Identification of key volatile and odor-active compounds in 10 main fragrance types of Fenghuang Dancong tea using HS-SPME/GC-MS combined with multivariate analysis.

Fenghuang Dancong tea (FHDC), a famous oolong tea originating from Guangdong Province in China, is known for its rich and unique fragrance. Nevertheless, the identification of the key aroma compounds with the difference fragrance types of FHDC remains uncertain. In order to characteristic the volatile components in different fragrance types of FHDC, 10 well-known fragrance types of FHDC and Tieguanyin (TGY) as a control were analyzed by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography mass spectrometry (GC-MS). Results indicated that 172 volatile compounds were identified as common volatile compounds among all the tea samples. A total of 16 compounds were identified as key compounds that could be used to distinguish between FHDC and TGY. Among the 10 FHDC fragrance types, indole, hotrienol, benzyl nitrile, and jasmine lactone were found to be the most abundant compounds. Despite the presence of certain similarities in aroma components, each type exhibits unique fragrance characteristics as a result of variation in compound composition content and proportion. Furthermore, using statistical and odor activity value analysis, 20 aroma-active compounds were recognized as potential characteristic markers accountable for the diverse fragrance types of FHDC. This research enhances our comprehension of the various fragrance types of FHDC and provides reference values for their rapid identification in the market.

Characterization of the Aroma Profiles of Guangdong Black Teas Using Non-Targeted Metabolomics.

Guangdong black teas have diverse flavors and aromas. To explore the molecular basis of these aromas, we extracted and analyzed the volatile flavor compounds of 31 black tea samples from 7 districts (Yingde, Luokeng, Renhua, Meizhou, Chaozhou, Lianshan, and Heyuan) in Guangdong Province with headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Then, 135 volatile flavor compounds (VFCs) were identified and grouped into 12 classes according to their chemical structure. Notably, alcohols accounted for 31.40-44.43% of total VFCs. The score plot of supervised partial least squares-discriminant analysis (PLS-DA) revealed good discrimination for most black tea samples. Additionally, 64 compounds with variable importance in projection > 1.0 were identified as differential odorants. Through an odor activity value analysis, eight volatile compounds were identified as the key active differential VFCs: linalool, methyl salicylate, phenylethyl alcohol, p-cresol, 3-methyl-butanoic acid, geraniol, benzaldehyde, and benzeneacetaldehyde. Thus, benzeneacetaldehyde and linalool in YJ-Yingde samples, benzaldehyde in Luokeng samples with an almond-like aroma, phenylethyl alcohol in the Heyuan samples, and p-cresol and 3-methyl-butanoic acid in the Chaozhou samples were the key volatile flavor compounds that could differentiate local black teas from other black teas. These findings will enrich the research in tea aroma chemistry and provide a method for identifying the origins of Guangdong black teas.

Identification and characterization of the key volatile flavor compounds in black teas from distinct regions worldwide.

Volatile flavor compounds in 112 black teas from seven countries were analyzed by untargeted metabolomics using headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS). Multivariate statistical analysis and odor activity values (OAVs) were used to classify these samples and identify key odorants. A total of 140 volatile flavor compounds (VFCs), including 12 different groups, were identified, and alcohols were prevalent in China and India samples, accounting for 40.83% and 34.96% of the total VFCs, respectively. Eight volatile compounds with OAVs > 1 were identified as key active differential odorants in Chinese, Indian, and Sri Lankan samples, including linalool, pentanoic acid, methyl salicylate, hexanoic acid, 1-methyl-naphthalene, phenylethyl alcohol, geraniol, and ß-ionone. Linalool, pentanoic acid, and hexanoic acid in Indian black teas, phenylethyl alcohol in Chinese black teas, and 1-methyl-naphthalene, ß-ionone in Sri Lankan black teas could be used to discriminate different black tea groups. A total of 12-14 VFCs with OAVs > 1 were identified as key active aromatics in Chinese black tea sample. Linalool and benzeneacetaldehyde in Yingde (Guangdong) black tea, methyl salicylate in Taiwanese samples, and benzeneacetic acid in Anhui black tea could be used as biomarkers to distinguish them from other Chinese samples. Sensory evaluation results showed that most black teas presented the common sweet, floral odors, which were consistent with GC-MS analysis. These results will contribute to characterize the odor metabolome of black teas and provide biochemical basis for identifying the authenticity of different black teas. PRACTICAL APPLICATION: Linalool, pentanoic acid, and hexanoic acid in Indian black teas, phenylethyl alcohol in Chinese black teas, 1-methyl-naphthalene, ß-ionone, and methyl salicylate in Sri Lankan black teas could be used to discriminate black teas from the three countries. Linalool and benzeneacetaldehyde in Yingde black teas, methyl salicylate in Taiwanese black teas, and benzeneacetic acid in Anhui black tea are the potential biomarkers to distinguish these teas from other Chinese black teas.

OsCER1 regulates humidity-sensitive genic male sterility through very-long-chain (VLC) alkane metabolism of tryphine in rice.

Humidity-sensitive genic male sterility (HGMS) is a novel type of environment-sensitive male sterility (EGMS) which plants are male sterile at low humidity and male fertile at high humidity. Previous studies have revealed that OsCER1 contributes to very-long-chain (VLC) alkanes biosynthesis in rice (Oryza sativa L.). Here, applying the CRISPR/Cas9 technique, we obtained two independent OsCER1 knockout lines (OsCER1Cas). Both OsCER1Cas lines exhibited HGMS. Mutant pollen showed defects in adhesion and germination on stigmas at low humidity, whereas high humidity enhanced the pollen germination rate. Transmission electron microscopy (TEM) observations of mutant pollen revealed abnormal tryphine structure, potentially representing the basis of HGMS. Furthermore, co-pollination with mixed OsCER1Cas mutant and maize (Zea mays L.) pollen could rescue the fertility of the mutant, thereby establishing the key role of tryphine in germination on stigmas. OsCER1 knockout might affect VLC alkane metabolism and therefore alter the lipid composition of tryphine. It could lead to the defects in pollen grain adhesion, hydration and germination, resulting in HGMS. This work identified the mechanism of HGMS induced by VLC alkanes in rice and the generality of tryphine in different species of Gramineae.

Ubiquitinome Profiling Reveals the Landscape of Ubiquitination Regulation in Rice Young Panicles.

Ubiquitination, an essential post-transcriptional modification (PTM), plays a vital role in nearly every biological process, including development and growth. Despite its functions in plant reproductive development, its targets in rice panicles remain unclear. In this study, we used proteome-wide profiling of lysine ubiquitination in rice (O. sativa ssp. indica) young panicles. We created the largest ubiquitinome dataset in rice to date, identifying 1638 lysine ubiquitination sites on 916 unique proteins. We detected three conserved ubiquitination motifs, noting that acidic glutamic acid (E) and aspartic acid (D) were most frequently present around ubiquitinated lysine. Enrichment analysis of Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these ubiquitinated proteins revealed that ubiquitination plays an important role in fundamental cellular processes in rice young panicles. Interestingly, enrichment analysis of protein domains indicated that ubiquitination was enriched on a variety of receptor-like kinases and cytoplasmic tyrosine and serine-threonine kinases. Furthermore, we analyzed the crosstalk between ubiquitination, acetylation, and succinylation, and constructed a potential protein interaction network within our rice ubiquitinome. Moreover, we identified ubiquitinated proteins related to pollen and grain development, indicating that ubiquitination may play a critical role in the physiological functions in young panicles. Taken together, we reported the most comprehensive lysine ubiquitinome in rice so far, and used it to reveal the functional role of lysine ubiquitination in rice young panicles.

HMS1 interacts with HMS1I to regulate very-long-chain fatty acid biosynthesis and the humidity-sensitive genic male sterility in rice (Oryza sativa).

Environment-sensitive genic male sterility (EGMS) lines are used widely in two-line hybrid breeding in rice (Oryza sativa). At present, photoperiod-sensitive genic male sterility (PGMS) lines and thermo-sensitive genic male sterility (TGMS) lines are predominantly used in two-line hybrid rice, with humidity-sensitive genic male sterility (HGMS) lines rarely being reported. Here, it is shown that HUMIDITY-SENSITIVE GENIC MALE STERILITY 1 (HMS1), encoding a ß-ketoacyl-CoA synthase, plays key roles in the biosynthesis of very-long-chain fatty acids (VLCFAs) and HGMS in rice. The hms1 mutant displayed decreased seed setting under low humidity, but normal seed setting under high humidity. HMS1 catalyzed the biosynthesis of the C26 and C28 VLCFAs, contributing to the formation of bacula and tryphine in the pollen wall, which protect the pollen from dehydration. Under low-humidity conditions, hms1 pollen showed poor adhesion and reduced germination on the stigmas, which could be rescued by increasing humidity. HMS1-INTERACTING PROTEIN (HMS1I) interacted with HMS1 to coregulate HGMS. Furthermore, both japonica and indica rice varieties with defective HMS1 exhibited HGMS, suggesting that hms1 potentially could be used in hybrid breeding. The results herein reveal the novel mechanism of VLCFA-mediated pollen wall formation, which protects pollen from low-humidity stress in rice, and has a potential use in hybrid crop breeding.

OsAGO2 controls ROS production and the initiation of tapetal PCD by epigenetically regulating OsHXK1 expression in rice anthers.

Proteins of the ARGONAUTE (AGO) family function in the epigenetic regulation of gene expression. Although the rice (Oryza sativa) genome encodes 19 predicted AGO proteins, few of their functions have thus far been characterized. Here, we show that the AGO protein OsAGO2 regulates anther development in rice. OsAGO2 was highly expressed in anthers. Knockdown of OsAGO2 led to the overaccumulation of reactive oxygen species (ROS) and abnormal anther development, causing premature initiation of tapetal programmed cell death (PCD) and pollen abortion. The expression level of Hexokinase 1 (OsHXK1) increased significantly, and the methylation levels of its promoter decreased, in plants with knocked-down OsAGO2 expression. Overexpression of OsHXK1 also resulted in the overaccumulation of ROS, premature initiation of PCD, and pollen abortion. Moreover, knockdown of OsHXK1 restored pollen fertility in OsAGO2 knockdown plants. Chromatin immunoprecipitation assays demonstrated that OsAGO2 binds directly to the OsHXK1 promoter region, suggesting that OsHXK1 is a target gene of OsAGO2. These results indicate that OsHXK1 controls the appropriate production of ROS and the proper timing of tapetal PCD and is directly regulated by OsAGO2 through epigenetic regulation.

OsCER1 Plays a Pivotal Role in Very-Long-Chain Alkane Biosynthesis and Affects Plastid Development and Programmed Cell Death of Tapetum in Rice (Oryza sativa L.).

Cuticle waxes, which are primarily comprised of very-long-chain (VLC) alkanes, play an important role in plant reproductive development. ECERIFERUM1 (CER1) is recognized as the core element for VLC alkane biosynthesis in Arabidopsis (Arabidopsis thaliana). However, genes involved in the VLC alkane biosynthesis in rice remain unclear, and the alkane-form pathway in rice has still to be further explored. Here, we show that OsCER1, a homology of CER1, functions in VLC alkanes biosynthesis, which also could regulate anther development and plastids differentiation in rice. OsCER1 was highly expressed in the tapetum (stage 10) and bicellular pollen cells (stage 11). The decreased content of VLC alkanes (C25 and C27) in the OsCER1 knocked down plants as well as the increased content of C27 alkanes in the OsCER1 overexpression plants indicates that OsCER1 participates in VLC alkane biosynthesis. Downregulation of OsCER1 in rice led to sterility, and fewer amyloplasts within the mature pollen grains. In addition, the downregulation of OsCER1 in rice caused delayed tapetal programmed cell death and abnormal development of plastids in the tapetal cells. Furthermore, significantly altered levels of expression of genes involved in the pollen development were exhibited in the OsCER1 knocked down plants. These results indicate that OsCER1 is critical for VLC alkanes biosynthesis, plastids differentiation, and pollen development. This work provides insights into the VLC alkanes biosynthesis in anther development in rice.

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System.

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new "transgene clean" TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable "transgene clean" TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new "transgene clean" TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

RNase Z(S1) processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice.

Thermosensitive genic male-sterile (TGMS) lines, which are male-sterile at restrictive (high) temperatures but male-fertile at permissive (low) temperatures, have been widely used in breeding two-line hybrid rice (Oryza sativa L.). Here we find that mutation of thermosensitive genic male sterile 5 (tms5) in rice causes the TGMS trait through a loss of RNase Z(S1) function. We show that RNase Z(S1) processes the mRNAs of three ubiquitin fusion ribosomal protein L40 (UbL40) genes into multiple fragments in vitro and in vivo. In tms5 mutants, high temperature results in increased levels of UbL40 mRNAs. Overaccumulation of UbL40 mRNAs causes defective pollen production and male sterility. Our results uncover a novel mechanism of RNase Z(S1)-mediated UbL40 mRNA regulation and shows that loss of this regulation produces TGMS in rice, a finding with potential applications in hybrid crop breeding.

Rice OsGL1-6 is involved in leaf cuticular wax accumulation and drought resistance.

Cuticular wax is a class of organic compounds that comprises the outermost layer of plant surfaces. Plant cuticular wax, the last barrier of self-defense, plays an important role in plant growth and development. The OsGL1-6 gene, a member of the fatty aldehyde decarbonylase gene family, is highly homologous to Arabidopsis CER1, which is involved in cuticular wax biosynthesis. However, whether OsGL1-6 participates in cuticular wax biosynthesis remains unknown. In this study, an OsGL1-6 antisense-RNA vector driven by its own promoter was constructed and introduced into the rice variety Zhonghua11 by Agrobacterium-mediated transformation to obtain several independent transgenic plants with decreased OsGL1-6 expression. These OsGL1-6 antisense-RNA transgenic plants showed droopy leaves at the booting stage, significantly decreased leaf cuticular wax deposition, thinner cuticle membrane, increased chlorophyll leaching and water loss rates, and enhanced drought sensitivity. The OsGL1-6 gene was constitutively expressed in all examined organs and was very highly expressed in leaf epidermal cells and vascular bundles. The transient expression of OsGL1-6-GFP fusion indicated that OsGL1-6 is localized in the endoplasmic reticulum. Qualitative and quantitative analysis of the wax composition using gas chromatography-mass spectrometry revealed a significantly reduced total cuticular wax load on the leaf blades of the OsGL1-6 antisense-RNA transgenic plants as well as markedly decreased alkane and aldehyde contents. Their primary alcohol contents increased significantly compared with those in the wild type plants, suggesting that OsGL1-6 is associated with the decarbonylation pathways in wax biosynthesis. We propose that OsGL1-6 is involved in the accumulation of leaf cuticular wax and directly impacts drought resistance in rice.