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Abstract Background Anguillid eels spend their larval period as leptocephalus larvae that have a unique and specialized body form with leaf-like and transparent features, and they undergo drastic metamorphosis to juvenile glass eels. Less is known about the transition of leptocephali to the glass eel stage, because it is difficult to catch the metamorphosing larvae in the open ocean. However, recent advances in rearing techniques for the Japanese eel have made it possible to study the larval metamorphosis of anguillid eels. In the present study, we investigated the dynamics of gene expression during the metamorphosis of Japanese eel leptocephali using RNA sequencing. Results During metamorphosis, Japanese eels were classified into 7 developmental stages according to their morphological characteristics, and RNA sequencing was used to collect gene expression data from each stage. A total of 354.8 million clean reads were generated from the body and 365.5 million from the head, after the processing of raw reads. For filtering of genes that characterize developmental stages, a classification model created by a Random Forest algorithm was built. Using the importance of explanatory variables feature obtained from the created model, we identified 46 genes selected in the body and 169 genes selected in the head that were defined as the “most characteristic genes” during eel metamorphosis. Next, network analysis and subsequently gene clustering were conducted using the most characteristic genes and their correlated genes, and then 6 clusters in the body and 5 clusters in the head were constructed. Then, the characteristics of the clusters were revealed by Gene Ontology (GO) enrichment analysis. The expression patterns and GO terms of each stage were consistent with previous observations and experiments during the larval metamorphosis of the Japanese eel. Conclusion Genome and transcriptome resources have been generated for metamorphosing Japanese eels. Genes that characterized metamorphosis of the Japanese eel were identified through statistical modeling by a Random Forest algorithm. The functions of these genes were consistent with previous observations and experiments during the metamorphosis of anguillid eels.

Understanding the mechanisms, especially those associated with phytohormones, of plant drought adaptation is crucial for sustaining agricultural production in the era of climate change. Arabidopsis histidine kinases (AHKs), an integral part of the cytokinin signaling pathway, and more axillary growth 2 (MAX2), a key component of the strigolactone and karrikin signaling pathways are reported to act as negative and positive regulators, respectively, in plant adaption to drought. However, the potential interaction between these singaling pathways in plant drought adaptation is not fully understood. To address this query, we assessed drought tolerance levels and associated phenotypic and physiological traits of the max2 single mutant, ahk2 ahk3 double mutant, ahk2 ahk3 max2 triple mutant, and wild-type (WT) Arabidopsis thaliana plants. Our findings revealed a distinct hierarchy in drought tolerance among these genotypes, as indicated by the differences in plant growth and stress survival rates. Specifically, the max2 mutant displayed the lowest drought tolerance level, followed by WT, ahk2 ahk3 max2, and ahk2 ahk3 plants. Additionally, the observed changes in leaf relative water content, leaf surface temperature, and cuticle formation were coherently aligned with the observed hierarchy of drought tolerance levels. Under drought conditions, the max2 mutant exhibited higher oxidative stress and membrane damage, as evidenced by increased levels of reactive oxygen species (ROS), malondialdehyde, and electrolyte leakage. In contrast, the ahk2 ahk3 and ahk2 ahk3 max2 mutants showed low and intermediate levels, respectively, for these parameters. The max2 mutant displayed reduced sensitivity, whereas ahk2 ahk3 and ahk2 ahk3 max2 mutants demonstrated high and intermediate sensitivities, respectively, to exogenous abscisic acid (ABA) treatments. Additionally, the expression analysis of several genes associated with the investigated drought tolerance-related traits showed a positive correlation between the transcript levels and corresponding trait(s) in both mutant and WT plants under drought conditions. Our results collectively indicate the presence of an antagonistic interaction between AHK and MAX2 signaling pathways in plant drought adaptation, impacting ABA responsiveness, leaf water retention, cuticle development, and ROS homestasis. The findings of this study provide a valuable foundation for developing agricultural methods to improve plant drought resilience.

A transcriptome analysis was used to compare the nodule transcriptomes of the model soybean ‘Williams 82’ inoculated with two Bradyrhizobium diazoefficiens strains (USDA110 vs. CB1809) under phosphate (Pi) deficiency. The entire dataset revealed a core set of low-Pi-responsive genes and recognized enormous differential transcriptional changes between the Pi-deprived USDA110-nodules and CB1809-nodules. The lower symbiotic efficiency of the Pi-starved USDA110 nodules was ascribed to the downregulation of an F1-ATPase gene engaged in oxidative phosphorylation, more likely contributing to diminished ATP production. To cope with energy shortage caused by Pi stress, the Pi-deprived USDA110-nodules preferentially upregulated the expression of a large number of genes encoding enzymes implicated in specialized energy-demanding pathways, such as the mitochondrial respiratory chain (i.e., cytochrome c oxidase), alcoholic fermentation (i.e., pyruvate decarboxylase and alcohol dehydrogenase) and glycolysis (e.g., hexokinase, phosphofructokinase, glyceraldehyde‐3‐phosphate dehydrogenase and pyruvate kinase). These respiratory adjustments were likely associated with higher metabolic cost and redox imbalance, thereby, negatively affecting nodule symbiosis under Pi deprivation. In contrast, the Pi-starved CB1809-nodules reduced the metabolic cost by regulating a lower number of genes and increasing the expression of genes encoding proteins implicated in non-phosphorylating bypasses (e.g., flavoprotein alpha and flavoprotein:ubiqionone oxidoreductase), which could promote the carbohydrate utilization efficiency and energy metabolism. Notably, the upregulation of a transcript encoding a malate dehydrogenase could boost the CB1809-nodules under Pi stress. The dynamic shifts in energy metabolism in the Pi-deprived USDA110-nodules and CB1809-nodules could be transformative to upgrade the mechanistic/conceptual understandings of soybean adaptation to Pi deficiency at the transcriptional level.

Abstract Transgene‐free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high‐efficiency, transgene‐free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR‐associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP‐based method. Through the direct delivery of LbCas12a‐containing RNPs, our method reached mutagenesis rates of approximately 77.2–94.5% at two different E. gracilis target genes, Glucan synthase‐like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock‐in and base editing at the target site using LbCas12a‐based RNPs with a single‐stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.

Photosynthetic organisms biosynthesize various carotenoids, a group of light-absorbing isoprenoid pigments that have key functions in photosynthesis, photoprotection, and phototaxis. Microalgae, in particular, contain diverse carotenoids and carotenoid biosynthetic pathways as a consequence of the various endosymbiotic events in their evolutionary history. Carotenoids such as astaxanthin, diadinoxanthin, and fucoxanthin are unique to algae. In microalgae, carotenoids are concentrated in the eyespot, a pigmented organelle that is important for phototaxis. A wide range of microalgae, including chlorophytes, euglenophytes, ochrophytes, and haptophytes, have an eyespot. In the chlorophyte Chlamydomonas reinhardtii, carotenoid layers in the eyespot reflect light to amplify the photosignal and shield photoreceptors from light, thereby enabling precise phototaxis. Our recent research revealed that, in contrast to the β-carotene-rich eyespot of C. reinhardtii, the euglenophyte Euglena gracilis relies on zeaxanthin for stable eyespot formation and phototaxis. In this review, we highlight recent advancements in the study of eyespot carotenoids and phototaxis in these microalgae, placing special emphasis on the diversity of carotenoid-dependent visual systems among microalgae.

A genome-wide association study (GWAS), which uses information on single nucleotide polymorphisms (SNPs) from many accessions, has become a powerful approach to gene identification. A metabolome GWAS (mGWAS), which relies on phenotypic information based on metabolite accumulation, can identify genes that contribute to primary and secondary metabolite contents. In this study, we carried out a mGWAS using seed metabolomic data from Arabidopsis thaliana accessions obtained by liquid chromatography–mass spectrometry to identify SNPs highly associated with the contents of metabolites such as glucosinolates. These SNPs were present in genes known to be involved in glucosinolate biosynthesis, thus confirming the effectiveness of our analysis. We subsequently focused on SNPs detected in an unknown methyltransferase gene associated with N-methylhistidine content. Knockout and overexpression of A. thaliana lines of this gene had significantly decreased and increased N-methylhistidine contents, respectively. We confirmed that the overexpressing line exclusively accumulated histidine methylated at the pi position, not at the tau position. Our findings suggest that the identified methyltransferase gene encodes a key enzyme for N-methylhistidine biosynthesis in A. thaliana.

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction.

Abstract Background The developmental gradient in monocot leaves has been exploited to uncover leaf developmental gene expression programs and chloroplast biogenesis processes. However, the relationship between the two is barely understood, which limits the value of transcriptome data to understand the process of chloroplast development. Results Taking advantage of the developmental gradient in the bread wheat leaf, we provide a simultaneous quantitative analysis for the development of mesophyll cells and of chloroplasts as a cellular compartment. This allows us to generate the first biologically-informed gene expression map of this leaf, with the entire developmental gradient from meristematic to fully differentiated cells captured. We show that the first phase of plastid development begins with organelle proliferation, which extends well beyond cell proliferation, and continues with the establishment and then the build-up of the plastid genetic machinery. The second phase is marked by the development of photosynthetic chloroplasts which occupy the available cellular space. Using a network reconstruction algorithm, we predict that known chloroplast gene expression regulators are differentially involved across those developmental stages. Conclusions Our analysis generates both the first wheat leaf transcriptional map and one of the most comprehensive descriptions to date of the developmental history of chloroplasts in higher plants. It reveals functionally distinct plastid and chloroplast development stages, identifies processes occurring in each of them, and highlights our very limited knowledge of the earliest drivers of plastid biogenesis, while providing a basis for their future identification.

The endoplasmic reticulum (ER), a eukaryotic organelle, is the major site of protein biosynthesis. The disturbance of ER function by biotic or abiotic stress triggers the accumulation of misfolded or unfolded proteins in the ER. The unfolded protein response (UPR) is the best-studied ER stress response. This transcriptional regulatory system senses ER stress, activates downstream genes that function to mitigate stress, and restores homeostasis. In addition to its conventional role in stress responses, recent reports indicate that the UPR is involved in plant growth and development. In this review, we summarize the current knowledge of ER stress sensing and the activation and downstream regulation of the UPR. We also describe how the UPR modulates both plant growth and stress tolerance by maintaining ER homeostasis. Lastly, we propose that the UPR is a major component of the machinery that balances the trade-off between plant growth and survival in a dynamic environment.

Anastomosis group AG-1 IA of Rhizoctonia solani Khün has a wide host range and threatens crop production. Various glycosyltransferases secreted by phytopathogenic fungi play an essential role in pathogenicity. Previously, we identified a glycosyltransferase RsIA_GT (AG11A_09161) as a secreted protein-encoding gene of R. solani AG-1 IA, whose expression levels increased during infection in rice. In this study, we further characterized the virulence function of RsIA_GT. It is conserved not only in Basidiomycota, including multiple anastomosis groups of R. solani, but also in other primary fungal taxonomic categories. RsIA_GT possesses a signal peptide (SP) for protein secretion, and its functionality was proven using yeast and Nicotiana benthamiana. The SP-truncated form of RsIA_GT (RsIA_GT(ΔS)) expressed in Escherichia coli-induced lesion-like phenotype in rice leaves when applied to punched leaves. However, Agrobacterium-mediated transient expressions of both the full-length RsIA_GT and RsIA_GT(ΔS) did not induce cell death in N. benthamiana leaves. Instead, only RsIA_GT(ΔS) suppressed the cell death induced by two reference cell death factors BAX and INF1 in N.benthamiana. RsIA_GT(ΔS)R154A D168A D170A, a mutant RsIA_GT(ΔS) for the glycosyltransferase catalytic domain, still suppressed the BAX- or INF1-induced cell death, suggesting that the cell death suppression activity of RsIA_GT(ΔS) would be independent from its enzymatic activity. RsIA_GT(ΔS) also suppressed the H2O2 production and callose deposition and showed an effect on the induction of defense genes associated with the expression of BAX and INF1. The transient expression of RsIA_GT(ΔS) in N. benthamiana enhanced the lesion area caused by R. solani AG-1 IA. The secreted glycosyltransferase, RsIA_GT, of R. solani AG-1 IA is likely to have a dual role in virulence inside and outside of host cells.

Plants in Mongolian grasslands are exposed to short, dry summers and long, cold winters. These plants should be prepared for fast germination and growth activity in response to the limited summer rainfall. The wild plant species adapted to the Mongolian grassland environment may allow us to explore useful genes, as a source of unique genetic codes for crop improvement. Here, we identified the Chloris virgata Dornogovi accession as the fastest germinating plant in major Mongolian grassland plants. It germinated just 5 h after treatment for germination initiation and showed rapid growth, especially in its early and young development stages. This indicates its high growth potential compared to grass crops such as rice and wheat. By assessing growth recovery after animal bite treatment (mimicked by cutting the leaves with scissors), we found that C. virgata could rapidly regenerate leaves after being damaged, suggesting high regeneration potential against grazing. To analyze the regulatory mechanism involved in the high growth potential of C. virgata, we performed RNA-seq-based transcriptome analysis and illustrated a comprehensive gene expression map of the species. Through de novo transcriptome assembly with the RNA-seq reads from whole organ samples of C. virgata at the germination stage (2 days after germination, DAG), early young development stage (8 DAG), young development stage (17 DAG), and adult development stage (28 DAG), we identified 21,589 unified transcripts (contigs) and found that 19,346 and 18,156 protein-coding transcripts were homologous to those in rice and Arabidopsis, respectively. The best-aligned sequences were annotated with gene ontology groups. When comparing the transcriptomes across developmental stages, we found an over-representation of genes involved in growth regulation in the early development stage in C. virgata. Plant development is tightly regulated by phytohormones such as brassinosteroids, gibberellic acid, abscisic acid, and strigolactones. Moreover, our transcriptome map demonstrated the expression profiles of orthologs involved in the biosynthesis of these phytohormones and their signaling networks. We discuss the possibility that C. virgata phytohormone signaling and biosynthesis genes regulate early germination and growth advantages. Comprehensive transcriptome information will provide a useful resource for gene discovery and facilitate a deeper understanding of the diversity of the regulatory systems that have evolved in C. virgata while adapting to severe environmental conditions.

The sustainable development goals (SDGs) adopted at the 2015 United Nations Summit are globally applicable goals designed to help countries realize a sustainable future. To achieve these SDGs, it is necessary to utilize renewable biological resources. In recent years, bioeconomy has been an attractive concept for achieving the SDGs. Microalgae are one of the biological resources that show promise in realizing the “5F”s (food, fiber, feed, fertilizer, and fuel). Among the microalgae, Euglena gracilis has the potential for achieving the “5F”s strategy owing to its unique features, such as production of paramylon, that are lacking in other microalgae. E. gracilis has already been produced on an industrial scale for use as an ingredient in functional foods and cosmetics. In recent years, genetic engineering methods for breeding E. gracilis have been researched and developed to achieve higher yields. In this article, we summarize how microalgae contribute toward achieving the SDGs. We focus on the contribution of E. gracilis to the bioeconomy, including its advantages in industrial use as well as its unique characteristics. In addition, we review genetic engineering-related research trends centered on E. gracilis, including a complete nuclear genome determination project, genome editing technology using the CRISPR-Cas9 system, and the development of a screening method for selecting useful strains. In particular, genome editing in E. gracilis could be a breakthrough for molecular breeding of industrially useful strains because of its high efficiency.

Summary: Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, and biofuels. However, targeted mutagenesis in this species has been a long-standing challenge. We recently developed a transgene-free, highly efficient, genome editing method for E. gracilis using CRISPR/Cas9 ribonucleoproteins (RNPs). Our method achieved mutagenesis rates of approximately 80% or more through an electroporation-based direct delivery of Cas9 RNPs. Therefore, this method is suitable for basic research and industrial applications, such as the breeding of Euglena.For complete details on the use and execution of this protocol, please refer to Nomura et al. (2019).

Summary: Heading time is a key trait in cereals affecting the maturation period for optimal grain filling before harvest. Here, we aimed to understand the factors controlling heading time in barley (Hordeum vulgare). We characterized a set of 274 barley accessions collected worldwide by planting them for 20 seasons under different environmental conditions at the same location in Kurashiki, Japan. We examined interactions among accessions, known genetic factors, and an environmental factor to determine the factors controlling heading response. Locally adapted accessions have been selected for genetic factors that stabilize heading responses appropriate for barley cultivation, and these accessions show stable heading responses even under varying environmental conditions. We identified vernalization requirement and PPD-H1 haplotype as major stabilizing mechanisms of the heading response for regional adaptation in Kurashiki.

Eukaryotic microalgae have been classified into several biological divisions and have evolutionarily acquired diverse morphologies, metabolisms, and life cycles. They are naturally exposed to environmental stresses that cause oxidative damage due to reactive oxygen species accumulation. To cope with environmental stresses, microalgae contain various antioxidants, including carotenoids, ascorbate (AsA), and glutathione (GSH). Carotenoids are hydrophobic pigments required for light harvesting, photoprotection, and phototaxis. AsA constitutes the AsA-GSH cycle together with GSH and is responsible for photooxidative stress defense. GSH contributes not only to ROS scavenging, but also to heavy metal detoxification and thiol-based redox regulation. The evolutionary diversity of microalgae influences the composition and biosynthetic pathways of these antioxidants. For example, α-carotene and its derivatives are specific to Chlorophyta, whereas diadinoxanthin and fucoxanthin are found in Heterokontophyta, Haptophyta, and Dinophyta. It has been suggested that AsA is biosynthesized via the plant pathway in Chlorophyta and Rhodophyta and via the Euglena pathway in Euglenophyta, Heterokontophyta, and Haptophyta. The GSH biosynthetic pathway is conserved in all biological kingdoms; however, Euglenophyta are able to synthesize an additional thiol antioxidant, trypanothione, using GSH as the substrate. In the present study, we reviewed and discussed the diversity of microalgal antioxidants, including recent findings.

Abstract Seed priming is a commercially used technique for improving seed performance including germination. However, the treatment sometimes reduces seed longevity as a side effect, limiting the storable period or longevity of the seeds. To overcome this problem, molecular mechanisms involved in the loss of seed longevity during priming were analyzed using natural variations of Arabidopsis thaliana. We found that the Est-1 accession retained longevity for longer after priming compared to the reference accession Col-0. QTL analysis using 279 recombinant inbred lines (RILs) derived from the Est-1 × Col-0 detected three QTL regions associated with the loss of seed longevity during priming. Bulked transcriptome analysis (RNA-Seq with bulked RIL populations) revealed that genes related to brassinosteroid (BR) biosynthesis/signaling and cell wall modification were highly expressed in primed seeds with shorter longevity. After priming, BR-deficient mutants cyp85a1/a2 and det2 showed significantly longer longevity than the wild type (WT). Moreover, tetrazolium staining indicated that mutant seed coats were less permeable after priming than those of WT. We suggest that the loss of seed longevity in primed seed is due to increased seed coat permeability, which is positively regulated, at least partly, via BR signaling.

Statistical and machine learning (ML)-based methods have recently advanced in construction of gene regulatory network (GRNs) based on high-throughput biological datasets. GRNs underlie almost all cellular phenomena; hence, comprehensive GRN maps are essential tools to elucidate gene function, thereby facilitating the identification and prioritization of candidate genes for functional analysis. High-throughput gene expression datasets have yielded various statistical and ML-based algorithms to infer causal relationship between genes and decipher GRNs. This review summarizes the recent advancements in the computational inference of GRNs, based on large-scale transcriptome sequencing datasets of model plants and crops. We highlight strategies to select contextual genes for GRN inference, and statistical and ML-based methods for inferring GRNs based on transcriptome datasets from plants. Furthermore, we discuss the challenges and opportunities for the elucidation of GRNs based on large-scale datasets obtained from emerging transcriptomic applications, such as from population-scale, single-cell level, and life-course transcriptome analyses.

Climate resilience of crops is critical for global food security. Understanding the genetic basis of plant responses to ambient environmental changes is key to developing resilient crops. To detect genetic factors that set flowering time according to seasonal temperature conditions, we evaluated differences of flowering time over years by using chromosome segment substitution lines (CSSLs) derived from japonica rice cultivars “Koshihikari” × “Khao Nam Jen”, each with different robustness of flowering time to environmental fluctuations. The difference of flowering times in 9 years’ field tests was large in “Khao Nam Jen” (36.7 days) but small in “Koshihikari” (9.9 days). Part of this difference was explained by two QTLs. A CSSL with a “Khao Nam Jen” segment on chromosome 11 showed 28.0 days’ difference; this QTL would encode a novel flowering-time gene. Another CSSL with a segment from “Khao Nam Jen” in the region around Hd16 on chromosome 3 showed 23.4 days” difference. A near-isogenic line (NIL) for Hd16 showed 21.6 days’ difference, suggesting Hd16 as a candidate for this QTL. RNA-seq analysis showed differential expression of several flowering-time genes between early and late flowering seasons. Low-temperature treatment at panicle initiation stage significantly delayed flowering in the CSSL and NIL compared with “Koshihikari”. Our results unravel the molecular control of flowering time under ambient temperature fluctuations.

Genome duplications aid in the formation of novel molecular networks through regulatory differentiation of the duplicated genes and facilitate adaptation to environmental change. Hexaploid wheat, Triticum aestivum, contains three homoeologous chromosome sets, the A-, B-, and D-subgenomes, which evolved through interspecific hybridization and subsequent whole-genome duplication. The divergent expression patterns of the homoeologs in hexaploid wheat suggest that they have undergone transcriptional and/or functional differentiation during wheat evolution. However, the distribution of transcriptionally differentiated homoeologs in gene regulatory networks and their related biological functions in hexaploid wheat are still largely unexplored. Therefore, we retrieved 727 publicly available wheat RNA-sequencing (RNA-seq) datasets from various tissues, developmental stages, and conditions, and identified 10,415 expressed homoeologous triplets. Examining the co-expression modules in the wheat transcriptome, we found that 66% of the expressed homoeologous triplets possess all three homoeologs grouped in the same co-expression modules. Among these, 15 triplets contain co-expressed homoeologs with differential expression levels between homoeoalleles across ≥ 95% of the 727 RNA-seq datasets, suggesting a consistent trend of homoeolog expression bias. In addition, we identified 2,831 differentiated homoeologs that showed gene expression patterns that deviated from those of the other two homoeologs. We found that seven co-expression modules contained a high proportion of such differentiated homoeologs, which accounted for ≥ 20% of the genes in each module. We also found that five of the co-expression modules are abundantly composed of genes involved in biological processes such as chloroplast biogenesis, RNA metabolism, putative defense response, putative posttranscriptional modification, and lipid metabolism, thereby suggesting that, the differentiated homoeologs might highly contribute to these biological functions in the gene network of hexaploid wheat.

Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range of biological fields. In this study, we developed a simple, custom, targeted re-sequencing method, designated “multiplex PCR targeted amplicon sequencing (MTA-seq),” and applied it to the genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to the genotyping of genome-wide single-nucleotide polymorphisms (SNPs) identified in natural accessions (Bd1-1, Bd3-1, Bd21-3, Bd30-1, Koz-1, Koz-3, and Koz-4) by comparing the re-sequencing data with that of reference accession Bd21. Examination of SNP-genotyping accuracy in 443 amplicons from eight parental accessions and an F1 progeny derived by crossing of Bd21 and Bd3-1 revealed that ~95% of the SNPs were correctly called. The assessment suggested that the method provided an efficient framework for accurate and robust SNP genotyping. The method described here enables easy design of custom target SNP-marker panels in various organisms, facilitating a wide range of high-throughput genetic applications, such as genetic mapping, population analysis, molecular breeding, and genomic diagnostics.

We report the comprehensive identification of periodic genes and their network inference, based on a gene co-expression analysis and an Auto-Regressive eXogenous (ARX) model with a group smoothly clipped absolute deviation (SCAD) method using a time-series transcriptome dataset in a model grass, Brachypodium distachyon. To reveal the diurnal changes in the transcriptome in B. distachyon, we performed RNA-seq analysis of its leaves sampled through a diurnal cycle of over 48 h at 4 h intervals using three biological replications, and identified 3,621 periodic genes through our wavelet analysis. The expression data are feasible to infer network sparsity based on ARX models. We found that genes involved in biological processes such as transcriptional regulation, protein degradation, and post-transcriptional modification and photosynthesis are significantly enriched in the periodic genes, suggesting that these processes might be regulated by circadian rhythm in B. distachyon. On the basis of the time-series expression patterns of the periodic genes, we constructed a chronological gene co-expression network and identified putative transcription factors encoding genes that might be involved in the time-specific regulatory transcriptional network. Moreover, we inferred a transcriptional network composed of the periodic genes in B. distachyon, aiming to identify genes associated with other genes through variable selection by grouping time points for each gene. Based on the ARX model with the group SCAD regularization using our time-series expression datasets of the periodic genes, we constructed gene networks and found that the networks represent typical scale-free structure. Our findings demonstrate that the diurnal changes in the transcriptome in B. distachyon leaves have a sparse network structure, demonstrating the spatiotemporal gene regulatory network over the cyclic phase transitions in B. distachyon diurnal growth.

Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.

In plants, the Nuclear Factor-Y (NF-Y) transcription factors (TFs), which include three distinct types of NF-YA, NF-YB, and NF-YC TFs, have been identified to play key roles in the regulation of various plant growth and developmental processes under both normal and environmental stress conditions. In this work, a total of 40 CaNF-Y-encoding genes, including eight CaNF-YAs, 21 CaNF-YBs, and 11 CaNF-YCs, were identified in chickpea, and their major gene and protein characteristics were subsequently obtained using various web-based tools. Of our interest, a phylogenetically-based analysis predicted 18 CaNF-Ys (eight CaNF-YAs, seven CaNF-YBs, and three CaNF-YCs) that potentially play roles in chickpea responses to dehydration according to their close relationship with the well-characterized GmNF-Ys in soybean. These results were in good agreement with the enrichment of drought-responsive cis-regulatory motifs and expression patterns obtained from in silico analyses using publically available transcriptome data. Most of the phylogenetically predicted drought-responsive CaNF-Y genes (15 of 18) were quantitatively validated to significantly respond to dehydration treatment in leaves and/or roots, further supporting the results of in silico analyses. Among these CaNF-Y genes, the transcript levels of CaNF-YA01 and CaNF-YC10 were the most highly accumulated in leaves (by approximately eight-fold) and roots (by approximately 18-fold), respectively, by dehydration. Furthermore, 12 of the 18 CaNF-Y genes were found to be responsive to the most well-known stress hormone, namely abscisic acid (ABA), in leaves and/or roots, suggesting that these genes may act in chickpea response to dehydration in ABA-dependent manner. Taken together, our study has provided a comprehensive and fundamental information for further functional analyses of selected CaNF-Y candidate genes, ultimately leading to the improvement of chickpea growth under water-limited conditions.

To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

The plant-specific NAC transcription factors (TFs) play important roles in regulation of diverse biological processes, including development, growth, cell division and responses to environmental stimuli. In this study, we identified the members of the NAC TF family of chickpea (Cicer arietinum) and assess their expression profiles during plant development and under dehydration and abscisic acid (ABA) treatments in a systematic manner. Seventy-one CaNAC genes were detected from the chickpea genome, including 8 membrane-bound members of which many might be involved in dehydration responses as judged from published literature. Phylogenetic analysis of the chickpea and well-known stress-related Arabidopsis and rice NACs enabled us to predict several putative stress-related CaNACs. By exploring available transcriptome data, we provided a comprehensive expression atlas of CaNACs in various tissues at different developmental stages. With the highest interest in dehydration responses, we examined the expression of the predicted stress-related and membrane-bound CaNACs in roots and leaves of chickpea seedlings, subjected to well-watered (control), dehydration and ABA treatments, using real-time quantitative PCR (RT-qPCR). Nine-teen of the 23 CaNACs examined were found to be dehydration-responsive in chickpea roots and/or leaves in either ABA-dependent or -independent pathway. Our results have provided a solid foundation for selection of promising tissue-specific and/or dehydration-responsive CaNAC candidates for detailed in planta functional analyses, leading to development of transgenic chickpea varieties with improved productivity under drought.

Cassava (Manihot esculenta Crantz) is an important crop that provides food security and income generation in many tropical countries, and is known for its adaptability to various environmental conditions. Its draft genome sequence and many expressed sequence tags are now publicly available, allowing the development of cassava polymorphism information. Here, we describe the genome-wide discovery of cassava DNA polymorphisms. Using the alignment of predicted transcribed sequences from the cassava draft genome sequence and ESTs from GenBank, we discovered 10,546 single-nucleotide polymorphisms and 647 insertions and deletions. To facilitate molecular marker development for cassava, we designed 9,316 PCR primer pairs to amplify the genomic region around each DNA polymorphism. Of the discovered SNPs, 62.7% occurred in protein-coding regions. Disease-resistance genes were found to have a significantly higher ratio of nonsynonymous-to-synonymous substitutions. We identified 24 read-through (changes of a stop codon to a coding codon) and 38 premature stop (changes of a coding codon to a stop codon) single-nucleotide polymorphisms, and found that the 5 gene ontology terms in biological process were significantly different in genes with read-through single-nucleotide polymorphisms compared with all cassava genes. All data on the discovered DNA polymorphisms were organized into the Cassava Online Archive database, which is available at http://cassava.psc.riken.jp/.

A comprehensive collection of full-length cDNAs is essential for correct structural gene annotation and functional analyses of genes. We constructed a mixed full-length cDNA library from 21 different tissues of Brachypodium distachyon Bd21, and obtained 78,163 high quality expressed sequence tags (ESTs) from both ends of ca. 40,000 clones (including 16,079 contigs). We updated gene structure annotations of Brachypodium genes based on full-length cDNA sequences in comparison with the latest publicly available annotations. About 10,000 non-redundant gene models were supported by full-length cDNAs; ca. 6,000 showed some transcription unit modifications. We also found ca. 580 novel gene models, including 362 newly identified in Bd21. Using the updated transcription start sites, we searched a total of 580 plant cis-motifs in the -3 kb promoter regions and determined a genome-wide Brachypodium promoter architecture. Furthermore, we integrated the Brachypodium full-length cDNAs and updated gene structures with available sequence resources in wheat and barley in a web-accessible database, the RIKEN Brachypodium FL cDNA database. The database represents a "one-stop" information resource for all genomic information in the Pooideae, facilitating functional analysis of genes in this model grass plant and seamless knowledge transfer to the Triticeae crops.