Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions.

The interaction between legumes and rhizobia leads to the establishment of a beneficial symbiotic relationship. Recent advances in legume - rhizobium symbiosis revealed that various reactive oxygen and nitrogen species including nitric oxide (NO) play important roles during this process. Nodule development occurs with a transition from a normoxic environment during the establishment of symbiosis to a microoxic environment in functional nodules. Such oxygen dynamics are required for activation and repression of various NO production and scavenging pathways. Both the plant and bacterial partners participate in the synthesis and degradation of NO. However, the pathways of NO production and degradation as well as their cross-talk and involvement in the metabolism are still a matter of debate. The plant-originated reductive pathways are known to contribute to the NO production in nodules under hypoxic conditions. Non-symbiotic hemoglobin (phytoglobin) (Pgb) possesses high NO oxygenation capacity, buffers and scavenges NO. Its operation, through a respiratory cycle called Pgb-NO cycle, leads to the maintenance of redox and energy balance in nodules. The role of Pgb/NO cycle under fluctuating NO production from soil needs further investigation for complete understanding of NO regulatory mechanism governing nodule development to attain optimal food security under changing environment.

The Solanum lycopersicum WRKY3 Transcription Factor SlWRKY3 Is Involved in Salt Stress Tolerance in Tomato.

Salinity threatens productivity of economically important crops such as tomato (Solanum lycopersicum L.). WRKY transcription factors appear, from a growing body of knowledge, as important regulators of abiotic stresses tolerance. Tomato SlWRKY3 is a nuclear protein binding to the consensus CGTTGACC/T W box. SlWRKY3 is preferentially expressed in aged organs, and is rapidly induced by NaCl, KCl, and drought. In addition, SlWRKY3 responds to salicylic acid, and 35S::SlWRKY3 tomatoes showed under salt treatment reduced contents of salicylic acid. In tomato, overexpression of SlWRKY3 impacted multiple aspects of salinity tolerance. Indeed, salinized (125 mM NaCl, 20 days) 35S::SlWRKY3 tomato plants displayed reduced oxidative stress and proline contents compared to WT. Physiological parameters related to plant growth (shoot and root biomass) and photosynthesis (stomatal conductance and chlorophyll a content) were retained in transgenic plants, together with lower Na+ contents in leaves, and higher accumulation of K+ and Ca2+. Microarray analysis confirmed that many stress-related genes were already up-regulated in transgenic tomatoes under optimal conditions of growth, including genes coding for antioxidant enzymes, ion and water transporters, or plant defense proteins. Together, these results indicate that SlWRKY3 is an important regulator of salinity tolerance in tomato.

Phytohormone profiling in relation to osmotic adjustment in NaCl-treated plants of the halophyte tomato wild relative species Solanum chilense comparatively to the cultivated glycophyte Solanum lycopersicum.

A holistic approach was used to investigate the hormonal profile in relation with osmotic adjustment under salinity in Solanum lycopersicum and its halophyte wild relative Solanum chilense. Plants were subjected to 125mM NaCl for 7days. Solanum chilense displayed a contrasting behaviour comparatively to S. lycopersicum, not only for mineral nutrition, but also regarding the modalities of osmotic adjustment and phytohormonal profiling. The extent of osmotic adjustment was higher in S. chilense than in S. lycopersicum. Ions K+ and Na+ were the major contributors of osmotic adjustment in S. chilense, accounting respectively for 47 and 60% of osmotic potential. In contrast the contributions of proline and soluble sugars remained marginal for the two species although salt-induced accumulation of proline was higher in S. lycopersicum than in S. chilense. Both species also differed for their hormonal status under salinity and concentrations of most hormonal compounds were higher in S. chilense than in S. lycopersicum. Interestingly, salicylic acid, ethylene and cytokinins were positively correlated with osmotic potential in S. chilense under salinity while these hormones were negatively correlated with osmotic adjustment in S. lycopersicum. Our results suggested that the capacity to use inorganic ions as osmotica may improve salt resistance in S.chilense and that phytohormones could be involved in this process.

SlDREB2, a tomato dehydration-responsive element-binding 2 transcription factor, mediates salt stress tolerance in tomato and Arabidopsis.

To counter environmental cues, cultivated tomato (Solanum lycopersicum L.) has evolved adaptive mechanisms requiring regulation of downstream genes. The dehydration-responsive element-binding protein 2 (DREB2) transcription factors regulate abiotic stresses responses in plants. Herein, we isolated a novel DREB2-type regulator involved in salinity response, named SlDREB2. Spatio-temporal expression profile together with investigation of its promoter activity indicated that SlDREB2 is expressed during early stages of seedling establishment and in various vegetative and reproductive organs of adult plants. SlDREB2 is up-regulated in roots and young leaves following exposure to NaCl, but is also induced by KCl and drought. Its overexpression in WT Arabidopsis and atdreb2a mutants improved seed germination and plant growth in presence of different osmotica. In tomato, SlDREB2 affected vegetative and reproductive organs development and the intronic sequence present in the 5' UTR drives its expression. Physiological, biochemical and transcriptomic analyses showed that SlDREB2 enhanced plant tolerance to salinity by improvement of K(+) /Na(+) ratio, and proline and polyamines biosynthesis. Exogenous hormonal treatments (abscisic acid, auxin and cytokinins) and analysis of WT and 35S::SlDREB2 tomatoes hormonal contents highlighted SlDREB2 involvement in abscisic acid biosynthesis/signalling. Altogether, our results provide an overview of SlDREB2 mode of action during early salt stress response.

Tomato (Solanum lycopersicum L.) SlIPT3 and SlIPT4 isopentenyltransferases mediate salt stress response in tomato.

BACKGROUND: Cytokinins (CKs) are involved in response to various environmental cues, including salinity. It has been previously reported that enhancing CK contents improved salt stress tolerance in tomato. However, the underlying mechanisms of CK metabolism and signaling under salt stress conditions remain to be deciphered. RESULTS: Two tomato isopentenyltransferases, SlIPT3 and SlIPT4, were characterized in tomato and Arabidopsis. Both proteins displayed isopentenyltransferase (IPT) activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. SlIPT3 and SlIPT4 were affected by the endogenous CK status, tightly connected with CKs feedback regulation, as revealed by hormonal treatements. In response to salt stress, SlIPT3 and SlIPT4 were strongly repressed in tomato roots, and differently affected in young and old leaves. SlIPT3 overexpression in tomato resulted in high accumulation of different CK metabolites, following modifications of CK biosynthesis-, signaling- and degradation-gene expression. In addition, 35S::SlIPT3 tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and K(+)/Na(+) ratio retention. Involvement of SlIPT3 and SlIPT4 in salt stress response was also observed in Arabidopsis ipt3 knock-out complemented plants, through maintenance of CK homeostasis. CONCLUSIONS: SlIPT3 and SlIPT4 are functional IPTs encoded by differently expressed genes, distinctively taking part in the salinity response. The substantial participation of SlIPT3 in CK metabolism during salt stress has been determined in 35S::SlIPT3 tomato transformants, where enhancement of CKs accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response.

Nitric oxide: a multifaceted regulator of the nitrogen-fixing symbiosis.

The specific interaction between legumes and Rhizobium-type bacteria leads to the establishment of a symbiotic relationship characterized by the formation of new differentiated organs named nodules, which provide a niche for bacterial nitrogen (N2) fixation. In the nodules, bacteria differentiate into bacteroids with the ability to fix atmospheric N2 via nitrogenase activity. As nitrogenase is strongly inhibited by oxygen, N2 fixation is made possible by the microaerophilic conditions prevailing in the nodules. Increasing evidence has shown the presence of NO during symbiosis, from early interaction steps between the plant and the bacterial partners to N2-fixing and senescence steps in mature nodules. Both the plant and the bacterial partners participate in NO synthesis. NO was found to be required for the optimal establishment of the symbiotic interaction. Transcriptomic analysis at an early stage of the symbiosis showed that NO is potentially involved in the repression of plant defence reactions, favouring the establishment of the plant-microbe interaction. In mature nodules, NO was shown to inhibit N2 fixation, but it was also demonstrated to have a regulatory role in nitrogen metabolism, to play a beneficial metabolic function for the maintenance of the energy status under hypoxic conditions, and to trigger nodule senescence. The present review provides an overview of NO sources and multifaceted effects from the early steps of the interaction to the senescence of the nodule, and presents several approaches which appear to be particularly promising in deciphering the roles of NO in N2-fixing symbioses.

The Solanum lycopersicum Zinc Finger2 cysteine-2/histidine-2 repressor-like transcription factor regulates development and tolerance to salinity in tomato and Arabidopsis.

The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed.

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity.

BACKGROUND: Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. RESULTS: In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b(L), the native protein being referred as VvMYB5b(R)) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b(L) exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5b(R) and VvMYB5b(L) genes in tobacco. Flowers from 35S::VvMYB5b(L) transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5b(R) and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. CONCLUSIONS: Taken together, our findings indicate that VvMYB5b(L) is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5(L) observed in the tobacco model.

Root-targeted biotechnology to mediate hormonal signalling and improve crop stress tolerance.

Since plant root systems capture both water and nutrients essential for the formation of crop yield, there has been renewed biotechnological focus on root system improvement. Although water and nutrient uptake can be facilitated by membrane proteins known as aquaporins and nutrient transporters, respectively, there is a little evidence that root-localised overexpression of these proteins improves plant growth or stress tolerance. Recent work suggests that the major classes of phytohormones are involved not only in regulating aquaporin and nutrient transporter expression and activity, but also in sculpting root system architecture. Root-specific expression of plant and bacterial phytohormone-related genes, using either root-specific or root-inducible promoters or grafting non-transformed plants onto constitutive hormone producing rootstocks, has examined the role of root hormone production in mediating crop stress tolerance. Root-specific traits such as root system architecture, sensing of edaphic stress and root-to-shoot communication can be exploited to improve resource (water and nutrients) capture and plant development under resource-limited conditions. Thus, root system engineering provides new opportunities to maintain sustainable crop production under changing environmental conditions.

Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway.

Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine.

Previous results indicated that in grapevine (Vitis vinifera), regulation of the flavonoid pathway genes by MYB transcription factors depends on their interaction with basic helix-loop-helix proteins (bHLHs). The present study describes the first functional characterization of a bHLH factor from grapevine named VvMYC1. This transcription factor is phylogenetically related to Arabidopsis bHLH proteins, which participate in the control of flavonoid biosynthesis and epidermal cell fate. Transient promoter and yeast two-hybrid assays demonstrated that VvMYC1 physically interacts with MYB5a, MYB5b, MYBA1/A2, and MYBPA1 to induce promoters of flavonoid pathway genes involved in anthocyanin and/or proanthocyanidin (PA) synthesis. Additionally, transient promoter assays revealed that VvMYC1 is involved in feedback regulation of its own expression. Transcript levels of VvMYC1 during berry development correlate with the synthesis of anthocyanins and PAs in skins and seeds of berries, suggesting that VvMYC1 is involved in the regulation of anthocyanins and PA synthesis in these organs. Likewise, transient expression of VvMYC1 and VvMYBA1 induces anthocyanin synthesis in grapevine suspension cells. These results suggest that VvMYC1 is part of the transcriptional cascade controlling anthocyanin and PA biosynthesis in grapevine.

The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco.

Previous work has shown that transgenic tobacco plants constitutively over-expressing the Vitis vinifera L. transcription factor VvWRKY2 exhibit reduced susceptibility to necrotrophic fungal pathogens, suggesting that this transcription factor plays a role in grapevine response to phytopathogens. The work presented here characterizes the modifications in cell wall structure observed in the stems and petioles of these transgenic plants. Histochemical stainings of stem and petiole cross-sections using phloroglucinol or Maüle reagents revealed a delay in xylem formation, particularly in the petioles, and differences in lignin composition. Evaluation of lignin quantity and quality showed a decrease in the syringyl/guaiacyl ratio in both stem and petioles. Expression analysis using RT-PCR and potato microarrays showed that tobacco plants over-expressing VvWRKY2 exhibited altered expression of genes involved in lignin biosynthesis pathway and cell wall formation. The ability of VvWRKY2 to activate the promoter of the VvC4H gene, which is involved in the lignin biosynthetic pathway, was confirmed by transient transcriptional activation assays in tobacco protoplasts. Moreover, in situ hybridization revealed that VvWRKY2 is specifically expressed in cells undergoing lignification in young grapevine stems. Together, these results confirm that VvWRKY2 plays a role in regulating lignification in grapevine, possibly in response to biotic or abiotic stresses.