Jéremy Astier

Nitric oxide and reactive oxygen species in plant biotic interactions.

Nitric oxide (NO) and reactive oxygen species (ROS) are important signaling molecules in plants. Recent progress has been made in defining their role during plant biotic interactions. Over the last decade, their function in disease resistance has been highlighted and focused a lot of investigations. Moreover, NO and ROS have recently emerged as important players of defense responses after herbivore attacks. Besides their role in plant adaptive response development, NO and ROS have been demonstrated to be involved in symbiotic interactions between plants and microorganisms. Here we review recent data concerning these three sides of NO and ROS functions in plant biotic interactions.

S-nitrosylation: an emerging post-translational protein modification in plants.

Increasing evidences support the assumption that nitric oxide (NO) acts as a physiological mediator in plants. Understanding its pleiotropic effects requires a deep analysis of the molecular mechanisms underlying its mode of action. In the recent years, efforts have been made in the identification of plant proteins modified by NO at the post-translational level, notably by S-nitrosylation. This reversible process involves the formation of a covalent bond between NO and reactive cysteine residues. This research has now born fruits and numerous proteins regulated by S-nitrosylation have been identified and characterized. This review describes the basic principle of S-nitrosylation as well as the Biotin Switch Technique and its recent adaptations allowing the identification of S-nitrosylated proteins in physiological contexts. The impact of S-nitrosylation on the structure/function of selected proteins is further discussed.

Nitric Oxide-Dependent Posttranslational Modification in Plants: An Update

Nitric oxide (NO) has been demonstrated as an essential regulator of several physiological processes in plants. The understanding of the molecular mechanism underlying its critical role constitutes a major field of research. NO can exert its biological function through different ways, such as the modulation of gene expression, the mobilization of second messengers, or interplays with protein kinases. Besides this signaling events, NO can be responsible of the posttranslational modifications (PTM) of target proteins. Several modifications have been identified so far, whereas metal nitrosylation, the tyrosine nitration and the S-nitrosylation can be considered as the main ones. Recent data demonstrate that these PTM are involved in the control of a wide range of physiological processes in plants, such as the plant immune system. However, a great deal of effort is still necessary to pinpoint the role of each PTM in plant physiology. Taken together, these new advances in proteomic research provide a better comprehension of the role of NO in plant signaling.

Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity

Several studies focusing on elucidating the mechanism of NO (nitric oxide) signalling in plant cells have highlighted that its biological effects are partly mediated by protein kinases. The identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. In the present study, we have attempted to clarify the mechanisms underlying NO action in the regulation of NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SNF1 (sucrose non-fermenting 1)-related protein kinase 2 family. We found that in tobacco BY-2 (bright-yellow 2) cells exposed to salt stress, NtOSAK is rapidly activated, partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO (diethylamine-NONOate), involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its cysteine residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase in its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not have an impact on the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO.

There's more to the picture than meets the eye: Nitric oxide cross-talk with Ca2+ signaling

The complex cross talk between Ca2+ and nitric oxide involves components of the Ca2+ signaling machinery regulated through a nitric oxide-dependent process at the posttranslational and/or transcriptional levels. Calcium and nitric oxide (NO) are two important biological messengers. Increasing evidence indicates that Ca2+ and NO work together in mediating responses to pathogenic microorganisms and microbe-associated molecular patterns. Ca2+ fluxes were recognized to account for NO production, whereas evidence gathered from a number of studies highlights that NO is one of the key messengers mediating Ca2+ signaling. Here, we present a concise description of the current understanding of the molecular mechanisms underlying the cross talk between Ca2+ and NO in plant cells exposed to biotic stress. Particular attention will be given to the involvement of cyclic nucleotide-gated ion channels and Ca2+ sensors. Notably, we provide new evidence that calmodulin might be regulated at the posttranslational level by NO through S-nitrosylation. Furthermore, we report original transcriptomic data showing that NO produced in response to oligogalacturonide regulates the expression of genes related to Ca2+ signaling. Deeper insight into the molecules involved in the interplay between Ca2+ and NO not only permits a better characterization of the Ca2+ signaling system but also allows us to further understand how plants respond to pathogen attack.

Nitric oxide inhibits the ATPase activity of the chaperone-like AAA+ ATPase CDC48, a target for S-nitrosylation in cryptogein signalling in tobacco cells

NO has important physiological functions in plants, including the adaptative response to pathogen attack. We previously demonstrated that cryptogein, an elicitor of defence reaction produced by the oomycete Phytophthora cryptogea, triggers NO synthesis in tobacco. To decipher the role of NO in tobacco cells elicited by cryptogein, in the present study we performed a proteomic approach in order to identify proteins undergoing S-nitrosylation. We provided evidence that cryptogein induced the S-nitrosylation of several proteins and identified 11 candidates, including CDC48 (cell division cycle 48), a member of the AAA+ ATPase (ATPase associated with various cellular activities) family. In vitro, NtCDC48 (Nicotiana tabacum CDC48) was shown to be poly-S-nitrosylated by NO donors and we could identify Cys(110), Cys(526) and Cys(664) as a targets for S-nitrosylation. Cys(526) is located in the Walker A motif of the D2 domain, that is involved in ATP binding and was previously reported to be regulated by oxidative modification in Drosophila. We investigated the consequence of NtCDC48 S-nitrosylation and found that NO abolished NtCDC48 ATPase activity and induced slight conformation changes in the vicinity of Cys(526). Similarly, substitution of Cys(526) by an alanine residue had an impact on NtCDC48 activity. More generally, the present study identified CDC48 as a new candidate for S-nitrosylation in plants facing biotic stress and further supports the importance of Cys(526) in the regulation of CDC48 by oxidative/nitrosative agents.

Arabidopsis thaliana nicotianamine synthase 4 is required for proper response to iron deficiency and to cadmium exposure

The nicotianamine synthase (NAS) enzymes catalyze the formation of nicotianamine (NA), a non-proteinogenic amino acid involved in iron homeostasis. We undertook the functional characterization of AtNAS4, the fourth member of the Arabidopsis thaliana NAS gene family. A mutant carrying a T-DNA insertion in AtNAS4 (atnas4), as well as lines overexpressing AtNAS4 both in the atnas4 and the wild-type genetic backgrounds, were used to decipher the role of AtNAS4 in NA synthesis, iron homeostasis and the plant response to iron deficiency or cadmium supply. We showed that AtNAS4 is an important source for NA. Whereas atnas4 had normal growth in iron-sufficient medium, it displayed a reduced accumulation of ferritins and exhibited a hypersensitivity to iron deficiency. This phenotype was rescued in the complemented lines. Under iron deficiency, atnas4 displayed a lower expression of the iron uptake-related genes IRT1 and FRO2 as well as a reduced ferric reductase activity. Atnas4 plants also showed an enhanced sensitivity to cadmium while the transgenic plants overexpressing AtNAS4 were more tolerant. Collectively, our data, together with recent studies, support the hypothesis that AtNAS4 displays an important role in iron distribution and is required for proper response to iron deficiency and to cadmium supply.

Nitric oxide production in plants: an update

Nitric oxide (NO) is a key signaling molecule in plant physiology. However, its production in photosynthetic organisms remains partially unresolved. The best characterized NO production route involves the reduction of nitrite to NO via different non-enzymatic or enzymatic mechanisms. Nitrate reductases (NRs), the mitochondrial electron transport chain, and the new complex between NR and NOFNiR (nitric oxide-forming nitrite reductase) described in Chlamydomonas reinhardtii are the main enzymatic systems that perform this reductive NO production in plants. Apart from this reductive route, several reports acknowledge the possible existence of an oxidative NO production in an arginine-dependent pathway, similar to the nitric oxide synthase (NOS) activity present in animals. However, no NOS homologs have been found in the genome of embryophytes and, despite an increasing amount of evidence attesting to the existence of NOS-like activity in plants, the involved proteins remain to be identified. Here we review NO production in plants with emphasis on the presentation and discussion of recent data obtained in this field.

Copper amine oxidase 8 regulates arginine-dependent nitric oxide production in Arabidopsis thaliana

Highlight Copper amine oxidase in polyamine metabolism modulates arginine-dependent nitric oxide production by affecting arginase activity in Arabidopsis thaliana.

Nitric oxide synthase in plants: The surprise from algae

Nitric oxide (NO) is a key signalling molecule involved in various plant physiological processes. Recent investigations showed that nitric oxide synthase (NOS), the main enzymatic source for NO in animals, is absent in land plants but present in several algal species including the green algal Ostreococcus tauri. In a recent issue of Plant Science, Weisslocker-Schaetzel et al. reported the in depth structural and functional analysis of OtNOS using a combination of phylogenetic, structural, spectroscopic and biochemical approaches. Their investigation highlights that OtNOS is a prototype of highly performant NOSs, producing NO as a yet unseen rate. Also, this original work opens new opportunities for research investigating the structure/function of members of this family, the physiology and ecology of algae and, more generally, the evolution of NO signalling.