Arabidopsis thaliana egy2 mutants display altered expression level of genes encoding crucial photosystem II proteins.

EGY2 is a zinc-containing, intramembrane protease located in the thylakoid membrane. It is considered to be involved in the regulated intramembrane proteolysis - a mechanism leading to activation of membrane-anchored transcription factors through proteolytic cleavage, which causes them to be released from the membrane. The physiological functions of EGY2 in chloroplasts remains poorly understood. To answer the question of what the significance is of EGY2 in chloroplast functioning, two T-DNA insertion lines devoid of EGY2 protein were obtained and the mutant phenotype and photosystem II parameters were analyzed. Chlorophyll fluorescence measurements revealed that the lack of EGY2 protease caused changes in non-photochemical quenching (NPQ) and minimum fluorescence yield (F0) as well as a higher sensitivity of photosystem II (PSII) to photoinhibition. Further immunoblot analysis revealed significant changes in the accumulation levels of the three chloroplast-encoded PSII core apoproteins: PsbA (D1) and PsbD (D2) forming the PSII reaction center and PsbC - a protein component of CP43, a part of the inner PSII antenna. The accumulation levels of nuclear-encoded proteins,Lhcb1-3, components of the major light-harvesting complex II (LHCII) as well as proteins forming minor peripheral antennae complexes, namely Lhcb4 (CP29), Lhcb5 (CP26), and Lhcb6 (CP24) remain, however, unchanged. The lack of EGY2 led to a significant increase in the level of PsbA (D1) with a simultaneous decrease in the accumulation levels of PsbC (CP43) and PsbD (D2). To test the hypothesis that the observed changes in the abundance of chloroplast-encoded proteins are a consequence of changes in gene expression levels, real-time PCR was performed. The results obtained show that egy2 mutants display an increased expression of PSBA and a reduction in the PSBD and PSBC genes. Simultaneously pTAC10, pTAC16 and FLN1 proteins were found to accumulate in thylakoid membranes of analyzed mutant lines. These proteins interact with the core complex of plastid-encoded RNA polymerase and may be involved in the regulation of chloroplast gene expression.

Physio-Genetic Dissection of Dark-Induced Leaf Senescence and Timing Its Reversal in Barley.

Barley crop model was analyzed for early and late events during the dark-induced leaf senescence (DILS) as well as for deciphering critical time limit for reversal of the senescence process. Chlorophyll fluorescence vitality index Rfd was determined as the earliest parameter that correlated well with the cessation of photosynthesis prior to microautophagy symptoms, initiation of DNA degradation, and severalfold increase in the endonuclease BNUC1. DILS was found characterized by up-regulation of processes that enable recycling of degraded macromolecules and metabolites, including increased NH4 + remobilization, gluconeogenesis, glycolysis, and partial up-regulation of glyoxylate and tricarboxylate acid cycles. The most evident differences in gene medleys between DILS and developmental senescence included hormone-activated signaling pathways, lipid catabolic processes, carbohydrate metabolic processes, low-affinity ammonia remobilization, and RNA methylation. The mega-autophagy symptoms were apparent much later, specifically on day 10 of DILS, when disruption of organelles-nucleus and mitochondria -became evident. Also, during this latter-stage programmed cell death processes, namely, shrinking of the protoplast, tonoplast interruption, and vacuole breakdown, chromatin condensation, more DNA fragmentation, and disintegration of the cell membrane were prominent. Reversal of DILS by re-exposure of the plants from dark to light was possible until but not later than day 7 of dark exposure and was accompanied by regained photosynthesis, increase in chlorophyll, and reversal of Rfd, despite activation of macro-autophagy-related genes.

A Role for Barley Calcium-Dependent Protein Kinase CPK2a in the Response to Drought.

Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L.), one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index (NBI), an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

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.

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.

Expression of modified Cry1Ac gene of Bacillus thuringiensis in transgenic tobacco plants.

Several mutations were introduced into the Cry1Ac toxin gene, resulting in four variants with altered sequences that were responsible for low expression of the toxin in transgenic plants. These variants were as follows: V1, with modified three A/T-rich regions, including the first signal of transcription termination; V2, with modified five putative polyadenylation signals (polyadenylation signals PAS) and the second signal of transcription termination; V3, with four initial AUUUA motifs; V4, with modification of six PASs, four AUUUA motifs, as well as the first and the second signals of transcription termination. The modified variants and the initial WT gene were cloned into the binary vector pBI121 and introduced into tobacco plants (Nicotiana tabacum) by Agrobacterium tumefaciens-mediated transformation. The presence of transgenes in the tobacco plants was confirmed by the polymerase chain reaction (PCR) method. The expression of particular variants of the Cry1Ac gene in tobacco was assayed using Western blotting with antibodies against the domain II of the Cry1Ac toxin. The average expression of WT was estimated to be 0.0025% of soluble proteins, and the expression levels of modified variants were 0.004%, 0.0098%, 0.0125%, and 0.0043% for V1, V2, V3, and V4, respectively. In this article we described the construction of a variant of the Cry1Ac gene (V3) with 12 point mutations leading to an average level of expression in transgenic plants five times higher than that observed in the case of the WT gene. Our results have shown for the first time that the modification of AUUUA sequences has a significant effect on the expression of the Cry1Ac gene in transgenic plants.