Signal transduction by nitric oxide in cellular stress responses

Nitric oxide (NO) has received wide attention as a biological signaling molecule that uses cyclic GMP as a cellular second messenger. Other work has supported roles for cysteine oxidation or nitrosylation as signaling events. Recent studies in bacteria and mammalian cells now point to the existence of at least two other pathways independent of cGMP. For the E. coli SoxR protein, signaling occurs by nitrosylation of its binuclear iron-sulfur clusters, a reaction that is unprecedented in gene activation. In intact cells, these nitrosylated centers are very rapidly replaced by unmodified iron-sulfur clusters, a result that points to the existence of an active repair pathway for this type of protein damage. Exposure of mammalian cells to NO elicits an adaptive resistance that confers elevated resistance of the cells to higher levels of NO. This resistance in many cell types involves the important defense protein heme oxygenase 1, although the mechanism by which this enzyme mediates NO resistance remains unknown. Induction of heme oxygenase in some cell types occurs through the stabilization of its mRNA. NO-induced stabilization of mRNA is mediated by pre-existing proteins and points to the existence of an important new signaling pathway that counteracts the damage and stress exerted by this free radical.     

Nitric oxide is required for the auxin-induced activation of NADPH-dependent thioredoxin reductase and protein denitrosylation during root growth responses in arabidopsis

Background and Aims Auxin is the main phytohormone controlling root development in plants. This study uses pharmacological and genetic approaches to examine the role of auxin and nitric oxide (NO) in the activation of NADPH-dependent thioredoxin reductase (NTR), and the effect that this activity has on root growth responses in Arabidopsis thaliana.Methods Arabidopsis seedlings were treated with auxin with or without the NTR inhibitors auranofin (ANF) and 1-chloro-2, 4-dinitrobenzene (DNCB). NTR activity, lateral root (LR) formation and S-nitrosothiol content were measured in roots. Protein S-nitrosylation was analysed by the biotin switch method in wild-type arabidopsis and in the double mutant ntra ntrb.Key Results The auxin-mediated induction of NTR activity is inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors ANF and DNCB prevent auxin-mediated activation of NTR and LR formation. Moreover, ANF and DNCB also inhibit auxin-induced DR5 : : GUS and BA3 : : GUS gene expression, suggesting that the auxin signalling pathway is compromised without full NTR activity. Treatment of roots with ANF and DNCB increases total nitrosothiols (SNO) content and protein S-nitrosylation, suggesting a role of the NTR-thioredoxin (Trx)-redox system in protein denitrosylation. In agreement with these results, the level of S-nitrosylated proteins is increased in the arabidopsis double mutant ntra ntrb as compared with the wild-type.Conclusions The results support for the idea that NTR is involved in protein denitrosylation during auxin-mediated root development. The fact that a high NO concentration induces NTR activity suggests that a feedback mechanism to control massive and unregulated protein S-nitrosylation could be operating in plant cells.     

ZIFL1.1 transporter modulates polar auxin transport by stabilizing membrane abundance of multiple PINs in Arabidopsis root tip

Cell-to-cell directional flow of the phytohormone auxin is primarily established by polar localization of the PIN auxin transporters, a process tightly regulated at multiple levels by auxin itself. We recently reported that, in the context of strong auxin flows, activity of the vacuolar ZIFL1.1 transporter is required for fine-tuning of polar auxin transport rates in the Arabidopsis root. In particular, ZIFL1.1 function protects plasma-membrane stability of the PIN2 carrier in epidermal root tip cells under conditions normally triggering PIN2 degradation. Here, we show that ZIFL1.1 activity at the root tip also promotes PIN1 plasma-membrane abundance in central cylinder cells, thus supporting the notion that ZIFL1.1 acts as a general positive modulator of polar auxin transport in roots.     

WD40‐REPEAT 5a represses root meristem growth by suppressing auxin synthesis through changes of nitric oxide accumulation in Arabidopsis

Although nitric oxide (NO) is known to regulate root growth, the factor(s) modulating NO during this process have not yet been elucidated. Here, we identified Arabidopsis WD40‐REPEAT 5a (WDR5a) as a novel factor that functions in root growth by modulating NO accumulation. The wdr5a‐1 mutant accumulated less NO and produced longer roots than the wild type, whereas the WDR5a overexpression lines had the opposite phenotype. The role of NO was further supported by our observation that the NO donor sodium nitroprusside (SNP) and the NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) rescued the root meristem growth phenotypes of the wdr5a‐1 and WDR5a overexpression lines, respectively. The regulation of root growth by WDR5a was found to involve auxin because the auxin levels were similar in SNP‐treated wdr5a‐1 and wild‐type roots, but higher in untreated wdr5a‐1 roots than in wild‐type roots. In addition, the wdr5a‐1 mutant had higher production and activity levels of the auxin biosynthetic enzyme TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), in contrast to its reduced expression and activity in the WDR5a overexpression lines, and the increased root meristem growth in wdr5a‐1 was suppressed by treatment with l ‐kynurenine, which inhibits TAA1, as well as by mutating TAA1. WDR5a therefore functions in root meristem growth by maintaining NO homeostasis, and thus TAA1‐mediated auxin biosynthesis.     

Effects of nitric oxide on sunflower seedlings: A balance between defense and development

Nitric oxide (NO) is a major plant signaling molecule that plays key roles during plant-pathogen interactions and plant development. Previous work showed the participation of NO in the development and lignin composition of sunflower roots. Thereby, we have hypothesized that NO applications could control the attack of the fungal pathogen Verticillium dahliae in sunflowers. Seedlings growing hydroponically were pretreated with NO donors and further inoculated with the fungus. Evaluation of disease symptoms showed that NO pretreatments could not reduce Verticillium wilt. Strikingly, NO donors appear to promote the fungal infection. These results indicate that NO applications were unable to protect sunflowers from Verticillium attack and highlight the role played by the fine tuning regulation of NO levels required to balance plant responses between development and defense.     

Pharmacological and genetical evidence supporting nitric oxide requirement for 2,4-epibrassinolide regulation of root architecture in Arabidopsis thaliana

Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.     

Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus

In a recent study, we demonstrated that although the auxin efflux carrier PIN-FORMED (PIN) proteins, such as PIN3 and PIN7, are required for the pulse-induced first positive phototropism in etiolated Arabidopsis hypocotyls, they are not necessary for the continuous-light-induced second positive phototropism when the seedlings are grown on the surface of agar medium, which causes the hypocotyls to separate from the agar surface. Previous reports have shown that hypocotyl phototropism is slightly impaired in pin3 single mutants when they are grown along the surface of agar medium, where the hypocotyls always contact the agar, producing some friction. To clarify the possible involvement of PIN3 and PIN7 in continuous-light-induced phototropism, we investigated hypocotyl phototropism in the pin3 pin7 double mutant grown along the surface of agar medium. Intriguingly, the phototropic curvature was slightly impaired in the double mutant when the phototropic stimulus was presented on the adaxial side of the hook, but was not impaired when the phototropic stimulus was presented on the abaxial side of the hook. These results indicate that PIN proteins are required for continuous-light-induced second positive phototropism, depending on the direction of the light stimulus, when the seedlings are in contact with agar medium.     

A new temperature-insensitive allele of the Arabidopsis AXR6/CUL1 locus derived from a missense mutation in the C-terminal RBX1 binding region

We isolated a new recessive allele at the AUXIN RESISTANT6/CULLIN1 (AXR6/CUL1) locus, axr6–101, from an EMS-mutagenized population of Arabidopsis thaliana, the Landsberg erecta ecotype. axr6–101 is auxin resistant and semi-dwarf similar to the other recessive axr6 mutants. The axr6–101 phenotype is caused by the E716K substitution of the CUL1 protein, which is likely to affect its ability to bind to the C-terminal RING domain of RING-box 1 (RBX1). The previously reported allele of AXR6, cul1–7, is caused by a substitution at T510 that binds to the N-terminal β-strand of RBX1. Although cul1–7 shows temperature-sensitive phenotype, the axr6–101 phenotype is largely unaffected by temperature. axr6–101 may provide an important genetic resource for study of the structure−function relationship of the CUL1 protein.     

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd²⁺ treatment. NO contributed caspase-3-like protease activation in Cd²⁺ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd²⁺-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd²⁺-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd²⁺-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd²⁺-induced plant PCD.     

Involvement of cotton gene GhFPF1 in the regulation of shade avoidance responses in Arabidopsis thaliana

Phytochrome system perceives the reduction in the ratio of red to far-red light when plants are grown under dense canopy. This signal, regarded as a warning of competition, will trigger a series of phenotypic changes to avoid shade. Progress has been made for several phytochrome signaling intermediates acting as positive regulators of accelerated elongation growth and promotion of flowering in shade-avoidance has been identified. Recently, a FPF1 homolog GhFPF1 was identified in upland cotton. Our data supported that transgenic Arabidopsis of over-expressing GhFPF1 displayed a constitutive shade-avoiding phenotype resembling phyB mutants in several respects such as accelerated elongation of hypocotyl and petioles, upward of leaf movement, and promoted flowering. In this addendum, by dissection of GhFPF1 acting as a component of shade-avoidance responses we suppose that GhFPF1 might influence the timing of the floral transition independently of shade-mediated early flowering. Furthermore, the opposite changes of IAA content in transgenic leaves and stems suggested that alteration of IAA storage and release took place during shade-avoidance responses.     

Nitric oxide accumulation is required to protect against iron-mediated oxidative stress in frataxin-deficient Arabidopsis plants

Frataxin is a mitochondrial protein that is conserved throughout evolution. In yeast and mammals, frataxin is essential for cellular iron (Fe) homeostasis and survival during oxidative stress. In plants, frataxin deficiency causes increased reactive oxygen species (ROS) production and high sensitivity to oxidative stress. In this work we show that a knock-down T-DNA frataxin-deficient mutant of Arabidopsis thaliana (atfh-1) contains increased total and organellar Fe levels. Frataxin deficiency leads also to nitric oxide (NO) accumulation in both, atfh-1 roots and frataxin null mutant yeast. Abnormally high NO production might be part of the defence mechanism against Fe-mediated oxidative stress.     

Homology modeling of Ferredoxin-nitrite reductase from Arabidopsis thaliana

Different subtypes of Influenza A virus are associated with species specific, zoonotic or pandemic Influenza. The cause of its severity underlies in complicated evolution of its segmented RNA genome. Although genetic shift and genetic drift are well known in the evolution of this virus, we reported the significant role of unique RNA palindromes in its evolution. Our computational approach identified the existence of unique palindromes in each subtype of Influenza A virus with its absence in Influenza B relating the fact of virulence and vigorous genetic hitchhiking in Influenza A. The current study focused on the re-assortment event responsible for the emergence of pandemic-2009 H1N1 virus, which is associated with outgrow of new palindrome and in turn, changing its RNA structure. We hypothesize that the change in RNA structure due to the presence of palindrome facilitates the event of re-assortment in Influenza A. Thus the evolutionary process of Influenza A is much more complicated as previously known, and that has been demonstrated in this study.     

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.¹ The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.     

The recombination landscape in Arabidopsis thaliana F2 populations

Recombination during meiosis shapes the complement of alleles segregating in the progeny of hybrids, and has important consequences for phenotypic variation. We examined allele frequencies, as well as crossover (XO) locations and frequencies in over 7000 plants from 17 F(2) populations derived from crosses between 18 Arabidopsis thaliana accessions. We observed segregation distortion between parental alleles in over half of our populations. The potential causes of distortion include variation in seed dormancy and lethal epistatic interactions. Such a high occurrence of distortion was only detected here because of the large sample size of each population and the number of populations characterized. Most plants carry only one or two XOs per chromosome pair, and therefore inherit very large, non-recombined genomic fragments from each parent. Recombination frequencies vary between populations but consistently increase adjacent to the centromeres. Importantly, recombination rates do not correlate with whole-genome sequence differences between parental accessions, suggesting that sequence diversity within A. thaliana does not normally reach levels that are high enough to exert a major influence on the formation of XOs. A global knowledge of the patterns of recombination in F(2) populations is crucial to better understand the segregation of phenotypic traits in hybrids, in the laboratory or in the wild.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.