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The 12-oxophytoeienoic acid reductase (OPR) is a kind of enzyme in the octadecanoid biosynthesis pathway that determines the biosynthesis of jasmonic acid. Although the roles of OPRs have been extensively studied in several crop plants, little is known about the biological functions of OPR-encoding genes in Capsicum annuum plants. In this study, seven OPR family genes ( CaOPR1–7) were identified from the C. annuum genome. The physical and chemical properties of CaOPR1–7 were further analyzed, including gene expression patterns, promoter elements, and chromosomal locations. The results showed that the seven CaOPR homologues could be divided into two subgroups, and CaOPR6 was highly similar to AtOPR3 in Arabidopsis. The expression of CaOPR6 was significantly induced by various stresses such as cold, salt, and pathogen infection, indicating that CaOPR6 plays important roles in response to abiotic and biotic stresses. Overall, these findings improve the understanding of the biological functions of CaOPR6 in the development of pepper fruit and stress response of pepper plants, and facilitate further studies on the molecular biology of OPR proteins in Solanaceae vegetables.

Thioredoxins (TRXs) are ubiquitous oxidoreductases and present as a multigenic family. TRXs determine the thiol redox balance, which is crucial for plants in the response to cold stress. However, limited knowledge is available about the role of TRXs in watermelon (Citrullus lanatus), which is highly sensitive to chilling stress in agricultural practice. Here, we identified 18 genes encoding 14 typical and 4 atypical TRXs from the watermelon genome, and found that ClTRX h2 localized at the plasma membrane was largely induced by chilling. Virus-induced gene silencing of ClTRX h2 resulted in watermelon plants that were more sensitive to chilling stress. We further found that ClTRX h2 physically interacted with mitogen-activated protein kinase kinase 5 (ClMPKK5), which was confirmed to phosphorylate and activate ClMPK3 in vitro, and the activation of ClMPK3 by ClMPKK5 was blocked by a point mutation of the Cys-229 residue to Ser in ClMPKK5. Additionally, ClTRX h2 inhibited the chilling-induced activation of ClMPK3, suggesting that the ClMPKK5–ClMPK3 cascade is regulated in a redox-dependent manner. We showed that ClMPK3-silenced plants had increased tolerance to chilling, as well as enhanced transcript abundances of the C-repeat/DREB binding factor (ClCBF) and cold-responsive (ClCOR) genes. Taken together, our results indicate that redox status mediated by ClTRX h2 inhibits ClMPK3 phosphorylation through the interaction between ClTRX h2 and ClMPKK5, which subsequently regulates the CBF–COR signaling pathway when submitted to chilling stress. Hence, our results provide a link between thiol redox balance and MAPK cascade signaling, revealing a conceptual framework to understand how TRX regulates chilling stress tolerance in watermelon.

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.

Maintenance of cell wall integrity is of great importance not only for plant growth and development, but also for the adaptation of plants to adverse environments. However, how the cell wall integrity is modulated under salt stress is still poorly understood. Here, we report that a nuclear-localized Agenet domain-containing protein SWO1 (SWOLLEN 1) is required for the maintenance of cell wall integrity in Arabidopsis under salt stress. Mutation in SWO1 gene results in swollen root tips, disordered root cell morphology, and root elongation inhibition under salt stress. The swo1 mutant accumulates less cellulose and pectin but more lignin under high salinity. RNA-seq and ChIP-seq assays reveal that SWO1 binds to the promoter of several cell wall-related genes and regulates their expression under saline conditions. Further study indicates that SWO1 interacts with importin ɑ IMPA1 and IMPA2, which are required for the import of nuclear-localized proteins. The impa1 impa2 double mutant also exhibits root growth inhibition under salt stress and mutations of these two genes aggravate the salt-hypersensitive phenotype of the swo1 mutant. Taken together, our data suggest that SWO1 functions together with importin ɑ to regulate the expression of cell wall-related genes, which enables plants to maintain cell wall integrity under high salinity.

The conserved histone variant H2A.Z is essential for transcriptional regulation; defense responses; and various biological processes in plants, such as growth, development, and flowering. However, little is known about how H2A.Z affects the developmental process and ripening of tomato fruits. Here, we utilized the CRISPR/Cas9 gene-editing system to generate a sl_hta9 sl_hta11 double-mutant, designated sl_h2a.z, and found that these two mutations led to a significant reduction in the fresh weight of tomato fruits. Subsequent messenger RNA (mRNA)-seq results showed that dysfunction of Sl_H2A.Z has profound effects on the reprogramming of genome-wide gene expression at different developmental stages of tomato fruits, indicating a ripening-dependent correlation between Sl_H2A.Z and gene expression regulation in tomato fruits. In addition, the expression of three genes, SlPSY1, SlPDS, and SlVDE, encoding the key enzymes in the biosynthesis pathway of carotenoids, was significantly upregulated in the later ripening stages, which was consistent with the increased contents of carotenoids in sl_h2a.z double-mutant fruits. Overall, our study reveals a role of Sl_H2A.Z in the regulation of carotenoids and provides a resource for the study of Sl_H2A.Z-dependent gene expression regulation. Hence, our results provide a link between epigenetic regulation via histone variants and fruit development, suggesting a conceptual framework to understand how histone variants regulate tomato fruit quality.

DNA methylation is critical for tuning gene expression to prevent potentially deleterious gene-silencing. The Arabidopsis DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic loci. However, the mechanism recruiting ROS1 to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor required for ROS1-mediated DNA demethylation, and targets ROS1 to specific loci. We found that AGDP3 could bind to the H3K9me2 mark by its AGD12 cassette. The crystal structure of the AGDP3 AGD12 in complex with an H3K9me2 peptide reveals the molecular basis for the specific recognition, that the dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. Interestingly, a histidine residue located in the methylysine binding aromatic cage enables AGDP3 pH-dependent H3K9me2 binding capacity. Considering the intracellular pH correlates with the histone acetylation status, our results provide the molecular mechanism for the regulation of ROS1 DNA demethylase by the gene silencing H3K9me2 mark and the potential crosstalk with active histone acetylation mark.

As a subgroup of horticultural crops, vegetable food is a kind of indispensable energy source for human beings, providing necessary nutritional components including vitamins, carbohydrates, dietary fiber, and active substances such as carotenoids and flavonoids. The developmental process of vegetable crops is not only regulated by environmental stimulations, but also manipulated by both genetic and epigenetic modifications. Epigenetic modifications are composed by several regulatory mechanisms, including DNA methylation, histone modification, chromatin remodeling, and non-coding RNAs. Among these modifications, DNA methylation functions in multiple biological pathways ranging from fundamental development to environmental stimulations by mediating transcriptomic alterations, resulting in the activation or silencing of target genes. In recent years, intensive studies have revealed that DNA methylation is essential to fruit development and ripening, indicating that the epigenome of fruit crops could be dynamically modified according to the specific requirements in the commercial production. Firstly, this review will present the mechanisms of DNA methylation, and update the understanding on active DNA demethylation in Arabidopsis thaliana. Secondly, this review will summarize the recent progress on the function of DNA methylation in regulating fruit ripening. Moreover, the possible functions of DNA methylation on controlling the expansion of edible organs, senescence of leafy vegetables, and anthocyanin pigmentation in several important vegetable crops will be discussed. Finally, this review will highlight the intractable issues that need to be resolved in the application of epigenome in vegetable crops, and provide perspectives for the potential challenges in the further studies.

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base-excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1 000s of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo. Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

Abiotic stress adversely inhibits the growth and development of plants, by changing the expression of multiple genes. Circular RNAs (circRNAs), as a class of non-coding RNAs, function in transcriptional and posttranscriptional regulation. Yet, the involvement of circRNAs in abiotic stress response is rarely reported. In this study, the participation and function of circRNAs in low-temperature (LT)-induced stress response were investigated in tomato leaves. We generated genome-wide profiles of circRNAs and mRNAs in tomato leaves grown at 25°C room temperature (RT) and 12°C LT. Our results show that 1,830 circRNAs were identified in tomato leaves in both RT and LT treatments, among which 1,759 were differentially induced by the LT treatment. We find that the identified circRNAs are mainly located at exons of genes, but less distributed at introns of genes or intergenic regions. Our results suggest that there are 383 differentially expressed circRNAs predicted to function as putative sponges of 266 miRNAs to target 4,476 mRNAs in total. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis assays indicate that multiple pathways were enriched in both differentially expressed genes induced by LT and parental genes of differentially expressed circRNAs induced by LT, revealing the key functions of circRNAs and the corresponding targeted genes in response to LT stress. Our results suggest that circRNAs may be involved in regulating metabolism (i.e., carbohydrate, amino acid, lipid, and energy), signal transduction, and environmental adaptation-related pathways and that these circRNAs were predicted to regulate the expression of transcription factors, genes in signal transduction pathways, and genes related to the Ca2+ channel through targeting the corresponding proteins, such as WRKY, NAC, cytochrome P450, and calmodulin binding protein. Taken together, our study uncovers that multiple circRNAs are isolated and differently regulated in response to LT stress and provides the resource and potential networks of circRNA–miRNA–mRNA under LT stress for further investigations in tomato leaves.

SUMMARYActive DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo.Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

Active DNA demethylation is critical for controlling the DNA methylomes in plants and mammals. However, little is known about how DNA demethylases are recruited to target loci, and the involvement of chromatin marks in this process. Here, we identify 2 components of the SWR1 chromatin-remodeling complex, PIE1 and ARP6, as required for ROS1-mediated DNA demethylation, and discover 2 SWR1-associated bromodomain-containing proteins, AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognize histone acetylation marks established by increased DNA methylation 1 (IDM1), a known regulator of DNA demethylation, redundantly facilitating H2A.Z deposition at IDM1 target loci. We show that at some genomic regions, H2A.Z and DNA methylation marks coexist, and H2A.Z physically interacts with ROS1 to regulate DNA demethylation and antisilencing. Our results unveil a mechanism through which DNA demethylases can be recruited to specific target loci exhibiting particular histone marks, providing a conceptual framework to understand how chromatin marks regulate DNA demethylation.

Brassinosteroids (BRs) are involved in the regulation of plant growth, development and stress responses. While the signalling pathways for BR-regulated plant growth and development are well studied, the mechanisms by which BRs regulate plant stress tolerance remain largely unclear. Here we showed that 24-epibrassinolide (EBR), which induced tolerance to oxidative and heat stress in tomato, was also capable of elevating the transcript levels of RBOH1, MPK1 and MPK2, increasing apoplastic H2 O2 accumulation, and enhancing activation of MPK1/2. Virus-induced gene silencing of RBOH1, MPK1, MPK2 and MPK1/2 resulted in reduced stress tolerance. Silencing of RBOH1 had no effect on the transcripts of MPK1 and MPK2 but inhibited MPK1/2 activation and H2 O2 accumulation. Silencing of either MPK1 or MPK2, on the other hand, reduced RBOH1 transcript, H2 O2 accumulation and MPK1/2 activity. BR-induced tolerance and MPK1/2 activation were compromised in RBOH1-, MPK2- and MPK1/2-silenced plants but not in MPK1-silenced plants. These results suggested that MPK2 played a more critical role than MPK1 in EBR-induced apoplastic H2 O2 accumulation. RBOH1, MPK1 and MPK2 were involved in the stress tolerance and BR-induced stress tolerance likely involved a positive feedback loop among RBOH1, H2 O2 and MPK2, leading to sustained apoplastic accumulation of H2 O2 and related signalling processes.

Brassinosteroids (BRs) are involved in the regulation of plant growth, development and stress responses. While the signalling pathways for BR-regulated plant growth and development are well studied, the mechanisms by which BRs regulate plant stress tolerance remain largely unclear. Here we showed that 24-epibrassinolide (EBR), which induced tolerance to oxidative and heat stress in tomato, was also capable of elevating the transcript levels of RBOH1, MPK1 and MPK2, increasing apoplastic H2 O2 accumulation, and enhancing activation of MPK1/2. Virus-induced gene silencing of RBOH1, MPK1, MPK2 and MPK1/2 resulted in reduced stress tolerance. Silencing of RBOH1 had no effect on the transcripts of MPK1 and MPK2 but inhibited MPK1/2 activation and H2 O2 accumulation. Silencing of either MPK1 or MPK2, on the other hand, reduced RBOH1 transcript, H2 O2 accumulation and MPK1/2 activity. BR-induced tolerance and MPK1/2 activation were compromised in RBOH1-, MPK2- and MPK1/2-silenced plants but not in MPK1-silenced plants. These results suggested that MPK2 played a more critical role than MPK1 in EBR-induced apoplastic H2 O2 accumulation. RBOH1, MPK1 and MPK2 were involved in the stress tolerance and BR-induced stress tolerance likely involved a positive feedback loop among RBOH1, H2 O2 and MPK2, leading to sustained apoplastic accumulation of H2 O2 and related signalling processes.