The Diospyros genus, which includes both wild and cultivated species such as Diospyros lotus and Diospyros kaki, represents a diverse genetic pool with significant agricultural value. In this study, we present a high-quality, haplotype-resolved, chromosome-level genome assembly for Diospyros deyangensis (hereinafter referred to as ‘Deyangshi’), an autotetraploid wild species notable for its short juvenile phase, by integrating high-fidelity single-molecule, nanopore sequencing, and high-throughput chromosome conformation capture techniques. The assembled genome size is ~3.01 Gb, anchored onto 60 pseudochromosomes. Comparative genomic analysis revealed that the D. deyangensis genome underwent an additional whole-genome duplication (WGD) event following the eudicots shared ancient hexaploidy event. Resequencing and clustering on 63 samples representing 11 geographically diverse Diospyros accessions revealed significant genetic differentiation between D. deyangensis and D. kaki, as well as between D. kaki and other Diospyros species using population genomic analyses, suggesting that D. kaki followed an independent evolutionary pathway. Additionally, we identified DdELF4 (EARLY FLOWERING 4) from the ‘Deyangshi’ backcross population using bulked segregant RNA sequencing (BSR-seq) with 50 early-flowering and 50 non-early-flowering individuals. Overexpression of DdELF4 in Arabidopsis resulted in delayed flowering and downregulation of FT gene expression, indicating its role as a flowering repressor. This high-quality genome assembly of ‘Deyangshi’ provides an essential genomic resource for the Diospyros genus, particularly for breeding programs focused on developing early-flowering persimmon varieties.
Citric acid accumulation is an essential process in citrus fruits that determines fruit flavor and marketability. The MBW complex transcription factor genes, CsAN11, CsAN1, and CsPH4 play key roles in regulating citric acid accumulation. Although how to regulate CsAN1 or CsPH4 was widely investigated, studies on the regulation of CsAN11 are scarce. In this study, we characterized the AP2/ERF (APETALA2/ethylene response factor) transcription factor gene CsAIL6, which is lowly expressed in high-acid citrus varieties and highly expressed in low-acid citrus varieties. Overexpressing CsAIL6 obviously decreased the citric acid content in citrus fruits, calli, or tomatoes, whereas silencing CsAIL6 significantly increased the fruit citric acid content. Additionally, transcript levels of CsAN11, CsAN1, and CsPH4 were significantly increased by silencing CsAIL6; only the CsAN11 transcript level was significantly decreased by overexpressing CsAIL6. Similarly, only the tomato AN11 (SIAN11) transcript level in CsAIL6 stably overexpressing fruits was markedly lower than that in wild-type (WT) fruits. Further experiments revealed that overexpressing CsAN11 significantly increased the organic acid content but had no obvious influence on the CsAIL6 transcript level; in addition, CsAIL6 only interacts with CsAN11, rather than with CsAN1 and CsPH4 of the MBW complex. Taken together, our findings verified that CsAIL6 negatively regulates citric acid accumulation through directly interacting with the WD40 protein CsAN11, which provides a new mechanism for citric acid accumulation in fruits through the regulation of the MBW complex.
In tomato, SlNOR and SlNOR-like1, members of the NAC family of transcription factors (TFs), are known to play critical roles in regulating fruit ripening and are highly expressed in floral organs. However, their role in flower development remains unclear. In this study, we generated and functionally characterized a double knockout mutant, nor/nor-like1. Our findings reveal that the pollen abortion of the nor/nor-like1 impedes ovarian enlargement, resulting in fruit formation failure. Histological analyses demonstrate that the pollen wall collapse occurs during the mature pollen stage and leads to the abnormal pollen wall component deposition at the microspore stage, resulting in the male sterility in the double knockout mutant lines. Kyoto Encyclopedia of Genes and Genomes enrichment pathway analyses further suggest that the loss of SlNOR and SlNOR-like1 function affects several metabolic pathways related to pollen development, including ‘ABC transporters’, ‘lipid metabolism’, ‘phenylpropanoid biosynthesis’, ‘hormone signal transduction’, ‘starch and sucrose metabolism’, and ‘cutin, suberine, and wax biosynthesis’. Furthermore, our results demonstrate that SlNOR and SlNOR-like1 could directly bind to the promoters of key genes associated with pollen wall formation and activate their expression, including ATP-binding cassette transporters of the G family (SlABCG8/9/23), ECERIFERUM (SlCER1), and glycine-rich protein (SlGRP92). These findings suggest that SlNOR and SlNOR-like1 may play a redundant role in the biosynthesis and transport of sporopollenin precursors, cuticular wax biosynthesis, and exine formation. In summary, our study highlights a previously uncharacterized role of SlNOR and SlNOR-like1 in tomato pollen wall formation and male fertility.
Brassinosteroids (BRs) are extensively distributed in plants and play crucial roles throughout all stages of plant growth. Nevertheless, the molecular mechanism through which BRs influence postharvest senescence in pakchoi remains elusive. Previous studies have demonstrated that the application of 1.5 μM of the BRs analog 2,4-epibrassinolide (EBR) delayed the leaf senescence in harvested pakchoi. In this study, we constructed the EBR-delayed senescence transcriptome in pakchoi leaves and discovered that EBR modulates the expression of genes involved in the chlorophyll (Chl) metabolism pathway and the BRs pathway in pakchoi. Notably, we identified and characterized an EBR-suppressed, nucleus-localized WRKY transcription factor called BrWRKY8. BrWRKY8 is a highly expressed transcriptional activator in senescent leaves, targeting the promoters of the Chl degradation-associated gene BrSGR2 and the BRs degradation-associated gene BrCHI2, thereby promoting their expression. Overexpression of the BrWRKY8 gene accelerated the senescence process in Arabidopsis leaves, while EBR treatment mitigated the leaf senescence phenotype induced by BrWRKY8 overexpression. Conversely, silencing of BrWRKY8 through the virus-induced gene silencing extended the postharvest storage period of pakchoi. In conclusion, the newly discovered BRs-BrWRKY8 regulatory model in this study provides novel insights into BRs-mediated leaf senescence in pakchoi.
The ubiquitin-26S proteasome system (UPS) is associated with protein stability and activity, regulation of hormone signaling, and the production of secondary metabolites in plants. Though the mechanism of action of SmMYB36 on the tanshinone and phenolic acid biosynthesis is well understood, its regulation through post-translational modifications is unclear. A constitutive photomorphogenesis 9 (COP9) signalosome subunit 5 (SmCSN5), which interacted with SmMYB36 and inhibited its ubiquitination-based degradation, was identified in Salvia miltiorrhiza. SmCSN5 promoted tanshinone biosynthesis but inhibited phenolic acid biosynthesis in the hairy roots of S. miltiorrhiza. SmMYB36 also activated the transcription of the target genes SmDXS2 and SmCPS1 but repressed that of SmRAS in a SmCSN5-dependent manner. SmCSN5 acts as a positive regulator in MeJA-induced biosynthesis of tanshinones and phenolic acids. Specifically, SmCSN5 alone, when expressed transiently in tobacco and rice protoplasts, was localized to the cytoplasm, cell membrane, and nucleus, whereas when coexpressed with SmMYB36, it was detected only in the nucleus. Additionally, the degradation of SmMYB361-153 by ubiquitination was lowered after truncation of the self-activating structural domain of SmMYB36154-160. Collectively, these results suggest that SmCSN5 affected the transcriptional activation of SmMYB36 and stabilized SmMYB36, providing insights into the SmMYB36-based regulation of the accumulation of tanshinone and phenolic acid at the transcriptional and post-translational levels.
Plant epicuticular waxes (EW) play a critical role in defending against biotic and abiotic stresses. Notably, onions (Allium cepa L.) present a distinctive case where the mutant with defect in leaf and stalk EW showed resistance to thrips compared with the wild type with integral EW. We identified a premature stop codon mutation in the AcCER2 gene, an ortholog of CER2 gene in Arabidopsis thaliana that has been proved essential for the biosynthesis of very long-chain fatty acids (VLCFAs), in the onions with glossy leaf and stalks in our experiments. The data hinted at the possibility that this mutation might impede the elongation process of VLCFAs from C28 to C32, thereby hindering the production of 16-hentriacontanone, a primary constituent of onion EW. Transcriptomic analysis revealed substantial alterations in expression of genes in the pathways related not only to lipid synthesis and transport but also to signal transduction and cell wall modification in glossy mutants. Meanwhile, metabolomic profiling indicates a remarkable increase in flavonoid accumulation and a significant reduction in soluble sugar content in glossy mutants. These findings suggested that the enhanced resistance of glossy mutants to thrips might be a consequence of multiple physiological changes, and our integrated multiomics analysis highlighting the regulatory role of AcCER2 in these processes. Our study has yielded valuable insights into the biosynthesis of onion EW and has provided an initial hypothesis for the mechanisms underlying thrip resistance. These findings hold significant promise for the breeding programs of thrip-resistant onion.
Trichomes are tiny outgrowths on the plant epidermis that serve defensive purposes against various stresses. While the regulatory mechanisms underlying unicellular trichome development are well understood, those governing multicellular trichome formation remain largely unexplored. In this study, we reveal a new regulatory pathway involving the Hair3 (H3) and H4 genes, which encode C2H2 zinc finger proteins that participate in multicellular trichome development in tomato (Solanum lycopersicum). Using CRISPR-Cas9 to generate single- and double-knockout lines, we found that h3 and h4 single-mutant plants did not show altered trichome characteristics compared to wild-type plants. However, h3/h4 double-knockout plants displayed decreased densities of Types I, VI, and VII trichomes, increased densities of Types III and V trichomes, and reduced leaf and stem lengths of Type I trichomes, revealing that H3 and H4 redundantly regulate trichome development. Notably, protein interaction assays demonstrated that H3 and H4 formed both homo- and heterodimers, supporting their cooperative role. Transcriptome and gene expression analyses identified H3 and H4 as key regulators of several genes involved in trichome development, including Woolly (Wo) and its downstream targets, such as Wox3b, MX1, H, and HD8. Protein-promoter assays showed that H3 and H4 did not directly bind to the Wo promoter but rather interacted with Wo, thereby enhancing the expression of Wo and Wox3b. These findings establish H3 and H4 as key regulators of trichome development and provide novel insights into the mechanisms controlling multicellular trichome development in tomato plants.
Benzoates, particularly salicylic acid (SA) and its derivatives, play critical roles in plant immune responses and basal defense through hydroxylation and glycosylation. Anthracnose is one of the most common and devastating diseases in tea plants (Camellia sinensis). However, the role of SA and its derivatives in tea plant immunity and resistance to anthracnose remains largely unexplored. In the present study, we identified and characterized a glycosyltransferase, CsUGT74B5, which was significantly downregulated in tea seedlings upon anthracnose infection. CsUGT74B5 was preferentially expressed in mature leaves and stem, and responded rapidly to exogenous SA treatment. Phylogenetic analysis suggested CsUGT74B5 might possess the catalytic activity toward benzoates. Enzymatic assays and molecular docking demonstrated recombinant CsUGT74B5 specifically glycosylated at the ortho hydroxyl groups of SA and 2, 6-dihydroxybenzoic acid (2, 6-DHBA), but did not glycosylate 2, 3-DHBA, 2, 5-DHBA, or other substrates in vitro. Overexpression of CsUGT74B5 in Arabidopsis thaliana and tobacco (Nicotiana tabacum) reduced SA level while promoting the accumulation of SA 2-O-β-D-glucoside (SAG), further validating the in vivo function of CsUGT74B5. Moreover, transient overexpression of CsUGT74B5 in two tea plant cultivars increased their sensitivity to anthracnose and accelerated lesion development, which was attributed to decreased SA levels. Overall, our finding demonstrated that CsUGT74B5-mediated biosynthesis of SAG regulated tea plant immunity against anthracnose by fine-tuning free SA levels, providing new progress into the immunity response of tea plants.
Bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease affecting plants in the Solanaceae family. In our previous study, CaHDZ27 was shown to act crucially in the pepper defense response to R. solanacearum. However, the molecular basis underlying CaHDZ27 function remains unexplored. In this study, we demonstrate that CaHDZ27 is post-translationally regulated by the 14-3-3 protein CaTFT7, which functions as a positive regulator in pepper immunity against R. solanacearum. RT-qPCR analysis revealed that CaTFT7 is transcriptionally induced by R. solanacearum infection. The data from virus-induced gene silencing revealed that CaTFT7 positively affects pepper immunity, which was further confirmed by the data of CaTFT7-overexpressing Nicotiana benthamiana. CaTFT7 interacted with CaHDZ27, thereby promoting the stability of CaHDZ27 and enhancing CaHDZ27 binding to the promoter of cysteine-rich receptor-like protein kinase 5 (CaCRK5), a gene that positively affects pepper defense against R. solanacearum. The above data indicated that CaTFT7 enhanced CaHDZ27 stability and promoted its ability to activate pepper immunity, shedding light on the mechanisms underlying pepper resistance to bacterial wilt.
The quality of traditional herbs depends on organ morphogenesis and the accumulation of active pharmaceutical ingredients. While recent research highlights the significance of cell mechanobiology in model plant morphogenesis, our understanding of mechanical signal initiation and transduction in traditional herbs remains incomplete. Recent studies reveal a close correlation between cell wall (CW) biosynthesis and active ingredient production, yet the role of cell mechanics in balancing morphogenesis and secondary metabolism is often overlooked. This review explores how the cell wall, plasma membrane, cytoskeleton, and vacuole collaborate to regulate cell mechanics and respond to mechanical changes. We propose CW biosynthesis as a hub in connecting cell mechanics with secondary metabolism and emphasize that understanding the relationship between mechanical remodeling and secondary metabolism could provide new insights into plant cell mechanobiology and the breeding of high-quality herbs.
Populus trichocarpa, a pivotal model organism for woody transgenic research, not only garners substantial scientific interest but plays an integral role in forestry economics. Previous genomic assemblies of P. trichocarpa predominantly treated its heterozygous genome as homozygous, thereby neglecting crucial haplotypic diversity. Leveraging the high-fidelity (HiFi) sequencing capabilities of PacBio sequencing and the chromosome conformation capture insights provided by Illumina's Hi-C technique, this study is the first to achieve a near telomere-to-telomere assembly of both paternal and maternal haplotypes in P. trichocarpa. Comparative genomic analysis between these haplotypes has uncovered several allelic variants and pathways critical for trait determination through allele-specific expression. Furthermore, utilizing RNA-seq data from multiple tissues, this investigation has detailed the tissue-specific expression patterns of the leucine-rich repeat gene family, which are essential in mediating plant signal transduction and developmental regulation. Our results not only illuminate the functional genomics landscape of P. trichocarpa but also provide invaluable theoretical underpinnings for the genetic improvement of woody plants and a robust framework for exploring genetic variability and allelic expression disparities in arboreal species.
Internal browning (IB) is a common chilling injury (CI) feature in peach fruit after prolonged cold storage. Our previous study demonstrated that low O2 and elevated CO2 (eCO2) condition of modified atmosphere (MA) storage alleviated CI by facilitating the accumulation of jasmonic acids (JAs) and γ-aminobutyric acid (GABA) in ‘Zhonghuashoutao’ (‘ZHST’) peach fruit. Here we show that 10% CO2 alone can improve cold tolerance, with ethylene response factor 17 (PpERF17) identified as a pivotal transcription factor (TF) that promotes biosynthesis of JAs and GABA. Stable transformation of PpERF17 in tobacco resulted in reduced cold damage, attributed to decreased levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA), as well as enhanced accumulation of JAs and GABA. Moreover, under eCO2, PpMYC2.1, the master regulator of JA signaling, was found to activate transcription of 13S-lipoxygenase (Pp13S-LOX), allene oxide synthase (PpAOS), 12-oxophytodienoate reductase 3 (PpOPR3), and glutamate decarboxylase (PpGAD), while also inducing the expression of the upstream TF PpERF17, thereby establishing positive feedback loops upregulating JA and GABA biosynthesis. Finally, application of methyl jasmonate (MeJA) to fruit before shelf transfer from cold storage alleviated chilling injury development, due to increased accumulation of JAs and GABA as a result of raised expression of related biosynthetic genes. Collectively, our results suggest that eCO2-induced PpERF17 enhances JAs and GABA accumulation while activating the JA signaling pathway. This contributes to a positive feedback loop mediated by PpMYC2.1, ultimately alleviating CI of peach fruit through the sustained accumulation of JAs and GABA.
In flowering plants, pollen tube growth is essential for delivering immotile sperm cells during double fertilization, directly influencing seed yield. This process relies on vesicle-mediated trafficking to sustain tip growth and fertility. However, investigating pollen tube growth is challenging in non-model plants due to the lack of transgenic tools. Here, we developed a method to transiently inhibit vesicle activity in pollen tubes of the wishbone flower (Torenia fournieri), a classic plant for sexual reproduction studies, using brefeldin A (BFA) and antisense oligodeoxynucleotides (AS-ODNs) targeting key genes. BFA broadly disrupted vesicle gradient homeostasis in T. fournieri pollen tubes, leading to widespread changes in cell wall deposition, ROS distribution, and pollen tube morphology. To assess the role of specific genes, we designed AS-ODNs against TfANX, the sole ANXUR homolog in T. fournieri, which successfully penetrated cell membranes and suppressed TfANX expression. This inhibition impaired pollen tube tip growth, causing pollen tube leakage at the shank region and, in some cases, multiple leakages. Similarly, AS-ODN targeting TfRABA4D, a pollen-specific vesicle regulator, induced a bulging phenotype and disrupted pectin deposition and reduced ROS distribution, mirroring BFA effects. These findings elucidate vesicle-mediated regulation in pollen tube tip growth and introduce an accessible method for genetic manipulation in reproductive research of non-model plants.
Tomato spotted wilt virus (TSWV) poses a significant threat as a devastating pathogen to the global production and quality of tomato (Solanum lycopersicum). Mining novel resistance genes within the tomato germplasm is an effective and environmentally friendly approach to combat TSWV. In this study, we investigated the mechanisms underlying high TSWV resistance in a specific tomato line after experimental inoculation, despite not possessing any known TSWV resistance genes. The candidate causal genes of disease resistance traits were finely mapped by constructing different genetic populations and performing bulk segregant analysis sequencing. This approach identified SlDnaJ (Solyc10g081220) as a key locus potentially regulating TSWV resistance. We determined a structural variant of SlDnaJ (designated Sldnaj) containing a 61-bp promoter sequence deletion that was specifically present in the germplasm of the susceptible M82 tomato plant lines. Sldnaj-knockout transgenic plants were significantly more resistant to TSWV than wild-type plants. Up-regulated expression of Sldnaj affected the salicylic acid/jasmonic acid signaling pathway, which induced and promoted the systemic infection of TSWV in M82 susceptible plants. In summary, this study identified a new candidate TSWV susceptibility gene with a natural deletion variation in tomato. These findings provide insights into the molecular mechanism underlying pathogen resistance while offering a target for breeding strategies of tomato with TSWV resistance.
Strawberry fruits, known for their excellent taste and potential health benefits, are particularly valued for their rich content of hydrolyzable tannins (HTs). These compounds play key roles in regulating growth and development. However, the molecular mechanisms underlying HT synthesis in plants remains poorly elucidated. In this study, based on a correlation analysis between the transcriptome and metabolome of HTs, galloyl glucosyltransferase (UGT84A22), serine carboxypeptidase-like acyltransferases (SCPL-ATs), and carboxylesterases (CXEs) were screened. Furthermore, in vitro enzymatic assays confirmed that FaSCPL3-1 acted as a hydrolyzable tannins synthase (HTS), catalyzing the continuous galloylation of glucose to form simple gallotannins (GTs). Additionally, FaCXE1/FaCXE3/FaCXE7 catalyzed the degalloylation of simple GTs and ellagitannins (ETs), and FaUGT84A22 catalyzed the glycosylation of gallic acid (GA) to produce 1-O-β-glucogallin (βG), a galloyl donor. Moreover, in FvSCPL3-1-RNAi transgenic strawberry plants, the contents of simple GT and some ET compounds were reduced, whereas, in FaCXE7 overexpressing strawberry plants, these compounds were increased. These enzymes constituted a biosynthetic pathway of galloyl derivatives, termed the “galloylation-degalloylation cycle” (G-DG cycle). Notably, the overexpression of FaCXE7 in strawberry plants not only promoted HT synthesis but also interfered with plant growth and development by reducing lignin biosynthesis. These findings offer new insights into the mechanisms of HT accumulation in plants, contributing to improving the quality of berry fruits quality and enhancing plant resistance.
Tea plant is a fluoride (F)-hyperaccumulator, which poses a potential threat to human health via tea consumption. Reducing F accumulation in fresh tea leaves is crucial for enhancing the safety of tea production at its source. This study aims to isolate novel genes responsible for F accumulation or transport in tea plants. We identified an aluminum (Al)-activated malate transporter gene, CsALMT6, which was hypothesized to be a candidate for differential F accumulation in Camellia sinensis, by employing a combination of transcriptome-wide association study and genome-wide identification of the CsALMT gene family. CsALMT6 exhibited high expression levels in old leaves, and its expression was significantly upregulated in tea plants subjected to F-stress conditions. Furthermore, heterologous expression of CsALMT6 in yeast, Arabidopsis, and Populus conferred F tolerance. However, the expression of F-tolerant hub genes, CsFEX1 and CsFEX2, remained unaffected in CsALMT6-silenced tea plants. Additionally, under F toxicity conditions, the transcription of CsALMT6 was negatively associated with F accumulation in tea plants. In conclusion, CsALMT6 plays a vital role in reducing F accumulation in C. sinensis, thus conferring F tolerance to plant cells.
Nitrate (NO3−), a key form of inorganic nitrogen (N) in soils, is typically lost in tea gardens through leaching. However, NO3− utilization efficiency (NiUE) and its characteristic mechanism in tea plants remain unclear. This study screened contrastive genotypes of NiUE using leaf chlorate sensitivity and explored the potential genes that regulate this process. Fresh branches of 10 cultivars were hydroponically cultivated and subjected to potassium nitrate (KNO3) and potassium chlorate (KClO3) treatments, with the former as the control group. The sensitive cultivar, Zhenong 117 (ZN117), showed a decrease in SPAD and Fv/Fm values following KClO3 treatment, while the tolerant cultivar, Teiguanyin (TGY), exhibited minimal significant changes. After 5 days of cultivation, the 15N concentration and proportion in new shoots of ZN117 were significantly higher than those in TGY. Transcriptome analysis revealed that the expression of genes responsible for NO3− transport, including the nitrate transporters NRT2.4, NPF4.6, NPF6.1, NPF1.10, and NPF1.11, significantly increased in ZN117 after NO3− supply. Genes involved in NO3− reduction, chlorophyll synthesis, and photosynthesis were progressively induced. Coexpression network analysis indicated that the squamosa promoter-binding protein activated the onset of NO3− signaling, while basic helix-loop-helix transcripts were triggered to higher levels during NO3− supply. This study proposes a rapid characterization method of NiUE in woody plants and a speculative molecular regulatory mechanism for the NO3− transfer and remobilization of tea plants. A set of specific genes involved in NO3− transport, reduction, and mobilization were identified and proposed as marker genes for NiUE in tea plants.
Lobularia maritima (sweet alyssum) is a popular ornamental plant that displays a range of flower colors, particularly white and purple. However, the genetic underpinning and evolutionary history of flower colors have remained unknown. To address this, we performed a de novo assembly of a chromosome-level genome for this species and conducted comparative population genomic analyses of both domestic and wild representatives. These analyses revealed distinct genetic clusters corresponding to wild and domestic groups, with further subdivisions based on geographic and phenotypic differences. Importantly, all cultivars originated from a single domestication event within the Tunisia group. One wild group did not contribute genetically to the current cultivars. The new mutations in key gene of the anthocyanin biosynthetic pathway, PAP1, that arose following domestication led to the origin of purple flower coloration in the cultivars. Moreover, the contrasting PAP1 haplotypes in white and purple varieties lead to differential expression of CHS and DFR, which in turn contributes to the observed flower color differences. These findings provide key insights into the domestication history and genetic regulation of flower color in L. maritima, laying the groundwork for future genetic breeding efforts focused on this plant, especially introducing genetic sources from other wild groups.
Tea (Camellia sinensis) is widely cultivated throughout the world for its unique flavor and health benefits. Galloylated catechins in tea plants serve as important secondary metabolites that play a pivotal role in tea taste determination and pharmacological effects. However, the genetic basis of galloylated catechins traits remains elusive. We identified a stable and major-effect quantitative trait locus (QTL) associated with galloylated catechins index (GCI), designated qGCI6.2. Within the QTL’s confidence interval, two shikimate dehydrogenases (CsSDH4, CsSDH3) were identified. These enzymes catalyze gallic acid (GA) production from 3-dehydroquinate dehydratase, thereby contributing to galloylated catechins accumulation. Quantitative real-time PCR (RT-qPCR) analysis revealed that CsSDH4 and CsSDH3 expression levels and GA and galloylated catechins contents were positively correlated. Furthermore, overexpressing CsSDH4 and CsSDH3 in transgenic tomato plants markedly increased GA and galloylated catechin contents. RNA-seq analysis of transgenic tomato indicated that CsSDH4 and CsSDH3 primarily regulate genes related to shikimic acid and flavonoid pathways, and jointly promote galloylated catechins synthesis. Our findings have further elucidated the galloylated catechins synthesis pathway and provided a theoretical basis for cultivation of tea cultivars with high galloylated catechin contents.
Acer pentaphyllum Diels (Sapindaceae), a highly threatened maple endemic to the dry-hot valleys of the Yalong River in western Sichuan, China, represents a valuable resource for horticulture and conservation. This study presents the first chromosomal-scale genome assembly of A. pentaphyllum (~626 Mb, 2n = 26), constructed using PacBio HiFi and Hi-C sequencing technologies. Comparative genomic analyses revealed significant recent genomic changes through rapid amplification of transposable elements, particularly long terminal repeat retrotransposons, coinciding with the dramatic climate change during recent uplift of the Hengduan Mountains. Genes involved in photosynthesis, plant hormone signal transduction, and plant-pathogen interaction showed expansion and/or positive selection, potentially reflecting adaptation to the species’ unique dry-hot valley habitat. Population genomic analysis of 227 individuals from 28 populations revealed low genetic diversity (1.04 ± 0.97 × 10−3) compared to other woody species. Phylogeographic patterns suggest an unexpected upstream colonization along the Yalong River, while Quaternary climate fluctuations drove its continuous lineage diversification and population contraction. Assessment of genetic diversity, inbreeding, and genetic load across populations revealed concerning levels of inbreeding and accumulation of deleterious mutations in small, isolated populations, particularly those at range edges (TKX, CDG, TES). Based on these results, we propose conservation strategies, including the identification of management units and recommendations for genetic rescue. These findings not only facilitate the conservation of A. pentaphyllum but also serve as a valuable resource for future horticultural development and as a model for similar studies on other endangered plant species adapted to extreme environments.
Pear ring rot disease (Botryosphaeria dothidea) is a significant threat to the healthy development of the pear industry. Recent research has identified the functional role of long non-coding RNAs (lncRNAs) in various biological processes of plants. The role of lncRNAs in the pear defense response remains unknown. In this study, transcriptome sequencing was used to analyze lncRNAs in pear stem infected with B. dothidea. It identified 3555 lncRNAs, of which 286 were significantly differentially expressed. GO and KEGG analyses showed that cis- and trans-regulated target genes were enriched in multiple disease resistance-related pathways. More specifically, MSTRG.32189, predicted as an endogenous target mimic (eTM), was significantly down-regulated in response to B. dothidea infection, and was confirmed to inhibit the cleavage effect of PcmiR399b on PcUBC24. OE-MSTRG.32189 transgenic Arabidopsis exhibited lower Pi content and weaker disease resistance to Botrytis cinerea compared with wild type. In pear callus, overexpression of MSTRG.32189 negatively regulated PcmiR399b, which decreased Pi content and reduced disease resistance. Overexpressing PcmiR399b in pear callus exhibited the opposite effects compared with OE-MSTRG.32189. Overexpression and knockout of PcUBC24 further clarified that PcUBC24 negatively regulates Pi content and disease resistance to B. dothidea infection. Furthermore, the ROS levels and expressions of disease resistance pathway-related genes were regulated by the MSTRG.32189-PcmiR399b-PcUBC24 module in transgenic pear callus, which contributed to disease resistance. Overall, our results demonstrated the role of lncRNAs in the pear defense response, revealing that the MSTRG.32189-PcmiR399b-PcUBC24 module regulates phosphate accumulation and disease resistance to B. dothidea infection in pear.
Potatoes are valued as reliable crops due to their high carbohydrate content and relatively low farming demands. Consequently, significant attention has been directed towards understanding and controlling the life cycle of potato tubers in recent years. Notably, recent studies have identified self-pruning 6A (StSP6A) as a key component of the tuberigen, the mobile signal for tuber formation, produced in leaves and then transported underground to induce tuber formation in potatoes. Recent progress in comprehending the signaling mechanisms that regulate StSP6A by photoperiod and ambient temperature components, its long-distance transport into underground tissue, and its involvement in regulating stolon tuberization has advanced significantly. Consequently, the modulation of StSP6A and other possible tuberigen signals, along with their regulatory pathways, significantly impacts potato domestication and crop yield. This progress highlights the differential regulation of tuberigen signals and their potential functions in promoting tuber formation.
Fungi produce microRNA-like RNAs (milRNAs) with functional importance in various biological processes. Our previous research identified a new milRNA Foc-milR87 from Fusarium oxysporum f. sp. cubense, which contributes to fungal virulence by targeting the pathogen glycosyl hydrolase encoding gene. However, the potential roles of fungal milRNAs in interactions with hosts are not well understood. This study demonstrated that Foc-milR87 specifically suppressed the expression of MaPTI6L, a pathogenesis-related gene that encodes a transcriptional activator in the banana (Musa acuminata Cavendish group cv. ‘Baxi Jiao’) genome, by targeting the 3'untranslated region (UTR) of MaPTI6L. Transient overexpression of MaPTI6L activated plant defense responses that depend on its nuclear localization, yet co-expression with Foc-milR87 attenuated these responses. MaPTI6L enhanced plant resistance by promoting transcription of the salicylic acid signaling pathway marker gene MaEDS1. Sequence analysis of the MaPTI6L gene in 19 banana varieties, particularly those resistant to Fusarium wilt, uncovered single nucleotide polymorphisms (SNPs) at Foc-milR87 target sites. Experimental validation showed that these SNPs significantly reduce the microRNA's ability to suppress target gene expression. Our findings reveal that Foc-milR87 plays an important role in impairing plant resistance by targeting MaPTI6L mRNA and reducing MaEDS1 transcription during the early infection stage, suggesting the 3'UTR of MaPTI6L as a promising target for genome editing in generation of disease-resistant banana cultivars.
Salicylic acid (SA) is a phenolic phytohormone widely believed to regulate plant growth and stress response. Despite its significance, the genetic basis of SA-mediated resistance to biotic stressors in tea plants is little understood. Our study investigated the genetic diversity, population structure, and linkage disequilibrium (LD) patterns of 299 tea accessions using 79 560 high-quality single nucleotide polymorphisms (SNPs) obtained from genotyping-by-sequencing (GBS) data. Our genome-wide association study identified CSS0033791.1, an essential gene encoding 9-cis-epoxycarotenoid dioxygenase (CsNCED1), which catalyzes a vital step in abscisic acid (ABA) biosynthesis. Exogenous ABA treatment and transgenic overexpression of the CsNCED1 gene lowered SA content in the respective tea plants by inhibiting the expression of the ICS gene. Further analysis revealed that ABA could reduce the expression levels of the SA receptor gene (NPR1) and NPR1 target genes (PR1 and WRKY18), increasing the plant’s susceptibility to biotic stressors. Furthermore, the feeding behavior of Spodoptera litura revealed that the insect bite area on transgenic leaves was substantially more extensive than that in wild type (WT), implying that the CsNCED1 gene had a negative regulatory role in SA-mediated immune response. This study thus provides the foundation for future insect resistance breeding, sustainable tea plant resource usage, and molecular marker-assisted (MAS) tea plant breeding.
High temperatures increase the sugar concentration of grape (Vitis vinifera L.) berries, which can negatively affect the composition and quality of wine, and global climate change is expected to exacerbate this problem. Modifying the source-to-sink ratio of grapevines by selective pruning is a potential strategy to mitigate this. To investigate the effects of low source-to-sink ratio (retaining three leaves per cluster) on carbon metabolism of grape (cv. Cabernet Sauvignon) berries, we conducted an analysis of 42 metabolites and 21 enzyme activities at nine berry developmental stages，as well as transcriptomes from berries grown under two leaves per cluster. The results revealed that the metabolic pathways were coordinately regulated to maintain homeostasis under low source-to-sink ratio conditions. Because of a delay between metabolites and enzyme activities, the metabolites were loosely correlated with enzyme activities, and a lower density of connectivity between them appeared in low source-to-sink conditions. Otherwise, transcripts of the carbohydrate and amino acid metabolism pathways were enriched by carbon limitation. In summary, this integrated analysis reveals a coordinated regulation of various metabolic pathways that maintains the balance of carbon metabolism and ensures survival in challenging environments, highlighting the high metabolic plasticity of grape berries.
The CRISPR-Cas9 system can be used to introduce site-specific mutations into the genome of tomato (Solanum lycopersicum) plants. However, the direct application of this revolutionary technology to desirable tomato cultivars has been hindered by the challenges of generating transgenic plants. To address this issue, we developed an efficient and heritable genome editing system using tobacco rattle virus (TRV) for an elite tomato cultivar (the paternal line of Saladette). Notably, this virus-induced genome editing (VIGE) system enables the rapid production of various mutant seeds without the need for additional plant transformation and tissue culture, once a Cas9-expressing tomato line is established. This VIGE system consists of transgenic tomato plants that express Cas9 under the control of the tomato ubiquitin 10 (SlUbi10) gene promoter and a mobile guide RNA scaffold (gRNA:SlmFT) generated using the sequence of the tomato Flowering Locus T (SlFT) gene. We determined its editing efficiency by targeting the tomato phytoene desaturase (SlPDS) gene, which causes photobleaching symptoms when disrupted. Most transgenic seedlings infected with the TRV vectors carrying the SlPDS-targeting sgRNA developed chimeric albino leaves associated with a high frequency of indel mutations in the SlPDS gene. Remarkably, fruits from these plants yielded homozygous SlPDS knockout seeds at rates ranging from 15% to 100%. These results demonstrate the exceptional effectiveness of our VIGE system in rapidly generating heritable genome edits in tomato.
The garlic bulb comprises several cloves, the swelling growth of which is significantly hindered by the accumulation of viruses. Herein, we describe a single-cell transcriptomic atlas of swelling cloves with virus accumulation, which comprised 19 681 high-quality cells representing 11 distinct cell clusters. Cells of two clusters, clusters 7 (C7) and 11 (C11), were inferred to be from the meristem. Cell trajectory analysis suggested the differentiation of clove cells to start from the meristem cells, along two pseudo-time paths. Investigation into the cell-specific activity of invasive viruses demonstrated that garlic virus genes showed relatively low-expression activity in cells of the clove meristem. There were 2060 garlic genes co-expressed with virus genes, many of which showed an association with the defense response. Five glutathione synthase/reductase genes co-expressed with virus genes displayed up-regulated expression, and the glutathione and related metabolites level showed an alteration in virus-invasive garlic clove, implying the role of glutathione in viral immunity of garlic. Our study offers valuable insights into the clove organogenesis and interaction between garlic and virus at single-cell resolution.
Cold stress profoundly affects the growth, development, and productivity of horticultural crops. Among the diverse strategies plants employ to mitigate the adverse effects of cold stress, flavonoids have emerged as pivotal components in enhancing plant resilience. This review was written to systematically highlight the critical role of flavonoids in plant cold tolerance, aiming to address the increasing need for sustainable horticultural practices under climate stress. We provide a comprehensive overview of the role of flavonoids in the cold tolerance of horticultural crops, emphasizing their biosynthesis pathways, molecular mechanisms, and regulatory aspects under cold stress conditions. We discuss how flavonoids act as antioxidants, scavenging reactive oxygen species (ROS) generated during cold stress, and how they regulate gene expression by modulating stress-responsive genes and pathways. Additionally, we explore the application of flavonoids in enhancing cold tolerance through genetic engineering and breeding strategies, offering insights into practical interventions for improving crop resilience. Despite significant advances, a research gap remains in understanding the precise molecular mechanisms by which specific flavonoids confer cold resistance, especially across different crop species. By addressing current knowledge gaps, proposing future research directions and highlighting implications for sustainable horticulture, we aim to advance strategies to enhance cold tolerance in horticultural crops.
Although the significance of some plant WRKYs in response to cold stress have been identified, the molecular mechanisms of most WRKYs remain unclear in grapevine. In this study, we demonstrate that cold-induced expression of VaBAM3 in Vitis amurensis executes a beneficial role in enhancing resistance by the regulating starch decomposition. VaWRKY65 was identified as an upstream transcriptional activator of VaBAM3 through yeast one-hybrid library screening and validated to directly interact with the W-box region inside the VaBAM3 promoter. Transgenic Arabidopsis thaliana plants and grapevine roots overexpression VaWRKY65 exhibited improved cold tolerance along with higher BAM activity and soluble sugar levels, whereas opposite changes were observed in VaWRKY65 knockdown lines created by virus-induced gene silencing (VIGS) in grapevine plants and in the knockout wrky65 mutants generated by CRISPR/Cas9 technology in grapevine roots. The transcriptome data show that overexpression of VaWRKY65 led to significant alteration of a diverse set of stress-related genes at the transcriptional level. One of the genes, Peroxidase 36 (VaPOD36), was further verified as a direct target of VaWRKY65. Consistently, VaWRKY65-overexpressing plants had higher VaPOD36 transcript levels and POD activity but a reduced ROS level, while silencing VaWRKY65 results in contrary changes. Collectively, these results reveal that VaWRKY65 enhanced cold tolerance through modulating soluble sugars produced from starch breakdown and ROS scavenging.