Claudia A. Casalongué

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor.

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.

Expression of a Solanum tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress conditions

Abstract Cyclophilins (CyPs) are ubiquitous proteins with an intrinsic enzymatic activity of peptidyl-prolyl cis-trans isomerase that catalyzes the rotation of X-Pro peptide bonds. These enzymes are believed to play a role in the folding of certain proteins. In addition, CyPs might be important in signal transduction processes. A cDNA library was prepared from potato (Solanum tuberosusm) tubers infected with the fungus Fusarium solani f. sp eumartii. Using a PCR-amplified subtracted cDNA probe, a clone encoding a cytosolic form of CyP, called StCyP (Solanum ṯuberosum CyP), was isolated. Except in tubers, StCyP is expressed at high levels in tissues of healthy potato plants. Northern blot analyses revealed that both wounding and fungal infection increased the level of StCyP mRNA in tubers. However, whereas wounding causes a transient accumulation of StCyP mRNA, fungal infection results in a maintained accumulation of this transcript. StCyP mRNA accumulation is also stimulated by the application of absicic acid (ABA) and methyl jasmonate (MeJA) in tubers. Treatment with fungal elicitor or salicilic acid (SA) has no effect on the level of StCyP mRNA accumulation. Together these results indicate that the observed accumulation of StCyP mRNAs in fungal-infected potato tubers might be a response to the wound produced by the penetration and colonization of the tissue by the pathogen. Furthermore, accumulation of StCyP transcripts was also detected when the potato tubers were exposed to heat-shock treatment. These findings support a role for cyclophilins in the plant response to environmental stresses.

Auxin signaling participates in the adaptative response against oxidative stress and salinity by interacting with redox metabolism in Arabidopsis

Auxin regulates gene expression through direct physical interaction with TIR1/AFB receptor proteins during different processes of growth and development in plants. Here we report the contribution of auxin signaling pathway to the adaptative response against abiotic stress in Arabidopsis. Phenotypic characterization of tir1/afb auxin receptor mutants indicates a differential participation of each member under abiotic stress. In particular, tir1 afb2 and tir1 afb3 mutants resulted more tolerant to oxidative stress. In addition, tir1 afb2 showed increased tolerance against salinity measured as chlorophyll content, germination rate and root elongation compared with wild-type plants. Furthermore, tir1 afb2 displayed a reduced accumulation of hydrogen peroxide and superoxide anion, as well as enhanced antioxidant enzymes activities under stress. A higher level of ascorbic acid was detected in tir1 afb2 compared with wild-type plants. Thus, adaptation to salinity in Arabidopsis may be mediated in part by an auxin/redox interaction.

Extracellular ATP induces nitric oxide production in tomato cell suspensions

In plant as well as in animal systems, extracellular ATP (eATP) regulates a broad range of physiological processes. In animals, downstream signaling events regulated by eATP have been described before. Among the second messengers reported to be involved in eATP signaling, there are inositol

Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins

Proteases have been proposed as part of the invasion strategies of some pathogenic fungi. In this work, a serine protease produced by the phytopathogenic fungus Fusarium solani f.sp. eumartii was purified and characterized. Purification of the enzyme was accomplished by gel filtration through a Superose 12 column, followed by hydrophobic interaction chromatography in Phenyl Superose and gel filtration chromatography through Superdex 75. Analysis of the purified enzyme by SDS/PAGE without heat treatment, revealed a single band, which corresponded to the proteolytic activity detected by zymogram. When this protein was subjected to denaturing conditions, two major polypeptides of approximately 30 and 33kDa were revealed. The N-terminal amino acid sequence of one of these polypeptides showed a high similarity with fungal mature serine proteases of the subtilisin family. This protease hydrolysed in vitro, specific polypeptides of potato intercellular washing fluids and cell walls. The protease was also able to degrade pathogenesis-related proteins from the intercellular washing fluids. The role of this serine protease as part of the fungal strategy to colonize potato tuber tissues is discussed.

MiR393 regulation of auxin signaling and redox-related components during acclimation to salinity in Arabidopsis.

One of the most striking aspects of plant plasticity is the modulation of development in response to environmental changes. Plant growth and development largely depend on the phytohormone auxin that exerts its function through a partially redundant family of F-box receptors, the TIR1-AFBs. We have previously reported that the Arabidopsis double mutant tir1 afb2 is more tolerant to salt stress than wild-type plants and we hypothesized that down-regulation of auxin signaling might be part of Arabidopsis acclimation to salinity. In this work, we show that NaCl-mediated salt stress induces miR393 expression by enhancing the transcription of AtMIR393A and leads to a concomitant reduction in the levels of the TIR1 and AFB2 receptors. Consequently, NaCl triggers stabilization of Aux/IAA repressors leading to down-regulation of auxin signaling. Further, we report that miR393 is likely involved in repression of lateral root (LR) initiation, emergence and elongation during salinity, since the mir393ab mutant shows reduced inhibition of emergent and mature LR number and length upon NaCl-treatment. Additionally, mir393ab mutant plants have increased levels of reactive oxygen species (ROS) in LRs, and reduced ascorbate peroxidase (APX) enzymatic activity compared with wild-type plants during salinity. Thus, miR393 regulation of the TIR1 and AFB2 receptors could be a critical checkpoint between auxin signaling and specfic redox-associated components in order to coordinate tissue and time-specific growth responses and tolerance during acclimation to salinity in Arabidopsis.

Phosphatidic acid formation is required for extracellular ATP‐mediated nitric oxide production in suspension‐cultured tomato cells

*In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca(2+), nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. *In vivo phospholipid signaling upon extracellular ATP treatment was studied in (32)P(i)-labeled suspension-cultured tomato (Solanum lycopersicum) cells. *Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. *Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.

Extracellular ATP, nitric oxide and superoxide act coordinately to regulate hypocotyl growth in etiolated Arabidopsis seedlings

Etiolated Arabidopsis thaliana seedlings germinated in the presence of reducing buffers such as reduced gluthathione (GSH) and dithiothreitol (DTT) have altered morphology. GSH and DTT inhibited hypocotyl elongation in a dose-dependent manner. The GSH-mediated effect was prevented by the simultaneous addition of extracellular ATP (eATP). NADPH oxidase (NOX) activity and endogenous nitric oxide (NO) generation were required to mediate eATP action on the hypocotyl elongation. A correlation was observed between hypocotyl length, eATP concentration and NO production. The action of eATP and NO on superoxide (O(2)(-)) accumulation and peroxidase activity was investigated. The O(2)(-) distribution was regulated by eATP and NO during hypocotyl elongation. Our data suggest that a finely tuned balance of redox status and optimal levels of ATP and NO are essential to regulate the hypocotyl elongation in the dark.

The analysis of an Arabidopsis triple knock-down mutant reveals functions for MBF1 genes under oxidative stress conditions.

Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) type belong to a small multigenic family that controls gene expression by connecting transcription factors and the basal transcription machinery. In this report, a triple knock-down mutant (abc-) for the Arabidopsis thaliana MBF1 genes AtMBF1a, AtMBF1b and AtMBF1c was generated. The phenotypic characterization using oxidative agents such as hydrogen peroxide and methyl viologen revealed that the abc- mutant was more sensitive to oxidative stress. The triple knock-down mutant, abc- was also sensitive to osmotic stress mediated by high concentrations of sorbitol. Furthermore, the abc- phenotype was partially or completely rescued by AtMBF1c cDNA over-expression (abc- +c) depending on physiological and developmental conditions. AtMBF1s regulate the expression of ABR1, which is a member of the ethylene-response factor family and acts as ABA repressor. Thus, we conclude that AtMBF1 gene family may function as a regulatory component of the cross-talk node between ethylene, ABA and stress signal pathways. Furthermore, higher levels of a HSP70 mRNA and an immunoreactive HSP70 protein were detected in the abc- mutant. The participation of MBF1c as a possible negative regulator of HSP genes was discussed.

Expression of the tetrahydrofolate-dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis.

Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well-described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb-4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions.