An R-R-type MYB transcription factor promotes non-climacteric pepper fruit carotenoid pigment biosynthesis.

Carotenoids are major accessory pigments in the chloroplast, and they also act as phytohormones and volatile compound precursors to influence plant development and confer characteristic colours, affecting both the aesthetic and nutritional value of fruits. Carotenoid pigmentation in ripening fruits is highly dependent on developmental trajectories. Transcription factors incorporate developmental and phytohormone signalling to regulate the biosynthesis process. By contrast to the well-established pathways regulating ripening-related carotenoid biosynthesis in climacteric fruit, carotenoid regulation in non-climacteric fruit is poorly understood. Capsanthin is the primary carotenoid of non-climacteric pepper (Capsicum) fruit; its biosynthesis is tightly associated with fruit ripening, and it confers red pigmentation to the ripening fruit. In the present study, using a coexpression analysis, we identified an R-R-type MYB transcription factor, DIVARICATA1, and demonstrated its role in capsanthin biosynthesis. DIVARICATA1 encodes a nucleus-localised protein that functions primarily as a transcriptional activator. Functional analyses showed that DIVARICATA1 positively regulates carotenoid biosynthetic gene (CBG) transcript levels and capsanthin levels by directly binding to and activating CBG promoter transcription. Furthermore, an association analysis revealed a significant positive association between DIVARICATA1 transcription level and capsanthin content. ABA promotes capsanthin biosynthesis in a DIVARICATA1-dependent manner. Comparative transcriptomic analysis of DIVARICATA1 in Solanaceae plants showed that its function likely differs among species. Moreover, the pepper DIVARICATA1 gene could be regulated by the ripening regulator MADS-RIN. The present study illustrates the transcriptional regulation of capsanthin biosynthesis and offers a target for breeding peppers with high red colour intensity.

The Physiology of Postharvest Tea (Camellia sinensis) Leaves, According to Metabolic Phenotypes and Gene Expression Analysis.

Proper postharvest storage preserves horticultural products, including tea, until they can be processed. However, few studies have focused on the physiology of ripening and senescence during postharvest storage, which affects the flavor and quality of tea. In this study, physiological and biochemical indexes of the leaves of tea cultivar 'Yinghong 9' preserved at a low temperature and high relative humidity (15-18 °C and 85-95%, PTL) were compared to those of leaves stored at ambient conditions (24 ± 2 °C and relative humidity of 65% ± 5%, UTL). Water content, chromatism, chlorophyll fluorescence, and key metabolites (caffeine, theanine, and catechins) were analyzed over a period of 24 h, and volatilized compounds were determined after 24 h. In addition, the expression of key biosynthesis genes for catechin, caffeine, theanine, and terpene were quantified. The results showed that water content, chromatism, and chlorophyll fluorescence of preserved leaves were more similar to fresh tea leaves than unpreserved tea leaves. After 24 h, the content of aroma volatiles and caffeine significantly increased, while theanine decreased in both groups. Multiple catechin monomers showed distinct changes within 24 h, and EGCG was significantly higher in preserved tea. The expression levels of CsFAS and CsTSI were consistent with the content of farnesene and theanine, respectively, but TCS1 and TCS2 expression did not correlate with caffeine content. Principal component analysis considered results from multiple indexes and suggested that the freshness of PTL was superior to that of UTL. Taken together, preservation conditions in postharvest storage caused a series of physiological and metabolic variations of tea leaves, which were different from those of unpreserved tea leaves. Comprehensive evaluation showed that the preservation conditions used in this study were effective at maintaining the freshness of tea leaves for 2-6 h. This study illustrates the metabolic changes that occur in postharvest tea leaves, which will provide a foundation for improvements to postharvest practices for tea leaves.

Dihydromyricetin Imbues Antiadipogenic Effects on 3T3-L1 Cells via Direct Interactions with 78-kDa Glucose-Regulated Protein.

BACKGROUND: Obesity is among the most serious public health problems worldwide, with few safe pharmaceutical interventions. Natural products have become an important source of potential anti-obesity therapeutics. Dihydromyricetin (DHM) exerts antidiabetic effects. The biochemical target of DHM, however, has been unknown. It is crucial to identify the biochemical target of DHM for elucidating its physiological function and therapeutic value. OBJECTIVES: The objective of this study was to identify the biochemical target of DHM. METHODS: An abundant antiadipogenic flavanonol was extracted from the herbal plant Ampelopsis grossedentata through bioassay-guided fractionation and characterized with high-resolution LC-MS and 1H and 13C nuclear magnetic resonance. Antiadipogenic experiments were done with mouse 3T3-L1 preadipocytes. A biochemical target of the chemical of interest was identified with drug affinity responsive target stability assay. Direct interactions between the chemical of interest and the protein target in vitro were predicted with molecular docking and subsequently confirmed with surface plasmon resonance. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ), which is associated with 78-kDa glucose-regulated protein (GRP78), were measured with real-time qPCR. RESULTS: DHM was isolated, purified, and structurally characterized. Cellular studies showed that DHM notably reduced intracellular oil droplet formation in 3T3-L1 cells with a median effective concentration of 294 µM (i.e., 94 µg/mL). DHM targeted the ATP binding site of GRP78, which is associated with adipogenesis. An equilibrium dissociation constant between DHM and GRP78 was 21.8 µM. In 3T3-L1 cells upon treatment with DHM at 50 µM (i.e., 16 µg/mL), the expression level of PPARγ was downregulated to 53.9% of the solvent vehicle control's level. CONCLUSIONS: DHM targets GRP78 in vitro. DHM is able to reduce lipid droplet formation in 3T3-L1 cells through a mode of action that is plausibly associated with direct interactions between GRP78 and DHM, which is a step forward in determining potential applications of DHM as an anti-obesity agent.

Natural variations in the MYB transcription factor MYB31 determine the evolution of extremely pungent peppers.

Plants produce countless specialized metabolites crucial for their development and fitness, and many are useful bioactive compounds. Capsaicinoids are intriguing genus-specialized metabolites that confer a pungent flavor to Capsicum fruits, and they are widely applied in different areas. Among the five domesticated Capsicum species, Capsicum chinense has a high content of capsaicinoids, which results in an extremely hot flavor. However, the species-specific upregulation of capsaicinoid-biosynthetic genes (CBGs) and the evolution of extremely pungent peppers are not well understood. We conducted genetic and functional analyses demonstrating that the quantitative trait locus Capsaicinoid1 (Cap1), which is identical to Pun3 contributes to the level of pungency. The Cap1/Pun3 locus encodes the Solanaceae-specific MYB transcription factor MYB31. Capsicum species have evolved placenta-specific expression of MYB31, which directly activates expression of CBGs and results in genus-specialized metabolite production. The capsaicinoid content depends on MYB31 expression. Natural variations in the MYB31 promoter increase MYB31 expression in C. chinense via the binding of the placenta-specific expression of transcriptional activator WRKY9 and augmentation of CBG expression, which promotes capsaicinoid biosynthesis. Our findings provide insights into the evolution of extremely pungent C. chinense, which is due to natural variations in the master regulator, and offers targets for engineering or selecting flavor in Capsicum.

Transcriptomic Insights into the Enhanced Aroma of Guangdong Oolong Dry Tea (Camellia sinensis cv. Yashixiang Dancong) in Winter.

Winter dry tea (WDT) exhibits a more intense and lasting aroma compared to dry tea from other seasons; however, this conclusion is solely based on sensory outcomes and lacks corroborative theoretical evidence. Our study aimed to analyze the aroma compounds in WDT and investigate the causes behind the formation of WDT's aroma by analyzing the volatile organic compounds (VOCs) in WDT, spring dry tea (SDT), winter fresh leaves (WFLs) and spring fresh leaves (SFLs) by gas chromatography-mass spectrometry (GC-MS), complemented by an analysis of gene expression pertinent to WFLs and SFLs by using transcriptomic analysis. The results revealed a significant increase in total VOCs in WDT compared to SDT, with WDT exhibiting distinct woody aromas as indicated by a higher α-muurolene content. In WFL, the contents of aldehydes and ketones were richer than those in SFL. Notably, the study found that UDP-glycosyltransferase genes in WFLs were significantly up-regulated, potentially promoting the synthesis of terpene glycosides. These terpene glycosides can release terpene aroma compounds during processing, contributing significantly to the intense and lasting aroma of WDT. Overall, this research provides valuable insights into the mechanism behind aroma formation in Guangdong oolong tea harvested during winter.

Comprehensive analysis of fresh tea (Camellia sinensis cv. Lingtou Dancong) leaf quality under different nitrogen fertilization regimes.

Our study investigated the impact of nitrogen fertilization at 0, 150, 300, and 450 kg/ha on the non-volatile and volatile substances, as well as gene expression in fresh leaves from Lingtou tea plants. We found that applying nitrogen at 450 kg/ha notably increased total polyphenols (TPs) and free amino acids (AAs) while decreasing the TP to AA ratio (TP/AA) and total catechins (TC) contents. Chlorophyll, caffeine (CAF) and theanine accumulated to a greater extent with nitrogen application rates of 150, 300, and 450 kg/ha, respectively, six substances - TP, CAF, TC, theanine, epigallocatechin (EGC), and AA - as key contributors to the taste quality of LTDC. Additionally, five substances with variable importance in projections (VIP) ≥ 1 and odor activation values (OAV) ≥ 1, notably linalool and cis-linalool oxide (furanoid), significantly contributed to the tea's overall aroma. Furthermore, applying 300 kg/ha nitrogen upregulated the dihydroflavonol reductase (DFR)gene, likely causing catechin decrease.

Microplastics in tea from planting to the final tea product: Traceability, characteristics and dietary exposure risk analysis.

Tea, sold as tea bags or loose tea, is a popular drink worldwide. We quantified microplastics in loose tea during various stages of production, from planting to processing and brewing. The quantity of microplastics in tea ranged from (70-3472 pcs/kg), with the highest abundance detected during processing, mainly in the rolling stage (2266 ± 1206 pcs/kg tea). Scanning electron microcopy revealed scratches and pits on the surface of microplastics fibers from tea plantation soil and processed tea, and their degradation was characterized by cracks and fractures. Exposure risks, based on an estimated dietary intake of 0.0538-0.0967 and 0.0101-0.0181 pcs /kg body weight /day for children and adults, respectively, are considered very low. This study not only evaluates the extent of research on microplastics pollution in tea, but also assess the risk of people's exposure to microplastics through drinking tea.

The Effects of Nitrogen Fertilizer on the Aroma of Fresh Tea Leaves from Camellia sinensis cv. Jin Xuan in Summer and Autumn.

Nitrogen fertilization level and harvesting season significantly impact tea aroma quality. In this study, we analyzed the volatile organic compounds of fresh Jin Xuan (JX) tea leaves under different nitrogen application levels (N0, N150, N300, N450) during summer and autumn. A total of 49 volatile components were identified by gas chromatography-mass spectrometry (GC-MS). Notably, (E)-2-hexenal, linalool, and geraniol were the main contributors to the aroma of fresh JX leaves. The no-nitrogen treatment (N0) presented the greatest quantity and variety of volatiles in both seasons. A greater difference in volatile compounds was observed between nitrogen treatments in summer vs. autumn. The N0 treatment had a greater total volatile concentration in summer, while the opposite was observed in the nitrogen application treatments (N150, N300, N450). Summer treatments appeared best suited to black tea production. The concentration of herbaceous aroma-type volatiles was higher in summer, while the concentration of floral volatiles was higher in autumn. Volatile concentrations were highest in the N0 and N450 treatments in autumn and appeared suitable for making black tea and oolong tea. Overall, this research provides valuable insights into how variations in N application rates across different harvesting seasons impact the aroma characteristics of tea leaves.

Characterization of Small Molecule-Protein Interactions Using SPR Method.

Surface plasmon resonance (SPR) is an optical phenomenon being used to monitor molecular binding events. With the advantages of being label-free, real-time, and sensitive, SPR assays have become one of the most commonly used techniques to measure binding kinetics, affinity, specificity, and concentration of molecular interactions. In an SPR experiment to measure small molecule-protein interactions, the protein is immobilized on the biosensor surface, while the small molecule flows through the surface of the sensor chip. The interactions between the small molecules and proteins are monitored by subsequent changes in the refractive index and quantified with resonance units. In this chapter, we have utilized an SPR assay to study the interaction of flavonoids and the glucose-regulated protein 78. Assay steps are detailed for immobilization optimization, SPR instrument setup, operation, sample injection, and affinity data analysis.

Virus-Induced Gene Silencing in the Tea Plant (Camellia sinensis).

The recent availability of a number of tea plant genomes has sparked substantial interest in using reverse genetics to explore gene function in tea (Camellia sinensis). However, a hurdle to this is the absence of an efficient transformation system, and virus-induced gene silencing (VIGS), a transient transformation system, could be an optimal choice for validating gene function in the tea plant. In this study, phytoene desaturase (PDS), a carotenoid biosynthesis gene, was used as a reporter to evaluate the VIGS system. The injection sites of the leaves (leaf back, petiole, and stem) for infiltration were tested, and the results showed that petiole injection had the most effective injection, without leading to necrotic lesions that cause the leaves to drop. Tea leaves were inoculated with Agrobacterium harboring a tobacco rattle virus plasmid (pTRV2) containing a CsPDS silencing fragment. The tea leaves exhibited chlorosis symptoms 7-14 days after inoculation, depending on the cultivar. In the chlorosis plants, the coat protein (CP) of tobacco rattle virus (TRV) was detected and coincided with the lower transcription of CsPDS and reduced chlorophyll content compared with the empty vector control, with 81.82% and 54.55% silencing efficiency of 'LTDC' and 'YSX', respectively. These results indicate that the VIGS system with petiole injection could quickly and effectively silence a gene in tea plants.

The Cultivar Effect on the Taste and Aroma Substances of Hakka Stir-Fried Green Tea from Guangdong.

The flavor and quality of tea largely depends on the cultivar from which it is processed; however, the cultivar effect on the taste and aroma characteristics of Hakka stir-fried green tea (HSGT) has received little attention. High-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and sensory evaluations were used to detect and predict the essential taste and aroma-contributing substances of HSGTs made from Huangdan (HD), Meizhan (MZ) and Qingliang Mountain (QL) cultivars. Orthogonal partial least squares data analysis (OPLS-DA) ranked four substances that putatively distinguished the tastes of the HSGTs, epigallocatechin gallate (EGCG) > theanine > epigallocatechin (EGC) > epicatechin gallate (ECG). Ten substances with variable importance in projections (VIPs) ≥ 1 and odor activation values (OAVs) ≥ 1 contributed to their overall aromas, with geranylacetone having the most significant effect on HD (OAV 1841), MZ (OAV 4402), and QL (OAV 1211). Additionally, sensory evaluations found that HD was relatively equivalent to QL in quality, and both were superior to MZ. HD had a distinct floral aroma, MZ had a distinct fried rice aroma, and QL had a balance of fried rice and fresh aromas. The results provide a theoretical framework for evaluating the cultivar effect on the quality of HSGT and put forward ideas for future HSGT cultivar development.

The Capsicum MYB31 regulates capsaicinoid biosynthesis in the pepper pericarp.

Pepper (Capsicum) are consumed worldwide as vegetables and food additives due to their pungent taste. Capsaicinoids are the bioactive compounds that confer the desired pungency to pepper fruits. Capsaicinoid biosynthesis was thought to occur exclusively in fruit placenta. Recently, biosynthesis in the pericarp of extremely pungent varieties was discovered, however, the mechanism of capsaicinoid biosynthesis regulation in the pericarp remains largely unknown. Here, the capsaicinoid contents of placenta and pericarp were analyzed. The results indicated that the Capsicum chinense pericarp accumulated a vast amount of capsaicinoids. Expression of the master regulator MYB31 and capsaicinoid biosynthesis genes (CBGs) were significantly upregulated in the pericarp in C. chinense accessions compared to accessions in other tested species. Moreover, in fruit of extremely-pungent 'Trinidad Moruga Scorpion' (C. chinense) and low-pungent '59' inbred line (C. annuum), the capsaicinoid accumulation patterns in the pericarp were consistent with expression levels of CBGs and MYB31. Silencing MYB31 in 'Trinidad Moruga Scorpion' pericarp leads to a significantly decreased CBGs transcription level and capsaicinoids content. Taken together, our results provide insights into the molecular mechanism arising from the expression of MYB31 in the pericarp that results in exceedingly hot peppers.

Genome-wide analysis of histone acetyltransferase and histone deacetylase families and their expression in fruit development and ripening stage of pepper (Capsicum annuum).

The fruit development and ripening process involve a series of changes regulated by fine-tune gene expression at the transcriptional level. Acetylation levels of histones on lysine residues are dynamically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), which play an essential role in the control of gene expression. However, their role in regulating fruit development and ripening process, especially in pepper (Capsicum annuum), a typical non-climacteric fruit, remains to understand. Herein, we performed genome-wide analyses of the HDAC and HAT family in the pepper, including phylogenetic analysis, gene structure, encoding protein conserved domain, and expression assays. A total of 30 HAT and 15 HDAC were identified from the pepper genome and the number of gene differentiation among species. The sequence and phylogenetic analysis of CaHDACs and CaHATs compared with other plant HDAC and HAT proteins revealed gene conserved and potential genus-specialized genes. Furthermore, fruit developmental trajectory expression profiles showed that CaHDAC and CaHAT genes were differentially expressed, suggesting that some are functionally divergent. The integrative analysis allowed us to propose CaHDAC and CaHAT candidates to be regulating fruit development and ripening-related phytohormone metabolism and signaling, which also accompanied capsaicinoid and carotenoid biosynthesis. This study provides new insights into the role of histone modification mediate development and ripening in non-climacteric fruits.

Specific-Locus Amplified Fragment Sequencing (SLAF-Seq) as High-Throughput SNP Genotyping Methods.

Most plant agronomic traits are quantitatively inherited. Identification of quantitative trait loci (QTL) is a challenging target for most scientists and crop breeders as large-scale genotyping is difficult. Molecular marker technology has continuously evolved from hybridization-based technology to PCR-based technology, and finally, sequencing-based high-throughput single-nucleotide polymorphisms (SNPs). High-throughput sequencing technologies can provide strategies for sequence-based SNP genotyping. Here we describe the SLAF-seq that can be applied as the SNP genotyping approach. The high-throughput SNP genotyping methods will prove useful for the construction of high-density genetic maps and identification of QTLs for their deployment in plant breeding and facilitate genome-wide selection (GWS) and genome-wide association studies (GWAS).

Genome-wide identification of the tea plant bHLH transcription factor family and discovery of candidate regulators of trichome formation.

Leaf trichomes play vital roles in plant resistance and the quality of tea. Basic helix-loop-helix (bHLH) transcription factors (TFs) play an important role in regulating plant development and growth. In this study, a total of 134 CsbHLH proteins were identified in the Camellia sinensis var. sinensis (CSS) genome. They were divided into 17 subgroups according to the Arabidopsis thaliana classification. Phylogenetic tree analysis indicated that members of subgroups IIIc-I and IIIc-II might be associated with trichome formation. The expression patterns of CsbHLH116, CsbHLH133, CsbHLH060, CsbHLH028, CsbHLH024, CsbHLH112 and CsbHLH053 from clusters 1, 3 and 5 were similar to the trichome distribution in tea plants. CsbHLH024 and CsbHLH133 were located in the cell nucleus and possessed transcriptional activation ability. They could interact with CsTTG1, which is a regulator of tea trichome formation. This study provides useful information for further research on the function of CsbHLHs in trichome formation.

Systematic Analysis of the R2R3-MYB Family in Camellia sinensis: Evidence for Galloylated Catechins Biosynthesis Regulation.

The R2R3-MYB transcription factor (TF) family regulates metabolism of phenylpropanoids in various plant lineages. Species-expanded or specific MYB TFs may regulate species-specific metabolite biosynthesis including phenylpropanoid-derived bioactive products. Camellia sinensis produces an abundance of specialized metabolites, which makes it an excellent model for digging into the genetic regulation of plant-specific metabolite biosynthesis. The most abundant health-promoting metabolites in tea are galloylated catechins, and the most bioactive of the galloylated catechins, epigallocatechin gallate (EGCG), is specifically relative abundant in C. sinensis. However, the transcriptional regulation of galloylated catechin biosynthesis remains elusive. This study mined the R2R3-MYB TFs associated with galloylated catechin biosynthesis in C. sinensis. A total of 118 R2R3-MYB proteins, classified into 38 subgroups, were identified. R2R3-MYB subgroups specific to or expanded in C. sinensis were hypothesized to be essential to evolutionary diversification of tea-specialized metabolites. Notably, nine of these R2R3-MYB genes were expressed preferentially in apical buds (ABs) and young leaves, exactly where galloylated catechins accumulate. Three putative R2R3-MYB genes displayed strong correlation with key galloylated catechin biosynthesis genes, suggesting a role in regulating biosynthesis of epicatechin gallate (ECG) and EGCG. Overall, this study paves the way to reveal the transcriptional regulation of galloylated catechins in C. sinensis.

Coexpression network analysis reveals an MYB transcriptional activator involved in capsaicinoid biosynthesis in hot peppers.

Plant biosynthesis involves numerous specialized metabolites with diverse chemical natures and biological activities. The biosynthesis of metabolites often exclusively occurs in response to tissue-specific combinatorial developmental cues that are controlled at the transcriptional level. Capsaicinoids are a group of specialized metabolites that confer a pungent flavor to pepper fruits. Capsaicinoid biosynthesis occurs in the fruit placenta and combines its developmental cues. Although the capsaicinoid biosynthetic pathway has been largely characterized, the regulatory mechanisms that control capsaicinoid metabolism have not been fully elucidated. In this study, we combined fruit placenta transcriptome data with weighted gene coexpression network analysis (WGCNA) to generate coexpression networks. A capsaicinoid-related gene module was identified in which the MYB transcription factor CaMYB48 plays a critical role in regulating capsaicinoid in pepper. Capsaicinoid biosynthetic gene (CBG) and CaMYB48 expression primarily occurs in the placenta and is consistent with capsaicinoid biosynthesis. CaMYB48 encodes a nucleus-localized protein that primarily functions as a transcriptional activator through its C-terminal activation motif. CaMYB48 regulates capsaicinoid biosynthesis by directly regulating the expression of CBGs, including AT3a and KasIa. Taken together, the results of this study indicate ways to generate robust networks optimized for the mining of CBG-related regulators, establishing a foundation for future research elucidating capsaicinoid regulation.

Jasmonate-Inducible R2R3-MYB Transcription Factor Regulates Capsaicinoid Biosynthesis and Stamen Development in Capsicum.

Jasmonates (JAs) play an important role in plant developmental processes and regulate the biosynthesis of various specialized metabolites, and transcription factors are crucial in mediating JA signaling to regulate these processes. Capsaicinoids (Caps) are intriguing specialized metabolites produced uniquely by Capsicum species that give their fruits a pungent flavor to defend against herbivory and pathogens. In this study, we identify a R2R3-MYB transcription factor CaMYB108 and demonstrate its roles in regulating the biosynthesis of Caps and stamen development. Transcriptional analysis indicated that CaMYB108 was preferentially expressed in the flower and fruit, while the subcellular localization of CaMYB108 was shown to be the nucleus. Virus-induced gene silencing of CaMYB108 led to the expression of capsaicinoid biosynthetic genes (CBGs), and the contents of Caps dramatically reduce. Moreover, the CaMYB108-silenced plants showed delayed anther dehiscence and reduced pollen viability. Transient overexpression of CaMYB108 caused the expression of CBGs to be upregulated, and the Caps content significantly increased. The results of dual-luciferase reporter assays showed that CaMYB108 targeted CBG promoters. In addition, the expression of CaMYB108 and CBGs was inducible by methyl jasmonate and was consistent with the increased content of Caps. Overall, our results indicate that CaMYB108 is involved in the regulation of Caps biosynthesis and stamen development.

Construction of a high density genetic map of an interspecific cross of Capsicum chinense and Capsicum annuum and QTL analysis of floral traits.

The yield of pepper plants (Capsicum spp.) is their most important trait and is affected by the flower number and flowering time. Capsicum annuum produces a single flower per node and has an early flowering habit. By contrast, Capsicum chinense yields multiple flowers per node and has a late flowering character. However, the genetic mechanism underlying the control of these floral traits remains largely unknown. In this study, 150 F2 populations from an interspecific cross between the inbred lines 740 (C. chinense) and CA1 (C. annuum) and their parents were used to construct a molecular genetic linkage map using the specific length amplified fragment sequencing (SLAF-seq) technique. This linkage map, spanning 1,586.78 cM in length, contained 9,038 markers on 12 chromosomes, with a mean marker distance of 0.18 cM. Phenotypic data on the flowering time and flower number per node were collected over multiple years, and QTL analysis identified 6 QTLs for the flowering time and flower number per node by composite interval mapping (CIM) and genome-wide composite interval mapping (GCIM) methods at least in two environments. The candidate genes within the major QTL were predicted. In the major flowering time QTL, the candidate gene Capana02g000700, which encodes the homeotic protein APETALA2, was identified. Quantitative reverse-transcription PCR (qRT-PCR) analysis indicated that its expression level in 740 was higher than that in CA1. Gene expression analysis indicated that the expression of Capana02g000700 was significantly upregulated in flowers, and many floral development-related genes were found to be coexpressed with Capana02g000700, supporting the function of this gene in association with flowering time in C. chinense and C. annuum species.

Overexpression of AtEDT1/HDG11 in Chinese Kale (Brassica oleracea var. alboglabra) Enhances Drought and Osmotic Stress Tolerance.

Plants are constantly challenged by environmental stresses, including drought and high salinity. Improvement of drought and osmotic stress tolerance without yield decrease has been a great challenge in crop improvement. The Arabidopsis ENHANCED DROUGHT TOLERANCE1/HOMEODOMAIN GLABROUS11 (AtEDT1/HDG11), a protein of the class IV HD-Zip family, has been demonstrated to significantly improve drought tolerance in Arabidopsis, rice, and pepper. Here, we report that AtEDT1/HDG11 confers drought and osmotic stress tolerance in the Chinese kale. AtEDT1/HDG11-overexpression lines exhibit auxin-overproduction phenotypes, such as long hypocotyls, tall stems, more root hairs, and a larger root system architecture. Compared with the untransformed control, transgenic lines have significantly reduced stomatal density. In the leaves of transgenic Chinese kale plants, proline (Pro) content and reactive oxygen species-scavenging enzyme activity was significantly increased after drought and osmotic stress, particularly compared to wild kale. More importantly, AtEDT1/HDG11-overexpression leads to abscisic acid (ABA) hypersensitivity, resulting in ABA inhibitor germination and induced stomatal closure. Consistent with observed phenotypes, the expression levels of auxin, ABA, and stress-related genes were also altered under both normal and/or stress conditions. Further analysis showed that AtEDT1/HDG11, as a transcription factor, can target the auxin biosynthesis gene YUCC6 and ABA response genes ABI3 and ABI5. Collectively, our results provide a new insight into the role of AtEDT1/HDG11 in enhancing abiotic stress resistance through auxin- and ABA-mediated signaling response in Chinese kale.