Long Non-Coding RNAs: Discoveries, Mechanisms, and Research Strategies in Seeds.

Seeds provide nutrients for the embryo and allow for dormancy in stressed environments to better adapt the plant to its environment. In addition, seeds are an essential source of food for human survival and are the basis for the formation of food production and quality. Therefore, the research on the genetic mechanism of seed development and germination will provide a theoretical basis and technical support for the improvement of crop yield and quality. Recent studies have shown that long non-coding RNAs (lncRNAs) occupy a pivotal position in seed development and germination. In this review, we describe the key processes in seed biology and examine discoveries and insights made in seed lncRNA, with emphasis on lncRNAs that regulate seed biology through multiple mechanisms. Given that thousands of lncRNAs are present in the seed transcriptome, characterization has lagged far behind identification. We provide an overview of research strategies and approaches including some exciting new techniques that may uncover the function of lncRNAs in seed. Finally, we discuss the challenges facing the field and the opening questions. All in all, we hope to provide a clear perspective on discoveries of seed lncRNA by linking discoveries, mechanisms, and technologies.

Linolenic Acid-Derived Oxylipins Inhibit Aflatoxin Biosynthesis in Aspergillus flavus through Activation of Imizoquin Biosynthesis.

Oxylipins play important signaling roles in aflatoxin (AF) biosynthesis in Aspergillus flavus. We previously showed that exogenous supply of autoxidated linolenic acid (AL) inhibited AF biosynthesis in A. flavus via oxylipins, but the molecular mechanism is still unknown. Here, we performed multiomics analyses of A. flavus grown in media with or without AL. Targeted metabolite analyses and quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) showed that the imizoquin (IMQ) biosynthetic pathway was distinctly upregulated in the presence of AL. 13C-glucose labeling confirmed in parallel that the tricarboxylic acid cycle was also enhanced by AL, consistent with observed increases in mycelial growth. Moreover, we integrated thermal proteome profiling and molecular dynamics simulations to identify a potential receptor of AL; AL was found to interact with a transporter (ImqJ) located in the IMQ gene cluster, primarily through hydrophobic interactions. Further analyses of strains with an IMQ pathway transcription factor overexpressed or knocked out confirmed that this pathway was critical for AL-mediated inhibition of AF biosynthesis. Comparison of 22 assembled A. flavus and Aspergillus oryzae genomes showed that genes involved in the IMQ pathway were positively selected in A. oryzae. Taken together, the results of our study provide novel insights into oxylipin-mediated regulation of AF biosynthesis and suggest potential methods for preventing AF contamination of crops.

Identification and Functional Analysis of Long Non-Coding RNA (lncRNA) in Response to Seed Aging in Rice.

Many lncRNAs have been shown to play a vital role in aging processes. However, how lncRNAs regulate seed aging remains unknown. In this study, we performed whole transcriptome strand-specific RNA sequencing of samples from rice embryos, analyzed the differences in expression of rice seed lncRNAs before and after artificial aging treatment (AAT), and systematically screened 6002 rice lncRNAs. During the AAT period, the expression levels of most lncRNAs (454) were downregulated and only four were upregulated among the 458 differentially expressed lncRNAs (DELs). Cis- or trans-regulated target genes of the four upregulated lncRNAs were mainly related to base repair, while 454 downregulated lncRNAs were related to plant-pathogen interaction, plant hormones, energy metabolism, and secondary metabolism. The pathways of DEL target genes were similar with those of differentially expressed mRNAs (DEGs). A competing endogenous RNA (ceRNA) network composed of 34 lncRNAs, 24 microRNAs (miRNA), and 161 mRNAs was obtained. The cDNA sequence of lncRNA LNC_037529 was obtained by rapid amplification of cDNA ends (RACE) cloning with a total length of 1325 bp, a conserved 5' end, and a non-conserved 3' end. Together, our findings indicate that genome-wide selection for lncRNA downregulation was an important mechanism for rice seed aging. LncRNAs can be used as markers of seed aging in rice. These findings provide a future path to decipher the underlying mechanism associated with lncRNAs in seed aging.

OsGLYI3, a glyoxalase gene expressed in rice seed, contributes to seed longevity and salt stress tolerance.

The glyoxalase pathway plays a vital role in the chemical detoxification of methylglyoxal (MG) in biological systems. Our previous study suggested that OsGLYI3 may be effective in seed natural aging. In this study, the rice OsGLYI3 gene was cloned and characterized as specifically expressed in the seed. The accelerated aging (AA) treatment results indicated significant roles of OsGLYI3 in seed longevity and vigor, as the seeds of the transgenic lines with overexpressed and knocked-out OsGLYI3 exhibited higher and lower germination, respectively. The AA treatment also increased the superoxide dismutase (SOD) activity in the overexpressed transgenic seeds compared to the wild-type seeds yet lowered the SOD activity in the CRISPR/Cas9-derived transgenic rice lines. Rice OsGLYI3 was markedly upregulated in response to NaCl induced stress conditions. Compared to wild-type plants, overexpressed transgenic rice lines exhibited increased GLYI activity, decreased MG levels and improved salt stress tolerance, while CRISPR/Cas9 knockout transgenic rice lines showed decreased glyoxalase I activity, increased MG levels, and greater sensitivity to stress treatments with NaCl. Collectively, our results confirmed for the first time that OsGLYI3 is specifically expressed in rice seeds and contributes to seed longevity and salt stress tolerance.

Lipid profile variations in high olecic acid peanuts by following different cooking processes.

High oleic acid (OA) peanut seed (PS), contains a higher ratio of oleic acid (C18:1) compared to general PS, which is favored by consumers due to its health benefits. However, comprehensive lipid metabolite profiles of high-OA PS, once they have been processed via domestic cooking methods, have never been produced. To establish a scientific guide for the selection of the most appropriate processing method for high-OA PS, lipidomics was performed to identify 706 lipid metabolites in high-OA PS following boiling, baking and frying, between the three groups, 75, 175 and 242 lipid metabolites were differentially expressed respectively. Additionally, 46 glycerolipids with C18:1 molecular were observed in the lipid profiles of the treatment groups compared to the raw sample. Further evaluation of seven lipid peroxides and six antioxidant status of each testing group suggested that boiled PS retained the highest levels of lipids and antioxidant activity. Following these findings, boiling appears to be an appropriate processing method when attempting to conserve the beneficial substances found in the PS. Finally, the levels of major free fatty acids present in high-OA PS, were jointly quantified by conventional methods (GC-MS) and lipidomic analysis. FA/C16:0 levels were similar, FA/C18:0, FA/C18:1 displayed opposite results, FA/C18:2 levels increased following frying and FA/C18:3 levels were down regulated once the PS was boiled. This indicates that GC-MS is a potential method of validation for the results of lipidomic analysis. Conclusively, this in depth understanding of lipid content in relation to domestic cooking methods has provided a foundation for the processing of high-OA peanut products.

Poly ADP-ribose polymerase-1 promotes seed-setting rate by facilitating gametophyte development and meiosis in rice (Oryza sativa L.).

Poly(ADP-ribose) polymerases (PARPs), which transfer either monomer or polymer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+ ) onto target proteins, are required for multiple processes in DNA damage repair, cell cycle, development, and abiotic stress in animals and plants. Here, the uncharacterized rice (Oryza sativa) OsPARP1, which has been predicted to have two alternative OsPARP1 mRNA splicing variants, OsPARP1.1 and OsPARP1.2, was investigated. However, bimolecular fluorescence complementation showed that only OsPARP1.1 interacted with OsPARP3 paralog, suggesting that OsPARP1.1 is a functional protein in rice. OsPARP1 was preferentially expressed in the stamen primordial and pollen grain of mature stamen during flower development. The osparp1 mutant and CRISPR plants were delayed in germination, indicating that defective DNA repair machinery impairs early seed germination. The mutant displayed a normal phenotype during vegetative growth but had a lower seed-setting rate than wild-type plants under normal conditions. Chromosome bridges and DNA fragmentations were detected in male meiocytes at anaphase I to prophase II. After meiosis II, malformed tetrads or tetrads with micronuclei were formed. Meanwhile, the abnormality was also found in embryo sac development. Collectively, these results suggest that OsPARP1 plays an important role in mediating response to DNA damage and gametophyte development, crucial for rice yield in the natural environment.

Comparative Multi-Omics of Tender Shoots from a Novel Evergrowing Tea Cultivar Provide Insight into the Winter Adaptation Mechanism.

Tea (Camellia sinensis [L.] O. Kuntze) tree is a perennial plant in which winter dormancy is an important biological adaptation to environmental changes. We discovered and reported a novel tea tree cultivar that can generate tender shoots in winter several years ago, but the molecular mechanism for this unique phenotype remains unknown . Here, we conducted comparative transcriptomics, proteomics and metabolomics along with phytohormone quantitation between the winter and spring tender shoots to investigate the physiological basis and putative regulatory mechanisms of its evergrowing character during winter. Our multi-omics study has led to the following findings. Gibberellin (GA) levels and key enzymes for GA biosynthesis and the signal transduction pathway were increased in the winter shoots, causing the ABA/GA content ratio to decrease, which might play a key regulatory role in maintaining normal growth during winter. The abundance of proteins, genes and metabolites involved in energy metabolism was all increased in winter shoots, indicating that energy is critical for continuous growth under the relatively weak-light and low-temperature environment. Abiotic resistance-related proteins and free amino acids were also increased in abundance in the winter shoots, which possibly represents an adaptation response to winter conditions. These results allowed us to hypothesize a novel molecular mechanism of adaptation for this unique tender shoot evergrowing in winter.

A Comparative Transcriptome Analysis of Volvariella volvacea Identified the Candidate Genes Involved in Fast Growth at the Mycelial Growth Stage.

The edible straw mushroom, Volvariella volvacea, is one of the most important cultivated mushrooms in tropical and sub-tropical regions. Strain improvement for V. volvacea is difficult because of the unknown mechanisms involved in its growth regulation and substrate utilization. A comparative physiological and transcriptomic study was conducted between two commercially available straw mushroom strains (v9 and v26) to explore their fast-growth regulation mechanism(s). The physiological study showed that V. volvacea v9 had a shorter growth cycle and higher biological efficiency (4% higher) than that in v26. At least 14,556 unigenes were obtained from the four cDNA libraries (two replicates per strain). Among them, the expression of 1597 unigenes was up-regulated while 1352 were down-regulated. Four heat-shock proteins were highly expressed in v9, showing that v9 has the better ability to handle stresses and/or environmental changes. Moreover, up to 14 putative transporter genes were expressed at a higher level in v9 than those in v26, implying that v9 has a better ability to transport nutrients or export xenobiotics efficiently. Our report allows to identify the candidate genes involved in the fast growth requirement of V. volvacea, which represents a valuable resource for strain improvement in this commercially important edible mushroom.

Coumarin-Induced Delay of Rice Seed Germination Is Mediated by Suppression of Abscisic Acid Catabolism and Reactive Oxygen Species Production.

Abscisic acid (ABA) is a crucial phytohormone for the regulation of seed germination. The ABA content of seeds is regulated by synthesis and catabolic pathways. Coumarin, an important plant allelochemical, can inhibit seed germination effectively, although whether it is involved in the regulation of ABA content during seed germination has not been elucidated. For the study reported herein, we show that coumarin effectively inhibits rice seed germination and vivipary. We found that the ABA content gradually decreased in water-imbibed rice seeds and that the content and activity of the Oryza sativa 9-cis epoxycarotenoid dioxygenases (OsNCEDs), which are ABA synthases, decreased during seed germination. At the transcription level, the expression of OsNCED1-3 appeared to decrease, whereas the expression of the ABA 8'-hydroxylase 2 and 3 genes (OsABA8'ox2/3) first appeared to increase and then decrease. Samples of rice seeds were also imbibed in water containing coumarin, which increased their ABA content but did not significantly increase the activity or content of their OsNCEDs or OsNCED1-3 transcription. Interestingly, coumarin imbibition remarkably reduced OsABA8'ox2/3 expression in rice embryos, which partially explained how coumarin increased the ABA content of germinating rice embryos. Coumarin also inhibited the accumulation of reactive oxygen species (ROS) in rice embryos and increased the activity of superoxide dismutase and catalase, which are indispensable for seed germination. These results indicate that coumarin delays seed germination by inhibiting ABA catabolism, particularly by decreasing the expression of OsABA8'ox2/3 rather than by increasing ABA synthesis. Moreover, coumarin increases the ABA content while decreasing the ROS content in rice embryos. Our results enhance our understanding of the regulation of ABA and ROS during seed germination and provide theoretical support for application of coumarin to prevent sprouting before crop harvesting.

Corrigendum: Involvement of Polyamine Oxidase-Produced Hydrogen Peroxide during Coleorhiza-Limited Germination of Rice Seeds.

[This corrects the article on p. 1219 in vol. 7, PMID: 27570530.].

Studying the Differences of Bacterial Metabolome and Microbiome in the Colon between Landrace and Meihua Piglets.

This study was conducted to compare the microbiome and metabolome differences in the colon lumen from two pig breeds with different genetic backgrounds. Fourteen weaned piglets at 30 days of age, including seven Landrace piglets (a lean-type pig breed with a fast growth rate) and seven Meihua piglets (a fatty-type Chinese local pig breed with a slow growth rate), were fed the same diets for 35 days. Untargeted metabolomics analyses showed that a total of 401 metabolites differed between Landrace and Meihua. Seventy of these 401 metabolites were conclusively identified. Landrace accumulated more short-chain fatty acids (SCFAs) and secondary bile acids in the colon lumen. Moreover, expression of the SCFAs transporter (solute carrier family 5 member 8, SLC5A8) and receptor (G protein-coupled receptor 41, GPR41) in the colon mucosa was higher, while the bile acids receptor (farnesoid X receptor, FXR) had lower expression in Landrace compared to Meihua. The relative abundances of 8 genera and 16 species of bacteria differed significantly between Landrace and Meihua, and were closely related to the colonic concentrations of bile acids or SCFAs based on Pearson's correlation analysis. Collectively, our results demonstrate for the first time that there were differences in the colonic microbiome and metabolome between Meihua and Landrace piglets, with the most profound disparity in production of SCFAs and secondary bile acids.

Involvement of Polyamine Oxidase-Produced Hydrogen Peroxide during Coleorhiza-Limited Germination of Rice Seeds.

Seed germination is a complicated biological process that requires regulated enzymatic and non-enzymatic reactions. The action of polyamine oxidase (PAO) produces hydrogen peroxide (H2O2), which promotes dicot seed germination. However, whether and, if so, how PAOs regulate monocot seed germination via H2O2 production is unclear. Herein, we report that the coleorhiza is the main physical barrier to radicle protrusion during germination of rice seed (a monocot seed) and that it does so in a manner similar to that of dicot seed micropylar endosperm. We found that H2O2 specifically and steadily accumulated in the coleorhizae and radicles of germinating rice seeds and was accompanied by increased PAO activity as the germination percentage increased. These physiological indexes were strongly decreased in number by guazatine, a PAO inhibitor. We also identified 11 PAO homologs (OsPAO1-11) in the rice genome, which could be classified into four subfamilies (I, IIa, IIb, and III). The OsPAO genes in subfamilies I, IIa, and IIb (OsPAO1-7) encode PAOs, whereas those in subfamily III (OsPAO8-11) encode histone lysine-specific demethylases. In silico-characterized expression profiles of OsPAO1-7 and those determined by qPCR revealed that OsPAO5 is markedly upregulated in imbibed seeds compared with dry seeds and that its transcript accumulated to a higher level in embryos than in the endosperm. Moreover, its transcriptional abundance increased gradually during seed germination in water and was inhibited by 5 mM guazatine. Taken together, these results suggest that PAO-generated H2O2 is involved in coleorhiza-limited rice seed germination and that OsPAO5 expression accounts for most PAO expression and activity during rice seed germination. These findings should facilitate further study of PAOs and provide valuable information for functional validation of these proteins during seed germination of monocot cereals.

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.

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA.

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.