Background: The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR.

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.

Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signalling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidising equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signalling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (O) and hydrogen peroxide (HO), initiate distinct signalling pathways when photosynthesis is perturbed. O, because of its potent reactivity means that it initiates but does not transduce signalling. In contrast, the lower reactivity of HO means that it can also be a mobile messenger in a spatially-defined signalling pathway. How plants translate a HO message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells.

No abstract

No abstract

No abstract

Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastid–nuclear complexes, which provide close physical contact between these organelles. Plastid-nuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H 2 O 2 to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid–nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid–nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state, the two modes of connection contribute to varying extents. We hypothesize that this association is dynamic and that there may be a link between plastid–nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves.To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly upregulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14.Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.