Accumulation Of D1 Protein Research Articles

VAR2/AtFtsH2 and EVR2/BCM1/CBD1 synergistically regulate the accumulation of PSII reaction centre D1 protein during de-etiolation in Arabidopsis.

Thylakoid FtsH complex participates in PSII repair cycle during high light-induced photoinhibition. The Arabidopsis yellow variegated2 (var2) mutants are defective in the VAR2/AtFtsH2 subunit of thylakoid FtsH complex. Taking advantage of the var2 leaf variegation phenotype, dissections of genetic enhancer loci have yielded novel paradigms in understanding functions of thylakoid FtsH complex. Here, we report the isolation of a new var2 enhancer, enhancer of variegation2-1 (evr2-1). We confirmed that EVR2 encodes a chloroplast protein that was known as BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1), or CHLOROPHYLL BIOSYNTHETIC DEFECT 1 (CBD1). We showed that EVR2/BCM1/CBD1 was involved in the oligomerization of photosystem I complexes. Genetic assays indicated that general defects in chlorophyll biosynthesis and the accumulation of photosynthetic complexes do not necessarily enhance var2 leaf variegation. In addition, we found that VAR2/AtFtsH2 is required for the accumulation of photosynthetic proteins during de-etiolation. Moreover, we identified PSII core proteins D1 and PsbC as potential EVR2-associated proteins using Co-IP/MS. Furthermore, the accumulation of D1 protein was greatly compromised in the var2-5 evr2-1 double mutant during de-etiolation. Together, our findings reveal a functional link between VAR2/AtFtsH2 and EVR2/BCM1/CBD1 in regulating chloroplast development and the accumulation of PSII reaction centre D1 protein during de-etiolation.

Activity of the photosynthetic apparatus in dehydrated leaves of a perennial shrub Rosa rubiginosa L. with different levels of drought memory

The aim of the study was to analyze the activity of the photosynthetic apparatus during induced dehydration of leaves harvested from perennial shrubs of Rosa rubiginosa growing at wet and dry sites and differing in the level of drought memory. The markers of drought memory included accumulation of D1 (PsbA) protein, chaperone Cpn60, Rubisco activase (RA), and Rubisco larger subunit (RbcL). The study investigated the levels of soluble carbohydrates and phenolic compounds, of cell wall-bound phenolics, non-enzymatic antioxidants, and assimilation pigments.During induced dehydration, leaves from the dry site exhibited slower water loss and greater activity of the photosynthetic apparatus, particularly in terms of effectiveness of electron transport between PSII and PSI. They also effectively dissipated excessive excitation energy in the form of heat and far-red fluorescence. Higher level of D1 protein in the leaves collected from the dry site was accompanied by lower accumulation of Cpn60, RA and RbcL proteins and considerably higher content of soluble carbohydrates and carotenoids.Drought memory in R. rubiginosa may be associated with a low level of proteins responsible for photosynthetic fixation of CO2 and reduced content of chlorophyll a and b, and also with enhanced accumulation of D1 protein and soluble carbohydrates and carotenoids.

Antioxidative response and photosynthetic regulatory mechanisms in common fig leaves after short‐term chilling stress

AbstractCommon fig (Ficus carica L.) is widely cultivated Mediterranean species. Such warm‐climate species are adapted to elevated temperatures and are susceptible to chilling stress (0–12°C). However, occasional short chilling periods are common during growing season in temperate areas that can affect functionality of the plant. The aim of this work was to investigate influence of short‐term chilling stress on antioxidative response and PSII photochemistry in developing leaves of two common fig varieties: Zamorčica and Green matalon. Leaves were detached from the trees, acclimated at room temperature in dark for 16 hr and then exposed to low temperature (10°C) at low irradiation (50 μmol m−2 s−1) for 4 hr. Dark adapted leaves were considered as the control. The damage to the membranes was determined as lipid peroxidation (thiobarbituric acid reactive substances) while the antioxidative response was evaluated by determining activities of the selected antioxidative enzymes. Photosynthetic performance was analysed by measuring in vivo chlorophyll a fluorescence (ChlF) increase. Relative accumulation of the main photosynthetic proteins (D1 and Rubisco LSU) was determined, as well. Due to efficient antioxidative activity, neither of investigated variety showed damage of the membrane lipids. Both varieties revealed functional antennae and its good connectivity to their reaction centres shown as negative L and K bands as well as stable D1 protein accumulation suggesting functional electron transport through photosystem II (PSII) and efficient primary photochemistry. Blocked electron flow further than QA resulted in limitation of the Photosystem I (PSI) functionality in both varieties. Due to differential relative accumulation of D1 and Rubisco LSU proteins upon the chilling stress, Zamorčica revealed greater decrease of the main photosynthetic parameters derived from in vivo ChlF induction (PItotal, PIABS and φP0) in comparison to Green matalon. In addition, increased specific energy fluxes through PSII in Zamorčica suggested its higher susceptibility to photoinhibition caused by chilling stress in comparison to Green matalon.

PAP90, a novel rice protein plays a critical role in regulation of D1 protein stability of PSII

IntroductionPhotosystem II (PSII) protein complex plays an essential role in the entire photosynthesis process. Various known and unknown protein factors are involved in the dynamics of the PSII complex that need to be characterized in crop plants for enhancing photosynthesis efficiency and productivity. ObjectivesThe experiments were conducted to decipher the regulatory proteins involved in PSII dynamics of rice crop. MethodsA novel rice regulatory protein PAP90 (PSII auxiliary protein ~90 kDa) was characterized by generating a loss-of-function mutant pap90. The mutation was characterized at molecular level followed by various experiments to analyze the morphological, physiological and biochemical processes of mutant under control and abiotic stresses. ResultsThe pap90 mutant showed reduced photosynthesis due to D1 protein instability that subsequently causes inadequate accumulation of thylakoid membrane complexes, especially PSII and decreases PSII functional efficiency. Expression of OsFtsH family genes and proteins were induced in the mutant, which are known to play a key role in D1 protein degradation and turnover. The reduced D1 protein accumulation in the mutant increased the production of reactive oxygen species (ROS). The accumulation of ROS along with the increased activity of antioxidant enzymes and induced expression of stress-associated genes and proteins in pap90 mutant contributed to its water-limited stress tolerance ability. ConclusionWe propose that PAP90 is a key auxiliary protein that interacts with D1 protein and maintains its stability, thereby promoting subsequent assembly of the PSII and associated membrane complexes.

Antioxidant protection and PSII regulation mitigate photo-oxidative stress induced by drought followed by high light in cashew plants

We tested the hypothesis that effective antioxidant and photoprotective mechanisms are able to avoid photodamage induced by prolonged water deficit (WD) followed by high light (HL). We employed cashew plants (Anacardium occidentale L.), a semiarid adapted species, as a model plant. WD-plants exposed to HL did not show alterations in maximum quantum efficiency of photosystem II (Fv/Fm), cellular integrity, H2O2 and thiobarbituric acid reactive species (TBARS) contents, evidencing that they did not suffered photoinhibition and oxidative stress. These responses were associated with increases in ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities, glutathione (GSH) oxidation and ascorbate (ASC) synthesis. This effective oxidative protection occurred in parallel to strong decrease in photosystem II and I (PSII and PSI) activities, increase in heat dissipation (qE), which was related to enhancement in cyclic electron flux. These favorable photoprotective changes were associated with efficient water use in response to HL, all contributing to avoid excess energy in chloroplasts of drought-exposed leaves. These protective features were associated with a tight regulation in D1 protein accumulation during HL, contributing to avoid reactive oxygen species over-accumulation and a subsequent effective PSII recovery during darkness. Our results indicate that cashew plants are able to avoid photoinhibition, tolerating extreme conditions of drought combined with HL. The displayed mechanisms involve essentially integrated responses to balance energy input and output, avoiding oxidative stress.

Dunaliella tertiolecta (Chlorophyta) Avoids Cell Death Under Ultraviolet Radiation By Triggering Alternative Photoprotective Mechanisms.

The effect of different ultraviolet radiation (UVR) treatments combining PAR (P), UVA (A) and UVB (B) on the molecular physiology of Dunaliella tertiolecta was studied during 6 days to assess the response to chronic UVR exposure. UVR reduced cell growth but did not cause cell death, as shown by the absence of SYTOX Green labeling and cellular morphology. However, caspase-like enzymatic activities (CLs), (regarded as cell death proteases), were active even though the cells were not dying. Maximal quantum yield of fluorescence (Fv /Fm ) and photosynthetic electron transport rate (ETR) dropped. Decreased nonphotochemical quenching (NPQ) paralleled a drop in xanthophyll cycle de-epoxidation under UVB. Reactive oxygen species (ROS) and D1 protein accumulation were inversely correlated. PAB exhibited elevated ROS production at earlier times. Once ROS decayed, D1 protein recovered two-fold compared with P and PA at later stages. Therefore, PsbA gene was still transcribed, suggesting ROS involvement in D1 recovery by its direct effect on mRNA-translation. We add evidence of an UVB-induced positive effect on the cells when P is present, providing photoprotection and resilience, by means of D1 repair. This allowed cells to survive. The photoprotective mechanisms described here (which are counterintuitive in principle) conform to an important ecophysiological response regarding light stress acclimation.

Analysis of heterologous regulatory and coding regions in algal chloroplasts

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II (psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5' UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5' UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts.

Cyclin D1 expression and its possible regulation in chewing tobacco mediated oral squamous cell carcinoma progression

Objective Proto-oncogene cyclin D1 is a G1 phase specific cell cycle regulator and known for its role in various cancers. The aim of the study was to understand oral cancer progression by observing the mRNA and protein expression of cyclin D1. Methods Different oral tissue samples were selected as a model to study oral cancer progression. Those include healthy oral mucosa, premalignant lesions (Leukoplakia, Erythroplakia, Oral SubMucous Fibrosis) and oral cancer (OSCC) samples. Cyclin D1 mRNA and protein expression were detected by slot-blot and by immunohistochemical methods, respectively. Results Premalignant lesions (PML) showed average 3-fold increase in the mRNA expression than normal oral mucosa ( p = 0.001) whereas only 1.3-fold increase in mRNA has been observed in OSCC samples over the PML. On the other hand OSCC showed average 4-fold increase in mRNA expression than normal oral mucosa ( p < 0.001). Cyclin D1 protein accumulation has been observed in 31.3% (16/51) of the OSCC samples whereas the normal oral mucosa and the PML showed no immunoreactivity. Oral cancer samples showing positive cyclin D1 immunoreactivity has increased from 15.0% (3/20) well differentiated SCC to 31.2% (5/16) moderately differentiated SCC to 53.3% (8/15) poorly differentiated SCC, found statistically significant ( p = 0.05). Conclusion By observing the expression of cyclin D1 in different stages, we have noticed two major transitions that occur in normal oral mucosa that leads to oral cancer. The first transitional event transforms the normal oral mucosa to PML whereas the second transition drives the PML to OSCC. These findings give evidence that the first transition induces cyclin D1 mRNA with no detectable cyclin D1 protein. The induction of mRNA is maintained with increased cyclin D1 protein accumulation in the second transition.

Inhibition of peptide deformylase in Nicotiana tabacum leads to decreased D1 protein accumulation, ultimately resulting in a reduction of photosystem II complexes

Eukaryotic homologs of bacterial peptide deformylases were recently found in several vascular plants and may be essential in chloroplast protein processing. Treating tobacco seedlings with the peptide deformylase inhibitor actinonin resulted in leaf chlorosis and reduced growth and development, indicative of a systemic movement of the inhibitor. Photosystem II (PSII) activity was reduced, manifested as a significant decrease in the maximum quantum efficiency of photosystem II. Accumulation and assembly of nascent D1 protein into PSII monomers was also reduced, eventually leading to PSII disassembly and leaf necrosis. Processing and assembly of D1 protein in tobacco was a major and potentially critical target of peptide deformylase inhibition. These results confirm that N-terminal deformylation is an essential step in the accumulation and assembly of PSII subunit polypeptides in the chloroplasts of vascular plants.

Biochemical and molecular studies on declining and decline-resistant spruce in the north-east of France.

In declining forests of the Vosges mountains (northeast of France), we previously observed that the yellowing of spruce (Picea abies L. cv. Karsten) needles was associated with impairment of the free radical scavenging capacity of the cells and coincided with chronic exposure to ozone. Chloroplasts of yellow needles were characterized by an abnormal accumulation of photosystem II (PSII) D1-protein in the thylakoids. Further experiments carried out on declining and decline-resistant individual spruce trees characterized in previous studies showed that needle yellowing was associated with impairment of the overall anti-oxidative defense in both the cytosol and the chloroplasts. Both enzymic (peroxidases) and non-enzymic (carotenoids) oxidant scavengers were shown to be affected in the declining spruce. PSII D1-protein accumulation seemed to result from a stabilization of the polypeptide, which led us to hypothesize that oxidative processes might interfere with the specific degradation of this protein in declining spruce, with destructive consequences for the photosystems.

Cyclin-dependent Kinase 2 Nucleocytoplasmic Translocation Is Regulated by Extracellular Regulated Kinase

Activation of cyclin-dependent kinase 2 (CDK2)-cyclin E in the late G(1) phase of the cell cycle is important for transit into S phase. In Chinese hamster embryonic fibroblasts (IIC9) phosphatidylinositol 3-kinase and ERK regulate alpha-thrombin-induced G(1) transit by their effects on cyclin D1 protein accumulation (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053). Here, we show that ERK also affects CDK2-cyclin E activation by regulating the subcellular localization of CDK2. Ectopic expression of cyclin E rescues the inhibition of alpha-thrombin-induced activation of CDK2-cyclin E and transit into S phase brought about by treatment of IIC9 cells with LY29004, a selective inhibitor of mitogen stimulation of phosphatidylinositol 3-kinase activity. However, cyclin E expression is ineffectual in rescuing these effects when ERK activation is blocked by treatment with PD98059, a selective inhibitor of MEK activation of ERK. Investigation into the mechanistic reasons for this difference found the following. 1) Although treatment with LY29004 inhibits alpha-thrombin-stimulated nuclear localization, ectopic expression of cyclin E rescues CDK2 translocation. 2) In contrast to treatment with LY29004, ectopic expression of cyclin E fails to restore alpha-thrombin-stimulated nuclear CDK2 translocation in IIC9 cells treated with PD98059. 3) CDK2-cyclin E complexes are not affected by treatment with either inhibitor. These data indicate that, in addition to its effects on cyclin D1 expression, ERK activity is an important controller of the translocation of CDK2 into the nucleus where it is activated.

Pulse-Chase Experiments with [35S]Methionine Show D1 Reaction II Protein Turnover in Variously Herbicide Tolerant Species

D1 protein of Photosystem II, the photosynthetic herbicide target, is rapidly turned over compared to the other chloroplastic proteins. However, the importance of D1 turnover for herbicide action is unclear. In short- and long-term experiments we treated various plant species with atrazine herbicide in combination with chloramphenicol (CAP), an inhibitor of D1 protein synthesis. A synergistic effect which inhibited plant growth was seen primarily in moderately resistant plants treated with herbicide and CAP. D1 protein turnover was studied by [ 35 S]methionine incorporation in seedlings treated in the light with these inhibitors. The effect of the two inhibitors on D1 content varied; CAP inhibited D1 synthesis and retarded its degradation, resulting in a depletion of D1 from the thylakoid membranes of about 50% in all tested species. The herbicide altered both D1 synthesis and degradation but the extent of inhibition varied significantly among species and resulted in either a reduction or an accumulation of D1 protein. Simultaneous treatment with both CAP and herbicide resulted in a significant decrease in D1 content in moderately tolerant plants only. It is supposed that tolerance may be due to faster degradation or overproduction of the D1 herbicide binding protein.

Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA.

The plastid psbA mRNA is present in all tissues, while the encoded 32 kDa D1 protein of photosystem II accumulates tissue-specifically and in response to light. To study the regulation of D1 accumulation, a chimeric uidA gene encoding beta-glucuronidase (GUS) under control of the psbA 5'- and 3'-regulatory regions (224 and 393 bp, respectively), was integrated into the tobacco plastid genome. A high level of GUS accumulation in leaves and the lack of GUS in roots, with uidA mRNA present in both tissues, indicated tissue-specific accumulation of the chimeric gene product. Light-regulated accumulation of GUS in seedlings was shown. (i) Light-induced accumulation (100-fold) of GUS in etiolated cotyledons was accompanied by only a modest increase in mRNA levels. (ii) Inhibition of GUS synthesis was observed in cotyledons when light-grown seedlings were transferred to the dark, with no reduction in mRNA levels. Tissue-specific and light-regulated accumulation of GUS indicates that D1 accumulation is controlled via cis-acting regulatory elements in the untranslated region of the psbA mRNA. We propose that in tobacco, control of translation initiation is the primary mechanism regulating D1 protein accumulation.