Histone modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

Unraveling the maternal and paternal origins of allotetraploid Vigna reflexo-pilosa.

The genomic structures of Vigna hirtella Ridl. and Vigna trinervia (B.Heyne ex Wight & Arn.) Tateishi & Maxted, key ancestral species of the allotetraploid Vigna reflexo-pilosa var. glabra (Roxb.) N.Tomooka & Maxted, remain poorly understood. This study presents a comprehensive genomic comparison of these species to deepen our knowledge of their evolutionary trajectories. By comparing the genomic profiles of V. hirtella and V. trinervia with those of V. reflexo-pilosa, we investigate the complex genomic mechanisms underlying allopolyploid evolution within the genus Vigna. Comparison of the chloroplast genome revealed that V. trinervia is closely related to V. reflexo-pilosa. De novo assembly of the whole genome, followed by synteny analysis and Ks value calculations, confirms that V. trinervia is closely related to the A genome of V. reflexo-pilosa, and V. hirtella to its B genome. Furthermore, the comparative analyses reveal that V. reflexo-pilosa retains residual signatures of a previous polyploidization event, particularly evident in higher gene family copy numbers. Our research provides genomic evidence for polyploidization within the genus Vigna and identifies potential donor species of allotetraploid species using de novo assembly techniques. Given the Southeast Asian distribution of both V. hirtella and V. trinervia, natural hybridization between these species, with V. trinervia as the maternal ancestor and V. hirtella as the paternal donor, seems plausible.

The complete mitochondrial genome of the biodiesel plant Jatropha curcas L.

Jatropha curcas (Linnaeus, 1753) is a plant species in the order Malpighiales and the family Euphorbiaceae and is native to the tropical regions of America, such as Mexico and Argentina. Currently, this plant species inhabits tropical and subtropical regions of the world. Jatropha has been widely used as a biofuel plant to produce high-quality diesel engine fuel. In this study, the complete mitochondrial genome sequence of J. curcas was assembled into 561,839 bp circular nucleotides with a GC content of 44.6%. The mitochondrial genome of J. curcas comprises 33 known protein-coding genes, 22 tRNA genes, three rRNA genes, one ncRNA gene, and 85 open reading frame genes. Phylogenetic analysis showed this species is closely related to the castor bean (Ricinus communis).

De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

Dynamic changes in DNA methylation occur in TE regions and affect cell proliferation during leaf-to-callus transition in Arabidopsis.

Plant somatic cells can be reprogrammed into pluripotent cell mass, called callus, through a two-step in vitro tissue culture method. Incubation on callus-inducing medium triggers active cell proliferation to form a pluripotent callus. Notably, DNA methylation is implicated during callus formation, but a detailed molecular process regulated by DNA methylation remains to be fully elucidated. Here, we compared genome-wide DNA methylation profiles between leaf and callus tissues in Arabidopsis using whole-genome bisulphite-sequencing. Global distribution of DNA methylation showed that CHG methylation was increased, whereas CHH methylation was reduced especially around transposable element (TE) regions during the leaf-to-callus transition. We further analysed differentially expressed genes around differentially methylated TEs (DMTEs) during the leaf-to-callus transition and found that genes involved in cell cycle regulation were enriched and also constituted a coexpression gene network along with pluripotency regulators. In addition, a conserved DNA sequence analysis for upstream cis-elements led us to find a putative transcription factor associated with cell fate transition. CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) was newly identified as a regulator of plant regeneration, and consistently, the cca1lhy mutant displayed altered phenotypes in callus proliferation. Overall, these results suggest that DNA methylation coordinates cell cycle regulation during callus formation, and CCA1 may act as a key upstream coordinator at least in part in the processes.

iRegNet: an integrative Regulatory Network analysis tool for Arabidopsis thaliana.

Gene expression is delicately controlled via multilayered genetic and/or epigenetic regulatory mechanisms. Rapid development of the high-throughput sequencing (HTS) technology and its derivative methods including chromatin immunoprecipitation sequencing (ChIP-seq) and DNA affinity purification sequencing (DAP-seq) have generated a large volume of data on DNA-protein interactions (DPIs) and histone modifications on a genome-wide scale. However, the ability to comprehensively retrieve empirically validated upstream regulatory networks of genes of interest (GOIs) and genomic regions of interest (ROIs) remains limited. Here, we present integrative Regulatory Network (iRegNet), a web application that analyzes the upstream regulatory network for user-queried GOIs or ROIs in the Arabidopsis (Arabidopsis thaliana) genome. iRegNet covers the largest empirically proven DNA-binding profiles of Arabidopsis transcription factors (TFs) and non-TF proteins, and histone modifications obtained from all currently available Arabidopsis ChIP-seq and DAP-seq data. iRegNet not only catalogs upstream regulomes and epigenetic chromatin states for single-query gene/genomic region but also suggests significantly overrepresented upstream genetic regulators and epigenetic chromatin states of user-submitted multiple query genes/genomic regions. Furthermore, gene-to-gene coexpression index and protein-protein interaction information were also integrated into iRegNet for a more reliable identification of upstream regulators and realistic regulatory networks. Thus, iRegNet will help discover upstream regulators as well as molecular regulatory networks of GOI(s) and/or ROI(s), and is freely available at http://chromatindynamics.snu.ac.kr:8082/iRegNet_main.

MET1-Dependent DNA Methylation Represses Light Signaling and Influences Plant Regeneration in Arabidopsis.

Plant somatic cells can be reprogrammed into a pluripotent cell mass, called callus, which can be subsequently used for de novo shoot regeneration through a two-step in vitro tissue culture method. MET1-dependent CG methylation has been implicated in plant regeneration in Arabidopsis, because the met1-3 mutant exhibits increased shoot regeneration compared with the wild-type. To understand the role of MET1 in de novo shoot regeneration, we compared the genome-wide DNA methylomes and transcriptomes of wild-type and met1-3 callus and leaf. The CG methylation patterns were largely unchanged during leaf-to-callus transition, suggesting that the altered regeneration phenotype of met1-3 was caused by the constitutively hypomethylated genes, independent of the tissue type. In particular, MET1-dependent CG methylation was observed at the blue light receptor genes, CRYPTOCHROME 1 (CRY1) and CRY2, which reduced their expression. Coexpression network analysis revealed that the CRY1 gene was closely linked to cytokinin signaling genes. Consistently, functional enrichment analysis of differentially expressed genes in met1-3 showed that gene ontology terms related to light and hormone signaling were overrepresented. Overall, our findings indicate that MET1-dependent repression of light and cytokinin signaling influences plant regeneration capacity and shoot identity establishment.

Genome of the world's smallest flowering plant, Wolffia australiana, helps explain its specialized physiology and unique morphology.

Watermeal, Wolffia australiana, is the smallest known flowering monocot and is rich in protein. Despite its great potential as a biotech crop, basic research on Wolffia is in its infancy. Here, we generated the reference genome of a species of watermeal, W. australiana, and identified the genome-wide features that may contribute to its atypical anatomy and physiology, including the absence of roots, adaxial stomata development, and anaerobic life as a turion. In addition, we found evidence of extensive genome rearrangements that may underpin the specialized aquatic lifestyle of watermeal. Analysis of the gene inventory of this intriguing species helps explain the distinct characteristics of W. australiana and its unique evolutionary trajectory.

Major QTLs, qARO1 and qARO9, Additively Regulate Adaxial Leaf Rolling in Rice.

Moderate leaf rolling is considered optimal for the ideal plant type in rice (Oryza sativa L.), as it improves photosynthetic efficiency and, consequently, grain yield. Determining the genetic basis of leaf rolling via the identification of quantitative trait loci (QTLs) could facilitate the development of high-yielding varieties. In this study, we identified three stable rice QTLs, qARO1, qARO5, and qARO9, which control adaxial leaf rolling in a recombinant inbred line (RIL) population derived from a cross between Tong 88-7 (T887) and Milyang 23 (M23), using high-density SNP markers. These QTLs controlled the rolling phenotype of both the flag leaf (FL) and secondary leaf (SL), and different allelic combinations of these QTLs led to a wide variation in the degree of leaf rolling. Additive gene actions of qARO1 and qARO9 on leaf rolling were observed in a backcross population. In addition, qARO1 (markers: 01id4854718 and 01asp4916781) and qARO9 (markers: 09id19650402 and 09id19740436) were successfully fine-mapped to approximately 60- and 90-kb intervals on chromosomes 1 and 9, respectively. Histological analysis of near-isogenic lines (NILs) revealed that qARO1 influences leaf thickness across the small vein, and qARO9 affects leaf thickness in the entire leaf and bulliform cell area, thus leading to adaxial leaf rolling. The results of this study advance our understanding of the genetic and molecular bases of adaxial leaf rolling, and this information can be used for the development of rice varieties with the ideal plant type.

Transcriptome comparison between pluripotent and non-pluripotent calli derived from mature rice seeds.

In vitro plant regeneration involves a two-step practice of callus formation and de novo organogenesis. During callus formation, cellular competence for tissue regeneration is acquired, but it is elusive what molecular processes and genetic factors are involved in establishing cellular pluripotency. To explore the mechanisms underlying pluripotency acquisition during callus formation in monocot plants, we performed a transcriptomic analysis on the pluripotent and non-pluripotent rice calli using RNA-seq. We obtained a dataset of differentially expressed genes (DEGs), which accounts for molecular processes underpinning pluripotency acquisition and maintenance. Core regulators establishing root stem cell niche were implicated in pluripotency acquisition in rice callus, as observed in Arabidopsis. In addition, KEGG analysis showed that photosynthetic process and sugar and amino acid metabolism were substantially suppressed in pluripotent calli, whereas lipid and antioxidant metabolism were overrepresented in up-regulated DEGs. We also constructed a putative coexpression network related to cellular pluripotency in rice and proposed potential candidates conferring pluripotency in rice callus. Overall, our transcriptome-based analysis can be a powerful resource for the elucidation of the molecular mechanisms establishing cellular pluripotency in rice callus.

EAT-UpTF: Enrichment Analysis Tool for Upstream Transcription Factors of a Group of Plant Genes.

EAT-UpTF (Enrichment Analysis Tool for Upstream Transcription Factors of a group of plant genes) is an open-source Python script that analyzes the enrichment of upstream transcription factors (TFs) in a group of genes-of-interest (GOIs). EAT-UpTF utilizes genome-wide lists of TF-target genes generated by DNA affinity purification followed by sequencing (DAP-seq) or chromatin immunoprecipitation followed by sequencing (ChIP-seq). Unlike previous methods based on the two-step prediction of cis-motifs and DNA-element-binding TFs, our EAT-UpTF analysis enabled a one-step identification of enriched upstream TFs in a set of GOIs using lists of empirically determined TF-target genes. The tool is designed particularly for plant researches, due to the lack of analytic tools for upstream TF enrichment, and available at https://github.com/sangreashim/EAT-UpTF and http://chromatindynamics.snu.ac.kr:8080/EatupTF.

Transcriptomic and biochemical analyses of the accumulation of sucrose in mungbean (Vigna radiata (L.) Wilczek) leaves after pod removal.

KEY MESSAGE: Synchronous pod maturity is critical for increasing grain yield. The candidate genes involved in synchronous pod maturity were identified through RNA-seq and HPLC. Mungbean (Vigna radiata [L.] Wilczek), an important source of carbohydrate and protein in Asia, is characterized by nonsynchronous pod maturity; consequently, harvesting is labor intensive. Because pod maturity is associated with synthesis and remobilization of sucrose, we examined changes in sucrose levels and transcriptome in leaf (source) tissues after pod (sink) removal using two genotypes, VC1973A and V2984; VC1973A had higher synchronicity in pod maturity than V2984. After pod removal, much higher number of pods were produced in V2984 than VC1973A. The sucrose content of leaf tissues significantly decreased in V2984 because it continued to utilize assimilates from leaves for producing new pods, but significantly increased in VC1973A because of the loss of sink. Transcriptome analysis revealed that the number of differentially expressed genes was approximately fourfold higher in VC1973A than in those of V2984 after pod removal. The expression of two paralogous genes (Vradi01g05010 and Vradi10g08240), encoding beta-glucosidase enzymes, significantly decreased in VC1973A after pod removal and was significantly lower in depodded VC1973A than depodded V2984, indicating these two genes may participate in sucrose utilization for seed development by regulating the level of glucose. The results of this study will help elucidate the genetic basis of synchronous pod maturity in mungbean and facilitate the development of new cultivars with synchronous pod maturity.

H3K36me2 is highly correlated with m6 A modifications in plants.

The intimate linkage between H3K36me3 and m6 A modifications has been demonstrated in mammals. In this issue, Shim et al. (2020) show that similar crosstalk between histone modification and mRNA methylation is conserved in plants, but H3K36me2 is more important for m6A deposition in plants.

GmBRC1 is a Candidate Gene for Branching in Soybean (Glycine max (L.) Merrill).

Branch number is one of the main factors affecting the yield of soybean (Glycine max (L.)). In this study, we conducted a genome-wide association study combined with linkage analysis for the identification of a candidate gene controlling soybean branching. Five quantitative trait nucleotides (QTNs) were associated with branch numbers in a soybean core collection. Among these QTNs, a linkage disequilibrium (LD) block qtnBR6-1 spanning 20 genes was found to overlap a previously identified major quantitative trait locus qBR6-1. To validate and narrow down qtnBR6-1, we developed a set of near-isogenic lines (NILs) harboring high-branching (HB) and low-branching (LB) alleles of qBR6-1, with 99.96% isogenicity and different branch numbers. A cluster of single nucleotide polymorphisms (SNPs) segregating between NIL-HB and NIL-LB was located within the qtnBR6-1 LD block. Among the five genes showing differential expression between NIL-HB and NIL-LB, BRANCHED1 (BRC1; Glyma.06G210600) was down-regulated in the shoot apex of NIL-HB, and one missense mutation and two SNPs upstream of BRC1 were associated with branch numbers in 59 additional soybean accessions. BRC1 encodes TEOSINTE-BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS 1 and 2 transcription factor and functions as a regulatory repressor of branching. On the basis of these results, we propose BRC1 as a candidate gene for branching in soybean.

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed.

Mapping of QTLs for Seed Phorbol Esters, a Toxic Chemical in Jatropha curcas (L.).

Jatropha (Jatropha curcas L.) is an oil-bearing plant that has potential to be cultivated as a biodiesel crop. The seed cake after oil extraction has 40-50% protein that can be used in animal feeds. A major limitation in utilizing the cake is the presence of phorbol esters (PE), a heat-tolerant toxic chemical. To identify the quantitative trait loci (QTLs) for PE, we constructed a genetic linkage map from an F2 population of 95 individuals from a cross "Chai Nat" × "M10" using 143 simple sequence repeat (SSR) markers. M10 is low in seed PE while Chai Nat is high. Seeds from each F2 individual were quantified for PE content by high performance liquid chromatography. A single marker analysis revealed five markers from linkage group 3 (LG3) and nine markers from LG8 associated with seed PE. Inclusive composite interval mapping identified two QTLs, each on LG3 (qPE3.1) and LG8 (qPE8.1) responsible for the PE. qPE3.1 and qPE8.1 accounted for 14.10%, and 15.49% of total variation in seed PE, respectively. Alelle(s) from M10 at qPE3.1 increased seed PE, while at qPE8.1 decreased seed PE. qPE3.1 is a new loci for PE, while qPE8.1 is the same locus with that reported recently for PE.

Genome-wide DNA methylation profile in mungbean.

DNA methylation on cytosine residues is known to affect gene expression and is potentially responsible for the phenotypic variations among different crop cultivars. Here, we present the whole-genome DNA methylation profiles and assess the potential effects of single nucleotide polymorphisms (SNPs) for two mungbean cultivars, Sunhwanogdu (VC1973A) and Kyunggijaerae#5 (V2984). By measuring the DNA methylation levels in leaf tissue with the bisulfite sequencing (BSseq) approach, we show both the frequencies of the various types of DNA methylation and the distribution of weighted gene methylation levels. SNPs that cause nucleotide changes from/to CHH - where C is cytosine and H is any other nucleotide - were found to affect DNA methylation status in VC1973A and V2984. In order to better understand the correlation between gene expression and DNA methylation levels, we surveyed gene expression in leaf tissues of VC1973A and V2984 using RNAseq. Transcript expressions of paralogous genes were controlled by DNA methylation within the VC1973A genome. Moreover, genes that were differentially expressed between the two cultivars showed distinct DNA methylation patterns. Our mungbean genome-wide methylation profiles will be valuable resources for understanding the phenotypic variations between different cultivars, as well as for molecular breeding.

Translational genomics for plant breeding with the genome sequence explosion.

The use of next-generation sequencers and advanced genotyping technologies has propelled the field of plant genomics in model crops and plants and enhanced the discovery of hidden bridges between genotypes and phenotypes. The newly generated reference sequences of unstudied minor plants can be annotated by the knowledge of model plants via translational genomics approaches. Here, we reviewed the strategies of translational genomics and suggested perspectives on the current databases of genomic resources and the database structures of translated information on the new genome. As a draft picture of phenotypic annotation, translational genomics on newly sequenced plants will provide valuable assistance for breeders and researchers who are interested in genetic studies.

Draft genome sequence of adzuki bean, Vigna angularis.

Adzuki bean (Vigna angularis var. angularis) is a dietary legume crop in East Asia. The presumed progenitor (Vigna angularis var. nipponensis) is widely found in East Asia, suggesting speciation and domestication in these temperate climate regions. Here, we report a draft genome sequence of adzuki bean. The genome assembly covers 75% of the estimated genome and was mapped to 11 pseudo-chromosomes. Gene prediction revealed 26,857 high confidence protein-coding genes evidenced by RNAseq of different tissues. Comparative gene expression analysis with V. radiata showed that the tissue specificity of orthologous genes was highly conserved. Additional re-sequencing of wild adzuki bean, V. angularis var. nipponensis, and V. nepalensis, was performed to analyze the variations between cultivated and wild adzuki bean. The determined divergence time of adzuki bean and the wild species predated archaeology-based domestication time. The present genome assembly will accelerate the genomics-assisted breeding of adzuki bean.

Genome sequence of mungbean and insights into evolution within Vigna species.

Mungbean (Vigna radiata) is a fast-growing, warm-season legume crop that is primarily cultivated in developing countries of Asia. Here we construct a draft genome sequence of mungbean to facilitate genome research into the subgenus Ceratotropis, which includes several important dietary legumes in Asia, and to enable a better understanding of the evolution of leguminous species. Based on the de novo assembly of additional wild mungbean species, the divergence of what was eventually domesticated and the sampled wild mungbean species appears to have predated domestication. Moreover, the de novo assembly of a tetraploid Vigna species (V. reflexo-pilosa var. glabra) provides genomic evidence of a recent allopolyploid event. The species tree is constructed using de novo RNA-seq assemblies of 22 accessions of 18 Vigna species and protein sets of Glycine max. The present assembly of V. radiata var. radiata will facilitate genome research and accelerate molecular breeding of the subgenus Ceratotropis.