Emmanuel Baudouin

Nitric oxide participates in cold‐responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.

Glutathione synthesis is regulated by nitric oxide in Medicago truncatula roots

Glutathione (GSH) is one of the main antioxidants in plants. Legumes have the specificity to produce a GSH homolog, homoglutathione (hGSH). We have investigated the regulation of GSH and hGSH synthesis by nitric oxide (NO) in Medicago truncatula roots. Analysis of the expression level of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) after treatment with sodium nitroprusside (SNP) and nitrosoglutathione (GSNO), two NO-donors, showed that γ-ecs and gshs genes are up regulated by NO treatment whereas hgshs expression is not. Differential accumulation of GSH was correlated to gene expression in SNP-treated roots. Our results provide the first evidence that GSH synthesis pathway is regulated by NO in plants and that there is a differential regulation between gshs and hgshs in M. truncatula.

Expression of Medicago truncatula Genes Responsive to Nitric Oxide in Pathogenic and Symbiotic Conditions

Nitric oxide (NO) is involved in diverse physiological processes in plants, including growth, development, response to pathogens, and interactions with beneficial microorganisms. In this work, a dedicated microarray representing the widest database available of NO-related transcripts in plants has been produced with 999 genes identified by a cDNA amplified fragment length polymorphism analysis as modulated in Medicago truncatula roots treated with two NO donors. The microarray then was used to monitor the expression of NO-responsive genes in M. truncatula during the incompatible interaction with the foliar pathogen Colletotrichum trifolii race 1 and during the symbiotic interaction with Sinorhizobium meliloti 1,021. A wide modulation of NO-related genes has been detected during the hypersensitive reaction or during nodule formation and is discussed with special emphasis on the physiological relevance of these genes in the context of the two biotic interactions. This work clearly shows that NO-responsive genes behave differently depending on the plant organ and on the type of interaction, strengthening the need to consider regulatory networks, including different signaling molecules.

Phytosphingosine‐phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling

• Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling. • PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. • Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. • Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants.

Identification of endogenously S-nitrosylated proteins in Arabidopsis plantlets: effect of cold stress on cysteine nitrosylation level.

S-nitrosylation is a nitric oxide (NO)-based post-translational modification regulating protein function and signalling. We used a combination between the biotin switch method and labelling with isotope-coded affinity tag to identify endogenously S-nitrosylated peptides in Arabidopsis thaliana proteins extracted from plantlets. The relative level of S-nitrosylation in the identified peptides was compared between unstressed and cold-stress seedlings. We thereby detected 62 endogenously nitrosylated peptides out of which 20 are over-nitrosylated following cold exposure. Taken together these data provide a new repertoire of endogenously S-nitrosylated proteins in Arabidopsis with cysteine S-nitrosylation site. Furthermore they highlight the quantitative modification of the S-nitrosylation status of specific cysteine following cold stress.

Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination

Sunflower (Helianthus annuus L.) seed dormancy is regulated by reactive oxygen species (ROS) and can be alleviated by incubating dormant embryos in the presence of methylviologen (MV), a ROS-generating compound. Ethylene alleviates sunflower seed dormancy whereas abscisic acid (ABA) represses germination. The purposes of this study were to identify the molecular basis of ROS effect on seed germination and to investigate their possible relationship with hormone signalling pathways. Ethylene treatment provoked ROS generation in embryonic axis whereas ABA had no effect on their production. The beneficial effect of ethylene on germination was lowered in the presence of antioxidant compounds, and MV suppressed the inhibitory effect of ABA. MV treatment did not alter significantly ethylene nor ABA production during seed imbibition. Microarray analysis showed that MV treatment triggered differential expression of 120 probe sets (59 more abundant and 61 less abundant genes), and most of the identified transcripts were related to cell signalling components. Many transcripts less represented in MV-treated seeds were involved in ABA signalling, thus suggesting an interaction between ROS and ABA signalling pathways at the transcriptional level. Altogether, these results shed new light on the crosstalk between ROS and plant hormones in seed germination.

Nitric oxide signaling in plants

Nitric oxide (NO) is now seen as a vitally important molecule in many biological systems. Once it was identified in mammalian systems in 1987 (Palmer et al., 1987) it was only a matter of time before researchers hunted for its presence in plants, with the first such reports being published in 1998 (Delledonne et al., 1998; Durner et al., 1998). Now 15 years later the interest in NO and its roles in plants is as eagerly investigated as ever, with an ever increasing number of papers published in the area each year. However, the field is not short of controversies, and it would certainly be fair to say that there is still much to be learnt in the area of NO biology. Here, a collection of papers by the many of the most active research groups in the field has been brought together. Some have contributed original research, others have written reviews to enable readers to get a up-to-date view of specialist areas in the field, while others still have written opinion articles, which give their views of the state-of-play in NO research as they see it.

New clues for a cold case: nitric oxide response to low temperature

Low temperature is among the most frequent stresses met by plants during their lifespan, and a plants ability to cold-acclimate is a determinant for further growth and development. Although intensive research has provided a good picture of the molecular and metabolic changes triggered by cold, the underlying regulatory mechanisms remain elusive and are thus being actively sought. Recent studies have shed light on the importance of nitric oxide (NO), a ubiquitous signalling molecule in eukaryotes, for plant tolerance to chilling and freezing. Indeed, NO formation following cold exposure has been reported in a range of plant species, and a series of proteins targeted by NO-based post-translational modifications have been identified. Moreover, key cold-regulated genes have been characterized as NO-dependent, suggesting the crucial importance of NO signalling for cold-responsive gene expression. This review provides a picture of our current understanding of the function of NO in the context of plant response to cold. Particular attention is dedicated to the open questions left by the fragmented data currently available concerning NO formation, transduction and biological significance for plant adaptation to low temperature.

Long chain base changes triggered by a short exposure of Arabidopsis to low temperature are altered by AHb1 non-symbiotic haemoglobin overexpression.

Long chain bases (LCB) are both precursors of complex sphingolipids (SL) and cellular signals in eukaryotic cells. Increasing evidence support a function for SL and/or LCBs in plant responses to environmental cues. In this study we analysed the impact of a short exposure to cold on the global LCB content and composition in Arabidopsis thaliana seedlings. We report that the total LCB amount significantly decreased after low temperature exposure. The decline was essentially due to reduction of t18:1 isomer content. On the other hand, chilling led to the increase of LCB content in a mutant over-expressing the non-symbiotic haemoglobin AHb1. Furthermore, this mutant was impaired in cold-dependent root growth inhibition and anthocyanin synthesis. As AHb1 is an element of nitric oxide turnover, our data suggest a possible link between nitric oxide, SL content and cold stress response.

A matter of fat: interaction between nitric oxide and sphingolipid signaling in plant cold response.

We recently evidenced that plant response to cold stress includes a rapid formation of nitric oxide (NO) that participates in the control of cold-responsive gene expression. Unexpectedly we also shed light on a novel downstream element of NO signalling that is phosphosphingolipid (PS) metabolism. Indeed, two phosphosphingolipid species, phytosphingosine phosphate (PHS-P) and a ceramide phosphate (Cer-P) are specifically synthesized upon cold exposure. Manipulating NO levels by pharmacological or genetic means dramatically modified the cold-triggered synthesis of PHS-P and Cer-P, but did not affect the cold-responsive formation of phosphatidic acid (PtdOH), a ubiquitous lipid signal derived from phospholipid degradation. So far no crosstalk between NO and PS signalling had been reported in plants. How NO might modulate PS formation and whether this regulation might be extended to other physiological processes are further discussed.