Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

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.     

Expression of the <Emphasis Type="Italic">Vicia faba VfPIP1</Emphasis> gene in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants improves their drought resistance

Plant aquaporins are believed to facilitate water transport across cell membranes. However, the relationship between aquaporins and drought resistance in plants remains unclear. VfPIP1, a putative aquaporin gene, was isolated from Vicia faba leaf epidermis, and its expression was induced by abscisic acid (ABA). Our results indicated that the VfPIP1 protein was localized in the plasma membrane, and its expression in V. faba was induced by 20% polyethylene glycol 6000. To further understand the function of VfPIP1, we obtained VfPIP1-expressing transgenic Arabidopsis thaliana plants under the control of the CaMV35S promoter. As compared to the wild-type control plants, the transgenic plants exhibited a faster growth rate, a lower transpiration rate, and greater drought tolerance. In addition, the stomata of the transgenic plants closed significantly faster than those of the control plants under ABA or dark treatment. These results suggest that VfPIP1 expression may improve drought resistance of the transgenic plants by promoting stomatal closure under drought stress.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains. Genes encoding many of these cysteine-rich RLKs (CRKs) are induced by pathogen infection, suggesting a possible role in plant defense responses. We have previously generated Arabidopsis plants expressing four pathogen-regulated CRK genes (CRK5, 6, 10 and 11) under control of a steroid-inducible promoter and found that induced expression of CRK5, but not the other three CRK genes, triggered hypersensitive response-like cell death in transgenic plants. In the present study, we have analyzed the structural relationship of the CRK family and identified three CRKs (CRK4, 19 and 20) that are structurally closely related to CRK5. Genes encoding these three CRKs are all induced by salicylic acid and pathogen infection. Furthermore, induced expression of CRK4, 19and 20 all activates rapid cell death in transgenic plants. Thus, the activity of inducing rapid cell death is shared by these structurally closely related CRKs. We have also performed yeast two-hybrid screens and identified proteins that interact with the kinase domains of CRKs. One of the identified CRK-interacting proteins is the kinase-associated type 2C protein phospohatase known to interact with a number of other RLKs through its kinase-interacting FHA domain. Other CRK-interacting proteins include a second protein with a FHA domain and another type 2C protein phosphatase. Interactions of CRKs with these three proteins in vivo were demonstrated through co-immunoprecipitation. These CRK-interacting proteins may play roles in the regulation and signaling of CRKs.     

<Emphasis Type="Italic">FORMOSA</Emphasis> controls cell division and expansion during floral development in <Emphasis Type="Italic">Antirrhinum</Emphasis><Emphasis Type="Italic">majus</Emphasis>

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

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.     

Proteomics Analysis of Drought Stress-Responsive Proteins in <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.

Hippophae rhamnoides L. is uniquely capable of growing well under extreme environmental conditions such as water deficit, low temperature, and high altitude. Such tolerance invokes much interest in understanding the biology of this plant species and its utilization potential. In this study, analysis of drought stress-responsive proteins in H. rhamnoides was conducted wherein greenhouse-grown seedlings were subjected to drought stress. By using proteomic techniques, proteins, extracted from leaves, were analyzed using two-dimensional electrophoresis and MALDI-TOF MS. Altogether, 55 proteins exhibited changes in abundance under stress. Of these, 13 proteins were identified, including three that disappeared under drought (a putative ABC transporter ATP-binging protein, a heat shock protein HslU, and a hypothetical protein XP-515578), seven that were up-regulated (three large subunits of rubisco, a hypothetical protein DSM3645–23351, a putative acyl-CoA dehydrogenase, a nesprin-2, and a J-type co-chaperone HSC20), and three that were only detected under drought (a probable nitrogen regulation protein (NtrX), a 4-hydroxyphenylpyruvate dioxygenase, and an unnamed protein product). These proteins may function in β-oxidation pathways in mitochondria, across membranes transport, abnormal protein removal, or prevent protein aggregation arrest, cell division, cytoskeleton stabilization, iron–sulfur cluster assembly, nitrogen metabolism regulation, and antioxidant substance biosynthesis. Four proteins (J-type co-chaperone Hsc20, a putative ABC transporter ATP-binging protein, NtrX, and HslU) were deemed as new discoveries in higher plants, and their functions were predicted either from their conserved domains or homologies to other organisms. These results provide new insights into our understanding of the mechanism of drought tolerance in plants.     

Differential responses of lipid peroxidation and antioxidants in <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis> subjected to drought stress

To evaluate oxidative stress and the plant antioxidant system of Alternanthera philoxeroides [Mart.] Griseb and Oryza sativa L. in the response to drought, root and leaf tissues of drought-treated A. philoxeroides and O. sativa were collected and relative water content, stomatal conductance, the concentrations of malondialdehyde, proline and the activities of superoxide dismutase, peroxidases, catalase and total antioxidative activity investigated. The results showed that drought treatment had almost no effect on relative water content in A. philoxeroides but reduced relative water content in O. sativa. A. philoxeroides maintained a greater stomatal conductance than O. sativa under drought stress. In A. philoxeroides levels of lipid peroxidation were lower than in O. sativa and did not change during the experiment. After exposure to drought, concentrations of proline and activities of superoxide dismutase, peroxidases and catalase in A. philoxeroides were between 10% and 30% higher than in O. sativa, whereas total antioxidative activity in A. philoxeroides was several-fold higher than in O. sativa.     

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.     

Diverse bacteria isolated from root nodules of <Emphasis Type="Italic">Trifolium</Emphasis>, <Emphasis Type="Italic">Crotalaria</Emphasis> and <Emphasis Type="Italic">Mimosa</Emphasis> grown in the subtropical regions of China

We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems. Accession numbers: DQ173160 (hrcA operon); DQ173161 (rpoH gene).     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Colonization of the <Emphasis Type="Italic">Arabidopsis</Emphasis> rhizosphere by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. activates a root-specific,ethylene-responsive <Emphasis Type="Italic">PR-5</Emphasis> gene in the vascular bundle

Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISR-inducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.