Nuclear lamina component KAKU4 regulates chromatin states and transcriptional regulation in the Arabidopsis genome.

BACKGROUND: The nuclear lamina links the nuclear membrane to chromosomes and plays a crucial role in regulating chromatin states and gene expression. However, current knowledge of nuclear lamina in plants is limited compared to animals and humans. RESULTS: This study mainly focused on elucidating the mechanism through which the putative nuclear lamina component protein KAKU4 regulates chromatin states and gene expression in Arabidopsis leaves. Thus, we constructed a network using the association proteins of lamin-like proteins, revealing that KAKU4 is strongly associated with chromatin or epigenetic modifiers. Then, we conducted ChIP-seq technology to generate global epigenomic profiles of H3K4me3, H3K27me3, and H3K9me2 in Arabidopsis leaves for mutant (kaku4-2) and wild-type (WT) plants alongside RNA-seq method to generate gene expression profiles. The comprehensive chromatin state-based analyses indicate that the knockdown of KAKU4 has the strongest effect on H3K27me3, followed by H3K9me2, and the least impact on H3K4me3, leading to significant changes in chromatin states in the Arabidopsis genome. We discovered that the knockdown of the KAKU4 gene caused a transition between two types of repressive epigenetics marks, H3K9me2 and H3K27me3, in some specific PLAD regions. The combination analyses of epigenomic and transcriptomic data between the kaku4-2 mutant and WT suggested that KAKU4 may regulate key biological processes, such as programmed cell death and hormone signaling pathways, by affecting H3K27me3 modification in Arabidopsis leaves. CONCLUSIONS: In summary, our results indicated that KAKU4 is directly and/or indirectly associated with chromatin/epigenetic modifiers and demonstrated the essential roles of KAKU4 in regulating chromatin states, transcriptional regulation, and diverse biological processes in Arabidopsis.

KAKU4 regulates leaf senescence through modulation of H3K27me3 deposition in the Arabidopsis genome.

Lamins are the major components of the nuclear lamina, which regulate chromatin structure and gene expression. KAKU4 is a unique nuclear lamina component in the nuclear periphery, modulates nuclear shape and size in Arabidopsis. The knowledge about the regulatory role of KAKU4 in leaf development remains limited. Here we found that knockdown of KAKU4 resulted in an accelerated leaf senescence phenotype, with elevated levels of H2O2 and hormones, particularly SA, JA, and ABA. Our results demonstrated the importance of KAKU4 as a potential negative regulator in age-triggered leaf senescence in Arabidopsis. Furthermore, we conducted combination analyses of transcriptomic and epigenomic data for the kaku4 mutant and WT leaves. The knockdown of KAKU4 lowered H3K27me3 deposition in the up-regulated genes associated with hormone pathways, programmed cell death, and leaf senescence, including SARD1, SAG113/HAI1, PR2, and so forth. In addition, we found the functional crosstalks between KAKU4 and its associated proteins (CRWN1/4, PNET2, GBPL3, etc.) through comparing multiple transcriptome datasets. Overall, our results indicated that KAKU4 may inhibit the expression of a series of genes related to hormone signals and H2O2 metabolism by affecting the deposition of H3K27me3, thereby suppressing leaf senescence.

Genome-Wide Investigation and Co-Expression Network Analysis of SBT Family Gene in Gossypium.

Subtilases (SBTs), which belong to the serine peptidases, control plant development by regulating cell wall properties and the activity of extracellular signaling molecules, and affect all stages of the life cycle, such as seed development and germination, and responses to biotic and abiotic environments. In this study, 146 Gossypium hirsutum, 138 Gossypium barbadense, 89 Gossypium arboreum and 84 Gossypium raimondii SBTs were identified and divided into six subfamilies. Cotton SBTs are unevenly distributed on chromosomes. Synteny analysis showed that the members of SBT1 and SBT4 were expanded in cotton compared to Arabidopsis thaliana. Co-expression network analysis showed that six Gossypium arboreum SBT gene family members were in a network, among which five SBT1 genes and their Gossypium hirsutum and Arabidopsis thaliana direct homologues were down-regulated by salt treatment, indicating that the co-expression network might share conserved functions. Through co-expression network and annotation analysis, these SBTs may be involved in the biological processes of auxin transport, ABA signal transduction, cell wall repair and root tissue development. In summary, this study provides valuable information for the study of SBT genes in cotton and excavates SBT genes in response to salt stress, which provides ideas for cotton breeding for salinity resistance.

Systems biology-based analysis indicates that PHO1;H10 positively modulates high light-induced anthocyanin biosynthesis in Arabidopsis leaves.

Arabidopsis PHO1;H10 is a member of the PHO1 gene family with SPX and EXS domains, and its functions remain largely unknown. As shown in PCSD database, the upstream region of PHO1;H10 gene is in the active chromatin states, with high DHS accessibility and binding sites of multiple transcription factors, especially ABI5, SPCH and HY5. Co-expression network and data-mining analyses showed PHO1;H10 and co-expression genes were with activation under high light stress. We did wet-lab experiments, and found that the detached leaves of PHO1;H10 overexpression lines accumulated more anthocyanin than those of WT and mutant under high light treatment. RNA-seq results showed overexpression of PHO1;H10 up-regulated many anthocyanin biosynthetic genes. The GSEA analysis result showed that the functional module related to anthocyanin pathway was significantly enriched. In summary, we conducted systems biology approach, combining dry- and wet-lab analyses, and discovered that PHO1;H10 might play an essential role during modulating high light-induced anthocyanin accumulation in the Arabidopsis detached leaves.

KNOX II transcription factor HOS59 functions in regulating rice grain size.

Plant Knotted1-like homeobox (KNOX) genes encode homeodomain-containing transcription factors. In rice (Oryza sativa L.), little is known about the downstream target genes of KNOX Class II subfamily proteins. Here we generated chromatin immunoprecipitation (ChIP)-sequencing datasets for HOS59, a member of the rice KNOX Class II subfamily, and characterized the genome-wide binding sites of HOS59. We conducted trait ontology (TO) analysis of 9705 identified downstream target genes, and found that multiple TO terms are related to plant structure morphology and stress traits. ChIP-quantitative PCR (qPCR) was conducted to validate some key target genes. Meanwhile, our IP-MS datasets showed that HOS59 was closely associated with BELL family proteins, some grain size regulators (OsSPL13, OsSPL16, OsSPL18, SLG, etc.), and some epigenetic modification factors such as OsAGO4α and OsAGO4ß, proteins involved in small interfering RNA-mediated gene silencing. Furthermore, we employed CRISPR/Cas9 editing and transgenic approaches to generate hos59 mutants and overexpression lines, respectively. Compared with wild-type plants, the hos59 mutants have longer grains and increased glume cell length, a loose plant architecture, and drooping leaves, while the overexpression lines showed smaller grain size, erect leaves, and lower plant height. The qRT-PCR results showed that mutation of the HOS59 gene led to upregulation of some grain size-related genes such as OsSPL13, OsSPL18, and PGL2. In summary, our results indicate that HOS59 may be a repressor of the downstream target genes, negatively regulating glume cell length, rice grain size, plant architecture, etc. The identified downstream target genes and possible interaction proteins of HOS59 improve our understanding of the KNOX regulatory networks.

PlantGSAD: a comprehensive gene set annotation database for plant species.

With the accumulation of massive data sets from high-throughput experiments and the rapid emergence of new types of omics data, gene sets have become more diverse and essential for the refinement of gene annotation at multidimensional levels. Accordingly, we collected and defined 236 007 gene sets across different categories for 44 plant species in the Plant Gene Set Annotation Database (PlantGSAD). These gene sets were divided into nine main categories covering many functional subcategories, such as trait ontology, co-expression modules, chromatin states, and liquid-liquid phase separation. The annotations from the collected gene sets covered all of the genes in the Brassicaceae species Arabidopsis and Poaceae species Oryza sativa. Several GSEA tools are implemented in PlantGSAD to improve the efficiency of the analysis, including custom SEA for a flexible strategy based on customized annotations, SEACOMPARE for the cross-comparison of SEA results, and integrated visualization features for ontological analysis that intuitively reflects their parent-child relationships. In summary, PlantGSAD provides numerous gene sets for multiple plant species and highly efficient analysis tools. We believe that PlantGSAD will become a multifunctional analysis platform that can be used to predict and elucidate the functions and mechanisms of genes of interest. PlantGSAD is publicly available at http://systemsbiology.cau.edu.cn/PlantGSEAv2/.

Refinement of bamboo genome annotations through integrative analyses of transcriptomic and epigenomic data.

Bamboo, one of the most crucial nontimber forest resources worldwide, has the capacity for rapid growth. In recent years, the genome of moso bamboo (Phyllostachys edulis) has been decoded, and a large amount of transcriptome data has been published. In this study, we generated the genome-wide profiles of the histone modification H3K4me3 in leaf, stem, and root tissues of bamboo. The trends in the distribution patterns were similar to those in rice. We developed a processing pipeline for predicting novel transcripts to refine the structural annotation of the genome using H3K4me3 ChIP-seq data and 29 RNA-seq datasets. As a result, 12,460 novel transcripts were predicted in the bamboo genome. Compared with the transcripts in the newly released version 2.0 of the bamboo genome, these novel transcripts are tissue-specific and shorter, and most have a single exon. Some representative novel transcripts were validated by semiquantitative RT-PCR and qRT-PCR analyses. Furthermore, we put these novel transcripts back into the ChIP-seq analysis pipeline and discovered that the percentages of H3K4me3 in genic elements were increased. Overall, this work integrated transcriptomic data and epigenomic data to refine the annotation of the genome in order to discover more functional genes and study bamboo growth and development, and the application of this predicted pipeline may help refine the structural annotation of the genome in other species.

HpeNet: Co-expression Network Database for de novo Transcriptome Assembly of Paeonia lactiflora Pall.

The herbaceous peony (Paeonia lactiflora Pall.) is a well-known ornamental flowering and pharmaceutical plant found in China. Its high medicinal value has long been recognized by traditional Chinese medicine (as Radix paeoniae Alba and Radix paeoniae Rubra), and it has become economically valued for its oilseed in recent years; like other Paeonia species, it has been identified as a novel resource for the α-linolenic acid used in seed oil production. However, its genome has not yet been sequenced, and little transcriptome data on Paeonia lactiflora are available. To obtain a comprehensive transcriptome for Paeonia lactiflora, RNAs from 10 tissues of the Paeonia lactiflora Pall. cv Shaoyou17C were used for de novo assembly, and 416,062 unigenes were obtained. Using a homology search, it was found that 236,222 (approximately 57%) unigenes had at least one BLAST hit in one or more public data resources. The construction of co-expression networks is a feasible means for improving unigene annotation. Using in-house transcriptome data, we obtained a co-expression network covering 95.13% of the unigenes. Then we integrated co-expression network analyses and lipid-related pathway genes to study lipid metabolism in Paeonia lactiflora cultivars. Finally, we constructed the online database HpeNet (http://bioinformatics.cau.edu.cn/HpeNet) to integrate transcriptome data, gene information, the co-expression network, and so forth. The database can also be searched for gene details, gene functions, orthologous matches, and other data. Our online database may help the research community identify functional genes and perform research on Paeonia lactiflora more conveniently. We hope that de novo transcriptome assembly, combined with co-expression networks, can provide a feasible means to predict the gene function of species that do not have a reference genome.

Over-Expression of HDA710 Delays Leaf Senescence in Rice (Oryza sativa L.).

Histone deacetylases (HDACs) influence chromatin state and gene expression. Eighteen HDAC genes with important biological functions have been identified in rice. In this study, we surveyed the gene presence frequency of all 18 rice HDAC genes in 3,010 rice accessions. HDA710/OsHDAC2 showed insertion/deletion (InDel) polymorphisms in almost 98.8% japonica accessions but only 1% indica accessions. InDel polymorphism association analysis showed that accessions with partial deletions in HDA710 tended to display early leaf senescence. Further transgenic results confirmed that HDA710 delayed leaf senescence in rice. The over-expression of HDA710 delayed leaf senescence, and the knock-down of HDA710 accelerated leaf senescence. Transcriptome analysis showed that photosynthesis and chlorophyll biosynthesis related genes were up-regulated in HDA710 over-expression lines, while some programmed cell death and disease resistance related genes were down-regulated. Co-expression network analysis with gene expression view revealed that HDA710 was co-expressed with multiple genes, particularly OsGSTU12, which was significantly up-regulated in 35S::HDA710-sense lines. InDels in the promoter region of OsGSTU12 and in the gene region of HDA710 occurred coincidentally among more than 90% accessions, and we identified multiple W-box motifs at the InDel position of OsGSTU12. Over-expression of OsGSTU12 also delayed leaf senescence in rice. Taken together, our results suggest that both HDA710 and OsGSTU12 are involved in regulating the process of leaf senescence in rice.

AtSPX1-mediated transcriptional regulation during leaf senescence in Arabidopsis thaliana.

Leaf senescence is the final stage of leaf growth, a highly coordinated and complicated process. Phosphorus as an essential macronutrient for plant growth is remobilized from senescing leaves to other vigorous parts of the plant. In this study, through data mining, we found some phosphate starvation induced genes such as AtSPX1, were significantly induced in aging leaves in Arabidopsis. We applied a reverse genetics approach to investigate the phenotypes of transgenic plants and mutant plants, and the results showed that the overexpression of AtSPX1 accelerated leaf senescence, suppressed Pi accumulation, promoted SA production and H2O2 levels in leaves, while the mutant lines of AtSPX1 showed slightly delayed leaf senescence. We conducted RNA-seq-based transcriptome analysis together with GO and GSEA enrichment analyses for transgenic vs. wild-type plants to elucidate the possible underlying regulatory mechanism. The 558 genes that were up-regulated in the overexpression plants 35S::AtSPX1/WT, were significantly enriched in the process of leaf senescence, Pi starvation responses and SA signaling pathways, as were the target genes of some transcription factors such as WRKYs and NACs. In a word, we characterized AtSPX1 as a key regulator, which mediated the crosstalks among leaf senescence, Pi starvation and SA signaling pathways in Arabidopsis thaliana.