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Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

Lipid peroxidation in leaves and roots of A. thaliana wild-type and spl7 knockout plants. Nineteen-days-old plants were exposed for 24 h to 2 μM CuSO4, 5 μM CdSO4 or grown under control conditions. Data are mean ± S.E. of 4 biological replicates. Significant treatment differences (P

Forward and reverse primers used to determine gene expression levels via quantitative real-time PCR. E-E-jn, exon-exon junction; E-I-b, exon-intron boundary; YLS, yellow-leaf-specific; UBQ10, ubiquitin; ACT, actin ; EF, elongation factor; APS, ATP sulfurylase; SULTR, sulfate transporter; LAC, laccase; CSD, Cu/Zn superoxide dismutase; FSD, Fe superoxide dismutase. *Pri-miRNA primer concentrations were increased to 900 nM. (DOCX 18 kb)

Verification of the T-DNA insertion in the spl7 mutant. Primers were designed using T-DNA primer design. (DOCX 32 kb)

Quantitative real-time PCR parameters according to the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines derived from Bustin et al. [51]. (DOCX 16 kb)

Plant survival under abiotic stress conditions requires morphological and physiological adaptations. Adverse soil conditions directly affect root development, although the underlying mechanisms remain largely to be discovered. Plant hormones regulate normal root growth and mediate root morphological responses to abiotic stress. Hormone synthesis, signal transduction, perception and cross-talk create a complex network in which metal stress can interfere, resulting in root growth alterations. We focus on Arabidopsis thaliana, for which gene networks in root development have been intensively studied, and supply essential terminology of anatomy and growth of roots. Knowledge of gene networks, mechanisms and interactions related to the role of plant hormones is reviewed. Most knowledge has been generated for auxin, the best-studied hormone with a pronounced primary role in root development. Furthermore, cytokinins, gibberellins, abscisic acid, ethylene, jasmonic acid, strigolactones, brassinosteroids and salicylic acid are discussed. Interactions between hormones that are of potential importance for root growth are described. This creates a framework that can be used for investigating the impact of abiotic stress factors on molecular mechanisms related to plant hormones, with the limited knowledge of the effects of the metals cadmium, copper and zinc on plant hormones and root development included as case example.

A general status of oxidative stress in plants caused by exposure to elevated metal concentrations in the environment coincides with a constraint on mitochondrial electron transport, which enhances ROS accumulation at the mitochondrial level. As mitochondria are suggested to be involved in redox signaling under environmental stress conditions, mitochondrial ROS can initiate a signaling cascade mediating the overall stress response, i.e., damage versus adaptation. This review highlights our current understanding of metal-induced responses in plants, with focus on the production and detoxification of mitochondrial ROS. In addition, the potential involvement of retrograde signaling in these processes will be discussed. The authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by the Research Foundation—Flanders (FWO) by a PhD grant for Els Keunen and the project G.0807.09. Support was also obtained from the Methusalem project 08G03VGRJ.

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. The authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by Hasselt University through BOF (Bijzonder Onderzoeksfonds) project BOF08G01 and the Methusalem project 08G03VGRJ.

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; gamma-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. The authors apologize to any researcher whose work is not cited here due to limitations of space and scope. This work was supported by the Agency for Innovation by Science and Technology with a PhD grant for Marijke Jozefczak. Additional financial support was obtained from the Research Foundation-Flanders (FWO) project G.0807.09 and from the Methusalem project 08G03VGRJ.

International audience

International audience