T2T genomes of carrot and Alternaria dauci and their utility for understanding host-pathogen interactions during carrot leaf blight disease.

Carrot (Daucus carota) is one of the most popular and nutritious vegetable crops worldwide. However, significant yield losses occur every year due to leaf blight, a disease caused by a fungal pathogen (Alternaria dauci). Past research on resistance to leaf blight disease in carrots has been slow because of the low-quality genome assemblies of both carrot and the pathogen. Here, we report the greatly improved assemblies and annotations of telomere-to-telomere (T2T) reference genomes of carrot DH13M14 (451.04 Mb) and A. dauci A2016 (34.91 Mb). Compared with the previous carrot genome versions, our assembly featured notable improvements in genome size, continuity, and completeness of centromeres and telomeres. In addition, we generated a time course transcriptomic atlas during the infection of carrots by A. dauci and captured their dynamic gene expression reprogramming during the interaction process. During infection, A. dauci genes encoding effectors and enzymes responsible for the degradation of plant cell wall components, e.g., cellulose and pectin, were identified, which appeared to increase pathogenic ability through upregulation. In carrot, the coordinated gene expression of components of pattern- and effector-triggered immunity (PTI and ETI) in response to A. dauci attack was characterized. The biosynthesis or signal transduction of plant hormones, including JA, SA, and ethylene, was also involved in the carrot response to A. dauci. This work provides a foundation for understanding A. dauci pathogenic progression and carrot defense mechanisms to improve carrot resistance to leaf blight disease. The Carrot Database (CDB) developed also provides a useful resource for the carrot community.

Hormone-mediated disassembly and inactivation of a plant E3 ubiquitin ligase complex.

Phytohormone abscisic acid (ABA) regulates key plant development and environmental stress responses. The ubiquitin-proteasome system tightly controls ABA signaling. CULLIN4-RING (CRL4) E3 ubiquitin ligases use the substrate receptor module CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP10)-DDB1-DET1-DDA1 (CDDD) to target Arabidopsis ABA receptor PYL8, acting as negative regulators of ABA responses. Conversely, ABA treatment attenuates PYL8 receptor degradation, although the molecular mechanism remained elusive. Here, we show that ABA promotes the disruption of CRL4-CDDD complexes, leading to PYL8 stabilization. ABA-mediated CRL4-CDDD dissociation likely involves an altered association between DDA1-containing complexes and the COP9 signalosome (CSN), a master regulator of the assembly of cullin-based E3 ligases, including CRL4-CDDD. Indeed, treatment with CSN inhibitor CSN5i-3 suppresses the ABA effect on CRL4-CDDD assembly. Our findings indicate that ABA stabilizes PYL8 by altering the dynamics of the CRL4-CDDD-CSN complex association, showing a regulatory mechanism by which a plant hormone inhibits an E3 ubiquitin ligase to protect its own receptors from degradation.

Light-induced remodeling of phytochrome B enables signal transduction by phytochrome-interacting factor.

Phytochrome B (phyB) and phytochrome-interacting factors (PIFs) constitute a well-established signaling module critical for plants adapting to ambient light. However, mechanisms underlying phyB photoactivation and PIF binding for signal transduction remain elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of the photoactivated phyB or the constitutively active phyBY276H mutant in complex with PIF6, revealing a similar trimer. The light-induced configuration switch of the chromophore drives a conformational transition of the nearby tongue signature within the phytochrome-specific (PHY) domain of phyB. The resulting α-helical PHY tongue further disrupts the head-to-tail dimer of phyB in the dark-adapted state. These structural remodelings of phyB facilitate the induced-fit recognition of PIF6, consequently stabilizing the N-terminal extension domain and a head-to-head dimer of activated phyB. Interestingly, the phyB dimer exhibits slight asymmetry, resulting in the binding of only one PIF6 molecule. Overall, our findings solve a key question with respect to how light-induced remodeling of phyB enables PIF signaling in phytochrome research.

Green light mediates atypical photomorphogenesis by dual modulation of Arabidopsis phytochromes B and A.

Although green light (GL) is located in the middle of the visible light spectrum and regulates a series of plant developmental processes, the mechanism by which it regulates seedling development is largely unknown. In this study, we demonstrated that GL promotes atypical photomorphogenesis in Arabidopsis thaliana via the dual regulations of phytochrome B (phyB) and phyA. Although the Pr-to-Pfr conversion rates of phyB and phyA under GL were lower than those under red light (RL) in a fluence rate-dependent and time-dependent manner, long-term treatment with GL induced high Pfr/Pr ratios of phyB and phyA. Moreover, GL induced the formation of numerous small phyB photobodies in the nucleus, resulting in atypical photomorphogenesis, with smaller cotyledon opening angles and longer hypocotyls in seedlings compared to RL. The abundance of phyA significantly decreased after short- and long-term GL treatments. We determined that four major PHYTOCHROME-INTERACTING FACTORs (PIFs: PIF1, PIF3, PIF4, and PIF5) act downstream of phyB in GL-mediated cotyledon opening. In addition, GL plays opposite roles in regulating different PIFs. For example, under continuous GL, the protein levels of all PIFs decreased, whereas the transcript levels of PIF4 and PIF5 strongly increased compared with dark treatment. Taken together, our work provides a detailed molecular framework for understanding the role of the antagonistic regulations of phyB and phyA in GL-mediated atypical photomorphogenesis.

Light-stabilized GIL1 suppresses PIN3 activity to inhibit hypocotyl gravitropism.

Light and gravity coordinately regulate the directional growth of plants. Arabidopsis Gravitropic in the Light 1 (GIL1) inhibits the negative gravitropism of hypocotyls in red and far-red light, but the underlying molecular mechanisms remain elusive. Our study found that GIL1 is a plasma membrane-localized protein. In endodermal cells of the upper part of hypocotyls, GIL1 controls the negative gravitropism of hypocotyls. GIL1 directly interacts with PIN3 and inhibits the auxin transport activity of PIN3. Mutation of PIN3 suppresses the abnormal gravitropic response of gil1 mutant. The GIL1 protein is unstable in darkness but it is stabilized by red and far-red light. Together, our data suggest that light-stabilized GIL1 inhibits the negative gravitropism of hypocotyls by suppressing the activity of the auxin transporter PIN3, thereby enhancing the emergence of young seedlings from the soil.

Telomere-to-telomere Citrullus super-pangenome provides direction for watermelon breeding.

To decipher the genetic diversity within the cucurbit genus Citrullus, we generated telomere-to-telomere (T2T) assemblies of 27 distinct genotypes, encompassing all seven Citrullus species. This T2T super-pangenome has expanded the previously published reference genome, T2T-G42, by adding 399.2 Mb and 11,225 genes. Comparative analysis has unveiled gene variants and structural variations (SVs), shedding light on watermelon evolution and domestication processes that enhanced attributes such as bitterness and sugar content while compromising disease resistance. Multidisease-resistant loci from Citrullus amarus and Citrullus mucosospermus were successfully introduced into cultivated Citrullus lanatus. The SVs identified in C. lanatus have not only been inherited from cordophanus but also from C. mucosospermus, suggesting additional ancestors beyond cordophanus in the lineage of cultivated watermelon. Our investigation substantially improves the comprehension of watermelon genome diversity, furnishing comprehensive reference genomes for all Citrullus species. This advancement aids in the exploration and genetic enhancement of watermelon using its wild relatives.

Maize smart-canopy architecture enhances yield at high densities.

Increasing planting density is a key strategy for enhancing maize yields1-3. An ideotype for dense planting requires a 'smart canopy' with leaf angles at different canopy layers differentially optimized to maximize light interception and photosynthesis4-6, among other features. Here we identified leaf angle architecture of smart canopy 1 (lac1), a natural mutant with upright upper leaves, less erect middle leaves and relatively flat lower leaves. lac1 has improved photosynthetic capacity and attenuated responses to shade under dense planting. lac1 encodes a brassinosteroid C-22 hydroxylase that predominantly regulates upper leaf angle. Phytochrome A photoreceptors accumulate in shade and interact with the transcription factor RAVL1 to promote its degradation via the 26S proteasome, thereby inhibiting activation of lac1 by RAVL1 and decreasing brassinosteroid levels. This ultimately decreases upper leaf angle in dense fields. Large-scale field trials demonstrate that lac1 boosts maize yields under high planting densities. To quickly introduce lac1 into breeding germplasm, we transformed a haploid inducer and recovered homozygous lac1 edits from 20 diverse inbred lines. The tested doubled haploids uniformly acquired smart-canopy-like plant architecture. We provide an important target and an accelerated strategy for developing high-density-tolerant cultivars, with lac1 serving as a genetic chassis for further engineering of a smart canopy in maize.

Light regulates nuclear detainment of intron-retained transcripts through COP1-spliceosome to modulate photomorphogenesis.

Intron retention (IR) is the most common alternative splicing event in Arabidopsis. An increasing number of studies have demonstrated the major role of IR in gene expression regulation. The impacts of IR on plant growth and development and response to environments remain underexplored. Here, we found that IR functions directly in gene expression regulation on a genome-wide scale through the detainment of intron-retained transcripts (IRTs) in the nucleus. Nuclear-retained IRTs can be kept away from translation through this mechanism. COP1-dependent light modulation of the IRTs of light signaling genes, such as PIF4, RVE1, and ABA3, contribute to seedling morphological development in response to changing light conditions. Furthermore, light-induced IR changes are under the control of the spliceosome, and in part through COP1-dependent ubiquitination and degradation of DCS1, a plant-specific spliceosomal component. Our data suggest that light regulates the activity of the spliceosome and the consequent IRT nucleus detainment to modulate photomorphogenesis through COP1.

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 aboveground and fruits belowground. Subterranean fruit-pod development, which significantly affects peanut production, involves complex molecular mechanisms that likely require the coordinated regulation of multiple genes in different tissues. To investigate the molecular mechanisms that underlie peanut fruit-pod development, we characterized the anatomical features of early fruit-pod development and integrated single-nucleus RNA-sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) data at the single-cell level. 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 used to examine potential molecular changes unique to each cell type during pivotal stages of fruit-pod development. snRNA-seq analyses of differentially expressed genes revealed cell-type-specific insights that were not previously obtainable from transcriptome analyses of bulk RNA. For instance, we identified MADS-box genes that contributes to the formation of parenchyma cells and gravity-related genes that are present in the vascular cells, indicating an essential role for the vascular cells in peg gravitropism. Overall, our single-nucleus analysis provides comprehensive and novel information on specific cell types, gene expression, and chromatin accessibility during the early stages of fruit-pod development. This information will enhance our understanding of the mechanisms that underlie fruit-pod development in peanut and contribute to efforts aimed at improving peanut production.

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.

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.

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.

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.