Roles In Plant Defense Responses Research Articles

Nicotiana tabacum overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid-mediated pathway

The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced along with NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.

Structure and expression profiling of a novel calcium-dependent protein kinase gene PgCDPK1a in roots, leaves, and cell cultures of Panax ginseng

Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study, we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator.

Quercetin-induced H 2O 2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H 2O 2 under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana ( Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H 2O 2 burst, callose deposition, cell death, PR1 ( pathogenesis-related 1) and PAL1 ( Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H 2O 2 may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H 2O 2 burst and involvement of SA and NPR1.

Cloning and Characterization of a Pathogenesis-Related Protein Gene TaPR10 from Wheat Induced by Stripe Rust Pathogen

Pathogenesis-related proteins (PRs) play many important roles in plant defense response against pathogen attack. To better understand the molecular mechanism of PR genes involved in wheat adult plant resistance (APR) to stripe rust, based on a differentially expressed transcribed derived fragment (TDF), a novel PR gene from wheat cv. Xingzi 9104 infected by the Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32, which was highly similar to the maize ZmPR10 gene and designated as TaPR10, was identified using in silico cloning and RT-PCR method. This novel TaPR10 gene was predicted to encode a 160-amino acid protein with a deduced molecular weight of 17.06 kDa and an isoelectronic point ( pI) of 5.19. An amino acid sequence analysis of TaPR10 demonstrated the presence of a typical conserved domain of pathogenesis related protein Bet_v_I family. Multiple alignment analysis based on the amino acids encoded by 10 different PR10 genes from maize ( Zea mays), rice ( Oryza sativa), broomcorn ( Sorghum bicolor), and wheat ( Triticum aestivum) indicated that PR proteins of class 10 was conserved among the 4 plant species with about 80% similarity. DNA sequence of TaPR10 suggested the presence of one 84-bp intron with the splicing sites of GT-AT bi-nucleotide sequence between 188 and 271 bp. Using a real-time quantitative RT-PCR (qRT-PCR), expression profiles of TaPR10 revealed that at the adult-plant stage, TaPR10 transcript was up-regulated as early as 12 h post-inoculation (hpi), with the occurrence of maximum induction at 24 hpi. At the seedling stage, TaPR10 was also slightly induced 18 hpi. However, the transcript amount was relatively lower than that of the adult-plant stage. Taken together, these results suggest that TaPR10 may participate in wheat defense response of APR to stripe rust.

GhWRKY3, a novel cotton (Gossypium hirsutum L.) WRKY gene, is involved in diverse stress responses

WRKY proteins play important roles in plant defense responses. In the present study, a novel WRKY gene, nominated as GhWRKY3, was isolated from cotton (Gossypium hirsutum L.). The full-length cDNA of GhWRKY3 is 1,705bp in length and encodes a protein with 507 amino acids containing two typical WRKY domains and two zinc finger motifs. Amino acid sequence alignment revealed that GhWRKY3 shares a high degree of identity with other higher plant WRKY proteins. The subcellular localization assay indicated that GhWRKY3 is localized to the nucleus. Analysis of 5'-flanking region of GhWRKY3 revealed a group of putative cis-acting elements. The results of expression analysis indicated that GhWRKY3 is constitutively expressed in roots, stems and leaves. Semi-quantitative RT-PCR showed that GhWRKY3 is up-regulated by application of various phytohormones including salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), gibberellins (GAs) and ethylene (ET). Furthermore, the transcripts of GhWRKY3 are enhanced after infection with Rhizoctonia solani, Colletotrichum gossypii and Fusarium oxysporum f. sp. vasinfectum, respectively. Also, GhWRKY3 can be induced by wounding treatment, but not by cytokinin (6-benzylaminopurine, 6-BA), auxin analogue, drought, NaCl, and cold (4°C). These data suggested that GhWRKY3 might play an important role in plant defense responses and fulfill a pivotal role in plant development.

Silencing OsHI‐LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder

The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plant's JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H(2)O(2) and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests.

Expression of ROP/RAC GTPase genes in postharvest loquat fruit in association with senescence and cold regulated lignification

ROP/RAC GTPases regulate various development processes and play important roles in plant defense responses. Recently, lignification or secondary cell wall formation related ROP members were reported in rice, zinnia, cotton and Eucalyptus. The present study aimed to investigate the possible association of loquat ROPs with flesh lignification under different temperatures. Four ROP cDNA fragments, EjROP1.1, EjROP1.2, EjROP2 and EjROP3, were isolated from ‘Luoyangqing’ (LYQ) loquat fruit, and all of them shared over 80% nucleotide identity with known ROPs from other plants. Sequence analysis revealed that EjROP1.1, EjROP2 and EjROP3 might be functional while EjROP1.2, with mutated C-terminal resulted from a 65 bp deletion in the corresponding nucleotide sequence as compared with EjROP1.1, might be dominant-negative and consequently act as a negative regulator of ROP signal transduction. Increase in expression of EjROP1.1, EjROP2 and EjROP3 was observed during first 4 or 6 d of storage at 20 °C and was positively correlated with the increase in flesh firmness. Expression of EjROP1.2 was constantly low under 20 °C but was quickly, within 6 h, induced under 0 °C, and it increased by about 20 times within 24 h. The expression was induced under 5 °C as well but not so strong as that under 0 °C, and transfer of fruit from 5 °C to 0 °C re-stimulated the expression. The possible roles of EjROPs played during senescence and cold regulated lignification was discussed, and the simultaneous increase in the expression of three functional EjROPs and the negative regulator EjROP1.2 was suggested to be important for maintaining a ROP rheostat to protect cells from excessive lignification. To our knowledge, this is the first study on a dominant-negative ROP resulted from a deletion mutation, and a ROP responded to low temperature.

Suppression of the Rice Fatty-Acid Desaturase Gene OsSSI2 Enhances Resistance to Blast and Leaf Blight Diseases in Rice

Fatty acids and their derivatives play important signaling roles in plant defense responses. It has been shown that suppressing a gene for stearoyl acyl carrier protein fatty-acid desaturase (SACPD) enhances the resistance of Arabidopsis (SSI2) and soybean to multiple pathogens. In this study, we present functional analyses of a rice homolog of SSI2 (OsSSI2) in disease resistance of rice plants. A transposon insertion mutation (Osssi2-Tos17) and RNAi-mediated knockdown of OsSSI2 (OsSSI2-kd) reduced the oleic acid (18:1) level and increased that of stearic acid (18:0), indicating that OsSSI2 is responsible for fatty-acid desaturase activity. These plants displayed spontaneous lesion formation in leaf blades, retarded growth, slight increase in endogenous free salicylic acid (SA) levels, and SA/benzothiadiazole (BTH)-specific inducible genes, including WRKY45, a key regulator of SA/BTH-induced resistance, in rice. Moreover, the OsSSI2-kd plants showed markedly enhanced resistance to the blast fungus Magnaporthe grisea and leaf-blight bacteria Xanthomonas oryzae pv. oryzae. These results suggest that OsSSI2 is involved in the negative regulation of defense responses in rice, as are its Arabidopsis and soybean counterparts. Microarray analyses identified 406 genes that were differentially expressed (>or=2-fold) in OsSSI2-kd rice plants compared with wild-type rice and, of these, approximately 39% were BTH responsive. Taken together, our results suggest that induction of SA-responsive genes, including WRKY45, is likely responsible for enhanced disease resistance in OsSSI2-kd rice plants.

An S-locus receptor-like kinase plays a role as a negative regulator in plant defense responses

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.

Involvement of nitric oxide in ultraviolet B-induced activation of phenylalanine ammoniumlyase and stimulation of flavonoid biosynthesis in Ginkgo biloba leaves

Previous studies have shown that ultraviolet B (UV-B) irradiation can stimulate biosynthesis of secondary metabolites. Several other studies have demonstrated that nitric oxide (NO) is an important signal molecule that plays a key role in plant defence responses to UV-B irradiation. However, the signalling mechanism of NO involvement in UV-B-induced secondary-metabolite synthesis is unclear. The purpose of the present study is to investigate the role of NO in the accumulation UV-B-induced secondary metabolite in the leaves of Ginkgo biloba. Our results showed that UV-B irradiation induced multiple biological responses in the leaves of G. biloba, including increases in both NO production and nitric oxide synthase (NOS) activity, and subsequent activation of phenylalanine ammoniumlyase (PAL) and synthesis of flavonoids. Application of NO via the donor sodium nitroprusside (SNP) enhanced UV-B-induced PAL activity and increased accumulation of flavonoids in G. biloba leaves. Both the NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) and the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO) reduced the production of NO. Moreover, UV-B-induced increase in PAL activity and flavonoid accumulation were suppressed by L-NAME and c-PTIO. These findings suggest a causal relationship between NO release and both PAL activity and flavonoid accumulation under UV-B irradiation. In addition, the results also indicate that NO, produced via NOS-like activity in ginkgo leaves subjected to UV-B irradiation, might act as an essential signal molecule for triggering the activation of PAL and synthesis of flavonoids.

The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

Messenger RNA (mRNA) turnover in eukaryotic cells begins with shortening of the poly (A) tail at the 3' end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4-associated factor 1 (CAF1), two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast caf1 mutant in the presence of caffeine or at high temperatures. The expression of AtCAF1a and AtCAF1b is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAF1b show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAF1b show reduced expression of pathogenesis-related (PR) genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection, whereas transgenic plants over-expressing AtCAF1a show elevated expression of PR1 and PR2 and increased resistance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro‐Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)‐dependent defence response in Micro‐Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA‐ and JA‐dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA‐inducible PR‐1(P6) gene was not up‐regulated in either Micro‐Tom or Moneymaker. SA did not accumulate in homogenate‐treated roots in comparison with distilled water‐treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA‐non‐accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA‐responsive gene for the basic PR‐6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate‐treated jai1‐1 mutant plants defective in JA signalling. These results indicated that the JA‐dependent signalling pathway is required for PO‐induced resistance against R. solanacearum in tomato.

Stress- and Pathogen-Induced Arabidopsis WRKY48 is a Transcriptional Activator that Represses Plant Basal Defense

Plant WRKY transcription factors can function as either positive or negative regulators of plant basal disease resistance. Arabidopsis WRKY48 is induced by mechanical and/or osmotic stress due to infiltration and pathogen infection and, therefore, may play a role in plant defense responses. WRKY48 is localized to the nucleus, recognizes the TTGACC W-box sequence with a high affinity in vitro and functions in plant cells as a strong transcriptional activator. To determine the biological functions directly, we have isolated loss-of-function T-DNA insertion mutants and generated gain-of-function transgenic overexpression plants for WRKY48 in Arabidopsis. Growth of a virulent strain of the bacterial pathogen Pseudomonas syringae was decreased in the wrky48 T-DNA insertion mutants. The enhanced resistance of the loss-of-function mutants was associated with increased induction of salicylic acid-regulated PR1 by the bacterial pathogen. By contrast, transgenic WRKY48-overexpressing plants support enhanced growth of P. syringae and the enhanced susceptibility was associated with reduced expression of defense-related PR genes. These results suggest that WRKY48 is a negative regulator of PR gene expression and basal resistance to the bacterial pathogen P. syringae.

Fractional changes in phenolic acids composition in root nodules of Arachis hypogaea L.

Phenolic acids are active antimicrobial compounds and root signaling molecules that play important roles in plant defense responses. They are generally present in plants as glycosides or esters. A range of soluble and bound phenolic acids were detected in roots and root nodules of Arachis hypogaea L., among which five were identified by high performance liquid chromatography (HPLC) coupled with UV–Vis diode array detector (DAD), viz., p-coumaric acid (p-com), p-hydroxybenzaldehyde (HBAld), p-hydroxybenzoic acid (HBA), caffeic acid (CA) and protocatechuic acid (PA). Para-coumaric acid was constitutively present in all fractions whereas HBA was present in the soluble form only in young nodules. CA and PA were mostly present in the wall bound fraction. The root nodules contain higher concentration of phenolic acids than non-nodulated roots and presence of peroxidase and polyphenol oxidase indicate the metabolism of phenolic acids in roots and root nodules. These results indicate that phenolic acids (p-com and CA) in bound-glycosidic or ester forms were major components in cell wall fortification which provide protection to the root nodule from pathogen attack.

Distinct Roles of Jasmonates and Aldehydes in Plant-Defense Responses

BackgroundMany inducible plant-defense responses are activated by jasmonates (JAs), C6-aldehydes, and their corresponding derivatives, produced by the two main competing branches of the oxylipin pathway, the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches, respectively. In addition to competition for substrates, these branch-pathway-derived metabolites have substantial overlap in regulation of gene expression. Past experiments to define the role of C6-aldehydes in plant defense responses were biased towards the exogenous application of the synthetic metabolites or the use of genetic manipulation of HPL expression levels in plant genotypes with intact ability to produce the competing AOS-derived metabolites. To uncouple the roles of the C6-aldehydes and jasmonates in mediating direct and indirect plant-defense responses, we generated Arabidopsis genotypes lacking either one or both of these metabolites. These genotypes were subsequently challenged with a phloem-feeding insect (aphids: Myzus persicae), an insect herbivore (leafminers: Liriomyza trifolii), and two different necrotrophic fungal pathogens (Botrytis cinerea and Alternaria brassicicola). We also characterized the volatiles emitted by these plants upon aphid infestation or mechanical wounding and identified hexenyl acetate as the predominant compound in these volatile blends. Subsequently, we examined the signaling role of this compound in attracting the parasitoid wasp (Aphidius colemani), a natural enemy of aphids.Principal FindingsThis study conclusively establishes that jasmonates and C6-aldehydes play distinct roles in plant defense responses. The jasmonates are indispensable metabolites in mediating the activation of direct plant-defense responses, whereas the C6-aldehyes are not. On the other hand, hexenyl acetate, an acetylated C6-aldehyde, is the predominant wound-inducible volatile signal that mediates indirect defense responses by directing tritrophic (plant-herbivore-natural enemy) interactions.SignificanceThe data suggest that jasmonates and hexenyl acetate play distinct roles in mediating direct and indirect plant-defense responses. The potential advantage of this “division of labor” is to ensure the most effective defense strategy that minimizes incurred damages at a reduced metabolic cost.

Nitric Oxide Elicitation Induces the Accumulation of Secondary Metabolites and Antioxidant Defense in Adventitious Roots ofEchinacea purpurea

Nitric oxide and reactive oxygen species are important signal molecules that play key roles in plant defense responses. We investigated the involvement of nitric oxide elicitation in the synthesis of secondary metabolites within the adventitious roots ofEchinacea purpurea. When roots were treated with 100 μM sodium nitroprusside (SNP), an exogenous nitric oxide producer, the accumulation of phenolics, flavonoids, and caffeic acid derivatives was enhanced. This level of SNP also induced an antioxidant defense, as indicated by increases in Superoxide dismutase, ascorbate peroxidase, and ascorbic acid, along with decreases in hydrogen peroxide, lipid peroxidation, and dehydroascorbate/ascorbic acid. However, a higher concentration (250 μM SNP) acted as a pro-oxidant, thereby raising the levels of hydrogen peroxide, lipid peroxidation, and dehydroascorbate/ascorbic acid while diminishing ascorbic acid, ascorbate peroxidase, and the accumulation of secondary metabolites compared with our observations at 100 μM SNP. Therefore, we conclude that elicitingE. purpurea adventitious roots with a concentration of 100 μM SNP is beneficial to their accumulation of secondary metabolites.

Elevated thiol levels affect virus infection inCucurbita pepoplants regenerated via somatic embryogenesis

Thiols are known to play crucial roles in plant defense responses, e.g. against pathogens. In the present study, the efficiency of these molecules was evaluated in regenerated Cucurbita pepo L. subsp. pepo var. styriaca Greb. plants in response to compatible Zucchini yellow mosaic virus infection. Thiol contents were modulated by applying the cysteine precursor L-2-oxothiazolidine-4-carboxylic acid (OTC) to callus cells. Cytological and biochemical methods were used for determination of glutathione within callus cells and leaves of plants regenerated via somatic embryogenesis. Reduced glutathione levels (GSH) significantly increased in callus cells one week after 1.0 mM OTC treatment by about 56% and resulted in enhanced GSH levels throughout the investigation period of 13 weeks. Three weeks after ZYMV infection untreated plants regenerated from callus culture exhibited stunted growth forms, yellowing, mosaic and blistering on the leaves whereas 68% of 1.0 mM OTC pretreated regenerated plants were characterized by a complete absence of symptoms. Treatment with OTC stimulated the synthesis, conversion and translocation of thiols during advanced ZYMV infection. Compared to untreated infected leaves, a graduate increase in cysteine (cys) and γ-glutamyl-cysteine (γ-glucys) contents was measured in 0.5 mM and 1.0 mM OTC treated older leaves, whereas total glutathione (tGSH) decreased significantly after infection. In OTC treated younger leaves cys and tGSH concentrations remained at control levels and were unaffected by ZYMV. In contrast, OTC treatment caused a dramatic induction of γ-glucys, up to 191% in younger infected leaves. The results show that altered levels of thiols by OTC were accompanied by enhanced tolerance against ZYMV, suppression of ZYMV induced symptoms as well as delayed symptom development.

Implication of a pepper h-type thioredoxin in type I- and II-nonhost resistance to Xanthomonas axonopodis

Thioredoxins (TRXs) are distributed ubiquitously in prokaryotic and eukaryotic organisms. Plants have the most complex forms of TRXs. The functional roles of such TRXs have been studied in abiotic stress but their roles in plant defense responses against biotic stresses have been less well studied. Here, we identified an h-type TRX gene from pepper, CaTRXh1, and characterized its possible effect on Type II nonhost resistance, which entails localized programmed cell death in response to nonhost pathogens. Peptide sequences of CaTRXh1 showed a high degree of similarity with TRXhs from tobacco and Arabidopsis thaliana. Southern blot analyses revealed that CaTRXh1 was present as a single copy in the pepper genome. Intriguingly, leaf infiltration by Xanthomonas axonopodis pv. glycines 8ra, eliciting a visible type II nonhost hypersensitive response (HR), and its type III secretion-system null mutant 8–13, eliciting a type I nonhost non-HR, both induced CaTRXh1 at a level similar to that of pathogenesis-related protein 4, an HR marker gene in pepper. More surprisingly, expression of CaTRXh1 was significantly increased when X. axonopodis pv. vesicatoria race 3 infiltrated the leaf of a pepper cultivar containing a resistance gene, but not with infiltration of a susceptible pepper cultivar. Taken together, our study suggests that the expression of CaTRXh1 has a critical role in HR-mediated active defense responses in pepper.

Identification of a new AT-rich-element binding factor PsATF1 and its combined effect with PsGBF on the activation of PsCHS1 promoter.

Chaltone synthase (CHS) is a key speed-limiting enzyme in the phenylpropanoid pathway which plays an important role in plant defense response against pathogens. In the PsCHS1 promoter, there is an AT-rich element (ATRE) which is required for the maximal elicitor-mediated activation. However, the transcription activator of the ATRE and its regulation mechanism in pea keep unclear. In this paper, a new ATRE-binding factor was isolated from an elicitor-induced pea cDNA expression library and was designated as PsATF1. Electrophoretic mobility shift assay (EMSA) indicated the ATRE-specific binding activity of PsATF1. Beta-galactosidase assays in yeast cells suggested that PsATF1 possessed transcription-activating activity because PsATF1 activated the expression of the reporter gene even without the GAL4 activation domain (AD). The current study also examined the co-activation effects of PsATF1 with another transcription factor PsGBF on ATRE or PsCHS1 promoter through a transient expression system. The present work reports that PsATF1 acts as a complete transcription activator and first indicates that there are combined effects of PsATF1 with PsGBF on the activation of PsCHS1 promoter. These results provide theoretical basis to the plant defense gene expression mechanism regulated by multiple activators.

Plant Seed Peroxygenase Is an Original Heme-oxygenase with an EF-hand Calcium Binding Motif

A growing body of evidence indicates that phytooxylipins play important roles in plant defense responses. However, many enzymes involved in the biosynthesis of these metabolites are still elusive. We have purified one of these enzymes, the peroxygenase (PXG), from oat microsomes and lipid droplets. It is an integral membrane protein requiring detergent for its solubilization. Proteinase K digestion showed that PXG is probably deeply buried in lipid droplets or microsomes with only about 2 kDa at the C-terminal region accessible to proteolytic digestion. Sequencing of the N terminus of the purified protein showed that PXG had no sequence similarity with either a peroxidase or a cytochrome P450 but, rather, with caleosins, i.e. calcium-binding proteins. In agreement with this finding, we demonstrated that recombinant thale cress and rice caleosins, expressed in yeast, catalyze hydroperoxide-dependent mono-oxygenation reactions that are characteristic of PXG. Calcium was also found to be crucial for peroxygenase activity, whereas phosphorylation of the protein had no impact on catalysis. Site-directed mutagenesis studies revealed that PXG catalytic activity is dependent on two highly conserved histidines, the 9 GHz EPR spectrum being consistent with a high spin pentacoordinated ferric heme.