The Arabidopsis thioredoxin TRXh5regulates the S-nitrosylation pattern of the TIRK receptor being both proteins essential in the modulation of defences to Tetranychus urticae.

The interaction between plants and phytophagous arthropods encompasses a complex network of molecules, signals, and pathways to overcome defences generated by each interacting organism. Although most of the elements and modulators involved in this interplay are still unidentified, plant redox homeostasis and signalling are essential for the establishment of defence responses. Here, focusing on the response of Arabidopsis thaliana to the spider mite Tetranychus urticae, we demonstrate the involvement in plant defence of the thioredoxin TRXh5, a small redox protein whose expression is induced by mite infestation. TRXh5 is localized in the cell membrane system and cytoplasm and is associated with alterations in the content of reactive oxygen and nitrogen species. Protein S-nitrosylation signal in TRXh5 over-expression lines is decreased and alteration in TRXh5 level produces changes in the JA/SA hormonal crosstalk of infested plants. Moreover, TRXh5 interacts and likely regulates the redox state of an uncharacterized receptor-like kinase, named THIOREDOXIN INTERACTING RECEPTOR KINASE (TIRK), also induced by mite herbivory. Feeding bioassays performed withTRXh5 over-expression plants result in lower leaf damage and reduced egg accumulation after T. urticae infestation than in wild-type (WT) plants. In contrast, mites cause a more severe injury in trxh5 mutant lines where a greater number of eggs accumulates. Likewise, analysis of TIRK-gain and -loss-of-function lines demonstrate the defence role of this receptor in Arabidopsis against T. urticae. Altogether, our findings demonstrate the interaction between TRXh5 and TIRK and highlight the importance of TRXh5 and TIRK in the establishment of effective Arabidopsis defences against spider mite herbivory.

Is Autophagy Involved in Pepper Fruit Ripening?

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles; however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.

Unravelling how plants benefit from ROS and NO reactions, while resisting oxidative stress.

BACKGROUND AND AIMS: Reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO), play crucial roles in the signal transduction pathways that regulate plant growth, development and defence responses, providing a nexus of reduction/oxidation (redox) control that impacts on nearly every aspect of plant biology. Here we summarize current knowledge and concepts that lay the foundations of a new vision for ROS/RNS functions ­ particularly through signalling hubs ­ for the next decade. SCOPE: Plants have mastered the art of redox control using ROS and RNS as secondary messengers to regulate a diverse range of protein functions through redox-based, post-translational modifications that act as regulators of molecular master-switches. Much current focus concerns the impact of this regulation on local and systemic signalling pathways, as well as understanding how such reactive molecules can be effectively used in the control of plant growth and stress responses. CONCLUSIONS: The spectre of oxidative stress still overshadows much of our current philosophy and understanding of ROS and RNS functions. While many questions remain to be addressed ­ for example regarding inter-organellar regulation and communication, the control of hypoxia and how ROS/RNS signalling is used in plant cells, not only to trigger acclimation responses but also to create molecular memories of stress ­ it is clear that ROS and RNS function as vital signals of living cells.

Salinity-induced changes in S-nitrosylation of pea mitochondrial proteins.

Together with reactive oxygen species, nitric oxide is an essential part of the signal transduction induced by stress conditions. In this work we study the pattern of S-nitrosylated proteins from mitochondria of pea plants subjected to 150mM NaCl for 5 and 14days. A differential pattern of target proteins was found during plant development and salt stress, with a minor number of S-nitrosylated proteins at 14 days specifically some key enzymes related to respiration and photorespiration. At this time of stress, only ATP synthase ß subunit, peroxiredoxin and Hsp90 were S-nitrosylated and no changes in protein levels were observed, although the activity of PrxII F may be reduced by S-nitrosylation. The NADH/NAD(+) ratio was also high at 14days but not the NADPH/NADP(+). An enhancement in NO measured by fluorimetry and confocal microscopy was observed in leaves, being part of the NO localized in mitochondria. An increase in mitochondrial GSNOR activity was produced in response to short and long-term NaCl treatment, where a higher number of nitrated proteins were also observed. The results indicated that posttranslational modifications seem to modulate respiratory and photorespiratory pathways, as well as some antioxidant enzymes, through differential S-nitrosylation/denitrosylation in control conditions and under salt stress.

Role of actin cytoskeleton in brassinosteroid signaling and in its integration with the auxin response in plants.

In plants, developmental programs and tropisms are modulated by the phytohormone auxin. Auxin reconfigures the actin cytoskeleton, which controls polar localization of auxin transporters such as PIN2 and thus determines cell-type-specific responses. In conjunction with a second growth-promoting phytohormone, brassinosteroid (BR), auxin synergistically enhances growth and gene transcription. We show that BR alters actin configuration and PIN2 localization in a manner similar to that of auxin. We describe a BR constitutive-response mutant that bears an allele of the ACTIN2 gene and shows altered actin configuration, PIN2 delocalization, and a broad array of phenotypes that recapitulate BR-treated plants. Moreover, we show that actin filament reconfiguration is sufficient to activate BR signaling, which leads to an enhanced auxin response. Our results demonstrate that the actin cytoskeleton functions as an integration node for the BR signaling pathway and auxin responsiveness.

Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts.

Photosynthesis is a process that inevitably produces reactive oxygen species, such as hydrogen peroxide, which is reduced by chloroplast-localized detoxification mechanisms one of which involves 2-Cys peroxiredoxins (2-Cys Prxs). Arabidopsis chloroplasts contain two very similar 2-Cys Prxs (denoted A and B). These enzymes are reduced by two pathways: NADPH thioredoxin reductase C (NTRC), which uses NADPH as source of reducing power; and plastidial thioredoxins (Trxs) coupled to photosynthetically reduced ferredoxin of which Trx chi is the most efficient reductant in vitro. With the aim of establishing the functional relationship between NTRC, Trx x, and 2-Cys Prxs in vivo, an Arabidopsis Trx chi knock-out mutant has been identified and a double mutant (denoted Delta 2cp) with <5% of 2-Cys Prx content has been generated. The phenotypes of the three mutants, ntrc, trxx, and Delta 2cp, were compared under standard growth conditions and in response to continuous light or prolonged darkness and oxidative stress. Though all mutants showed altered redox homeostasis, no difference was observed in response to oxidative stress treatment. Moreover, the redox status of the 2-Cys Prx was imbalanced in the ntrc mutant but not in the trxx mutant. These results show that NTRC is the most relevant pathway for chloroplast 2-Cys Prx reduction in vivo, but the antioxidant function of this system is not essential. The deficiency of NTRC caused a more severe phenotype than the deficiency of Trx chi or 2-Cys Prxs as determined by growth, pigment content, CO(2) fixation, and F(v)/F(m), indicating additional functions of NTRC.

The quaternary structure of NADPH thioredoxin reductase C is redox-sensitive.

NADPH thioredoxin reductase C (NTRC) is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase (NTR) and thioredoxin (TRX) activities for the efficient reduction of 2-Cys peroxiredoxin (2-Cys PRX). Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G-V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.

cpFBPaseII, a novel redox-independent chloroplastic isoform of fructose-1,6-bisphosphatase.

A full-length FBPase cDNA has been isolated from Fragaria x ananassa (strawberry) corresponding to a novel putative chloroplastic FBPase but lacking the regulatory redox domain, a characteristic of the plastidial isoenzyme (cpFBPaseI). Another outstanding feature of this novel isoform, called cpFBPaseII, is the absence of the canonical active site. Enzymatic assays with cpFBPaseII evidenced clear Mg(2+)-dependent FBPase activity and a K(m) for fructose-1,6-bisphosphate (FBP) of 1.3 mM. Immunolocalization experiments and chloroplast isolation confirmed that the new isoenzyme is located in the stroma. Nevertheless, unlike cpFBPaseI, which is redox activated, cpFBPaseII did not increase its activity in the presence of either DTT or thioredoxin f (TRX f) and is resistant to H(2)O(2) inactivation. Additionally, the novel isoform was able to complement the growth deficiency of the yeast FBP1 deletion fed with a non-fermentable carbon source. Furthermore, orthologues are restricted to land plants, suggesting that cpFBPaseII is a novel and an intriguing chloroplastic FBPase that emerged late in the evolution of photosynthetic organisms, possibly because of a pressing need of land plants.

Peroxisome proliferation, wound-activated responses and expression of peroxisome-associated genes are cross-regulated but uncoupled in Arabidopsis thaliana.

Plant peroxisomes are multifunctional organelles that show plasticity in number, size, morphology, cellular location and metabolic functions. Many of these changes occur in response to environmental factors and are decisive for the development and defence of the plant. Among them, peroxisomal beta-oxidation-mediated synthesis of jasmonic acid (JA) is a key process in regulating development as well as wound- or pathogen-triggered defence responses. This work seeks for the connection between wound, JA and the proliferation of peroxisomes in Arabidopsis thaliana. The hypolipidemic drug clofibrate (CFB) induced the proliferation of peroxisomes and the expression of the beta-oxidation 3-ketoacyl-CoA thiolase 2 (KAT2) gene, coding for a key enzyme in the biosynthesis of JA, among other wound- and JA-responsive gene transcripts in Arabidopsis leaves. The CFB-activated expression of wound-responsive genes was not dependent on JA synthesis or perception and those responsive to JA required the function of the F-box protein COI1. In turn, wounding neither triggered peroxisome proliferation nor required peroxisome integrity to activate gene expression. Interestingly, cells from JA-treated leaves contained fewer but larger peroxisomes than cells from untreated leaves. The proliferation of peroxisomes, the synthesis of JA and the activation of wound-responsive genes by CFB, although functionally connected, were uncoupled in Arabidopsis.