In intact plants, Cd-induced Fe deficiency is thought to play a role in the toxic effects of Cd on photosynthesis. To investigate the contribution of the Cd-induced Fe deficiency to Cd stress symptoms we studied the composition and organization changes of thylakoid pigment-protein complexes by twodimensional Blue Native-SDS gel electrophoresis and mass spectrometry, in parallel to functional changes, using Beta vulgaris plants grown in hydroponics. Plants were treated by withdrawing of Fe or with 10 µM CdCl2 for 10 days. Both metal stresses caused a marked decline in leaf chlorophyll concentration and chloroplast Fe content, as well as a loss in photosystem I (PSI) and light harvesting complex II (LHCII) particles. Furthermore, organizational changes of the photosynthetic apparatus were found, including a decrease in the ratio of the PSII mega-/supercomplexes and an increase in the monomeric form of the LHCII antennae, with the extent of these changes being similar under both stresses. This supports that Fe deficiency responses have a major role in the responses of plants under Cd stress. In the Fe-deficient thylakoids, an increase in the ratio of PSI supercomplexes and degrading PSII particles was more pronounced, together with a higher zeaxanthin content. Under Cd stress, a stronger inhibition of PSII activity and enhancement of thermal dissipation of the inactive PSII complexes were observed. The differences detected under the two metal stresses lead to the conclusion that both local Fe deficiency in chloroplasts and other direct or indirect inhibitory effects of Cd are behind the response mechanisms of plants grown under Cd stress. Our results appreciably contribute to the sparse structural information on thylakoid complexes affected by Cd toxicity and Fe deficiency.
EÖTVÖS LORÁND UNIVERSITY
INSTITUTE OF BIOLOGY
DEPARTMENTAll previously published work cited in the manuscript has been fully acknowledged.I am looking forward to hearing from you at your earliest convenience.With best regards, Yours faithfully,
Brigitta BasaHighlights:-Cd-induced chloroplast Fe deficiency is a prime trigger for thylakoid acclimation to Cd excess.-Both Cd stress and Fe deficiency induce PSII supercomplex and LHCII disassembly.-Thermal dissipation by inactive PSII complexes is markedly induced by Cd stress.-The amounts of PSI supercomplexes and zeaxanthin rise with Fe deficiency.-Cyclic electron flow and zeaxanthin are likely to be energy quenchers under low Fe.
Real-time RT–PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin β-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.
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