Transgenic Arabidopsis Thaliana Plants Research Articles

Discovery and functional characterization of novel cotton promoters with potential application to pest control.

pGhERF105andpGhNc-HARBI1promoters are highly responsive to CBW infestation and exhibit strong activity in vegetative and reproductive tissues, increasing their potential application in GM crop plants for pest control. The main challenge to cotton (Gossypium hirsutum) crop productivity is the constant attack of several pests, including the cotton boll weevil (CBW, Anthonomus grandis), which uses cotton floral buds for feeding and egg-laying. The endophytic nature of the early developmental stages of CBW makes conventional pesticide-based control poorly efficient. Most biotechnological assets used for pest control are based on Bacillus thurigiensis insecticidal Cry toxins or the silencing of insect-pest essential genes using RNA-interference technology. However, suitable plant promoter sequences are required to efficientlydrive insecticidal molecules to the target plant tissue. This study selected the Ethylene Responsive Factor 105 (GhERF105) and Harbinger transposase-derivednuclease (GhNc-HARBI1) genes based on available transcriptome-wide data from cotton plants infested by CBW larvae. The GhERF105 and GhNc-HARBI1 genes showed induction kinetics from 2 to 96h under CBW's infestation in cotton floral buds, uncovering the potential application of their promoters. Therefore, the promoter regions (1,500 base pairs) were assessed and characterized using Arabidopsis thaliana transgenic plants. The pGhERF105 and pGhNc-HARBI1 promoters showed strong activity in plant vegetative (leaves and roots) and reproductive (flowers and fruits) tissues, encompassing higher GUS transcriptional activity than the viral-constitutive Cauliflower Mosaic Virus 35S promoter (pCaMV35S). Notably, pGhERF105 and pGhNc-HARBI1 promoters demonstrated more efficiency in driving reporter genes in flowers than other previously characterized cotton flower-specific promoters. Overall, the present study provides a new set of cotton promoters suitable for biotechnological application in cotton plants for pest resistance.

The Wheat Gene TaVQ14 Confers Salt and Drought Tolerance in Transgenic Arabidopsis thaliana Plants.

Wheat is one of the most widely cultivated food crops worldwide, and the safe production of wheat is essential to ensure food security. Soil salinization and drought have severely affected the yield and quality of wheat. Valine-glutamine genes play important roles in abiotic stress response. This study assessed the effect of the gene TaVQ14 on drought and salt stresses resistance. Sequence analysis showed that TaVQ14 encoded a basic unstable hydrophobic protein with 262 amino acids. Subcellular localization showed that TaVQ14 was localized in the nucleus. TaVQ14 was upregulated in wheat seeds under drought and salt stress. Under NaCl and mannitol treatments, the percentage of seed germination was higher in Arabidopsis lines overexpressing TaVQ14 than in wild-type lines, whereas the germination rate was significantly lower in plants with a mutation in the atvq15 gene (a TaVQ14 homolog) than in WT controls, suggesting that TaVQ14 increases resistance to salt and drought stress in Arabidopsis seeds. Moreover, under salt and drought stress, Arabidopsis lines overexpressing TaVQ14 had higher catalase, superoxide dismutase, and proline levels and lower malondialdehyde concentrations than WT controls, suggesting that TaVQ14 improves salt and drought resistance in Arabidopsis by scavenging reactive oxygen species. Expression analysis showed that several genes responsive to salt and drought stress were upregulated in Arabidopsis plants overexpressing TaVQ14. Particularly, salt treatment increased the expression of AtCDPK2 in these plants. Moreover, salt treatment increased Ca2+ concentrations in plants overexpressing TaVQ14, suggesting that TaVQ14 enhances salt resistance in Arabidopsis seeds through calcium signaling. In summary, this study demonstrated that the heterologous expression of TaVQ14 increases the resistance of Arabidopsis seeds to salt and drought stress.

Toxicity and degradation of 2,4,6-trinitrotoluene in transgenic Arabidopsis expressing Citrobacter freundii nitroreductase

The degradation of organic pollutants in plants involves uptake and diffusion through the roots, trunk or leaves, transformation, accumulation and/or volatilization of soil- and aqueous-phase contaminants. 2,4,6-trinitrotoluene (TNT), the most widely used explosive, is toxic to a number of photosynthetic organisms, poses hazards to human health and pollutes the environment because it is recalcitrant to degradation. In this study, we investigated the nitroreductase gene from Citrobacter freundii in transgenic Arabidopsis thaliana plants. Transgenic plants showed a promising ability to tolerate, take up and detoxify TNT. Our results suggest that transgenic plants show higher potential for removing and transformation of TNT. The expression of nitroreductase in Arabidopsis could be useful for phytoremediation and could be explored for the effective cleanup of TNT-contaminated sites.

Host induced gene silencing of Sclerotinia sclerotiorum effector genes for the control of white mold

Sclerotinia sclerotiorum (Lib.), the causal agent of white mold, is a necrotrophic fungus with worldwide distribution. This fungus can infect more than 600 agricultural crops, causing damage worth hundreds of millions of dollars annually. The control of white mold is usually performed using integrated management practices. Nevertheless, after the establishment of the disease, chemical compounds need to be used, increasing production costs and offering environmental risks. In this study, we used Host Induced Gene Silencing (HIGS) in an attempt to control white mold. Specific vectors were constructed to express a silencing hairpin (dsRNAs) of the pathogen effector genes Ss-caF1 (putative Ca2+ binding protein), SspG1d (endopolygalacturonase) and SsiTL (integrin). The results showed a reduction in the severity of the symptoms in Arabidopsis thaliana transgenic plants and a delay in the occurrence of early symptoms. The results obtained for the control of S. sclerotiorum support the potential of HIGS in the generation of plants resistant to phytopathogenic fungi. It is also possible to suggest that the effector genes Ss-caF1, SspG1d and SsiTL are not only involved in the interaction but also play important roles during the host colonization and infection process.

Transcriptomic Analysis of the Interaction Between FLOWERING LOCUS T Induction and Photoperiodic Signaling in Response to Spaceflight.

Spaceflight has an impact on the growth and development of higher plants at both the vegetative stage and reproductive stage. A great deal of information has been available on the vegetative stage in space, but relatively little is known about the influence of spaceflight on plants at the reproductive stage. In this study, we constructed transgenic Arabidopsis thaliana plants expressing the flowering control gene, FLOWERING LOCUS T (FT), together with the green fluorescent protein gene (GFP) under control of a heat shock-inducible promoter (HSP17.4), by which we induced FT expression inflight through remote controlling heat shock (HS) treatment. Inflight photography data showed that induction of FT expression in transgenic plants in space under non-inductive short-day conditions could promote flowering and reduce the length of the inflorescence stem in comparison with that of wild-type plants under the same conditions. Whole-genome microarray analysis of gene expression changes in leaves of wild-type and these transgenic plants grown under the long-day and short-day photoperiod conditions in space indicated that the function of the photoperiod-related spaceflight responsive genes is mainly involved in protein synthesis and post-translation protein modulation, notably protein phosphorylation. In addition, changes of the circadian component of gene expression in response to spaceflight under different photoperiods indicated that roles of the circadian oscillator could act as integrators of spaceflight response and photoperiodic signals in Arabidopsis plants grown in space.

Characterization of a strong constitutive promoter from paper mulberry vein banding virus.

Paper mulberry vein banding virus (PMVBV), a member of the genus Badnavirus in the family Caulimoviridae, infects paper mulberry (Broussonetia papyrifera), a dicotyledonous plant. Putative promoter regions in the PMVBV genome were tested using recombinant plant expression vectors, revealing that the promoter activity of three genome fragments was about 1.5-fold higher than that of the 35S promoter of cauliflower mosaic virus in Nicotiana benthamiana. In transformed transgenic Arabidopsis thaliana plants, these promoter constructs showed constitutive expression. Based on the activity and gene expression patterns of these three promoter constructs, a fragment of 384 bp (named PmVP) was deduced to contain the full-length promoter of the PMVBV genome. The results suggest that the PMVBV-derived promoter can be used for the constitutive expression of transgenes in dicotyledonous plants.

Overexpression of \u03b3-glutamylcysteine synthetase gene from Caragana korshinskii decreases stomatal density and enhances drought tolerance

BackgroundGamma-glutamylcysteine synthetase (γ-ECS) is a rate-limiting enzyme in glutathione biosynthesis and plays a key role in plant stress responses. In this study, the endogenous expression of the Caragana korshinskiiγ-ECS (Ckγ-ECS) gene was induced by PEG 6000-mediated drought stress in the leaves of C. korshinskii. and the Ckγ-ECS overexpressing transgenic Arabidopsis thaliana plants was constructed using the C. korshinskii. isolated γ-ECS.ResultsCompared with the wildtype, the Ckγ-ECS overexpressing plants enhanced the γ-ECS activity, reduced the stomatal density and aperture sizes; they also had higher relative water content, lower water loss, and lower malondialdehyde content. At the same time, the mRNA expression of stomatal development-related gene EPF1 was increased and FAMA and STOMAGEN were decreased. Besides, the expression of auxin-relative signaling genes AXR3 and ARF5 were upregulated.ConclusionsThese changes suggest that transgenic Arabidopsis improved drought tolerance, and Ckγ-ECS may act as a negative regulator in stomatal development by regulating the mRNA expression of EPF1 and STOMAGEN through auxin signaling.

Determining the Phase Separation Characteristics of Plant Proteins.

Eukaryotic cells contain numerous membrane-bound and membraneless organelles that provide spatiotemporal control for diverse biological processes. The liquid-liquid phase separation of proteins has been proposed as the driving force behind the formation of membraneless organelles. Here, we describe a method to determine the phase separation activities of proteins in plants. This basic method includes protocols for an in vivo fluorescence recovery after photobleaching assay in Nicotiana benthamiana using transient expression, an in vitro liquid droplet reconstitution assay using purified recombinant proteins, and an in vivo fluorescence recovery after photobleaching assay in Arabidopsis thaliana using stable transgenic plants. With these assays, the phase separation characteristics of a protein can be determined. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Detection of protein phase separation activities in N. benthamiana Support Protocol: Fluorescence recovery after photobleaching assay Basic Protocol 2: Detection of protein phase separation in vitro Basic Protocol 3: Detection of protein phase separation in stable transgenic Arabidopsis thaliana plants.

The Effect of the Anticipated Nuclear Localization Sequence of 'Candidatus Phytoplasma mali' SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor.

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, which results in a crinkled leaf and silique phenotype in transgenic A. thaliana. Moreover, we provide evidence that the SAP11-like protein has a destabilizing effect on some TCPs in vivo.

A rice transcription factor, OsMADS57, positively regulates high salinity tolerance in transgenic Arabidopsis thaliana and Oryza sativa plants.

MADS-box transcription factors (TFs) play indispensable roles in various aspects of plant growth, development as well as in response to environmental stresses. Several MADS-box genes have been reported to be involved in the salt tolerance in different plant species. However, the role of the transcription factor OsMADS57 under salinity stress is still unknown. Here, the results of this study showed that OsMADS57 was mainly expressed in roots and leaves of rice plants (Oryza sativa). Gene expression pattern analysis revealed that OsMADS57 was induced by NaCl. Overexpression of OsMADS57 in both Arabidopsis thaliana (A. thaliana) and rice could improve their salt tolerance, which was demonstrated by higher germination rates, longer root length and better growth status of overexpression plants than wild type (WT) under salinity conditions. In contrast, RNA interference (RNAi) lines of rice showed more sensitivity towards salinity. Moreover, less reactive oxygen species (ROS) accumulated in OsMADS57 overexpressing lines when exposed to salt stress, as measured by 3, 3'-diaminobenzidine (DAB) or nitroblue tetrazolium (NBT) staining. Further experiments exhibited that overexpression of OsMADS57 in rice significantly increased the tolerance ability of plants to oxidative damage under salt stress, mainly by increasing the activities of antioxidative enzymes such as superoxide dismutase (SOD) and peroxidase (POD), reducing malonaldehyde (MDA) content and improving the expression of stress-related genes. Taken together, these results demonstrated that OsMADS57 plays a positive role in enhancing salt tolerance by activating the antioxidant system.

Isolation and Characterization of Endomycorrhizal Fungi Associated with Growth Promotion of Blueberry Plants.

Despite their notable root mutualism with blueberries (Vaccinium spp.), studies related to Ericoid mycorrhizal (ERM) are relatively limited. In this study, we report the isolation of 14 endomycorrhizal fungi and their identification by fungal colony morphology characterization combined with PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. Six of the isolated strains were confirmed as beneficial mycorrhizal fungi for blueberry plants following inoculation. We observed the formation of typical ERM hyphae coil structures—which promote and nutritionally support growth—in blueberry seedlings and significant nitrogen and phosphorous content increases in diverse tissues. QRT-PCRs confirmed changes in VcPHT1s expression patterns. After the formation of ERM, PHT1-1 transcription in roots was upregulated by 1.4- to threefold, whilst expression of PHT1-3 and PHT1-4 in roots were downregulated 72% and 60%, respectively. Amino acid sequence analysis of all four VcPHT1s genes from the blueberry variety “Sharpblue” revealed an overall structural similarity of 67% and predicted transmembrane domains. Cloning and overexpression of PHT1-1 and PHT1-3 genes in transgenic Arabidopsis thaliana plants significantly enriched total phosphorus and chlorophyll content, confirming that PHT1-1 and PHT1-3 gene functions are associated with the transport and absorption of phosphorus.

Biochemical and functional characterization of two microbial type terpene synthases from moss Stereodon subimponens

Terpenes constitute a large class of plant secondary metabolites. Usually, there is only one type of terpene synthase in seed plants, which is called typical plant terpene synthase. Currently, as a new family of plant terpene synthases, microbial terpene synthase-like (MTPSL) is identificated in nonseed plants. However, our knowledge about the biological function of most MTPSLs in nonseed plants is very limited. Here, we investigated the biochemical and functional characterization of the enzymes encoded by two MTPSLs from moss Stereodon subimponens, SsMTPSL1 and SsMTPSL2. A phylogenetic tree analysis showed that SsMTPSL1 and SsMTPSL2 are homologous to AaMTPSL1, AaMTPSL3, ApMTPSL1, and ApMTPSL3 from hornworts. The enzyme activity experiment demonstrated that SsMTPSL1 has monoterpene synthase and sesquiterpene synthase activity, and SsMTPSL2 has monoterpene synthase activity. Next, we selected SsMTPSL1 to study its biochemical functions. Anti-bacterial activity test in vitro showed that the products of SsMTPSL1 have an anti-bacterial effect on Escherichia coli, Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000), and Staphylococcus aureus. To further understand the function of SsMTPSL1, the transgenic Arabidopsis thaliana plant of SsMTPSL1 is inoculated by Pst DC3000, and the result showed that SsMTPSL1 enhances the resistance of A. thaliana to Pst DC3000. All in all, this study provides new information about the functions of moss MTPSLs.

Preferential ammonium: nitrate ratio of blueberry is regulated by nitrogen transport and reduction systems

Blueberry (Vaccinium spp., Vc) is inefficient in uptake and utilizing a single nitrogen (N) form (ammonium or nitrate), and prefers the presence of both N forms; however, its preferential ammonium: nitrate ratios and the underlying molecular mechanism remain elusive. To reveal the mechanism of blueberry preference in the composition of N forms, blueberries had grown over three months in pure coconut bran irrigated with modified Hoagland nutrient solution containing ammonium N (NH4+-N) alone or nitrate N (NO3−-N) alone at two pHs (5.0 and 7.5), and NH4+-N:NO3−-N ratios of 4:1, 2:1 and 1:1 at pH 5.0. After three months, the physiological responses and the expression of key genes involved in N metabolism were analyzed. Afterwards, the genes were then genetically transformed into Arabidopsis thaliana for functional verification. The results showed that (i) plants grew better at pH 5.0 than pH 7.5; (ii) at pH 5.0, when NH4+-N:NO3−-N ratio was applied at 2:1, plant growth was at the best performance (i.e., 11% higher in the crown width compared to the control(nil-N applied). In the presence of both NH4+ and NO3−, the transcriptional levels of VcAMT3, VcNRT1.5, VcNRT2, VcNR and VcNiR were significantly upregulated. The highest value appeared in the 2:1 of NH4+-N:NO3−-N ratio where, VcAMT3 and VcNRT2 were raised by 4 and 5 times, respectively. Hence, the results suggested that the mixed N forms at 2:1 NH4+-N:NO3−-N ratio at pH 5.0 induced the expression of the related gene, which may have increased root absorption and transportation of NH4+-N and NO3−-N, resulting in the better plant growth; and (iii) under NH4+-N alone treatments, the transgenic VcNiR Arabidopsis thaliana plants were the only survival; under NO3−-N alone treatments, compared to the wild-type plants, the transgenic VcAMT3 Arabidopsis thaliana had 11 and 2 times higher in the plant height and leaf disc size. The results showed that VcAMT3, VcNRT2, VcNiR genes could improve the nitrogen absorption ability of Arabidopsis thaliana plants. Our study improved the understanding of the underlying molecular mechanism of N speciation preference of blueberry, and the findings can be a great guidance for better N fertilizer management to promote blueberry productivity.

Gateway cloning and in-planta transformation of drought stress responsive Ecmyb1 gene isolated from Eleusine coracana var.PRM 6107

Drought is one of the key abiotic stress that critically influences the crops by restraining their growth and yield potential. Being sessile, plants tackle the detrimental effects of drought stress via modulating the cellular state by changing the gene expression. Such alteration of gene expression is essentially driven by the transcriptional syndicate. Transcription factors (TF) are the key regulatory protein that controls the expression of their target gene by binding to the cis-regulatory elements present in the promoter region. Myb-TFs ubiquitously present in all eukaryotes belong to one of the largest TF family, and play wide array of biological functions in plants including anthocyanin biosynthesis, vasculature system, cell signaling, seed maturation and abiotc stress responses. In the present study the full length Myb TF from Eleusine corocana was subcloned using Gateway cloning system and further transformed into Arabidopsis thaliana through floral dip method. Transgenic Arabidopsis thaliana plants harbouring Ecmyb1 gene were screened and grown in transgenic glasshouse under controlled conditions.

Functional analysis and expression patterns of members of the FLOWERING LOCUS T (FT) gene family in Lilium

Lilium is an important commercial flowering species, and there are many varieties and more than 100 species of wild Lilium. Lilium × formolongi is usually propagated from seedlings, and the flowering of these plants is driven mainly by the photoperiodic pathway. Most of the other lily plants are propagated via bulblets and need to be vernalized; these plants can be simply divided into pretransplantation types and posttransplantation types according to the time at which the floral transition occurs. We identified three Lilium FLOWERING LOCUS T (LFT) family members in 7 Lilium varieties, and for each gene, the coding sequence of the different varieties was identical. Among these genes, the LFT1 gene of Lilium was most homologous to the AtFT gene, which promotes flowering in Arabidopsis. We analyzed the expression patterns of LFT genes in Lilium × formolongi seedlings and in different Lilium varieties, and the results showed that LFT1 and LFT3 may promote floral induction. Compared with LFT3, LFT1 may have a greater effect on floral induction in Lilium, which is photoperiod sensitive, while LFT3 may play a more important role in the floral transition of lily plants, which have a high requirement for vernalization. LFT2 may be involved in the differentiation of bulblets, which was verified by tissue culture experiments, and LFT1 may have other functions involved in promoting bulblet growth. The functions of LFT genes were verified by the use of transgenic Arabidopsis thaliana plants, which showed that both the LFT1 and LFT3 genes can promote early flowering in Arabidopsis. Compared with LFT3, LFT1 promoted flowering more obviously, and thus, this gene could be an important promoter of floral induction in Lilium.

Volatile DMNT directly protects plants against Plutella xylostella by disrupting the peritrophic matrix barrier in insect midgut.

Insect pests negatively affect crop quality and yield; identifying new methods to protect crops against insects therefore has important agricultural applications. Our analysis of transgenic Arabidopsis thaliana plants showed that overexpression of pentacyclic triterpene synthase 1, encoding the key biosynthetic enzyme for the natural plant product (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), led to a significant resistance against a major insect pest, Plutella xylostella. DMNT treatment severely damaged the peritrophic matrix (PM), a physical barrier isolating food and pathogens from the midgut wall cells. DMNT repressed the expression of PxMucin in midgut cells, and knocking down PxMucin resulted in PM rupture and P. xylostella death. A 16S RNA survey revealed that DMNT significantly disrupted midgut microbiota populations and that midgut microbes were essential for DMNT-induced killing. Therefore, we propose that the midgut microbiota assists DMNT in killing P. xylostella. These findings may provide a novel approach for plant protection against P. xylostella.

Dual function of VvWRKY18 transcription factor in the β-aminobutyric acid-activated priming defense in grapes.

Induction of phytoalexin production after invading pathogens is recognized as an essential aspect of the plant-induced resistance. The WRKY family includes plant-specific transcriptional factors associated with plant defense responses, but the comprehensive mechanisms are poorly understood. Here, we attempted to elaborate the regulatory function of VvWRKY18 from the group IIa of WRKY transcription factor (TF) from Vitis vinifera, in the regulation of β-aminobutyric acid (BABA)-activated stilbene phytoalexins biosynthesis and PATHOGENESIS-RELATED (PR) genes expressions in grapes. BABA at 10 mmol L-1 triggered a priming protection in grapes and conferred a potentiation of the expression levels of VvWRKY18, VvNPR1, and several salicylic acid (SA)-responsive genes, which was accompanied by enhanced stilbene production upon Botrytis cinerea infection. In addition, a physical interaction between VvWRKY18 and the regulatory protein VvNPR1 was detected in vivo and in vitro by yeast-2-hybrid (Y2H), pull-down and co-immunoprecipitation assay (Co-IP) assays. Furthermore, yeast-1-hybrid (Y1H) and dual-luciferase reporter (DLR) assays indicated that VvWRKY18 activated the transcription of STILBENE SYNTHASE (STS) genes, including VvSTS1 and VvSTS2, by directly binding the W-box elements within the specific promoters and resultantly enhancing stilbene phytoalexins biosynthesis. Further investigation demonstrated that heterologous expression of VvWRKY18 elevated the transcriptions of STS and PR genes, thus contributing to potentiating the defense of transgenic Arabidopsis thaliana plants and resultantly inhibiting B. cinerea invasion. Hence, VvWRKY18 serves as a singular effector involved in the synthesis of stilbene phytoalexins in grapes and its interaction with VvNPR1 provided DNA binding ability required for VvNPR1 to initiate systemic acquired resistance (SAR) defense.

Genome-Wide Analysis of Ribosomal Protein GhRPS6 and Its Role in Cotton Verticillium Wilt Resistance.

Verticillium wilt is threatening the world’s cotton production. The pathogenic fungus Verticillium dahliae can survive in the soil in the form of microsclerotia for a long time, colonize through the root of cotton, and invade into vascular bundles, causing yellowing and wilting of cotton leaves, and in serious cases, leading to plant death. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. In previous studies, proteomic analysis was carried out on different cotton varieties inoculated with V. dahliae strain Vd080. It was found that GhRPS6 was phosphorylated after inoculation, and the phosphorylation level in resistant cultivars was 1.5 times than that in susceptible cultivars. In this study, knockdown of GhRPS6 expression results in the reduction of SA and JA content, and suppresses a series of defensive response, enhancing cotton plants susceptibility to V. dahliae. Overexpression in Arabidopsis thaliana transgenic plants was found to be more resistant to V. dahliae. Further, serines at 237 and 240 were mutated to phenylalanine, respectively and jointly. The transgenic Arabidopsis plants demonstrated that seri-237 compromised the plant resistance to V. dahliae. Subcellular localization in Nicotiana benthamiana showed that GhRPS6 was localized in the nucleus. Additionally, the pathogen inoculation and phosphorylation site mutation did not change its localization. These results indicate that GhRPS6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.

The NAC-type transcription factor CaNAC46 regulates the salt and drought tolerance of transgenic Arabidopsis thaliana

BackgroundThe NAC (NAM, ATAF1/ATAF2, and CUC2) transcription factors belong to a large family of plant-specific transcription factors in monocot and dicot species. These transcription factors regulate the expression of stress tolerance-related genes that protect plants from various abiotic stresses, including drought, salinity, and low temperatures.ResultsIn this study, we identified the CaNAC46 transcription factor gene in Capsicum annuum. Its open reading frame was revealed to comprise 921 bp, encoding a protein consisting of 306 amino acids, with an isoelectric point of 6.96. A phylogenetic analysis indicated that CaNAC46 belongs to the ATAF subfamily. The expression of CaNAC46 was induced by heat, cold, high salt, drought, abscisic acid, salicylic acid, and methyl jasmonate treatments. Thus, CaNAC46 may be important for the resistance of dry pepper to abiotic stresses. A subcellular localization analysis confirmed that CaNAC46 is localized in the nucleus. The overexpression of CaNAC46 improved the tolerance of transgenic Arabidopsis thaliana plants to drought and salt stresses. The CaNAC46-overexpressing lines had longer roots and more lateral roots than wild-type lines under prolonged drought and high salt stress conditions. Additionally, CaNAC46 affected the accumulation of reactive oxygen species (ROS). Moreover, CaNAC46 promoted the expression of SOD, POD, RD29B, RD20, LDB18, ABI, IAA4, and P5CS. The malondialdehyde contents were higher in TRV2-CaNAC46 lines than in wild-type plants in response to drought and salt stresses. Furthermore, the expression levels of stress-responsive genes, such as ABA2, P5CS, DREB, RD22, CAT, and POD, were down-regulated in TRV2-CaNAC46 plants.ConclusionsUnder saline and drought conditions, CaNAC46 is a positive regulator that activates ROS-scavenging enzymes and enhances root formation. The results of our study indicate CaNAC46 is a transcriptional regulator responsible for salinity and drought tolerance and suggest the abiotic stress-related gene regulatory mechanisms controlling this NAC transcription factor are conserved between A. thaliana and pepper.

Expression of the Medicago truncatula MtDof32 transcription factor regulates plant growth and enhances abiotic stress tolerances in transgenic Arabidopsis

The DNA-binding one Zinc Finger (Dof) protein is an important transcription factor unique in plants, which plays important roles in regulating plant biological rhythm, flowering and nitrogenium storage. In this study, the MtDof32 gene was isolated from Medicago truncatula and the gene function was characterized in trangenic plants. The expression pattern exhibited that MtDof32 gene might be involved in stress responses and tolerances. Transgenic Arabidopsis thaliana plants were obtained by agrobacterium tumefaciens-mediated transformation. Compared with wild-type plants, the MtDof32 transgenic Arabidopsis showed reduced total branch number by 40 % on average, and increased rosettes number, the number gap was growing with plant growth. These phenotypes have never been found in previous studies. In addition, transgenic plants also showed delayed flowering, enlarged flower and leaf organs, and enhanced tolerances to osmotic and salt stresses. The expression level of some branching and flowering regulation genes in transgenic plants changed significantly, compared to those of wild-type plants. Taken together, these results demonstrated novel functions of MtDof32 in reducing branch number and increasing rosettes number, which is beneficial for further studies about the plant growth and development regulation network mediated by Dof protein family.