Virus-induced Gene Silencing Technology Research Articles

Transcriptomic profiling of the floral fragrance biosynthesis of Iris germanica ‘Harvest of Memories’ and functional characterization of the IgTPS14 gene

Iris (Iris germanica) is a very popular ornamental plant and is known for the precious spice irone produced from its roots. Many iris varieties can also release fragrances through their flowers. However, the composition of iris aroma and the molecular mechanism of its synthesis have not been reported. In this study, we analyzed the volatile floral compounds of I. germanica ‘Harvest of Memories’ at different stages and in different tissues through headspace solid-phase microextraction gas chromatography-mass spectrometry. A total of 36 volatile compounds were identified, and linalool was the dominant component. Transcriptome analysis showed that 35 differentially expressed genes and 218 differentially expressed transcription factors were positively correlated with linalool release. According to the results of qRT-qPCR, the expression level of the IgTPS14 gene was consistent with the release trend of linalool, suggesting that IgTPS14 may play a certain role in linalool synthesis. Phylogenetic analysis showed that the IgTPS14 protein belonged to the TPS-g subfamily. An in vitro enzymatic assay of IgTPS14 and its transient and stable overexpression in tobacco indicated that this gene produced linalool. The transient silencing of IgTPS14 in petals by virus-induced gene silencing technology revealed a significant reduction in the release of linalool. These results will help explore and reveal the molecular mechanism of monoterpenoid synthesis and provide a certain reference for studying the formation of iris aroma.

Metabolite and Transcriptome Profiling Analysis Provides New Insights into the Distinctive Effects of Exogenous Melatonin on Flavonoids Biosynthesis in Rosa rugosa.

Although the petals of Rosa rugosa are rich in flavonoids and their bioactivity has a significant impact on human health, the flavonoid content decreases during flower development. In this study, R. rugosa 'Feng hua' was used to investigate the effects of the melatonin foliar spray on enhancing the quality of rose by focusing on major flavonoids. The results showed that the contents of total flavonoids in rose petals at the full bloom stage induced by melatonin obeyed a bell-shaped curve, with a maximum at 0.3 mM, indicating the concentration-dependent up-regulation of flavonoid biosynthesis. In the treatment with 0.3 mM melatonin, metabolomic analyses showed that the concentrations of ten main flavonoids were identified to be increased by melatonin induction, with high levels and increases observed in three flavonols and two anthocyanins. KEGG enrichment of transcriptomic analysis revealed a remarkable enrichment of DEGs in flavonoid and flavonol biosynthesis, such as Rr4CL, RrF3H, and RrANS. Furthermore, functional validation using virus-induced gene silencing technology demonstrated that Rr4CL3 is the crucial gene regulating flavonoid biosynthesis in response to the stimulant of melatonin. This study provides insights into the exogenous melatonin regulation mechanism of biosynthesis of flavonoids, thereby offering potential industrial applications.

The characteristic analysis of TaTDF1 reveals its function related to male sterility in wheat (Triticum aestivum L.)

BackgroundThe male sterile lines are an important foundation for heterosis utilization in wheat (Triticum aestivum L.). Thereinto, pollen development is one of the indispensable processes of wheat reproductive development, and its fertility plays an important role in wheat heterosis utilization, and are usually influencing by genes. However, these key genes and their regulatory networks during pollen abortion are poorly understood in wheat.ResultsDEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) is a member of the R2R3-MYB family and has been shown to be essential for early tapetal layer development and pollen grain fertility in rice (Oryza sativa L.) and Arabidopsis thaliana. In order to clarify the function of TDF1 in wheat anthers development, we used OsTDF1 gene as a reference sequence and homologous cloned wheat TaTDF1 gene. TaTDF1 is localized in the nucleus. The average bolting time of Arabidopsis thaliana overexpressed strain (TaTDF1-OE) was 33 d, and its anther could be colored normally by Alexander staining solution, showing red. The dominant Mosaic suppression silence-line (TaTDF1-EAR) was blue-green in color, and the anthers were shrimpy and thin. The TaTDF1 interacting protein (TaMAP65) was confirmed using Yeast Two-Hybrid Assay (Y2H) and Bimolecular-Fluorescence Complementation (BiFC) experiments. The results showed that downregulated expression of TaTDF1 and TaMAP65 could cause anthers to be smaller and shrunken, leading to pollen abortion in TaTDF1 wheat plants induced by virus-induced gene-silencing technology. The expression pattern of TaTDF1 was influenced by TaMAP65.ConclusionsThus, systematically revealing the regulatory mechanism of wheat TaTDF1 during anther and pollen grain development may provide new information on the molecular mechanism of pollen abortion in wheat.

Zinc Treatment of Tea Plants Improves the Synthesis of Trihydroxylated Catechins via Regulation of the Zinc-Sensitive Protein CsHIPP3.

The tea plant (Camellia sinensis [L.] O. Kussntze) is a global economic crop. Zinc treatment of tea plants can enhance catechin biosynthesis. However, the underlying molecular mechanism behind catechin formation through zinc regulation remains unclear. This study identified a zinc-responsive protein, C. sinensis heavy metal-associated isoprenylated plant protein 3 (CsHIPP3), from zinc-treated tea seedlings. CsHIPP3 expression was positively correlated with trihydroxylated catechin (TRIC) content. CsF3'5'H1 is a crucial regulator of the TRIC synthesis pathway. The interaction between CsHIPP3 and CsF3'5'H1 was assessed using bimolecular fluorescence complementation, firefly luciferase complementation imaging, and pulldown experiments. CsHIPP3 knockdown using virus-induced gene silencing technology decreased the content of each component of TRICs. Compared with the control, the relative catechin content was reduced by 40.12-55.39%. Co-overexpression of CsHIPP3 and CsF3'5'H1 significantly elevated the TRIC content in tea leaves and calli. Moreover, the TRIC content in transient co-overexpression leaves was 1.44-fold higher than that of the control group, and tea callus was 50.83% higher in transient co-overexpression than in the wild type. Thus, zinc-regulated TRIC synthesis in a zinc-rich environment was mediated by binding CsHIPP3 with CsF3'5'H1 to promote TRIC synthesis and accumulation.

SlZIP11 mediates zinc accumulation and sugar storage in tomato fruits.

Zinc (Zn) is a vital micronutrient essential for plant growth and development. Transporter proteins of the ZRT/IRT-like protein (ZIP) family play crucial roles in maintaining Zn homeostasis. Although the acquisition, translocation, and intracellular transport of Zn are well understood in plant roots and leaves, the genes that regulate these pathways in fruits remain largely unexplored. In this study, we aimed to investigate the function of SlZIP11 in regulating tomato fruit development. We used Solanum lycopersicum L. 'Micro-Tom' SlZIP11 (Solanum lycopersicum) is highly expressed in tomato fruit, particularly in mature green (MG) stages. For obtaining results, we employed reverse transcription-quantitative polymerase chain reaction (RT-qPCR), yeast two-hybrid assay, bimolecular fluorescent complementation, subcellular localization assay, virus-induced gene silencing (VIGS), SlZIP11 overexpression, determination of Zn content, sugar extraction and content determination, and statistical analysis. RT-qPCR analysis showed elevated SlZIP11 expression in MG tomato fruits. SlZIP11 expression was inhibited and induced by Zn deficiency and toxicity treatments, respectively. Silencing SlZIP11 via the VIGS technology resulted in a significant increase in the Zn content of tomato fruits. In contrast, overexpression of SlZIP11 led to reduced Zn content in MG fruits. Moreover, both silencing and overexpression of SlZIP11 caused alterations in the fructose and glucose contents of tomato fruits. Additionally, SlSWEEET7a interacted with SlZIP11. The heterodimerization between SlSWEET7a and SlZIP11 affected subcellular targeting, thereby increasing the amount of intracellularly localized oligomeric complexes. Overall, this study elucidates the role of SlZIP11 in mediating Zn accumulation and sugar transport during tomato fruit ripening. These findings underscore the significance of SlZIP11 in regulating Zn levels and sugar content, providing insights into its potential implications for plant physiology and agricultural practices.

Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies.

Gene function verification is a crucial step in studying the molecular mechanisms regulating various plant life activities. However, a stable and efficient homologous genetic transgenic system for herbaceous peonies has not been established. In this study, using virus-induced gene silencing technology (VIGS), a highly efficient homologous transient verification system with distinctive advantages was proposed, which not only achieves true "intact-plant" infiltration but also minimizes the operation. One-year-old roots of the representative species, Paeonia lactiflora Pall., were used as the materials; prechilling (4 °C) treatment for 3-5 weeks was applied as a critical precondition for P. lactiflora to acquire a certain chilling accumulation. A dormancy-related gene named HOMEOBOX PROTEIN 31 (PlHB31), believed to negatively regulate bud endodormancy release (BER), was chosen as the target gene in this study. GFP fluorescence was detected in directly infiltrated and newly developed roots and buds; the transgenic plantlets exhibited remarkably earlier budbreak, and PlHB31 was significantly downregulated in silenced plantlets. This study established a homologous transient silencing system featuring intact-plant infiltration and minimized manipulation for gene function research, and also offers technical support and serves as a theoretical basis for gene function discovery in numerous other geophytes.

Isolation and functional analysis of drought related PobZIP4 transcription factor in Paeonia ostii

Drought, as a natural disaster of force majeure, significantly disrupts the physiological balance of plants and crop yields. The basic leucine zipper (bZIP) transcription factors assume the pivotal role of paramount regulators in orchestrating drought response. In this study, a bZIP transcription factor PobZIP4 was isolated on the base of Paeonia ostii transcriptome, and its function in response to drought stress was verified. PobZIP4 had a 579 bp open reading frame, encoded 192 amino acids, and existed in a conserved structural domain typical of bZIP, and was highly expressed when P. ostii was stressed by drought. Moreover, the subcellular localization of PobZIP4 was observed with fluorescent protein labeling method and it was found that PobZIP4 localized in the Nicotiana benthamiana epidermal cell nucleus. Then, we silenced PobZIP4 in P. ostii using virus-induced gene silencing technology, and found that PobZIP4-silenced plants showed significantly less tolerant to drought stress than the negative control. The accumulation of reactive oxygen species was found to be heightened, while the photosynthetic capacity, proline content, and protective enzyme activities exhibited a significant decline upon silencing PobZIP4, which unequivocally demonstrated that PobZIP4 amplifies the drought tolerance of P. ostii. This study highlighted the positive role of PobZIP4 in mitigating drought stress in P. ostii, thereby establishing a molecular basis for enhancing the drought tolerance of P. ostii.

The glycoside hydrolase 7 member VdGH7a regulates Verticillium dahliae pathogenicity and induces host defenses by interacting with GhOLP11

Pathogens secrete multiple enzymes that can degrade the cell wall, thereby weakening the host’s cell wall and facilitating the penetration of the pathogen into the plant. In this study, we identified VdGH7a, a glycoside hydrolase family 7 (GH7) cellobiohydrolase from Verticillium dahliae, which exhibited hydrolytic activity against 1,4-β-glucan. Interestingly, we found that VdGH7a induced cell death in Nicotiana benthamiana when signal peptides were present. However, this phenomenon was effectively prevented by the carbohydrate-binding type-1 (CBM1) protein domain. Furthermore, we observed that the knockout of VdGH7a significantly reduced the pathogenicity of V. dahliae to cotton plant, as evidenced by the inability of the knockout mutants to penetrate cellophane membrane. Additionally, these knockout mutants displayed diminished ability to exploit carbon sources, rendering them more susceptible to osmotic and cell wall stresses. Moreover, VdGH7a interacted with an osmotin-like protein (GhOLP1) in cotton through yeast two-hybrid screening, and further confirmed using bi-molecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI). Furthermore, virus-induced gene silencing technology was employed to silence GhOLP1, causing cotton’s salicylic acid (SA) content and resistance to V. dahliae were both reduced, whereas heterologous overexpression of GhOLP1 in Arabidopsis increased both resistance and the expression of genes involved in the SA signaling pathway. Collectively, these findings demonstrate a virulence strategy whereby the secreted protein VdGH7a from V. dahliae interacts with GhOLP1 to stimulate host immunity and play a significant role in plant resistance against V. dahliae.

AnWRKY29 and AnHSP90 synergistically modulate trehalose levels in a desert shrub leaves during osmotic stress.

Trehalose, a biological macromolecule with osmotic adjustment properties, plays a crucial role during osmotic stress. As a psammophyte, Ammopiptanthus nanus relies on the accumulation of organic solutes to respond to osmotic stress. We utilized virus-induced gene silencing technology for the first time in the desert shrub A. nanus to confirm the central regulatory role of AnWRKY29 in osmotic stress, as it controls the transcription of AnTPS11 (trehalose-6-phosphate synthase 11). Further investigation has shown that AnHSP90 may interact with AnWRKY29. The AnHSP90 gene is sensitive to osmotic stress, underscoring its pivotal role in orchestrating the response to such adverse conditions. By directly targeting the W-box element within the AnTPS11 promoter, AnWRKY29 effectively enhances the transcriptional activity of AnTPS11, which is facilitated by AnHSP90. This discovery highlights the critical role of AnWRKY29 and AnHSP90 in enabling organisms to adapt to and cope effectively with osmotic stress, which can be a crucial factor in A. nanus survival and overall ecological resilience. Collectively, uncovering the molecular mechanisms underlying the osmotic responses of A. nanus is paramount for comprehending and augmenting the osmotic tolerance mechanisms of psammophyte shrub plants.

Genome-Wide Analysis of C-Repeat Binding Factor Gene Family in Capsicum baccatum and Functional Exploration in Low-Temperature Response.

Capsicum baccatum is a close relative of edible chili peppers (Capsicum annuum) with high economic value. The CBF gene family plays an important role in plant stress resistance physiology. We detected a total of five CBF genes in the C. baccatum genome-wide sequencing data. These genes were scattered irregularly across four chromosomes. The genes were categorized into three groupings according to their evolutionary relationships, with genes in the same category showing comparable principles for motif composition. The 2000 bp upstream of CbCBF contains many resistance-responsive elements, hormone-responsive elements, and transcription factor binding sites. These findings emphasize the crucial functions of these genes in responding to challenging conditions and physiological regulation. Analysis of tissue-specific expression revealed that CbCBF3 exhibited the greatest level of expression among all tissues. Under conditions of low-temperature stress, all CbCBF genes exhibited different levels of responsiveness, with CbCBF3 showing a considerable up-regulation after 0.25 h of cold stress, indicating a high sensitivity to low-temperature response. The importance of the CbCBF3 gene in the cold response of C. baccatum was confirmed by the use of virus-induced gene silencing (VIGS) technology, as well as the prediction of its protein interaction network. To summarize, this study conducts a thorough bioinformatics investigation of the CbCBF gene family, showcases the practicality of employing VIGS technology in C. baccatum, and confirms the significance of the CbCBF3 gene in response to low temperatures. These findings provide significant references for future research on the adaptation of C. baccatum to low temperatures.

Silencing the CsSnRK2.11 Gene Decreases Drought Tolerance of Cucumis sativus L.

Drought stress restricts vegetable growth, and abscisic acid plays an important role in its regulation. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) is a key enzyme in regulating ABA signal transduction in plants, and it plays a significant role in response to multiple abiotic stresses. Our previous experiments demonstrated that the SnRK2.11 gene exhibits a significant response to drought stress in cucumbers. To further investigate the function of SnRK2.11 under drought stress, we used VIGS (virus-induced gene silencing) technology to silence this gene and conducted RNA-seq analysis. The SnRK2.11-silencing plants displayed increased sensitivity to drought stress, which led to stunted growth and increased wilting speed. Moreover, various physiological parameters related to photosynthesis, chlorophyll fluorescence, leaf water content, chlorophyll content, and antioxidant enzyme activity were significantly reduced. The intercellular CO2 concentration, non-photochemical burst coefficient, and malondialdehyde and proline content were significantly increased. RNA-seq analysis identified 534 differentially expressed genes (DEGs): 311 were upregulated and 223 were downregulated. GO functional annotation analysis indicated that these DEGs were significantly enriched for molecular functions related to host cells, enzyme activity, and stress responses. KEGG pathway enrichment analysis further revealed that these DEGs were significantly enriched in phytohormone signalling, MAPK signalling, and carotenoid biosynthesis pathways, all of which were associated with abscisic acid. This study used VIGS technology and transcriptome data to investigate the role of CsSnRK2.11 under drought stress, offering valuable insights into the mechanism of the SnRK2 gene in enhancing drought resistance in cucumbers.

MsCYP71 is a positive regulator for drought resistance in alfalfa

Cytochrome P450 (CYP450) family proteins play key roles in plant growth, development, stress responses, and other physiological processes. Here, we cloned the cytochrome P450 gene MsCYP71 in alfalfa and found that the expression of MsCYP71 was induced by drought stress. Silencing the MsCYP71 gene using virus-induced gene silencing technology significantly decreased the drought resistance of alfalfa, as indicated by their lower relative water content, net photosynthetic rate, and chlorophyll fluorescence maximum (Fm); further, the heterologous overexpression of MsCYP71 in tobacco significantly enhanced the drought resistance and Fm of transgenic tobacco. Furthermore, the expression of MsCYP71 across 45 alfalfa accessions under drought stress was investigated. A significant positive correlation between drought resistance and MsCYP71 expression was observed. The 45 alfalfa accessions were clustered into four groups, and drought resistance, Fm, and MsCYP71 were higher in group I than in the other groups, indicating that group I accessions can be used as candidate germplasm resources for the breeding of drought-resistant alfalfa varieties. Overall, our findings indicated that MsCYP71 is a positive regulator of drought resistance in alfalfa, and its expression can be used to evaluate the drought resistance of alfalfa.

Establishment of virus-induced gene silencing (VIGS) system in Luffa acutangula using Phytoene desaturase (PDS) and tendril synthesis related gene (TEN)

BackgroundVirus-induced gene silencing (VIGS) is a reverse genetics technology that can efficiently and rapidly identify plant gene functions. Although a variety of VIGS vectors have been successfully used in plants, only a few reports on VIGS technology in Luffa exist.ResultsIn the present study, a new cucumber green mottle mosaic virus (CGMMV)-based VIGS vector, pV190, was applied to establish the CGMMV-VIGS to investigate the feasibility of the silencing system for Luffa. Phytoene desaturase (PDS) gene was initially selected as a VIGS marker gene to construct a recombinant vector. Plants infected with Agrobacterium harboring pV190-PDS successfully induced effective silencing in Luffa, and an effective gene silencing phenotype with obvious photobleaching was observed. To further validate the efficiency, we selected TEN for gene-silencing, which encodes a CYC/TB1-like transcription factor and is involved in tendril development. Luffa plants inoculated with the pV190-TEN exhibited shorter tendril length and nodal positions where tendrils appear are higher compared to those of non-inoculated plants. RT-qPCR showed that the expression levels of PDS and TEN were significantly reduced in the CGMMV-VIGS plants. Moreover, we evaluated the CGMMV-VIGS efficiency in three cucurbits, including cucumber, ridge gourd, and bottle gourd.ConclusionWe successfully established a CGMMV-based VIGS system on ridge gourd and used marker genes to identify the feasibility of the silencing system in Luffa leaves and stems.

Application and Expansion of Virus-Induced Gene Silencing for Functional Studies in Vegetables

Increased consumption of vegetables has been recommended worldwide as a part of a healthy diet; therefore, determining gene function among breeding materials is crucial for vegetable improvement to meet the sustainable development of new vegetable varieties. However, genetic transformation is time-consuming and laborious, which limits the exploration of gene function for various vegetable crops. Virus-Induced Gene Silencing (VIGS) can perform large-scale and rapid gene silencing in plants due to a reduction in the experimental period and its independence from the stable genetic transformation, providing an excellent opportunity for functional research. VIGS can accelerate model plant research and make it easier to analyze gene function and validation in vegetable crops. Moreover, with the advent of technologies such as virus-mediated heterologous protein expression and the development of CRISPR/Cas9 technology, virus-mediated genetic tools have ushered in a new era in genetics and crop improvement. This study summarizes recent achievements in VIGS and Virus-Induced Gene Editing (VIGE) in vegetables. We also identify several challenges in the current state of VIGS technology in vegetables, serving as a guide for future research.

A novel Tartary buckwheat gene FtIST1, associated with salt tolerance, was isolated and identified via over-expression and VIGS

FtIST1 is a novel SBP gene isolated from salt-responsive Tartary buckwheat, and its molecular characteristics and function in salt tolerance are unclear. With the use of DNAMAN and MEGA7.0 software and online analysis program SOPMA, we performed the bioinformatics analysis of the novel FtIST1 gene. We also identified the function of FtIST1 gene via over-expression and VIGS (virus-induced gene silencing). FtIST1 gene was cloned from Tartary buckwheat, and its GenBank accession number is MK799640. FtIST1 belongs to the SBP transcription factor gene family, it is 374 bp long and encodes 99 amino acids. The molecular weight and isoelectric point of FtIST1 protein are 11.7 kDa and 8.78, which is predicted to be a soluble hydrophilic protein. Through analyzing the physiological characteristics of transgenic Arabidopsis, it was found that the activities of antioxidant enzymes (SOD, POD and CAT), chlorophyll content, Fv/Fm, Fv/F0, net photosynthetic rate, fresh weight and dry weight, K+ and Mg2+ content increased significantly in Arabidopsis with over-expression of FtIST1 gene, while the plasmalemma permeability, chlorophyll a fluorescence F0 and Na+ content of leaves decreased significantly, indicating that the over-expression of FtIST1 gene can obviously improve the physiological characteristics of Arabidopsis under salt stress and the salt tolerance of transgenic Arabidopsis. After successfully inhibiting the expression of FtIST1 gene in Tartary buckwheat by VIGS technology, the activities of antioxidant enzymes such as SOD, POD and CAT in Tartary buckwheat decreased significantly, while the plasmalemma permeability and Na+ content of leaves increased significantly, indicating that the loss of FtIST1 gene expression in Tartary buckwheat led to the decrease of antioxidant capacity, the increase of cell membrane damage, the increase of Na+ content in shoot and the obvious decrease of salt tolerance. Our data indicate that FtIST1 is a salt tolerance gene, which is closely associated with salt tolerance of Tartary buckwheat.

Sugar composition and transcriptome analysis in developing ‘Fengtang’ plum (Prunus salicina Lindl.) reveal candidate genes regulating sugar accumulation

Sweetness is an important attribute of fruit quality, which directly affects consumers' preference for fresh fruit and is mostly determined by carbohydrate composition. ‘Fengtang’ plum (Prunus salicina Lindl.) is recognized for its high soluble sugar content, but the sugar composition and the molecular mechanisms underlying sugar overproduction are not fully understood. In this work, the sugar components were analyzed using gas chromatography-mass spectrometry combined with transcription profiles from RNA-sequencing and Quantitative Real-time PCR during fruit development. The target metabolic group showed that sucrose was the dominant sugar component in mature fruit, followed by glucose, fructose, and sorbitol. Based on the transcriptome data and qRT-PCR validation, we identified 12 key structural genes that significantly responded to corresponding component accumulation: sucrose synthase (PsSUS4), sucrose phosphate synthase (PsSPS2), neutral invertase (PsNINV1/3/4), phosphoglucomutase (PsPGM1), UTP-glucose-1-phosphate uridylyl transferase (PsUGP1/2), hexose kinase (PsHXK1/3), sugar transport protein (PsSTP1), and Sugars Will Eventually be Exported Transporter (PsSWEET4). In which PsSUS4 and PsSPS2, whose encoding proteins immediately catalyze sucrose synthesis, were selected to be silenced using the virus-induced gene silencing technology. Silencing of PsSUS4 or PsSPS2 resulted in decreased sucrose content by 27.6% and 8%, respectively, compared with the control, verifying their important roles in sucrose accumulation. Subsequently, sugar metabolism networks in this high-sugar plum were constructed with 12 key structural genes, 72 putative transcription factors, and 4 major sugar components. These results might facilitate a better understanding of the molecular mechanisms of sugar accumulation in ‘Fengtang’ plum and provide a framework for future fruit quality improvement.

Application of virus-induced gene silencing technology to investigate the phytochrome metabolism mechanism: a review

Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.

Research Progress of Virus-Induced Gene Silencing and its Application in Gramineae

With the proliferation of genome sequence information, biological science research has entered the era of big data, but how to annotate the function of genome information has become an important research goal. Virus-induced gene silencing (VIGS) is a powerful tool that has been used to study key genes in multiple plant growth and development processes. This technology uses the innate antiviral defense system of plants. And generates small interfering RNA (siRNA) in the plant by inserting the target gene fragment into the viral vector, so that the transcript of the endogenous gene of the plant becomes a target for degradation, resulting in the expression of the target gene being down-regulated. This paper systematically reviews the development, mechanism, and advantages of VIGS technology, focuses on the vectors that can be applied in grasses, and finally discusses the limitations and development trend of VIGS technology in the application of Gramineae.

Cloning and functional analysis of GhDFR1, a key gene of flavonoid synthesis pathway in naturally colored cotton.

The naturally colored brown cotton fiber is the most widely used environmentally friendly textile material, which primarily contains proanthocyanidins and their derivatives. Many structural genes in the flavonoid synthesis pathway are known to improve the genetic resources of naturally colored cotton. Among them, DFR is a crucial late enzyme to synthesis both anthocyanins and proanthocyanidins in the plant flavonoid pathway. The protein sequences of GhDFRs were analyzed using bioinformatic tools. The expression levels of GhDFRs in various tissues and organs of upland cotton Zongxu1 (ZX1), were analyzed by quantitative real-time PCR, and the expression pattern of GhDFR1 during fiber development of white cotton and brown cotton was analyzed further. The function of GhDFR1 in NCC ZX1 was preliminarily analyzed by virus induced gene silencing (VIGS) technology. Bioinformatic analysis revealed that GhDFRs sequences in upland cotton genome were extremely conserved. Furthermore, evolutionary tree analysis revealed that the functions of GhDFR1 and GhDFR2, and GhDFR3 and GhDFR4, presented different and shared some similarities. Our study showed GhDFR1 and GhDFR2 were specifically expressed in fibers, while GhDFR3 and GhDFR4 were specifically expressed in petals. GhDFR1 was exclusively expressed in brown cotton fiber at various stages of development and progressively increased with the growth of fiber, but the trend of expression in white cotton was quite the opposite. We silenced GhDFR1 expression in brown cotton fiber using VIGS technology, and observed the VIGS-interference plants. After reducing the expression level of GhDFR1, the period for significant GhDFR1 expression in the developing fibers changed, reducing the content of anthocyanins, and lightening the color of mature cotton fibers. GhDFR1 was preferentially expressed in brown cotton during fiber development. The timing of GhDFR1 expression for flavonoid synthesis altered, resulting in anthocyanin contents reduced and the fiber color of the GhDFR1i lines lightened. These findings showed the role of GhDFR1 in fiber coloration of NCC and provided a new candidate for NCC genetic improvement.

PlMAPK1 facilitates growth and photosynthesis of herbaceous peony (Paeonia lactiflora Pall.) under high-temperature stress

Herbaceous peony (Paeonia lactiflora Pall.) is a traditional famous flower in China that prefers cold climates and is not tolerant of high summer temperatures. Mitogen-activated protein kinase (MAPK) is widely involved in plant signal transduction to various environmental stresses. In order to clarify the role of PlMAPK1 in response to high-temperature stress, it was cloned, and its open reading frame was 1215 bp, encoding 405 amino acids. Quantitative real-time PCR (qRT-PCR) analysis showed that when the plant was severely damaged by high temperatures, the expression level of PlMAPK1 was at its peak. Moreover, based on a systematic study of exogenous yeast, it was found that the yeast strain overexpressing PlMAPK1 grew better than the control under high-temperature stress. In addition, PlMAPK1 was transiently silenced by virus induced gene silencing technology (VIGS) in P. lactiflora. And under high-temperature stress, the leaves of transgenic plants wilted significantly, the accumulation of reactive oxygen species increased and the photosynthetic capacity decreased significantly. These findings suggested that PlMAPK1 might play a positive regulatory role in the response of P. lactiflora to high-temperature stress, laying a theoretical groundwork for P. lactiflora high-temperature tolerance breeding.