Maintaining an optimal leaf and stem orientation to yield a maximum photosynthetic output is accomplished by terrestrial plants using sophisticated mechanisms to balance their orientation relative to the Earth's gravity vector and the direction of sunlight. Knowledge of the signal transduction chains of both gravity and light perception and how they influence each other is essential for understanding plant development on Earth and plant cultivation in space environments. However, in situ analyses of cellular signal transduction processes in weightlessness, such as live cell imaging of signaling molecules using confocal fluorescence microscopy, require an adapted experimental setup that meets the special requirements of a microgravity environment. In addition, investigations under prolonged microgravity conditions require extensive resources, are rarely accessible, and do not allow for immediate sample preparation for the actual microscopic analysis. Therefore, supply concepts are needed that ensure both the viability of the contained plants over a longer period of time and an unhindered microscopic analysis in microgravity. Here, we present a customized supply unit specifically designed to study gravity-induced Ca2+ mobilization in roots of Arabidopsis thaliana. The unit can be employed for ground-based experiments, in parabolic flights, on sounding rockets, and probably also aboard the International Space Station.

Plant organs that are exposed to continuous unilateral light reach in the steady-state a photogravitropic bending angle that results from the mutual antagonism between the photo- and gravitropic responses. To characterize the interaction between the two tropisms and their quantitative relationship we irradiated seedlings of Arabidopsis thaliana that were inclined at various angles and determined the fluence rates of unilateral blue light required to compensate the gravitropism of the inclined hypocotyls. We found the compensating fluence rates to increase with the tangent of the inclination angles (0° < γ < 90° or max. 120°) and decrease with the cotangent (90°< γ < 180° or max. 120°of the inclination angles. The tangent dependence became also evident from analysis of previous data obtained with Avena sativa and the phycomycete fungus, Phycomyces blakesleeanus. By using loss-of function mutant lines of Arabidopsis, we identified EHB1 (enhanced bending 1) as an essential element for the generation of the tangent and cotangent relationships. Because EHB1 possesses a C2-domain with two putative calcium binding sites, we propose that the ubiquitous calcium dependence of gravi- and phototropism is in part mediated by Ca2+-bound EHB1. Based on a yeast-two-hybrid analysis we found evidence that EHB1 does physically interact with the ARF-GAP protein AGD12. Both proteins were reported to affect gravi- and phototropism antagonistically. We further showed that only AGD12, but not EHB1, interacts with its corresponding ARF-protein. Evidence is provided that AGD12 is able to form homodimers as well as heterodimers with EHB1. On the basis of these data we present a model for a mechanism of early tropism events, in which Ca2+-activated EHB1 emerges as the central processor-like element that links the gravi- and phototropic transduction chains and that generates in coordination with NPH3 and AGD12 the tangent / cotangent algorithm governing photogravitropic equilibrium.

The aim of this study was to investigate the effect of NaCl salinity (0, 100 and 300 mM) on the individual response of the quinoa varieties Kcoito (Altiplano Ecotype) and UDEC-5 (Sea-level Ecotype) with physiological and proteomic approaches. Leaf protein profile was performed using two dimensional gel electrophoresis (2-DE). UDEC-5 showed an enhanced capacity to withstand salinity stress compared to Kcoito. In response to salinity, we detected overall the following differences between both genotypes: Toxicity symptoms, plant growth performance, photosynthesis performance and intensity of ROS-defense. We found a mirroring of these differences in the proteome of each genotype. Among the 700 protein spots reproducibly detected, 24 exhibited significant abundance variations between samples. These proteins were involved in energy and carbon metabolism, photosynthesis, ROS scavenging and detoxification, stress defense and chaperone functions, enzyme activation and ATPases. A specific set of proteins predominantly involved in photosynthesis and ROS scavenging showed significantly higher abundance under high salinity (300 mM NaCl). The adjustment was accompanied by a stimulation of various metabolic pathways to balance the supplementary demand for energy or intermediates. However, the more salt-resistant genotype UDEC-5 presented a beneficial and significantly higher expression of nearly all stress-related altered enzymes than Kcoito.

The aim of this study was to investigate the effect of NaCl salinity (0, 100 and 300 mM) on the individual response of the quinoa varieties Kcoito (Altiplano Ecotype) and UDEC-5 (Sea-level Ecotype) with physiological and proteomic approaches. UDEC-5 showed an enhanced capacity to withstand salinity stress compared to Kcoito. In response to salinity, we detected overall the following differences between both genotypes: Toxicity symptoms, plant growth performance, photosynthesis performance and intensity of ROS-defense. We found a mirroring of these differences in the proteome of each genotype. Among the 700 protein spots reproducibly detected, 24 exhibited significant abundance variations between samples. These 24 proteins were involved in energy and carbon metabolism, photosynthesis, ROS scavenging and detoxification, stress defense and chaperone functions, enzyme activation and ATPases. A specific set of proteins predominantly involved in photosynthesis and ROS scavenging showed significantly higher abundance under high salinity (300 mM NaCl). The adjustment was accompanied by a stimulation of various metabolic pathways to balance the supplementary demand for energy or intermediates. However, the more salt-resistant genotype UDEC-5 presented a beneficial and significantly higher expression of nearly all stress-related altered enzymes than Kcoito. Salinity, halophyte, quinoa, proteomic, photosynthesis, antioxidant, salt resistance, oxidative stress.

In Arabidopsis gravitropism is affected by two antagonistically interacting proteins, AGD12 (ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN) and EHB1 (ENHANCED BENDING 1). While AGD12 enhances gravitropic bending, EHB1 functions as a negative element. To further characterize their cellular function, we analyzed the location of AGD12-GFP and EHB1-GFP fusion proteins in the root apex by confocal laser-scanning microscopy after gravitropic stimulation. For this purpose, a novel method of microscopic visualization was developed with the objective and root axes aligned allowing an improved and comparable discernment of the fluorescence gradient across the columella. In vertical roots, both proteins were localized symmetrically and occurred preferentially in the outer layers of the columella. After reorienting roots horizontally, EHB1-GFP accumulated in the upper cell layers of the columella, that is, opposite to the gravity vector. The gravity-induced EHB1-GFP asymmetry disappeared after reorienting the roots back into the vertical position. No such asymmetry occurred with AGD12-GFP. Our findings reveal that after a gravitropic stimulus the cellular ratio between EHB1 and AGD12 is affected differently in the upper and lower part of the root. Its impact as a significant signaling event that ultimately affects the redirection of the lateral auxin flux toward the lower site of the root is discussed.

Previous findings concerning two proteins involved in gravity and light-induced signal transduction, (AGD12-ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN 12 and EHB1 (ENHANCED BENDING 1)), in Arabidopsis provided evidence for an antagonistic regulation of a gravitropic stimulus on roots and shoots. Both proteins share high C-terminal homology containing a canonical calcium-binding C2-domain, but EHB1 lacks a functional N-terminally located GTP-binding domain. Physiological analysis using loss-of-function mutants under clinostat conditions, where any gravitropic stimulus could be ruled out, provided evidence for diminished phototropism of agd12 mutants, but enhanced phototropism of ehb1-mutants. Data could be confirmed by seedlings overexpressing either AGD12 or EHB1, showing enhanced bending in AGD12 and reduced bending in EHB1 overexpressing seedlings. We conclude that EHB1 and AGD12 are early signaling elements of the gravitropic phototropic bending response, because both of them bind to NPH3 (NON PHOTOTROPIC HYPOCOTYL), which was described as the first element of the phototropic signal transduction chain, directly interacting with phot1. Both proteins affect phototropic bending antagonistically. In contrast to the total incapability of agd12 and ehb1 mutants to respond to increased calcium concentration in response to gravity, agd12 nor ehb1 seedlings differ only gradually in their calcium induced bending response to blue light. This would indicate that EHB1 and AGD12 might be triggered by other signal elements within the phototropism signal transduction chain.

In experimental work with Arabidopsis thaliana, sucrose is routinely used in growth media as an energy source assuring vigorous growth and stable development. We investigated the impact of sucrose on the phenotypic stability of two early tropism mutants of Arabidopsis with defects in either the EHB1 or AGD12 gene. Database analyses show that both these genes possess in their upstream promoter regions sucrose- and light-responsive elements. We show that exogenously applied sucrose is able to revert the gravitropic and phototropic phenotypes associated with the two mutants. Depending on the specific tropism assays and the mutant employed, sucrose elicits either a change from hypertropism to hypotropism or vice versa from hypotropism to hypertropism. The observations serve as a caveat to view sucrose exclusively under the aspect of energy supply. The capability of sucrose to elicit phenotypic reversals argues strongly for its role as an essential signaling molecule. The association of the tropism genes, EHB1 and AGD12, with several sucrose-responsive elements indicates that these cis-acting elements, in conjunction with the requisite transcription factors, constitute very likely the physical basis for the observed sucrose effects.

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca2+-activated states.

Gravitropic bending of seedlings of Arabidopsis thaliana in response to centrifugal accelerations was determined in a range between 0.0025 and 4×g to revisit and validate the so-called resultant law, which claims that centrifugation causes gravitropic organs to orient parallel to the resultant stimulus vector. We show here for seedlings of A. thaliana that this empirical law holds for hypocotyls but surprisingly fails for roots. While the behavior of hypocotyls could be modeled by an arc tangent function predicted by the resultant law, roots displayed a sharp maximum at 1.8×g that substantially overshoots the predicted value and that represents a novel phenomenon, diagravitropism elicited by centrifugal acceleration. The gravitropic bending critically depended on the orientation of the seedling relative to the centrifugal acceleration. If the centrifugal vector pointed toward the cotyledons, gravitropic bending of hypocotyls and roots was substantially enhanced. The complex behavior of Arabidopsis seedlings provides strong evidence that gravitropic bending entails a cosine component (longitudinal stimulus) to which the seedlings were more sensitive than to the classical sine component. The absolute gravitropic thresholds of hypocotyls and roots were determined in a clinostat-centrifuge and found to be below 0.015×g. A tropism mutant lacking the EHB1 protein, which interacts with ARF-GAP (ARF GTPase-activating protein) and thus indirectly with a small ARF-type G protein, displayed a lower gravitropic threshold for roots and also enhanced bending, while the responses of the hypocotyls remained nearly unaffected.

sHSPs interact with clients under denaturing conditions. CPH1Δ2, a truncated version of cyanobacterial phytochrome CPH1, was introduced as a new reporter (client). Comparative analyses of At17.8 and At17.6B as cytosolic class I sHSP representatives demonstrated the advantages of a chromophore-bearing photoreversible protein as new client for analyzing sHSP holdase function in addition to malate dehydrogenase (MDH). The tested sHSPs protected both clients in similar ways but with different efficiencies. Bis-ANS binding studies with sHSPs suggested that the bis-ANS binding is dependent on interactions between different sHSPs and MDH under denaturing temperatures.

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.

Although sessile, plants are able to grow toward or away from an environmental stimulus. Important examples are stem or leaf orientation of higher plants in response to the direction of the incident light. The responsible photoreceptors belong to the phototropin photoreceptor family. Although the mode of phototropin action is quite well understood, much less is known of how the light signal is transformed into a bending response. Several lines of evidence indicate that a lateral auxin gradient is responsible for asymmetric cell elongation along the light gradient within the stem. However, some of the molecular key players leading to this asymmetric auxin distribution are, as yet, unidentified. Previously, it was shown that phototropin gets autophosphorylated upon illumination and binds to a scaffold protein termed NPH3 (for nonphototropic hypocotyl 3). Using a yeast three-hybrid approach with phototropin and NPH3 as a bait complex, we isolated a protein, termed EHB1 (for enhanced bending 1), with a so far unknown function, which binds to this binary complex. This novel interacting factor negatively affects hypocotyl bending under blue light conditions in Arabidopsis (Arabidopsis thaliana) and thus seems to be an important component regulating phototropism. Interestingly, it could be shown that the gravitropic response was also affected. Thus, it cannot be ruled out that this protein might also have a more general role in auxin-mediated bending toward an environmental stimulus.

Singlet oxygen ( 1 O 2 ) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of 1 O 2 generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP‐driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1 min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to 1 O 2 than CF1CFo in its reduced state, a new insight on the mechanism of 1 O 2 interaction with the γ subunit.

it is of fundamental importance to understand the physiological differences leading to salt resistance and to get access to the molecular mechanisms underlying this physiological response. The aim of this work was to investigate the effects of short-term salt exposure on the proteome of maize chloroplasts in the initial phase of salt stress (up to 4h). It could be shown that sodium ions accumulate quickly and excessively in chloroplasts in the initial phase of moderate salt stress. A change in the chloroplast protein pattern was observed without a change in water potential of the leaves. 2-DE revealed that 12 salt-responsive chloroplast proteins increased while eight chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1 epsilon and a Ca2+-sensing receptor show a rather transient response for the first 4 h of salt exposure. The enhanced abundance of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the photosystem 11, and the FtsH-like protein might reflect mechanism to attenuate the detrimental effects of Na+ on the photosynthetic machinery. The observed transient increase and subsequent decrease of selected proteins may exhibit a counterbalancing effect of target proteins in this context. Intriguingly, several subunits of the CF1-CF0 complex are unequally affected, whereas others do not respond at all.

Using gene targeting by homologous recombination in Ceratodon purpureus , we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1 , which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene.

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana . In flu , the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of 1 O 2 . Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its γ subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to 1 O 2 .

Any cellular function is based upon the proper function of native proteins. However, protein function requires a specific 3D structure. Establishing the correct three-dimensional conformation is therefore a premise for the exclusive biochemical function of a given protein. But how can this structure be achieved and maintained throughout the entire lifetime of a protein? In 1972, Christian Anfinsen was awarded with the Nobel Prize for chemistry for showing that the primary amino acid sequence contains all the information required for the native structure and that folding is an autonomous process, which does not require any additional energy. Based on in-vitro experiments with purified proteins, he suggested that the correct three-dimensional structure can form spontaneously in vivo once the newly synthesized protein leaves the ribosome. However, this view changed considerably over the last decade. It has become clear that a complex and sophisticated collection of proteins exists, which assists nascent protein folding and allows the functional state of proteins to be maintained under stress conditions in which they would normally unfold and aggregate. These proteins are collectively called “molecular chaperones”, because they prevent unwanted interactions between their immature clients, like their human counterparts in Victorian society of the 19th century, who as chaperones had to prevent bourgeoisie female adolescents from unwanted encounters with men. Experimental data indicate that several members of this group of proteins form heterooligomeric complexes, sometimes termed “chaperone machines”, interacting with each other to generate a network for maturation, assembly and intracellular targeting of proteins. In this review, the essential information on the structure and function of chaperone and chaperone complexes are summarized and the impact on the cellular stress response in plants will be discussed.

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures.

The mechanism of nuclear export of RNAs in yeast and animal cells is rapidly being uncovered, but RNA export in plants has received little attention. We introduced capped and uncapped fluorescent mRNAs into tobacco ( Nicotiana plumbaginifolia ) protoplasts and studied their cellular localization. Following insertion, capped transcripts were found in the cytoplasm, while uncapped messengers transiently appeared in the nucleus in about one-quarter to one-third of the cells. These mRNAs were trapped by the nuclear export-inhibiting drug leptomycin B, pointing to an export mechanism in plants similar to Rev-NES-mediated RNP export in other organisms.