High-density genetic map construction and QTL mapping of a zigzag-shaped stem trait in tea plant (Camellia sinensis).

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement.

A key mutation in magnesium chelatase I subunit leads to a chlorophyll-deficient mutant of tea (Camellia sinensis).

Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.

Bulked Segregant RNA-Seq Reveals Different Gene Expression Patterns and Mutant Genes Associated with the Zigzag Pattern of Tea Plants (Camellia sinensis).

The unique zigzag-patterned tea plant is a rare germplasm resource. However, the molecular mechanism behind the formation of zigzag stems remains unclear. To address this, a BC1 genetic population of tea plants with zigzag stems was studied using histological observation and bulked segregant RNA-seq. The analysis revealed 1494 differentially expressed genes (DEGs) between the upright and zigzag stem groups. These DEGs may regulate the transduction and biosynthesis of plant hormones, and the effects on the phenylpropane biosynthesis pathways may cause the accumulation of lignin. Tissue sections further supported this finding, showing differences in cell wall thickness between upright and curved stems, potentially due to lignin accumulation. Additionally, 262 single-nucleotide polymorphisms (SNPs) across 38 genes were identified as key SNPs, and 5 genes related to zigzag stems were identified through homologous gene function annotation. Mutations in these genes may impact auxin distribution and content, resulting in the asymmetric development of vascular bundles in curved stems. In summary, we identified the key genes associated with the tortuous phenotype by using BSR-seq on a BC1 population to minimize genetic background noise.

Comprehensive analysis and expression profiles of the AP2/ERF gene family during spring bud break in tea plant (Camellia sinensis).

BACKGROUND: AP2/ERF transcription factors (AP2/ERFs) are important regulators of plant physiological and biochemical metabolism. Evidence suggests that AP2/ERFs may be involved in the regulation of bud break in woody perennials. Green tea is economically vital in China, and its production value is significantly affected by the time of spring bud break of tea plant. However, the relationship between AP2/ERFs in tea plant and spring bud break remains largely unknown. RESULTS: A total of 178 AP2/ERF genes (CsAP2/ERFs) were identified in the genome of tea plant. Based on the phylogenetic analysis, these genes could be classified into five subfamilies. The analysis of gene duplication events demonstrated that whole genome duplication (WGD) or segmental duplication was the primary way of CsAP2/ERFs amplification. According to the result of the Ka/Ks value calculation, purification selection dominated the evolution of CsAP2/ERFs. Furthermore, gene composition and structure analyses of CsAP2/ERFs indicated that different subfamilies contained a variety of gene structures and conserved motifs, potentially resulting in functional differences among five subfamilies. The promoters of CsAP2/ERFs also contained various signal-sensing elements, such as abscisic acid responsive elements, light responsive elements and low temperature responsive elements. The evidence presented here offers a theoretical foundation for the diverse functions of CsAP2/ERFs. Additionally, the expressions of CsAP2/ERFs during spring bud break of tea plant were analyzed by RNA-seq and grouped into clusters A-F according to their expression patterns. The gene expression changes in clusters A and B were more synchronized with the spring bud break of tea plant. Moreover, several potential correlation genes, such as D-type cyclin genes, were screened out through weighted correlation network analysis (WGCNA). Temperature and light treatment experiments individually identified nine candidate CsAP2/ERFs that may be related to the spring bud break of tea plant. CONCLUSIONS: This study provides new evidence for role of the CsAP2/ERFs in the spring bud break of tea plant, establishes a theoretical foundation for analyzing the molecular mechanism of the spring bud break of tea plant, and contributes to the improvement of tea cultivars.

GoNe encoding a class VIIIb AP2/ERF is required for both extrafloral and floral nectary development in Gossypium.

The floral nectary, first recognized and described by Carl Linnaeus, is a remarkable organ that serves to provide carbohydrate-rich nectar to visiting pollinators in return for gamete transfer between flowers. Therefore, the nectary has indispensable biological significance in plant reproduction and even in evolution. Only two genes, CRC and STY, have been reported to regulate floral nectary development. However, it is still unknown what genes contribute to extrafloral nectary development. Here, we report that a nectary development gene in Gossypium (GoNe), annotated as an APETALA 2/ethylene-responsive factor (AP2/ERF), is responsible for the formation of both floral and extrafloral nectaries. GoNe plants that are silenced via virus-induced gene silencing technology and/or knocked out by Cas9 produce a nectariless phenotype. Point mutation and gene truncation simultaneously in duplicated genes Ne1 Ne2 lead to impaired nectary development in tetraploid cotton. There is no difference in the expression of the CRC and STY genes between the nectary TM-1 and the nectariless MD90ne in cotton. Therefore, the GoNe gene responsible for the formation of floral and extrafloral nectaries may be independent of CRC and STY. A complex mechanism might exist that restricts the nectary to a specific position with different genetic factors. Characterization of these target genes regulating nectary production has provided insights into the development, evolution, and function of nectaries and insect-resistant breeding.

Construction of a high-density genetic map and identification of QTLs related to agronomic and physiological traits in an interspecific (Gossypium hirsutum × Gossypium barbadense) F2 population.

BACKGROUND: Advances in genome sequencing technology, particularly restriction-site associated DNA sequence (RAD-seq) and whole-genome resequencing, have greatly aided the construction of cotton interspecific genetic maps based on single nucleotide polymorphism (SNPs), Indels, and other types of markers. High-density genetic maps can improve accuracy of quantitative trait locus (QTL) mapping, narrow down location intervals, and facilitate identification of the candidate genes. RESULT: In this study, 249 individuals from an interspecific F2 population (TM-1 and Hai7124) were re-sequenced, yielding 6303 high-confidence bin markers spanning 5057.13 cM across 26 cotton chromosomes. A total of 3380 recombination hot regions RHRs were identified which unevenly distributed on the 26 chromosomes. Based on this map, 112 QTLs relating to agronomic and physiological traits from seedling to boll opening stage were identified, including 15 loci associated with 14 traits that contained genes harboring nonsynonymous SNPs. We analyzed the sequence and expression of these ten candidate genes and discovered that GhRHD3 (GH_D10G0500) may affect fiber yield while GhGPAT6 (GH_D04G1426) may affect photosynthesis efficiency. CONCLUSION: Our research illustrates the efficiency of constructing a genetic map using binmap and QTL mapping on the basis of a certain size of the early-generation population. High-density genetic map features high recombination exchanges in number and distribution. The QTLs and the candidate genes identified based on this high-density genetic map may provide important gene resources for the genetic improvement of cotton.

Role of phasiRNAs from two distinct phasing frames of GhMYB2 loci in cis- gene regulation in the cotton genome.

BACKGROUND: Phased small interfering RNA (phasiRNA) is primarily derived from the 22-nt miRNA targeting loci. GhMYB2, a gene with potential roles in cotton fiber cell fate determination, is a target gene of miR828 and miR858 in the generation of phasiRNAs. RESULTS: In the presented work, through the evaluation of phasing scores and phasiRNA distribution pattern, we found that phasiRNAs from GhMYB2 were derived from the 3' cleavage fragments of 22-nt miR828 and 21-nt miR858 respectively. These two miRNA targeting sites initiated two phasing frames on transcripts of one locus. By means of RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we further demonstrated that phasiRNAs derived from the two phasing frames played a role in cis-regulation of GhMYB2. The phasiRNAs derived from GhMYB2 were expressed in the somatic tissues, especially in anther and hypocotyl. We further employed our previous small RNA sequencing data as well as the degradome data of cotton fiber bearing ovules, anthers, hypocotyls and embryogenic calli tissues published in public databases, to validate the expression, phasing pattern and functions of phasiRNAs. CONCLUSIONS: The presenting research provide insights of the molecular mechanism of phasiRNAs in regulation of GhMYB2 loci.

Divergence and evolution of cotton bHLH proteins from diploid to allotetraploid.

BACKGROUND: Polyploidy is considered a major driving force in genome expansion, yielding duplicated genes whose expression may be conserved or divergence as a consequence of polyploidization. RESULTS: We compared the genome sequences of tetraploid cotton (Gossypium hirsutum) and its two diploid progenitors, G. arboreum and G. raimondii, and found that the bHLH genes were conserved over the polyploidization. Oppositely, the expression of the homeolgous gene pairs was diversified. The biased homeologous proportion for bHLH family is significantly higher (64.6%) than the genome wide homeologous expression bias (40%). Compared with cacao (T. cacao), orthologous genes only accounted for a small proportion (41.7%) of whole cotton bHLHs family. The further Ks analysis indicated that bHLH genes underwent at least two distinct episodes of whole genome duplication: a recent duplication (1.0-60.0 million years ago, MYA, 0.005 < Ks < 0.312) and an old duplication (> 60.0 MYA, 0.312 < Ks < 3.0). The old duplication event might have played a key role in the expansion of the bHLH family. Both recent and old duplicated pairs (68.8%) showed a divergent expression profile, indicating specialized functions. The expression diversification of the duplicated genes suggested it might be a universal feature of the long-term evolution of cotton. CONCLUSIONS: Overview of cotton bHLH proteins indicated a conserved and divergent evolution from diploids to allotetraploid. Our results provided an excellent example for studying the long-term evolution of polyploidy.

Genetics and evolution of MIXTA genes regulating cotton lint fiber development.

Cotton, with cellulose-enriched mature fibers, is the largest source of natural textiles. Through a map-based cloning strategy, we isolated an industrially important lint fiber development gene (Li3 ) that encodes an MYB-MIXTA-like transcription factor (MML) on chromosome D12 (GhMML4_D12). Virus-induced gene silencing or decreasing the expression of the GhMML4_D12 gene in n2 NSM plants resulted in a significant reduction in epidermal cell prominence and lint fiber production. GhMML4_D12 is arranged in tandem with GhMML3, another MIXTA gene responsible for fuzz fiber development. These two very closely related MIXTA genes direct fiber initiation production in two specialized cell forms: lint and fuzz fibers. They may control the same metabolic pathways in different cell types. The MIXTAs expanded in Malvaceae during their evolution and produced a Malvaceae-specific family that regulates epidermal cell differentiation, different from the gene family that regulates leaf hair trichome development. Cotton has developed a unique transcriptional regulatory network for fiber development. Characterization of target genes regulating fiber production has provided insights into the molecular mechanisms underlying cotton fiber development and has allowed the use of genetic engineering to increase lint yield by inducing more epidermal cells to develop into lint rather than fuzz fibers.

Mutation of SELF-PRUNING homologs in cotton promotes short-branching plant architecture.

In cotton, the formation of fruiting branches affects both plant architecture and fiber yield. Here, we report map-based cloning of the axillary flowering mutation gene (GbAF) that causes bolls to be borne directly on the main plant stem in Gossypium barbadense, and of the clustered boll mutation gene (cl1) in G. hirsutum. Both mutant alleles were found to represent point mutations at the Cl1 locus. Therefore, we propose that the GbAF mutation be referred to as cl1b. These Cl1 loci correspond to homologs of tomato SELF-PRUNING (SP), i.e. Gossypium spp. SP (GoSP) genes. In tetraploid cottons, single monogenic mutation of either duplicate GoSP gene (one in the A and one in the D subgenome) is associated with the axillary cluster flowering phenotype, although the shoot-indeterminate state of the inflorescence is maintained. By contrast, silencing of both GoSPs leads to the termination of flowering or determinate plants. The architecture of axillary flowering cotton allows higher planting density, contributing to increased fiber yield. Taken together the results provide new insights into the underlying mechanism of branching in cotton species, and characterization of GoSP genes may promote the development of compact cultivars to increase global cotton production.

Rapid mapping and cloning of the virescent-1 gene in cotton by bulked segregant analysis-next generation sequencing and virus-induced gene silencing strategies.

Map-based gene cloning is a vital strategy for the identification of the quantitative trait loci or genes underlying important agronomic traits. The conventional map-based cloning method is powerful but generally time-consuming and labor-intensive. In this context, we introduce an improved bulked segregant analysis method in combination with a virus-induced gene silencing (VIGS) strategy for rapid and reliable gene mapping, identification and functional verification. This method was applied to a multiple recessive marker line of upland cotton, Texas 582 (T582), and identified unique genomic positions harboring mutant loci, showing the reliability and efficacy of this method. The v1 locus was further fine-mapped. Only one gene, GhCHLI, which encodes one of the subunits of Mg chelatase, was differentially down-regulated in T582 compared with TM-1. A point mutation occurred in the AAA+ conserved region of GhCHLI and led to an amino acid substitution. Suppression of its expression by VIGS in TM-1 resulted in a yellow blade phenotype that was similar to T582. This integrated approach provides a paradigm for the rapid mapping and identification of the candidate genes underlying the genetic traits in plants with large and complex genomes in the future.

Small interfering RNAs from bidirectional transcripts of GhMML3_A12 regulate cotton fiber development.

Natural antisense transcripts (NATs) are commonly observed in eukaryotic genomes, but only a limited number of such genes have been identified as being involved in gene regulation in plants. In this research, we investigated the function of small RNA derived from a NAT in fiber cell development. Using a map-based cloning strategy for the first time in tetraploid cotton, we cloned a naked seed mutant gene (N1 ) encoding a MYBMIXTA-like transcription factor 3 (MML3)/GhMYB25-like in chromosome A12, GhMML3_A12, that is associated with fuzz fiber development. The extremely low expression of GhMML3_A12 in N1 is associated with NAT production, driven by its 3' antisense promoter, as indicated by the promoter-driven histochemical staining assay. In addition, small RNA deep sequencing analysis suggested that the bidirectional transcriptions of GhMML3_A12 form double-stranded RNAs and generate 21-22 nt small RNAs. Therefore, in a fiber-specific manner, small RNA derived from the GhMML3_A12 locus can mediate GhMML3_A12 mRNA self-cleavage and result in the production of naked seeds followed by lint fiber inhibition in N1 plants. The present research reports the first observation of gene-mediated NATs and siRNA directly controlling fiber development in cotton.

Identification of centromeric regions on the linkage map of cotton using centromere-related repeats.

Centromere usually contains high-copy-number retrotransposons and satellite repeats, which are difficult to map, clone and sequence. Currently, very little is known about the centromere in cotton. Here, we sequenced a bacterial artificial chromosome (BAC) mapping to the centromeric region and predicted four long-terminal-repeat (LTR) retrotransposons. They were located in the heterochromatic centromeric regions of all 52 pachytene chromosomes in Gossypium hirsutum. Fiber-FISH mapping revealed that these retrotransposons span an area of at least 1.8Mb in the centromeric region. Comparative analysis showed that these retrotransposons generated similar, strong fluorescent signals in the D progenitor Gossypium raimondii but not in the A progenitor Gossypium herbaceum, suggesting that the centromere sequence of tetraploid cotton might be derived from the D progenitor. Centromeric regions were anchored on 13 chromosomes of D-genome sequence. Characterization of these centromere-related repeats and regions will enhance cotton centromere mapping, sequencing and evolutionary studies.

Cotton fiber elongation network revealed by expression profiling of longer fiber lines introgressed with different Gossypium barbadense chromosome segments.

BACKGROUND: Cotton fiber, a highly elongated, thickened single cell of the seed epidermis, is a powerful cell wall research model. Fiber length, largely determined during the elongation stage, is a key property of fiber quality. Several studies using expressed sequence tags and microarray analysis have identified transcripts that accumulate preferentially during fiber elongation. To further show the mechanism of fiber elongation, we used Digital Gene Expression Tag Profiling to compare transcriptome data from longer fiber chromosome introgressed lines (CSILs) containing segments of various Gossypium barbadense chromosomes with data from its recurrent parent TM-1 during fiber elongation (from 5 DPA to 20 DPA). RESULTS: A large number of differentially expressed genes (DEGs) involved in carbohydrate, fatty acid and secondary metabolism, particularly cell wall biosynthesis, were highly upregulated during the fiber elongation stage, as determined by functional enrichment and pathway analysis. Furthermore, DEGs related to hormone responses and transcription factors showed upregulated expression levels in the CSILs. Moreover, metabolic and regulatory network analysis indicated that the same pathways were differentially altered, and distinct pathways exhibited altered gene expression, in the CSILs. Interestingly, mining of upregulated DEGs in the introgressed segments of these CSILs based on D-genome sequence data showed that these lines were enriched in glucuronosyltransferase, inositol-1, 4, 5-trisphosphate 3-kinase and desulfoglucosinolate sulfotransferase activity. These results were similar to the results of transcriptome analysis. CONCLUSIONS: This report provides an integrative network about the molecular mechanisms controlling fiber length, which are mainly tied to carbohydrate metabolism, cell wall biosynthesis, fatty acid metabolism, secondary metabolism, hormone responses and Transcription factors. The results of this study provide new insights into the critical factors associated with cell elongation and will facilitate further research aimed at understanding the mechanisms underlying cotton fiber elongation.

CsEXL3 regulate mechanical harvest-related droopy leaves under the transcriptional activation of CsBES1.2 in tea plant.

Due to a labor shortage, the mechanical harvesting of tea plantations has become a focal point. However, mechanical harvest efficiency was hampered by droopy leaves, leading to a high rate of broken tea shoots and leaves. Here, we dissected the genetic structure of leaf droopiness in tea plants using genome-wide association studies (GWAS) on 146 accessions, combined with transcriptome from two accessions with contrasting droopy leaf phenotypes. A set of 16 quantitative trait loci (QTLs) containing 54 SNPs and 34 corresponding candidate genes associated with droopiness were then identified. Among these, CsEXL3 (EXORDIUM-LIKE 3) from Chromosome 1 emerged as a candidate gene. Further investigations revealed that silencing CsEXL3 in tea plants resulted in weaker vascular cell malformation and brassinosteroid-induced leaf droopiness. Additionally, brassinosteroid signal factor CsBES1.2 was proved to participate in CsEXL3-induced droopiness and vascular cell malformation via using the CsBES1.2-silencing tea plant. Notably, CsBES1.2 bound on the E-box of CsEXL3 promoter to transcriptionally activate CsEXL3 expression as CUT&TAG based ChIP-qPCR and ChIP-seq suggested in vivo as well as EMSA and Y1H indicated in vitro. Furthermore, CsEXL3 instead of CsBES1.2 decreased lignin content and the expressing levels of lignin biosynthesis genes. Overall, our findings suggest that CsEXL3 regulates droopy leaves, partially through the transcriptional activation of CsBES1.2, with the potential to improve mechanical harvest efficiency in tea plantations.

Identification of QTL controlling volatile terpene contents in tea plant (Camellia sinensis) using a high-aroma 'Huangdan' x 'Jinxuan' F1 population.

Aroma is an important factor affecting the character and quality of tea. The improvement of aroma trait is a crucial research direction of tea plant breeding. Volatile terpenes, as the major contributors to the floral odors of tea products, also play critical roles in the defense responses of plants to multiple stresses. However, previous studies have largely focused on the aroma formation during the manufacture of tea or the comparison of raw tea samples. The mechanisms causing different aroma profiles between tea cultivars have remained underexplored. In the current study, a high-density genetic linkage map of tea plant was constructed based on an F1 population of 'Huangdan' × 'Jinxuan' using genotyping by sequencing. This linkage map covered 1754.57 cM and contained 15 linkage groups with a low inter-marker distance of 0.47 cM. A total of 42 QTLs associated with eight monoterpene contents and 12 QTLs associated with four sesquiterpenes contents were identified with the average PVE of 12.6% and 11.7% respectively. Furthermore, six candidate genes related to volatile terpene contents were found in QTL cluster on chromosome 5 by RNA-seq analysis. This work will enrich our understanding of the molecular mechanism of volatile terpene biosynthesis and provide a theoretical basis for tea plant breeding programs for aroma quality improvement.

Deeply functional identification of TCS1 alleles provides efficient technical paths for low-caffeine breeding of tea plants.

Caffeine is an important functional component in tea, which has the effect of excitement and nerve stimulation, but excessive intake can cause insomnia and dysphoria. Therefore, the production of tea with low-caffeine content can meet the consumption needs of certain people. Here, in addition to the previous alleles of the tea caffeine synthase (TCS1) gene, a new allele (TCS1h) from tea germplasms was identified. Results of in vitro activity analysis showed that TCS1h had both theobromine synthase (TS) and caffeine synthase (CS) activities. Site-directed mutagenesis experiments of TCS1a, TCS1c, and TCS1h demonstrated that apart from the 225th amino acid residue, the 269th amino acid also determined the CS activity. GUS histochemical analysis and dual-luciferase assay indicated the low promoter activity of TCS1e and TCS1f. In parallel, insertion and deletion mutations in large fragments of alleles and experiments of site-directed mutagenesis identified a key cis-acting element (G-box). Furthermore, it was found that the contents of purine alkaloids were related to the expression of corresponding functional genes and alleles, and the absence or presence and level of gene expression determined the content of purine alkaloids in tea plants to a certain extent. In summary, we concluded TCS1 alleles into three types with different functions and proposed a strategy to effectively enhance low-caffeine tea germplasms in breeding practices. This research provided an applicable technical avenue for accelerating the cultivation of specific low-caffeine tea plants.

The potential effects of chlorophyll-deficient mutation and tree_age on the accumulation of amino acid components in tea plants.

Albino tea has been receiving growing attention on the tea market due to its attractive appearance and fresh taste, mainly caused by high amino acid contents. Here, variations in the contents of five free amino acids in relation to pigment contents and tree age in two hybrid populations'Longjin 43'(♀) × 'Baijiguan'(♂) and 'Longjin 43'(♀) ×'Huangjinya'(♂) with 334 first filial generation individuals including chlorophyll-deficient and normal tea plants were investigated. The data showed that the contents of main amino acids in all filial generation gradually decreased as plant age increased. Principal component analysis indicated that the amino acid content of individual plant tended to be stable with the growth of plants. Correlation analysis clarified that several main amino acids were significantly negatively correlated with chlorophyll a, chlorophyll b and carotenoid contents. Our results showed that the accumulation of amino acids in tea plant was closely related to leaf color variation and the tree age during growing period.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Cotton genes GhMML1 and GhMML2 control trichome branching when ectopically expressed in tobacco.

Trichomes exhibit extraordinary diversity in shape, ultrastructure, distribution, secretion capability, biological functions, and morphological differences, which are strongly associated with their multifunction. Previous researches showed MIXTA-like transcription factors involved in regulating trichome initiation and patterning via forming MYB-bHLH-WD40 transcriptional activator complex to induce the expression of downstream genes. Here, we report the characteristics and role of GhMML1 and GhMML2, members of subgroup 9 of the R2R3-type MYB TFs. GhMML1 and GhMML2 were preferentially targeted to the nucleus and prominently expressed in the early stage during fiber development. Ectopic expression of GhMML1 and GhMML2 respectively in the transgenic tobacco plants changed the morphological characteristics of leaf trichomes; that is, the unbranched trichomes turned into multiple branched, and in the meantime, the density of trichomes was reduced on the surface of the leaf. Y2H and LCI assay revealed that both GhMML1 and GhMML2 could physically interact with a bZIP transcription factor family protein (GhbZIP) in vivo and in vitro. It has been reported that GhbZIP's homolog TAG3 in Arabidopsis is involved in the asymmetric growth of leaves and flowers via direct interaction with BOP1. Taken together, our results demonstrated that two MYB MIXTA-like proteins, GhMML1 and GhMML2, together with GhbZIP might form a multimeric complex to involve in trichome development. This study highlights the importance of MIXTA-like genes from TF subgroup 9 and will help to uncover the molecular mechanism underlying differential trichomes and their development.