome Formation.

Tea plant trichomes not only contribute to the unique flavor and high quality of tea products but also provide physical and biochemical defenses for tea plants. Transcription factors play crucial roles in regulating plant trichome formation. However, limited information about the regulatory mechanism of transcription factors underlying tea plant trichome formation is available. Here, the investigation of trichome phenotypes among 108 cultivars of Yunwu Tribute Tea, integrated with a transcriptomics analysis of both hairy and hairless cultivars, revealed the potential involvement of CsGeBPs in tea trichome formation. In total, six CsGeBPs were identified from the tea plant genome, and their phylogenetic relationships, as well as the structural features of the genes and proteins, were analyzed to further understand their biological functions. The expression analysis of CsGeBPs in different tissues and in response to environmental stresses indicated their potential roles in regulating tea plant development and defense. Moreover, the expression level of CsGeBP4 was closely associated with a high-density trichome phenotype. The silencing of CsGeBP4 via the newly developed virus-induced gene silencing strategy in tea plants inhibited trichome formation, indicating that CsGeBP4 was required for this process. Our results shed light on the molecular regulatory mechanisms of tea trichome formation and provide new candidate target genes for further research. This should lead to an improvement in tea flavor and quality and help in breeding stress-tolerant tea plant cultivars.

Genome-wide characterization of the U-box gene in Camellia sinensis and functional analysis in transgenic tobacco under abiotic stresses.

Plants U-box genes are crucial for plant survival, and they extensively regulate plant growth, reproduction and development as well as coping with stress and other processes. In this study, we identified 92 CsU-box genes through genome-wide analysis in the tea plant (Camellia sinensis), all of them contained the conserved U-box domain and were divided into 5 groups, which supported by the further genes structure analysis. The expression profiles in eight tea plant tissues and under abiotic and hormone stresses were analyzed using the TPIA database. 7 CsU-box genes (CsU-box27/28/39/46/63/70/91) were selected to verify and analyze expression patterns under PEG-induced drought and heat stress in tea plant respectively, the qRT-PCR results showed consistent with transcriptome datasets; and the CsU-box39 were further heterologous expressed in tobacco to perform gene function analysis. Phenotypic analyses of overexpression transgenic tobacco seedlings and physiological experiments revealed that CsU-box39 positively regulated the plant response to drought stress. These results lay a solid foundation for studying the biological function of CsU-box, and will provide breeding strategy basis for tea plant breeders.

Study of Camellia sinensis diploid and triploid leaf development mechanism based on transcriptome and leaf characteristics.

Polyploidization results in significant changes in the morphology and physiology of plants, with increased growth rate and genetic gains as the number of chromosomes increases. In this study, the leaf functional traits, photosynthetic characteristics, leaf cell structure and transcriptome of Camellia sinensis were analyzed. The results showed that triploid tea had a significant growth advantage over diploid tea, the leaf area was 59.81% larger, and the photosynthetic capacity was greater. The morphological structure of triploid leaves was significantly different, the xylem of the veins was more developed, the cell gap between the palisade tissue and the sponge tissue was larger and the stomata of the triploid leaves were also larger. Transcriptome sequencing analysis revealed that in triploid tea, the changes in leaf morphology and physiological characteristics were affected by the expression of certain key regulatory genes. We identified a large number of genes that may play important roles in leaf development, especially genes involved in photosynthesis, cell division, hormone synthesis and stomata development. This research will enhance our understanding of the molecular mechanism underlying tea and stomata development and provide a basis for molecular breeding of high-quality and high-yield tea varieties.

Chromosome-scale genome assembly of Camellia sinensis combined with multi-omics provides insights into its responses to infestation with green leafhoppers.

The tea plant (Camellia sinensis) is an important economic crop, which is becoming increasingly popular worldwide, and is now planted in more than 50 countries. Tea green leafhopper is one of the major pests in tea plantations, which can significantly reduce the yield and quality of tea during the growth of plant. In this study, we report a genome assembly for DuyunMaojian tea plants using a combination of Oxford Nanopore Technology PromethION™ with high-throughput chromosome conformation capture technology and used multi-omics to study how the tea plant responds to infestation with tea green leafhoppers. The final genome was 3.08 Gb. A total of 2.97 Gb of the genome was mapped to 15 pseudo-chromosomes, and 2.79 Gb of them could confirm the order and direction. The contig N50, scaffold N50 and GC content were 723.7 kb, 207.72 Mb and 38.54%, respectively. There were 2.67 Gb (86.77%) repetitive sequences, 34,896 protein-coding genes, 104 miRNAs, 261 rRNA, 669 tRNA, and 6,502 pseudogenes. A comparative genomics analysis showed that DuyunMaojian was the most closely related to Shuchazao and Yunkang 10, followed by DASZ and tea-oil tree. The multi-omics results indicated that phenylpropanoid biosynthesis, α-linolenic acid metabolism, flavonoid biosynthesis and 50 differentially expressed genes, particularly peroxidase, played important roles in response to infestation with tea green leafhoppers (Empoasca vitis Göthe). This study on the tea tree is highly significant for its role in illustrating the evolution of its genome and discovering how the tea plant responds to infestation with tea green leafhoppers will contribute to a theoretical foundation to breed tea plants resistant to insects that will ultimately result in an increase in the yield and quality of tea.

High-quality chromosome-level genome assembly of Litsea coreana L. provides insights into Magnoliids evolution and flavonoid biosynthesis.

The magnoliid Litsea coreana has been the subject of a substantial amount of research owing to its production of many flavonoid metabolites, high food processing value, and a controversial phylogenetic position. For this study, we assembled a high-grade genome at the chromosome scale and annotation of L. coreana that was anchored to 12 chromosomes. The total genome was 1139.45 Mb, while the N50 scaffold was 97.18 Mb long. The analysis of phylogenetic trees constructed by different methods show that the phylogeny of Magnoliids is inconsistent, indicating that the differentiation process of monocots, eudicots, and Magnoliids still remains in dispute. An ancient whole-genome duplication (WGD) event was shown to have occurred before the Magnoliales and Laurels had differentiated. Subsequently, an independent WGD appeared in the Lauralean lineage. A total of 27 types of flavonoids were detected in all five tissues of L. coreana. Chalcone synthases (CHSs) that are responsible for production of flavonoids have been validated at the bioinformatics level. The retention of comparative genomic analyses of the CHS gene family showed that this family had contracted significantly in L. coreana. Our research further elaborated the evolution of Lauraceae and perfected the genetic basis of flavonoid biosynthesis in L. coreana. SIGNIFICANCE STATEMENT: Provides evidence that determines the evolutionary status of Magnoliids. The chalcone synthase gene family was significantly contracted in Litsea coreana.

Establishment of a Virus-Induced Gene-Silencing (VIGS) System in Tea Plant and Its Use in the Functional Analysis of CsTCS1.

Tea (Camellia sinensis [L.] O. Kuntze) is an important global economic crop and is considered to enhance health. However, the functions of many genes in tea plants are unknown. Virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) is an effective tool for the analysis of gene functions, although this method has rarely been reported in tea plants. In this study, we established an effective VIGS-mediated gene knockout technology to understand the functional identification of large-scale genomic sequences in tea plants. The results showed that the VIGS system was verified by detecting the virus and using a real-time quantitative reverse transcription PCR (qRT-PCR) analysis. The reporter gene CsPOR1 (protochlorophyllide oxidoreductase) was silenced using the vacuum infiltration method, and typical photobleaching and albino symptoms were observed in newly sprouted leaves at the whole plant level of tea after infection for 12 d and 25 d. After optimization, the VIGS system was successfully used to silence the tea plant CsTCS1 (caffeine synthase) gene. The results showed that the relative caffeine content was reduced 6.26-fold compared with the control, and the level of expression of CsPOR1 decreased by approximately 3.12-fold in plants in which CsPOR1 was silenced. These results demonstrate that VIGS can be quickly and efficiently used to analyze the function of genes in tea plants. The successful establishment of VIGS could eliminate the need for tissue culture by providing an effective method to study gene function in tea plants and accelerate the process of functional genome research in tea.

Genome-level diversification of eight ancient tea populations in the Guizhou and Yunnan regions identifies candidate genes for core agronomic traits.

The ancient tea plant, as a precious natural resource and source of tea plant genetic diversity, is of great value for studying the evolutionary mechanism, diversification, and domestication of plants. The overall genetic diversity among ancient tea plants and the genetic changes that occurred during natural selection remain poorly understood. Here, we report the genome resequencing of eight different groups consisting of 120 ancient tea plants: six groups from Guizhou Province and two groups from Yunnan Province. Based on the 8,082,370 identified high-quality SNPs, we constructed phylogenetic relationships, assessed population structure, and performed genome-wide association studies (GWAS). Our phylogenetic analysis showed that the 120 ancient tea plants were mainly clustered into three groups and five single branches, which is consistent with the results of principal component analysis (PCA). Ancient tea plants were further divided into seven subpopulations based on genetic structure analysis. Moreover, it was found that the variation in ancient tea plants was not reduced by pressure from the external natural environment or artificial breeding (nonsynonymous/synonymous = 1.05). By integrating GWAS, selection signals, and gene function prediction, four candidate genes were significantly associated with three leaf traits, and two candidate genes were significantly associated with plant type. These candidate genes can be used for further functional characterization and genetic improvement of tea plants.

Overexpression of DBF-Interactor Protein 6 Containing an R3H Domain Enhances Drought Tolerance in Populus L. (Populus tomentosa).

Drought is the primary disaster that endangers agricultural production, including animal husbandry, and affects the distribution, growth, yield, and quality of crops. Previous study had revealed that DIP, as a potential regulator of DBF activity, played an important role in response to drought stress in maize. In this study, a total of 67 DIPs were identified from seventeen land plants, including six tobacco DIPs (NtDIPs). NtDIP6 gene was further selected as a candidate gene for subsequent experiments based on the phylogenetic analysis and structural analysis. The transgenic tobacco and poplar plants over-expressing NtDIP6 gene were generated using the Agrobacterium- mediated method. Although there was not phenotypic difference between transgenic plants and wild-type plants under normal conditions, overexpression of the NtDIP6 gene in transgenic tobacco and poplar plants enhanced the drought tolerance under drought treatments in comparison with the wild type. The content of antioxidant defense enzymes peroxidase (POD), catalase (CAT), and the photosynthetic rate increased in NtDIP6-Ox transgenic tobacco and poplar plants, while the content of malondialdehyde decreased, suggesting that the overexpression of NtDIP6 enhances the antioxidant capacity of transgenic poplar. Furthermore, the results of qRT-PCR showed that the level of expression of drought-related response genes significantly increased in the NtDIP6-Ox transgenic plants. These results indicated that NtDIP6, as a positive response regulator, improves drought stress tolerance by scavenging superoxide via the accumulation of antioxidant defense enzymes.