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.

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.

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.

Keeping it cool.

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

Reviving grain quality in wheat through non-destructive phenotyping techniques like hyperspectral imaging.

A long-term goal of breeders and researchers is to develop crop varieties that can resist environmental stressors and produce high yields. However, prioritising yield often compromises improvement of other key traits, including grain quality, which is tedious and time-consuming to measure because of the frequent involvement of destructive phenotyping methods. Recently, non-destructive methods such as hyperspectral imaging (HSI) have gained attention in the food industry for studying wheat grain quality. HSI can quantify variations in individual grains, helping to differentiate high-quality grains from those of low quality. In this review, we discuss the reduction of wheat genetic diversity underlying grain quality traits due to modern breeding, key traits for grain quality, traditional methods for studying grain quality and the application of HSI to study grain quality traits in wheat and its scope in breeding. Our critical review of literature on wheat domestication, grain quality traits and innovative technology introduces approaches that could help improve grain quality in wheat.

Volatile profiles of commercial vetch prepared via different processing methods.

Vicia sativa (Common Vetch) is currently an underutilised leguminous crop species with high protein content and superior drought tolerance. This study aimed to understand the mechanisms behind vetch flavor development following processing to facilitate its uptake as a future source of dietary protein. A total of 95 volatile compounds were identified by solid-phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) for a range of vetches processed by dehulling, soaking, germination, microwaving, and fermentation.2-pentyl furan, benzyl alcohol, benzaldehyde, 1-octen-3-ol and 1-hexanol were found to be characteristic aroma compounds of V. sativa. Analysis of a V. sativa landrace demonstrated significant intraspecies variation in volatile abundance, three-fold that of commercial varieties. Both natto and tempeh fermentation produced significant quantities of alcohols, esters, and carboxylic acids with specifically natto generating significant pyrazines. Concentrations of 1-octen-3-ol significantly decreased after tempeh fermentation indicating its potential to reduce documented off flavor generating volatiles within V. sativa.

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 modify inflorescence branching and increase grain protein content of wheat.

Plant and inflorescence architecture determine the yield potential of crops. Breeders have harnessed natural diversity for inflorescence architecture to improve yields, and induced genetic variation could provide further gains. Wheat is a vital source of protein and calories; however, little is known about the genes that regulate the development of its inflorescence. Here, we report the identification of semidominant alleles for a class III homeodomain-leucine zipper transcription factor, HOMEOBOX DOMAIN-2 (HB-2), on wheat A and D subgenomes, which generate more flower-bearing spikelets and enhance grain protein content. These alleles increase HB-2 expression by disrupting a microRNA 165/166 complementary site with conserved roles in plants; higher HB-2 expression is associated with modified leaf and vascular development and increased amino acid supply to the inflorescence during grain development. These findings enhance our understanding of genes that control wheat inflorescence development and introduce an approach to improve the nutritional quality of grain.

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.

Chromosome-level assembly of the common vetch (Vicia sativa) reference genome.

Vicia sativa L. (common vetch, n = 6) is an annual, herbaceous, climbing legume, originating in the Fertile Crescent of the Middle East and now widespread in the Mediterranean basin, West, Central and Eastern Asia, North and South America. V. sativa is of economic importance as a forage legume in countries such as Australia, China, and the USA, and contributes valuable nitrogen to agricultural rotation cropping systems. To accelerate precision genome breeding and genomics-based selection of this legume, we present a chromosome-level reference genome sequence for V. sativa, constructed using a combination of long-read Oxford Nanopore sequencing, short-read Illumina sequencing, and high-throughput chromosome conformation data (CHiCAGO and Hi-C) analysis. The chromosome-level assembly of six pseudo-chromosomes has a total genome length of 1.65 Gbp, with a median contig length of 684 Kbp. BUSCO analysis of the assembly demonstrated very high completeness of 98% of the dicotyledonous orthologs. RNA-seq analysis and gene modelling enabled the annotation of 53,218 protein-coding genes. This V. sativa assembly will provide insights into vetch genome evolution and be a valuable resource for genomic breeding, genetic diversity and for understanding adaption to diverse arid environments.

Near-Complete Genome Assembly of the Grapevine Crown Gall Pathogen Allorhizobium vitis Strain K377.

Here, we report the annotated, near-complete genome sequence of Allorhizobium vitis K377, a phytopathogenic Rhizobiales strain isolated from a grapevine in South Australia. The assembled genome sequence is 6.40 Mb long, with 5,855 predicted protein-coding sequences, 56 tRNAs, and 12 rRNAs, and contains ttuC (tartrate metabolism; chromosomal) and nopaline synthesis, uptake, and catabolic genes (tumor-inducing plasmid-encoded).

Novel allele elh of the UBP14 gene affects plant organ size via cell expansion in Arabidopsis thaliana.

Plant organ size control is an essential process of plant growth and development. The regulation of plant organ size involves a complicated network of genetic, molecular interactions, as well as the interplay of environmental factors. Here, we report a temperature-sensitive hypocotyl elongation EMS-generated mutant, hereby referred to as elongated hypocotyl under high-temperature (elh). The elongated hypocotyl phenotype was prominent when the elh seedlings were grown at high temperature, 28°C, but not under the growth temperature of 21°C. We observed significantly larger organ sizes in elh plants, including cotyledons, petals and seeds. In elh plants, the cell sizes in cotyledons and petals were significantly larger than wild type. By measuring the cell density and organ area of cotyledons, petals and mature dissected embryos, we found no differences in total cell numbers in any organ indicating that cell expansion rather than cell proliferation was perturbed in elh. elh plants produced leaves at a slower rate than wild type plants, suggesting that perturbing the balance between cell division and cell expansion is linked to the developmental rate at which leaves are produced.

Quantitative and Single-Nucleotide Resolution Profiling of RNA 5-Methylcytosine.

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (m5C) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of m5C in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.

A High Amylose Wheat Diet Improves Gastrointestinal Health Parameters and Gut Microbiota in Male and Female Mice.

High amylose wheat (HAW) contains more resistant starch than standard amylose wheat (SAW) and may have beneficial effects on gastrointestinal health. However, it is currently unclear whether these effects differ according to the level of HAW included in the diet or between males and females. Male and female C57BL/6 mice (n = 8/group/sex) were fed SAW65 (65% SAW; control), HAW35 (35% HAW), HAW50 (50% HAW) or HAW65 (65% HAW) diet for eight weeks. Female but not male, mice consuming any amount of HAW exhibited accelerated gastric emptying compared to SAW65 group. In both sexes, relative colon weights were higher in the HAW65 group compared to SAW65 group and in females, relative weights of the small intestine and cecum were also higher in the HAW65 group. In females only, colonic expression of Pyy and Ocln mRNAs were higher in the HAW65 group compared to HAW35 and HAW50 groups. In both sexes, mice consuming higher amounts of HAW (HAW50 or HAW65) had increased fecal bacterial load and relative abundance of Bacteroidetes phylum and reduced relative abundance of Firmicutes compared to SAW65 group. These data are consistent with a beneficial impact of HAW on gastrointestinal health and indicate dose-dependent and sex-specific effects of HAW consumption.

An Efficient Root Transformation System for Recalcitrant Vicia sativa.

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop a simple, efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. At first, we infected the hypocotyls of 5-day-old in vitro or in vivo, soil-grown seedlings with Rhizobium rhizogenes K599 using a stabbing method and produced transgenic hairy roots after 24 days at 19 and 50% efficiency, respectively. We later improved the hairy root transformation in vitro by infecting different explants (seedling, hypocotyl-epicotyl, and shoot) with R. rhizogenes. We observed hairy root formation at the highest efficiency in shoot and hypocotyl-epicotyl explants with 100 and 93% efficiency, respectively. In both cases, an average of four hairy roots per explant were obtained, and about 73 and 91% of hairy roots from shoot and hypocotyl-epicotyl, respectively, showed stable expression of a co-transformed marker ß-glucuronidase (GUS). In summary, we developed a rapid, highly efficient, hairy root transformation method by using R. rhizogenes on vetch explants, which could facilitate functional gene analysis in common vetch.

Complete Genome Sequence of Allorhizobium vitis Strain K306, the Causal Agent of Grapevine Crown Gall.

Here, we present the annotated complete genome sequence of Allorhizobium vitis K306, a phytopathogenic strain causing crown gall of grapevine. The A. vitis K306 genome is 5.79 Mb long with 5,199 predicted protein-coding genes and contains 2 circular chromosomes of 3.8 Mb and 1.1 Mb and 2 plasmids, namely, pTiK306 and pTrK306, that are 262 kb and 581 kb, respectively.

Common Vetch: A Drought Tolerant, High Protein Neglected Leguminous Crop With Potential as a Sustainable Food Source.

Global demand for protein is predicted to increase by 50% by 2050. To meet the increasing demand whilst ensuring sustainability, protein sources that generate low-greenhouse gas emissions are required, and protein-rich legume seeds have the potential to make a significant contribution. Legumes like common vetch (Vicia sativa) that grow in marginal cropping zones and are drought tolerant and resilient to changeable annual weather patterns, will be in high demand as the climate changes. In common vetch, the inability to eliminate the γ-glutamyl-ß-cyano-alanine (GBCA) toxin present in the seed has hindered its utility as a human and animal food for many decades, leaving this highly resilient species an "orphan" legume. However, the availability of the vetch genome and transcriptome data together with the application of CRISPR-Cas genome editing technologies lay the foundations to eliminate the GBCA toxin constraint. In the near future, we anticipate that a zero-toxin vetch variety will become a significant contributor to global protein demand.

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.