Characterization of Defense Signaling Pathways of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Brassica carinata</Emphasis> in Response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.     

Characterization of gene expression of <Emphasis Type="Italic">QM</Emphasis> from <Emphasis Type="Italic">Caragana jubata</Emphasis>, a plant species that grows under extreme cold

Caragana [Caragana jubata (Pall.) Poir] is a temperate plant that thrives well under extremes of cold in high altitude of Himalaya and hence the plant is expected to be a source of genes that might play an important role in tolerance to low temperature (LT). In order to identify LT inducible gene(s), differential display of mRNA (DD) was performed using the apical buds growing under snow as well as growing in the near vicinity without snow, and a LT inducible QM gene (CjQM) homologue was identified. Realizing the importance of QM gene (which encodes human Wilms’ tumor suppressor QM protein) in aggregation of 40 and 60S ribosomal subunit and that not much has been reported on this gene in plant systems in relation to its relationship with LT, full length cDNA of CjQM was cloned through rapid amplification of cDNA ends. The gene (977 bp), encoded by small gene family, had an open reading frame of 651 bp and was found to be intronless. The gene exhibited up-regulation within 20 min of exposure to LT and abscisic acid (ABA), but no significant change in gene expression was observed in response to drought stress (DS), salicylic acid (SA) and methyl jasmonate (MJ) application. Up-regulation of CjQM was obtained in the tissues growing in situ under snow. Non-responsiveness of CjQM towards DS, SA and MJ, but up-regulation in response to LT and ABA suggested a specific regulation of the gene in Caragana under varied cues.     

Identification and expression analysis of <Emphasis Type="Italic">CjLTI</Emphasis>, a novel low temperature responsive gene from <Emphasis Type="Italic">Caragana jubata</Emphasis>

Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of 351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database, therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices, 4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure to low temperature (LT, 4 ± 3°C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid (ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115% reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression.     

Maize<Emphasis Type="Italic"> ZmMEK1</Emphasis> is a single-copy gene

Mitogen-activated protein kinase (MAPK) cascade constitutes a conserved signaling module in eukaryotes. MAPK kinase (MAPKK) plays a crucial role in a MAPK cascade. ZmMEK1 is the first characterized MAPKK gene in maize. Although ZmMEK1 has been studied in detail in biochemical level, the genomic organization of ZmMEK1 gene is obscure. In this research, we clarified ZmMEK1 is a single-copy gene in the maize genome. Southern blot analysis using 3′ specific region of ZmMEK1 cDNA as a probe revealed the presence of distinct single bands in each lane of EcoRI and HindIII. Although previous Southern blot analysis using full-length ZmMEK1 cDNA as a probe revealed several hybridizing bands, we showed here that all bands come from one genomic fragment corresponding to ZmMEK1 gene. Furthermore, ZmMEK1 was induced by PEG, abscisic acid (ABA), and salicylic acid (SA) and was down-regulated by NaCl.     

Salicylic acid and salicylic acid glucoside in xylem sap of <Emphasis Type="Italic">Brassica napus</Emphasis> infected with <Emphasis Type="Italic">Verticillium longisporum</Emphasis>

Salicylic acid (SA) and its glucoside (SAG) were detected in xylem sap of Brassica napus by HPLC–MS. Concentrations of SA and SAG in xylem sap from the root and hypocotyl of the plant, and in extracts of shoots above the hypocotyl, increased after infection with the vascular pathogen Verticillium longisporum. Both concentrations were correlated with disease severity assessed as the reduction in shoot length. Furthermore, SAG levels in shoot extracts were correlated with the amount of V. longisporum DNA in the hypocotyls. Although the concentration of SAG (but not SA) in xylem sap of infected plants gradually declined from 14 to 35 days post infection, SAG levels remained significantly higher than in uninfected plants during the whole experiment. Jasmonic acid (JA) and abscisic acid (ABA) levels in xylem sap were not affected by infection with V. longisporum. SA and SAG extend the list of phytohormones potentially transported from root to shoot with the transpiration stream. The physiological relevance of this transport and its contribution to the distribution of SA in plants remain to be elucidated.     

<Emphasis Type="Italic">Fusarium</Emphasis> Infection Causes Phenolic Accumulations and Hormonal Disorders in <Emphasis Type="Italic">Orobanche</Emphasis> spp.

The physiological effects of Fusarium oxysporum on in-root parasitic weed, Orobanche spp. (broomrape) with references to change in plant hormones and secondary plant constituents were investigated. The levels of IAA, GA, ABA and JA in the experimental group were significantly lower than those in the control group, while the level of SA was higher in the experimental group. In secondary metabolic studies, the quantities of various phenols were measured in the two groups and catechin, syringic acid and p-coumaric acid amounts were significantly higher in the experimental group than in the control group, unlike gallic acid which have a lower amount. Consequently, in the light of all data, it was concluded that Fusarium oxysporum (1) causes heavy hormonal disorder, (2) triggered only SA-mediated defense and (3) induced intensively accumulation of phenolic substances in orobanche. Fusarium oxysporum causes lethal physiological damage on Orobanche spp.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Expression analysis and functional characterization of a novel cold-responsive gene <Emphasis Type="Italic">CbCOR15a</Emphasis> from <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd’s purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA₃ accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA₃ regulation and confers enhanced cold acclimation in transgenic plants.     

Aridity promotes differences in proline and phytohormone levels in <Emphasis Type="Italic">Pinus pinaster</Emphasis> populations from contrasting environments

We analysed proline, abscisic acid, (ABA), jasmonic acid (JA), indole acetic acid (IAA) and salicylic acid (SA) accumulation after summer drought at two Pinus pinaster provenance-progeny trial sites. The aim of the study was to evaluate P. pinaster phenotypic plasticity and intraspecific variation in the endogenous concentrations of these metabolites and to determine the best stress indicators for family and population discrimination. The environmental effect was remarkable, as striking differences between the sites were obtained for all indicators except for SA, which was unaffected by the environmental conditions. The levels of proline, ABA and IAA were higher in the xeric than in the mesic site. In contrast, JA was higher in the mesic site. The higher variation displayed at the family level led to family differences for all parameters and sites. Differences in proline and ABA between populations were exclusively found in the xeric site, where the population from the wet climate showed higher accumulation. This study provides evidence for differentiation among P. pinaster populations and families in their plastic responses to drought and highlights the importance of considering intraspecific variability when evaluating biochemical stress indicators in environmental studies.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

Endophytic bacteria in sunflower (<Emphasis Type="Italic">Helianthus annuus </Emphasis>L.): isolation,characterization, and production of jasmonates and abscisic acid in culture medium

This study was designed to isolate and characterize endophytic bacteria from sunflower (Helianthus annuus) grown under irrigation and water stress (drought) conditions, to analyze growth of isolated bacteria under drought condition, and to evaluate the ability of bacteria isolated from plants cultivated under drought to produce jasmonates (JAs) and abscisic acid (ABA). Bacteria were isolated from soil samples collected when sunflower plants were at the end of the vegetative stage. A total of 29 endophytic strains were isolated from plants grown under irrigation or drought condition. Eight strains (termed SF1 through SF8) were selected based on nitrogen-fixing ability. All eight strains showed positive catalase and oxidase activities; five strains (SF2, SF3, SF4, SF5, SF7) solubilized phosphates; none of the strains produced siderophores. Strains SF2, SF3, SF4, and SF5, the ones with the highest phosphate solubilization ability, strongly inhibited growth of the pathogenic fungi Verticillum orense and Sclerotinia sclerotiorum but had less inhibitory effect on Alternaria sp. Among the eight strains, SF2 showed 99.9% sequence homology with Achromobacter xiloxidans or Alcaligenes sp., while the other seven showed 99.9% homology with Bacillus pumilus. Strains SF2, SF3, and SF4 grown in control medium produced jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), and ABA. These three strains did not differ in amount of JA or OPDA produced. ABA content was higher than that of JA, and production of both ABA and JA increased under drought condition. The characteristics of these isolated bacterial strains have technological implications for inoculant formulation and improved growth of sunflower crops.     

Cloning of a 9-<Emphasis Type="Italic">cis</Emphasis>-epoxycarotenoid dioxygenase gene (<Emphasis Type="Italic">SgNCED1</Emphasis>) from <Emphasis Type="Italic">Stylosanthes guianensis </Emphasis>and its expression in response to abiotic stresses

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. Oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is the main regulatory step in the biosynthesis of ABA in higher plants. A NCED gene, SgNCED1, was cloned from the dehydrated leaves of Stylosanthes guianensis. The 2,241-bp full-length SgNCED1 had a 1,809-bp ORF, which encodes a peptide of 602 amino acids. The deduced amino acid sequence of SgNCED1 protein shared high identity with other NCEDs. At the N-terminus of the SgNCED1 located a chloroplast transit peptide sequence. DNA blot analysis revealed that SgNCED1 was a single copy gene in the genome of S. guianensis. The relationship between expression of SgNCED1 and endogenous ABA level was investigated. The expression of SgNCED1 was induced in both leaves and roots of S. guianensis under drought stress. Dehydration and salt stress induced the expression of SgNCED1 strongly and rapidly. The ABA accumulation was coincidently induced with the SgNCED1 mRNA under drought, dehydration and salt stress. The expression of SgNCED1 and ABA accumulation were also induced under chilling condition.