RMS2 Encoding a GDSL Lipase Mediates Lipid Homeostasis in Anthers to Determine Rice Male Fertility.

Plant male gametogenesis is a coordinated effort involving both reproductive tissues and sporophytic tissues, in which lipid metabolism plays an essential role. Although GDSL esterases/lipases have been well known as key enzymes for many plant developmental processes and stress responses, their functions in reproductive development remain unclear. Here, we report the identification of a rice male sterile2 (rms2) mutant in rice (Oryza sativa), which is completely male sterile due to the defects in tapetum degradation, cuticle formation in sporophytic tissues, and impaired exine and central vacuole development in pollen grains. RMS2 was map-based cloned as an endoplasmic reticulum-localized GDSL lipase gene, which is predominantly transcribed during early anther development. In rms2, a three-nucleotide deletion and one base substitution (TTGT to A) occurred within the GDSL domain, which reduced the lipid hydrolase activity of the resulting protein and led to significant changes in the content of 16 lipid components and numerous other metabolites, as revealed by a comparative metabolic analysis. Furthermore, RMS2 is directly targeted by the male fertility regulators Undeveloped Tapetum1 and Persistent Tapetal Cell1 both in vitro and in vivo, suggesting that RMS2 may serve as a key node in the rice male fertility regulatory network. These findings shed light on the function of GDSLs in reproductive development and provide a promising gene resource for hybrid rice breeding.

OsUGT88C3 Encodes a UDP-Glycosyltransferase Responsible for Biosynthesis of Malvidin 3-O-Galactoside in Rice.

The diversity of anthocyanins is largely due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. Although a number of glycosyltransferases have been identified to glycosylate anthocyanidin in plants, the enzyme that catalyzes malvidin galactosylation remains unclear. In this study, we identified three rice varieties with different leaf color patterns, different anthocyanin accumulation patterns, and different expression patterns of anthocyanin biosynthesis genes (ABGs) to explore uridine diphosphate (UDP)-glycosyltransferases (UGTs) responsible for biosynthesis of galactosylated malvidin. Based on correlation analysis of transcriptome data, nine candidate UGT genes coexpressed with 12 ABGs were identified (r values range from 0.27 to 1.00). Further analysis showed that the expression levels of one candidate gene, OsUGT88C3, were highly correlated with the contents of malvidin 3-O-galactoside, and recombinant OsUGT88C3 catalyzed production of malvidin 3-O-galactoside using UDP-galactose and malvidin as substrates. OsUGT88C3 was closely related to UGTs with flavone and flavonol glycosylation activities in phylogeny. Its plant secondary product glycosyltransferase (PSPG) motif ended with glutamine. Haplotype analysis suggested that the malvidin galactosylation function of OsUGT88C3 was conserved among most of the rice germplasms. OsUGT88C3 was highly expressed in the leaf, pistil, and embryo, and its protein was located in the endoplasmic reticulum and nucleus. Our findings indicate that OsUGT88C3 is responsible for the biosynthesis of malvidin 3-O-galactoside in rice and provide insight into the biosynthesis of anthocyanin in plants.

The tRNA 3'-end processing enzyme tRNase Z2 contributes to chloroplast biogenesis in rice.

tRNase Z (TRZ) is a ubiquitous endonuclease that removes the 3'-trailer from precursor tRNAs during maturation. In yeast and animals, TRZ regulates the cell cycle via its (t)RNA processing activity; however, its physiological function in higher plants has not been well characterized. This study describes the identification of a rice (Oryza sativa) TRZ2 mutant; plants homozygous for the osatrz2 mutation were albinos with deficient chlorophyll content. A microscopic analysis of the mutant plants revealed that the transition of proplastids to chloroplasts was arrested at an early stage, and the number and size of the plastids in callus cells was substantially decreased. A genetic complementation test and an RNA interference analysis confirmed that disruption of OsaTRZ2 was responsible for the mutant phenotype. OsaTRZ2 is expressed in all rice tissues, but is preferentially expressed in leaves, sheathes, and calli. OsaTRZ2 was subcellularly localized in chloroplasts, and displayed tRNA 3'-end processing activity in both in vitro and in vivo assays. In the osatrz2 mutants, transcription of plastid-encoded and nucleus-encoded RNA polymerases was severely reduced and moderately increased, respectively. These results suggest that the tRNA 3' processing activity of OsaTRZ2 contributes to chloroplast biogenesis.

Integrated Transcriptomic and Metabolomic Analyses Reveal the Molecular and Metabolic Basis of Flavonoids in Areca catechu L.

Areca catechu L., of the Arecaceae family, is widely distributed in tropical Asia. In A. catechu, the extracts and compounds, including flavonoids, have various pharmacological activities. Although there are many studies of flavonoids, the molecular mechanism of their biosynthesis and regulation remains unclear in A. catechu. In this study, 331 metabolites were identified from the root, stem, and leaf of A. catechu using untargeted metabolomics, including 107 flavonoids, 71 lipids, 44 amino acids and derivatives, and 33 alkaloids. The transcriptome analysis identified 6119 differentially expressed genes, and some were enriched in the flavonoid pathway. To analyze the biosynthetic mechanism of the metabolic differences in A. catechu tissues, 36 genes were identified through combined transcriptomic and metabolomic analysis, in which glycosyltransferase genes Acat_15g017010 and Acat_16g013670 were annotated as being involved in the glycosylation of kaempferol and chrysin by their expression and in vitro activities. Flavonoid biosynthesis could be regulated by the transcription factors, AcMYB5 and AcMYB194. This study laid a foundation for further research on the flavonoid biosynthetic pathway of A. catechu.

Comprehensive Analysis of Metabolome and Transcriptome Reveals the Regulatory Network of Coconut Nutrients.

Coconut flesh is widely consumed in the market for its good flavor. However, a comprehensive and dynamic assessment of the nutrients in coconut flesh and their molecular regulatory mechanisms is lacking. In this study, the metabolite accumulation and gene expression of three representative coconut cultivars belonging to two subspecies were investigated using ultra performance liquid chromatography/tandem mass spectrometry. A total of 6101 features were detected, of which 52, 8, and 158 were identified as amino acids and derivatives, polyamines, and lipids, respectively. The analysis of the metabolite pathway showed that glutathione and α-linolenate were the main differential metabolites. Transcriptome data revealed significant differences in the expression of five glutathione structural genes and thirteen polyamine-regulated genes, consistent with trends in metabolite accumulation. Weighted correlation network and co-expression analyses showed that a novel gene WRKY28 was implicated in the regulation of lipid synthesis. These results broaden our understanding of coconut nutrition metabolism and provide new insights into the molecular basis of coconut nutrition metabolism.

Effort and contribution of T-DNA Insertion mutant library for rice functional genomics research in China: review and perspective.

With the completion of the rice (Oryza sativa L.) genome-sequencing project, the rice research community proposed to characterize the function of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Regulation Mechanism of Plant Pigments Biosynthesis: Anthocyanins, Carotenoids, and Betalains.

Anthocyanins, carotenoids, and betalains are known as the three major pigments in the plant kingdom. Anthocyanins are flavonoids derived from the phenylpropanoid pathway. They undergo acylation and glycosylation in the cytoplasm to produce anthocyanin derivatives and deposits in the cytoplasm. Anthocyanin biosynthesis is regulated by the MBW (comprised by R2R3-MYB, basic helix-loop-helix (bHLH) and WD40) complex. Carotenoids are fat-soluble terpenoids whose synthetic genes also are regulated by the MBW complex. As precursors for the synthesis of hormones and nutrients, carotenoids are not only synthesized in plants, but also synthesized in some fungi and bacteria, and play an important role in photosynthesis. Betalains are special water-soluble pigments that exist only in Caryophyllaceae plants. Compared to anthocyanins and carotenoids, the synthesis and regulation mechanism of betalains is simpler, starting from tyrosine, and is only regulated by MYB (myeloblastosis). Recently, a considerable amount of novel information has been gathered on the regulation of plant pigment biosynthesis, specifically with respect to aspects. In this review, we summarize the knowledge and current gaps in our understanding with a view of highlighting opportunities for the development of pigment-rich plants.

Integrated Metabolomics and Transcriptomics Analyses Reveal the Metabolic Differences and Molecular Basis of Nutritional Quality in Landraces and Cultivated Rice.

Rice (Oryza sativa L.) is one of the most globally important crops, nutritionally and economically. Therefore, analyzing the genetic basis of its nutritional quality is a paramount prerequisite for cultivating new varieties with increased nutritional health. To systematically compare the nutritional quality differences between landraces and cultivated rice, and to mine key genes that determine the specific nutritional traits of landraces, a seed metabolome database of 985 nutritional metabolites covering amino acids, flavonoids, anthocyanins, and vitamins by a widely targeted metabolomic approach with 114 rice varieties (35 landraces and 79 cultivars) was established. To further reveal the molecular mechanism of the metabolic differences in landrace and cultivated rice seeds, four cultivars and six landrace seeds were selected for transcriptome and metabolome analysis during germination, respectively. The integrated analysis compared the metabolic profiles and transcriptomes of different types of rice, identifying 358 differentially accumulated metabolites (DAMs) and 1982 differentially expressed genes (DEGs), establishing a metabolite-gene correlation network. A PCA revealed anthocyanins, flavonoids, and lipids as the central differential nutritional metabolites between landraces and cultivated rice. The metabolite-gene correlation network was used to screen out 20 candidate genes postulated to be involved in the structural modification of anthocyanins. Five glycosyltransferases were verified to catalyze the glycosylation of anthocyanins by in vitro enzyme activity experiments. At the same time, the different mechanisms of the anthocyanin synthesis pathway and structural diversity in landrace and cultivated rice were systematically analyzed, providing new insights for the improvement and utilization of the nutritional quality of rice landrace varieties.

COVID-19 impacts, opportunities and challenges for wildlife farms in Binh Duong and Ba Ria Vung Tau, Vietnam.

The wildlife trade is a major cause of species loss and can trigger disease transmission. While the COVID-19 pandemic sparked public interest in eliminating the wildlife trade, a better understanding is needed of the economic repercussions of COVID-19 on those who rely on wildlife farming for their livelihoods. Using the case studies of Ba Ria Vung Tau and Binh Duong provinces in Vietnam - a country seen as Asia's wildlife trade hotspot - this paper explores COVID-19's impacts on wildlife farms and their owners. Understanding these impacts is important, both in order to design appropriate interventions to support local people in mitigating COVID-19's impacts as well as to inform effective policymaking around wildlife conservation in Vietnam. In this study, we adopted mixed research methods (including a literature and policy review, stakeholder consultation with government agencies and NGOs engaged in designing and monitoring wildlife conservation policies, a wildlife farming household survey, and research validation workshop) to understand the status of Vietnamese wildlife farms, as well as the impacts of COVID-19, and any opportunities and challenges for wildlife conservation and management in Vietnam. Our paper shows that, across the two studied provinces, numbers of wildlife farms and farmed wildlife animals have both declined since the pandemic, with declining market demand and wildlife farm owners experiencing difficulties accessing markets due to travel restrictions. Although this affected wildlife-related income, this represented less than 30 % of families' overall income on average, and thus households were able to maintain their livelihoods through other sources. Most wildlife is raised as an additional food source for farming families and plays an important role in the diets of surveyed households. Findings also highlighted that most surveyed households' post-pandemic recovery strategies involved expanding their wildlife farms in scope and scale; these households perceived a stable domestic market and high prices for wildlife products in future. Our study found several opportunities for sustainable wildlife farming practices, including greater political commitment, an increasing number of wildlife conservation policies, and stronger law enforcement mechanisms. Challenges remain, however; including an unclear and inconsistent policy framework, the presence of an illegal market, and wildlife farm owners' limited knowledge and understanding of wildlife policies. Our paper also shows a lack of comprehensive data and understanding around actual wildlife transactions during the pandemic, leading to challenges in confirming whether COVID-19 had any real impact on wildlife trade. Further research is required to address this knowledge gap.

RID1, encoding a Cys2/His2-type zinc finger transcription factor, acts as a master switch from vegetative to floral development in rice.

Transition from the vegetative phase to reproductive phase is a crucial process in the life cycle of higher plants. Although the molecular mechanisms of flowering regulation have been extensively characterized in a number of plant species, little is known regarding how the transition process initiates. Here, we show that the Rice Indeterminate 1 (RID1) gene acts as the master switch for the transition from the vegetative to reproductive phase. RID1 encodes a Cys-2/His-2-type zinc finger transcription factor that does not have an ortholog in Arabidopsis spp. A RID1 knockout (rid1), mutated by T-DNA insertion, never headed after growing for >500 days under a range of growth conditions and is thus referred to as a never-flowering phenotype. This mutation-suppressed expression of the genes is known to be involved in flowering regulation, especially in the Ehd1/Hd3a pathway and a series of RFT homologs. RID1 seems to be independent of the circadian clock. A model was proposed to place RID1 in the molecular pathways of flowering regulation in rice, for which there are two indispensable elements. In the first, RID1 is controlling the phase transition and initiation of floral induction. In the other, the Hd3a/RFL1/FTL complex acts as the immediate inducer of flowering. Loss of function in either element would cause never-flowering. Once the phase transition is induced with the activation of RID1, flowering signal is transduced and regulated through the various pathways and eventually integrated with FT-like proteins to induce flowering.

Rice RBH1 Encoding A Pectate Lyase is Critical for Apical Panicle Development.

Panicle morphology is one of the main determinants of the rice yield. Panicle abortion, a typical panicle morphological defect results in yield reduction due to defective spikelet development. To further elucidate the molecular mechanism of panicle abortion in rice, a rice panicle bald head 1 (rbh1) mutant with transfer DNA (T-DNA) insertion showing severely aborted apical spikelets during panicle development was identified and characterized. The rbh1-1 mutant showed obviously altered cell morphology and structure in the degenerated spikelet. Molecular genetic studies revealed that RBH1 encodes a pectate lyase protein. Pectate lyase-specific activity of Rice panicle Bald Head 1 (RBH1) protein assay using polygalacturonic acid (PGA) as substrates illustrated that the enzyme retained a significant capacity to degrade PGA. In addition, immunohistochemical analysis showed that the degradation of pectin is inhibited in the rbh1-1 mutant. Further analysis revealed that a significant increase in reactive oxygen species (ROS) level was found in degenerated rbh1-1 spikelets. Taken together, our findings suggest that RBH1 is required for the formation of panicle and for preventing panicle abortion.

Mutant resources for the functional analysis of the rice genome.

Rice is one of the most important crops worldwide, both as a staple food and as a model system for genomic research. In order to systematically assign functions to all predicted genes in the rice genome, a large number of rice mutant lines, including those created by T-DNA insertion, Ds/dSpm tagging, Tos17 tagging, and chemical/irradiation mutagenesis, have been generated by groups around the world. In this study, we have reviewed the current status of mutant resources for functional analysis of the rice genome. A total of 246 566 flanking sequence tags from rice mutant libraries with T-DNA, Ds/dSpm, or Tos17 insertion have been collected and analyzed. The results show that, among 211 470 unique hits, inserts located in the genic region account for 68.16%, and 60.49% of nuclear genes contain at least one insertion. Currently, 57% of non-transposable-element-related genes in rice have insertional tags. In addition, chemical/irradiation-induced rice mutant libraries have contributed a lot to both gene identification and new technology for the identification of mutant sites. In this review, we summarize how these tools have been used to generate a large collection of mutants. In addition, we discuss the merits of classic mutation strategies. In order to achieve saturation of mutagenesis in rice, DNA targeting, and new resources like RiceFox for gene functional identification are reviewed from a perspective of the future generation of rice mutant resources.

Rice functional genomics research: progress and implications for crop genetic improvement.

Rice is a staple food crop and has become a reference of monocot plant for functional genomic research. With the availability of high quality rice genome sequence, there has been rapid accumulation of functional genomic resources, including: large mutant libraries by T-DNA insertion, transposon tagging, and chemical mutagenesis; global expression profiles of the genes in the entire life cycle of rice growth and development; full-length cDNAs for both indica and japonica rice; sequences from resequencing large numbers of diverse germplasm accessions. Such resource development has greatly accelerated gene cloning. By the end of 2010, over 600 genes had been cloned using various methods. Many of the genes control agriculturally useful traits such as yield, grain quality, resistances to biotic and abiotic stresses, and nutrient-use efficiency, thus have potential utility in crop genetic improvement. This review was aimed to provide a comprehensive summary of such progress. We also presented our perspective for future studies.