A synthetic antimicrobial peptide BTD-S expressed in Arabidopsis thaliana confers enhanced resistance to Verticillium dahliae.

BTD-S is a synthetic non-cyclic θ-defensin derivative which was previously designed in our laboratory based on baboon θ-defensins (BTDs). It shows robust antimicrobial activity against economically important phytopathogen, Verticillium dahliae. Here, we deduced the coding nucleotide sequence of BTD-S and introduced the gene into wild-type (ecotype Columbia-0) Arabidopsis thaliana plants. Results demonstrated that BTD-S-transgenic lines displayed in bioassays inhibitory effects on the growth of V. dahliae in vivo and in vitro. Based on symptom severity, enhanced resistance was found in a survey of BTD-S-transgenic lines. Besides, crude protein extracts from root tissues of BTD-S-transformed plants significantly restricted the growth of fungal hyphae and the germination of conidia. Also, fungal biomass over time determined by real-time PCR demonstrated the overgrowth of V. dahliae in wild-type plants 2-3 weeks after inoculation, while almost no fungal DNA was detected in aerial tissues of their transgenic progenitors. The result suggested that fungus failed to invade and progress acropetally up to establish a systemic infection in BTD-S-transgenic plants. Moreover, the assessment of basal defense responses was performed in the leaves of WT and BTD-S-transgenic plants. The mitigated oxidative stress and low antioxidase level in BTD-S-transgenic plants revealed that BTD-S acts via permeabilizing target microbial membranes, which is in a category different from hypersensitive response-dependent defense. Taken together, our results demonstrate that BTD-S is a promising gene to be explored for transgenic engineering for plant protection against Verticillium wilt.

A novel NAP member GhNAP is involved in leaf senescence in Gossypium hirsutum.

Premature leaf senescence has a negative influence on the yield and quality of cotton, and several genes have been found to regulate leaf senescence. Howeer, many underlying transcription factors are yet to be identified. In this study, a NAP-like transcription factor (GhNAP) was isolated from Gossypium hirsutum. GhNAP has the typical NAC structure and a conserved novel subdomain in its divergent transcription activation region (TAR). GhNAP was demonstrated to be a nuclear protein, and it showed transcriptional activation activity in yeast. Furthermore, the expression of GhNAP was closely associated with leaf senescence. GhNAP could rescue the delayed-senescence phenotype of the atnap null mutant. Overexpression of GhNAP could cause precocious senescence in Arabidopsis. However, down-regulation of GhNAP delayed leaf senescence in cotton, and affected cotton yield and its fibre quality. Moreover, the expression of GhNAP can be induced by abscisic acid (ABA), and the delayed leaf senescence phenotype in GhNAPi plants might be caused by the decreased ABA level and reduced expression level of ABA-responsive genes. All of the results suggested that GhNAP could regulate the leaf senescence via the ABA-mediated pathways and was further related to the yield and quality in cotton.

Molecular evolution and species-specific expansion of the NAP members in plants.

The NAP (NAC-Like, Activated by AP3 /PI) subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants, but no NAP members were found in eight non-vascular plants. All NAP proteins were phylogenetically classified into two groups (NAP I and NAP II), and the origin time of the NAP I group might be relatively later than that of the NAP II group. Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns. Besides, functional divergence was limited after the gene duplication. Abscisic acid (ABA) might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants.

A non-cyclic baboon θ-defensin derivative exhibiting antimicrobial activity against the phytopathogen Verticillium dahliae.

θ-Defensins are the only natural cyclic proteins found in primates. They have strong antimicrobial activity related to their trisulfide ladders and macrocyclic conformation. A non-cyclic baboon θ-defensin (BTD) was synthesized by substituting valine with phenylalanine at position 17, at the C-terminal end of the BTD; this was termed "BTD-S." The antimicrobial activities of this synthetic peptide were investigated against Escherichia coli and two cotton phytopathogens: Verticillium dahliae and Fusarium oxysporum. The minimum inhibitory concentration (MIC) of BTD-S for E. coli was 10 µg/mL and for V. dahliae was 5 µg/mL, significantly lower than that for F. oxysporum (40.0 µg/mL). A time course analysis of fungal cultures indicated that the growth of V. dahliae was completely inhibited after 96 h of BTD-S treatment. Furthermore, hemolysis assays revealed that BTD-S was not toxic to mammalian cells as it could not induce lysis of sheep red blood cells even at ten times the MIC (50 µg/mL). Scanning electron microscopy and double-stained (calcofluor white and propidium iodide binding) fluorescence microscopy showed that exposure of spores of V. dahliae to BTD-S either disabled normal germination or disintegrated the spores. The size of cells exposed to BTD-S was significantly reduced compared with controls, and their number increased in a dose-dependent curve when measured by flow cytometry. These findings suggest that BTD-S has great potential to inhibit the growth of V. dahliae and can be utilized as an effective remedy to control economic losses caused by Verticillium wilt in the development of wilt-resistant cotton.

Role of brassinosteroids in alleviating toxin-induced stress of Verticillium dahliae on cotton callus growth.

Brassinosteroids are well known to mitigate biotic stresses; however, their role to induce tolerance against Verticillium dahliae is unknown. The current study employed V. dahliae (Vd) toxin as pathogen-free model system to induce stress on cotton callus growth, and its amelioration was investigated using 24-epibrassinolide (EBR). Results revealed that EBR has ameliorative effects against Vd toxin with greater seen effect when callus was treated with EBR prior to its exposure to Vd toxin (pre-EBR treatment) than EBR applied along with Vd toxin simultaneously (co-EBR treatment). Pre-EBR-treated calli remained green, while 65 and 90% callus browning was observed in co-EBR- and Vd toxin-alone-treated callus, respectively. Likewise, the fresh weight of the pre-EBR-treated callus was 52% higher than Vd toxin-alone treatment, whereas this increase was only 23% in co-EBR-treated callus. Meanwhile, EBR treatment of the cotton callus has also increased the contents of chlorophylls a and b, carotenoids, total phenols, flavonoids, soluble sugars, and proteins and increased the activity of enzymes involved in secondary metabolism like polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL), cinnamyl alchol dehydrogenase (CAD), and shikimate dehydrogenase (SKDH) over Vd toxin-alone treatment with higher increments being observed in pre-EBR-treated callus. Furthermore, EBR treatment mimicked the DNA damage and improved the structure of mitochondria, granum, stroma thylakoids, and the attachment of ribosomes with the endoplasmic reticulum. This EBR-mediated mitigation was primarily associated with substantially increased contents of photosynthetic pigments and regulation of secondary metabolism.

Genomic Identification and Comparative Expansion Analysis of the Non-Specific Lipid Transfer Protein Gene Family in Gossypium.

Plant non-specific lipid transfer proteins (nsLTPs) are involved in many biological processes. In this study, 51, 47 and 91 nsLTPs were identified in Gossypium arboreum, G. raimondii and their descendant allotetraploid G. hirsutum, respectively. All the nsLTPs were phylogenetically divided into 8 distinct subfamilies. Besides, the recent duplication, which is considered cotton-specific whole genome duplication, may have led to nsLTP expansion in Gossypium. Both tandem and segmental duplication contributed to nsLTP expansion in G. arboreum and G. hirsutum, while tandem duplication was the dominant pattern in G. raimondii. Additionally, the interspecific orthologous gene pairs in Gossypium were identified. Some GaLTPs and GrLTPs lost their orthologs in the At and Dt subgenomes, respectively, of G. hirsutum. The distribution of these GrLTPs and GaLTPs within each subfamily was complementary, suggesting that the loss and retention of nsLTPs in G. hirsutum might not be random. Moreover, the nsLTPs in the At and Dt subgenomes might have evolved symmetrically. Furthermore, both intraspecific and interspecific orthologous genes showed considerable expression variation, suggesting that their functions were strongly differentiated. Our results lay an important foundation for expansion and evolutionary analysis of the nsLTP family in Gossypium, and advance nsLTP studies in other plants, especially polyploid plants.

Molecular evolution and expansion analysis of the NAC transcription factor in Zea mays.

NAC (NAM, ATAF1, 2 and CUC2) family is a plant-specific transcription factor and it controls various plant developmental processes. In the current study, 124 NAC members were identified in Zea mays and were phylogenetically clustered into 13 distinct subfamilies. The whole genome duplication (WGD), especially an additional WGD event, may lead to expanding ZmNAC members. Different subfamily has different expansion rate, and NAC subfamily preference was found during the expansion in maize. Moreover, the duplication events might occur after the divergence of the lineages of Z. mays and S. italica, and segmental duplication seemed to be the dominant pattern for the gene duplication in maize. Furthermore, the expansion of ZmNAC members may be also related to gain and loss of introns. Besides, the restriction of functional divergence was discovered after most of the gene duplication events. These results could provide novel insights into molecular evolution and expansion analysis of NAC family in maize, and advance the NAC researches in other plants, especially polyploid plants.

In vitro inhibition of pigmentation and fiber development in colored cotton.

Colored cotton has naturally pigmented fibers. The mechanism of pigmentation in cotton fiber is not well documented. This experiment was conducted to study the effects of respiratory chain inhibitors, i.e., rotenone and thiourea, on pigmentation and fiber development in colored cotton. After 1 d post-anthesis, ovaries were harvested and developing ovules were cultured on the liquid medium containing different concentrations of rotenone and thiourea for 30 d. The results demonstrate that both respiratory inhibitors reduced fiber length and ovule development under ovule culture conditions, and the inhibition efficiency of rotenone was much higher than that of thiourea. Rotenone and thiourea also showed significant effects on fiber pigment (color) development in colored cotton. In green cotton fiber, rotenone advanced fiber pigment development by 7 d at 200 µmol/L, while thiourea inhibited fiber pigmentation at all treatment levels (400, 600, 800, 1000, and 2000 µmol/L). Both respiratory inhibitors, however, had no significant effects on pigmentation of brown cotton fibers. The activities of cytochrome c oxidase (COX) and polyphenol oxidase (PPO) decreased significantly with increasing levels of both respiratory inhibitors. It is suggested that both respiratory inhibitors have important roles in deciphering the mechanism of pigmentation and fiber development in colored cotton.

Differential changes in antioxidants, proteases, and lipid peroxidation in flag leaves of wheat genotypes under different levels of water deficit conditions.

Changes in enzymatic antioxidants and oxidative injury were evaluated in flag leaves of seven wheat genotypes under well watered (WW), medium watered (MW), low watered (LW) and soil stored moisture (SSM) conditions maintained in lysimeters through neutron moisture prob. Genotypes behaved differentially in terms of antioxidant response and stress induced injury under above indicated water deficit levels. In general, antioxidant enzymes were rarely enhanced under MW condition, often increased under LW condition while remained unchanged, elevated or diminished under SSM condition (severe stress). Higher CAT and POD activities were observed in NR-234 and in Pfau followed by FD-83 respectively under LW conditions. Under SSM condition, APX and POD increased significantly in Nesser and Pfau and CAT in NR-234, Nesser and Pfau, while remained at control level or decreased in other genotypes. In NR-234, SOD activity enhanced only under LW condition. However, SOD rose in Nesser, FD-83 and Sarsabz while remained unaffected in NR-241, Sitta and Pfau under all water deficit conditions. Lipid peroxidation increased significantly in FD-83 only under MW condition along with raised protease activity and protein contents. However, peroxidation of lipids was significantly enhanced in all genotypes under LW and SSM conditions. It was concluded that response of genotypes vary under different levels of water deficit. Hydrogen peroxide scavenging system was more actively involved in detoxification of oxidative stress induced by water deficit. Raised antioxidants (CAT, POD) resulting in comparatively lower lipid peroxidation in Pfau under SSM condition and in Sitta under LW condition confer stress tolerance in these genotypes.