The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pKa values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pKa1 = 5.70 ± 0.28; pKa2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed

Verfolgung und Leiden der katholischen Kirche in Russland : mit noch ungedruckten Dokumenten

von einem ehemaligen russischen Staatsrath, Ritter des St. Stanislaus- und Wladimir-Ordens [=Marie Joseph d'Horrer] ; aus dem Französischen übersetzt von M. Zürcher, KaplanOriginaltitel: Persécutions et souffrances de l'église catholique en Russi

Verfolgung und Leiden der Katholischen Kirche in Rußland : mit noch ungedruckten Dokumenten

von einem ehemaligen russischen Staatsrath ... [d.i. Marie-Joseph Comte de Horrer]. Aus dem Franz. übers. von M. ZürcherVerf. ermitteltAus dem Franz. übers.In Fraktu

Gibberellic acid signaling is required for ambient temperature-mediated induction of flowering in Arabidopsis thaliana.

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature-dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process

Spatial control of flowering by DELLA proteins in Arabidopsis thaliana

The transition from vegetative to reproductive development is a central event in the plant life cycle. To time the induction of flowering correctly, plants integrate environmental and endogenous signals such as photoperiod, temperature and hormonal status. The hormone gibberellic acid (GA) has long been known to regulate flowering. However, the spatial contribution of GA signaling in flowering time control is poorly understood. Here we have analyzed the effect of tissue-specific misexpression of wild-type and GA-insensitive (dellaΔ17) DELLA proteins on the floral transition in Arabidopsis thaliana. We demonstrate that under long days, GA affects the floral transition by promoting the expression of flowering time integrator genes such as FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) in leaves independently of CONSTANS (CO) and GIGANTEA (GI). In addition, GA signaling promotes flowering independently of photoperiod through the regulation of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in both the leaves and at the shoot meristem. Our data suggest that GA regulates flowering by controlling the spatial expression of floral regulatory genes throughout the plant in a day-length-specific manner

DELLA-Mediated Control of Flowering in Arabidopsis Thaliana

The transition from vegetative to reproductive development is a critical event in the plant life cycle. To ensure the correct timing of flowering, plants perceive and integrate environmental and endogenous signals such as photoperiod, temperature and hormonal status. The hormone gibberellic acid (GA) has long been known to contribute to the regulation of flowering in many plant species. However, where in the plant and how GA signaling is integrated into the flowering time pathways is poorly understood. To address this question we have performed a systematic analysis of the role of the five Arabidopsis thaliana DELLA proteins, which have recently emerged as important components of the GA signaling cascade in the regulation of flowering. We demonstrate that misexpression of GA-insensitive DELLA either in vasculature or shoot meristem using tissue-specific promoter delays flowering under long day condition. Interestingly, we demonstrate that GA regulates the expression of flowering time integrators such as FLOWERING LOCUS T (FT) and TWIN SYSTER OF FT (TSF) independently of CONSTANS (CO) and GIGANTEA (GI) in leaves. In contrast, under non-inductive short days GA-signaling contributes to the induction of flowering specifically at the shoot meristem. Expression analysis revealed that DELLA proteins control the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) expression under both long and short day conditions independently of miR-156