The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis ‘Hongyang’ and A.eriantha ‘White’). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.
Viral infections in plants pose major challenges to agriculture and global food security in the twenty-first century. Plants have evolved a diverse range of specialized metabolites (PSMs) for defenses against pathogens. Although, PSMs-mediated plant-microorganism interactions have been widely discovered, these are mainly confined to plant-bacteria or plant-fungal interactions. PSM-mediated plant-virus interaction, however, is more complicated often due to the additional involvement of virus spreading vectors. Here, we review the major classes of PSMs and their emerging roles involved in antiviral resistances. In addition, evolutionary scenarios for PSM-mediated interactions between plant, virus and virus-transmitting vectors are presented. These advancements in comprehending the biochemical language of PSMs during plant-virus interactions not only lay the foundation for understanding potential co-evolution across life kingdoms, but also open a gateway to the fundamental principles of biological control strategies and beyond.
Plant Myeloblastosis (MYB) proteins function crucially roles upon variegated abiotic stresses. Nonetheless, their effects and mechanisms in rose (Rosa chinensis) are not fully clarified. In this study, we characterized the effects of rose RcMYB8 under salt and drought tolerances. For induction of the RcMYB8 expression, NaCl and drought stress treatment were adopted. Rose plants overexpressing RcMYB8 displayed enhanced tolerance to salinity and drought stress, while silencing RcMYB8 resulted in decreased tolerance, as evidenced by lowered intra-leaf electrolyte leakage and callose deposition, as well as photosynthetic sustainment under stressed conditions. Here, we further show that RcMYB8 binds similarly to the promoters of RcPR5/1 and RcP5C51 in vivo and in vitro. Inhibiting RcP5CS1 by virus-induced gene silencing led to decreased drought tolerance through the reactive oxygen species (ROS) homeostatic regulation. RcP5CS1-silenced plants showed an increase in ion leakage and reduce of proline content, together with the content of malondialdehyde (MDA) increased, lowered activities of Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Our study highlights the transcriptional modulator role of RcMYB8 in drought and salinity tolerances, which bridges RcPR5/1 and RcP5CS1 by promoting ROS scavenging. Besides, it is probably applicable to the rose plant engineering for enhancing their abiotic stress tolerances.
Actinidia arguta, known as hardy kiwifruit, is a widely cultivated species with distinct botanical characteristics such as small and smooth-fruited, rich in beneficial nutrients, rapid softening and tolerant to extremely low temperatures. It contains the most diverse ploidy types, including diploid, tetraploid, hexaploid, octoploid, and decaploid. Here we report a haplotype-resolved tetraploid genome (A. arguta cv. ‘Longcheng No.2’) containing four haplotypes, each with 40,859, 41,377, 39,833 and 39,222 protein-coding genes. We described the phased genome structure, synteny, and evolutionary analyses to identify and date possible WGD events. Ks calculations for both allelic and paralogous genes pairs throughout the assembled haplotypic individuals showed its tetraploidization is estimated to have formed ~ 1.03 Mya following Ad-α event occurred ~ 18.7 Mya. Detailed annotations of NBS-LRRs or CBFs highlight the importance of genetic variations coming about after polyploidization in underpinning ability of immune responses or environmental adaptability. WGCNA analysis of postharvest quality indicators in combination with transcriptome revealed several transcription factors were involved in regulating ripening kiwi berry texture. Taking together, the assembly of an A. arguta tetraploid genome provides valuable resources in deciphering complex genome structure and facilitating functional genomics studies and genetic improvement for kiwifruit and other crops.
N4-acetylcytidine (ac4C) modification of mRNA has been shown to be present in plant RNAs, but its regulatory function in plant remains largely unexplored. In this study, we investigated the differentially expressed mRNAs, lncRNAs and acetylation modifications of mRNAs in tomato fruits from both genotypes. By comparing wild-type (AC) tomato and the ethylene receptor-mutant (Nr) tomato from mature green (MG) to six days after the breaker (Br6) stage, we identified differences in numerous key genes related to fruit ripening and observed the corresponding lncRNAs positively regulated the target genes expression. At the post-transcriptional level, the acetylation level decreased and increased in AC and Nr tomatoes from MG to Br6 stage, respectively. The integrated analysis of RNA-seq and ac4C-seq data revealed the potential positive role of acetylation modification in regulating gene expression. Furthermore, we found differential acetylation modifications of certain transcripts (ACO, ETR, ERF, PG, CesA, β-Gal, GAD, AMY, and SUS) in AC and Nr fruits which may explain the differences in ethylene production, fruit texture, and flavor during their ripening processes. The present study provides new insights into the molecular mechanisms by which acetylation modification differentially regulates the ripening process of wild-type and mutant tomato fruits deficient in ethylene signaling.
Putrescine plays a role in superficial scald development during the cold storage of pear fruit. However, the molecular mechanism behind this phenomenon has not been un-fully clarified until recently. In this study, a conjoint analysis of metabolites and gene expression profiles in the putrescine-metabolic pathway of P. bretschneideri Rehd. fruit followed by experimental validation revealed that PbrADC1, forming a homodimer in the chloroplast, was involved in putrescine biosynthesis and thus fruit chilling resistance. Additionally, the substrate-binding residue Cys546 in PbrADC1, whose activity was modified by H2O2, played a crucial role in arginine decarboxylation into agmatine. Through a combined analysis of the distribution of cis-acting elements in the PbrADC1 promoter as well as the expression profiles of related transcription factors (TFs), several TFs were identified as upstream regulators of PbrADC1 gene. Further investigation revealed that the nuclear PbrWRKY62 could directly bind to the W-box elements in the PbrADC1 promoter, activate its expression, enhance putrescine accumulation, and thus increase fruit chilling tolerance. In conclusion, our results suggest that the PbrWRKY62-PbrADC1 module is involved in the development of superficial scald in P. bretschneideri Rehd. fruit via regulating putrescine biosynthesis. Consequently, these findings could serve as valuable genetic resources for breeding scald-resistant pear fruit.
Botrytis cinerea is one of the most destructive phytopathogenic fungi, causing significant losses to horticultural crops. As a necrotrophic fungus, B. cinerea obtains nutrients by killing host cells. Secreted cell death-inducing proteins (CDIPs) play a crucial role in necrotrophic infection; however, only a limited number have been reported. For high-throughput CDIP screening, we optimized the prokaryotic expression system and compared its efficiency with other commonly used protein expression systems. The optimized prokaryotic expression system showed superior effectiveness and efficiency and was selected for subsequent CDIP screening. The screening system verified fifty-five candidate proteins and identified two novel SGNH family CDIPs: BcRAE and BcFAT. BcRAE and BcFAT exhibited high expression levels throughout the infection process. Site-directed mutagenesis targeting conserved Ser residues abolished the cell death-inducing activity of both BcRAE and BcFAT. Moreover, the transient expression of BcRAE and BcFAT in plants enhanced plant resistance against B. cinerea without inducing cell death, independent of their enzymatic activities. Our results suggest a high-efficiency screening system for high-throughput CDIP screening and provide new targets for further study of B. cinerea-plant interactions.
Artemisinin is primarily synthesized and stored in the subepidermal space of the glandular trichomes of Artemisia annua. The augmentation of trichome density has been demonstrated to enhance artemisinin yield. However, existing literature lacks insights into the correlation between the stratum corneum and trichomes. This study aims to unravel the involvement of TrichomeLess Regulator 3 (TLR3), which encodes the transcription factor, in artemisinin biosynthesis and its potential association with the stratum corneum. TLR3 was identified as a candidate gene through transcriptome analysis. The role of TLR3 in trichome development and morphology was investigated using yeast two-hybrid, pull-down analysis, and RNA electrophoresis mobility assay. Our research revealed that TLR3 negatively regulates trichome development. It modulates the morphology of Arabidopsis thaliana trichomes by inhibiting branching and inducing the formation of abnormal trichomes in Artemisia annua. Overexpression of the TLR3 gene disrupts the arrangement of the stratum corneum and reduces artemisinin content. Simultaneously, TLR3 possesses the capacity to regulate stratum corneum development and trichome follicle morphology by interacting with TRICHOME AND ARTEMISININ REGULATOR 1, and CycTL. Consequently, our findings underscore the pivotal role of TLR3 in the development of glandular trichomes and stratum corneum biosynthesis, thereby influencing the morphology of Artemisia annua trichomes.
Although there is increasing evidence suggesting that DNA methylation regulates seed development, the underlying mechanisms remain poorly understood. Therefore, we aimed to shed light on this by conducting whole-genome bisulfite sequencing using seeds from the large-seeded cultivar 'HZ' and the abortive-seeded cultivar 'NMC'. Our analysis revealed that the 'HZ' seeds exhibited a hypermethylation level compared to the 'NMC' seeds. Furthermore, we found that the genes associated with differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were mainly enriched in the reactive oxygen species (ROS) metabolic pathway. To investigate this further, we conducted nitroblue tetrazolium (NBT) and 2,7-Dichlorodihydrofluorescein (DCF) staining, which demonstrated a significantly higher amount of ROS in the 'NMC' seeds compared to the 'HZ' seeds. Moreover, we identified that the gene LcGPX6, involved in ROS scavenging, exhibited hypermethylation levels and parallelly lower expression levels in 'NMC' seeds compared to 'HZ' seeds. Interestingly, the ectopic expression of LcGPX6 in Arabidopsis enhanced ROS scavenging and resulted in lower seed production. Together, we suggest that DNA methylation-mediated ROS production plays a significant role in seed development in litchi, during which hypermethylation levels of LcGPX6 might repress its expression, resulting in the accumulation of excessive ROS and ultimately leading to seed abortion.
The auxin response factor (ARF) and auxin/indole-3-acetic acid (Aux/IAA) family of genes are central components of the auxin signaling pathway and play essential roles in plant growth and development. Their large-scale analysis and evolutionary trajectory of origin are currently not known. Here, we identified the corresponding ARF and Aux/IAA family members and performed a large-scale analysis by scanning 406 plant genomes. The results showed that the ARF and Aux/IAA gene families originated from charophytes. The ARF family sequences were more conserved than the Aux/IAA family sequences. Dispersed duplications were the common expansion mode of ARF and Aux/IAA families in bryophytes, ferns, and gymnosperms; however, whole-genome duplication was the common expansion mode of the ARF and Aux/IAA families in basal angiosperms, magnoliids, monocots, and dicots. Expression and regulatory network analyses revealed that the Arabidopsis thaliana ARF and Aux/IAA families responded to multiple hormone, biotic, and abiotic stresses. The APETALA2 and serum response factor-transcription factor gene families were commonly enriched in the upstream and downstream genes of the ARF and Aux/IAA gene families. Our study provides a comprehensive overview of the evolutionary trajectories, structural functions, expansion mechanisms, expression patterns, and regulatory networks of these two gene families.
Roses are consistently ranked at the forefront in cut flower production. Increasing demands of market and changing climate conditions have resulted in the need to further improve the diversity and quality of traits. However, frequent hybridization leads to highly heterozygous nature, including the allelic variants. Therefore, the absence of comprehensive genomic information leads to them making it challenging to molecular breeding. Here, two haplotype-resolved chromosome genomes for Rosa chinensis ‘Chilong Hanzhu’ (2n = 14) which is high heterozygous diploid old Chinese rose are generated. An amount of genetic variation (1,605,616 SNPs, 209,575 indels) is identified. 13,971 allelic genes show differential expression patterns between two haplotypes. Importantly, these differences hold valuable insights into regulatory mechanisms of traits. RcMYB114b can influence cyanidin-3-glucoside accumulation and the allelic variation in its promoter leads to differences in promoter activity, which as a factor control petal color. Moreover, gene family expansion may contribute to the abundance of terpenes in floral scents. Additionally, RcANT1, RcDA1, RcAG1 and RcSVP1 genes are involved in regulation of petal number and size under heat stress treatment. This study provides a foundation for molecular breeding to improve important characteristics of roses.
The molecular basis of orchid flower development involves a specific regulatory program in which MADS-box transcription factors play a central role. The recent ‘perianth code’ model hypothesizes that two types of higher-order heterotetrameric complexes, namely SP complex and L complex, play pivotal roles in the orchid perianth organ formation. Therefore, we explored their roles and searched for other components of the regulatory network.
Through the combined analysis for transposase-accessible chromatin with high-throughput sequencing and RNA sequencing of the lip-like petal and lip from Phalaenopsis equestris var.trilip, transcription factor-(TF) genes involved in lip development were revealed. PeNAC67 encoding a NAC-type TF and PeSCL23 encoding a GRAS-type TF were differentially expressed between the lip-like petal and the lip. PeNAC67 interacted with and stabilized PeMADS3, which positively regulated the development of lip-like petal to lip. PeSCL23 and PeNAC67 competitively bound with PeKAN2 and positively regulated the development of lip-like petal to petal by affecting the level of PeMADS3. PeKAN2 as an important TF that interacts with PeMADS3 and PeMADS9 can promote lip development. These results extend the ‘perianth code’ model and shed light on the complex regulation of orchid flower development.
The plant genome exhibits a significant amount of transcriptional activity, with most of the resulting transcripts lacking protein-coding potential. Non-coding RNAs play a pivotal role in the development and regulatory processes in plants. Long non-coding RNAs (lncRNAs), which exceed 200 nucleotides, may play a significant role in enhancing plant resilience to various abiotic stresses, such as excessive heat, drought, cold, and salinity. In addition, the exogenous application of chemicals, such as abscisic acid and salicylic acid, can augment plant defense responses against abiotic stress. While how lncRNAs play a role in abiotic stress tolerance is relatively well-studied in model plants, this review provides a comprehensive overview of the current understanding of this function in horticultural crop plants. It also delves into the potential role of lncRNAs in chemical priming of plants in order to acquire abiotic stress tolerance, although many limitations exist in proving lncRNA functionality under such conditions.
Salicylic acid (SA) is a multi-functional phytohormone, regulating diverse processes of plant growth and development, especially triggering plant immune responses and initiating leaf senescence. However, the early SA signaling events remain elusive in most plant species apart from Arabidopsis, and even less is known about the multi-facet mechanism underlying SA-regulated processes. Here, we report the identification of a novel regulatory module in cucumber, CsNPR1-CsWRKY11, which mediates the regulation of SA-promoted leaf senescence and ROS burst. Our analyses demonstrate that under SA treatment, CsNPR1 recruits CsWRKY11 to bind to the promoter of CsWRKY11 to activate its expression, thus amplifying the primary SA signal. Then, CsWRKY11 cooperates with CsNPR1 to directly regulate the expression of both chlorophyll degradation and ROS biosynthesis related genes, thereby inducing leaf de-greening and ROS burst. Our study provides a solid line of evidence that CsNPR1 and CsWRKY11 constitute a key module in SA signaling pathway in cucumber, and gains an insight into the interconnected regulation of SA-triggered processes.
Most of the carbon found in fruits at harvest is imported by the phloem. Imported carbon provide the material needed for the accumulation of sugars, organic acids, secondary compounds, in addition to the material needed for the synthesis of cell walls. The accumulation of sugars during fruit development influences not only sweetness but also various parameters controlling fruit composition (fruit “quality”). The accumulation of organic acids and sugar in grape berry flesh cells is a key process for berry development and ripening. The present review presents an update of the research on grape berry development, anatomical structure, sugar and acid metabolism, sugar transporters, and regulatory factors.
Michelia alba DC is a highly valuable ornamental plant of the Magnoliaceae family. This evergreen tropical tree commonly grows in Southeast Asia and is adored for its delightful fragrance. Our study assembled the M. alba haplotype genome MC and MM by utilizing Nanopore ultralong reads, Pacbio Hifi long reads and parental second-generation data. Moreover, the first methylation map of Magnoliaceae was constructed based on the methylation site data obtained using Nanopore data. Metabolomic datasets were generated from the flowers of three different species to assess variations in pigment and volatile compound accumulation. Finally, transcriptome data were generated to link genomic, methylation, and morphological patterns to reveal the reasons underlying the differences between M. alba and its parental lines in petal color, flower shape, and fragrance. We found that the AP1 and AP2 genes are crucial in M. alba petal formation, while the 4CL, PAL, and C4H genes control petal color. The data generated in this study serve as a foundation for future physiological and biochemical research on M. alba, facilitate the targeted improvement of M. alba varieties, and offer a theoretical basis for molecular research on Michelia L.
