Robert Luciński

Lhca5 interaction with plant photosystem I.

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light‐harvesting protein of photosystem I (Lhca)‐type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross‐linking studies on PSI‐LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light‐harvesting protein with LHCI in the Lhca2/Lhca3‐site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding‐competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.

The thylakoid protease Deg2 is involved in stress-related degradation of the photosystem II light-harvesting protein Lhcb6 in Arabidopsis thaliana

• The thylakoid protease Deg2 is a serine-type protease peripherally attached to the stromal side of the thylakoid membrane. Given the lack of knowledge concerning its function, two T-DNA insertion lines devoid of Deg2 were prepared to study the functional importance of this protease in Arabidopsis thaliana. • The phenotypic appearance of deg2 mutants was studied using a combination of stereo and transmission electron microscopy, and short-stress-mediated degradation of apoproteins of minor light-harvesting antennae of photosystem II (PSII) was analysed by immunoblotting in the mutants in comparison with wild-type plants. • Deg2 repression produced a phenotype in which reduced leaf area and modified chloroplast ultrastructure of older leaves were the most prominent features. In contrast to the wild type, the chloroplasts of second-whorl leaves of 4-wk-old deg2 mutants did not display features typical of the early senescence phase, such as undulation of the chloroplast envelope and thylakoids. The ability to degrade the photosystem II light-harvesting protein Lhcb6 apoprotein in response to brief high-salt, wounding, high-temperature and high-irradiance stress was demonstrated to be impaired in deg2 mutants. • Our results suggest that Deg2 is required for normal plant development, including the chloroplast life cycle, and has an important function in the degradation of Lhcb6 in response to short-duration stresses.

Aerobic and anaerobic nitrate and nitrite reduction in free-living cells of Bradyrhizobium sp. (Lupinus)

Induction, energy gain, effect on growth, and interaction of nitrate and nitrite reduction of Bradyrhizobium sp. (Lupinus) USDA 3045 were characterized. Both nitrate and nitrite were reduced in air, although nitrite reduction was insensitive to ammonium inhibition. Anaerobic reduction of both ions was shown to be linked with energy conservation. A dissimilatory ammonification process was detected, which has not been reported in rhizobia so far. Nevertheless, anaerobic conversion of nitrate to ammonium was lower than 40%, which suggests the presence of an additional, nitrite reductase of denitrifying type. Nitrite toxicity caused a non-linear relationship between biomass produced and >2 mM concentrations of each N oxyanion consumed. At > or =5 mM initial concentrations of nitrate, a stoichiometric nitrite accumulation occurred and nitrite remained in the medium. This suggests an inhibition of nitrite reductase activity by nitrate, presumably due to competition with nitrate reductase for electron donors. Lowering of growth temperature almost completely diminished nitrite accumulation and enabled consumption as high as 10 mM nitrate, which confirms such a conclusion.

Involvement of Deg5 protease in wounding-related disposal of PsbF apoprotein.

Deg5 is a serine-type protease peripherally attached to luminal side of thylakoid membrane. Given the lack of knowledge concerning its function homozygous T-DNA insertion line depleted in Deg5 was prepared to study the functional importance of this protease in Arabidopsis thaliana. deg5 mutants displayed a pleiotropic phenotype with regard to fourth whorl leaves of four-weeks old plants. The alterations involved an increased leaf area, reduced leaf thickness, reduced cross-sectional area of palisade mesophyll cells as well as changed chloroplast ultrastructure including lack of signs of entering the senescence phase (e.g. presence of much smaller plastoglobules) and the accumulation of large starch grains at the end of the dark period. It was shown that whereas PsbA, C and F apoproteins of photosystem II reaction center undergo an extensive disappearance in response to a set of brief stresses deg5 mutant was fully resistant to the disappearance of PsbF apoprotein which follows an exposition of leaves to wounding. Our results demonstrate that Deg5 is of seminal importance for normal plant development and degradation of PsbF which occurs following brief wounding.

Functional similarities of nitrate reductase from yellow lupine bacteroids to bacterial denitrification systems

Summary The nitrate-reducing activity of root nodules of field-grown yellow lupine plants ( Lupinus luteus cv. Ventus) was found to be about 25 and 100 times higher than that of roots and leaves, respectively. At least 97 percnt; of nodule nitrate reductase (NR) activity proved to be of bacteroidal origin. Data from the present work indicates that nitrate reductase (EC 1. 7. 99. 4) of symbiotic Bradyrhizobium sp. ( Lupinus ) cells possess some functional similarities to bacterial denitrification systems. The enzyme occurs in association with membranes, and electron transport from NADH to nitrate is dependent on the mediation of membrane components. Another feature in common with bacterial nitrate dissimilation is inhibition of bacteroidal nitrate reductase activity under aerobic conditions. Membrane permeabilization significantly alleviated this inhibitory effect. It was found that enzyme activity is not nitrate-dependent and peaks at pH 5.5-6.5. Rotenone inhibition indicated that NADH-quinol oxidoreductase, not succinate dehydrogenase, is the major participant in a very efficient succinate-dependent nitrate reductase activity. Pyruvate and malate were also highly effective in nitrate reduction. By contrast, oxaloacetate and 2-oxoglutarate were not efficient, even in combination with pyruvate, which suggests that this effect could not be caused by acetyl-SCoA limitation. These results suggest that a large part of the TCA cycle may not be necessary for nitrate reduction in yellow lupine bacteroids. The possible alternative pathways, which could provide electrons from pyruvate and malate to nitrate reductase, are discussed.

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1-3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1-3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1-3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.

Dissimilatory Nitrate Reductase from Bradyrhizobium sp. (Lupinus): Subcellular Location, Catalytic Properties, and Characterization of the Active Enzyme Forms

Subcellular location, chlorate specificity, and sensitivity to micromolar concentrations of azide suggest that most of the anaerobically induced nitrate reductase (NR) activity in Bradyrhizobium sp. (Lupinus) could be ascribed to the membrane type of bacterial dissimilatory NRs. Two active complexes of the enzyme, NRI of 140 kDa and NRII of 190 kDa, were detected in membranes of the nitrate-respiring USDA strain 3045. Both enzyme forms were purified to homogeneity. Obtained specific antibodies showed that these native species were immunologically closely related and composed of largely similar 126-kDa, 65-kDa, and 25-kDa subunits. The finding that NRI and NRII share common epitopes suggests that they may not be different species, but rather two forms of the same enzyme.

The irradiance dependent transcriptional regulation of AtCLPB3 expression.

Transcript abundance analysis was applied to determine whether expression of genes coding for 50 principal constituents of chloroplast and mitochondria proteolytic machinery, i.e. isoforms of proteases and regulatory subunits of Clp and FtsH families as well as Deg group of chymotrypsin family are differentially expressed in response to acclimation to elevated irradiance. Of 50 genes analysed only a single one coding for ClpB3 regulatory subunit was found to be up-regulated and gene coding for Deg2 to be down-regulated significantly during acclimation to excessive irradiance conditions. Hierarchical clustering of transcript abundance data revealed that CLPB3 co-expressed tightly with genes coding for PAP1, GBF6 and bHLH family member transcription factors during the acclimation. It was found that CLPB3 contains cis-regulatory elements able to bind all three transcription factors. By performing analyses of publicly available transcriptomic data sets from a range of long-term abiotic stresses we suggest that PAP1 may mediate condition-dependent transcriptional response of CLPB3, induced by a group of long-term abiotic stresses.

A reversible decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase carboxylation activity caused by the aggregation of the enzyme's large subunit is triggered in response to the exposure of moderate irradiance-grown plants to low irradiance.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is highly regulated in response to fluctuations in the environment, including changes in irradiance. However, no complex data are available on Rubisco regulatory mechanisms triggered in plants which are submitted to moderate-low irradiance shift. Therefore, we investigated in a comprehensive way the changes at the level of amount of Rubisco protein, its structural organization and carboxylase activity of the holoenzyme as triggered by exposure of moderate irradiance-grown Arabidopsis thaliana plants to low irradiance conditions. An exposure of moderate irradiance-grown plants to low irradiance for a single photoperiod caused the exclusion of a certain pool of Rubisco under altered conditions owing to oxidative modifications resulting in the formation of protein aggregates involving Rubisco large subunit (LS). As a result, both initial and total Rubisco carboxylase activities were reduced, whereas Rubisco activation state remained largely unchanged. The results of the determination of reactive oxygen species indicated that a moderate/low irradiance transition had stimulated (1) O2 accumulation and we strongly suggest that Rubisco oxidative modifications leading to formation of aggregates encompassing Rubisco-LS were triggered by (1) O2 . When moderate irradiance regime was resumed, the majority of Rubisco-LS containing aggregates tended to be resolubilized, and this allowed Rubisco carboxylation activities to be largely recovered, without changes in the activation state of the enzyme. In the longer term, these results allow us to better understand a complexity of Rubisco regulatory mechanisms activated in response to abiotic stresses and during recovery from the stresses.

Arabidopsis thaliana intramembrane proteases

Proteolysis is considered as a crucial factor determining the proper development of the plant and its efficient functioning in variable environmental conditions. The role of proteases in protein quality control and protein turnover processes is well documented. The results of studies performed in recent years reveal; however, that proteolytic enzymes also participate in signal transduction pathways by releasing membrane-anchored transcription factors in the process known as regulated intramembrane proteolysis (RIP). The first described intramembrane protease was identified in human cells in 1997. In turn, the first plant intramembrane protease was identified in 2005, in Arabidopsis thaliana. To date, most studies concerning the RIP process in plants have been performed on this model plant. The knowledge concerning the potential physiological role of RIP is very limited. However, continuously accumulating information concerning this issue indicates that RIP, like the other proteolytic mechanisms, has a significant effect on plant ontogenesis, acclimatization and fertility. The aim of this article is to gather and systemize the present knowledge concerning the intramembrane proteases in A. thaliana.