Genome-wide characterization and function analysis of ginger (Zingiber officinale Roscoe) ZoGRFs in responding to adverse stresses.

Growth-regulating factors (GRFs) play crucial roles in plant growth, development, hormone signaling, and stress response. Despite their significance, the roles of GRFs in ginger remain largely unknown. Herein, 31 ginger ZoGRFs were identified and designated as ZoGRF1-ZoGRF31 according to their phylogenetic relationships. All ZoGRFs were characterized as unstable, hydrophilic proteins, with 29 predicted to be located in the nucleus. Functional cis-elements related to growth and development were enriched in ZoGRF's promoter regions. RNA-seq and RT-qPCR analysis revealed that ZoGRF12, ZoGRF24, and ZoGRF28 were highly induced in various growth and development stages, displaying differential regulation under waterlogging, chilling, drought, and salt stresses, indicating diverse expression patterns of ZoGRFs. Transient expression analysis in Nicotiana benthamiana indicated that overexpressing ZoGRF28 regulated the transcription levels of salicylic acid, jasmonic acid, and pattern-triggered immunity-related genes, increased chlorophyll content and contributed to reduced disease lesions and an increased net photosynthetic rate. This research lays the foundation for further understanding the biological roles of ZoGRFs.

Morphological and Molecular Identification of Fusarium ipomoeae as the Causative Agent of Leaf Spot Disease in Tobacco from China.

Tobacco (Nicotiana tabacum L.), which creates jobs for 33 million people and contributes two trillion dollars' tax annually, is one of the most important economic plants globally. However, tobacco is seriously threatened by numerous diseases during production. Previously, the field survey of tobacco diseases was conducted in the Guizhou and Guangxi provinces, the two main tobacco-producing areas in China. A serious leaf spot disease, with a 22% to 35% incidence, was observed in farming plants. In order to determine the causal agents, we collected the disease samples and isolated the pathogenic fungi. The pathogen was identified as Fusariumipomoeae, based on the morphological characteristics and phylogenetic analysis. Pathogenicity tests showed that F. ipomoeae could induce tobacco leaf spot and blight. To our knowledge, this is the first report worldwide of F. ipomoeae causing leaf spots and stems on tobacco. Our study reveals the serious consequences of F. ipomoeae on tobacco filed production and provides information for future diagnosis and management of the Fusarium disease.

Antibacterial activity and mechanism of ginger extract against Ralstonia solanacearum.

AIMS: The current study aimed to determine the chemical compositions of ginger extract (GE) and to assess the antibacterial activities of GE against the ginger bacterial wilt pathogen Ralstonia solanacearum and to screen their mechanisms of action. METHODS AND RESULTS: A total of 393 compounds were identified by using ultra-performance liquid chromatography and tandem-mass spectrometry. The antibacterial test indicated that GE had strong antibacterial activity against R. solanacearum and that the bactericidal effect exhibited a dose-dependent manner. The minimum inhibitory concentration and minimum bactericidal concentration of R. solanacearum were 3.91 and 125 mg/ml, respectively. The cell membrane permeability and integrity of R. solanacearum were destroyed by GE, resulting in cell content leakage, such as electrolytes, nucleic acids, proteins, extracellular adenosine triphosphate and exopoly saccharides. In addition, the activity of cellular succinate dehydrogenase and alkaline phosphatase of R. solanacearum decreased gradually with an increase in the GE concentration. Scanning electron microscopy analysis revealed that GE treatment changed the morphology of the R. solanacearum cells. Further experiments demonstrated that GE delayed or slowed the occurrence of bacterial wilt on ginger. CONCLUSIONS: GE has a significant antibacterial effect on R. solanacearum, and the antibacterial effect is concentration dependent. The GE treatments changed the morphology, destroyed membrane permeability and integrity, reduced key enzyme activity and inhibit the synthesis of the virulence factor EPS of R. solanacearum. GE significantly controlled the bacterial wilt of ginger during infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This research provides insight into the antimicrobial mechanism of GE against R. solanacearum, which will open a new application field for GE.

Genome-wide characterization and function analysis uncovered roles of wheat LIMs in responding to adverse stresses and TaLIM8-4D function as a susceptible gene.

The Lin-11, Isl-1, and Mec-3 domains (LIM) transcription factors play essential roles in regulating plant biological processes. Despite that, there is a lack of a full understanding of LIMs in wheat (Triticum aestivum L.). In this study, 28 wheat LIM s (TaLIMs) were identified and designated as TaLIM1-1A to TaLIM12-7D. The cis-regulatory element analysis showed that TaLIMs were rich in elements related to biological and abiotic stresses. Expression profiling analysis showed that certain members of TaLIMs were responsive to biotic and abiotic stresses, such as TaLIM1-1A, TaLIM3-2B, TaLIM8-4D, and TaLIM10-5D, were significantly induced by heat, drought, sodium chloride (NaCl), abscisic acid (ABA) and Fusarium graminearum stresses. Furthermore, the biological function of TaLIM8-4D was analyzed and results showed that it was subcellular localization in the nucleus and could induce weak cell death in Nicotiana benthamiana leaves. Additionally, overexpression of TaLIM8-4D could upregulate plant pathogenesis-related (PR) genes, promoting the infection of hemibiotrophic pathogen, implying that TaLIM8-4D could function as susceptible gene in the nucleus by upregulating PR genes and inducing cell death to promote the colonization of hemibiotrophic agent F. graminearum. Overall, the systematic identification, characterization, expression profiling, evolutionary, and function analyses provided the ability to understand TaLIMs and laid a foundation for the further function study of LIM family members in wheat.

Stem-end Rot Caused by Lasiodiplodia brasiliense on Tobacco in China.

Tobacco (Nicotiana tabacum L.), is a major cash crop grown worldwide for its leaves, which are dried and fermented before being put in tobacco products. Tobacco production is seriously threatened by numerous diseases (Qiu et al. 2021). In August 2021, plants with stem-end rot were observed in a tobacco field in Zunyi City, Guizhou Province, China. Surveys indicated a 22 to 35% disease incidence in five counties. Symptoms of black necrosis appeared at the base of stems, and leaves turned yellow. To isolate the pathogen, diseased stems were cut into small segments and placed on potato dextrose agar (PDA) at 25°C in darkness for 3 to 5 days. To obtain pure cultures, hyphal tips from colonies were transferred to fresh PDA plates. A representative strain, GZAX 110, was used for further identification. The fungal colonies were initially gray, later deepening to smoke-gray. Conidiogenous cells were fully divided, discrete, transparent, thin-walled, smooth and cylindrical. Conidia matured slowly, were ellipsoid to ovoid, containing granular content, with a rounded apex. The base was largely truncated, and conidia became dark brown with one central septum, 21.0-30.0 × 12.0-18.0 µm. On water-agar medium, teleomorph structures were not observed. These characteristics suggested the fungus was Lasiodiplodia sp. (Netto et al. 2014). For further confirmation, genomic DNA was extracted using the CTAB method (Watanabe et al. 2010); and the ITS (internal transcribed spacer region of rDNA) and EF-1α (translation-elongation factor) regions were amplified with primer pairs ITS1/ITS4 and EF1-688F/EF1-1251R (Cruywagen et al. 2017), respectively. The ITS and EF-1α sequences (OM534558 and OM632673) were analyzed by BLASTN searches. The ITS sequence showed 100% identity (490/490 bp) to L. brasiliense strain AGQMy0011 (MW274145) and the EF-1ɑ sequence showed 100% identity (551/551 bp) to L. brasiliense strain EX1 (MF580811). A multilocus phylogenetic tree was constructed via the Maximum-likelihood (RAxML v.7.2.8) and Bayesian Inference (MrBayes v.3.2.1) analyses (Elsie et al. 2017) using combined ITS and EF1-α reference sequences of Lasiodiplodia species. Phylogenetic analysis showed that GZAX 110 clustered monophyletically with strains of L. brasiliense. Thus, the isolate GZAX 110 was confirmed as L. brasiliense. Pathogenicity of GZAX 110 was tested on tobacco plants at the eight leaf stage by attaching mycelial plugs (5 mm in diameter) to stems and leaves according to Cruywagen et al. (2017). Inoculated plants were kept in a greenhouse (16 h light/8 h darkness, 22℃, relative humidity >85%). Control plants were inoculated with PDA plugs. The experiment was repeated three times with five plants. Seven days after inoculation, dark brown necrosis was observed at inoculation sites on stems and leaves, while the control plants remained healthy. The pathogen was re-isolated from the inoculated sites and further validated as the same fungus through morphological and phylogenetic analyses. Previously, this fungus has been reported on Mangifera indica (mango) in China (Zhang et al. 2018), and apple (Martins et al. 2018) and papaya (Netto et al. 2014) in Brazil. However, to our knowledge, this is the first worldwide report of L. brasiliense causing stem-end rot on tobacco. This report provides information for future diagnosis and management of the disease.

Silicon Nanoparticles Enhance Ginger Rhizomes Tolerance to Postharvest Deterioration and Resistance to Fusarium solani.

Postharvest deterioration of ginger rhizome caused by microorganisms or wound infections causes significant economic losses. Fusarium solani is one of the important causal agents of prevalent ginger disease soft rot across the world. The massive and continuous use of chemical fungicides in postharvest preservation pose risks to human health and produce environmental contamination. Hence, new alternative tools are required to reduce postharvest deterioration and extend the postharvest life of ginger. In this study, the use of silicon nanoparticles (SiNPs) on the storability of ginger rhizomes during postharvest storage and their resistance to Fusarium solani was investigated. The results showed that 50, 100, and 150 mg L-1 of SiNPs increased the firmness of the ginger rhizome during storage but decreased the decay severity, water loss, total color difference, and the reactive oxygen species (ROS; H2O2 and superoxide anion) accumulation. Specifically, 100 mg L-1 (SiNP100) demonstrated the best effect in the extension of postharvest life and improved the quality of the ginger rhizomes. SiNP100 application increased the activities of antioxidant enzymes (SOD and CAT) and the total phenolics and flavonoid contents, thereby reducing the ROS accumulation and malondialdehyde (MDA) content. Meanwhile, SiNP100 treatment negatively impacts the peroxidase (POD) and polyphenol oxidase (PPO) activities, which may have contributed to the lower level of lignin and decreased total color difference. SiNP100 likely decreased water loss and the transfer of water by altering the expression of aquaporin genes. Moreover, SiNP100 modulated the expression of lignin synthesis and phytopathogenic responses genes including MYB and LysM genes. Furthermore, SiNP100 inhibited Fusarium solani by preventing the penetration of hyphae into cells, thus decreasing the severity of postharvest pathogenic decay. In summary, this study revealed the physiology and molecular mechanisms of SiNPs-induced tolerance to postharvest deterioration and resistance to disease, which provides a foundation for using SiNPs resources as a promising alternative tool to maintain ginger quality and control postharvest diseases.

Genome-wide characterization of the C2H2 zinc-finger genes in Cucumis sativus and functional analyses of four CsZFPs in response to stresses.

BACKGROUNDS: C2H2-type zinc finger protein (ZFPs) form a relatively large family of transcriptional regulators in plants, and play many roles in plant growth, development, and stress response. However, the comprehensive analysis of C2H2 ZFPs in cucumber (CsZFPs) and their regulation function in cucumber are still lacking. RESULTS: In the current study, the whole genome identification and characterization of CsZFPs, including the gene structure, genome localization, phylogenetic relationship, and gene expression were performed. Functional analysis of 4 selected genes by transient transformation were also conducted. A total of 129 full-length CsZFPs were identified, which could be classified into four groups according to the phylogenetic analysis. The 129 CsZFPs unequally distributed on 7 chromosomes. Promoter cis-element analysis showed that the CsZFPs might involve in the regulation of phytohormone and/or abiotic stress response, and 93 CsZFPs were predicted to be targeted by one to 20 miRNAs. Moreover, the subcellular localization analysis indicated that 10 tested CsZFPs located in the nucleus and the transcriptome profiling analysis of CsZFPs demonstrated that these genes are involved in root and floral development, pollination and fruit spine. Furthermore, the transient overexpression of Csa1G085390 and Csa7G071440 into Nicotiana benthamiana plants revealed that they could decrease and induce leave necrosis in response to pathogen attack, respectively, and they could enhance salt and drought stresses through the initial induction of H2O2. In addition, Csa4G642460 and Csa6G303740 could induce cell death after 5 days transformation. CONCLUSIONS: The identification and function analysis of CsZFPs demonstrated that some key individual CsZFPs might play essential roles in response to biotic and abiotic stresses. These results could lay the foundation for understanding the role of CsZFPs in cucumber development for future genetic engineering studies.

Genome-wide identification, characterization analysis and expression profiling of auxin-responsive GH3 family genes in wheat (Triticum aestivum L.).

Auxin affects many aspects of plant growth and development by regulating the expression of auxin-responsive genes. As one of the three major auxin-responsive families the Gretchen Hagen3 (GH3) gene family maintains hormonal homeostasis by conjugating excess indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA) to amino acids during hormone and stress-related signaling. Although some work has been carried out the functions of wheat GH3 (TaGH3) family genes in response to abiotic stresses (including salt stress and osmotic stress) are largely unknown. Access to the complete wheat genome sequence permits genome-wide studies on TaGH3s. We performed a systematic identification of the TaGH3 gene family at the genome level and detected 36 members on 14 wheat chromosomes. Many of the genes were segmentally duplicated and Ka/Ks and inter-species synthetic analyses indicated that polyploidization was the contributor to the increased number of TaGH3 members. Phylogenetic analyses revealed that TaGH3 proteins could divided into three major categories (TaGH3-I, TaGH3-II, and TaGH3-III). Diversified cis-elements in the promoters of TaGH3 genes were predicted as essential players in regulating TaGH3 expression patterns. Gene structure and motif analyses indicated that most TaGH3 genes have relatively conserved exon/intron arrangements and motif compositions. Analysis of multiple transcriptome data sets indicated that many TaGH3 genes are responsive to biological and abiotic stresses and possibly have important functions in stress response. qRT-PCR analysis revealed that TaGH3s were induced by salt and osmotic stresses. Customized annotation results revealed that TaGH3s were widely involved in phytohormone response, defense, growth and development, and metabolism. Overall, our work provides a comprehensive insight into the TaGH3 family members, and a basis for the further study of their biological functions in wheat.

Genome-wide identification, structure characterization, and expression pattern profiling of aquaporin gene family in cucumber.

BACKGROUND: Aquaporin (AQP) proteins comprise a group of membrane intrinsic proteins (MIPs) that are responsible for transporting water and other small molecules, which is crucial for plant survival under stress conditions including salt stress. Despite the vital role of AQPs, little is known about them in cucumber (Cucumis sativus L.). RESULTS: In this study, we identified 39 aquaporin-encoding genes in cucumber that were separated by phylogenetic analysis into five sub-families (PIP, TIP, NIP, SIP, and XIP). Their substrate specificity was then assessed based on key amino acid residues such as the aromatic/Arginine (ar/R) selectivity filter, Froger's positions, and specificity-determining positions. The putative cis-regulatory motifs available in the promoter region of each AQP gene were analyzed and results revealed that their promoter regions contain many abiotic related cis-regulatory elements. Furthermore, analysis of previously released RNA-seq data revealed tissue- and treatment-specific expression patterns of cucumber AQP genes (CsAQPs). Three aquaporins (CsTIP1;1, CsPIP2;4, and CsPIP1;2) were the most transcript abundance genes, with CsTIP1;1 showing the highest expression levels among all aquaporins. Subcellular localization analysis in Nicotiana benthamiana epidermal cells revealed the diverse and broad array of sub-cellular localizations of CsAQPs. We then performed RNA-seq to identify the expression pattern of CsAQPs under salt stress and found a general decreased expression level of root CsAQPs. Moreover, qRT-PCR revealed rapid changes in the expression levels of CsAQPs in response to diverse abiotic stresses including salt, polyethylene glycol (PEG)-6000, heat, and chilling stresses. Additionally, transient expression of AQPs in N. benthamiana increased leaf water loss rate, suggesting their potential roles in the regulation of plant water status under stress conditions. CONCLUSIONS: Our results indicated that CsAQPs play important roles in response to salt stress. The genome-wide identification and primary function characterization of cucumber aquaporins provides insight to elucidate the complexity of the AQP gene family and their biological functions in cucumber.

Transcriptome Analysis Reveals the Accumulation Mechanism of Anthocyanins in Buckwheat (Fagopyrum esculentum Moench) Cotyledons and Flowers.

Buckwheat (Fagopyrum esculentum) is a valuable crop which can produce multiple human beneficial secondary metabolites, for example, the anthocyanins in sprouts and flowers. However, as the predominant group of visible polyphenols in pigmentation, little is known about the molecular mechanisms underlying the anthocyanin biosynthesis within buckwheat. In this study, a comparative transcriptome analysis of green and red common buckwheat cultivars was carried out through RNA sequencing. Overall, 3727 and 5323 differently expressed genes (DEGs) were identified in flowers and cotyledons, respectively. Through GO and KEGG analysis, we revealed that DEGs in flowers and cotyledons are predominately involved in biosynthesis of anthocyanin. A total of 42 unigenes encoding 11 structural enzymes of the anthocyanin biosynthesis were identified as DEGs. We also identified some transcription factor families involved in the regulation of anthocyanin biosynthesis. Real-time qPCR validation of candidate genes was performed in flowers and cotyledons, and the results suggested that the high expression level of structural genes involved in anthocyanin biosynthetic pathway promotes anthocyanin accumulation. Our results provide the insight understanding for coloration of red common buckwheat.

Silicon enhances the salt tolerance of cucumber through increasing polyamine accumulation and decreasing oxidative damage.

Silicon can increase salt tolerance, but the underlying mechanism has remained unclear. Here, we investigated the effect of silicon on polyamine metabolism and the role of polyamine accumulation in silicon-mediated salt tolerance in cucumber. Seedlings of cucumber 'JinYou 1' were subjected to salt stress (75 mM NaCl) in the presence or absence of added 0.3 mM silicon. Plant growth, polyamine metabolism and effects of exogenous polyamines and polyamine synthesis inhibitor dicyclohexylammonium sulphate on oxidative damage were investigated. The results showed that salt stress inhibited plant growth and decreased leaf chlorophyll levels and the maximum quantum yield of PSII, and added silicon ameliorated these negative effects. Salt stress increased polyamine accumulation in the leaves and roots. Compared with salt stress alone, overall, silicon addition decreased free putrescine concentrations, but increased spermidine and spermine concentrations in both leaves and roots under salt stress. Silicon application resulted in increased polyamine levels under salt stress by promoting the activities of S-adenosylmethionine decarboxylase and arginine decarboxylase while inhibiting the activity of diamine oxidase. Exogenous application of spermidine and spermine alleviated salt-stress-induced oxidative damage, whereas polyamine synthesis inhibitor eliminated the silicon-mediated decrease in oxidative damage. The results suggest that silicon-enhanced polyamine accumulation in cucumber under salt stress may play a role in decreasing oxidative damage and therefore increase the salt tolerance.

Conserved RXLR Effector Genes of Phytophthora infestans Expressed at the Early Stage of Potato Infection Are Suppressive to Host Defense.

Late blight has been the most devastating potato disease worldwide. The causal agent, Phytophthora infestans, is notorious for its capability to rapidly overcome host resistance. Changes in the expression pattern and the encoded protein sequences of effector genes in the pathogen are responsible for the loss of host resistance. Among numerous effector genes, the class of RXLR effector genes is well-known in mediating host genotype-specific resistance. We therefore performed deep sequencing of five genetically diverse P. infestans strains using in planta materials infected with zoospores (12 h post inoculation) and focused on the identification of RXLR effector genes that are conserved in coding sequences, are highly expressed in early stages of plant infection, and have defense suppression activities. In all, 245 RXLR effector genes were expressed in five transcriptomes, with 108 being co-expressed in all five strains, 47 of them comparatively highly expressed. Taking sequence polymorphism into consideration, 18 candidate core RXLR effectors that were conserved in sequence and with higher in planta expression levels were selected for further study. Agrobacterium tumefaciens-mediated transient expression of the selected effector genes in Nicotiana benthamiana and potato demonstrated their potential virulence function, as shown by suppression of PAMP-triggered immunity (PTI) or/and effector-triggered immunity (ETI). The identified collection of core RXLR effectors will be useful in the search for potential durable late blight resistance genes. Analysis of 10 known Avr RXLR genes revealed that the resistance genes R2, Rpi-blb2, Rpi-vnt1, Rpi-Smira1, and Rpi-Smira2 may be effective in potato cultivars. Analysis of 8 SFI (Suppressor of early Flg22-induced Immune response) RXLR effector genes showed that SFI2, SFI3, and SFI4 were highly expressed in all examined strains, suggesting their potentially important function in early stages of pathogen infection.