TuMV triggers stomatal closure but reduces drought tolerance in Arabidopsis.

Compatible plant viral infections are a common cause of agricultural losses worldwide. Characterization of the physiological responses controlling plant water management under combined stresses is of great interest in the current climate change scenario. We studied the outcome of TuMV infection on stomatal closure and water balance, hormonal balance and drought tolerance in Arabidopsis. TuMV infection reduced stomatal aperture concomitantly with diminished gas exchange rate, daily water consumption and rosette initial dehydration rate. Infected plants overaccumulated salicylic acid and abscisic acid and showed altered expression levels of key ABA homeostasis genes including biosynthesis and catabolism. Also the expression of ABA signalling gene ABI2 was induced and ABCG40 (which imports ABA into guard cells) was highly induced upon infection. Hypermorfic abi2-1 mutant plants, but no other ABA or SA biosynthetic, signalling or degradation mutants tested abolished both stomatal closure and low stomatal conductance phenotypes caused by TuMV. Notwithstanding lower relative water loss during infection, plants simultaneously subjected to drought and viral stresses showed higher mortality rates than mock-inoculated drought stressed controls, alongside downregulation of drought-responsive gene RD29A. Our findings indicate that despite stomatal closure triggered by TuMV, additional phenomena diminish drought tolerance upon infection.

Growth promotion and protection from drought in Eucalyptus grandis seedlings inoculated with beneficial bacteria embedded in a superabsorbent polymer.

Eucalyptus grandis is a globally important tree crop. Greenhouse-grown tree seedlings often face water deficit after outplanting to the field, which can affect their survival and establishment severely. This can be alleviated by the application of superabsorbent hydrophilic polymers (SAPs). Growth promoting bacteria can also improve crop abiotic stress tolerance; however, their use in trees is limited, partly due to difficulties in the application and viability loss. In this work, we evaluated the improvement of drought tolerance of E. grandis seedlings by inoculating with two Pseudomonas strains (named M25 and N33), carried by an acrylic-hydrocellulosic SAP. We observed significant bacterial survival in the seedling rhizosphere 50 days after inoculation. Under gradual water deficit conditions, we observed a considerable increase in the water content and wall elasticity of M25-inoculated plants and a trend towards growth promotion with both bacteria. Under rapid water deficit conditions, which caused partial defoliation, both strains significantly enhanced the formation of new leaves, while inoculation with M25 reduced the transpiration rate. Co-inoculation with M25 and N33 substantially increased growth and photosynthetic capacity. We conclude that the selected bacteria can benefit E. grandis early growth and can be easily inoculated at transplant by using an acrylic-hydrocellulosic SAP.

Single-stranded oligodeoxynucleotides induce plant defence in Arabidopsis thaliana.

BACKGROUND AND AIMS: Single-stranded DNA oligodeoxynucleotides (ssODNs) have been shown to elicit immune responses in mammals. In plants, RNA and genomic DNA can activate immunity, although the exact mechanism through which they are sensed is not clear. The aim of this work was to study the possible effect of ssODNs on plant immunity. KEY RESULTS: The ssODNs IMT504 and 2006 increased protection against the pathogens Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea but not against tobacco mosaic virus-Cg when infiltrated in Arabidopsis thaliana. In addition, ssODNs inhibited root growth and promoted stomatal closure in a concentration-dependent manner, with half-maximal effective concentrations between 0.79 and 2.06 µm. Promotion of stomatal closure by ssODNs was reduced by DNase I treatment. It was also diminished by the NADPH oxidase inhibitor diphenyleneiodonium and by coronatine, a bacterial toxin that inhibits NADPH oxidase-dependent reactive oxygen species (ROS) synthesis in guard cells. In addition it was found that ssODN-mediated stomatal closure was impaired in bak1-5, bak1-5/bkk1, mpk3 and npr1-3 mutants. ssODNs also induced early expression of MPK3, WRKY33, PROPEP1 and FRK1 genes involved in plant defence, an effect that was reduced in bak1-5 and bak1-5/bkk1 mutants. CONCLUSIONS: ssODNs are capable of inducing protection against pathogens through the activation of defence genes and promotion of stomatal closure through a mechanism similar to that of other elicitors of plant immunity, which involves the BAK1 co-receptor, and ROS synthesis.

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis.

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the ß-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Coronatine Inhibits Stomatal Closure through Guard Cell-Specific Inhibition of NADPH Oxidase-Dependent ROS Production.

Microbes trigger stomatal closure through microbe-associated molecular patterns (MAMPs). The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) synthesizes the polyketide toxin coronatine, which inhibits stomatal closure by MAMPs and by the hormone abscisic acid (ABA). The mechanism by which coronatine, a jasmonic acid-isoleucine analog, achieves this effect is not completely clear. Reactive oxygen species (ROS) are essential second messengers in stomatal immunity, therefore we investigated the possible effect of coronatine on their production. We found that coronatine inhibits NADPH oxidase-dependent ROS production induced by ABA, and by the flagellin-derived peptide flg22. This toxin also inhibited NADPH oxidase-dependent stomatal closure induced by darkness, however, it failed to prevent stomatal closure by exogenously applied H2O2 or by salicylic acid, which induces ROS production through peroxidases. Contrary to what was observed on stomata, coronatine did not affect the oxidative burst induced by flg22 in leaf disks. Additionally, we observed that in NADPH oxidase mutants atrbohd and atrbohd/f, as well as in guard cell ABA responsive but flg22 insensitive mutants mpk3, mpk6, npr1-3, and lecrk-VI.2-1, the inhibition of ABA stomatal responses by both coronatine and the NADPH oxidase inhibitor diphenylene iodonium was markedly reduced. Interestingly, coronatine still impaired ABA-induced ROS synthesis in mpk3, mpk6, npr1-3, and lecrk-VI.2-1, suggesting a possible feedback regulation of ROS on other guard cell ABA signaling elements in these mutants. Altogether our results show that inhibition of NADPH oxidase-dependent ROS synthesis in guard cells plays an important role during endophytic colonization by Pst through stomata.

Xanthomonas campestris attenuates virulence by sensing light through a bacteriophytochrome photoreceptor.

Phytochromes constitute a major photoreceptor family found in plants, algae, fungi, and prokaryotes, including pathogens. Here, we report that Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease which affects cruciferous crops worldwide, codes for a functional bacteriophytochrome (XccBphP). XccBphP possesses an N-terminal PAS2-GAF-PHY photosensory domain triad and a C-terminal PAS9 domain as its output module. Our results show that illumination of Xcc, prior to plant infection, attenuates its virulence in an XccBphP-dependent manner. Moreover, in response to light, XccBphP downregulates xanthan exopolysaccharide production and biofilm formation, two known Xcc virulence factors. Furthermore, the XccbphP null mutant shows enhanced virulence, similar to that of dark-adapted Xcc cultures. Stomatal aperture regulation and callose deposition, both well-established plant defense mechanisms against bacterial pathogens, are overridden by the XccbphP strain. Additionally, an RNA-Seq analysis reveals that far-red light or XccBphP overexpression produces genomewide transcriptional changes, including the inhibition of several Xcc virulence systems. Our findings indicate that Xcc senses light through XccBphP, eliciting bacterial virulence attenuation via downregulation of bacterial virulence factors. The capacity of XccBphP to respond to light both in vitro and in vivo was abolished by a mutation on the conserved Cys13 residue. These results provide evidence for a novel bacteriophytochrome function affecting an infectious process.

Xanthan Pyruvilation Is Essential for the Virulence of Xanthomonas campestris pv. campestris.

Xanthan, the main exopolysaccharide (EPS) synthesized by Xanthomonas spp., contributes to bacterial stress tolerance and enhances attachment to plant surfaces by helping in biofilm formation. Therefore, xanthan is essential for successful colonization and growth in planta and has also been proposed to be involved in the promotion of pathogenesis by calcium ion chelation and, hence, in the suppression of the plant defense responses in which this cation acts as a signal. The aim of this work was to study the relationship between xanthan structure and its role as a virulence factor. We analyzed four Xanthomonas campestris pv. campestris mutants that synthesize structural variants of xanthan. We found that the lack of acetyl groups that decorate the internal mannose residues, ketal-pyruvate groups, and external mannose residues affects bacterial adhesion and biofilm architecture. In addition, the mutants that synthesized EPS without pyruvilation or without the external mannose residues did not develop disease symptoms in Arabidopsis thaliana. We also observed that the presence of the external mannose residues and, hence, pyruvilation is required for xanthan to suppress callose deposition as well as to interfere with stomatal defense. In conclusion, pyruvilation of xanthan seems to be essential for Xanthomonas campestris pv. campestris virulence.

Brassinosteroids tailor stomatal production to different environments.

Two recent reports show that brassinosteroids control stomata production by regulating the GSK3-like kinase BIN2-mediated phosphorylation of two different stomatal signalling components resulting in opposite stomatal phenotypes. We discuss how these two mechanisms might differentially control stomatal generation under diverse growth conditions.

SPEECHLESS integrates brassinosteroid and stomata signalling pathways.

Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.

Arabidopsis α Aurora kinases function in formative cell division plane orientation.

To establish three-dimensional structures/organs, plant cells continuously have to adapt the orientation of their division plane in a highly regulated manner. However, mechanisms underlying switches in division plane orientation remain elusive. Here, we characterize a viable double knockdown mutant in Arabidopsis thaliana group α Aurora (AUR) kinases, AUR1 and AUR2, (aur1-2 aur2-2), with a primary defect in lateral root formation and outgrowth. Mutant analysis revealed that aur1-2 aur2-2 lateral root primordia are built from randomly oriented cell divisions instead of distinct cell layers. This phenotype could be traced back to cytokinesis defects and misoriented cell plates during the initial anticlinal pericycle cell divisions that give rise to lateral root primordia. Complementation assays showed that the Arabidopsis α group Aurora kinases are functionally divergent from the single ß group member AUR3 and that AUR1 functions in division plane orientation prior to cytokinesis. In addition to defective lateral root patterning, aur1-2 aur2-2 plants also show defects in orienting formative divisions during embryogenesis, divisions surrounding the main root stem cell niche, and divisions surrounding stomata formation. Taken together, our results put forward a central role for α Aurora kinases in regulating formative division plane orientation throughout development.

Plants grow on brassinosteroids.

Brassinosteroids (BRs) are plant steroid hormones known mainly for promoting organ growth through their combined effect on cell expansion and division. In addition, BRs regulate a broad spectrum of plant developmental and physiological responses, including plant architecture, vascular differentiation, male fertility, flowering, senescence, photomorphogenesis and tolerance to biotic and abiotic stresses. Recently, a complete core BR signaling pathway was defined in which BR signals are conveyed from the cell surface to the nucleus through sequential signaling modules. A major challenge now is to understand precisely how this signaling pathway controls the different BR-regulated actions. The current identification of direct targets of BRASSINAZOLE-RESISTANT1 (BRZ1) and BR-INSENSITIVE-EMS-SUPPRESSOR1 (BES1)/BZR2 transcription factors suggests that BR signaling pathway controls growth and interacts with other signaling pathways mainly at the transcriptional level.

Expression of ABA signalling genes and ABI5 protein levels in imbibed Sorghum bicolor caryopses with contrasting dormancy and at different developmental stages.

BACKGROUND AND AIMS: Pre-harvest sprouting susceptibility in grain sorghum (Sorghum bicolor) is related to low seed dormancy and reduced embryo sensitivity to inhibition of germination by abscisic acid (ABA). Intra-specific variability for pre-harvest sprouting might involve differential regulation of ABA signalling genes. METHODS: Sorghum genes encoding homologues for ABA signalling components from other species (ABI5, ABI4, VP1, ABI1 and PKABA1) were studied at the transcriptional and protein level (ABI5) during grain imbibition for two sorghum lines with contrasting sprouting phenotypes and in response to hormones. KEY RESULTS: Transcript levels of these genes and protein levels of ABI5 were higher in imbibed immature caryopses of the more dormant line. Dormancy loss was related to lower transcript levels of these genes and lower ABI5 protein levels in both genotypes. Exogenous ABA inhibited germination of isolated embryos but failed to prevent ABI5 rapid decrease supporting a role for the seed coat in regulating ABI5 levels. CONCLUSIONS: Several genes involved in ABA signalling are regulated differently in imbibed caryopses from two sorghum lines with contrasting pre-harvest sprouting response before - but not after - physiological maturity. A role for ABI5 in the expression of dormancy during grain development is discussed.

Xanthomonas campestris overcomes Arabidopsis stomatal innate immunity through a DSF cell-to-cell signal-regulated virulence factor.

Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host. In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA). We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense. By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system. In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pst coronatine-deficient mutants, showing that MPK3 is required for stomatal immune response. Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xcc culture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3.

Stomata and pathogens: Warfare at the gates.

Bacteria and fungi are capable of triggering stomatal closure through pathogen-associated molecular patterns (PAMPs), which prevents penetration through these pores. Therefore, the stomata can be considered part of the plant innate immune response. Some pathogens have evolved mechanisms to evade stomatal defense. The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc), which infects plants of the Brassicaceae family mainly through hydathodes, has also been reported to infect plants through stomata. A recent report shows that penetration of Xcc in Arabidopsis leaves through stomata depends on a secreted small molecule whose synthesis is under control of the rpf/diffusible signal factor (DSF) cell-to-cell signaling system, which also controls genes involved in biofilm formation and pathogenesis. The same reports shows that Arabidopsis ROS- and PAMP-activated MAP kinase 3 (MPK3) is essential for stomatal innate response. Other recent and past findings about modulation of stomatal behaviour by pathogens are also discussed. In all, these findings support the idea that PAMP-triggered stomatal closure might be a more effective and widespread barrier against phytopathogens than previously thought, which has in turn led to the evolution in pathogens of several mechanisms to evade stomatal defense.

Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide.

MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H(2)O(2), both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H(2)O(2) synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H(2)O(2). These results provide clear evidence for the important role of MPK3 in the perception of ABA and H(2)O(2) in guard cells.

Dimerization and DNA-binding of ASR1, a small hydrophilic protein abundant in plant tissues suffering from water loss.

The Asr gene family is present in Spermatophyta. Its members are generally activated under water stress. We present evidence that tomato ASR1, one of the proteins of the family, accumulates in seed during late stages of embryogenesis, a physiological process characterized by water loss. In vitro, electrophoretic assays show a homo-dimeric structure for ASR1 and highlight strong non-covalent interactions between monomers prone to self-assemble. Direct visualization of single molecules by atomic force microscopy (AFM) confirms that ASR1 forms homodimers and that uncovers both monomers and dimers bind double stranded DNA.

Arabidopsis MPK3, a Key Signalling Intermediate in Stomatal Function.

Regulation of stomatal aperture is of critical importance to plants to balance gas exchange and water loss, and also to control ingress of bacterial pathogens. MAP kinase signal transduction pathways are mediators of biotic and abiotic stress, and have been indicted in the control of stomatal movements. Cell-specific antisense was used to down-regulate MPK3 gene expression in Arabidopsis guard cells, resulting in ABA insensitivity during inhibition of stomatal opening, but a normal ABA response in promotion of closure assays. This response is similar to that of the heterotrimeric G protein alpha subunit mutant gpa1, as is the imposition of ABA insensitivity during stomatal closure by butyrate treatment, suggesting that MPK3 and GPA1 are in the same ABA signal transduction pathway and adding further evidence for parallel signalling pathways during ABA-induced closure. By contrast, antisense plants were less sensitive to H(2)O(2) in both promotion of closure and inhibition of opening assays, although H(2)O(2) production in response to ABA was not affected. Regulation of stomatal aperture by PAMPs has recently been shown to be an important plant defense mechanism; since MPK3 is also activated by such pathogen elicitors, we postulate that in addition to a signalling role in guard cell movements, MPK3 is involved in the active prevention of bacterial infection through stomata.