Protein Gel Blot Research Articles

A24 AN ULCERATIVE COLITIS-ISOLATED PATHOBIONT CAN DEGRADE MUCUS PRODUCED BY UC PATIENT-DERIVED COLONOIDS

Abstract Background Inflammatory bowel disease (IBD) pathobionts are commensal microbes with pathogenic potential that may cause or exacerbate IBD symptoms. Some pathobionts (ex. Escherichia coli) reside at low levels in the lumen of a healthy gut but can rapidly grow in the inflamed colons of ulcerative colitis (UC) patients. To promote disease, these pathobionts must cross the colonic mucus barrier (comprised of MUC2) that separates the epithelium from luminal microbes. It is currently unclear how bacterial pathobionts cross the mucus barrier of UC patients. Aims Using healthy and UC patient biopsy-derived colonic organoids (colonoids) and an air liquid interface (ALI) monolayer model, we investigated how the UC-isolated E. coli pathobiont p19A crosses the mucus barrier. Methods Apical out healthy and UC patient biopsy-derived colonoids were infected with p19A to confirm this pathobiont exerts direct cytopathic effects on human colonocytes. Sequencing p19A’s genome, we found it contains two mucus degrading proteins (mucinases). Healthy human and UC colonoids, as well as mouse colonoids, were used to generate mucus-producing ALI monolayers. To detect p19A-mediated mucus degradation, concentrated p19A supernatant was incubated with ALI-derived mucus and degraded MUC2 proteins were detected by protein gel and MUC2 Western blot. MUC2 glycosylation was analyzed by protein gel and PAS staining. ALI monolayers were infected with p19A to evaluate mucus degradation and p19A localization by immunostaining. Results The UC-isolated pathobiont p19A infected and exerted cytotoxic effects on apical out healthy and UC patient colonoids. By day 14, ALI monolayers were differentiated and covered by a thick mucus layer as assessed using brightfield microscopy. Mucus removed for mucinase assays was replenished within 7 days. p19A harbours proteins capable of degrading human ALI-, but not mouse ALI-derived mucus, in vitro, suggesting the presence of host-specific mucinases. Mucus produced by UC ALI monolayers showed reduced glycosylation and increased degradation both over time and by p19A proteins. Day 21 ALI-monolayers infected with p19A for 18 hours exhibited overt mucus degradation allowing p19A to infect the underlying epithelium. Conclusions Patient-derived ALI monolayers produce a thick mucus layer that can be used to study pathobiont-mucus interactions. The UC pathobiont p19A disrupts apical out organoids and produces proteins that degrade ALI-derived mucus in vitro, with UC mucus being more susceptible to degradation than mucus from healthy controls. The results from our model suggest a potential mechanism for pathobiont-mediated mucosal barrier disruption in UC patients. Patient-derived ALI monolayer (blue/white) produces a thick mucus layer (green) that can be degraded by the pathobiont p19A (red). Funding Agencies CCC, CIHR

Predominantly symplastic phloem unloading of photosynthates maintains efficient starch accumulation in the cassava storage roots (Manihot esculenta Crantz)

BackgroundCassava (Manihot esculenta Crantz) efficiently accumulates starch in its storage roots. However, how photosynthates are transported from the leaves to the phloem (especially how they are unloaded into parenchymal cells of storage roots) remains unclear.ResultsHere, we investigated the sucrose unloading pattern and its impact on cassava storage root development using microstructural and physiological analyses, namely, carboxyfluorescein (CF) and C14 isotope tracing. The expression profiling of genes involved in symplastic and apoplastic transport was performed, which included enzyme activity, protein gel blot analysis, and transcriptome sequencing analyses. These finding showed that carbohydrates are transported mainly in the form of sucrose, and more than 54.6% was present in the stem phloem. Sucrose was predominantly unloaded symplastically from the phloem into storage roots; in addition, there was a shift from apoplastic to symplastic unloading accompanied by the onset of root swelling. Statistical data on the microstructures indicated an enrichment of plasmodesmata within sieve, companion, and parenchyma cells in the developing storage roots of a cultivar but not in a wild ancestor. Tracing tests with CF verified the existence of a symplastic channel, and [14C] Suc demonstrated that sucrose could rapidly diffuse into root parenchyma cells from phloem cells. The relatively high expression of genes encoding sucrose synthase and associated proteins appeared in the middle and late stages of storage roots but not in primary fibrous roots, or secondary fibrous roots. The inverse expression pattern of sucrose transporters, cell wall acid invertase, and soluble acid invertase in these corresponding organs supported the presence of a symplastic sucrose unloading pathway. The transcription profile of genes involved in symplastic unloading and their significantly positive correlation with the starch yield at the population level confirmed that symplastic sucrose transport is vitally important in the development of cassava storage roots.ConclusionsIn this study, we revealed that the cassava storage root phloem sucrose unloading pattern was predominantly a symplastic unloading pattern. This pattern is essential for efficient starch accumulation in high-yielding varieties compared with low-yielding wild ancestors.

Biosynthesis pathway of indole-3-acetyl-myo-inositol during development of maize (Zea mays L.) seeds

Indole-3-acetic acid (IAA) conjugation is one of the mechanisms responsible for auxin homeostasis. IAA ester conjugates biosynthesis has been studied during development of maize seeds where IAA-inositol (IAInos) and its glycosidic forms make up about 50 % of its ester conjugates pool. 1-O-indole-3-acetyl-β-d-glucose (IAGlc) synthase and indole-3-acetyl transferase (IAInos synthase) are key enzymes in a two-step pathway of IAInos synthesis. In the first reaction, IAA is glucosylated to a high energy acetal, 1-O-indole-3-acetyl-β-d-glucose by IAGlc synthase, whereas in the second step, IAInos synthase transfers IAA moiety to myo-inositol forming a stable auxin ester, indole-3-acetyl-myo-inositol (IAInos). It should be mentioned that IAGlc synthase catalyzes a reversible reaction with unfavourable equilibrium that delivers IAGlc for favourable transacylation to IAInos. This is the first study where IAGlc synthase and IAInos synthase are simultaneously analyzed by enzymatic activity assay and quantitative RT-PCR in maize seeds at four stages of development (13, 26, 39 and 52 Days After Flowering). Activity of IAGlc/IAInos synthases as well as their expression profiles during seed development were different. While both enzymatic activities and ZmIAIn expression were the highest in seeds at 26 DAF, the highest expression of ZmIAGlc was observed at 13 DAF. Protein gel blot analysis showed that IAInos synthase exists as a mixture of several isoforms at a similar protein level at particular stages of seed development. Neither of other ester conjugates of IAA (IAA-mannose) nor IAA-amino acids were detected at the stages studied. Catalytic activity of l-tryptophan aminotransferase involved in IAA biosynthesis as well as UDPG pyrophosphorylase, synthesizing UDPG as a substrate for IAGlc synthase, were also analyzed. l-tryptophan aminotransferase activity was the highest at 26 DAF. Changes in enzyme activity of UDPG pyrophosphorylase are difficult to interpret. Expression levels of ZmIPS and ZmIPP encoding two enzymes of myo-inositol biosynthesis pathway: inositol-x-phosphate synthase (IPS) and inositol-x-phosphate phosphatase (IPP), respectively, were analyzed. 26 DAF seeds displayed the highest expression level of ZmIPS, whereas transcription of ZmIPP was the highest at 13 DAF.

Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene TaHsp90 in Indian wheat (Triticum aestivum L.) cv C306.

To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to Hsp90 was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of TaHsp90 (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of TaHsp90 was validated in E. coli BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene TaHsp90 isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.

The HSP70 chaperone machines of Chlamydomonas are induced by cold stress

The responses of Chlamydomonas reinhardtii cells to low temperatures have not been extensively studied compared with other stresses. Like other organisms, this green alga has heat shock protein 70s (HSP70s) that are located in chloroplast, mitochondrion and cytosol. To test whether temperature downshifts affected HSP70s synthesis, we used real-time PCR and protein gel blot analysis. C. reinhardtii cells exposed to cold stress show increased HSP70s mRNA levels. Genes encoding other components of HSP70 chaperone machines (e.g. CGE1, CDJ1, HSP90C and HSP90A) are also up-regulated in response to decreased temperature. We demonstrated that the accumulation of all analyzed mRNA occur more slowly and with reduced amplitude in cells exposed to cold than in cells treated with heat. Furthermore, C. reinhardtii cells display the splicing of the CGE1 transcript that was dependent on low temperature. Finally, the transcription regulator of C. reinhardtii HSF1 is also cold-responsive, suggesting its role in the transcriptional regulation of HSP genes at low temperature.

Differences in gene expression between natural and artificially induced leaf senescence in barley

Senescence is the last step of leaf development in the life span of an annual plant. Senescence can be induced prematurely by treating leaf tissues with jasmonic acid methyl ester (methyl jasmonate, MeJA). During both senescence programmes, drastic changes occur at the biochemical, cellular and ultra-structural levels that were compared here for primary leaves of barley (Hordeum vulgare L.). Our findings indicate that both types of senescence are similar with respect to the morphological changes including the loss of chlorophyll, disintegration of thylakoids, and formation of plastoglobules. However, the time elapsed for reaching senescence completion was different and ranged from 7 to 8 days for artificially senescing, MeJA-treated plants to 7–8 weeks for naturally senescing plants. Pulse-labelling studies along with RNA and protein gel blot analyses showed differential changes in the expression of both plastid and nuclear genes coding for photosynthetic proteins. Several unique messenger products accumulated in naturally and artificially senescing, MeJA-treated leaves. Detailed expression and crosslinking studies revealed that pheophorbide a oxygenase (PAO), a previously implicated key enzyme of chlorophyll breakdown, is most likely not rate-limiting for chlorophyll destruction under both senescence conditions. Metabolite profiling identified differential changes in the composition of carotenoid derivatives and prenyl-lipids to occur in naturally senescing and artificially senescing plants that underscored the differences between both senescence programmes.

Evidence for alternative splicing mechanisms in meadow fescue (Festuca pratensis) and perennial ryegrass (Lolium perenne) Rubisco activase gene

Rubisco activase is required to regulate the catalytic activity of Rubisco in plants, in an ATP-dependent manner. One or two Rubisco activase proteins have been identified in different plant species. In some species, the two isoforms are the products of alternative splicing of the Rubisco activase gene. The aim of this study was to confirm that Lolium perenne and Festuca pratensis plants have two isoforms of Rubisco activase and that they are the products of alternative splicing of common pre-mRNA. Protein gel blot analyses indicated that L. perenne and F. pratensis leaves contained two Rubisco activase proteins. Sequence analysis of cDNA and genomic DNA showed that differential splicing generated two mRNAs that differed in sequence only in the inclusion of 48bp. The insertion contains a stop codon leading to the synthesis of a shorter polypeptide. Under the conditions of our experiment, the shorter splicing variant of L. perenne and F. pratensis Rubisco activase gene was preferentially produced. Any further studies concerning Rubisco activase genes in L. perenne and/or F. pratensis plants should take into consideration the mechanism of its expression.

Possible involvement of heterotrimeric G-protein signaling in Al-induced secretion of organic acid anions in Arabidopsis and rye

Secretion of organic acid anions (OAs) from the roots in response to aluminum (Al) is a common mechanism for Al resistance in plants. Transporter genes involved in the Al-induced secretion of malate and citrate have been identified, but the signal cascades mediating the regulation of the secretion of OAs has not been understood. Here, we investigated the possible role of heterotrimeric G-proteins in the Al-induced secretion of OAs. Seedlings of rye and Arabidopsis thaliana were grown in an environment-controlled growth room. Organic acid anions in the exudates of roots were determined spectrophotometrically. Heterotrimeric G-proteins and GTPase activity were assayed using protein gel blot analyses with Gα antibody and spectrofluorometer, respectively. GPA1 gene expression level was determined by semi-quantitative RT-PCR. Immunoblotting revealed a heterotrimeric G-protein in the plasma membrane fraction of rye and Arabidopsis roots. Exposure of the rye roots to Al resulted in activation of GTPase. Al-induced secretion was inhibited by pertussis toxin, a heterotrimeric G-protein antagonist, but was stimulated by cholera toxin, a heterotrimeric G-protein agonist. Furthermore, Al-induced OA secretion was blocked in Gα- or Gβ-deficient mutants of Arabidopsis. Al induced GPA1 expression in Arabidopsis roots. Heterotrimeric G-proteins-coordinated transduction of Al-stress signals may be associated with the secretion of OAs from roots.

Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential.

Metallothioneins are essential in plants for metal detoxification in addition to their other roles in plant life cycle. This study reports the characterization of an olive (Olea europaea L. cv. Ayvalik) metallothionein with respect to molecular and functional properties. A cDNA encoding a type 2 metallothionein from olive was isolated from a leaf cDNA library, characterized and named OeMT2 after its molecular and functional properties. OeMT2 was expressed in Escherichia coli, and a single protein band was confirmed by protein gel blot analysis. Metal tolerance ability of bacterial cells expressing OeMT2 was determined against 0.2mM CdCl2, 0.4mM CdCl2 and 1mM CuSO4 in the growth medium. Metal ion contents of bacterial cells expressing OeMT2 were measured by ICP. Metal tolerance assays and ICP measurements suggested that OeMT2 effectively binds Cu and Cd. Molecular analysis of OeMT2 revealed two introns, three exons, a short 3' UTR and a long 5' UTR. Comparing the genomic sequences from 14 olive cultivars revealed OeMT2 had both intron and exon polymorphisms dividing the cultivars into three groups. Real-time PCR analysis demonstrated that OeMT2 expresses more or less the same amounts in all tissues of the olive tree examined. The genomic copy number of OeMT2 was also determined employing real-time PCR which suggested a single copy gene in the olive genome while three other MT2 members were determined from the draft olive genome sequences of Ayvalik cultivar and that of wild olive. This is the first report on molecular and functional characterization of an olive metallothionein and shows that OeMT2 expressed in E. coli has the capability of effectively binding toxic heavy metals. This may suggest that OeMT2 plays an important role in metal homeostasis in addition to a good potential for environmental and industrial usage.

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum.

The model marine diatom Phaeodactylum tricornutum can accumulate high levels of triacylglycerols (TAGs) under nitrogen depletion and has attracted increasing attention as a potential system for biofuel production. However, the molecular mechanisms involved in TAG accumulation in diatoms are largely unknown. Here, we employed a label-free quantitative proteomics approach to estimate differences in protein abundance before and after TAG accumulation. We identified a total of 1193 proteins, 258 of which were significantly altered during TAG accumulation. Data analysis revealed major changes in proteins involved in branched-chain amino acid (BCAA) catabolic processes, glycolysis, and lipid metabolic processes. Subsequent quantitative RT-PCR and protein gel blot analysis confirmed that four genes associated with BCAA degradation were significantly upregulated at both the mRNA and protein levels during TAG accumulation. The most significantly upregulated gene, encoding the β-subunit of methylcrotonyl-CoA carboxylase (MCC2), was selected for further functional studies. Inhibition of MCC2 expression by RNA interference disturbed the flux of carbon (mainly in the form of leucine) toward BCAA degradation, resulting in decreased TAG accumulation. MCC2 inhibition also gave rise to incomplete utilization of nitrogen, thus lowering biomass during the stationary growth phase. These findings help elucidate the molecular and metabolic mechanisms leading to increased lipid production in diatoms.

NADH-dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2-oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin-dependent glutamate synthase (Fd-GOGAT) and nicotinamide adenine dinucleotide (NADH)-GOGAT. While Fd-GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH-GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH-GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real-time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH-GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH-GOGAT and Fd-GOGAT did not fully overlap. Promoter-β-glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH-GOGAT was highly accumulated in non-green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH-GOGAT transfer DNA (T-DNA) insertion lines under normal CO2 condition. The data emphasize the importance of NADH-GOGAT in the ammonium assimilation in Arabidopsis roots.

Multistep involvement of glutathione with salicylic acid and ethylene to combat environmental stress

The role of glutathione (GSH) in plant defense is an established fact. However, the association of GSH with other established signaling molecules within the defense signaling network remains to be evaluated. Previously we have shown that GSH is involved in defense signaling network likely through NPR1-dependent salicylic acid (SA)-mediated pathway. In this study, to gain further insight, we developed chloroplast-targeted gamma-glutamylcysteine synthetase (γ-ECS) overexpressed transgenic Nicotiana tabacum (NtGp line) and constructed a forward subtracted cDNA (suppression subtractive hybridization (SSH)) library using NtGp line as a tester. Interestingly, in addition to SA-related transcripts like pathogenesis-related protein 1a (PR1a) and SAR8.2 m/2l, 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), a key enzyme of ethylene (ET) biosynthesis, was identified in the SSH library. Besides, transcription factors like WRKY transcription factor 3 (WRKY3), WRKY1 and ethylene responsive factor 4 (ERF4), associated with SA and ET respectively, were also identified thus suggesting an interplay of GSH with ET and SA. Furthermore, proteomic profiling of NtGp line, performed by employing two-dimensional gel electrophoresis (2-DE), corroborated with the transcriptomic profile and several defense-related proteins like serine/threonine protein kinase, and heat shock 70 protein (HSP70) were identified with increased accumulation. Fascinatingly, induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) was also noted thus demonstrating the active involvement of GSH with ET. Protein gel blot analysis confirmed the enhanced accumulation of ACC oxidase in NtGp line. Together, our data revealed that GSH is involved in the synergistic multiple steps crosstalk through ET as well as SA to combat environmental stress.

Cold stress causes rapid but differential changes in properties of plasma membrane H+-ATPase of camelina and rapeseed

Camelina (Camelina sativa) and rapeseed (Brassica napus) are well-established oil-seed crops with great promise also for biofuels. Both are cold-tolerant, and camelina is regarded to be especially appropriate for production on marginal lands. We examined physiological and biochemical alterations in both species during cold stress treatment for 3 days and subsequent recovery at the temperature of 25°C for 0, 0.25, 0.5, 1, 2, 6, and 24h, with particular emphasis on the post-translational regulation of the plasma membrane (PM) H+-ATPase (EC3.6.3.14). The activity and translation of the PM H+-ATPase, as well as 14-3-3 proteins, increased after 3 days of cold stress in both species but recovery under normal conditions proceeded differently. The increase in H+-ATPase activity was the most dramatic in camelina roots after recovery for 2h at 25°C, followed by decay to background levels within 24h. In rapeseed, the change in H+-ATPase activity during the recovery period was less pronounced. Furthermore, H+-pumping increased in both species after 15min recovery, but to twice the level in camelina roots compared to rapeseed. Protein gel blot analysis with phospho-threonine anti-bodies showed that an increase in phosphorylation levels paralleled the increase in H+-transport rate. Thus our results suggest that cold stress and recovery in camelina and rapeseed are associated with PM H+-fluxes that may be regulated by specific translational and post-translational modifications.

Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?

Trees necessarily experience changes in temperature, requiring efficient short-term strategies that become crucial in environmental change adaptability. DNA methylation and histone posttranslational modifications have been shown to play a key role in both epigenetic control and plant functional status under stress by controlling the functional state of chromatin and gene expression. Cork oak (Quercus suber L.) is a key stone of the Mediterranean region, growing at temperatures of 45°C. This species was subjected to a cumulative temperature increase from 25°C to 55°C under laboratory conditions in order to test the hypothesis that epigenetic code is related to heat stress tolerance. Electrolyte leakage increased after 35°C, but all plants survived to 55°C. DNA methylation and acetylated histone H3 (AcH3) levels were monitored by HPCE (high performance capillary electrophoresis), MS-RAPD (methylation-sensitive random-amplified polymorphic DNA) and Protein Gel Blot analysis and the spatial distribution of the modifications was assessed using a confocal microscope. DNA methylation analysed by HPCE revealed an increase at 55°C, while MS-RAPD results pointed to dynamic methylation-demethylation patterns over stress. Protein Gel Blot showed the abundance index of AcH3 decreasing from 25°C to 45°C. The immunohistochemical detection of 5-mC (5-methyl-2′-deoxycytidine) and AcH3 came upon the previous results. These results indicate that epigenetic mechanisms such as DNA methylation and histone H3 acetylation have opposite and particular dynamics that can be crucial for the stepwise establishment of this species into such high stress (55°C), allowing its acclimation and survival. This is the first report that assesses epigenetic regulation in order to investigate heat tolerance in forest trees.

Long distance movement of DIR1 and investigation of the role of DIR1-like during systemic acquired resistance in Arabidopsis

DIR1 is a lipid transfer protein (LTP) postulated to complex with and/or chaperone a signal(s) to distant leaves during Systemic Acquired Resistance (SAR) in Arabidopsis. DIR1 was detected in phloem sap-enriched petiole exudates collected from wild-type leaves induced for SAR, suggesting that DIR1 gains access to the phloem for movement from the induced leaf. Occasionally the defective in induced resistance1 (dir1-1) mutant displayed a partially SAR-competent phenotype and a DIR1-sized band in protein gel blots was detected in dir1-1 exudates suggesting that a highly similar protein, DIR1-like (At5g48490), may contribute to SAR. Recombinant protein studies demonstrated that DIR1 polyclonal antibodies recognize DIR1 and DIR1-like. Homology modeling of DIR1-like using the DIR1-phospholipid crystal structure as template, provides clues as to why the dir1-1 mutant is rarely SAR-competent. The contribution of DIR1 and DIR1-like during SAR was examined using an Agrobacterium-mediated transient expression-SAR assay and an estrogen-inducible DIR1-EGFP/dir1-1 line. We provide evidence that upon SAR induction, DIR1 moves down the leaf petiole to distant leaves. Our data also suggests that DIR1-like displays a reduced capacity to move to distant leaves during SAR and this may explain why dir1-1 is occasionally SAR-competent.

Oxidative stress, protein carbonylation, proteolysis and antioxidative defense system as a model for depicting water deficit tolerance in Indica rice seedlings

Water deficit is an important constraint to rice (Oryza sativa L.) productivity. The present study was undertaken to investigate whether the level of oxidative stress, carbonylation of proteins, proteolysis and status of antioxidative defense could serve as a model to distinguish water deficit tolerant and sensitive rice cultivars. When 10-day-grown seedlings of two rice cultivars, Malviya-36 (drought-sensitive) and Brown Gora (drought-tolerant) were subjected to −1.0 and −2.1 MPa water deficit treatments for 24–72 h with polyethylene glycol 6000 in the medium, a greater decline in the growth of the seedlings and levels of leaf water potential, relative water content, Chl a, Chl b, carotenoids and greater increase in leaf water loss were observed in the sensitive cultivar than the tolerant. Under similar level of water deficit seedlings of sensitive cultivar showed higher level of superoxide anion generation, H2O2, lipid peroxidation and proteolysis in roots as well as shoots compared to the tolerant. Drought-tolerant cultivar had higher constitutive level of antioxidative enzymes superoxide dismutase and catalase and the activities of these two enzymes alongwith of guaiacol peroxidase showed greater increase in this cultivar under water deficit compared to the sensitive. A significant decline in the level of protein thiol and a higher increase in protein carbonyls content, also confirmed by protein gel blot analysis with an antibody against 2,4-dinitrophenylhydrazine was observed in the seedlings of drought sensitive cv. Malviya-36 compared to the tolerant cv. Brown Gora when subjected to similar level of water deficit. Seedlings of drought sensitive cultivar, under water deficit, showed higher proteolytic activity, higher number of in-gel activity stained proteolytic bands and higher expression of oxidized proteins in roots compared to the tolerant cultivar. Results suggest that poor capacity of antioxidative enzymes could be, at least partly, correlated with water deficit sensitivity of sensitive cultivar and that higher activity of antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, low proteolytic activity, lower level of protein carbonyls and protein thiolation could serve as a model to depict water deficit tolerance in Indica rice seedlings.

Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.

Genetic transformation and analysis of rice OsAPx2 gene in Medicago sativa.

The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H2O2)concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H2O2 content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H2O2, sodium sulfite (Na2SO3), and sodium bicarbonate (NaHCO3). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H2O2 and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained.

Expression and Localization of Amorpha-4,11-diene Synthase in Artemisia annua L.

Artemisia annua L. is the only commercial source of the widely used antimalarial compound artemisinin. Biosynthesis of artemisinin has been proposed to take place in glandular trichomes. The first committed step in the conversion of farnesyl diphosphate (FDP) to artemisinin is conducted by the amorpha-4,11-diene synthase (ADS). To explore the organ-specific and developmental distributions of ADS, rabbit polyclonal antibodies were raised against recombinant ADS produced in Escherichia coli from the corresponding A. annua cDNA. Protein gel blot analysis of different A. annua organs showed that ADS was most abundant in young leaves and flower buds. Minor amounts of ADS were found in mature leaves. These findings were generally consistent with the analysis of the transcript level of the ADS gene. Immunolocalization of ADS showed strong positive staining in apical meristems, young leaves and glandular trichomes. No staining was observed in other cells of the leaf. The whole mount hybridization revealed that ADS was not expressed in all glandular trichomes of mature leaves. Specific staining of ADS could be detected in about 10–40 % of glandular trichomes.

Expression pattern of the psbO gene and its involvement in acclimation of the photosynthetic apparatus during abiotic stresses in Festuca arundinacea and F. pratensis

PsbO, the manganese-stabilizing protein, plays a crucial role in oxygen-evolving complex functioning and stabilization, by maintaining optimal manganese, calcium and chloride concentrations at the active state of PSII. In this paper we present a study focused on recognizing the relationship between psbO gene activity and acclimation of the photosynthetic apparatus under abiotic stresses in the grasses Festuca arundinacea and F. pratensis. PsbO expression was compared between two distinct genotypes within each species which differed in their levels of stress tolerance (drought and frost, respectively) during drought treatment (F. arundinacea) and cold acclimation (F. pratensis). The research involved: (1) the analyses of psbO gene expression profiles using real-time PCR, and (2) the analyses of PsbO protein accumulation profiles using protein gel blot hybridization. The results indicate that PsbO plays a protective function with respect to the photosynthetic apparatus during abiotic stresses. In cold-treated F. pratensis plants the accumulation of PsbO seems to be responsible for differences in the PSII photochemical efficiency. Higher stability of PSII during drought, observed in the high-drought tolerant F. arundinacea genotype, is not associated with PsbO accumulation, although the degradation of this protein affects destabilization of the oxygen-evolving complex in drought.