Hang He

Global Epigenetic and Transcriptional Trends among Two Rice Subspecies and Their Reciprocal Hybrids

This work examines the molecular basis of heterosis by comprehensively describing the epigenetic modifications and transcriptional output, including both mRNA and small RNAs, of two rice subspecies and their reciprocal hybrids. The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. Here, we report highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice (Oryza sativa) subspecies and their reciprocal hybrids. We found that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions. Differential epigenetic modifications correlated with changes in transcript levels among hybrids and parental lines. Distinct patterns in gene expression and epigenetic modifications in reciprocal hybrids were observed. Through analyses of single nucleotide polymorphisms from our sequence data, we observed a high correlation of allelic bias of epigenetic modifications or gene expression in reciprocal hybrids with their differences in the parental lines. The abundance of distinct small RNA size classes differed between the parents, and more small RNAs were downregulated than upregulated in the reciprocal hybrids. Together, our data reveal a comprehensive overview of transcriptional and epigenetic trends in heterotic rice crosses and provide a useful resource for the rice community.

High-Resolution Mapping of Epigenetic Modifications of the Rice Genome Uncovers Interplay between DNA Methylation, Histone Methylation, and Gene Expression

We present high-resolution maps of DNA methylation and H3K4 di- and trimethylation of two entire chromosomes and two fully sequenced centromeres in rice (Oryza sativa) shoots and cultured cells. This analysis reveals combinatorial interactions between these epigenetic modifications and chromatin structure and gene expression. Cytologically densely stained heterochromatin had less H3K4me2 and H3K4me3 and more methylated DNA than the less densely stained euchromatin, whereas centromeres had a unique epigenetic composition. Most transposable elements had highly methylated DNA but no H3K4 methylation, whereas more than half of protein-coding genes had both methylated DNA and di- and/or trimethylated H3K4. Methylation of DNA but not H3K4 was correlated with suppressed transcription. By contrast, when both DNA and H3K4 were methylated, transcription was only slightly reduced. Transcriptional activity was positively correlated with the ratio of H3K4me3/H3K4me2: genes with predominantly H3K4me3 were actively transcribed, whereas genes with predominantly H3K4me2 were transcribed at moderate levels. More protein-coding genes contained all three modifications, and more transposons contained DNA methylation in shoots than cultured cells. Differential epigenetic modifications correlated to tissue-specific expression between shoots and cultured cells. Collectively, this study provides insights into the rice epigenomes and their effect on gene expression and plant development.

Genome-Wide Analysis of DNA Methylation and Gene Expression Changes in Two Arabidopsis Ecotypes and Their Reciprocal Hybrids

This work analyzed DNA methylation and gene expression changes in two Arabidopsis ecotypes and their reciprocal hybrids and found that increased DNA methylation in both hybrid genomes, possibly directed by the RdDM pathway, may play a role in heterosis. Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single-base-pair resolution the DNA methylomes of Arabidopsis thaliana Landsberg erecta and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs, implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to methylome remodeling were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA ENHANCER1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in heterosis.

Dynamic Landscapes of Four Histone Modifications during Deetiolation in Arabidopsis

Although landscapes of several histone marks are now available for Arabidopsis thaliana and Oryza sativa, such profiles remain static and do not provide information about dynamic changes of plant epigenomes in response to developmental or environmental cues. Here, we analyzed the effects of light on four histone modifications (acetylation and trimethylation of lysines 9 and 27 on histone H3: H3K9ac, H3K9me3, H3K27ac, and H3K27me3, respectively). Our genome-wide profiling of H3K9ac and H3K27ac revealed that these modifications are nontransposable element gene-specific. By contrast, we found that H3K9me3 and H3K27me3 target nontransposable element genes, but also intergenic regions and transposable elements. Specific light conditions affected the number of modified regions as well as the overall correlation strength between the presence of specific modifications and transcription. Furthermore, we observed that acetylation marks not only ELONGATED HYPOCOTYL5 and HY5-HOMOLOG upon deetiolation, but also their downstream targets. We found that the activation of photosynthetic genes correlates with dynamic acetylation changes in response to light, while H3K27ac and H3K27me3 potentially contribute to light regulation of the gibberellin metabolism. Thus, this work provides a dynamic portrait of the variations in histone modifications in response to the plants changing light environment and strengthens the concept that histone modifications represent an additional layer of control for light-regulated genes involved in photomorphogenesis.

Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulation

LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology, and defective hormonal and stimulus responses. HY5 thus acts as an integrator that links various gene networks to coordinate plant development. Here we report the experimental mapping of HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate that HY5 binds to over 9000 genes, detectably affecting the expression of over 1100 genes, either positively or negatively. Further, HY5 indirectly regulate many other genes through sub-networks mediated by other regulators. In particular, HY5 regulates eight miRNA genes that in turn control the transcript abundance of specific target genes. Over-expressing HY5-targeted miR408 resulted in phenotypes that are opposite to the hy5 mutants. Together, our results reveal both transcriptional and post-transcriptional components of the HY5-mediated gene networks.

A High-Density SNP Genotyping Array for Rice Biology and Molecular Breeding

A high-density single nucleotide polymorphism (SNP) array is critically important for geneticists and molecular breeders. With the accumulation of huge amounts of genomic re-sequencing data and available technologies for accurate SNP detection, it is possible to design high-density and high-quality rice SNP arrays. Here we report the development of a high-density rice SNP array and its utility. SNP probes were designed by screening more than 10 000 000 SNP loci extracted from the re-sequencing data of 801 rice varieties and an array named RiceSNP50 was produced on the Illumina Infinium platform. The array contained 51 478 evenly distributed markers, 68% of which were within genic regions. Several hundred rice plants with parent/F1 relationships were used to generate a high-quality cluster file for accurate SNP calling. Application tests showed that this array had high genotyping accuracy, and could be used for different objectives. For example, a core collection of elite rice varieties was clustered with fine resolution. Genome-wide association studies (GWAS) analysis correctly identified a characterized QTL. Further, this array was successfully used for variety verification and trait introgression. As an accurate high-throughput genotyping tool, RiceSNP50 will play an important role in both functional genomics studies and molecular breeding.

A Genome-Wide Transcription Analysis Reveals a Close Correlation of Promoter INDEL Polymorphism and Heterotic Gene Expression in Rice Hybrids

Heterosis, or hybrid vigor, refers to the phenomenon in which hybrid progeny of two inbred varieties exhibits enhanced growth or agronomic performance. Although a century-long history of research has generated several hypotheses regarding the genetic basis of heterosis, the molecular mechanisms underlying heterosis and heterotic gene expression remain elusive. Here, we report a genome-wide gene expression analysis of two heterotic crosses in rice, taking advantage of its fully sequenced genomes. Approximately 7-9% of the genes were differentially expressed in the seedling shoots from two sets of heterotic crosses, including many transcription factor genes, and exhibited multiple modes of gene action. Comparison of the putative promoter regions of the ortholog genes between inbred parents revealed extensive sequence variation, particularly small insertions/deletions (INDELs), many of which result in the formation/disruption of putative cis-regulatory elements. Together, these results suggest that a combinatorial interplay between expression of transcription factors and polymorphic promoter cis-regulatory elements in the hybrids is one plausible molecular mechanism underlying heterotic gene action and thus heterosis in rice.

Development and application of a set of breeder-friendly SNP markers for genetic analyses and molecular breeding of rice (Oryza sativa L.)

Single nucleotide polymorphisms (SNPs) are the most abundant DNA markers in plant genomes. In this study, based on 54,465 SNPs between the genomes of two Indica varieties, Minghui 63 (MH63) and Zhenshan 97 (ZS97) and additional 20,705 SNPs between the MH63 and Nipponbare genomes, we identified and confirmed 1,633 well-distributed SNPs by PCR and Sanger sequencing. From these, a set of 372 SNPs were further selected to analyze the patterns of genetic diversity in 300 representative rice inbred lines from 22 rice growing countries worldwide. Using this set of SNPs, we were able to uncover the well-known Indica–Japonica subspecific differentiation and geographic differentiations within Indica and Japonica. Furthermore, our SNP results revealed some common and contrasting patterns of the haplotype diversity along different rice chromosomes in the Indica and Japonica accessions, which suggest different evolutionary forces possibly acting in specific regions of the rice genome during domestication and evolution of rice. Our results demonstrated that this set of SNPs can be used as anchor SNPs for large scale genotyping in rice molecular breeding research involving Indica–Japonica and Indica–Indica crosses.

Basic leucine zipper transcription factor OsbZIP16 positively regulates drought resistance in rice

Abiotic stress has been shown to limit the growth, development, and productivity of crops. Here, we characterized the function of a rice bZIP transcription factor OsbZIP16 in drought stress. Expression of OsbZIP16 was dramatically induced under drought conditions. Transient expression and transactivation assays demonstrated that OsbZIP16 was localized in the nucleus and had transactivation activity. At both the seedling and tillering stages, transgenic rice plants overexpressing OsbZIP16 exhibited significantly improved drought resistance, which was positively correlated with the observed expression levels of OsbZIP16. Representative downstream drought-inducible genes were observed to have significantly higher expression levels in transgenic rice plants than in the wild type plants under drought conditions. OsbZIP16 was shown to be induced by exogenous ABA treatment, while overexpression of OsbZIP16 was observed to make transgenic plants more sensitive to ABA than wild type plants were. Transcriptome analysis identified a number of differentially expressed genes between wild type plants and plants overexpressing OsbZIP16, many of which are involved in stress response according to their gene ontologies. Overall, our findings suggest that OsbZIP16 positively regulates drought resistance in rice.

Functional characterization of the Arabidopsis ubiquitin‐specific protease gene family reveals specific role and redundancy of individual members in development

SUMMARY Ubiquitin-specific proteases (UBPs) are a highly conserved family of proteins in eukaryotes, and play critical roles in protein de-ubiquitination. Here we report a systematic genetic and expression profiling analysis of the UBP gene family in the Arabidopsis thaliana genome. Mutation analysis of 25 of the 27 member genes representing 13 of the 14 sub-families of the UBP gene family revealed that single-gene mutants of three genes in two sub-families exhibit visible phenotypes. Two of these three genes belonging to the UBP15 sub-family were selected for further characterization. The ubp15 mutants display narrower, serrated and flat rosette leaves, partially due to a defect in cell proliferation, as well as other phenotypes such as early flowering, weak apical dominance and reduced fertility, while the line over-expressing UBP15 shows opposite phenotypes. We demonstrated that UPB15 has UBP activity in vitro, and that this biochemical activity is essential for its in vivo function. A genetic interaction analysis among members of this sub-family revealed that UBP15 and UBP16, but not UBP17, have functional redundancy. Our data thus suggest that distinct UBPs, even within a closely related sub-family, can function in different developmental pathways. Although there are clearly functional redundancies among related sub-family members, those redundancies cannot be inferred simply based on the amino acid identity of the family members.