Functional characterization of NBS-LRR genes reveals an NBS-LRR gene that mediates resistance against Fusarium wilt.

BACKGROUND: Most disease resistance (R) genes in plants encode proteins that contain leucine-rich-repeat (LRR) and nucleotide-binding site (NBS) domains, which belong to the NBS-LRR family. The sequenced genomes of Fusarium wilt-susceptible Vernicia fordii and its resistant counterpart, Vernicia montana, offer significant resources for the functional characterization and discovery of novel NBS-LRR genes in tung tree. RESULTS: Here, we identified 239 NBS-LRR genes across two tung tree genomes: 90 in V. fordii and 149 in V. montana. Five VmNBS-LRR paralogous were predicted in V. montana, and 43 orthologous were detected between V. fordii and V. montana. The orthologous gene pair Vf11G0978-Vm019719 exhibited distinct expression patterns in V. fordii and V. montana: Vf11G0978 showed downregulated expression in V. fordii, while its orthologous gene Vm019719 demonstrated upregulated expression in V. montana, indicating that this pair may be responsible for the resistance to Fusarium wilt in V. montana. Vm019719 from V. montana, activated by VmWRKY64, was shown to confer resistance to Fusarium wilt in V. montana by a virus-induced gene silencing (VIGS) experiment. However, in the susceptible V. fordii, its allelic counterpart, Vf11G0978, exhibited an ineffective defense response, attributed to a deletion in the promoter's W-box element. CONCLUSIONS: This study provides the first systematic analysis of NBS-LRR genes in the tung tree and identifies a candidate gene that can be utilized for marker-assisted breeding to control Fusarium wilt in V. fordii.

Transcriptomics and virus-induced gene silencing identify defence-related genes during Ralstonia solanacearum infection in resistant and susceptible tobacco.

Bacterial wilt (BW) caused by Ralstonia solanacearum is a globally prevalent bacterial soil-borne disease. In this study, transcriptome sequencing were subjected to roots after infection with the R. solanacearum in the resistant and susceptible tobacco variety. DEGs that responded to R. solanacearum infection in both resistant and susceptible tobacco contributed to pectinase and peroxidase development and were enriched in plant hormone signal transduction, signal transduction and MAPK signalling pathway KEGG terms. Core DEGs in the resistant tobacco response to R. solanacearum infection were enriched in cell wall, membrane, abscisic acid and ethylene terms. qRT-PCR indicated that Nitab4.5_0004899g0110, Nitab4.5_0004234g0080 and Nitab4.5_0001439g0050 contributed to the response to R. solanacearum infection in different resistant and susceptible tobacco. Silencing the p450 gene Nitab4.5_0001439g0050 reduced tobacco resistance to bacterial wilt. These results improve our understanding of the molecular mechanism of BW resistance in tobacco and solanaceous plants.

Genome-wide identification of the TIFY gene family in tobacco and expression analysis in response to Ralstonia solanacearum infection.

The TIFY gene family plays an essential role in plant development and abiotic and biotic stress responses. In this study, genome-wide identification of TIFY members in tobacco and their expression pattern analysis in response to Ralstonia solanacearum infection were performed. A total of 33 TIFY genes were identified, including the TIFY, PPD, ZIM&ZML and JAZ subfamilies. Promoter analysis results indicated that a quantity of light-response, drought-response, SA-response and JA-response cis-elements exist in promoter regions. The TIFY gene family exhibited expansion and possessed gene redundancy resulting from tobacco ploidy change. In addition, most NtTIFYs equivalently expressed in roots, stems and leaves, while NtTIFY1, NtTIFY4, NtTIFY18 and NtTIFY30 preferentially expressed in roots. The JAZ III clade showed significant expression changes after inoculation with R. solanacearum, and the expression of NtTIFY7 in resistant varieties, compared with susceptible varieties, was more stably induced. Furthermore, NtTIFY7-silenced plants, compared with the control plants, were more susceptible to bacterial wilt. These results lay a foundation for exploring the evolutionary history of TIFY gene family and revealing gene function of NtTIFYs in tobacco bacterial wilt resistance.

Interaction of Whitefly Effector G4 with Tomato Proteins Impacts Whitefly Performance.

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci. Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

PaWOX3 and PaWOX3B Regulate Flower Number and The Lip Symmetry of Phalaenopsis.

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL-related homeobox (WOX) genes, PaWOX3 and PaWOX3B, were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decrease in mature flower and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium orthologue, OnPRS, exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3/PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2, which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3/PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' can not be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to control lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3/PaWOX3B in maintaining the symmetric axis of lip.

Comprehensive identification and systematical characterization of BRX gene family and the functional of GhBRXL5A in response to salt stress.

BACKGROUND: BRVIS RADIX (BRX) family is a small gene family with the highly conserved plant-specific BRX domains, which plays important roles in plant development and response to abiotic stress. Although BRX protein has been studied in other plants, the biological function of cotton BRX-like (BRXL) gene family is still elusive. RESULT: In this study, a total of 36 BRXL genes were identified in four cotton species. Whole genome or segmental duplications played the main role in the expansion of GhBRXL gene family during evolutionary process in cotton. These BRXL genes were clustered into 2 groups, α and ß, in which structural and functional conservation within same groups but divergence among different groups were found. Promoter analysis indicated that cis-elements were associated with the phytohormone regulatory networks and the response to abiotic stress. Transcriptomic analysis indicated that GhBRXL2A/2D and GhBRXL5A/5D were up/down-regulated in response to the different stress. Silencing of GhBRXL5A gene via virus-induced gene silencing (VIGS) improved salt tolerance in cotton plants. Furthermore, yeast two hybrid analysis suggested homotypic and heterotypic interactions between GhBRXL1A and GhBRXL5D. CONCLUSIONS: Overall, these results provide useful and valuable information for understanding the evolution of cotton GhBRXL genes and their functions in salt stress.

Genome-wide expression analysis of LACS gene family implies GhLACS25 functional responding to salt stress in cotton.

BACKGROUND: Long-chain acyl-coenzyme A synthetase (LACS) is a type of acylating enzyme with AMP-binding, playing an important role in the growth, development, and stress response processes of plants. RESULTS: The research team identified different numbers of LACS in four cotton species (Gossypium hirsutum, Gossypium barbadense, Gossypium raimondii, and Gossypium arboreum). By analyzing the structure and evolutionary characteristics of the LACS, the GhLACS were divided into six subgroups, and a chromosome distribution map of the family members was drawn, providing a basis for further research classification and positioning. Promoter cis-acting element analysis showed that most GhLACS contain plant hormones (GA, MeJA) or non-biological stress-related cis-elements. The expression patterns of GhLACS under salt stress treatment were analyzed, and the results showed that GhLACS may significantly participate in salt stress response through different mechanisms. The research team selected 12 GhLACSs responsive to salt stress for tissue expression analysis and found that these genes are expressed in different tissues. CONCLUSIONS: There is a certain diversity of LACS among different cotton species. Analysis of promoter cis-acting elements suggests that GhLACS may be involved in regulating plant growth, development and stress response processes. GhLACS25 was selected for in-depth study, which confirmed its significant role in salt stress response through virus-induced gene silencing (VIGS) and induced expression in yeast cells.

Identification and virus-induced gene silencing (VIGS) analysis of methyltransferase affecting tomato (Solanum lycopersicum) fruit ripening.

MAIN CONCLUSION: Six methyltransferase genes affecting tomato fruit ripening were identified through genome-wide screening, VIGS assay, and expression pattern analysis. The data provide the basis for understanding new mechanisms of methyltransferases. Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases.

Indole-3 acetic acid negatively regulates rose black spot disease resistance through antagonizing the salicylic acid signaling pathway via jasmonic acid.

MAIN CONCLUSION: IAA cooperates with JA to inhibit SA and negatively regulates rose black spot disease resistance. Black spot disease caused by the fungus Marssonina rosae is the most prevalent and severe ailment in rose cultivation, leading to the appearance of black spots on leaves and eventual leaf fall, significantly impacting the utilization of roses in gardens. Salicylic acid (SA) and jasmonic acid (JA) are pivotal hormones that collaborate with indole-3 acetic acid (IAA) in regulating plant defense responses; however, the detailed mechanisms underlying the induction of black spot disease resistance by IAA, JA, and SA remain unclear. In this study, transcript analysis was conducted on resistant (R13-54) and susceptible (R12-26) lines following M. rosae infection. In addition, the impact of exogenous interference with IAA on SA- and JA-mediated disease resistance was examined. The continuous accumulation of JA, in synergy with IAA, inhibited activation of the SA signaling pathway in the early infection stage, thereby negatively regulating the induction of effective resistance to black spot disease. IAA administration alleviated the inhibition of SA on JA to negatively regulate the resistance of susceptible strains by further enhancing the synthesis and accumulation of JA. However, IAA did not contribute to the negative regulation of black spot resistance when high levels of JA were inhibited. Virus-induced gene silencing of RcTIFY10A, an inhibitor of the JA signaling pathway, further suggested that IAA upregulation led to a decrease in disease resistance, a phenomenon not observed when the JA signal was inhibited. Collectively, these findings indicate that the IAA-mediated negative regulation of black spot disease resistance relies on activation of the JA signaling pathway.

Co-localization and analysis of miR477b with fiber length quantitative trait loci in cotton.

Cotton is an important cash crop for the textile industry. However, the understanding of natural genetic variation of fiber elongation in relation to miRNA is lacking. A miRNA gene (miR477b) was found to co-localize with a previously mapped fiber length (FL) quantitative trait locus (QTL). The miR477b was differentially expressed during fiber elongation between two backcross inbred lines (BILs) differing in FL and its precursor sequences. Bioinformatics and qRT-PCR analysis were further used to analyse the miRNA genes, which could produce mature miR477b. Cotton plants with virus-induced gene silencing (VIGS) constructs to over-express the allele of miR477b from the BIL with longer fibers had significantly longer fibers as compared with negative control plants, while the VIGS plants with suppressed miRNA expression had significantly shorter fibers. The expression level of the target gene (DELLA) and related genes (RDL1 and EXPA1 for DELLA through HOX3 protein) in the two BILs and/or the VIGS plants were generally congruent, as expected. This report represents one of the first comprehensive studies to integrate QTL linkage mapping and physical mapping of small RNAs with both small and mRNA transcriptome analysis, followed by VIGS, to identify candidate small RNA genes affecting the natural variation of fiber elongation in cotton.

Gene identification, expression analysis, and molecular docking of SAT and OASTL in the metabolic pathway of selenium in Cardamine hupingshanensis.

KEY MESSAGE: Identification of selenium stress-responsive expression and molecular docking of serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) in Cardamine hupingshanensis. A complex coupled with serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) is the key enzyme that catalyzes selenocysteine (Sec) synthesis in plants. The functions of SAT and OASTL genes were identified in some plants, but it is still unclear whether SAT and OASTL are involved in the selenium metabolic pathway in Cardamine hupingshanensis. In this study, genome-wide identification and comparative analysis of ChSATs and ChOASTLs were performed. The eight ChSAT genes were divided into three branches, and the thirteen ChOASTL genes were divided into four branches by phylogenetic analysis and sequence alignment, indicating the evolutionary conservation of the gene structure and its association with other plant species. qRT-PCR analysis showed that the ChSAT and ChOASTL genes were differentially expressed in different tissues under various selenium levels, suggesting their important roles in Sec synthesis. The ChSAT1;2 and ChOASTLA1;2 were silenced by the VIGS system to investigate their involvement in selenium metabolites in C. hupingshanensis. The findings contribute to understanding the gene functions of ChSATs and ChOASTLs in the selenium stress and provide a reference for further exploration of the selenium metabolic pathway in plants.

GhiPLATZ17 and GhiPLATZ22, zinc-dependent DNA-binding transcription factors, promote salt tolerance in upland cotton.

KEY MESSAGE: The utilization of transcriptome analysis, functional validation, VIGS, and DAB techniques have provided evidence that GhiPLATZ17 and GhiPLATZ22 play a pivotal role in improving the salt tolerance of upland cotton. PLATZ (Plant AT-rich sequences and zinc-binding proteins) are known to be key regulators in plant growth, development, and response to salt stress. In this study, we comprehensively analyzed the PLATZ family in ten cotton species in response to salinity stress. Gossypium herbaceum boasts 25 distinct PLATZ genes, paralleled by 24 in G. raimondii, 25 in G. arboreum, 46 in G. hirsutum, 48 in G. barbadense, 43 in G. tomentosum, 67 in G. mustelinum, 60 in G. darwinii, 46 in G. ekmanianum, and a total of 53 PLATZ genes attributed to G. stephensii. The PLATZ gene family shed light on the hybridization and allopolyploidy events that occurred during the evolutionary history of allotetraploid cotton. Ka/Ks analysis suggested that the PLATZ gene family underwent intense purifying selection during cotton evolution. Analysis of synteny and gene collinearity revealed a complex pattern of segmental and dispersed duplication events to expand PLATZ genes in cotton. Cis-acting elements and gene expressions revealed that GhiPLATZ exhibited salt stress resistance. Transcriptome analysis, functional validation, virus-induced gene silencing (VIGS), and diaminobenzidine staining (DAB) demonstrated that GhiPLATZ17 and GhiPLATZ22 enhance salt tolerance in upland cotton. The study can potentially advance our understanding of identifying salt-resistant genes in cotton.

GhGASA14 regulates the flowering time of upland cotton in response to GA3.

KEY MESSAGE: The silencing of GhGASA14 and the identification of superior allelic variation in its coding region indicate that GhGASA14 may positively regulate flowering and the response to GA3. Gibberellic acid-stimulated Arabidopsis (GASA), a member of the gibberellin-regulated short amino acid family, has been extensively investigated in several plant species and found to be critical for plant growth and development. However, research on this topic in cotton has been limited. In this study, we identified 38 GhGASAs that were dispersed across 18 chromosomes in upland cotton, and all of these genes had a GASA core domain. Transcriptome expression patterns and qRT-PCR results revealed that GhGASA9 and GhGASA14 exhibited upregulated expression not only in the floral organs but also in the leaves of early-maturing cultivars. The two genes were functionally characterized by virus-induced gene silencing (VIGS), and the budding and flowering times after silencing the target genes were later than those of the control (TRV:00). Compared with that in the water-treated group (MOCK), the flowering period of the different fruiting branches in the GA3-treated group was more concentrated. Interestingly, allelic variation was detected in the coding sequence of GhGASA14 between early-maturing and late-maturing accessions, and the frequency of this favorable allele was greater in high-latitude cotton cultivars than in low-latitude ones. Additionally, a significant linear relationship was observed between the expression level of GhGASA14 and flowering time among the 12 upland cotton accessions. Taken together, these results indicated that GhGASA14 may positively regulate flowering time and respond to GA3. These findings could lead to the use of valuable genetic resources for breeding early-maturing cotton cultivars in the future.

Zinc Finger-Homeodomain Transcriptional Factors (ZHDs) in Cucumber (Cucumis sativus L.): Identification, Evolution, Expression Profiles, and Function under Abiotic Stresses.

Cucumber (Cucumis sativus L.) is a globally prevalent and extensively cultivated vegetable whose yield is significantly influenced by various abiotic stresses, including drought, heat, and salinity. Transcription factors, such as zinc finger-homeodomain proteins (ZHDs), a plant-specific subgroup of Homeobox, play a crucial regulatory role in stress resistance. In this study, we identified 13 CsZHDs distributed across all six cucumber chromosomes except chromosome 7. Phylogenetic analysis classified these genes into five clades (ZHDI-IV and MIF) with different gene structures but similar conserved motifs. Collinearity analysis revealed that members of clades ZHD III, IV, and MIF experienced amplification through segmental duplication events. Additionally, a closer evolutionary relationship was observed between the ZHDs in Cucumis sativus (C. sativus) and Arabidopsis thaliana (A. thaliana) compared to Oryza sativa (O. sativa). Quantitative real-time PCR (qRT-PCR) analysis demonstrated the general expression of CsZHD genes across all tissues, with notable expression in leaf and flower buds. Moreover, most of the CsZHDs, particularly CsZHD9-11, exhibited varying responses to drought, heat, and salt stresses. Virus-induced gene silencing (VIGS) experiments highlighted the potential functions of CsZHD9 and CsZHD10, suggesting their positive regulation of stomatal movement and responsiveness to drought stress. In summary, these findings provide a valuable resource for future analysis of potential mechanisms underlying CsZHD genes in response to stresses.

The Silencing of GhPIP5K2 and GhPIP5K22 Weakens Abiotic Stress Tolerance in Upland Cotton (Gossypium hirsutum).

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), essential enzymes in the phosphatidylinositol signaling pathway, are crucial for the abiotic stress responses and the overall growth and development of plants. However, the GhPIP5Ks had not been systematically studied, and their function in upland cotton was unknown. This study identified a total of 28 GhPIP5Ks, and determined their chromosomal locations, gene structures, protein motifs and cis-acting elements via bioinformatics analysis. A quantitative real-time PCR (qRT‒PCR) analysis showed that most GhPIP5Ks were upregulated under different stresses. A virus-induced gene silencing (VIGS) assay indicated that the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were significantly decreased, while malondialdehyde (MDA) content were significantly increased in GhPIP5K2- and GhPIP5K22-silenced upland cotton plants under abiotic stress. Furthermore, the expression of the stress marker genes GhHSFB2A, GhHSFB2B, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhBIN2, GhCBL3, GhNHX1, GhPP2C, GhCBF1, GhSnRK2.6 and GhCIPK6 was significantly decreased in the silenced plants after exposure to stress. These results revealed that the silencing of GhPIP5K2 and GhPIP5K22 weakened the tolerance to abiotic stresses. These discoveries provide a foundation for further inquiry into the actions of the GhPIP5K gene family in regulating the response and resistance mechanisms of cotton to abiotic stresses.

Genome-Wide Identification of the GhANN Gene Family and Functional Validation of GhANN11 and GhANN4 under Abiotic Stress.

Annexins (ANNs) are a structurally conserved protein family present in almost all plants. In the present study, 27 GhANNs were identified in cotton and were unevenly distributed across 14 chromosomes. Transcriptome data and RT-qPCR results revealed that multiple GhANNs respond to at least two abiotic stresses. Similarly, the expression levels of GhANN4 and GhANN11 were significantly upregulated under heat, cold, and drought stress. Using virus-induced gene silencing (VIGS), functional characterization of GhANN4 and GhANN11 revealed that, compared with those of the controls, the leaf wilting of GhANN4-silenced plants was more obvious, and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were lower under NaCl and PEG stress. Moreover, the expression of stress marker genes (GhCBL3, GhDREB2A, GhDREB2C, GhPP2C, GhRD20-2, GhCIPK6, GhNHX1, GhRD20-1, GhSOS1, GhSOS2 and GhSnRK2.6) was significantly downregulated in GhANN4-silenced plants after stress. Under cold stress, the growth of the GHANN11-silenced plants was significantly weaker than that of the control plants, and the activities of POD, SOD, and CAT were also lower. However, compared with those of the control, the elasticity and orthostatic activity of the GhANN11-silenced plants were greater; the POD, SOD, and CAT activities were higher; and the GhDREB2C, GhHSP, and GhSOS2 expression levels were greater under heat stress. These results suggest that different GhANN family members respond differently to different types of abiotic stress.

Genome-Wide and Expression Pattern Analysis of the DVL Gene Family Reveals GhM_A05G1032 Is Involved in Fuzz Development in G. hirsutum.

DVL is one of the small polypeptides which plays an important role in regulating plant growth and development, tissue differentiation, and organ formation in the process of coping with stress conditions. So far, there has been no comprehensive analysis of the expression profile and function of the cotton DVL gene. According to previous studies, a candidate gene related to the development of fuzz was screened, belonging to the DVL family, and was related to the development of trichomes in Arabidopsis thaliana. However, the comprehensive identification and systematic analysis of DVL in cotton have not been conducted. In this study, we employed bioinformatics approaches to conduct a novel analysis of the structural characteristics, phylogenetic tree, gene structure, expression pattern, evolutionary relationship, and selective pressure of the DVL gene family members in four cotton species. A total of 117 DVL genes were identified, including 39 members in G. hirsutum. Based on the phylogenetic analysis, the DVL protein sequences were categorized into five distinct subfamilies. Additionally, we successfully mapped these genes onto chromosomes and visually represented their gene structure information. Furthermore, we predicted the presence of cis-acting elements in DVL genes in G. hirsutum and characterized the repeat types of DVL genes in the four cotton species. Moreover, we computed the Ka/Ks ratio of homologous genes across the four cotton species and elucidated the selective pressure acting on these homologous genes. In addition, we described the expression patterns of the DVL gene family using RNA-seq data, verified the correlation between GhMDVL3 and fuzz development through VIGS technology, and found that some DVL genes may be involved in resistance to biotic and abiotic stress conditions through qRT-PCR technology. Furthermore, a potential interaction network was constructed by WGCNA, and our findings demonstrated the potential of GhM_A05G1032 to interact with numerous genes, thereby playing a crucial role in regulating fuzz development. This research significantly contributed to the comprehension of DVL genes in upland cotton, thereby establishing a solid basis for future investigations into the functional aspects of DVL genes in cotton.

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.

Heat shock protein HvHSP16.9 from wild barley enhances tolerance to salt stress.

Heat shock proteins (HSPs) are known to play a crucial role in the response of plants to environmental stress, particularly heat stress. Nevertheless, the function of HSPs in salt stress tolerance in plants, especially in barley, remains largely unexplored. Here, we aimed to investigate and compare the salt tolerance mechanisms between wild barley EC_S1 and cultivated barley RGT Planet through a comprehensive analysis of physiological parameters and transcriptomic profiles. Results demonstrated that the number of differentially expressed genes (DEGs) in EC_S1 was significantly higher than in RGT Planet, indicating that wild barley gene regulation is more adaptive to salt stress. KEGG enrichment analysis revealed that DEGs were mainly enriched in the processes of photosynthesis, plant hormone signal transduction, and reactive oxygen species metabolism. Furthermore, the application of weighted gene correlation network analysis (WGCNA) enabled the identification of a set of key genes, including small heat shock protein (sHSP), Calmodulin-like proteins (CML), and protein phosphatases 2C (PP2C). Subsequently, a novel sHSP gene, HvHSP16.9 encoding a protein of 16.9 kDa, was cloned from wild barley, and its role in plant response to salt stress was elucidated. In Arabidopsis, overexpression of HvHSP16.9 increased the salt tolerance. Meanwhile, barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) of HvHSP16.9 significantly reduced the salt tolerance in wild barley. Overall, this study offers a new theoretical framework for comprehending the tolerance and adaptation mechanisms of wild barley under salt stress. It provides valuable insights into the salt tolerance function of HSP, and identifies new candidate genes for enhancing cultivated barley varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01455-4.