Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression

Background: Anthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana. Results: Increased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production. Conclusions: Cadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants

Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana tabacum

Selection and use of appropriate reference genes as internal controls in real-time reverse transcription PCR (RT-PCR) assays is highly important for accurate quantification of gene expression levels. Since some photosynthetic genes are encoded in the nuclear genome and others in the chloroplast genome, we evaluated both nuclear- and plastid-encoded candidate reference genes. Six plastid-encoded candidate reference genes were derived from Arabidopsis microarray data and three plastid- and five nuclear-encoded reference genes were derived from literature. Cytokinins influence photosynthetic gene expression, so we evaluated the expression stability of the candidate reference genes in transgenic Nicotiana tabacum plants with elevated or diminished cytokinin content. We found that the most reliable strategy makes use of plastid-encoded genes for normalizing plastid photosynthetic genes and nuclear-encoded reference genes for normalizing nuclear photosynthetic genes. Compared to the use of nuclear reference genes only, this approach assimilates any effects on transcriptional activity of chloroplasts or number of chloroplast. The best expression stabilities in Nicotiana tabacum were observed for the plastid-encoded references genes Nt-RPS3, Nt-NDHI and Nt-IN1 and for the nuclear-encoded genes Nt-ACT9, Nt-αTUB and Nt-SSU. These genes may be suitable for normalization of photosynthetic genes under other experimental conditions in Nicotiana tabacum, and orthologues of these genes may be suitable candidates for normalizing photosynthetic gene expression in other species

Glutathione Is a Key Player in Metal-Induced Oxidative Stress Defenses

Since the industrial revolution, the production, and consequently the emission of metals, has increased exponentially, overwhelming the natural cycles of metals in many ecosystems. Metals display a diverse array of physico-chemical properties such as essential versus non-essential and redox-active versus non-redox-active. In general, all metals can lead to toxicity and oxidative stress when taken up in excessive amounts, imposing a serious threat to the environment and human health. In order to cope with different kinds of metals, plants possess defense strategies in which glutathione (GSH; γ-glu-cys-gly) plays a central role as chelating agent, antioxidant and signaling component. Therefore, this review highlights the role of GSH in: (1) metal homeostasis; (2) antioxidative defense; and (3) signal transduction under metal stress. The diverse functions of GSH originate from the sulfhydryl group in cysteine, enabling GSH to chelate metals and participate in redox cycling

Demographic, clinical and antibody characteristics of patients with digital ulcers in systemic sclerosis: data from the DUO Registry

OBJECTIVES: The Digital Ulcers Outcome (DUO) Registry was designed to describe the clinical and antibody characteristics, disease course and outcomes of patients with digital ulcers associated with systemic sclerosis (SSc). METHODS: The DUO Registry is a European, prospective, multicentre, observational, registry of SSc patients with ongoing digital ulcer disease, irrespective of treatment regimen. Data collected included demographics, SSc duration, SSc subset, internal organ manifestations, autoantibodies, previous and ongoing interventions and complications related to digital ulcers. RESULTS: Up to 19 November 2010 a total of 2439 patients had enrolled into the registry. Most were classified as either limited cutaneous SSc (lcSSc; 52.2%) or diffuse cutaneous SSc (dcSSc; 36.9%). Digital ulcers developed earlier in patients with dcSSc compared with lcSSc. Almost all patients (95.7%) tested positive for antinuclear antibodies, 45.2% for anti-scleroderma-70 and 43.6% for anticentromere antibodies (ACA). The first digital ulcer in the anti-scleroderma-70-positive patient cohort occurred approximately 5 years earlier than the ACA-positive patient group. CONCLUSIONS: This study provides data from a large cohort of SSc patients with a history of digital ulcers. The early occurrence and high frequency of digital ulcer complications are especially seen in patients with dcSSc and/or anti-scleroderma-70 antibodies

Gene expression studies in different genotypes of an ectomycorrhizal fungus require a 1 high number of reliable reference genes. 2 3

Abstract 13 Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the 14 expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-15 PCR measurements strongly depends on the normalization with appropriate endogenous 16 reference genes. In this study a set of candidate reference genes for the use in gene expression 17 studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or 18 cadmium was identified, evaluated and validated. Seven candidate genes were selected from 19 cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to 20 evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in 21 different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has 22 its own most stably expressed set of reference genes, regardless of the metal treatments, i

The Influence of Metal Stress on the Availability and Redox State of Ascorbate, and Possible Interference with Its Cellular Functions

Worldwide, metals have been distributed to excessive levels in the environment due to industrial and agricultural activities. Plants growing on soils contaminated with excess levels of metals experience a disturbance of the cellular redox balance, which leads to an augmentation of reactive oxygen species (ROS). Even though the increased ROS levels can cause cellular damage, controlled levels play an important role in modulating signaling networks that control physiological processes and stress responses. Plants control ROS levels using their antioxidative defense system both under non-stress conditions, as well as under stress conditions such as exposure to excess metals. Ascorbate (AsA) is a well-known and important component of the plant’s antioxidative system. As primary antioxidant, it can reduce ROS directly and indirectly via ascorbate peroxidase in the ascorbate–glutathione cycle. Furthermore, AsA fulfills an essential role in physiological processes, some of which are disturbed by excess metals. In this review, known direct effects of excess metals on AsA biosynthesis and functioning will be discussed, as well as the possible interference of metals with the role of AsA in physiological and biochemical processes