Drought is the most serious abiotic stress that hinders rice production under rainfed conditions. Breeding for deep rooting is a promising strategy to improve the root system architecture in shallow-rooting rice cultivars to avoid drought stress. We analysed the quantitative trait loci (QTLs) for the ratio of deep rooting (RDR) in three F₂ mapping populations derived from crosses between each of three shallow-rooting varieties ('ARC5955', 'Pinulupot1', and 'Tupa729') and a deep-rooting variety, 'Kinandang Patong'. In total, we detected five RDR QTLs on chromosomes 2, 4, and 6. In all three populations, QTLs on chromosome 4 were found to be located at similar positions; they explained from 32.0% to 56.6% of the total RDR phenotypic variance. This suggests that one or more key genetic factors controlling the root growth angle in rice is located in this region of chromosome 4.

Unlike animals, plants can pause their life cycle as dormant seeds. In both plants and animals, DNA methylation is involved in the regulation of gene expression and genome integrity. In animals, reprogramming erases and re-establishes DNA methylation during development. However, knowledge of reprogramming or reconfiguration in plants has been limited to pollen and the central cell. To better understand epigenetic reconfiguration in the embryo, which forms the plant body, we compared time-series methylomes of dry and germinating seeds to publicly available seed development methylomes.

The volume that the root system can occupy is associated with the efficiency of water and nutrient uptake from soil. Genetic improvement of root length, which is a limiting factor for root distribution, is necessary for increasing crop production. In this report, we describe identification of two quantitative trait loci (QTLs) for maximal root length, QUICK ROOTING 1 (QRO1) on chromosome 2 and QRO2 on chromosome 6, in cultivated rice (Oryza sativa L.). We measured the maximal root length in 26 lines carrying chromosome segments from the long-rooted upland rice cultivar Kinandang Patong in the genetic background of the short-rooted lowland cultivar IR64. Five lines had longer roots than IR64. By rough mapping of the target regions in BC4F2 populations, we detected putative QTLs for maximal root length on chromosomes 2, 6, and 8. To fine-map these QTLs, we used BC4F3 recombinant homozygous lines. QRO1 was mapped between markers RM5651 and RM6107, which delimit a 1.7-Mb interval on chromosome 2, and QRO2 was mapped between markers RM20495 and RM3430-1, which delimit an 884-kb interval on chromosome 6. Both QTLs may be promising gene resources for improving root system architecture in rice.

Genetic improvement of root system architecture is a promising approach for improved uptake of water and mineral nutrients distributed unevenly in the soil. To identify genomic regions associated with the length of different root types in rice, we quantified root system architecture in a set of 26 chromosome segment substitution lines derived from a cross between lowland indica rice, IR64, and upland tropical japonica rice, Kinandang Patong, (IK-CSSLs), using 2D & 3D root phenotyping platforms.

Osmotic stresses, such as drought and high salinity, adversely affect plant growth and productivity. The phytohormone abscisic acid (ABA) accumulates in response to osmotic stress and enhances stress tolerance in plants by triggering multiple physiological responses through ABA signaling. Subclass III SNF1-related protein kinases 2 (SnRK2s) are key regulators of ABA signaling. Although SnRK2s have long been considered to be self-activated by autophosphorylation after release from PP2C-mediated inhibition, they were recently revealed to be activated by two independent subfamilies of group B Raf-like kinases, B2-RAFs and B3-RAFs, under osmotic stress conditions. However, the relationship between SnRK2 phosphorylation by these RAFs and SnRK2 autophosphorylation and the individual physiological roles of each RAF subfamily remain unknown. In this study, we indicated that B2-RAFs are constantly active and activate SnRK2s when released from PP2C-mediated inhibition by ABA-binding ABA receptors, whereas B3-RAFs are activated only under stress conditions in an ABA-independent manner and enhance SnRK2 activity. Autophosphorylation of subclass III SnRK2s is not sufficient for ABA responses, and B2-RAFs are needed to activate SnRK2s in an ABA-dependent manner. Using plants grown in soil, we found that B2-RAFs regulate subclass III SnRK2s at the early stage of drought stress, whereas B3-RAFs regulate SnRK2s at the later stage. Thus, B2-RAFs are essential kinases for the activation of subclass III SnRK2s in response to ABA under mild osmotic stress conditions, and B3-RAFs function as enhancers of SnRK2 activity under severe stress conditions.

A new glutelin gene, designated GluD-1, has been discovered by comparing the seed storage proteins from 48 japonica and indica rice cultivars on SDS-PAGE gels. Evidence that GluD-1 is a member of the glutelin family was provided by Western blots using anti-glutelin antiserum and by mapping the gene to the chromosomal glutelin gene cluster. The limited GluD-1 size polymorphism among the rice varieties is due to amino acid substitutions rather than to post-transcriptional modification. GluD-1 is maximally expressed in the starchy endosperm starting at 5 d after flowering (DAF) and increasing through 30 DAF, a major difference from the other glutelins which are primarily expressed in the subaleurone from 10-16 DAF. Only about 0.2 kb of the GluD-1 promoter was sufficient to confer inner starchy endosperm-specific expression. The 0.2 kb truncated GluD-1 promoter contains a bifactorial endosperm box consisting of a truncated GCN4 motif (TGA(G/C)TCA) and AAAG Prolamin box (P box), and ACGT and AACA motifs as cis-regulatory elements. Gel retardation assays and trans-activation experiments indicated that the truncated GCN4 and P box are specifically recognized by RISBZ1 b-ZIP and RPBF Dof activators in vitro, respectively, and are synergistically transactivated, indicating that combinatorial interactions of these motifs are involved in essential endosperm-specific regulation. Furthermore, deviation from the cognate GCN4 motif alters tissue-specific expression in the inner starchy endosperm to include other endosperm tissues.

The root system architecture (RSA) of crops can affect their production, particularly in abiotic stress conditions, such as with drought, waterlogging, and salinity. Salinity is a growing problem worldwide that negatively impacts on crop productivity, and it is believed that yields could be improved if RSAs that enabled plants to avoid saline conditions were identified. Here, we have demonstrated, through the cloning and characterization of qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1), that a shallower root growth angle (RGA) could enhance rice yields in saline paddies. qSOR1 is negatively regulated by auxin, predominantly expressed in root columella cells, and involved in the gravitropic responses of roots. qSOR1 was found to be a homolog of DRO1 (DEEPER ROOTING 1), which is known to control RGA. CRISPR-Cas9 assays revealed that other DRO1 homologs were also involved in RGA. Introgression lines with combinations of gain-of-function and loss-of-function alleles in qSOR1 and DRO1 demonstrated four different RSAs (ultra-shallow, shallow, intermediate, and deep rooting), suggesting that natural alleles of the DRO1 homologs could be utilized to control RSA variations in rice. In saline paddies, near-isogenic lines carrying the qSOR1 loss-of-function allele had soil-surface roots (SOR) that enabled rice to avoid the reducing stresses of saline soils, resulting in increased yields compared to the parental cultivars without SOR. Our findings suggest that DRO1 homologs are valuable targets for RSA breeding and could lead to improved rice production in environments characterized by abiotic stress.

RNA extraction has been improved by integration of a variety of materials in the protocol, such as phenol, guanidine thiocyanate, and silica, according to the case-specific demands. However, few methods have been designed for high-throughput RNA preparation for large-scale transcriptome studies. In this study, we established a high-throughput guanidinium thiocyanate and isopropyl alcohol based RNA extraction method (HighGI). HighGI is based on simple and phenol-free homemade buffers and the cost is substantially lower than a column-based commercial kit. We demonstrated that the quality and quantity of RNA extracted with HighGI were comparable to those extracted with a conventional phenol/chloroform-based method and a column-based commercial kit. HighGI retained small RNAs less than 200 bp, which are lost with a commercial column-based kit. We also demonstrated that HighGI is readily applicable to semi-automated RNA extraction. HighGI enables high-throughput RNA extraction for large-scale RNA preparation with high yield and quality.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

In the evolutionary history of plants, variation in cis-regulatory elements (CREs) resulting in diversification of gene expression has played a central role in driving the evolution of lineage-specific traits. However, it is difficult to predict expression behaviors from CRE patterns to properly harness them, mainly because the biological processes are complex. In this study, we used cistrome datasets and explainable convolutional neural network (CNN) frameworks to predict genome-wide expression patterns in tomato (Solanum lycopersicum) fruit from the DNA sequences in gene regulatory regions. By fixing the effects of trans-acting factors using single cell-type spatiotemporal transcriptome data for the response variables, we developed a prediction model for crucial expression patterns in the initiation of tomato fruit ripening. Feature visualization of the CNNs identified nucleotide residues critical to the objective expression pattern in each gene, and their effects were validated experimentally in ripening tomato fruit. This cis-decoding framework will not only contribute to the understanding of the regulatory networks derived from CREs and transcription factor interactions, but also provides a flexible means of designing alleles for optimized expression.

Root system architecture plays a crucial role in nutrient and water absorption during rice production. Genetic improvement of the rice root system requires elucidating its genetic control. Genome-wide association studies (GWASs) have identified genomic regions responsible for rice root phenotypes. However, candidate gene prioritization around the peak region often suffers from low statistical power and resolution. Transcriptomics enables other statistical mappings, such as transcriptome-wide association study (TWAS) and expression GWAS (eGWAS), which improve candidate gene identification by leveraging the natural variation of the expression profiles. To explore the genes responsible for root phenotypes, we conducted GWAS, TWAS, and eGWAS for 12 root phenotypes in 57 rice accessions using 427,751 single nucleotide polymorphisms (SNPs) and the expression profiles of 16,901 genes expressed in the roots. The GWAS identified three significant peaks, of which the most significant peak responsible for seven root phenotypes (crown root length, crown root surface area, number of crown root tips, lateral root length, lateral root surface area, lateral root volume, and number of lateral root tips) was detected at 6,199,732 bp on chromosome 8. In the most significant GWAS peak region, OsENT1 was prioritized as the most plausible candidate gene because its expression profile was strongly negatively correlated with the seven root phenotypes. In addition to OsENT1, OsEXPA31, OsSPL14, OsDEP1, and OsDEC1 were identified as candidate genes responsible for root phenotypes using TWAS. Furthermore, a cis-eGWAS peak SNP was detected for OsDjA6, which showed the eighth strongest association with lateral root volume in the TWAS. The cis-eGWAS peak SNP for OsDjA6 was in strong linkage disequilibrium (LD) with a GWAS peak SNP on the same chromosome for lateral root volume and in perfect LD with another SNP variant in a putative cis-element at the 518 bp upstream of the gene. These candidate genes provide new insights into the molecular breeding of root system architecture.

Separating male and female sex organs is one of the main strategies used to maintain genetic diversity within a species. However, the genetic determinants and their regulatory mechanisms have been identified in only a few species. In dioecious persimmons, the homeodomain transcription factor, MeGI, which is the target of a Y chromosome-encoded small-RNA, OGI, can determine floral sexuality. The basic features of this system are conserved in the monoecious hexaploid Oriental persimmon, in which an additional epigenetic regulation of MeGI determines floral sexuality. The downstream regulatory pathways of MeGI remain uncharacterized. In this study, we examined transcriptomic data for male and female flowers from monoecious persimmon cultivars to unveil the gene networks orchestrated by MeGI. A network visualization and cistrome assessment suggested that class-1 KNOTTED-like homeobox (KNOX)/ovate family protein (OFP)/growth regulating factors (GRFs) and short vegetative phase (SVP) genes mediate the differences in gynoecium and androecium development between male and female flowers, respectively. The expression of these genes is directly controlled by MeGI. The gene networks also suggested that some cytokinin, auxin, and gibberellin signaling genes function cooperatively in the KNOX/OFP/GRF pathway during gynoecium differentiation. Meanwhile, SVP may repress PI expression in developing androecia. Overall, our results suggest that MeGI evolved the ability to promote gynoecium development and suppress androecium development by regulating KNOX/OFP/GRF and SVP expression levels, respectively. These insights may help to clarify the molecular mechanism underlying the production of unisexual flowers, while also elucidating the physiological background enabling a single-factor system to establish dioecy in plants.

Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions.

Root system architecture (RSA) is one of the most important traits determining water and nutrient availability for plants. Modification of RSA is known to be a useful approach for improving root performance of crops. However, for conducting root phenotyping, there are few alternatives for the rapid collection of root samples from a constant soil volume. In this report, we propose a rapid root-sampling method, which uses a steel cylinder known as round monolith and backhoes to reduce the physical effort. The monolith was set on the ground surrounding individual rice plants and vertically driven back by a backhoe. Soil samples with 20 cm width and 25 cm depth were excavated by the monolith, from which root samples were then isolated. This backhoe-assisted monolith method requires at most five minutes to collect root samples from one plant. Using this method, we quantified the root traits of three rice lines, reported to form different types of root system such as shallow-, intermediate-, and deep-roots, using a root image analysis software. The data obtained through this method, which showed the same trend as previously reported, clearly demonstrated that this method is useful for quantitative evaluation of roots in the soil.

The repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs may escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. Genome-wide DNA demethylation is also observed in the endosperm after fertilization. In the present study, we used a custom microarray to survey the transcripts generated from TEs during rice endosperm development and at selected time points in the embryo as a control. The expression patterns of TE transcripts are dynamically up- and downregulated during endosperm development, especially those of miniature inverted-repeat TEs (MITEs). Some TE transcripts were directionally controlled, whereas the other DNA transposons and retrotransposons were not. We also discovered the NUCLEAR FACTOR Y binding motif, CCAAT, in the region near the 5' terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

IR64 is a rice variety with high-yield that has been widely cultivated around the world. IR64 has been replaced by modern varieties in most growing areas. Given that modern varieties are mostly progenies or relatives of IR64, genetic analysis of IR64 is valuable for rice functional genomics. However, chromosome-level genome sequences of IR64 have not been available previously. Here, we sequenced the IR64 genome using synthetic long reads obtained by linked-read sequencing and ultra-long reads obtained by nanopore sequencing. We integrated these data and generated the de novo assembly of the IR64 genome of 367 Mb, equivalent to 99% of the estimated size. Continuity of the IR64 genome assembly was improved compared with that of a publicly available IR64 genome assembly generated by short reads only. We annotated 41,458 protein-coding genes, including 657 IR64-specific genes, that are missing in other high-quality rice genome assemblies IRGSP-1.0 of japonica cultivar Nipponbare or R498 of indica cultivar Shuhui498. The IR64 genome assembly will serve as a genome resource for rice functional genomics as well as genomics-driven and/or molecular breeding.

The epigenome orchestrates genome accessibility, functionality, and three-dimensional structure. Because epigenetic variation can impact transcription and thus phenotypes, it may contribute to adaptation. Here, we report 1,107 high-quality single-base resolution methylomes and 1,203 transcriptomes from the 1001 Genomes collection of Arabidopsis thaliana. Although the genetic basis of methylation variation is highly complex, geographic origin is a major predictor of genome-wide DNA methylation levels and of altered gene expression caused by epialleles. Comparison to cistrome and epicistrome datasets identifies associations between transcription factor binding sites, methylation, nucleotide variation, and co-expression modules. Physical maps for nine of the most diverse genomes reveal how transposons and other structural variants shape the epigenome, with dramatic effects on immunity genes. The 1001 Epigenomes Project provides a comprehensive resource for understanding how variation in DNA methylation contributes to molecular and non-molecular phenotypes in natural populations of the most studied model plant.

The functional allele of the rice gene DEEPER ROOTING 1 (DRO1) increases the root growth angle (RGA). However, wide natural variation in RGA is observed among rice cultivars with the functional DRO1 allele. To elucidate genetic factors related to such variation, we quantitatively measured RGA using the basket method and analyzed quantitative trait loci (QTLs) for RGA in three F2 mapping populations derived from crosses between the large RGA-type cultivar Kinandang Patong and each of three accessions with varying RGA: Momiroman has small RGA and was used to produce the MoK-F2 population; Yumeaoba has intermediate RGA (YuK-F2 population); Tachisugata has large RGA (TaK-F2 population). All four accessions belong to the same haplotype group of functional DRO1 allele.

To induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.), we expressed double-strand RNA of each promoter region and thus induced RNA-directed DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin (CNX), protein disulphide isomerase (PDIL1-1) and luminal binding protein (BiP1); an endoplasmic reticulum stress-inducible gene (OsbZIP50); and genes with seed-specific expression encoding α-globulin (Glb-1) and glutelin-B4 (GluB4). TGS of four genes was obtained with high efficiency (CNX, 66.7% of regenerated plants; OsBiP1, 67.4%; OsbZIP50, 63.4%; GluB4, 66.1%), whereas the efficiency was lower for PDIL1-1 (33.3%) and Glb-1 TGS lines (10.5%). The heredity of TGS, methylation levels of promoter regions and specificity of silencing of the target gene were investigated in some of the TGS lines. In progeny of CNX and OsbZIP50 TGS lines, suppression of the target genes was preserved (except in the endosperm) even after the removal of trigger genes (T-DNA) by segregation. TGS of CNX was reverted by demethylation treatment, and a significant difference in CG and CHG methylation levels in the -1 to -250 bp region of the CNX promoter was detected between the TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of promoter. TGS exhibited specific suppression towards the target gene compared with post-transcriptional gene silencing when GluB4 gene from glutelin multigene family was targeted. Based on these results, future perspectives and problems to be solved in the application of RdDM to new plant breeding techniques in rice are discussed.

DNA cytosine methylation is an epigenetic mark associated with silencing of transposable elements (TEs) and heterochromatin formation. In plants, it occurs in three sequence contexts: CG, CHG, and CHH (where H is A, T, or C). The latter does not allow direct inheritance of methylation during DNA replication due to lack of symmetry, and methylation must therefore be re-established every cell generation. Genome-wide association studies (GWAS) have previously shown that CMT2 and NRPE1 are major determinants of genome-wide patterns of TE CHH methylation. Here we instead focus on CHH methylation of individual TEs and TE-families, allowing us to identify the pathways involved in CHH methylation simply from natural variation and confirm the associations by comparing them with mutant phenotypes. Methylation at TEs targeted by the RNA-directed DNA methylation (RdDM) pathway is unaffected by CMT2 variation, but is strongly affected by variation at NRPE1, which is largely responsible for the longitudinal cline in this phenotype. In contrast, CMT2-targeted TEs are affected by both loci, which jointly explain 7.3% of the phenotypic variation (13.2% of total genetic effects). There is no longitudinal pattern for this phenotype, however, because the geographic patterns appear to compensate for each other in a pattern suggestive of stabilizing selection.