Cold stress is a major environmental challenge affecting the production of crops. Calcium-dependent protein kinases (CDPKs/CPKs) are crucial regulators relaying calcium (Ca) signals into cellular stress responses. However, the specific mechanisms of CPKs in regulating cold stress signaling are not well understood. In this study, through genetic, physiological and molecular biology assays, we characterized the function of CPK27 in enhancing tomato cold tolerance. We found that CPK27 stimulates flavonoid biosynthesis in a Ca-dependent manner, which in turn boosts the plant's tolerance. Tomato plants lacking CPK27 (cpk27) showed decreased flavonoid levels under cold stress, accompanied by the increased sensitivity to cold. Activated by cold stress, CPK27 accumulates within the nucleus, where it physically interacts and phosphorylates ELONGATED HYPOCOTYL 5 (HY5) protein at serine23 (S23) and S57 residues, contributing to the cold-induced accumulation of HY5 protein. HY5 directly binds to the promoter regions and stimulates the transcription of flavonoid biosynthesis genes. Further genetic analysis showed that CPK27 acts upstream of HY5, and the flavonoid biosynthesis pathway activated by CPK27 is HY5-dependent. Our study elucidates the regulatory mechanism whereby the CPK27-HY5 molecule integrates cold-triggered Ca signals with flavonoid biosynthesis pathways to confer cold stress tolerance, thereby uncovering the key strategy for cold signal transduction.

Plasmodesmata (PD) allow direct communication across the cellulosic plant cell wall, facilitating the intercellular movement of metabolites and signaling molecules within the symplast. In Arabidopsis thaliana embryos with reduced levels of the chloroplast RNA helicase ISE2, intercellular trafficking and the number of branched PD were increased. We therefore investigated the relationship between altered ISE2 expression and intercellular trafficking. Gene expression analyses in Arabidopsis tissues where ISE2 expression was increased or decreased identified genes associated with the metabolism of glucosinolates (GLSs) as highly affected. Concomitant with changes in the expression of GLS-related genes, plants with abnormal ISE2 expression contained altered GLS metabolic profiles compared with wild-type (WT) counterparts. Indeed, changes in the expression of GLS-associated genes led to altered intercellular trafficking in Arabidopsis leaves. Exogenous application of GLSs but not their breakdown products also resulted in altered intercellular trafficking. These changes in trafficking may be mediated by callose levels at PD as exogenous GLS treatment was sufficient to modulate plasmodesmal callose in WT plants. Furthermore, auxin metabolism was perturbed in plants with increased indole-type GLS levels. These findings suggest that GLSs, which are themselves transported between cells via PD, can act on PD to regulate plasmodesmal trafficking capacity.

In plants, the photoperiod sensitivity directly influences flowering time, which in turn affects latitudinal adaptation and yield. However, research into the mechanisms underlying photoperiod sensitivity, particularly those mediated by epigenetic regulation, is still in its nascent stages. In this study, we analyzed the regulation of photoperiod sensitivity in Arabidopsis thaliana. We demonstrate that the evening complex LUX ARRYTHMO (LUX) and the chromatin remodeling factor SWITCH/SUCROSE NONFERMENTING 3C (SWI3C) regulate GI locus chromatin compaction and H3K4me3 modification levels at the GIGANTEA locus under different photoperiod conditions. This mechanism is one of the key factors that allow plants to distinguish between long-day and short-day photoperiods. Our study provides insight into how the LUX-SWI3C module regulates photoperiod sensitivity at the epigenetic level.

Leaf senescence can be triggered by various abiotic stresses. Among these, heat stress emerges as a pivotal environmental factor, particularly in light of the predicted rise in global temperatures. However, the molecular mechanism underlying heat-induced leaf senescence remains largely unexplored. As a cool-season grass species, tall fescue (Festuca arundinacea) is an ideal and imperative material for investigating heat-induced leaf senescence because heat stress easily triggers leaf senescence to influence its forage yield and turf quality. Here, we investigated the role of FaNAC047 in heat-induced leaf senescence. Overexpression of FaNAC047 promoted heat-induced leaf senescence in transgenic tall fescue that was evidenced by a more seriously destructive photosystem and higher accumulation of reactive oxygen species (ROS), whereas knockdown of FaNAC047 delayed leaf senescence. Further protein-DNA interaction assays indicated that FaNAC047 directly activated the transcriptions of NON-YELLOW COLORING 1 (FaNYC1), NYC1-like (FaNOL), and STAY-GREEN (FaSGR) but directly inhibited Catalases 2 (FaCAT2) expression, thereby promoting chlorophyll degradation and ROS accumulation. Subsequently, protein-protein interaction assays revealed that FaNAC047 physically interacted with FaNAC058 to enhance its regulatory effect on FaNYC1, FaNOL, FaSGR, and FaCAT2. Additionally, FaNAC047 could transcriptionally activate FaNAC058 expression to form a regulatory cascade, driving senescence progression. Consistently, the knockdown of FaNAC058 significantly delayed heat-induced leaf senescence. Collectively, our results reveal that FaNAC047-FaNAC058 module coordinately mediates chlorophyll degradation and ROS production to positively regulate heat-induced leaf senescence. The findings illustrate the molecular network of heat-induced leaf senescence for breeding heat-resistant plants.

Excessive exposure to high light can lead to photoinhibition, which impairs photosynthetic efficiency and causes oxidative damage in plants, such as sunburn in grapevines. This study investigates the role of resveratrol (Res), a stilbenoid with antioxidant properties, in protecting plants from high light damage. We found that exposure to high light increased reactive oxygen species (ROS) accumulation and induced photoinhibition in grapevine leaves. In response, Res biosynthesis was upregulated, along with an increase in stilbene synthase (VvSTS) expression. Application of exogenous Res alleviated ROS accumulation and improved photosynthetic efficiency. Further analysis revealed that the VvHY5-VvBEE1 regulatory module plays a pivotal role in regulating VvSTS expression under high light conditions. Specifically, VvHY5 activated VvSTS expression, while VvBEE1 repressed it. Transgenic analysis showed that overexpression of VvHY5 enhanced Res production and photoprotection, whereas overexpression of VvBEE1 reduced Res levels and exacerbated light-induced damage. VvHY5 and VvBEE1 competed for binding to the VvSTS promoter, with brassinosteroids (BRs) modulating their interaction. Our findings reveal the interplay between light signaling and brassinosteroid pathways in regulating Res biosynthesis, providing insights for protecting grapevines from sunburn.

Plants have created an immense diversity of specialized metabolites to optimize fitness within a complex environment. Each plant lineage has created novel metabolites often using the classical duplication/neo-functionalization model, but this is constrained by undersampled genera and an absence of high-quality genomes. Phylogenetically resolved genomes, deeper chemical sampling and mechanistic assessment of glucosinolate diversity in the Brassicales is beginning to fill in a deeper understanding of how chemical novelty arises. This is showing that small-scale duplications like tandem or distal events may have more influence on the formation of metabolic novelty. Similarly, this is showing that gene loss is playing a significant role in metabolic diversity across the entire genera. Finally, mechanistic work is showing that the glucosinolate pathway is not a defined endpoint but is being used as a launching pad for the creation of other metabolites. In combination, this work is showing the potential in combining high-quality genomes with balanced phylogenetic sampling to develop improved models on how specialized metabolite gene evolution occurs.

Soil salinity is a severe abiotic stress that damages plant growth and development. As an antioxidant and free radical scavenger, melatonin is well known for helping plants survive abiotic conditions, including salinity stress. Here, we report that the salt-related gene MsSNAT1, encoding a rate-limiting melatonin biosynthesis enzyme, is located in the chloroplast and contributes to salinity stress tolerance in alfalfa. We found that the MsSNAT1 overexpressing alfalfa lines exhibited higher endogenous melatonin levels and increased tolerance to salt stress by promoting antioxidant systems and improving ion homeostasis. Furthermore, through a combination of transcriptome sequencing, dual-luciferase assays and transgenic analysis, we identified that the basic leucine zipper (bZIP) transcription factor, MsbZIP55, is associated with salt response and MsSNAT1 expression. EMSA analysis and ChIP-qPCR uncovered that MsbZIP55 can recognize and directly bind to the MsSNAT1 promoter in vitro and in vivo. MsbZIP55 acts as a negative regulator of MsSNAT1 expression, thereby reducing melatonin biosynthesis. Morphological analysis revealed that overexpressing MsbZIP55 conferred salt sensitivity to transgenic alfalfa through a higher Na/K ratio and lower antioxidant activities, which could be alleviated by applying exogenous melatonin. Silencing of MsbZIP55 by RNA interference in alfalfa resulted in higher expression of MsSNAT1 and promoted salt tolerance by enhancing the antioxidant system enzyme activities and ion homeostasis. Our findings indicate that the MsbZIP55-MsSNAT1 module plays a crucial role in regulating melatonin biosynthesis in alfalfa while facilitating protection against salinity stress. These results shed light on the regulatory mechanism of melatonin biosynthesis related to the salinity stress response in alfalfa.

Plant roots play myriad roles that include foraging for resources in complex soil environments. Within this highly dynamic soil environment roots must sense, interact with, and acclimate to factors such as water availability, microbiota, and heterogeneous distribution of nutrients. To aid their acclimation, roots alter their growth and development to optimize their architecture and actively regulate the physical, chemical, and biological properties of their rhizosphere. Understanding the complex interactions between roots and rhizosphere is critical for designing future crops with improved root traits better adapted to diverse and challenging soil conditions. However, studying roots and their interactions with soil under real-world conditions presents significant challenges. Addressing these challenges demands developing realistic laboratory-based model systems and innovative field-based root imaging techniques. Our review surveys the current knowledge and recent advances in understanding root-environment interactions while proposing future solutions to study roots under more "real-life" soil conditions.

Nitrate is the main source of nitrogen in plants. Nitrate stimulation causes changes in plant secondary metabolites, including anthocyanins. However, the molecular mechanism underlying how nitrate regulates anthocyanin biosynthesis remains unclear. In this study, we identified a nitrate response factor MdLBD36 in apple. This factor positively regulated nitrate deficiency-induced anthocyanin biosynthesis by promoting the transcriptional activity of MdABI5, an important regulator of anthocyanins, and directly activated MdABI5 expression. The E3 ubiquitin ligase MdBRG3 promoted the ubiquitinated degradation of MdLBD36 to reduce anthocyanin biosynthesis under nitrate-sufficient conditions. Nitrate deficiency-activated MdMPK7 maintained the stimulating effect of MdLBD36 on anthocyanin biosynthesis by counteracting the MdBRG3-mediated degradation of MdLBD36. Nitrate coordinated gibberellin (GA) signaling to regulate anthocyanin biosynthesis. The GA signaling repressor MdRGL2a contributed to MdLBD36-promoted anthocyanin biosynthesis by enhancing the MdLBD36-MdABI5 interaction and increasing the MdLBD36 transcriptional activation of MdABI5. In summary, our results elucidate the molecular framework of the coordinated regulation of the nitrate signaling response and anthocyanin biosynthesis by ubiquitination and phosphorylation. This study revealed the cross talk between nitrate and GA signaling in the regulation of anthocyanin biosynthesis and provides references for an in-depth exploration of the nitrate signal transduction pathway and its interactions with hormones.

Flavonoids are polyphenolic secondary metabolites in tomato fruit with important roles in nutritional quality. Dissecting the transcriptional regulatory network modulating flavonoid metabolism is the first step to improve the nutritional quality of tomato fruits through molecular breeding technology. In this study, we identified a transcription factor SlbHLH95 as a key regulator in flavonoid metabolism through analysis of the MicroTom Metabolic Network (MMN) data set. Functional analyses revealed that knockout of SlbHLH95 increased the accumulation of naringenin, while the levels of rutin and nictoflorin decreased. Conversely, overexpression of SlbHLH95 resulted in an opposite pattern of accumulation of flavonoids. Transactivation assays showed that SlbHLH95 positively activated the expression of SlF3H and SlFLS, two key enzyme-encoding genes in the flavonoid pathway, while repressing the expression of SlCHS1. Electrophoretic mobility shift assays (EMSA) demonstrated that SlbHLH95 could directly bind to the promoters of SlF3H and SlFLS, although it could not bind to the promoter of SlCHS1. Furthermore, SlbHLH95 interacted with the transcription factor SlMYB12 and coordinately regulated the expression of SlF3H and SlFLS. Beyond its role in flavonoid metabolism, SlbHLH95 positively regulated the grey mould resistance in tomato fruits by repressing SlBG10. Overall, our findings revealed the important role of bi-functional SlbHLH95 in flavonoid metabolism and grey mould resistance in tomato fruits by acting as both a transcriptional activator and a repressor. This study provides new insights into strategies for improving fruit quality and enhancing fruit disease resistance through targeted genetic modulation.

Anthocyanin biosynthesis and accumulation determines the colour of tuber flesh in potato (Solanum tuberosum) and influences nutritional quality. However, the regulatory mechanism behind anthocyanin biosynthesis in potato tuber flesh remains unclear. In this study, we identified the Pigmented tuber flesh (Pf) locus through a genome-wide association study using 135 diploid potato landraces. Genome editing of two tandem R2R3 MYB transcription factor genes, StMYB200 and StMYB210, within the Pf locus demonstrated that both genes are involved in anthocyanin biosynthesis in tuber flesh. Molecular and biochemical assays revealed that StMYB200 promotes StMYB210 transcription by directly binding to a 1.7-kb insertion present in the StMYB210 promoter, while StMYB210 also regulates its own expression. Furthermore, StMYB200 and StMYB210 both activated the expression of the basic helix-loop-helix transcription factor gene StbHLH1 and interacted with StbHLH1 to regulate anthocyanin biosynthesis. An analysis of the StMYB210 promoter in different diploid potato accessions showed that the 1.7-kb insertion is associated with flesh colour in potato. These findings reveal the genetic and molecular mechanism by which the Pf locus regulates anthocyanin accumulation in tuber flesh and provide an important reference for breeding new potato varieties with colourful flesh.

Global metabolic and transcriptional reprogramming is a common event in plant abiotic stress responses, however, the relevant molecular mechanisms remain largely unknown. Here, we characterized the physiological function and molecular mechanism for the rice UGT706F1. We found that UGT706F1 can be potently induced by high temperature. Its overexpression can markedly enhance the heat tolerance of rice through improving the capacity of scavenging reactive oxygen species, whereas its functional deletion results in heat sensitivity in rice. To investigate the regulatory mechanism of UGT706F1 in response to high temperature, we carried out extensive screening of the in vitro enzymatic activity of UGT706F1 and discovered that UGT706F1 exhibits broad-spectrum activity toward flavonoid compounds. Through targeted flavonoid metabolomics analysis, we further revealed that the overexpression of UGT706F1 elevated the content of diverse flavonoids and flavonoid glycosides in rice. Subsequently, via transcriptome analysis, we found that following heat treatment, the overexpression of UGT706F1 was capable of enhancing the transcriptional activity of those genes including the flavonoid synthases, heat shock factors, heat shock proteins, glutathione S-transferase, and various antioxidant enzymes. Furthermore, we identified an R2R3 MYB-type transcription factor MYB61 and demonstrated that MYB61 could directly bind the promoter of UGT706F1 and activate the transcription of UGT706F1. The overexpression of MYB61 also enhanced the heat tolerance and increased flavonoid glycosides. Overall, this study unveiled a novel pathway of the plant heat tolerance response mediated by MYB61-UGT706F1 module and identified a new UGT player for the metabolic and transcriptional regulation under high-temperature circumstance.

Anthocyanins, the important antioxidants and signaling molecules, are natural polyphenolic compounds widely present in plants and essential for plant defense. However, little is known about the mechanisms underlying plant anthocyanin accumulation in relation to drought stress. This study reveals that drought stress induces significant anthocyanin accumulation in Rosa chinensis, alongside an increase in the expression of the MYB transcription factor (TF) gene RcMYB75 and the glutathione S-transferase (GST) gene RcGSTFL11. When overexpressed, RcMYB75 markedly increases anthocyanin contents in both roses and tobaccos; conversely, reducing its expression significantly lowers anthocyanin contents in rose petals. RcGSTFL11 was confirmed as an anthocyanin transporter and overexpression of RcGSTFL11 can restore the anthocyanin-deficient phenotype in the Arabidopsis tt19 mutant. Transgenic roses overexpressing RcGSTFL11 exhibit enhanced anthocyanin accumulation, while those with downregulated RcGSTFL11 have reduced contents. Transcriptomic analysis indicates that RcMYB75 upregulates the expression of key genes in the anthocyanin biosynthetic pathway and the anthocyanin transport gene RcGSTFL11. Ultimately, we also found that anthocyanin accumulation in these transgenics further enhances plant resistance to drought stress. Taken together, RcMYB75 and RcGSTFL11 promote the synthesis and transport of anthocyanins and play a key role in the feedforward loop responding to drought stress in roses. This study provides insights into the molecular mechanisms by which MYB TFs contribute to anthocyanin biosynthesis and transport, as well as the adaptive strategies of roses in response to drought stress.

Water availability is a major determinant of crop production, and rising temperatures from climate change are leading to more extreme droughts. To combat the effects of climate change on crop yields, we need to develop varieties that are more tolerant to water-limited conditions. We aimed to determine how diverse crop types (winter/spring oilseed, tuberous, and leafy) of the allopolyploid Brassica napus, a species that contains the economically important rapeseed oilseed crop, respond to prolonged water limitation. We exposed plants to an 80% reduction in water and assessed growth and color on a high-throughput phenotyping system over 4 weeks and ended the experiment with tissue collection for a time course transcriptomic study. We found an overall reduction in growth across cultivars but to varying degrees. Diel transcriptome analyses revealed significant accession-specific changes in time-of-day regulation of photosynthesis, carbohydrate metabolism, and sulfur metabolism. Interestingly, there was extensive variation in which homoeologs from the two parental subgenomes responded to water limitation across crop types that could be due to differences in regulatory regions in these allopolyploid lines. Follow-up experiments on select cultivars confirmed that plants maintained photosynthetic health during the prolonged water limitation while slowing growth. In two cultivars examined, we found significant time of day changes in levels of glucosinolates, sulfur- and nitrogen -rich specialized metabolites, consistent with the diel transcriptomic responses. These results suggest that these lines are adjusting their sulfur and nitrogen stores under water-limited conditions through distinct time of day regulation.

DNA methylation plays an essential role in plant growth and development, however, its specific influence on maize kernel development remains uncertain. In this study, we investigated the gene responsible for the maize kernel mutant smk313 and identified it as the DNA methyltransferase ZmMET1. The smk313 mutants displayed a distinct small kernel phenotype and exhibited developmental abnormalities in the basal endosperm transfer layer (BETL), the endosperm adjacent to the scutellum cell (EAS), and the starchy endosperm cells (SEs). Compared with that of the wild-type (WT), we found that the mutants had lower CG methylation density across the whole genome through whole genome methylation sequencing (WGBS), and there were many accessible chromatin regions (ACRs) through assay for targeting accessible chromatin with high-throughout sequencing (ATAC-seq). Combining these findings with the transcriptome analysis revealed a cascade of effects caused by the loss of ZmMET1 function. This deficiency leads to alterations in genomic methylation and chromatin accessibility, which in turn influences the expression of genes related to starch and protein synthesis, as well as material transport processes. These alterations were consistent with the delayed development and dysplasia observed in EAS and BETLs of smk313 kernels. Consequently, our investigation emphasizes the vital role of ZmMET1 in maize seed development.

Plant CSN5 is widely recognized as the subunit of the COP9 signalosome and CSN5 is mainly involved in plant growth and development, and tolerance to biotic and abiotic stresses. However, the molecular mechanism of CSN5 regulating anthocyanin biosynthesis in plants is still largely unknown. Here, we identified FvCSN5 from the woodland strawberry yeast two-hybrid library using the anthocyanin pathway inhibitor MYB1 as bait. We demonstrated the interaction of FvCSN5 and FvMYB1 by H2Y, Pull-down, LCI, and BiFC assays. FvCSN5 was expressed in all test tissues and localized in the nucleus and cytosol with self-activation activity. Stable overexpression of FvCSN5 in woodland strawberries reduced anthocyanin accumulation in fruits. The protein level of FvMYB1 greatly decreased in overexpressing FvCSN5 plants compared with wild-type plants. Protein degradation assay and MG-132 treatment (a proteasome inhibitor blocking 26S proteasome activity) revealed FvCSN5 degraded FvMYB1 through the ubiquitination pathway. In addition, FvCSN5 also interacted with the anthocyanin activator FvBBX20 and FvBBX20 could be degraded by FvCSN5. Moreover, transient expression analysis showed the expression of anthocyanin biosynthetic genes FvCHS and FvF3H was greatly increased and decreased when FvCSN5 was co-expressed with FvMYB1 and FvBBX20, respectively. These results indicate that FvMYB1-FvCSN5-FvBBX20 is a novel ternary complex that regulates anthocyanin biosynthesis by the ubiquitination pathway.

Vitamin E (VE) is essential for plants and animals. Rapeseed oil is rich in α-tocopherol (α-T), which is the most bioactive form of VE in human body. This study demonstrated that VE in rapeseed seeds was mainly controlled by embryo genotype through incomplete diallel hybridization. By genome-wide association study, the QTL-qVE.C02 associated with VE and α-T contents was detected in a Brassica napus association population, and the phenotypic contribution rate was up to 18.71%. BnaC02.VTE4, encoding gama-tocopherol methyltransferase, was proved as the target gene of qVE.C02 by genetic complementation. Two BnaC02.VTE4 haplotypes were identified in the population. Compared with BnaC02.VTE4, a point mutation from A to G at the 3' splicing site of the second intron were found in BnaC02.VTE4, resulting in alternative splicing and early termination of translation. HapL, the site-directed mutagenesis fragment of BnaC02.VTE4, was introduced into Arabidopsis vte4 mutant and 8S088 (a BnaC02.VTE4 accession), and the contents of VE and α-T in atvte4-4 and 8S088 seeds were increased by 90.10% to 307.29%. These demonstrated the point mutation as the causal for the difference in VE biosynthesis in rapeseed. Further, this variation also led to the significant difference in glucosinolate content between BnaC02.VTE4 and BnaC02.VTE4 accessions. Multi-omics analysis suggested that the expression of some genes and the accumulation of several metabolites related to the glucosinolate biosynthesis pathway were significantly increased in BnaC02.VTE4 group. Moreover, by functional marker identification, the BnaC02.VTE4 was found to be selected during domestication. Our findings offered promising opportunities for enhancing rapeseed quality traits.

High temperature reduces anthocyanin accumulation in various horticultural plants. However, the molecular mechanisms underlying the high-temperature-induced reduction of anthocyanin in grape (Vitis vinifera) remain poorly understood. In this study, VvMYB44-1 was identified as a transcriptional repressor of anthocyanin biosynthesis in grape berries, and its gene expression was strongly induced by high-temperature treatment. Overexpression of VvMYB44-1 inhibited anthocyanin accumulation in both grape berries and tobacco (Nicotiana tabacum) by repressing the transcription of the anthocyanin biosynthesis genes dihydroflavonol-4-reductase (VvDFR) and UDP-glucose flavonoid-3-O-glucosyltransferase (VvUFGT). Furthermore, the interaction between VvMYB44-1 and VvWDR2 competitively inhibited the formation of the MYB-bHLH-WD40 (MBW) activation complex and weakened the transcriptional activity of the complex, thereby decreasing anthocyanin accumulation. Additionally, VvMYB44-1 facilitated cytokinin (CK) accumulation by upregulating the expression of the CK synthesis gene lonely guy 8 (VvLOG8) and inhibiting the CK degradation gene CK oxidase 4(VvCKX4), thus contributing to CK-mediated anthocyanin inhibition in grape berries. Moreover, the inhibitory effect of VvMYB44-1 on anthocyanin biosynthesis and its downstream target genes was weakened with the deletion of the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, indicating that the EAR motif is indispensable for the inhibitory effect of VvMYB44-1 on anthocyanin biosynthesis in grapes. These results provide insights into the regulatory network of VvMYB44-1 in high-temperature-mediated anthocyanin biosynthesis in grapes.

Chlorophylls and carotenoids are 2 pivotal photosynthetic pigments directly influencing the economic value of pepper (Capsicum annuum L.) fruits. However, the coordinated regulatory mechanisms governing the accumulation of both chlorophylls and carotenoids during pepper fruit development remain elusive. In this study, pepper B-box 10 (CaBBX10), a candidate hub transcription factor, was found to play dual roles in the early development of pepper fruit. CaBBX10 virus-induced gene silencing and overexpression experiments demonstrated that the encoded transcription factor promotes both chlorophyll and carotenoid accumulation in pepper fruit. Further comprehensive analyses showed that CaBBX10 directly binds to the promoter of magnesium chelatase subunit D subunit (CaCHLD) and phytoene synthase 1 (CaPSY1), thereby activating their expression in the chlorophyll and carotenoid biosynthesis pathways, respectively. Additionally, the photomorphogenic factor CaCOP1 was found to physically interact with CaBBX10 and lead to its degradation. Therefore, CaBBX10 may serve as a critical link connecting chlorophyll and carotenoid biosynthesis to light signaling. Altogether, our findings reveal a mechanism for the complex transcriptional regulation that simultaneously promotes chlorophyll and carotenoid accumulation in pepper fruit.

Wild citrus (Citrus L.) exhibits high disease resistance accompanied by high-acidity fruit, whereas cultivated citrus produces tastier fruit but is more susceptible to disease. This is a common phenomenon, but the underlying molecular mechanisms remain unknown. Citrus PH4 (CitPH4) is a key transcription factor promoting citric acid accumulation in fruits. Accordingly, CitPH4 expression decreased during citrus domestication, along with a reduction in citric acid levels. Here, we demonstrate that a CitPH4-knockout mutant exhibits an acidless phenotype and displays substantially lower resistance to citrus diseases. Metabolome and transcriptome analyses of CitPH4-overexpressing citrus callus, Arabidopsis, and CitPH4-knockout citrus fruits revealed that quercetin, pipecolic acid (Pip), and N-hydroxypipecolic acid (NHP) are pivotal defense-related metabolites. Application of quercetin and Pip inhibited the growth of Xcc and Penicillium italicum, while NHP inhibited the growth of P. italicum and Huanglongbing. Biochemical experiments demonstrated that CitPH4 enhances the expression of quercetin and NHP biosynthesis genes by binding to their promoters. Moreover, Pip and quercetin contents were positively associated with citric acid content in the pulp of fruits from natural citrus populations. Finally, the heterologous expression of CitPH4 in Arabidopsis promoted the expression of stress response genes and enhanced its resistance to the fungal pathogen Botrytis cinerea. The overexpression of CitPH4 in tobacco (Nicotiana tabacum) enhanced disease resistance. This study reveals the mechanism by which CitPH4 regulates disease resistance and fruit acidity, providing a conceptual strategy to control fruit acidity and resistance to devastating diseases.

Patterning of DNA methylation in eukaryotic genomes is controlled by de novo methylation, maintenance mechanisms and demethylation pathways. In Arabidopsis thaliana, DNA demethylation enzymes are clearly important for shaping methylation patterns, but how they are regulated is poorly understood. Here we show that the targeting of histone H3 lysine four trimethylation (H3K4me3) with the catalytic domain of the SDG2 histone methyltransferase potently erased DNA methylation and gene silencing at FWA and also erased CG DNA methylation in many other regions of the Arabidopsis genome. This methylation erasure was completely blocked in the ros1 dml2 dml3 triple mutant lacking DNA demethylation enzymes, showing that H3K4me3 promotes the active removal of DNA methylation. Conversely, we found that the targeted removal of H3K4me3 increased the efficiency of targeted DNA methylation. These results highlight H3K4me3 as a potent anti-DNA methylation mark and also pave the way for development of more powerful epigenome engineering tools.

Linalool not only is one of characteristic flavour volatiles of tea, contributing to floral aroma, but also a kind of defensive compounds, playing essential roles in resistance against biotic/abiotic stresses. Although the linalool synthases have been identified, much is unknown about the regulation mechanism in tea plants. We identified two pairs of MYB paralogs as linalool biosynthesis activators, in which one pair (CsMYB148/CsMYB193) specifically expressed in flowers, and another (CsMYB68/CsMYB147) highly expressed in flowers, leaves, fruits and roots. These activators interacted with CsMYC2 to form MYC2-MYB complexes to regulate linalool synthase. While Jasmonate ZIM-domain (JAZ) proteins served as the linalool biosynthesis repressors by interfering MYC2-MYB complex. Further, we found that the transcripts of CsMYB68/CsMYB147 were significantly upregulated by jasmonic acid (JA) to improve linalool products during tea processing and that linalool pathway may as one of the downstream pathways of JA signalling and DNA methylation processes to participate in cold resistance. Under cold stress, JA signalling was activated to elevate the abundance of MYC-MYB complexes; meanwhile, DNA demethylation was also activated, leading to declining methylation levels and increasing transcripts of CsMYB68/CsMYB147. Our study provides a new insight into synergistically improving tea quality and tea plant resistance.

Lettuce is one of the most important vegetables worldwide. Bolting time is an important agronomic trait in lettuce production. Premature bolting reduces crop quality and marketability. Here, we genetically clone the LsBLH2 gene controlling bolting time in lettuce. LsBLH2 encodes a BEL1-like homeodomain protein. In the late bolting parent, the LsBLH2 had a 1-bp deletion in exon 1 which leads to a premature stop codon. CRISPR/cas9 knocking out and complementary tests showed that the loss-of-function of LsBLH2 delays bolting in lettuce. ChIP-seq, gene expression and phytohormone analysis showed that LsBLH2 regulates the gibberellin (GA) biosynthesis and metabolism. LsBLH2 binds to the promoter of the LsGA20ox1 and LsGA2ox8 and regulates their expression, leading to the bioactive GA accumulation during the vegetative-to-reproductive phase transition. Both LsOFP6 and LsKNAT3 interact with LsBLH2 and regulate bolting in a LsBLH2-dependent manner. LsOFP6 promotes, while LsKNAT3 suppresses the effects of LsBLH2 on GA biosynthesis during the transition and rosette stage in lettuce, respectively. In summary, the LsBLH2-LsOFP6-LsKANT3 module orchestrates bioactive GA accumulation to regulate bolting in lettuce, which provides insight into the bolting development process and offers new approaches for lettuce breeding to prevent premature bolting.

Jasmonic acid (JA) and gibberellin (GA) coordinate many aspects of plant growth and development, including anthocyanin biosynthesis. However, the crossover points of JA and GA signals and the pathways through which they interact to regulate anthocyanin biosynthesis are poorly understood. Here, we investigated the molecular mechanism by which the zinc finger protein (ZFP) transcription factor Malus domestica ZFP7 (MdZFP7) regulates anthocyanin biosynthesis by integrating JA and GA signals at the transcriptional and post-translational levels. MdZFP7 is a positive regulator of anthocyanin biosynthesis, which fulfills its role by directly activating the expression of MdMYB1 and enhancing the transcriptional activation of MdWRKY6 on the target genes MdDFR and MdUF3GT. MdZFP7 integrates JA and GA signals by interacting with the JA repressor apple JASMONATE ZIM-DOMAIN2 (MdJAZ2) and the GA repressor apple REPRESSOR-of-ga1-3-like 3a (MdRGL3a). MdJAZ2 weakens the transcriptional activation of MdMYB1 by MdZFP7 and disrupts the MdZFP7-MdWRKY6 interaction, thereby reducing the anthocyanin biosynthesis promoted by MdZFP7. MdRGL3a contributes to the stimulation of anthocyanin biosynthesis by MdZFP7 by sequestering MdJAZ2 from the MdJAZ2-MdZFP7 complex. The E3 ubiquitin ligase apple BOI-related E3 ubiquitin-protein ligase 3 (MdBRG3), which is antagonistically regulated by JA and GA, targets the ubiquitination degradation of MdZFP7. The MdBRG3-MdZFP7 module moves the crosstalk of JA and GA signals from the realm of transcriptional regulation and into the protein post-translational modification. In conclusion, this study not only elucidates the node-role of MdZFP7 in the integration of JA and GA signals, but also describes the transcriptional and post-translational regulatory network of anthocyanin biosynthesis with MdZFP7 as the hub.

Anthracnose, caused by Colletotrichum gloeosporioides, is a significant fungal disease that affects poplar trees globally, leading to reduced yields and substantial economic losses. Proanthocyanidins (PAs) play a key role in resistance to fungal pathogens; however, the mechanisms by which PAs mediate resistance to anthracnose in poplar remain poorly understood. In this study, we identified PopbZIP2, a transcription factor-encoding gene that was initially expressed in infected leaves and subsequently in uninfected leaves in response to C. gloeosporioides infection. As a transcriptional activator, PopbZIP2 can bind to the promoters of target genes PopGRF3 and PopAPA1, increasing proanthocyanidin levels in cells to enhance defense against pathogens. It is noteworthy that the PopAPA1 protein can directly inhibit pathogen growth. We further demonstrated that PopMYB4 can interact with PopbZIP2, reducing its promoter binding activity and thereby inhibiting the expression of PopGRF3 and PopAPA1. Overexpression of PopMYB4 led to sensitivity to the pathogen C. gloeosporiodes. Under normal conditions, the soluble and insoluble proanthocyanidin contents in PopMYB4 transgenic plants were significantly lower compared to the control. The dual regulation of immune responses by the PopMYB4-PopbZIP2 module unveils a novel regulatory mechanism in Populus, enhancing our understanding of the complex networks governing immune responses.

Histones are far more than just the basic units of chromatin. Posttranslational modifications of histone tails have emerged as important regulatory mechanisms for diverse biological processes, including genome organization, gene expression, transposable element suppression, development, and environmental responses. This field is expanding rapidly with the development of new technologies and growing interest from both the basic and translational research communities. The past two decades have witnessed tremendous progress in our understanding of the complex, multilayered regulation and actions of histone modifications in plants. This review summarizes the characteristics, localization, and molecular functions of histone modifications with an emphasis on the well-studied marks in . We further discuss their functions in developmental transitions and environmental responses as well as their contributions to epigenomic diversity and plasticity. By highlighting the functions and fundamental mechanisms of epigenetic modifications in model plants, this review underscores the potential to harness epigenetic regulation for agricultural improvement.

Carotenoids are vital photosynthetic pigments for plants. Golden2-like transcription factors (GLKs) are widely recognized as major regulators of Chl biosynthesis and chloroplast development. However, despite GLKs being subjected to intensive investigations, whether GLKs directly regulate carotenoid biosynthesis and the molecular mechanisms by which GLKs transcriptionally activate their target genes remain unclear. Here, we report that GLKs directly regulate carotenoid biosynthesis and activate their target genes in a G-box binding factor (GBF)-dependent manner in Arabidopsis. Both in vitro and in vivo studies reveal that GLKs physically interact with GBFs to activate transcription of phytoene synthase (PSY), the gene encoding a rate-limiting enzyme for carotenoid biosynthesis. While GLKs possess transactivation activity, they depend on GBFs to directly bind to the G-box motif to modulate PSY expression. Loss of GBFs impairs GLK function in regulating carotenoid and Chl biosynthesis. Since the G-box motif is an enriched motif in the promoters of GLK-regulated genes, the GLK-GBF regulatory module likely serves as a common mechanism underlying GLK-regulated photosynthetic pigment biosynthesis and chloroplast development. Our findings uncover a novel regulatory machinery of carotenoid biosynthesis, discover a molecular mechanism of transcriptional regulation by GLKs, and divulge GLKs as important regulators to coordinate photosynthetic pigment synthesis in plants.

The snow lotus species Saussurea involucrata (Kar. & Kir.) Sch.Bip., an endangered traditional Chinese herb, belongs to a genus of the Asteraceae family. Syringin present in S. involucrata stands as one of the predominant bioactive compounds. However, the biosynthetic pathway of syringin remains largely elusive. Here, S. involucrata suspension cell culture was subjected to methyl jasmonate (MeJA) treatment, which stimulated the synthesis of syringin, increasing its content by up to 3.9-fold. Comparative transcriptome analysis revealed that genes involved in syringin biosynthesis were generally upregulated in response to MeJA. Furthermore, two candidate UDP-glycosyltransferase genes, SiUGT72BZ2 and SiUGT72CY1, were identified through phylogenetic tree and expression profiling analyses. Overexpression of SiUGT72BZ2 (BZ2_OE) and SiUGT72CY1 (CY1_OE) in S. involucrata suspension cell cultures led to 15.2- and 5.9-fold higher syringin levels than empty vector control cultures, respectively. Notably, upregulation of SiUGT72BZ2 enhanced the biosynthesis of coniferin as well. In contrast, only trace amounts of coniferin were present in control and CY1_OE cell cultures. Subsequent anti-inflammatory assays using lipopolysaccharide (LPS)-stimulated RAW264.7 cells demonstrated that the extracts from these cell cultures possessed remarkable anti-inflammatory properties. Most strikingly, the BZ2_OE cultures exhibited superior anti-inflammatory effects compared to the control and CY1_OE. In conclusion, our research has not only identified the key enzymes in syringin synthesis but also, through genetic engineering, has generated novel cell culture resources enriched with syringin and coniferin, and enhanced anti-inflammatory activities, highlighting the potential of S. involucrata cell culture as an alternative for wild snow lotus resources.

The pivotal role of ethylene (ETH) in fruit ripening has been extensively studied; however, the function of brassinosteroids (BRs) in regulating fruit ripening remains poorly understood. Specifically, the mechanism by which BRs interact with ETH to affect kiwifruit (Actinidia deliciosa) ripening is unclear. Our research showed that 2 genes encoding transcription factors, AdNAC3 and AdMYB19, and the fruit softening gene AdEXP3 (encoding a cell wall expansion protein, expansin 3) were upregulated by ETH and downregulated by BRs. Furthermore, AdNAC3 and AdMYB19 positively regulated the activity of the AdEXP3 promoter, and AdNAC3 positively regulated the promoter activity of AdMYB19. The physical interaction between AdNAC3 and the B-box-type zinc finger protein AdBBX32 affected fruit ripening. Transient overexpression and silencing experiments revealed that ETH upregulated and BRs downregulated the expression of AdNAC3 and AdMYB19, thereby regulating the expression level of AdEXP3 and participating in pectin degradation. Stable transformation of AdNAC3 in tomato fruits accelerated fruit color change and promoted fruit ripening. These results indicate that AdNAC3 and AdMYB19 are involved in the hormone interaction between BRs and ETH in regulating kiwifruit ripening, providing insights into the molecular mechanisms underlying the crosstalk between BRs and ETH.

[This corrects the article DOI: 10.1093/hr/uhab077.].

Soil salinity is a destructive environmental factor that inhibits plant growth and crop yield. Applying nitrogen fertilizer is a practical method to enhance salt tolerance. However, the underlying mechanisms remain largely unknown. Here, we demonstrated that NO3--enhanced salt tolerance in rice (Oryza sativa L.) seedlings is mediated by nitrate reductase (NR)-dependent nitric oxide (NO) production. Seedlings grown in nitrate condition (N) exhibited much greater salt tolerance compared with those grown in ammonium nitrate and ammonium (A) conditions, a pattern also observed in the MADS-box transcription factor 27 (mads27) mutant. NR activity was highly induced by NO3- under both normal and salt stress conditions. Only the double mutant nr1/2 and the triple mutant nr1/2/3 displayed a dramatic reduction in salt tolerance. Application of tungstate suppressed salt tolerance of wild-type seedlings but not the triple mutants. Furthermore, both NO3--enhanced salt tolerance and salt-induced NO production were totally blocked in triple mutants. However, treatment with exogenous sodium nitroprusside (an NO donor) significantly enhanced salt tolerance in both Nipponbare (NIP) and the triple mutants. Antioxidant enzyme activities in shoots were significantly inhibited in the triple mutants when compared with NIP. Furthermore, expression of OsAKT1 was specifically induced by NO3- but was inhibited in the roots of triple mutants, resulting in a lower potassium/sodium ratio in NR triple mutants. Our results revealed that NO3--conferred salt tolerance is mediated by NR-dependent NO production in rice seedlings.

Cold stress adversely affects crop growth and productivity. Resolving the genetic basis of freezing tolerance is important for crop improvement. Wild potato (Solanum commersonii) exhibits excellent freezing tolerance. However, the genetic factors underlying its freezing tolerance remain poorly understood. Here, we identified flavonoid 3'-hydroxylase (F3'H), a key gene in the flavonoid biosynthesis pathway, as highly expressed in S. commersonii compared with cultivated potato (S. tuberosum L.). Loss of ScF3'H function impaired freezing tolerance in S. commersonii, while ScF3'H overexpression in cultivated potato enhanced its freezing tolerance. Metabolic analysis revealed that F3'H generates more downstream products by adding hydroxyl (-OH) groups to the flavonoid ring structures. These flavonoids enhance reactive oxygen species scavenging, thereby contributing to freezing tolerance. Furthermore, the W-box element in the F3'H promoter plays a critical role in cold responses. Cold-induced transcription factor ScWRKY41 directly binds to the ScF3'H promoter region and recruits histone acetyltransferase 1 (ScHAC1), which enhances histone acetylation at the F3'H locus and activates its transcription. Overall, we identified the cold-responsive WRKY41-F3'H module that enhances freezing tolerance by augmenting the antioxidant capacity of flavonoids. This study reveals a valuable natural gene module for breeding enhanced freezing tolerance in potato and other crops.

Plant flavonols act primarily as ultraviolet radiation absorbers, reactive oxygen species scavengers, and phytoalexins, and they contribute to biotic and abiotic stress tolerance in plants. Banana (Musa acuminata), an herbaceous monocot and important fruit crop, accumulates flavonol derivatives in different organs, including the edible fruit pulp. Although flavonol content varies greatly in different organs, the molecular mechanisms involving transcriptional regulation of flavonol synthesis in banana are not known. Here, we characterized three SG7-R2R3 MYB transcription factors (MaMYBFA1, MaMYBFA2, and MaMYBFA3) and heat shock transcription factor (MaHSF11), to elucidate the molecular mechanism involved in transcriptional regulation of flavonol biosynthesis in banana. MaMYBFA positively regulates flavonol synthase 2 (MaFLS2) and downregulates MaFLS1. We show these transcription factors to be weak regulators of flavonol synthesis. Overexpression of MaHSF11 enhances flavonol contents, particularly that of myricetin, and promotes flavonol B-ring hydroxylation, which contributes to the diversity of flavonol derivatives. MaHSF11 directly interacts with the MaFLS1 and flavonoid 3',5'-hydroxylase1 (MaF3'5'H1) promoters, both in vitro and in vivo. MaHSF11 activates the expression of MaDREB1 directly, which is known to promote cold and chilling tolerance in banana fruit. Overall, our study elucidates a regulatory mechanism for flavonol synthesis in banana and suggests possible targets for genetic optimization to enhance nutritional value and stress responses in this globally important fruit crop.

Sugarcane, which provides 80% of global table sugar and 40% of biofuel, presents unique breeding challenges due to its highly polyploid, heterozygous, and frequently aneuploid genome. Significant progress has been made in developing genetic resources, including the recently completed reference genome of the sugarcane cultivar R570 and pan-genomic resources from sorghum, a closely related diploid species. Biotechnological approaches including RNA interference (RNAi), overexpression of transgenes, and gene editing technologies offer promising avenues for accelerating sugarcane improvement. These methods have successfully targeted genes involved in important traits such as sucrose accumulation, lignin biosynthesis, biomass oil accumulation, and stress response. One of the main transformation methods-biolistic gene transfer or Agrobacterium-mediated transformation-coupled with efficient tissue culture protocols, is typically used for implementing these biotechnology approaches. Emerging technologies show promise for overcoming current limitations. The use of morphogenic genes can help address genotype constraints and improve transformation efficiency. Tissue culture-free technologies, such as spray-induced gene silencing, virus-induced gene silencing, or virus-induced gene editing, offer potential for accelerating functional genomics studies. Additionally, novel approaches including base and prime editing, orthogonal synthetic transcription factors, and synthetic directed evolution present opportunities for enhancing sugarcane traits. These advances collectively aim to improve sugarcane's efficiency as a crop for both sugar and biofuel production. This review aims to discuss the progress made in sugarcane methodologies, with a focus on RNAi and gene editing approaches, how RNAi can be used to inform functional gene targets, and future improvements and applications.

In Arabidopsis, the outbreaks of reactive oxygen species (ROS) occur upon pathogen recognition by pattern- and effector-triggered immunity (PTI and ETI, respectively), which plays a significant role in disease resistance. Here, we found that Arabidopsis also experiences two outbreaks of ROS (oral secretion (OS)-induced ROS (ROS)) upon the perception of OS from cotton bollworm (Helicoverpa armigera) and other lepidopterans. Oral secretion-induced ROS burst requires the PTI machinery, including BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and BOTRYTIS-INDUCED KINASE1 (BIK1). Oral secretion-induced ROS are primarily produced by respiratory burst oxidase homologue D (RBOHD) in the apoplast, and the double mutant, rbohdf, exhibits reduced resistance to lepidopterans. Insect biting rather than wounding induces the gene expressions of plant defense-associated respiratory burst and toxin catabolic processes, facilitating the breakdown of leaf glucosinolates into bioactive intermediates, like sulforaphane, thereby impeding insect herbivory. Our investigation demonstrates that Arabidopsis perceives insect OS in a BAK1-BIK1-dependent manner and employs RBOHD to produce ROS in the apoplast, thereby enhancing its insect resistance by accelerating glucosinolate hydrolysis.

Regeneration represents a fundamental biological process wherein an organism's tissues or organs repair and replace themselves following damage or environmental stress. In plant systems, injured tree branches can regenerate adventitious buds and develop new crowns through propagation techniques like cuttings and canopy pruning, while transgenic plants emerge via tissue culture in genetic engineering processes intimately connected to plant regeneration mechanisms. The advancement of plant regeneration technology is critical for addressing complex and dynamic climate challenges, ultimately ensuring global agricultural sustainability. This review comprehensively synthesizes the latest genetic transformation technologies, including transformation systems across woody, herbaceous and algal species, organellar genetic modifications, crucial regeneration factors facilitating Agrobacterium-mediated transformations, the intricate hormonal networks regulating plant regeneration, comparative analyses of transient transformation approaches and marker gene dynamics throughout transformation processes. Ultimately, the review offers novel perspectives on current transformation bottlenecks and proposes future research trajectories.