Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Two WD-repeat genes from cotton are functional homologues of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis><Emphasis Type="Italic">TRANSPARENT TESTA GLABRA1</Emphasis> (<Emphasis Type="Italic">TTG1</Emphasis>) gene

Cotton fibres are single, highly elongated cells derived from the outer epidermis of ovules, and are developmentally similar to the trichomes of Arabidopsis thaliana. To identify genes involved in the molecular control of cotton fibre initiation, we isolated four putative homologues of the Arabidopsis trichome-associated gene TRANSPARENT TESTA GLABRA1 (TTG1). All four WD-repeat genes are derived from the ancestral D diploid genome of tetraploid cotton and are expressed in many tissues throughout the plant, including ovules and growing fibres. Two of the cotton genes were able to restore trichome formation in ttg1 mutant Arabidopsis plants. Both these genes also complemented the anthocyanin defect in a white-flowered Matthiola incana ttg1 mutant. These results demonstrate parallels in differentiation between trichomes in cotton and Arabidopsis, and indicate that these cotton genes may be functional homologues of AtTTG1.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

AtCPK23 functions in <Emphasis Type="Italic">Arabidopsis</Emphasis> responses to drought and salt stresses

Calcium-dependent protein kinases (CDPKs) are unique serine/threonine kinases in plants and there are 34 CDPKs in Arabidopsis genome alone. Although several CDPKs have been demonstrated to be critical calcium signaling mediators for plant responses to various environmental stresses, the biological functions of most CDPKs in stress signaling remain unclear. In this study, we provide the evidences to demonstrate that AtCPK23 plays important role in Arabidopsis responses to drought and salt stresses. The cpk23 mutant, a T-DNA insertion mutant for AtCPK23 gene, showed greatly enhanced tolerance to drought and salt stresses, while the AtCPK23 overexpression lines became more sensitive to drought and salt stresses and the complementary line of the cpk23 mutant displayed similar phenotype as wild-type plants. The results of stomatal aperture measurement showed that the disruption of AtCPK23 expression reduced stomatal apertures, while overexpression of AtCPK23 increased stomatal apertures. The alteration of stomatal apertures by changes in AtCPK23 expression may account, at least in partial, for the modified Arabidopsis response to drought stress. In consistent with the enhanced salt-tolerance by disruption of AtCPK23 expression, K⁺ content in the cpk23 mutant was not reduced under high NaCl stress compared with wild-type plants, which indicates that the AtCPK23 may also regulate plant K⁺-uptake. The possible mechanisms by which AtCPK23 mediates drought and salt stresses signaling are discussed.     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

<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.     

A rapid<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

Nucleosome transactions on the<Emphasis Type="Italic"> Hypocrea jecorina</Emphasis> (<Emphasis Type="Italic"> Trichoderma reesei</Emphasis>) cellulase promoter<Emphasis Type="Italic"> cbh2</Emphasis> associated with cellulase induction

The 5 regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes –1 and –2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome –1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5 regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome –1.Communicated by C. A. M. J. J. van den Hondel     

<Emphasis Type="Italic">PCC1</Emphasis>: a merging point for pathogen defence and circadian signalling in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Using a cDNA-array we identified expressed sequence tag 163B24T7 as rapidly up-regulated in Arabidopsis thaliana (L.) Heynh. after pathogen exposure. Detailed expression analysis revealed that the corresponding gene is up-regulated not only after exposure to avirulent Pseudomonas syringae pv. tomato but also to virulent strains. This up-regulation is dependent on functional salicylic acid defence-signalling pathways. Moreover, we found the gene was circadian-regulated, showing peaks of expression at the end of the day. Using plants overexpressing the clock component CCA1, we showed that the PCC1 gene is regulated by the inner clock of Arabidopsis. Accordingly, we named the gene PCC1, for pathogen and circadian controlled. PCC1 is a member of a novel family of six small polypeptides in Arabidopsis. A functional role for PCC1 in plant defence was demonstrated since plants overexpressing PCC1 are resistant against normally virulent oomycetes. Thus, PCC1 demonstrates a potential interrelationship between pathogen and circadian signalling pathways.Abbreviations cfu Colony-forming units - EST Expressed sequence tag - Pst Pseudomonas syringae pv. tomato - TAIR The Arabidopsis information resource     

<Emphasis Type="Italic">Arabidopsis</Emphasis> constitutive photomorphogenic mutant, <Emphasis Type="Italic">bls1</Emphasis>,displays altered brassinosteroid response and sugar sensitivity

We have isolated an Arabidopsis mutant impaired in light- and brassinosteroid (BR) induced responses, as well as in sugar signalling. The bls1 (brassinosteroid, light and sugar1) mutant displays short hypocotyl, expanded cotyledons, and de-repression of light-regulated genes in young seedlings, and leaf differentiation and silique formation on prolonged growth in dark. In light, the bls1 mutant is dwarf and develops a short root, compact rosette, with reduced trichome number, and exhibits delayed bolting. The activity of the BR inducible TCH4 and auxin inducible SAUR promoters, fused with GUS gene, is also altered in seedlings harbouring bls1 mutant background. In addition, the bls1 mutant is hypersensitive to metabolizable sugars. The short hypocotyl phenotype in dark, short root phenotype in light and sugar hypersensitivity could be rescued with BR application. Moreover, the bls1 mutant also showed higher expression of a BR biosynthetic pathway gene CPD, which is known to be feedback-regulated by BR. Using a genome-wide AFLP mapping strategy, the bls1 mutant has been mapped to a 1.4Mb region of chromosome 5. Since no other mutant with essentially a similar phenotype has been assigned to this region, we suggest that the bls1 mutant defines a novel locus involved in regulating endogenous BR levels, with possible ramifications in integrating light, hormone and sugar signalling.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Calmodulin-like Proteins from <Emphasis Type="Italic">Arabidopsis</Emphasis> and Tomato are Involved in Host Defense Against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca²⁺ is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca²⁺-binding proteins such as calmodulin (CaM) detect Ca²⁺ signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant’s immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca²⁺ in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca²⁺-dependent signals during the plant immune response to bacterial pathogens. This work was supported by a research grant (WAS) and postgraduate scholarships (DC, SLD) from the Natural Science and Engineering Research Council of Canada, the National Science Foundation (IBN-0109633; GBM), and the Swedish Research Council (SKE).     

<Emphasis Type="Italic"> Cis</Emphasis>-acting elements sufficient for induction of<Emphasis Type="Italic"> FDH1</Emphasis> expression by formate in the methylotrophic yeast<Emphasis Type="Italic"> Candida boidinii</Emphasis>

The FDH1 gene of Candida boidinii encodes an NAD⁺-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5 non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.Communicated by C. P. Hollenberg     

Expression analysis of four<Emphasis Type="Italic"> Pinus radiata</Emphasis> male cone promoters in the heterologous host<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Four male cone-specific promoters were isolated from the genome of Pinus radiata D. Don, fused to the -glucuronidase (GUS) reporter gene and analysed in the heterologous host Arabidopsis thaliana (L.) Heynh. The temporal and spatial activities of the promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1 during seven anther developmental stages are described in detail. The two promoters PrMC2 and PrMALE1 confer an identical GUS expression pattern on Arabidopsis anthers. DNA sequence analysis of the PrMC2 and PrMALE1 promoters revealed an 88% sequence identity over 276 bp and divergence further upstream (<40% sequence identity). GUS expression driven by a 276-bp PrMALE1 promoter fragment showed the same pattern in Arabidopsis anthers as observed for the full-length PrMALE1 promoter. Within the 276-bp promoter fragment a region of high homology to a previously described 16-bp anther-box was identified. In gain-of-function experiments the putative PrMALE1 anther-box was fused upstream of a 90-bp CaMV 35S minimal promoter, as a single copy in the sense direction and as an inverted repeat. No GUS expression was conferred to Arabidopsis anthers by either of these two constructs. In a loss-of-function experiment a 226-bp PrMALE1 deletion construct, which did not contain the putative PrMALE1 anther-box, still maintained the originally observed PrMALE1 GUS expression pattern. Hence, gain-of-function as well as loss-of-function experiments consistently showed that the putative anther-box of the PrMALE1 promoter is non-functional in the Arabidopsis genetic background. For the analysis of the four full-length pine promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1, transformation vectors based on pCAMBIA2200 and pCAMBIA1302 were used. It will also be demonstrated in this article that sequences within the T-DNA borders of these vectors caused a characteristic histological background expression in Arabidopsis, with staining observed in vascular tissue of leaves, sepals, roots, filaments of stamens and in stems and pistils.Abbreviation GUS -glucuronidaseGenBank accession numbers for the analysed promoters: AF 337656 (PrCHS1), AF 337655 (PrLTP2), AF 337657 (PrMC2) and AF 337658 (PrMALE1).     

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.     

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.