Comparative proteomic analysis of salt response proteins in seedling roots of two wheat varieties

A comparative proteomic analysis was made of salt response in seedling roots of wheat cultivars Jing-411 (salt tolerant) and Chinese Spring (salt sensitive) subjected to a range of salt stress concentrations (0.5%, 1.5% and 2.5%) for 2 days. One hundred and ninety eight differentially expressed protein spots (DEPs) were located with at least two-fold differences in abundance on 2-DE maps, of which 144 were identified by MALDI-TOF-TOF MS. These proteins were involved primarily in carbon metabolism (31.9%), detoxification and defense (12.5%), chaperones (5.6%) and signal transduction (4.9%). Comparative analysis showed that 41 DEPs were salt responsive with significant expression changes in both varieties under salt stress, and 99 (52 in Jing-411 and 47 in Chinese Spring) were variety specific. Only 15 and 9 DEPs in Jing-411 and Chinese Spring, respectively, were up-regulated in abundance under all three salt concentrations. All dynamics of the DEPs were analyzed across all treatments. Some salt responsive DEPs, such as guanine nucleotide-binding protein subunit beta-like protein, RuBisCO large subunit-binding protein subunit alpha and pathogenesis related protein 10, were up-regulated significantly in Jing-411 under all salt concentrations, whereas they were down-regulated in salinity-stressed Chinese Spring.     

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC–MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C₃–C₄ photosynthetic plant.     

Effects of light environment on the induction of chlorophyll fluorescence in leaves: a comparative study of Tradescantia species of different ecotypes

In this work, using a PAM-fluorimetry technique, we have compared effects of plant adaptation to the light or dark conditions on the kinetics of chlorophyll a fluorescence yield in Tradecantia leaves of several species (Tradescantia albiflora, Tradescantia fluminensis, Tradescantia navicularis, and Tradescantia sillamontana), which represent plants of different ecotypes. Two fluorescence parameters were used to assess photosynthetic performance in vivo: non-photochemical quenching (NPQ) of chlorophyll fluorescence (q_NPQ) determined by energy losses in the light-harvesting antenna of photosystem 2 (PS2), and PS2 operating efficiency (Φ_PSII). Comparative study of light-induced changes in q_NPQ and Φ_PSII has demonstrated that shade-tolerant Tradecantia species (T. albiflora Kunth, T. fluminensis Vell.) reveal higher capacities for NPQ and demonstrate slower transitions between the ‘light-adapted’ and ‘dark-adapted’ states than succulent species T. navicularis and T. sillamontana, which are typical habitats of semi-deserts. We analyze the photosynthetic performance of Tradescantia species in the context of their adaptabilities to variable environment conditions. The ability of shade-tolerant plants to retain a relatively long-term (∼40-60 min) ‘memory’ for illumination history may be associated with the regulatory mechanisms that provide the flexibility of photosynthetic apparatus in response to fluctuations of light intensity.     

Chlorophyll fluorescence in the leaves of Tradescantia species of different ecological groups: Induction events at different intensities of actinic light

Chlorophyll fluorescence analysis is one of the most convenient and widespread techniques used to monitor photosynthesis performance in plants. In this work, after a brief overview of the mechanisms of regulation of photosynthetic electron transport and protection of photosynthetic apparatus against photodamage, we describe results of our study of the effects of actinic light intensity on photosynthetic performance in Tradescantia species of different ecological groups. Using the chlorophyll fluorescence as a probe of photosynthetic activity, we have found that the shade-tolerant species Tradescantia fluminensis shows a higher sensitivity to short-term illumination (≤20 min) with low and moderate light (≤200 μE m⁻² s⁻¹) as compared with the light-resistant species Tradescantia sillamontana. In T. fluminensis, non-photochemical quenching of chlorophyll fluorescence (NPQ) and photosystem II operational efficiency (parameter Φ_PSII) saturate as soon as actinic light reaches ≈200 μE m⁻² s⁻¹. Otherwise, T. sillamontana revealed a higher capacity for NPQ at strong light (≥800 μE m⁻² s⁻¹). The post-illumination adaptation of shade-tolerant plants occurs slower than in the light-resistant species. The data obtained are discussed in terms of reactivity of photosynthetic apparatus to short-term variations of the environment light.     

Comparative analysis of zygospore transcripts during early germination in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has a haplontic life cycle, and forms diploid zygotes for reproduction. The zygospore, a sporulating zygote, begins germination in response to light signals, generating haploid progenies and inducing several cell-biological events; e.g., DNA synthesis and meiotic division, successively. Their regulatory mechanisms remain largely unknown, so we focused on the early stages of germination and analyzed the dynamics of gene expression associated with the germination process. The gene expression levels of zygospores at 1 and 6 h after light exposure were analyzed by a next-generation sequencing platform, the 454 GS Junior. At 6 h, the photosynthesis pathway, including its antenna proteins and two methionine metabolism-related genes (methionine synthase and sulfite reductase), were up-regulated compared to 1 h after light exposure. Meanwhile, three uncharacterized genes that contained an antibiotic biosynthesis monooxygenase domain and an HSP20/alpha crystallin family protein were specifically expressed at 1 h after light exposure. These gene expressions were also verified by quantitative real-time PCR analysis. These results suggest that the photosynthesis and methionine synthesis pathways, both of which occur in the chloroplast, are activated in zygospores at around 6 h after light exposure, and that some polyketides and/or a small heat shock protein may be related to the initiation of zygospore germination.     

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents

To understand the photosynthetic basis in a single seed descent line 10 (SSDL10) of wheat contained high ATP in leaves, the chloroplast proteome was compared to SSDL10 and its parents using a combination of 2-DE and MALDI-TOF MS and MS/MS. More than 300 protein spots could be reproducibly detected in the 2D gel. 18 spots were differentially expressed between SSDL10 and the parents, 16 of which were identified by MS with the localization in chloroplasts. These proteins are grouped into diverse functional categories, including Calvin cycle and electron transport in photosynthesis, redox homeostasis, metabolism, and regulation. In addition to Rubisco large subunit, the content of photosynthetic electron transfers such as chlorophyll a-b binding protein, ATP synthase δ subunit, ferredoxin-NADP⁺ oxidoreductase (FNR) was higher in SSDL10 than in its parents. Furthermore, cyclic electron transfer around photosystem I (CET) was faster in SSDL10 than in the parents. Analysis of NADPH-NBT oxidoreductase activity combined with immuno-detection further revealed that, the activity of two high molecular mass protein complexes containing FNR probably involved, the CET appeared higher in SSDL10 than in the parents. The possible mechanism for the regulative role of CET in photosynthesis in SSDL10 is discussed.     

Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function

Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70–80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either in the absence or in the presence of IFP.     

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis : Application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved.     

Mitochondrial defect and PGC-1α dysfunction in parkin-associated familial Parkinson's disease

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.     

PS II model based analysis of transient fluorescence yield measured on whole leaves of Arabidopsis thaliana after excitation with light flashes of different energies

Our recently presented PS II model (Belyaeva et al., 2008) was improved in order to permit a consistent simulation of Single Flash Induced Transient Fluorescence Yield (SFITFY) traces that were earlier measured by Steffen et al. (2005) on whole leaves of Arabidopsis (A.) thaliana at four different energies of the actinic flash. As the essential modification, the shape of the actinic flash was explicitly taken into account assuming that an exponentially decaying rate simulates the time dependent excitation of PS II by the 10 ns actinic flash. The maximum amplitude of this excitation exceeds that of the measuring light by 9 orders of magnitude. A very good fit of the SFITFY data was achieved in the time domain from 100 ns to 10 s for all actinic flash energies (the maximum energy of 7.5 × 10¹⁶ photons/(cm² flash) is set to 100%, the relative energies of weaker actinic flashes were of ∼8%, 4%, ∼1%). Our model allows the calculation and visualization of the transient PS II redox state populations ranging from the dark adapted state, via excitation energy and electron transfer steps induced by pulse excitation, followed by final relaxation into the stationary state eventually attained under the measuring light. It turned out that the rate constants of electron transfer steps are invariant to intensity of the actinic laser flash. In marked contrast, an increase of the actinic flash energy by more than two orders of magnitude from 5.4 × 10¹⁴ photons/(cm² flash) to 7.5 × 10¹⁶ photons/(cm² flash), leads to an increase of the extent of fluorescence quenching due to carotenoid triplet (³Car) formation by a factor of 14 and of the recombination reaction between reduced primary pheophytin (Phe⁻) and P680⁺ by a factor of 3 while the heat dissipation in the antenna complex remains virtually constant.The modified PS II model offers new opportunities to compare electron transfer and dissipative parameters for different species (e.g. for the green algae and the higher plant) under varying illumination conditions.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.     

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment-protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (> 200 μmol photons m⁻² s⁻¹) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment-protein complexes function as low light-induced mobile antennae. Further, we have detected α-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.