Genetic and epigenetic signatures for improved breeding of cultivated blueberry.

Blueberry belongs to the Vaccinium genus and is a highly popular fruit crop with significant economic importance. It was not until the early twentieth century that they began to be domesticated through extensive interspecific hybridization. Here, we collected 220 Vaccinium accessions from various geographical locations, including 154 from the United States, 14 from China, eight from Australia, and 29 from Europe and other countries, comprising 164 Vaccinium corymbosum, 15 Vaccinium ashei, 10 lowbush blueberries, seven half-high blueberries, and others. We present the whole-genome variation map of 220 accessions and reconstructed the hundred-year molecular history of interspecific hybridization of blueberry. We focused on the two major blueberry subgroups, the northern highbush blueberry (NHB) and southern highbush blueberry (SHB) and identified candidate genes that contribute to their distinct traits in climate adaptability and fruit quality. Our analysis unveiled the role of gene introgression from Vaccinium darrowii and V. ashei into SHB in driving the differentiation between SHB and NHB, potentially facilitating SHB's adaptation to subtropical environments. Assisted by genome-wide association studies, our analysis suggested VcTBL44 as a pivotal gene regulator governing fruit firmness in SHB. Additionally, we conducted whole-genome bisulfite sequencing on nine NHB and 12 SHB cultivars, and characterized regions that are differentially methylated between the two subgroups. In particular, we discovered that the ß-alanine metabolic pathway genes were enriched for DNA methylation changes. Our study provides high-quality genetic and epigenetic variation maps for blueberry, which offer valuable insights and resources for future blueberry breeding.

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.

Corrigendum: Triticeae crop genome biology: an endless frontier.

[This corrects the article DOI: 10.3389/fpls.2023.1222681.].

Triticeae crop genome biology: an endless frontier.

Triticeae, the wheatgrass tribe, includes several major cereal crops and their wild relatives. Major crops within the Triticeae are wheat, barley, rye, and oat, which are important for human consumption, animal feed, and rangeland protection. Species within this tribe are known for their large genomes and complex genetic histories. Powered by recent advances in sequencing technology, researchers worldwide have made progress in elucidating the genomes of Triticeae crops. In addition to assemblies of high-quality reference genomes, pan-genome studies have just started to capture the genomic diversities of these species, shedding light on our understanding of the genetic basis of domestication and environmental adaptation of Triticeae crops. In this review, we focus on recent signs of progress in genome sequencing, pan-genome analyses, and resequencing analysis of Triticeae crops. We also propose future research avenues in Triticeae crop genomes, including identifying genome structure variations, the association of genomic regions with desired traits, mining functions of the non-coding area, introgression of high-quality genes from wild Triticeae resources, genome editing, and integration of genomic resources.

Control of histone demethylation by nuclear-localized α-ketoglutarate dehydrogenase.

Methylations on nucleosomal histones play fundamental roles in regulating eukaryotic transcription. Jumonji C domain-containing histone demethylases (JMJs) dynamically control the level of histone methylations. However, how JMJ activity is generally regulated is unknown. We found that the tricarboxylic acid cycle-associated enzyme α-ketoglutarate (α-KG) dehydrogenase (KGDH) entered the nucleus, where it interacted with various JMJs to regulate α-KG-dependent histone demethylations by JMJs, and thus controlled genome-wide gene expression in plants. We show that nuclear targeting is regulated by environmental signals and that KGDH is enriched at thousands of loci in Arabidopsis thaliana. Chromatin-bound KGDH catalyzes α-KG decarboxylation and thus may limit its local availability to KGDH-coupled JMJs, inhibiting histone demethylation. Thus, our results uncover a regulatory mechanism for histone demethylations by JMJs.

Molecular epigenetic mechanisms for the memory of temperature stresses in plants.

The sessile plants encounter various stresses; some are prolonged, whereas some others are recurrent. Temperature is crucial for plant growth and development, and plants often encounter adverse high temperature fluctuations (heat stresses) as well as prolonged cold exposure such as seasonal temperature drops in winter when grown in temperate regions. Many plants can remember past temperature stresses to get adapted to adverse local temperature changes to ensure survival and/or reproductive success. Here, we summarize chromatin-based mechanisms underlying acquired thermotolerance or thermomemory in plants and review recent progresses on molecular epigenetic understanding of 'remembering of prolonged cold in winter' or vernalization, a process critical for various over-wintering plants to acquire competence to flower in the coming spring. In addition, perspectives on future study in temperature stress memories of economically-important crops are discussed.

SKIP Regulates ABA Signaling through Alternative Splicing in Arabidopsis.

Abscisic acid (ABA) plays key roles in plant development and responses to abiotic stresses. A wide number of transcriptional and posttranslational regulatory mechanisms of ABA signaling are known; however, less is known about the regulatory roles of alternative splicing. In this work, we found that SKIP, a splicing factor, positively regulates ABA signaling. SKIP binds to the pre-mRNA of ABA signaling-related genes, such as PYL7, PYL8, ABI1, HAB1 and ABI5, to regulate their splicing. The precursor mRNA alternative splicing of several PYL receptors, PP2C phosphatases and ABF transcriptional factors is disrupted by the skip-1 mutation. The abnormal alternative splicing in skip-1 represses the expression of ABA-positive regulators, including PYLs and ABFs, and activates the expression of ABA-negative regulators, such as PP2Cs, which confers ABA hyposensitive phenotype of skip-1. We also found that ABA-mediated genome-wide alternative splicing and differential gene expression are changed by the skip-1 mutation. The number of the differential splicing events is increased by skip-1; however, the number of differential expressed genes in response to ABA is reduced by skip-1. Our results reveal a principle on how a splicing factor regulates ABA signaling and ABA-mediated genome-wide alternative splicing.

Structural and Functional Analyses of Hub MicroRNAs in An Integrated Gene Regulatory Network of Arabidopsis.

MicroRNAs (miRNAs) are trans-acting small regulatory RNAs that work coordinately with transcription factors (TFs) to shape the repertoire of cellular mRNAs available for translation. Despite our growing knowledge of individual plant miRNAs, their global roles in gene regulatory networks remain mostly unassessed. Based on interactions obtained from public databases and curated from the literature, we reconstructed an integrated miRNA network in Arabidopsis that includes 66 core TFs, 318 miRNAs, and 1712 downstream genes. We found that miRNAs occupy distinct niches and enrich miRNA-containing feed-forward loops (FFLs), particularly those with miRNAs as intermediate nodes. Further analyses revealed that miRNA-containing FFLs coordinate TFs located in different hierarchical layers and that intertwined miRNA-containing FFLs are associated with party and date miRNA hubs. Using the date hub MIR858A as an example, we performed detailed molecular and genetic analyses of three interconnected miRNA-containing FFLs. These analyses revealed individual functions of the selected miRNA-containing FFLs and elucidated how the date hub miRNA fulfills multiple regulatory roles. Collectively, our findings highlight the prevalence and importance of miRNA-containing FFLs, and provide new insights into the design principles and control logics of miRNA regulatory networks governing gene expression programs in plants.

MicroRNA775 regulates intrinsic leaf size and reduces cell wall pectin levels by targeting a galactosyltransferase gene in Arabidopsis.

Plants possess unique primary cell walls made of complex polysaccharides that play critical roles in determining intrinsic cell and organ size. How genes responsible for synthesizing and modifying the polysaccharides in the cell wall are regulated by microRNAs (miRNAs) to control plant size remains largely unexplored. Here we identified 23 putative cell wall-related miRNAs, termed as CW-miRNAs, in Arabidopsis thaliana and characterized miR775 as an example. We showed that miR775 post-transcriptionally silences GALT9, which encodes an endomembrane-located galactosyltransferase belonging to the glycosyltransferase 31 family. Over-expression of miR775 and deletion of GALT9 led to significantly enlarged leaf-related organs, primarily due to increased cell size. Monosaccharide quantification, confocal Raman imaging, and immunolabeling combined with atomic force microscopy revealed that the MIR775A-GALT9 circuit modulates pectin levels and the elastic modulus of the cell wall. We also showed that MIR775A is directly repressed by the transcription factor ELONGATED HYPOCOTYL5 (HY5). Genetic analysis confirmed that HY5 is a negative regulator of leaf size that acts through the HY5-MIR775A-GALT9 repression cascade to control pectin levels. These findings demonstrate that miR775-regulated cell wall remodeling is an integral determinant of intrinsic leaf size in A. thaliana. Studying other CW-miRNAs would provide more insights into cell wall biology.

The PIF1-miR408-PLANTACYANIN repression cascade regulates light-dependent seed germination.

Light-dependent seed germination is a vital process for many seed plants. A decisive event in light-induced germination is degradation of the central repressor PHYTOCHROME INTERACTING FACTOR 1 (PIF1). The balance between gibberellic acid (GA) and abscisic acid (ABA) helps to control germination. However, the cellular mechanisms linking PIF1 turnover to hormonal balancing remain elusive. Here, employing far-red light-induced Arabidopsis thaliana seed germination as the experimental system, we identified PLANTACYANIN (PCY) as an inhibitor of germination. It is a blue copper protein associated with the vacuole that is both highly expressed in mature seeds and rapidly silenced during germination. Molecular analyses showed that PIF1 binds to the miR408 promoter and represses miR408 accumulation. This in turn posttranscriptionally modulates PCY abundance, forming the PIF1-miR408-PCY repression cascade for translating PIF1 turnover to PCY turnover during early germination. Genetic analysis, RNA-sequencing, and hormone quantification revealed that PCY is necessary and sufficient to maintain the PIF1-mediated seed transcriptome and the low-GA-high-ABA state. Furthermore, we found that PCY domain organization and regulation by miR408 are conserved features in seed plants. These results revealed a cellular mechanism whereby PIF1-relayed external light signals are converted through PCY turnover to internal hormonal profiles for controlling seed germination.

Time Series RNA-seq in Pigeonpea Revealed the Core Genes in Metabolic Pathways under Aluminum Stress.

Pigeonpea is an important economic crop in the world and is mainly distributed in tropical and subtropical regions. In order to further expand the scope of planting, one of the problems that must be solved is the impact of soil acidity on plants in these areas. Based on our previous work, we constructed a time series RNA sequencing (RNA-seq) analysis under aluminum (Al) stress in pigeonpea. Through a comparison analysis, 11,425 genes were found to be differentially expressed among all the time points. After clustering these genes by their expression patterns, 12 clusters were generated. Many important functional pathways were identified by gene ontology (GO) analysis, such as biological regulation, localization, response to stimulus, metabolic process, detoxification, and so on. Further analysis showed that metabolic pathways played an important role in the response of Al stress. Thirteen out of the 23 selected genes related to flavonoids and phenols were downregulated in response to Al stress. In addition, we verified these key genes of flavonoid- and phenol-related metabolism pathways by qRT-PCR. Collectively, our findings not only revealed the regulation mechanism of pigeonpea under Al stress but also provided methodological support for further exploration of plant stress regulation mechanisms.

Arabidopsis SAURs are critical for differential light regulation of the development of various organs.

During deetiolation of Arabidopsis seedlings, light promotes the expansion of cotyledons but inhibits the elongation of hypocotyls. The mechanism of this differential regulation of cell enlargement is unclear. Our organ-specific transcriptomic analysis identified 32 Small Auxin Up RNA (SAUR) genes whose transcripts were light-induced in cotyledons and/or repressed in hypocotyls. We therefore named these SAURs as lirSAURs Both overexpression and mutation analyses demonstrated that lirSAURs could promote cotyledon expansion and opening and enhance hypocotyl elongation, possibly by inhibiting phosphatase activity of D-clade type 2C protein phosphatases (PP2C-Ds). Light reduced auxin levels to down-regulate the expression of lirSAURs in hypocotyls. Further, phytochrome-interacting factors (PIFs) were shown to directly bind the genes encoding these SAURs and differentially regulate their expression in cotyledons and hypocotyls. Together, our study demonstrates that light mediates auxin levels and PIF stability to differentially regulate the expression of lirSAURs in cotyledons and hypocotyls, and these lirSAURs further mediate the differential growth of these two organs.

RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize.

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.

Arabidopsis DET1 represses photomorphogenesis in part by negatively regulating DELLA protein abundance in darkness.

Arabidopsis De-etiolated 1 (DET1) is one of the key repressors that maintain the etiolated state of seedlings in darkness. The plant hormone gibberellic acid (GA) also participates in this process, and plants deficient in GA synthesis or signaling show a partially de-etiolated phenotype in darkness. However, how DET1 and the GA pathway work in concert in repressing photomorphogenesis remains largely unknown. In this study, we found that the abundance of DELLA proteins in det1-1 was increased in comparison with that in the wild-type plants. Mutation in DET1 changed the sensitivity of hypocotyl elongation of mutant seedlings to GA and paclobutrazol (PAC), an inhibitor of GA synthesis. However, we did not find obvious differences between det1-1 and wild-type plants with regard to the bioactive GA content or the GA signaling upstream of DELLAs. Genetic data showed that removal of several DELLA proteins suppressed the det1-1 mutant phenotype more obviously than GA treatment, indicating that DET1 can regulate DELLA proteins via some other mechanisms. In addition, a large-scale transcriptomic analysis revealed that DET1 and DELLAs play antagonistic roles in regulating expression of photosynthetic and cell elongation-related genes in etiolated seedlings. Taken together, our results show that DET1 represses photomorphogenesis in darkness in part by reducing the abundance of DELLA proteins.

SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis.

Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 5' and 3' splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance.

Arabidopsis DE-ETIOLATED1 represses photomorphogenesis by positively regulating phytochrome-interacting factors in the dark.

Arabidopsis thaliana seedlings undergo photomorphogenic development even in darkness when the function of DE-ETIOLATED1 (DET1), a repressor of photomorphogenesis, is disrupted. However, the mechanism by which DET1 represses photomorphogenesis remains unclear. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genetic analysis showed that each pif single mutant could enhance the det1-1 phenotype, and ectopic expression of each PIF in det1-1 partially suppressed the det1-1 phenotype, based on hypocotyl elongation and cotyledon opening angles observed in darkness. Genomic analysis also revealed that DET1 may modulate the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs. The observed interaction and regulation between DET1 and PIFs not only reveal how DET1 represses photomorphogenesis, but also suggest a possible mechanism by which two groups of photomorphogenic repressors, CONSTITUTIVE PHOTOMORPHOGENESIS/DET/FUSCA and PIFs, work in concert to repress photomorphogenesis in darkness.