Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening

Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3) - enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H+-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light

Brechungsindexsensoren basierend auf metallischen Nanostrukturen - Untersuchung der Sensitivität, Anwendung als Biosensor und Integration in einen kompakten Aufbau

In metallischen Nanostrukturen können Elektronen zu kollektiven Schwingungen angeregt werden. Diese Schwingungen werden als lokalisierte Oberflächenplasmonen bezeichnet. Sie weisen eine Resonanzfrequenz auf, welche von Größe, Form und Material der Nanostruktur, sowie deren Umgebung abhängt. Eine Anregung solcher Plasmonen kann beispielsweise durch Wechselwirkung mit elektromagnetischer Strahlung geschehen. Für Metalle, wie Gold und Silber liegen die Plasmonenresonanzen im sichtbaren Bereich des Spektrums und können mit gängigen optischen Mikroskopen und Spektrometern untersucht werden. Die Sensitivität der Resonanz auf die Umgebung der Nanostruktur kann für sensorische Anwendungen genutzt werden. So können Änderungen des Brechungsindex des Mediums, in welchem sich die Nanostruktur befindet, durch Resonanzverschiebungen nachgewiesen werden. Da auch die Anlagerung von Biomolekülen den effektiven Brechungsindex in der Umgebung der Nanostrukturen ändert, eignen sie sich für die Anwendung als Biosensor. In dieser Arbeit werden verschiedene Nanostrukturen hergestellt und auf ihre Sensitivität hin untersucht. Dabei werden neben einfachen Strukturgeometrien auch Hybridstrukturen und Gitteranordnungen, welche aus mehreren wechselwirkenden Einzelelementen aufgebaut sind, betrachtet. Die Anwendbarkeit als Biosensor wird mit Hilfe eines Testosteron-Immunassays gezeigt. Dabei erfolgt der Nachweis von Antikörpern aus einer Lösung durch Resonanzverschiebung nach Anbindung an eine spezifische Erkennungsstruktur an den Nanostrukturen. Weiterhin wird ein Ansatz für einen kompakten Aufbau des Sensors verfolgt. Die Nanostrukturen werden dafür auf eine GRIN-Linse aufgebracht, deren Fokusebene direkt auf ihrer Oberfläche liegt. Dadurch können die Strukturen ohne zusätzliche Justage oder weitere teure optische Elemente abgebildet werden. Durch Integration der Linse in eine Mikrofluidikzelle wird die Herstellung von einfach zu handhabenden und kostengünstigen Biosensoren ermöglicht

Circulation in the upper mixed layer of the equatorial North Pacific

The circulation in the upper mixed layer in the equatorial region of the North Pacific is treated by solving the steady-state equations containing terms of Corioli\u27s force, pressure gradient, and horizontal as well as vertical mixing…

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

Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness.

This is the final version of the article. Available from the publisher via the DOI in this record.Stomata respond to darkness by closing to prevent excessive water loss during the night. Although the reorganisation of actin filaments during stomatal closure is documented, the underlying mechanisms responsible for dark-induced cytoskeletal arrangement remain largely unknown. We used genetic, physiological and cell biological approaches to show that reorganisation of the actin cytoskeleton is required for dark-induced stomatal closure. The opal5 mutant does not close in response to darkness but exhibits wild-type (WT) behaviour when exposed to abscisic acid (ABA) or CaCl2 . The mutation was mapped to At5g18410, encoding the PIR/SRA1/KLK subunit of the ArabidopsisSCAR/WAVE complex. Stomata of an independent allele of the PIR gene (Atpir-1) showed reduced sensitivity to darkness and F1 progenies of the cross between opal5 and Atpir-1 displayed distorted leaf trichomes, suggesting that the two mutants are allelic. Darkness induced changes in the extent of actin filament bundling in WT. These were abolished in opal5. Disruption of filamentous actin using latrunculin B or cytochalasin D restored wild-type stomatal sensitivity to darkness in opal5. Our findings suggest that the stomatal response to darkness is mediated by reorganisation of guard cell actin filaments, a process that is finely tuned by the conserved SCAR/WAVE-Arp2/3 actin regulatory module.This work was supported by grants from the BBSRC (BB/ N001168/1; BB/J002364/1; BBF001177/1), The Gatsby Charitable Foundation and the Leverhulme Trust to A.M.H. and grants from the National Natural Science Foundation of China (nos. 31300213 and 31670408 to K.J.). This work was also supported by the Centre National de la Recherche Scientifique and the Commissariat a l’Energie Atomique et aux Energies Alternatives (for the IR camera) and the European Union Marie Curie FP5 Research Training Network (program no. STRESSIMAGING HNRT-CT-2002-00254 to J.M.C. and B.G.). J.M.C. was in addition supported by a scholarship of the Fundac ~ao para a Ci^encia e Tecnologia, Portugal (grant no. SFRH/BPD/34429/2006)