Light control of three-dimensional chromatin organization in soybean.

Higher-order chromatin structure is critical for regulation of gene expression. In plants, light profoundly affects the morphogenesis of emerging seedlings as well as global gene expression to ensure optimal adaptation to environmental conditions. However, the changes and functional significance of chromatin organization in response to light during seedling development are not well documented. We constructed Hi-C contact maps for the cotyledon, apical hook and hypocotyl of soybean subjected to dark and light conditions. The resulting high-resolution Hi-C contact maps identified chromosome territories, A/B compartments, A/B sub-compartments, TADs (Topologically Associated Domains) and chromatin loops in each organ. We observed increased chromatin compaction under light and we found that domains that switched from B sub-compartments in darkness to A sub-compartments under light contained genes that were activated during photomorphogenesis. At the local scale, we identified a group of TADs constructed by gene clusters consisting of different numbers of Small Auxin-Upregulated RNAs (SAURs), which exhibited strict co-expression in the hook and hypocotyl in response to light stimulation. In the hypocotyl, RNA polymerase II (RNAPII) regulated the transcription of a SAURs cluster under light via TAD condensation. Our results suggest that the 3D genome is involved in the regulation of light-related gene expression in a tissue-specific manner.

Two telomere-to-telomere gapless genomes reveal insights into Capsicum evolution and capsaicinoid biosynthesis.

Chili pepper (Capsicum) is known for its unique fruit pungency due to the presence of capsaicinoids. The evolutionary history of capsaicinoid biosynthesis and the mechanism of their tissue specificity remain obscure due to the lack of high-quality Capsicum genomes. Here, we report two telomere-to-telomere (T2T) gap-free genomes of C. annuum and its wild nonpungent relative C. rhomboideum to investigate the evolution of fruit pungency in chili peppers. We precisely delineate Capsicum centromeres, which lack high-copy tandem repeats but are extensively invaded by CRM retrotransposons. Through phylogenomic analyses, we estimate the evolutionary timing of capsaicinoid biosynthesis. We reveal disrupted coding and regulatory regions of key biosynthesis genes in nonpungent species. We also find conserved placenta-specific accessible chromatin regions, which likely allow for tissue-specific biosynthetic gene coregulation and capsaicinoid accumulation. These T2T genomic resources will accelerate chili pepper genetic improvement and help to understand Capsicum genome evolution.

Liquid-liquid phase separation of TZP promotes PPK-mediated phosphorylation of the phytochrome A photoreceptor.

Phytochrome A (phyA) is the plant far-red (FR) light photoreceptor and plays an essential role in regulating photomorphogenic development in FR-rich conditions, such as canopy shade. It has long been observed that phyA is a phosphoprotein in vivo; however, the protein kinases that could phosphorylate phyA remain largely unknown. Here we show that a small protein kinase family, consisting of four members named PHOTOREGULATORY PROTEIN KINASES (PPKs) (also known as MUT9-LIKE KINASES), directly phosphorylate phyA in vitro and in vivo. In addition, TANDEM ZINC-FINGER/PLUS3 (TZP), a recently characterized phyA-interacting protein required for in vivo phosphorylation of phyA, is also directly phosphorylated by PPKs. We reveal that TZP contains two intrinsically disordered regions in its amino-terminal domain that undergo liquid-liquid phase separation (LLPS) upon light exposure. The LLPS of TZP promotes colocalization and interaction between PPKs and phyA, thus facilitating PPK-mediated phosphorylation of phyA in FR light. Our study identifies PPKs as a class of protein kinases mediating the phosphorylation of phyA and demonstrates that the LLPS of TZP contributes significantly to more production of the phosphorylated phyA form in FR light.

Single-nucleus RNA and ATAC sequencing analyses provide molecular insights into early pod development of peanut fruit.

Peanut (Arachis hypogaea L.) is an important leguminous oil and economic crop that produces flowers above ground and fruits underground. Subterranean fruit pod development, which significantly impacts peanut production, involves complex molecular mechanisms that likely require the coordinated regulation of multiple genes in different tissues. To investigate the molecular mechanisms underlying peanut fruit pod development, we characterized the anatomical features of early fruit pod development and integrated the snRNA-seq and snATAC-seq data at single-cell levels. Moreover, we identified distinct cell types, such as meristem, embryo, vascular tissue, cuticular layer, and stele cells within the shell wall. These specific cell types were employed to scrutinize potential molecular changes unique to each cell type during pivotal stages of peanut fruit pod development. The snRNA-seq analyses of DEGs (differential expressed genes) unveiled cell-type-specific insights that were not previously discernible through bulk-RNA transcriptome analysis. For instance, MADS-box genes contributing to the formation of parenchyma cells were identified, and gravity-related genes in the vascular cells were detected, indicating an essential role for vascular cells in peg gravitropism. Overall, our single-nucleus analysis provides comprehensive and novel information on specific cell types, gene expression chromatin accessibility during the early stages of fruit pod development. This information will enhance our comprehension of the mechanisms underlying peanut fruit pod development and contribute to efforts aimed at improving peanut production.

The molecular basis of COP1 action during photomorphogenesis.

COP1 (CONSTITUTIVE PHOTOMORPHOGENIC1), a repressor of seedling photomorphogenesis, is tightly controlled by light. In Arabidopsis, COP1 primarily acts as a part of large E3 ligase complexes and targets key light-signaling factors for ubiquitination and degradation. Upon light perception, the action of COP1 is precisely modulated by active photoreceptors. During seedling development, light plays a predominant role in modulating seedling morphogenesis, including inhibition of hypocotyl elongation, cotyledon opening and expansion, and chloroplast development. These visible morphological changes evidently are resulted from networks of molecular action. In this review, we summarize the current knowledge about the molecular role of COP1 in mediating light-controlled seedling development.

Toward understanding and utilizing crop heterosis in the age of biotechnology.

Heterosis, a universal phenomenon in nature, mainly reflected in the superior productivity, quality, and fitness of F1 hybrids compared with their inbred parents, has been exploited in agriculture and greatly benefited human society in terms of food security. However, the flexible and efficient utilization of heterosis has remained a challenge in hybrid breeding systems because of the limitations of "three-line" and "two-line" methods. In the past two decades, rapidly developed biotechnologies have provided unprecedented conveniences for both understanding and utilizing heterosis. Notably, "third-generation" (3G) hybrid breeding technology together with high-throughput sequencing and gene editing greatly promoted the efficiency of hybrid breeding. Here, we review emerging ideas about the genetic or molecular mechanisms of heterosis and the development of 3G hybrid breeding system in the age of biotechnology. In addition, we summarized opportunities and challenges for optimal heterosis utilization in the future.

Increased long-distance and homo-trans interactions related to H3K27me3 in Arabidopsis hybrids.

In plants, the genome structure of hybrids changes compared with their parents, but the effects of these changes in hybrids remain elusive. Comparing reciprocal crosses between Col × C24 and C24 × Col in Arabidopsis using high-throughput chromosome conformation capture assay (Hi-C) analysis, we found that hybrid three-dimensional (3D) chromatin organization had more long-distance interactions relative to parents, and this was mainly located in promoter regions and enriched in genes with heterosis-related pathways. The interactions between euchromatin and heterochromatin were increased, and the compartment strength decreased in hybrids. In compartment domain (CD) boundaries, the distal interactions were more in hybrids than their parents. In the hybrids of CURLY LEAF (clf) mutants clfCol × clfC24 and clfC24 × clfCol , the heterosis phenotype was damaged, and the long-distance interactions in hybrids were fewer than in their parents with lower H3K27me3. ChIP-seq data revealed higher levels of H3K27me3 in the region adjacent to the CD boundary and the same interactional homo-trans sites in the wild-type (WT) hybrids, which may have led to more long-distance interactions. In addition, the differentially expressed genes (DEGs) located in the boundaries of CDs and loop regions changed obviously in WT, and the functional enrichment for DEGs was different between WT and clf in the long-distance interactions and loop regions. Our findings may therefore propose a new epigenetic explanation of heterosis in the Arabidopsis hybrids and provide new insights into crop breeding and yield increase.

Construction of a Female Sterility Maintaining System Based on a Novel Mutation of the MEL2 Gene.

BACKGROUND: Hybrid rice has significant yield advantage and stress tolerance compared with inbred rice. However, production of hybrid rice seeds requires extensive manual labors. Currently, hybrid rice seeds are produced by crosspollination of male sterile lines by fertile paternal lines. Because seeds from paternal lines can contaminate the hybrid seeds, mechanized production by mixed-seeding and mixed-harvesting is difficult. This problem can be solved if the paternal line is female sterile. RESULTS: Here we identified a female infertile mutant named h569 carrying a novel mutation (A1106G) in the MEL2 gene that was previously reported to regulate meiosis entry both in male and female organs. h569 mutant is female infertile but male normal, suggesting that MEL2 regulates meiosis entry in male and female organs through distinct pathways. The MEL2 gene and h569 mutant gave us tools to construct female sterility maintaining systems that can be used for propagation of female sterile lines. We connected the wild-type MEL2 gene with pollen-killer gene ZmAA1 and seed-marker gene DsRed2 in one T-DNA cassette and transformed it into ZZH1607, a widely used restorer line. Transgenic line carrying a single transgene inserted in an intergenic region was selected to cross with h569 mutant. F2 progeny carrying homozygous A1106G mutation and hemizygous transgene displayed 1:1 segregation of fertile and infertile pollen grains and 1:1 segregation of fluorescent and non-fluorescent seeds upon self-fertilization. All of the non-fluorescent seeds generated female infertile plants, while the fluorescent seeds generated fertile plants that reproduced in the way as their previous generation. CONCLUSIONS: These results indicated that the female sterility maintaining system constructed in the study can be used to breed and propagate paternal lines that are female infertile. The application of this system will enable mechanized production of hybrid rice seed by using the mixed-seeding and mixed harvesting approach, which will significantly reduce the cost in hybrid rice seed production.

Mapping nucleosome-resolution chromatin organization and enhancer-promoter loops in plants using Micro-C-XL.

Although chromatin organizations in plants have been dissected at the scales of compartments and topologically associating domain (TAD)-like domains, there remains a gap in resolving fine-scale structures. Here, we use Micro-C-XL, a high-throughput chromosome conformation capture (Hi-C)-based technology that involves micrococcal nuclease (instead of restriction enzymes) and long cross-linkers, to dissect single nucleosome-resolution chromatin organization in Arabidopsis. Insulation analysis reveals more than 14,000 boundaries, which mostly include chromatin accessibility, epigenetic modifications, and transcription factors. Micro-C-XL reveals associations between RNA Pols and local chromatin organizations, suggesting that gene transcription substantially contributes to the establishment of local chromatin domains. By perturbing Pol II both genetically and chemically at the gene level, we confirm its function in regulating chromatin organization. Visible loops and stripes are assigned to super-enhancers and their targeted genes, thus providing direct insights for the identification and mechanistic analysis of distal CREs and their working modes in plants. We further investigate possible factors regulating these chromatin loops. Subsequently, we expand Micro-C-XL to soybean and rice. In summary, we use Micro-C-XL for analyses of plants, which reveal fine-scale chromatin organization and enhancer-promoter loops and provide insights regarding three-dimensional genomes in plants.

The MBD-ACD DNA methylation reader complex recruits MICRORCHIDIA6 to regulate ribosomal RNA gene expression in Arabidopsis.

DNA methylation is an important epigenetic mark implicated in selective rRNA gene expression, but the DNA methylation readers and effectors remain largely unknown. Here, we report a protein complex that reads DNA methylation to regulate variant-specific 45S ribosomal RNA (rRNA) gene expression in Arabidopsis (Arabidopsis thaliana). The complex, consisting of METHYL-CpG-BINDING DOMAIN PROTEIN5 (MBD5), MBD6, ALPHA-CRYSTALLIN DOMAIN PROTEIN15.5 (ACD15.5), and ACD21.4, directly binds to 45S rDNA. While MBD5 and MBD6 function redundantly, ACD15.5 and ACD21.4 are indispensable for variant-specific rRNA gene expression. These 4 proteins undergo phase separation in vitro and in vivo and are interdependent for their phase separation. The α-crystallin domain of ACD15.5 and ACD21.4, which is essential for their function, enables phase separation of the complex, likely by mediating multivalent protein interactions. The effector MICRORCHIDIA6 directly interacts with ACD15.5 and ACD21.4, but not with MBD5 and MBD6, and is recruited to 45S rDNA by the MBD-ACD complex to regulate variant-specific 45S rRNA expression. Our study reveals a pathway in Arabidopsis through which certain 45S rRNA gene variants are silenced, while others are activated.

Establishment and Advances of Third-Generation Hybrid Rice Technology: A Review.

Rice (Oryza sativa L.) is one of the most important food crops worldwide. The utilisation of heterosis (hybrid vigour) has played a significant role in increasing rice yield and ensuring food supply. Over the past 50 years, the first-generation three-line system based on cytoplasmic male sterility, and the second-generation two-line system based on environment-sensitive genic male sterility (EGMS), have been widely applied in hybrid rice production. However, the three-line system is restricted by the matching relationship among the three parental lines and allows only ~ 2-5% of germplasms to be explored for elite combinations. The environmental sensitivity of EGMS lines has posed serious risks to the production of hybrid seeds. These factors have hindered the development and applications of hybrid rice. Third-generation hybrid rice technology (TGHRT) is based on environment-insensitive genic male sterility, which can effectively overcome the intrinsic problems of the three-line and two-line systems. Since the establishment of TGHRT, numerous findings and innovations have been reported. This paper gives a brief review of traditional hybrid rice technologies and discusses the establishment of TGHRT, technical innovations in TGHRT, and future research that is necessary to promote the wide application of TGHRT in rice production.

Time series single-cell transcriptional atlases reveal cell fate differentiation driven by light in Arabidopsis seedlings.

Light serves as the energy source for plants as well as a signal for growth and development during their whole life cycle. Seedling de-etiolation is the most dramatic manifestation of light-regulated plant development processes, as massive reprogramming of the plant transcriptome occurs at this time. Although several studies have reported about organ-specific development and expression induced by light, a systematic analysis of cell-type-specific differentiation and the associated transcriptional regulation is still lacking. Here we obtained single-cell transcriptional atlases for etiolated, de-etiolating and light-grown Arabidopsis thaliana seedlings. Informative cells from shoot and root tissues were grouped into 48 different cell clusters and finely annotated using multiple markers. With the determination of comprehensive developmental trajectories, we demonstrate light modulation of cell fate determination during guard cell specialization and vasculature development. Comparison of expression atlases between wild type and the pifq mutant indicates that phytochrome-interacting factors (PIFs) are involved in distinct developmental processes in endodermal and stomatal lineage cells via controlling cell-type-specific expression of target genes. These results provide information concerning the light signalling networks at the cell-type resolution, improving our understanding of how light regulates plant development at the cell-type and genome-wide levels. The obtained information could serve as a valuable resource for comprehensively investigating the molecular mechanism of cell development and differentiation in response to light.

Cloning of the wheat leaf rust resistance gene Lr47 introgressed from Aegilops speltoides.

Leaf rust, caused by Puccinia triticina Eriksson (Pt), is one of the most severe foliar diseases of wheat. Breeding for leaf rust resistance is a practical and sustainable method to control this devastating disease. Here, we report the identification of Lr47, a broadly effective leaf rust resistance gene introgressed into wheat from Aegilops speltoides. Lr47 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that is both necessary and sufficient to confer Pt resistance, as demonstrated by loss-of-function mutations and transgenic complementation. Lr47 introgression lines with no or reduced linkage drag are generated using the Pairing homoeologous1 mutation, and a diagnostic molecular marker for Lr47 is developed. The coiled-coil domain of the Lr47 protein is unable to induce cell death, nor does it have self-protein interaction. The cloning of Lr47 expands the number of leaf rust resistance genes that can be incorporated into multigene transgenic cassettes to control this devastating disease.

Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants.

Gravity controls directional growth of plants, and the classical starch-statolith hypothesis proposed more than a century ago postulates that amyloplast sedimentation in specialized cells initiates gravity sensing, but the molecular mechanism remains uncharacterized. The LAZY proteins are known as key regulators of gravitropism, and lazy mutants show striking gravitropic defects. Here, we report that gravistimulation by reorientation triggers mitogen-activated protein kinase (MAPK) signaling-mediated phosphorylation of Arabidopsis LAZY proteins basally polarized in root columella cells. Phosphorylation of LAZY increases its interaction with several translocons at the outer envelope membrane of chloroplasts (TOC) proteins on the surface of amyloplasts, facilitating enrichment of LAZY proteins on amyloplasts. Amyloplast sedimentation subsequently guides LAZY to relocate to the new lower side of the plasma membrane in columella cells, where LAZY induces asymmetrical auxin distribution and root differential growth. Together, this study provides a molecular interpretation for the starch-statolith hypothesis: the organelle-movement-triggered molecular polarity formation.

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology.

Genetic and molecular regulation of increased photosynthetic cell number contributes to leaf size heterosis in Arabidopsis.

Heterosis is an important genetic phenomenon that has been observed and widely utilized in agriculture. However, the genetic and molecular bases of heterosis are unclear. Through transcriptome-wide association studies (TWAS) and expression quantitative trait locus (eQTL) analysis to integrate genome, transcriptome, and heterotic phenotype of a half-sibling Arabidopsis hybrid population, we report that the genetic and molecular bases of variations in leaf growth heterosis can be explained by the varied expression levels of growth-regulating genes resulting from distinct sets of heterozygous eQTLs carried by the half-sibling hybrids. In F1 versus parent, the degree of up-regulated gene expression in the cell cycle pathway in the shoot apex and the photosynthesis pathway in true leaf positively correlates with true leaf area heterosis level, and this is affected by the accumulation of superior heterozygous eQTLs. This was further corroborated by the major contribution of increased photosynthetic cell number to leaf area heterosis.

Reconstitution of phytochrome A-mediated light modulation of the ABA signaling pathways in yeast.

Abscisic acid (ABA), a classical plant hormone, plays an essential role in plant adaptation to environmental stresses. The ABA signaling mechanisms have been extensively investigated, and it was shown that the PYR1 (PYRABACTIN RESISTANCE1)/PYL (PYR1-LIKE)/RCAR (REGULATORY COMPONENT OF ABA RECEPTOR) ABA receptors, the PP2C coreceptors, and the SnRK2 protein kinases constitute the core ABA signaling module responsible for ABA perception and initiation of downstream responses. We recently showed that ABA signaling is modulated by light signals, but the underlying molecular mechanisms remain largely obscure. In this study, we established a system in yeast cells that was not only successful in reconstituting a complete ABA signaling pathway, from hormone perception to ABA-responsive gene expression, but also suitable for functionally characterizing the regulatory roles of additional factors of ABA signaling. Using this system, we analyzed the roles of several light signaling components, including the red and far-red light photoreceptors phytochrome A (phyA) and phyB, and the photomorphogenic central repressor COP1, in the regulation of ABA signaling. Our results showed that both phyA and phyB negatively regulated ABA signaling, whereas COP1 positively regulated ABA signaling in yeast cells. Further analyses showed that photoactivated phyA interacted with the ABA coreceptors ABI1 and ABI2 to decrease their interactions with the ABA receptor PYR1. Together, data from our reconstituted yeast ABA signaling system provide evidence that photoactivated photoreceptors attenuate ABA signaling by directly interacting with the key components of the core ABA signaling module, thus conferring enhanced ABA tolerance to light-grown plants.

Gene expression variations and allele-specific expression of two rice and their hybrid in caryopses at single-nucleus resolution.

Seeds are an indispensable part of the flowering plant life cycle and a critical determinant of agricultural production. Distinct differences in the anatomy and morphology of seeds separate monocots and dicots. Although some progress has been made with respect to understanding seed development in Arabidopsis, the transcriptomic features of monocotyledon seeds at the cellular level are much less understood. Since most important cereal crops, such as rice, maize, and wheat, are monocots, it is essential to study transcriptional differentiation and heterogeneity during seed development at a finer scale. Here, we present single-nucleus RNA sequencing (snRNA-seq) results of over three thousand nuclei from caryopses of the rice cultivars Nipponbare and 9311 and their intersubspecies F1 hybrid. A transcriptomics atlas that covers most of the cell types present during the early developmental stage of rice caryopses was successfully constructed. Additionally, novel specific marker genes were identified for each nuclear cluster in the rice caryopsis. Moreover, with a focus on rice endosperm, the differentiation trajectory of endosperm subclusters was reconstructed to reveal the developmental process. Allele-specific expression (ASE) profiling in endosperm revealed 345 genes with ASE (ASEGs). Further pairwise comparisons of the differentially expressed genes (DEGs) in each endosperm cluster among the three rice samples demonstrated transcriptional divergence. Our research reveals differentiation in rice caryopsis from the single-nucleus perspective and provides valuable resources to facilitate clarification of the molecular mechanism underlying caryopsis development in rice and other monocots.