Qualitative and quantitative evaluation of thylakoid complexes separated by Blue Native PAGE

Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging

Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis

The desiccation tolerance of plants relies on defense mechanisms that enable the protection of macromolecules, biological structures, and metabolism. Although the defense of leaf tissues exposed to solar irradiation is challenging, mechanisms that protect the viability of the roots, yet largely unexplored, are equally important for survival. Although the photosynthetic apparatus in leaves contributes to the generation of oxidative stress under drought stress, we hypothesized that oxidative stress and thus antioxidative defense is also predominant in the roots. Thus, we aimed for a comparative analysis of the protective mechanisms in leaves and roots during the desiccation of Haberlea rhodopensis. Consequently, a high content of non-enzymatic antioxidants and high activity of antioxidant enzymes together with the activation of specific isoenzymes were found in both leaves and roots during the final stages of desiccation of H. rhodopensis. Among others, catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves, whereas, unlike that in the leaves, superoxide dismutase activity was enhanced under severe but not under medium desiccation in roots. Nitric oxide accumulation in the root tips was found to be sensitive to water restriction but suppressed under severe desiccation. In addition to the antioxidative defense, desiccation induced an enhanced abundance of dehydrins, ELIPs, and sHSP 17.7 in leaves, but this was significantly better in roots. In contrast to leaf cells, starch remained in the cells of the central cylinder of desiccated roots. Taken together, protective compounds and antioxidative defense mechanisms are equally important in protecting the roots to survive desiccation. Since drought-induced damage to the root system fundamentally affects the survival of plants, a better understanding of root desiccation tolerance mechanisms is essential to compensate for the challenges of prolonged dry periods

Iron uptake machinery of chloroplasts tends to utilise stoichiometric ferric-citrate complexes in Brassica napus

In plant shoots, the majority of iron is found in the chloroplasts, incorporated into the photosynthetic, sulphur assimilatory and Fe-S cluster biogenesis apparatuses. Although some members of their Fe transport machinery related to both reduction-based and nicotianamine-complex uptake systems have already been identified, the in vivo substrate preference of the system remained unknown. To clarify the mechanism of action and the substrate preference of the uptake system, intact chloroplasts of oilseed rape (Brassica napus) were subjected to Fe uptake assays using natural and artificial Fe complexes and chelates: Fe(III)-citrate 1:1.1 and 1:10, Fe(III)-malate 1:1.1, Fe(II)- and Fe(III)-nicotianamine 1:1.2, Fe(III)-EDTA 1:1 and Fe(III)-o,o’EDDHA 1:1. Iron complexes were typified by the chemical microenvironment of Fe in the compounds by Mössbauer spectroscopy. Iron uptake by chloroplasts was assessed by determining chloroplast iron content spectrophotometrically. Putative homologue genes of major, Fe uptake related, chloroplast envelope membrane proteins were identified in Brassica napus using the Brassica Database and NCBI. The expression of BnFro7 (Bra037953), BnMar1 (Bra020559), BnNico (Bra037287), BnPic1 (Bra036409), and BnYsl4 (XM_009141995.2) was studied using ß-tubulin (XM_009125342.1) and 18S rRNA (KT225373) as for reference genes in leaves subjected to chloroplast Fe uptake assays. Chloroplast inner envelope ferric chelate reductase activity of the isolated chloroplasts were also monitored using Fe(III)-EDTA. Chloroplasts preferred stoichiometric Fe(III)-citrate compared to Fe(III)-citrate 1:10 and Fe(III)-malate complexes. Moreover, uptake from Fe(III)-NA and Fe(II)-NA but also from Fe(III)-EDTA and Fe(III)-o,o’EDDHA sources were negligible (with significantly higher KM) compared to Fe(III)-citrate complexes. For these latter complexes, the light-inducible component was also missing. Regarding the components of the chloroplast Fe uptake system, genes of the reduction-based Fe uptake system showed high expression only. Nevertheless, the Fe-nicotianamine transport related chloroplast transporter BnYsl4 was mainly expressed in generative tissues. In conclusion, chloroplasts in leaves can only effectively utilize stoichiometric Fe(III)-citrate complexes in their Fe uptake. This work was supported by the grant financed by the National Research, Development and Innovation Office, Hungary (NKFIH K-124159). Á.Solti was also supported by the Bolyai János Research Scholarship of the Hungarian Academy of Sciences (BO/00207/15/4)

A vasellátás szabályozó hatása a kloroplasztisz vas-kelát-oxidoreduktáz génexpressziójára

A kloroplasztisz vas-kelát-oxidoreduxtáz expressziós mintázatának kísérletes vizsgálata, változó vasellátású olajrepce növényekben

Iron uptake of etioplasts is independent from photosynthesis but applies the reduction based strategy

Iron (Fe) is one of the most important cofactors in the photosynthetic apparatus, and its uptake by chloroplasts have been also associated with the operation of the photosynthetic electron transport chain during reduction-based plastidial Fe uptake. Therefore, plastidial Fe uptake was considered not to be operational in the absence of the photosynthetic activity. Nevertheless, Fe is also required for enzymatic functions unrelated to photosynthesis, highlighting the importance of Fe acquisition by nonphotosynthetic plastids. Yet, it remains unclear how these plastids acquire Fe in the absence of photosynthetic function. Furthermore, plastids of etiolated tissues should already possess the ability to acquire Fe, since the biosynthesis of thylakoid membrane complexes requires a massive amount of readily available Fe. Thus, we aimed to investigate whether the reduction based plastidial Fe uptake solely relies on the functioning photosynthetic apparatus. We used Savoy cabbage plant as a model, which develops natural etiolation in the inner leaves of the heads due to the shading of the outer leaf layers. Foliar and plastidial Fe content of Savoy cabbage leaves decreased towards the inner leaf layers. The leaves of the innermost leaf layers proved to be etiolated, containing etioplasts which lacked the photosynthetic machinery and thus were photosynthetically inactive. However, we discovered that these etioplasts contained, and were able to take up Fe. Although, the relative transcript abundance of genes associated with plastidial Fe uptake and homeostasis decreased towards the inner leaf layers, both ferric chelate reductase FRO7 transcripts and activity was detected in the innermost leaf layer. Additionally, a significant NADP(H) pool and NAD(P)H dehydrogenase activity was detected in the etioplasts of the innermost leaf layer, indicating the presence of the reducing capacity that likely supports the reduction based Fe uptake of etioplasts. Based on these findings, the reduction based plastidial Fe acquisition should not be considered exclusively dependent on the photosynthetic functions

Iron uptake of etioplasts is independent from photosynthesis but applies the reduction-based strategy

Introduction: Iron (Fe) is one of themost important cofactors in the photosynthetic apparatus, and its uptake by chloroplasts has also been associated with the operation of the photosynthetic electron transport chain during reduction-based plastidial Fe uptake. Therefore, plastidial Fe uptake was considered not to be operational in the absence of the photosynthetic activity. Nevertheless, Fe is also required for enzymatic functions unrelated to photosynthesis, highlighting the importance of Fe acquisition by non-photosynthetic plastids. Yet, it remains unclear how these plastids acquire Fe in the absence of photosynthetic function. Furthermore, plastids of etiolated tissues should already possess the ability to acquire Fe, since the biosynthesis of thylakoid membrane complexes requires a massive amount of readily available Fe. Thus, we aimed to investigate whether the reduction-based plastidial Fe uptake solely relies on the functioning photosynthetic apparatus. Methods: In our combined structure, iron content and transcript amount analysis studies, we used Savoy cabbage plant as a model, which develops natural etiolation in the inner leaves of the heads due to the shading of the outer leaf layers. Results: Foliar and plastidial Fe content of Savoy cabbage leaves decreased towards the inner leaf layers. The leaves of the innermost leaf layers proved to be etiolated, containing etioplasts that lacked the photosynthetic machinery and thus were photosynthetically inactive. However, we discovered that these etioplasts contained, and were able to take up, Fe. Although the relative transcript abundance of genes associated with plastidial Fe uptake and homeostasis decreased towards the inner leaf layers, both ferric chelate reductase FRO7 transcripts and activity were detected in the innermost leaf layer. Additionally, a significant NADP(H) pool and NAD(P)H dehydrogenase activity was detected in the etioplasts of the innermost leaf layer, indicating the presence of the reducing capacity that likely supports the reduction-based Fe uptake of etioplasts. Discussion: Based on these findings, the reduction-based plastidial Fe acquisition should not be considered exclusively dependent on the photosynthetic functions