A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 - and abscisic acid-mediated stomatal movements in Arabidopsis.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2 , abscisic acid, darkness, vapor pressure deficit and ozone.

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO2 , light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO2 , light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO2 induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5-2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO2 and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).

Boolink: a graphical interface for open access Boolean network simulations and use in guard cell CO2 signaling.

Signaling networks are at the heart of almost all biological processes. Most of these networks contain large number of components, and often either the connections between these components are not known or the rate equations that govern the dynamics of soluble signaling components are not quantified. This uncertainty in network topology and parameters can make it challenging to formulate detailed mathematical models. Boolean networks, in which all components are either on or off, have emerged as viable alternatives to detailed mathematical models that contain rate constants and other parameters. Therefore, open-source platforms of Boolean models for community use are desirable. Here, we present Boolink, a freely available graphical user interface that allows users to easily construct and analyze existing Boolean networks. Boolink can be applied to any Boolean network. We demonstrate its application using a previously published network for abscisic acid (ABA)-driven stomatal closure in Arabidopsis spp. (Arabidopsis thaliana). We also show how Boolink can be used to generate testable predictions by extending the network to include CO2 regulation of stomatal movements. Predictions of the model were experimentally tested, and the model was iteratively modified based on experiments showing that ABA effectively closes Arabidopsis stomata at near-zero CO2 concentrations (1.5-ppm CO2). Thus, Boolink enables public generation and the use of existing Boolean models, including the prior developed ABA signaling model with added CO2 signaling components.

A role for calcium-dependent protein kinases in differential CO2 - and ABA-controlled stomatal closing and low CO2 -induced stomatal opening in Arabidopsis.

Low concentrations of CO2 cause stomatal opening, whereas [CO2 ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2 -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+ -binding proteins that function in the stomatal CO2 response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2 , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2 -induced stomatal closure and, unexpectedly, also in low CO2 -induced stomatal opening. Furthermore, K+ -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2 -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.

A pathogen type III effector with a novel E3 ubiquitin ligase architecture.

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

Activation of defense against Phytophthora infestans in potato by down-regulation of syntaxin gene expression.

The oomycete Phytophthora infestans is the causal agent of late blight, the most devastating disease of potato. The importance of vesicle fusion processes and callose deposition for defense of potato against Phytophthora infestans was analyzed. Transgenic plants were generated, which express RNA interference constructs targeted against plasma membrane-localized SYNTAXIN-RELATED 1 (StSYR1) and SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE FACTOR ADAPTOR PROTEIN 33 (StSNAP33), the potato homologs of Arabidopsis AtSYP121 and AtSNAP33, respectively. Phenotypically, transgenic plants grew normally, but showed spontaneous necrosis and chlorosis formation at later stages. In response to infection with Phytophthora infestans, increased resistance of StSYR1-RNAi plants, but not StSNAP33-RNAi plants, was observed. This increased resistance correlated with the constitutive accumulation of salicylic acid and PR1 transcripts. Aberrant callose deposition in Phytophthora infestans-infected StSYR1-RNAi plants coincided with decreased papilla formation at penetration sites. Resistance against the necrotrophic fungus Botrytis cinerea was not significantly altered. Infiltration experiments with bacterial solutions of Agrobacterium tumefaciens and Escherichia coli revealed a hypersensitive phenotype of both types of RNAi lines. The enhanced defense status and the reduced growth of Phytophthora infestans on StSYR1-RNAi plants suggest an involvement of syntaxins in secretory defense responses of potato and, in particular, in the formation of callose-containing papillae.

Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity.

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression. In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.

Suppression of the AvrBs1-specific hypersensitive response by the YopJ effector homolog AvrBsT from Xanthomonas depends on a SNF1-related kinase.

*Pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) depends on a type III secretion system that translocates a cocktail of > 25 type III effector proteins into the plant cell. *In this study, we identified the effector AvrBsT as a suppressor of specific plant defense. AvrBsT belongs to the YopJ/AvrRxv protein family, members of which are predicted to act as proteases and/or acetyltransferases. *AvrBsT suppresses the hypersensitive response (HR) that is elicited by the effector protein AvrBs1 from Xcv in resistant pepper plants. HR suppression occurs inside the plant cell and depends on a conserved predicted catalytic residue of AvrBsT. Yeast two-hybrid based analyses identified plant interaction partners of AvrBs1 and AvrBsT, including a putative regulator of sugar metabolism, SNF1-related kinase 1 (SnRK1), as interactor of AvrBsT. Intriguingly, gene silencing experiments revealed that SnRK1 is required for the induction of the AvrBs1-specific HR. *We therefore speculate that SnRK1 is involved in the AvrBsT-mediated suppression of the AvrBs1-specific HR.

Insights into the Molecular Mechanisms of CO2-Mediated Regulation of Stomatal Movements.

Plants must continually balance the influx of CO2 for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO2] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO2-perception mechanisms and CO2-mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.

Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1.

Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement.

Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.

The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood-brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC. The corresponding exchange of the tetanus neurotoxin HC-fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire HC-fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system.

Myoglobin as a prognostic indicator for outcome in dogs with gastric dilatation-volvulus.

OBJECTIVE: To determine whether myoglobin (Mb) is a useful prognostic indicator for outcome and to investigate any relationship between Mb and mortality in dogs with gastric dilatation-volvulus (GDV). DESIGN: Prospective study. SETTING: Veterinary teaching hospital. ANIMALS: Seventy-two dogs with GDV. INTERVENTIONS: Blood sampling. MEASUREMENTS AND MAIN RESULTS: Mb levels were measured at the time of diagnosis (Mbt0), 24 hours (Mbt1), and 48 hours (Mbt2) after signs of GDV were recognized. Fifty-seven dogs survived (group I) and 15 dogs did not survive (group II). Mbt0 differed significantly between groups (P=0.04). Mbt0 in group I ranged from <30 to >700 ng/mL (n=57, median 74 ng/mL), and in group II from 34 to >700 ng/mL (n=15, median 238 ng/mL). Analysis of a receiver operating characteristic curve of Mbt0 suggested that the best single cutpoint would be 168 ng/mL (sensitivity 60.0%, specificity 84.2%). Fifty percent of dogs with Mbt0>168 ng/mL were euthanized, while 88.9% with Mbt0<168 ng/mL survived. Mbt1 and Mbt2 differed significantly between groups I and II. Mbt1 in group I ranged from 32 to >700 ng/mL (n=55, median 123 ng/mL), and Mbt1 in group II ranged from 131 to 643 ng/mL (n=7, median 343 ng/mL) (P=0.006). Mbt2 in group I ranged from 30 to 597 ng/mL (n=54, median 101 ng/mL), and in group II from 141 to >700 ng/mL (n=8, median 203 ng/mL) (P=0.02). CONCLUSIONS: In this study, Mbt0 is a moderately sensitive and specific prognostic indicator. Almost 90% of the dogs below the cutpoint survived to discharge, whereas 50% with Mbt0 above the cutpoint did not survive.