Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (Lupinus luteus L. 'Mister') were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H2O2 was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed

Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

A protocol is presented for low-cost and fast isolation of intact and pure chloroplasts from leaves of plants acclimated to low irradiance. The protocol is based on a differential centrifugation of cleared leaf homogenate and omits a centrifugation on Percoll gradient step. The intactness and purity of the chloroplasts isolated from leaves of low irradiance-acclimated plants by using this protocol (confirmed by phase contrast microscopy as well as enzymatic and immunological approaches) allows plausible studies on low irradiance-dependent Rubisco regulation

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana : Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.Peer reviewe

Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L.

During daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b(6)f complex, ATP synthase and several isoforms of ferredoxin-NADP(+) oxidoreductase (FNR), and ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b(6)f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b(6)f complex, FNR and redox-inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone (PQ) and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd and cytochrome b(h) of the cytochrome b(6)f complex, and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both NADH and NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration that may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b(6)f complex during cyclic electron transport in chloroplasts.Significance Statement Chromorespiration takes place via two pathways, one depends on FNR, ferredoxin, the cytochrome b6f complex, and the other depends on the NDH complex and is ferredoxin independent. We propose an electron transport via the cytochrome b6f complex that involves neither a Q-cycle nor a high potential electron transport chai

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.</p

Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (Lupinus luteus L. ‘Mister’) were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H2O2 was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed