Poplar glutathione S-transferase PtrGSTF8 contributes to reactive oxygen species scavenging and salt tolerance.

Glutathione S-transferases (GSTs) constitute a protein superfamily encoded by a large gene family and play a crucial role in plant growth and development. However, their precise functions in wood plant responses to abiotic stress are not fully understood. In this study, we isolated a Phi class glutathione S-transferase-encoding gene, PtrGSTF8, from poplar (Populus alba × P. glandulosa), which is significantly up-regulated under salt stress. Moreover, compared with wild-type (WT) plants, transgenic tobacco plants exhibited significant salt stress tolerance. Under salt stress, PtrGSTF8-overexpressing tobacco plants showed a significant increase in plant height and root length, and less accumulation of reactive oxygen species. In addition, these transgenic tobacco plants exhibited higher superoxide dismutase, peroxidase, and catalase activities and reduced malondialdehyde content compared with WT plants. Quantitative real-time PCR experiments showed that the overexpression of PtrGSTF8 increased the expression of numerous genes related to salt stress. Furthermore, PtrMYB108, a MYB transcription factor involved in salt resistance in poplar, was found to directly activate the promoter of PtrGSTF8, as demonstrated by yeast one-hybrid assays and luciferase complementation assays. Taken together, these findings suggest that poplar PtrGSTF8 contributes to enhanced salt tolerance and confers multiple growth advantages when overexpressed in tobacco.

LpNAC5 positively regulates drought, salt and alkalinity tolerance of Lilium pumilum.

NACs (NAM、ATAF1/2、CUC1/2), as a large family of plant transcription factors, are widely involved in abiotic stress responses. This study aimed to isolate and clone a novel stress-responsive transcription factor LpNAC5 from Lilium pumilum bulbs. Drought, salt, alkali, and ABA stresses induced the expression of LpNAC5. Transgenic tobacco plants overexpressing LpNAC5 were generated using the Agrobacterium-mediated method to understand the role of this factor in stress response. These plants exhibited increased tolerance to drought, salt, and alkali stresses. The tobacco plants overexpressing LpNAC5 showed strong drought, salt, and alkaline tolerance. Under the three abiotic stresses, the activities of antioxidant enzymes were enhanced, the contents of proline and chlorophyll increased, and the contents of malondialdehyde decreased. The functional analysis revealed that LpNAC5 enabled plants to positively regulate drought and salt stresses. These findings not only provided valuable insights into stress tolerance mechanisms in L. pumilum but also offered a potential genetic resource for breedi.

Genome-Wide and Expression Pattern Analysis of the HIT4 Gene Family Uncovers the Involvement of GHHIT4_4 in Response to Verticillium Wilt in Gossypium hirsutum.

Chromatin remodelers are essential for regulating plant growth, development, and responses to environmental stresses. HIT4 (HEAT-INTOLERANT 4) is a novel stress-induced chromatin remodeling factor that has been less studied in abiotic stress and stress resistance, particularly in cotton. In this study, we conducted a comprehensive analysis of the members of the HIT4 gene family in Gossypium hirsutum using bioinformatics methods, including phylogenetic relationships, gene organization, transcription profiles, phylogenetic connections, selection pressure, and stress response. A total of 18 HIT4 genes were identified in four cotton species, with six HIT4 gene members in upland cotton. Based on the evolutionary relationships shown in the phylogenetic tree, the 18 HIT4 protein sequences were classified into four distinct subgroups. Furthermore, we conducted chromosome mapping to determine the genomic locations of these genes and visually represented the structural characteristics of HIT4 in G. hirsutum. In addition, we predicted the regulatory elements in HIT4 in G. hirsutum and conducted an analysis of repetitive sequences and gene collinearity among HIT4 in four cotton species. Moreover, we calculated the Ka/Ks ratio for homologous genes to assess the selection pressure acting on HIT4. Using RNA-seq, we explored the expression patterns of HIT4 genes in G. hirsutum and Gossypium barbadense. Through weighted gene co-expression network analysis (WGCNA), we found that GHHIT4_4 belonged to the MEblue module, which was mainly enriched in pathways such as DNA replication, phagosome, pentose and glucuronate interconversions, steroid biosynthesis, and starch and sucrose metabolism. This module may regulate the mechanism of upland cotton resistance to Verticillium wilt through DNA replication, phagosome, and various metabolic pathways. In addition, we performed heterologous overexpression of GH_D11G0591 (GHHIT4_4) in tobacco, and the results showed a significant reduction in disease index compared to the wild type, with higher expression levels of disease resistance genes in the transgenic tobacco. After conducting a VIGS (virus-induced gene silencing) experiment in cotton, the results indicated that silencing GHHIT4_4 had a significant impact, the resistance to Verticillium wilt weakened, and the internode length of the plants significantly decreased by 30.7% while the number of true leaves increased by 41.5%. qRT-PCR analysis indicated that GHHIT4_4 mainly enhanced cotton resistance to Verticillium wilt by indirectly regulating the PAL, 4CL, and CHI genes. The subcellular localization results revealed that GHHIT4_4 was predominantly distributed in the mitochondria and nucleus. This study offers preliminary evidence for the involvement of the GHHIT4_4 in cotton resistance to Verticillium wilt and lays the foundation for further research on the disease resistance mechanism of this gene in cotton.

Overexpression of the NbZFP1 encoding a C3HC4-type zinc finger protein enhances antiviral activity of Nicotiana benthamiana.

Viral diseases are crucial determinants affecting tobacco cultivation, leading to a substantial annual decrease in production. Previous studies have demonstrated the regulatory function of the C3HC4 family of plant zinc finger proteins in combating bacterial diseases. However, it remains to be clarified whether this protein family also plays a role in regulating resistance against plant viruses. In this study, the successful cloning of the zinc finger protein coding gene NbZFP1 from Nicotiana benthamiana has been achieved. The full-length coding sequence of NbZFP1 is 576 bp. Further examination and analysis of this gene revealed its functional properties. The induction of NbZFP1 transcription in N. benthamiana has been observed in response to TMV, CMV, and PVY. Transgenic N. benthamiana plants over-expressing NbZFP1 demonstrated a notable augmentation in the production of chlorophyll a (P < 0.05). Moreover, NbZFP1-overexpressing tobacco exhibited significant resistance to TMV, CMV, and PVY, as evidenced by a decrease in virus copies (P < 0.05). In addition, the defense enzymes activities of PAL, POD, and CAT experienced a significant increase (P < 0.05). The up-regulated expression of genes of NbPAL, NbNPR1 and NbPR-1a, which play a crucial role in SA mediated defense, indicated that the NbZFP1 holds promise in enhancing the virus resistance of tobacco plant. Importantly, the results demonstrate that NbZFP1 can be considered as a viable candidate gene for the cultivation of crops with enhanced virus resistance.

GhTPPA_2 enhancement of tobacco sugar accumulation and drought tolerance.

Trehalose metabolism plays an important role in plant growth and response to abiotic stress. Trehalose-6-phosphate (Tre6P) can help regulate sugar homeostasis and act as an indication signal for intracellular sugar levels. Crop productivity can be greatly increased by altering the metabolic level of endogenous trehalose in plants, which can optimize the source-sink connection. In this study, the upland cotton GhTPP protein family was first homologously compared and 24 GhTPP genes were found. Transcriptome analysis revealed that GhTPP members had obvious tissue expression specificity. Among them, GhTPPA_2 (Gh_A12G223300.1) was predominantly expressed in leaves and bolls. The results of subcellular localization showed that GhTPPA_2 is localized in the chloroplast. Via PlantCare, we analyzed the promoters and found that the expression of GhTPPA_2 may be induced by light, abiotic stress, and hormones such as abscisic acid, ethylene, salicylic acid and jasmonic acid. In addition, GhTPPA_2 was overexpressed (TPPAoe) in tobacco, and we found that the TPPase activity of TPPAoe tobacco increased by 66 %. Soluble sugar content increased by 39 % and starch content increased by 27 %. Whereas, the transgenic tobacco had obvious growth advantages under 100 mM mannitol stress. Transcriptome sequencing results showed that the differential genes between TPPAoe and control were considerably enriched in functions related to photosynthesis, phosphate group metabolism, and carbohydrate metabolism. This study shows that GhTPPA_2 is involved in regulating sugar metabolism, improving soluble sugar accumulation and drought stress tolerance of tobacco, which provides theoretical basis for research on high yield and drought tolerance of crops.

Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3'5'H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3'5'H. In this study, PgF3'5'H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3'5'H showed a higher expression level in all PgF3'5'H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3'5'H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3'5'H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3'5'H products have F3'5'H enzyme activity. In addition, PgF3'5'H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3'5'H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.

Identification of Dioscorea opposite Thunb. CDPK gene family reveals that DoCDPK20 is related to heat resistance.

Temperature affects the growth and yield of yam (Dioscorea opposite Thunb.), and calcium-dependent protein kinases (CDPKs) play an important role in the plant stress response. However, there has been a lack of system analyses of yam's CDPK gene family. In this study, 29 CDPK transcriptome sequences with complete open reading frames (ORFs) were identified from yam RNA sequencing data. The sequences were classified into four groups (I-VI) using phylogenetic analysis. Two DoCDPK genes were randomly selected from each group and the gene patterns of yam leaves were determined using quantitative real-time PCR (qRT-PCR) under high and low temperature stress in order to show their unique functions in mediating specific responses. Among them, DoCDPK20 was significantly induced in high temperatures. The pPZP221-DoCDPK20 was transformed into tobacco leaves using an agrobacterium-mediated method. Under high temperature stress, DoCDPK20 overexpression reduced photosynthesis and improved heat tolerance in transgenic tobacco. Our research offers meaningful perspectives into CDPK genes and new avenues for the genetic engineering and molecular breeding of yam.

Cloning and functional analysis prohibitins protein-coding gene EuPHB1 in Eucommia ulmoides Oliver.

As multifunctional proteins, prohibitins(PHBs) participate in many cellular processes and play essential roles in organisms. In this study, using rapid amplification of cDNA end (RACE) technology, EuPHB1 was cloned from Eucommia ulmoides Oliver (E. ulmoides). A subcellular localization assay preliminarily located EuPHB1 in mitochondria. Then EuPHB1 was transformed into tobacco, and phenotype analyses showed that overexpression of EuPHB1 caused leaves to become chlorotic and shrivel. Furthermore, genes related to hormone and auxin signal transduction, auxin binding, and transport, such as ethylene-responsive transcription factor CRF4-like and ABC transporter B family member 11-like, were significantly inhibited in response to EuPHB1 overexpression. Its overexpression disturbs the original signal transduction pathway, thus causing the corresponding phenotypic changes in transgenic tobacco. Indeed, such overexpression caused fading of palisade tissue and an increase in the number of certain mesophyll cells. It also increased adenosine triphosphate (ATP) synthase activity, mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS) levels in cells. Our results suggest that EuPHB1 expression promotes cellular energy metabolism by accelerating the oxidative phosphorylation of the mitochondrial respiratory chain. Elevated levels of EuPHB1 in the mitochondria, which helps supply the extra energy required to support rapid rates of cell division.

A pathogenesis-related protein 1 of Cucurbita moschata responds to powdery mildew infection.

Pumpkin (Cucurbita moschata Duch.) productivity is severely hindered by powdery mildew (PM) worldwide. The causative agent of pumpkin PM is Podosphaera xanthii, a biotrophic fungus. Pathogenesis-related protein 1 (PR1) homolog was previously identified from transcriptomic analysis of a PM-resistant pumpkin. Here, we investigated the effects of CmPR1 gene from pumpkin for resistance to PM. Subcellular localization assay revealed that CmPR1 is a cytoplasmic protein in plants. The expression of CmPR1 gene was strongly induced by P. xanthii inoculation at 48 h and exogenous ethylene (ET), jasmonic acid (JA) and NaCl treatments, but repressed by H2O2 and salicylic acid (SA) treatments. Visual disease symptoms, histological observations of fungal growth and host cell death, and accumulation of H2O2 in transgenic tobacco plants indicated that CmPR1 overexpression significantly enhanced the resistance to Golovinomyces cichoracearum compared to wild type plants during PM pathogens infection, possibly due to inducing cell death and H2O2 accumulation near infected sites. The expression of PR1a was significantly induced in transgenic tobacco plants in response to G. cichoracearum, suggesting that CmPR1 overexpression positively modulates the resistance to PM via the SA signaling pathway. These findings indicate that CmPR1 is a defense response gene in C. moschata and can be exploited to develop disease-resistant crop varieties.

Identification of the inducible activity in the promoter of the soybean BBI-DII gene exposed to abiotic stress or abscisic acid.

The expression of the soybean Bowman-Birk proteinase isoinhibitor DII (BBI-DII) gene and the inducible activity of its promoter were studied under salt, drought, low temperature, and abscisic acid (ABA) exposure conditions. The BBI-DII gene was induced by salt, drought, low temperature, and ABA, and the relative expression levels were 103.09-, 107.01-, 17.25- and 27.24-fold, respectively, compared with the untreated control. The putative promoter, designated BP1 (- 1255 to + 872 bp), located 5'-upstream of the BBI-DII gene was cloned. The expression of the GUS gene in pCAM-BP1 transgenic tobacco plants was highest at 5 h after treatment with salt, drought, low temperature and ABA, especially under salt and drought. Using histochemical staining and fluorescence analysis of GUS, BP1 activity under salt and drought conditions after 5 h was 1.03 and 1.07-fold, respectively, compared with that of the CaMV35S promoter. Based on a 5' deletion analysis, the segment (+ 41 to + 474 bp) was the basal region that responded to salt and drought, whereas the segment (- 820 to + 41 bp) was the area that responded to increased salt and drought activity. The BP2 (- 820 to + 872) activities were 0.98- and 1.02-fold compared with that of BP1 under salt and drought conditions and was 435 bp shorter than BP1. The salt- and drought-inducible activities of the BP2 promoter in the roots, stems, and leaves of transgenic tobacco plants were stable. Taken together, BP2 is more suitable than the BP1 promoter for the study and molecular breeding of stress-resistant soybean plants.

Mutant IAA21 genes from Dendrocalamus sinicus Chia et J. L. Sun inhibit stem and root growth in transgenic tobacco by interacting with ARF5.

Woody bamboos are important resource of industrial fibres. Auxin signaling plays a key role in multiple plant developmental processes, as yet the role of auxin/indole acetic acid (Aux/IAA) in culm development of woody bamboos has not been previously characterized. Dendrocalamus sinicus Chia et J. L. Sun is the largest woody bamboo documented in the world. Here, we identified two alleles of DsIAA21 gene (sIAA21 and bIAA21) from the straight- and bent-culm variants of D. sinicus, respectively, and studied how the domains I, i, and II of DsIAA21 affect the gene transcriptional repression. The results showed that bIAA21 expression was rapidly induced by exogenous auxin in D. sinicus. In transgenic tobacco, sIAA21 and bIAA21 mutated in domains i, and II significantly regulated plant architecture and root development. Stem cross sections revealed that parenchyma cells were smaller in transgenic plants than that in wild type plants. Domain i mutation changed the leucine and proline at position 45 to proline and leucine (siaa21L45P and biaa21P45L) strongly repressed cell expansion and root elongation by reducing the gravitropic response. Substitution of isoleucine with valine in domain II of the full length DsIAA21 resulted in dwarf stature in transgenic tobacco plants. Furthermore, the DsIAA21 interacted with auxin response factor 5 (ARF5) in transgenic tobacco plants, suggesting that DsIAA21 might inhibit stem and root elongation via interacting with ARF5. Taken together, our data indicated that DsIAA21 was a negative regulator of plant development and suggested that amino acid differences in domain i of sIAA21 versus bIAA21 affected their response to auxin, and might play a key role in the formation of the bent culm variant in D. sinicus. Our results not only shed a light on the morphogenetic mechanism in D. sinicus, but also provided new insights into versatile function of Aux/IAAs in plants.

Ectopic Expression of PsnNAC090 Enhances Salt and Osmotic Tolerance in Transgenic Tobacco.

The NAC transcription factor family is well known to play vital roles in plant development and stress responses. For this research, a salt-inducible NAC gene, PsnNAC090 (Po-tri.016G076100.1), was successfully isolated from Populus simonii × Populus nigra. PsnNAC090 contains the same motifs at the N-terminal end of the highly conserved NAM structural domain. The promoter region of this gene is rich in phytohormone-related and stress response elements. Transient transformation of the gene in the epidermal cells of both tobacco and onion showed that the protein was targeted to the whole cell including the cell membrane, cytoplasm and nucleus. A yeast two-hybrid assay demonstrated that PsnNAC090 has transcriptional activation activity with the activation structural domain located at 167-256aa. A yeast one-hybrid experiment showed that PsnNAC090 protein can bind to ABA-responsive elements (ABREs). The spatial and temporal expression patterns of PsnNAC090 under salt and osmotic stresses indicated that the gene was tissue-specific, with the highest expression level in the roots of Populus simonii × Populus nigra. We successfully obtained a total of six transgenic tobacco lines overexpressing PsnNAC090. The physiological indicators including peroxidase (POD) activity, superoxide dismutase (SOD) activity, chlorophyll content, proline content, malondialdehyde (MDA) content and hydrogen peroxide (H2O2) content were measured in three transgenic tobacco lines under NaCl and polyethylene glycol (PEG) 6000 stresses. The findings reveal that PsnNAC090 improves salt and osmotic tolerance by enhancing reactive oxygen species (ROS) scavenging and reducing membrane lipid peroxide content in transgenic tobacco. All the results suggest that the PsnNAC090 gene is a potential candidate gene playing an important role in stress response.

Genome-wide characterization of the U-box gene in Camellia sinensis and functional analysis in transgenic tobacco under abiotic stresses.

Plants U-box genes are crucial for plant survival, and they extensively regulate plant growth, reproduction and development as well as coping with stress and other processes. In this study, we identified 92 CsU-box genes through genome-wide analysis in the tea plant (Camellia sinensis), all of them contained the conserved U-box domain and were divided into 5 groups, which supported by the further genes structure analysis. The expression profiles in eight tea plant tissues and under abiotic and hormone stresses were analyzed using the TPIA database. 7 CsU-box genes (CsU-box27/28/39/46/63/70/91) were selected to verify and analyze expression patterns under PEG-induced drought and heat stress in tea plant respectively, the qRT-PCR results showed consistent with transcriptome datasets; and the CsU-box39 were further heterologous expressed in tobacco to perform gene function analysis. Phenotypic analyses of overexpression transgenic tobacco seedlings and physiological experiments revealed that CsU-box39 positively regulated the plant response to drought stress. These results lay a solid foundation for studying the biological function of CsU-box, and will provide breeding strategy basis for tea plant breeders.

AoSAP8-P encoding A20 and/or AN1 type zinc finger protein in asparagus officinalis L. Improving stress tolerance in transgenic Nicotiana sylvestris.

The full length CDS of an A20 and AN1 type zinc finger gene (named AoSAP8-P), located nearby the male specific Y chromosome (MSY) region of Asparagus officinalis (garden asparagus) was amplified by RT-PCR from purple passion. This gene, predicted as the stress associated protein (SAPs) gene families, encodes 172 amino acids with multiple cis elements including light, stress response box, MYB and ERF binding sites on its promoter. To analyze its function, the gene expression of different organs in different asparagus gender were analyzed and the overexpressed transgenic Nicotiana sylvestris lines were generated. The results showed the gene was highly expressed in both flower and root of male garden asparagus; the germination rate of seeds of the T2 transgenic lines (T2-5-4 and T2-7-1) under the stress conditions of 125 mM NaCl and 150 mM mannitol were significantly higher than the wild type (WT) respectively. When the potted T2-5-4, T2-7-1 lines and WT were subjected to drought stress for 24 days and the leaf discs immerged into 20 % PEG6000 and 300 mM NaCl solution for 48 h respectively, the T2-5-4 and T2-7-1 with AoSAP8-P expression showed stronger drought, salt and osmotic stress tolerance. When compared, the effects of AoSAP8-P overexpression on productive development showed that the flowering time of transgenic lines, were âˆ¼ 9 day earlier with larger but fewer pollens than its WT counterparts. However, there were no significant differences in anthers, stigmas and pollen viability between the transgenic lines and WT. Our results suggested that, the AoSAP8-P gene plays a role in improving the stress resistance and shortening seeds generation time for perianal survival during the growth and development of garden asparagus.

Ectopic expression of Jatropha curcas JcTAW1 improves the vegetative growth, yield, and drought resistance of tobacco.

BACKGROUND: Jatropha curcas is a promising alternative bio-energy resource. However, underrun limited its broad application in the industry. Luckily, TAW1 is a high-productivity promoting gene that increases the lateral branches by prolonging the identification of inflorescence meristems to generate more spikes and flowers. RESULTS: In the current study, we introduced the Jatropha JcTAW1 gene into tobacco to depict its functional profile. Ectopically expressed JcTAW1 increased the lateral branches and ultimate yield of the transgenic tobacco plants. Moreover, the JcTAW1 lines had significantly higher plant height, longer roots, and better drought resistance than those of wild-type (W.T.). We performed RNA sequencing and weighted gene co-expression network analysis to determine which biological processes were affected by JcTAW1. The results showed that biological processes such as carbon metabolism, cell wall biosynthesis, and ionization transport were extensively promoted by the ectopic expression of JcTAW1. Seven hub genes were identified. Therein, two up-regulated genes affect glucose metabolism and cell wall biosynthesis, five down-regulated genes are involved in DNA repair and negative regulation of TOR (target-of-rapamycin) signaling which was identified as a central regulator to promote cell proliferation and growth. CONCLUSIONS: Our study verified a new promising candidate for Jatropha productive breeding and discovered several new features of JcTAW1. Except for boosting flowering, JcTAW1 was found to promote stem and root growth. Additionally, transcriptome analysis indicated that JcTAW1 might promote glucose metabolism while suppressing the DNA repair system.

Ectopic expression of a grapevine alkaline α-galactosidase seed imbibition protein VvSIP enhanced salinity tolerance in transgenic tobacco plants.

Alpha-galactosidase seed imbibition protein (VvSIP) isolated from Vitis vinifera is up-regulated upon salt stress and mediates osmotic stress responses in a tolerant grapevine cultivar. So far, little is known about the putative role of this stress-responsive gene. In the present study, VvSIP function was investigated in model tobacco plants via Agrobacterium-mediated genetic transformation. Our results showed that overexpression of VvSIP exhibited increased tolerance to salinity at germination and late vegetative stage in transgenic Nicotiana benthamiana compared to the nontransgenic plants based on the measurement of the germination rate and biomass production. High salt concentrations of 200 and 400 mM NaCl in greenhouse-grown pot assay resulted in better relative water content, higher leaf osmotic potential, and leaf water potential in transgenic lines when compared to the wild-type (WT) plants. These physiological changes attributed to efficient osmotic adjustment improved plant performance and tolerance to salinity compared to the WT. Moreover, the VvSIP-expressing lines SIP1 and SIP2 showed elevated amounts of chlorophyll with lower malondialdehyde content indicating a reduced lipid peroxidation required to maintain membrane stability. When subjected to high salinity conditions, the transgenic tobacco VvSIP exhibited higher soluble sugar content, which may suggest an enhancement of the carbohydrate metabolism. Our findings indicate that the VvSIP is involved in plant salt tolerance by functioning as a positive regulator of osmotic adjustment and sugar metabolism, both of which are responsible for stress mitigation. Such a candidate gene is highly suitable to alleviate environmental stresses and thus could be a promising candidate for crop improvement.

FTX271: A potential gene resource for plant antiviral transgenic breeding.

Flammutoxin (FTX), as well as its precursor TDP, is a protein from Flammulina velutipes with antiviral activity. Transgenic tobacco with the FTX271 (gene of FTX or TDP) can not only delay the onset time of symptoms but also alleviate the symptoms caused by tobacco mosaic virus (TMV), but the mechanism is still unclear. In this study, FTX271 was introduced into Nicotiana benthamiana, and the disease resistance mechanism activated by FTX271 was speculated by transcriptomic and proteomic techniques. The results showed that TDP was detected, and some genes, proteins and pathways were significant upregulated or enriched in transgenic tobacco, including the mitogen-activated protein kinase (MAPK) cascade signal transduction pathway, the expression of hypersensitive response (HR) marker genes H1N1 and HSR203J, pathogenesis-related (PR) genes, and the key genes COI1 and lipoxygenase gene LOX2 of the jasmonic acid (JA) signaling pathway, indicating FTX271 may activate the MAPK pathway and increase the content of reactive oxygen species (ROS) and JA, which promoted the HR and inducible systemic resistance (ISR). ISR caused increased expression of peroxidase (POD) and other proteins involved in pathogen defense. In addition, transgenic tobacco may use sHSP-assisted photoreparation to alleviate the symptoms of TMV. In conclusion, JA-mediated ISR and sHSP-assisted photoreparation are activated by FTX271 to protect tobacco from TMV infection and alleviate the symptoms caused by the virus. The study provided a theoretical basis for the TMV resistance mechanism of FTX271, which may represent a potential gene resource for plant antiviral transgenic breeding.

Ectopic expression of NAC transcription factor HaNAC3 from Haloxylon ammodendron increased abiotic stress resistance in tobacco.

MAIN CONCLUSION: HaNAC3 is a transcriptional activator located in the nucleus that may be involved in the response to high temperature, high salt and drought stresses as well as phytohormone IAA and ABA treatments. Our study demonstrated that HaNAC3 increased the tolerance of transgenic tobacco to abiotic stress and was involved in the regulation of a range of downstream genes and metabolic pathways. This also indicates the potential application of HaNAC3 as a plant tolerance gene. NAC transcription factors play a key role in plant growth and development and plant responses to biotic and abiotic stresses. However, the biological functions of NAC transcription factors in the desert plant Haloxylon ammodendron are still poorly understood. In this study, the NAC transcription factor HaNAC3 was isolated and cloned from a typical desert plant H. ammodendron, and its possible biological functions were investigated. Bioinformatics analysis showed that HaNAC3 has the unique N-terminal NAC structural domain of NAC transcription factor. Quantitative real-time fluorescence analysis showed that HaNAC3 was able to participate in the response to simulated drought, high temperature, high salt, and phytohormone IAA and ABA treatments, and was very sensitive to simulated high temperature and phytohormone ABA treatments. Subcellular localization analysis showed that the GFP-HaNAC3 fusion protein was localized in the nucleus of tobacco epidermal cells. The transcriptional self-activation assay showed that HaNAC3 had transcriptional self-activation activity, and the truncation assay confirmed that the transcriptional activation activity was located at the C-terminus. HaNAC3 gene was expressed exogenously in wild-type Nicotiana benthamiana, and the physiological function of HaNAC3 was verified by simulating drought and other abiotic stresses. The results indicated that transgenic tobacco had better resistance to abiotic stresses than wild-type B. fuminata. Further transcriptome analysis showed that HaNAC3 was involved in the regulation of a range of downstream resistance genes, wax biosynthesis and other metabolic pathways. These results suggest that HaNAC3 may have a stress resistance role in H. ammodendron and has potential applications in plant molecular breeding.

Panax notoginseng WRKY Transcription Factor 9 Is a Positive Regulator in Responding to Root Rot Pathogen Fusarium solani.

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using ß-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen.

R2R3-MYB transcription factor FaMYB5 is involved in citric acid metabolism in strawberry fruits.

The citrate content of strawberry fruits affects their organoleptic quality. However, little is known about the transcriptional regulatory mechanisms of citric acid metabolism in strawberry fruits. In this study, the R2R3-MYB transcription factor FaMYB5 was identified and placed in the R2R3-MYB subfamily. FaMYB5 is found in the nucleus and shows tissue- and stage-specific expression levels. Citric acid content was positively correlated with FaMYB5 transcript levels. Upregulated FaMYB5 increased citric acid accumulation in transient FaMYB5-overexpressing strawberry fruits, whereas transient RNA silencing of FaMYB5 in strawberry fruits resulted in a reduction of citric acid content. The role of FaMYB5 was verified using stable transgenic NC89 tobacco. Furthermore, a yeast one-hybrid assay revealed that FaMYB5 influences citric acid accumulation by binding to the FaACO (aconitase), FaGAD (glutamate decarboxylase), and FaCS2 (citrate synthase) promoters. Dual-luciferase assays were used to demonstrate that FaMYB5 could activate FaCS2 expression and repress the transcription levels of FaACO and FaGAD. This study identified important roles of FaMYB5 in the regulation of citric acid metabolism and provided a potential target for improving strawberry fruit taste in horticultural crops.