The genome of Vicia sativa ssp. amphicarpa provides insights into the role of terpenoids in antimicrobial resistance within subterranean fruits.

Vicia sativa ssp. amphicarpa is a unique forage crop capable of simultaneously producing fruits above and below ground, representing a typical amphicarpic plant. In this study, we sequenced and assembled seven pseudo-chromosomes of the genome of V. sativa ssp. amphicarpa (n = 7) yielding a genome size of 1.59 Gb, with a total annotation of 48 932 protein-coding genes. Long terminal repeat (LTR) elements constituted 62.28% of the genome, significantly contributing to the expansion of genome size. Phylogenetic analysis revealed that the divergence between V. sativa ssp. amphicarpa and V. sativa was around 0.88 million years ago (MYA). Comparative transcriptomic and metabolomic analysis of aerial and subterranean pod shells showed biosynthesis of terpenoids in the subterranean pod shells indicating a correlation between the antimicrobial activity of subterranean pod shells and the biosynthesis of terpenoids. Furthermore, functional validation indicates that overexpression of VsTPS5 and VsTPS16 enhances terpenoid biosynthesis for antibacterial activity. Metabolomic analysis suggests the involvement of terpenoids in the antimicrobial properties of subterranean pod shells. Deciphering the genome of V. sativa ssp. amphicarpa elucidated the molecular mechanisms behind the antimicrobial properties of subterranean fruits in amphicarpic plants, providing valuable insights for the study of amphicarpic plant biology.

m6A demethylase OsALKBH5 is required for double-strand break formation and repair by affecting mRNA stability in rice meiosis.

N6-methyladenosine (m6A) RNA modification is the most prevalent messenger RNA (mRNA) modification in eukaryotes and plays critical roles in the regulation of gene expression. m6A is a reversible RNA modification that is deposited by methyltransferases (writers) and removed by demethylases (erasers). The function of m6A erasers in plants is highly diversified and their roles in cereal crops, especially in reproductive development essential for crop yield, are largely unknown. Here, we demonstrate that rice OsALKBH5 acts as an m6A demethylase required for the normal progression of male meiosis. OsALKBH5 is a nucleo-cytoplasmic protein, highly enriched in rice anthers during meiosis, that associates with P-bodies and exon junction complexes, suggesting that it is involved in regulating mRNA processing and abundance. Mutations of OsALKBH5 cause reduced double-strand break (DSB) formation, severe defects in DSB repair, and delayed meiotic progression, leading to complete male sterility. Transcriptome analysis and m6A profiling indicate that OsALKBH5-mediated m6A demethylation stabilizes the mRNA level of multiple meiotic genes directly or indirectly, including several genes that regulate DSB formation and repair. Our study reveals the indispensable role of m6A metabolism in post-transcriptional regulation of meiotic progression in rice.

Transcriptome and Coexpression Network Analyses Provide In-Sights into the Molecular Mechanisms of Hydrogen Cyanide Synthesis during Seed Development in Common Vetch (Vicia sativa L.).

The common vetch (Vicia sativa L.) seed is an ideal plant-based protein food for humans, but its edible value is mainly limited by the presence of cyanogenic glycosides that hydrolyze to produce toxic hydrogen cyanide (HCN), and the genes that regulate HCN synthesis in common vetch are unknown. In this study, seeds from common vetch at 5, 10, 15, 20, 25, 30, and 35 days after anthesis were sampled, and the seven stages were further divided into five developmental stages, S1, S2, S3, S4, and S5, based on morphological and transcriptome analyses. A total of 16,403 differentially expressed genes were identified in the five developmental stages. The HCN contents of seeds in these five stages were determined by alkaline titration, and weighted gene coexpression network analysis was used to explain the molecular regulatory mechanism of HCN synthesis in common vetch seeds. Eighteen key regulatory genes for HCN synthesis were identified, including the VsGT2, VsGT17 and CYP71A genes, as well as the VsGT1 gene family. VsGT1, VsGT2, VsGT17 and CYP71A jointly promoted HCN synthesis, from 5 to 25 days after anthesis, with VsGT1-1, VsGT1-4, VsGT1-11 and VsGT1-14 playing major roles. The HCN synthesis was mainly regulated by VsGT1, from 25 to 35 days after anthesis. As the expression level of VsGT1 decreased, the HCN content no longer increased. In-depth elucidation of seed HCN synthesis lays the foundations for breeding common vetch with low HCN content.

Two Nucleoporin98 homologous genes jointly participate in the regulation of starch degradation to repress senescence in Arabidopsis.

BACKGROUND: Starch is synthesized during daylight for temporary storage in leaves and then degraded during the subsequent night to support plant growth and development. Impairment of starch degradation leads to stunted growth, even senescence and death. The nuclear pore complex is involved in many cellular processes, but its relationship with starch degradation has been unclear until now. We previously identified that two Nucleoporin98 genes (Nup98a and Nup98b) redundantly regulate flowering via the CONSTANS (CO)-independent pathway in Arabidopsis thaliana. The double mutant also shows severe senescence phenotypes. RESULTS: We find that Nucleoporin 98 participates in the regulation of sugar metabolism in leaves and is also involved in senescence regulation in Arabidopsis. We show that Nup98a and Nup98b function redundantly at different stages of starch degradation. The nup98a-1 nup98b-1 double mutant accumulates more starch, showing a severe early senescence phenotype compared to wild type plants. The expression of marker genes related to starch degradation is impaired in the nup98a-1 nup98b-1 double mutant, and marker genes of carbon starvation and senescence express their products earlier and in higher abundance than in wild type plants, suggesting that abnormalities in energy metabolism are the main cause of senescence in the double mutant. Addition of sucrose to the growth medium rescues early senescence phenotypes of the nup98a-1 nup98b-1 mutant. CONCLUSIONS: Our results provide evidence for a novel role of the nuclear pore complex in energy metabolism related to growth and development, in which Nup98 functions in starch degradation to control growth regulation in Arabidopsis.

Genome-wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea.

Hybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24-nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid-MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down-regulated hybrid-MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Transcriptome-Wide Mapping of RNA 5-Methylcytosine in Arabidopsis mRNAs and Noncoding RNAs.

Posttranscriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification with diverse roles, such as regulating stress responses, stem cell proliferation, and RNA metabolism. Here, we used RNA bisulfite sequencing for transcriptome-wide quantitative mapping of m5C in the model plant Arabidopsis thaliana We discovered more than a thousand m5C sites in Arabidopsis mRNAs, long noncoding RNAs, and other noncoding RNAs across three tissue types (siliques, seedling shoots, and roots) and validated a number of these sites. Quantitative differences in methylated sites between these three tissues suggest tissue-specific regulation of m5C. Perturbing the RNA m5C methyltransferase TRM4B resulted in the loss of m5C sites on mRNAs and noncoding RNAs and reduced the stability of tRNAAsp(GTC) We also demonstrate the importance of m5C in plant development, as trm4b mutants have shorter primary roots than the wild type due to reduced cell division in the root apical meristem. In addition, trm4b mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50-nucleotide sequence flanking m5C C3349 in MAIGO5 mRNA is sufficient to confer methylation of a transgene reporter in Nicotiana benthamiana.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.

Experimental reconstruction of double-stranded break repair-mediated plastid DNA insertion into the tobacco nucleus.

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double-stranded DNA breaks into the nuclear genome of tobacco through inducible expression of I-SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double-stranded DNA break repair. Microhomology-mediated linking of disparate segments of chloroplast DNA occurs frequently during healing of induced nuclear double-stranded breaks (DSB) but the resulting nuclear integrants are often immediately unstable. Non-Mendelian inheritance of a selectable marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the screen. The instability of these de novo nuclear insertions of plastid DNA (nupts) was shown to be associated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of transfer. This deletion of pre-existing nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is potentially far greater than previously thought.

Transposable elements (TEs) contribute to stress-related long intergenic noncoding RNAs in plants.

Noncoding RNAs have been extensively described in plant and animal transcriptomes by using high-throughput sequencing technology. Of these noncoding RNAs, a growing number of long intergenic noncoding RNAs (lincRNAs) have been described in multicellular organisms, however the origins and functions of many lincRNAs remain to be explored. In many eukaryotic genomes, transposable elements (TEs) are widely distributed and often account for large fractions of plant and animal genomes yet the contribution of TEs to lincRNAs is largely unknown. By using strand-specific RNA-sequencing, we profiled the expression patterns of lincRNAs in Arabidopsis, rice and maize, and identified 47 611 and 398 TE-associated lincRNAs (TE-lincRNAs), respectively. TE-lincRNAs were more often derived from retrotransposons than DNA transposons and as retrotransposon copy number in both rice and maize genomes so did TE-lincRNAs. We validated the expression of these TE-lincRNAs by strand-specific RT-PCR and also demonstrated tissue-specific transcription and stress-induced TE-lincRNAs either after salt, abscisic acid (ABA) or cold treatments. For Arabidopsis TE-lincRNA11195, mutants had reduced sensitivity to ABA as demonstrated by longer roots and higher shoot biomass when compared to wild-type. Finally, by altering the chromatin state in the Arabidopsis chromatin remodelling mutant ddm1, unique lincRNAs including TE-lincRNAs were generated from the preceding untranscribed regions and interestingly inherited in a wild-type background in subsequent generations. Our findings not only demonstrate that TE-associated lincRNAs play important roles in plant abiotic stress responses but lincRNAs and TE-lincRNAs might act as an adaptive reservoir in eukaryotes.

Two SUMO Proteases SUMO PROTEASE RELATED TO FERTILITY1 and 2 Are Required for Fertility in Arabidopsis.

In plants, the posttranslational modification small ubiquitin-like modifier (SUMO) is involved in regulating several important developmental and cellular processes, including flowering time control and responses to biotic and abiotic stresses. Here, we report two proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2, that regulate male and female gamete and embryo development and remove SUMO from proteins in vitro and in vivo. spf1 mutants exhibit abnormal floral structures and embryo development, while spf2 mutants exhibit largely a wild-type phenotype. However, spf1 spf2 double mutants exhibit severe abnormalities in microgametogenesis, megagametogenesis, and embryo development, suggesting that the two genes are functionally redundant. Mutation of SPF1 and SPF2 genes also results in misexpression of generative- and embryo-specific genes. In vitro, SPF1 and SPF2 process SUMO1 precursors into a mature form, and as expected in vivo, spf1 and spf2 mutants accumulate SUMO conjugates. Using a yeast two-hybrid screen, we identified EMBRYO SAC DEVELOPMENT ARREST9 (EDA9) as an SPF1-interacting protein. In vivo, we demonstrate that EDA9 is sumolyated and that, in spf1 mutants, EDA9-SUMO conjugates increase in abundance, demonstrating that EDA9 is a substrate of SPF1. Together, our results demonstrate that SPF1 and SPF2 are two SUMO proteases important for plant development in Arabidopsis (Arabidopsis thaliana).

JMJ14, a JmjC domain protein, is required for RNA silencing and cell-to-cell movement of an RNA silencing signal in Arabidopsis.

JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.

Origin and Functional Prediction of Pollen Allergens in Plants.

Pollen allergies have long been a major pandemic health problem for human. However, the evolutionary events and biological function of pollen allergens in plants remain largely unknown. Here, we report the genome-wide prediction of pollen allergens and their biological function in the dicotyledonous model plant Arabidopsis (Arabidopsis thaliana) and the monocotyledonous model plant rice (Oryza sativa). In total, 145 and 107 pollen allergens were predicted from rice and Arabidopsis, respectively. These pollen allergens are putatively involved in stress responses and metabolic processes such as cell wall metabolism during pollen development. Interestingly, these putative pollen allergen genes were derived from large gene families and became diversified during evolution. Sequence analysis across 25 plant species from green alga to angiosperms suggest that about 40% of putative pollen allergenic proteins existed in both lower and higher plants, while other allergens emerged during evolution. Although a high proportion of gene duplication has been observed among allergen-coding genes, our data show that these genes might have undergone purifying selection during evolution. We also observed that epitopes of an allergen might have a biological function, as revealed by comprehensive analysis of two known allergens, expansin and profilin. This implies a crucial role of conserved amino acid residues in both in planta biological function and allergenicity. Finally, a model explaining how pollen allergens were generated and maintained in plants is proposed. Prediction and systematic analysis of pollen allergens in model plants suggest that pollen allergens were evolved by gene duplication and then functional specification. This study provides insight into the phylogenetic and evolutionary scenario of pollen allergens that will be helpful to future characterization and epitope screening of pollen allergens.

Deciphering the epitranscriptome: A green perspective.

The advent of high-throughput sequencing technologies coupled with new detection methods of RNA modifications has enabled investigation of a new layer of gene regulation - the epitranscriptome. With over 100 known RNA modifications, understanding the repertoire of RNA modifications is a huge undertaking. This review summarizes what is known about RNA modifications with an emphasis on discoveries in plants. RNA ribose modifications, base methylations and pseudouridylation are required for normal development in Arabidopsis, as mutations in the enzymes modifying them have diverse effects on plant development and stress responses. These modifications can regulate RNA structure, turnover and translation. Transfer RNA and ribosomal RNA modifications have been mapped extensively and their functions investigated in many organisms, including plants. Recent work exploring the locations, functions and targeting of N6 -methyladenosine (m6 A), 5-methylcytosine (m5 C), pseudouridine (Ψ), and additional modifications in mRNAs and ncRNAs are highlighted, as well as those previously known on tRNAs and rRNAs. Many questions remain as to the exact mechanisms of targeting and functions of specific modified sites and whether these modifications have distinct functions in the different classes of RNAs.

Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae.

BACKGROUND: Post-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m(5)C) is an important modification that regulates RNA metabolism and occurs in both eukaryotes and prokaryotes. Yet, to date, no transcriptome-wide identification of m(5)C sites has been undertaken in plants. Plants provide a unique comparative system for investigating the origin and evolution of m(5)C as they contain three different genomes, the nucleus, mitochondria and chloroplast. Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m(5)C sites in non-coding ribosomal RNAs and transfer RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba. RESULTS: Using the plant model Arabidopsis thaliana, we identified a total of 39 highly methylated m(5)C sites in predicted structural positions of nuclear tRNAs and 7 m(5)C sites in rRNAs from nuclear, chloroplast and mitochondrial transcriptomes. Both the nucleotide position and percent methylation of tRNAs and rRNAs m(5)C sites were conserved across all species analysed, from single celled algae N. oculata to multicellular plants. Interestingly the mitochondrial and chloroplast encoded tRNAs were devoid of m(5)C in A. thaliana and this is generally conserved across Plantae. This suggests independent evolution of organelle methylation in animals and plants, as animal mitochondrial tRNAs have m(5)C sites. Here we characterize 5 members of the RNA 5-methylcytosine family in Arabidopsis and extend the functional characterization of TRDMT1 and NOP2A/OLI2. We demonstrate that nuclear tRNA methylation requires two evolutionarily conserved methyltransferases, TRDMT1 and TRM4B. trdmt1 trm4b double mutants are hypersensitive to the antibiotic hygromycin B, demonstrating the function of tRNA methylation in regulating translation. Additionally we demonstrate that nuclear large subunit 25S rRNA methylation requires the conserved RNA methyltransferase NSUN5. Our results also suggest functional redundancy of at least two of the NOP2 paralogs in Arabidopsis. CONCLUSIONS: Our data demonstrates widespread occurrence and conservation of non-coding RNA methylation in the kingdom Plantae, suggesting important and highly conserved roles of this post-transcriptional modification.

Mobile microRNAs hit the target.

MicroRNAs (miRNAs) are negative regulators of gene expression in eukaryotic organisms, whereas small interfering RNAs (siRNAs) guide host-cell defence against viruses, transposons and transgenes. A key issue in plant biology is whether miRNAs act only in cells in which they are formed, or if, like siRNAs, they also function after passive diffusion or active transportation into other cells. Recent reports show that miRNAs are indeed able to move between plant cells to direct developmental programming of gene expression. In both leaf and root development, miRNAs establish intercellular gradients of gene expression that are essential for cell and tissue differentiation. Gradients in gene expression also play crucial roles in animal development, and there is strong evidence for intercellular movement of miRNAs in animals. Thus, intercellular movement of miRNAs may be crucial to animal developmental biology as well as plants.

Functional characterisation of HvCO1, the barley (Hordeum vulgare) flowering time ortholog of CONSTANS.

Variation in photoperiod response is a major factor determining plant development and the agronomic performance of crops. The genetic control of photoperiodic flowering has been elucidated in the model plant Arabidopsis, and many of the identified genes are structurally conserved in the grasses. In this study, HvCO1, the closest barley ortholog of the key photoperiod response gene CONSTANS in Arabidopsis, was over-expressed in the spring barley Golden Promise. Over-expression of HvCO1 accelerated time to flowering in long- and short-day conditions and caused up-regulation of HvFT1 mRNA under long-day conditions. However, the transgenic plants retained a response to photoperiod, suggesting the presence of photoperiod response factors acting downstream of HvCO1 transcription. Analysis of a population segregating for HvCO1 over-expression and natural genetic variation at Ppd-H1 demonstrated that Ppd-H1 acts downstream of HvCO1 transcription on HvFT1 expression and flowering. Furthermore, variation at Ppd-H1 did not affect diurnal expression of HvCO1 or HvCO2. Over-expression of HvCO1 increased transcription of the spring allele of Vrn-H1 in long- and short-day conditions, while genetic variation at Ppd-H1 did not affect Vrn-H1 expression. Over-expression of HvCO1 and natural genetic variation at Ppd-H1 accelerated inflorescence development and stem elongation. Thus, HvCO1 probably induces flowering by activating HvFT1 whilst Ppd-H1 regulates HvFT1 independently of HvCO1 mRNA, and all three genes also appear to have a strong effect in promoting inflorescence development.

Keeping it cool.

A well-established model for how plants start the process of flowering in periods of cold weather may need revisiting.

The role of epigenetic and epitranscriptomic modifications in plants exposed to non-essential metals.

Contamination of the soil with non-essential metals and metalloids is a serious problem in many regions of the world. These non-essential metals and metalloids are toxic to all organisms impacting crop yields and human health. Crop plants exposed to high concentrations of these metals leads to perturbed mineral homeostasis, decreased photosynthesis efficiency, inhibited cell division, oxidative stress, genotoxic effects and subsequently hampered growth. Plants can activate epigenetic and epitranscriptomic mechanisms to maintain cellular and organism homeostasis. Epigenetic modifications include changes in the patterns of cytosine and adenine DNA base modifications, changes in cellular non-coding RNAs, and remodeling histone variants and covalent histone tail modifications. Some of these epigenetic changes have been shown to be long-lasting and may therefore contribute to stress memory and modulated stress tolerance in the progeny. In the emerging field of epitranscriptomics, defined as chemical, covalent modifications of ribonucleotides in cellular transcripts, epitranscriptomic modifications are postulated as more rapid modulators of gene expression. Although significant progress has been made in understanding the plant's epigenetic changes in response to biotic and abiotic stresses, a comprehensive review of the plant's epigenetic responses to metals is lacking. While the role of epitranscriptomics during plant developmental processes and stress responses are emerging, epitranscriptomic modifications in response to metals has not been reviewed. This article describes the impact of non-essential metals and metalloids (Cd, Pb, Hg, Al and As) on global and site-specific DNA methylation, histone tail modifications and epitranscriptomic modifications in plants.

Challenges facing sustainable protein production: Opportunities for cereals.

Rising demands for protein worldwide are likely to drive increases in livestock production, as meat provides ∼40% of dietary protein. This will come at a significant environmental cost, and a shift toward plant-based protein sources would therefore provide major benefits. While legumes provide substantial amounts of plant-based protein, cereals are the major constituents of global foods, with wheat alone accounting for 15-20% of the required dietary protein intake. Improvement of protein content in wheat is limited by phenotyping challenges, lack of genetic potential of modern germplasms, negative yield trade-offs, and environmental costs of nitrogen fertilizers. Presenting wheat as a case study, we discuss how increasing protein content in cereals through a revised breeding strategy combined with robust phenotyping could ensure a sustainable protein supply while minimizing the environmental impact of nitrogen fertilizer.