Non-transformed Research Articles

Impairment in photosynthesis induced by CAT inhibition depends on the intensity of photorespiration and peroxisomal APX expression in rice

We have previously shown that rice plants silenced for peroxisomal ascorbate peroxidase (OsAPX4-RNAi) display higher resilience to photosynthesis under oxidative stress and photorespiratory conditions. However, the redox mechanisms underlying that intriguing response remain unknown. Here, we tested the hypothesis that favorable effects triggered by peroxisomal APX deficiency on photosynthesis resilience under CAT inhibition are dependent on the intensity of photorespiration associated with the abundance of photosynthetic and redox proteins. Non-transformed (NT) and OsAPX4-RNAi silenced rice plants were grown under ambient (AC) or high CO2 (HC) conditions and subjected to 3-amino-1,2,4-triazole (3-AT)-mediated CAT activity inhibition. Photosynthetic measurements evidenced that OsAPX4-RNAi plants simultaneously exposed to CAT inhibition and HC lost the previously acquired advantage in photosynthesis resilience displayed under AC. Silenced plants exposed to environment photorespiration and CAT inhibition presented lower photorespiration as indicated by smaller Gly/Ser and Jo/Jc ratios and glycolate oxidase activity. Interestingly, when these silenced plants were exposed to HC and CAT-inhibition, they exhibited an inverse response compared to AC in terms of photorespiration indicators, associated with higher accumulation of proteins. Multivariate and correlation network analyses suggest that the proteomics changes induced by HC combined with CAT inhibition are substantially different between NT and OsAPX4-RNAi plants. Our results suggest that the intensity of photorespiration and peroxisomal APX-mediated redox signaling are tightly regulated under CAT inhibition induced oxidative stress, which can modulate the photosynthetic efficiency, possibly via a coordinated regulation of protein abundance and rearrangement, ultimately triggered by crosstalk involving H2O2 levels related to CAT and APX activities in peroxisomes.

AhNRAMP1 Enhances Manganese and Zinc Uptake in Plants.

Manganese (Mn) and zinc (Zn) play essential roles in plants. Members of the natural resistance-associated macrophage protein (NRAMP) family transport divalent metal ions. In this research, the function of peanut (Arachis hypogaea L.) AhNRAMP1 in transporting Mn and Zn, as well as its potential for iron(Fe) and Zn biofortification was examined. AhNRAMP1 transcription was strongly induced by Mn- or Zn-deficiency in roots and stems of peanut. Yeast complementation assays suggested that AhNRAMP1 encoded a functional Mn and Zn transporter. Exogenous expression of AhNRAMP1 in tobacco and rice enhanced Mn or Zn concentrations, improving tolerance to Mn or Zn deficiency. With higher Mn concentration, transgenic plants exhibited inhibited growth under Mn excess condition; similar results were obtained under excessive Zn treatment. AhNRAMP1 expression increased biomass in transgenic tobacco and rice, as well as yield in transgenic rice grown on calcareous soil. Compared with non-transformed (NT) plants, Fe and Zn concentrations were elevated whereas concentrations of Mn, copper (Cu), and cadmium (Cd) were not enhanced. These results revealed that AhNRAMP1 contributes to Mn and Zn transport in plants and may be a candidate gene for Fe and Zn biofortification.

Validated comprehensive metabolomics and lipidomics analysis of colon tissue and cell lines

Current untargeted approaches for metabolic fingerprinting of colon tissue and cell lines lack validation of reproducibility and/or focus on a selection of metabolites as opposed to the entire metabolome. Yet, both are critical to ensure reliable results and pursue a fully holistic analysis. Therefore, we have optimized and validated a platform for analyzing the polar metabolome and lipidome of colon-derived cell and tissue samples based on a consecutive extraction of polar and apolar components. Peak areas of selected targeted analytes and the number of untargeted components were assessed. Analysis was performed using ultra-high performance liquid-chromatography (UHPLC) coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry (HRMS). This resulted in an optimized extraction protocol using 50% methanol/ultrapure water to obtain the polar fraction followed by a dichloromethane-based lipid extraction. Using this comprehensive approach, we have detected more than 15,000 components with CV < 30% in internal quality control (IQC) samples and were able to discriminate the non-transformed (NT) and transformed (T) state in human colon tissue and cell lines based on validated OPLS-DA models (R2Y > 0.719 and Q2 > 0.674). To conclude, our validated polar metabolomics and lipidomics fingerprinting approach could be of great value to reveal gastrointestinal disease-associated biomarkers and mechanisms.

Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill

Protein p68 is a prototype constituent of DEAD-box protein family, which is involved in RNA metabolism, induced during abiotic stress conditions. In order to address the salinity stress faced by economically important soybean crop, we have transformed soybean cv. PUSA 9712 via direct organogenesis with marker free construct of p68 gene by Agrobacterium-mediated genetic transformation. The putative transgenic plants were screened by Polymerase chain reaction (PCR), Dot blot analysis and Southern blot hybridization. Reverse transcriptase-PCR(RT-PCR) and Quantitative real-time PCR(qRT-PCR) established that the p68 gene expressed in three out of five southern positive (T1) plants. The transformed (T1) soybean plants survived irrigation upto 200mM of NaCl whereas the non-transformed (NT) plants could not survive even 150mM NaCl. The transgenic soybean (T1) plants showed a higher accumulation of chlorophyll, proline, CAT, APX, SOD, RWC, DHAR and MDHAR than the NT plants under salinity stress conditions. The transformed (T1) soybean plants also retained a higher net photosynthetic rate, stomatal conductance and CO2 assimilation as compared to NT plants. Further analysis revealed that (T1) soybean plants accumulated higher K+ and lower Na+ levels than NT plants. Yield performance of transformed soybean plants was estimated in the transgenic green house under salinity stress conditions. The transformed (T1) soybean plants expressing the p68 gene were morphologically similar to non-transformed plants and produced 22-24 soybean pods/plant containing 8-9g (dry weight) of seeds at 200mM NaCl concentration. The present investigation evidenced the role of the p68 gene against salinity, by enhancing the tolerance towards salinity stress in soybean plants.

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1.

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase.

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T3 and T4 generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T4 generation). The transgenic plants also showed increased tolerance to the externally applied OA (60mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1h and 4h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H2O2 at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.

Mitochondrial glutathione peroxidase (OsGPX3) has a crucial role in rice protection against salt stress

Rice is one of the world’s most important crops and an excellent model system for understanding the interaction between genes and environmental changes. However, its productivity is often challenged by abiotic stresses, which results in the accumulation of reactive oxygen species. Glutathione peroxidases are part of the mechanism by which plants face oxidative stress. These enzymes can control redox homeostasis and also play a role in redox signaling. Here, we investigate the role of rice GPX3 in plant responses to salt stress using OsGPX3-RNAi silenced rice plants (GPX3s). Our results indicate that GPX3s plants are more sensitive to salinity showing decreased biomass, CO2 assimilation rate, stomatal conductance, and intercellular CO2 partial pressure. Moreover, these plants present significant damage to photosystem II activity and decline in chlorophyll content. Salt stress induced ROS accumulation in both non-transformed (NT) and GPX3s plants, indicating that GPX3s sensibility to salt stress was not due to the significant impairment in redox equilibrium. Together, these results show GPX3 importance in rice to achieve salt stress tolerance via an independent ROS-scavenger mechanism. Moreover, it also provides new light into the cross-talk between chloroplasts and mitochondria, suggesting a novel role to this enzyme beyond its role as ROS-scavenger.

Over-expression of the Pseudomonas syringae harpin-encoding gene hrpZm confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean.

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseasesreducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T2-T4 generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.

Thylakoidal APX modulates hydrogen peroxide content and stomatal closure in rice (Oryza sativa L.)

In rice (Oryza sativa L.) there are two chloroplast ascorbate peroxidases isoforms, OsAPX7 (stromal) and OsAPX8 (thylakoidal). Previous works demonstrated that double silencing of OsAPX7 and OsAPX8 led to differences in photosynthetic parameters in stress conditions. Nevertheless, both APX encoding genes present contrasting expression patterns in response to abiotic stress and the individual role of them in chloroplast redox metabolism and in stress response remains largely unknown.Here, we showed that plants silenced to OsAPX8 (RNAiOsAPX8) have a lower growth rate and a higher H2O2 content, when compared to non-transformed (NT) plants. Furthermore, RNAiOsAPX8 plants show a decrease in stomatal opening with consequent increase in leaf surface temperature. However, these plants were not more susceptible to drought stress. RNAseq analysis showed that the silencing of OsAPX8 has impact on plant gene expression, mainly affecting genes related to photosynthesis and signal transduction. Our results indicate that the reduction of OsAPX8 expression led to a stress-like phenotype but it does not alter drought tolerance.

Ascorbic acid toxicity is related to oxidative stress and enhanced by high light and knockdown of chloroplast ascorbate peroxidases in rice plants

Toxicity caused by high concentrations of ascorbic acid (AA) has been widely reported in animal cells but is scarcely described in plants. In this study, rice plants deficient (knockdown) in two chloroplast ascorbate peroxidases (APX7/8) and non-transformed (NT) were exposed to wide exogenous AA concentrations in the presence of low light and high light (HL). Reduced (ASC) and oxidized (DHA) ascorbate reached much higher concentrations in symplast compared to the apoplastic space, and high redox states were found in both cellular compartments. Exogenous AA concentrations above 30 mM caused strong cellular and oxidative damage indicated by decreased cell integrity and increased lipid peroxidation in leaves. These toxic effects were strongly enhanced by HL and, to a small extent, by deficiency of both chloroplastic proteins APX7/8. The combination of HL and high AA concentration induced a strong increase in H2O2, associated with decrease in the content of chlorophylls and carotenoids. High AA concentrations strongly induced stomatal closure and impairment in CO2 assimilation, in combination with decreased quantum efficiency of photosystem II (PSII) and PSI. We postulate that oxidative stress caused by AA toxicity in the presence of HL was induced by over-production of reactive oxygen species due to an imbalance between excess energy in the photosystems and low CO2 assimilation, which was related closely to strong decrease in stomatal conductance. In addition, high ASC levels might have acted as a pro-oxidant in the presence of high H2O2 concentrations, stimulating the Fenton reaction and contributing to the intensification of oxidative stress in rice leaves.

Dehydrins Impart Protection against Oxidative Stress in Transgenic Tobacco Plants.

Environmental stresses generate reactive oxygen species (ROS) which might be detrimental to the plants when produced in an uncontrolled way. However, the plants ameliorate such stresses by synthesizing antioxidants and enzymes responsible for the dismutation of ROS. Additionally, the dehydrins were also able to protect the inactivation of the enzyme lactate dehydrogenase against hydroxyl radicals (OH⋅) generated during Fenton’s reaction. SbDhn1 and SbDhn2 overexpressing transgenic tobacco plants were able to protect against oxidative damage. Transgenic tobacco lines showed better photosynthetic efficiency along with high chlorophyll content, soluble sugar and proline. However, the malonyl dialdehyde (MDA) content was significantly lower in transgenic lines. Experimental evidence demonstrates the protective effect of dehydrins on electron transport chain in isolated chloroplast upon methyl viologen (MV) treatment. The transgenic tobacco plants showed significantly lower superoxide radical generation () upon MV treatment. The accumulation of the H2O2 was also lower in the transgenic plants. Furthermore, in the transgenic plants the expression of ROS scavenging enzymes was higher compared to non-transformed (NT) or vector transformed (VT) plants. Taken together these data, during oxidative stress dehydrins function by scavenging the () directly and also by rendering protection to the enzymes responsible for the dismutation of () thereby significantly reducing the amount of hydrogen peroxides formed. Increase in proline content along with other antioxidants might also play a significant role in stress amelioration. Dehydrins thus function co-operatively with other protective mechanisms under oxidative stress conditions rendering protection in stress environment.

Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter.

Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6–43.2 fold in the PANSBP_DP plants and 2.0–124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean.

Robust RNAi-mediated resistance to multiple Potyvirus strains and isolates, but not to Secovirus BPMV, was conferred by expressing a short SMV P3 hairpin in soybean plants. Engineering resistance to multiple Potyvirus strains is of great interest because of a wide variability of the virus strains, and mixed infections of multiple viruses or strains commonly associated with field grown soybean. In this study, RNAi-mediated silencing of the soybean mosaic virus (SMV) P3 cistron, which is reported to participate in virus movements and pathogenesis and to be the putative determinant of SMV virulence, was used to induce resistance to multiple Potyvirus strains and isolates in soybean. A 302bp inverted repeat (IR) of the P3 cistron, isolated from the SMV strain SC3, was introduced into soybean. The transgenic lines exhibited stable and enhanced resistance to SMV SC3 under field conditions over 3 consecutive years. The transgenic lines also showed significantly enhanced resistance to four other SMV strains (SC7, SC15, SC18, and SMV-R, a novel recombinant found in China), the soybean-infecting bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV). Nevertheless, no significant differences were found between transgenic plants and their non-transformed (NT) counterparts in terms of resistance to bean pod mottle virus (BPMV, Secoviridae). Consistent with the results of resistance evaluations, the expression of the respective viral CP cistrons and virus accumulation were significantly lower in seven Potyvirus strains and isolates than in the NT plants, but not in BCMV-inoculated transgenic lines. The results demonstrate the effectiveness of engineering resistance to multiple Potyvirus strains and isolates via RNAi-mediated SMV P3 cistron silencing, and thus provide an effective control strategy against Potyvirus infections in soybean and other crops.

RNAi-mediated suppression of dihydroflavonol 4-reductase in tobacco allows fine-tuning of flower color and flux through the flavonoid biosynthetic pathway

To examine flux regulation in the flavonoid pathway of tobacco flowers, we suppressed two genes for dihydroflavonol 4-reductase (NtDFR 1 and 2) by RNA interference (Ri)-mediated post transcriptional gene silencing in pink-flowered tobacco. Two phenotypes were observed, pale pink (DFR-Ri_PP)- and white (DFR-Ri_W)-flowered lines. The relative mRNA levels of NtDFR genes in DFR-Ri_PP and DFR-Ri_W lines were reduced by 79%–95% relative to non-transformed (NT) plants. DFR-Ri_W lines had five-fold higher levels of small interference RNAs compared to DFR-Ri_PP lines. Expression of eight structural genes in the flavonoid pathway was significantly increased in DFR-Ri_W lines but not in DFR-Ri_PP lines based on quantitative RT-PCR. Anthocyanin contents correlated with flower color, with a reduction of 72%–97% in DFR-Ri_PP and DFR-Ri_W lines. Decreases in anthocyanin in flower were proportional with reductions of proanthocyanidin content in seeds. Two pale pink lines, DFR-Ri_PP 17 and 20, with anthocyanin decreases and the lowest level of DFR gene silencing, had higher (dihydro) flavonol production than a white flowered line, DFR-Ri_W 67. This finding suggests that suppression of DFR can increase the total levels of flavonoids due to (dihydro) flavonol biosynthesis. Our observations that higher suppression of DFR had a greater influence on the expression of flavonoid biosynthetic genes demonstrates the key role of DFR in the pathway and allows selection among DFR-Ri lines for plants with specific gene expression profiles to fine-tune flux through the pathway.

Salinity and osmotic stress trigger different antioxidant responses related to cytosolic ascorbate peroxidase knockdown in rice roots

Salinity and osmotic stress trigger distinct signals in roots, which might induce differences in antioxidant responses. To clarify these relationships, transgenic rice plants silenced in both cytosolic ascorbate peroxidases (apx1/2) and non-transformed (NT) were exposed to iso-osmotic concentrations of NaCl and mannitol. Under both stress conditions, apx1/2 roots did not suffer oxidative stress, revealing that cytosolic APXs were not crucial to oxidative protection. Silenced and non-transformed roots triggered different responses to high salinity and osmotic stress and these stressful factors induced also distinct antioxidant changes. High salinity up-regulated expression of important OsAPX isoforms and these changes were related to increased APX activity, especially in NT roots. Intriguingly, salt stress triggered up-regulation of OsCAT isoforms but CAT activity did not change in both genotypes. In contrast, mannitol trigged very low increment in expression of OsAPX isoforms but induced substantial up-regulation in APX activity in NT roots. Mannitol also remarkably up-regulated OsCATB expression in parallel to CAT activity, in both apx1/2 and NT roots. POD and GPX (glutathione peroxidases) activities were strongly increased by high salinity but did not change in response to mannitol, in both genotypes. The two stress types as well as apx1/2 and NT roots displayed different response in terms of modulation in the H2O2 levels but lipid peroxidation did not change. Membrane integrity was drastically affected by both stressful factors and similarly in both genotypes, whereas root fresh matter was affected only by salt stress. Altogether, the obtained data reveal that high salinity and osmotic stress trigger different antioxidant responses and these strategies were genotype-dependent. The different antioxidant molecular-biochemical mechanisms employed by cytosolic APX knockdown and non-transformed roots allowed reaching similar physiological performance.

Transgenic potato plants expressing the cold‐inducible transcription factor SCOF‐1 display enhanced tolerance to freezing stress

AbstractPotato (Solanum tuberosum) is a frost‐sensitive species that is incapable of cold acclimation. Brief exposure to frost can significantly reduce yield, and hard frost can completely destroy entire crops. Thus, enhancing freezing tolerance by even a few degrees would considerably improve crop hardiness to frost damage. Transgenic potato plants expressing the soybean cold‐inducible zinc finger transcription factor (SCOF‐1) under control of the oxidative stress‐inducible sweet potato peroxidase (SWPA2) promoter (designated as SF plants) were generated, and leaf discs and whole plants were evaluated for enhanced tolerance to cold and freezing conditions. SF plants were treated with 4°C for 5 days, and SCOF‐1 expression positively correlated with tolerance to cold stress. SF plants treated with cold stress exhibited greater photosynthetic activity and total chlorophyll levels than those in cold‐stressed non‐transformed (NT) plants. During freeze–thaw treatment, lipid peroxidation levels in SF plants recovered to approximately prestress levels after 12 h of recovery at 25°C, whereas lipid peroxidation remained at an increased level in NT plants after the same recovery period. The results of our study indicate that freezing stress can be efficiently enhanced in potato by overexpressing SCOF‐1.

Mitochondrial GPX1 silencing triggers differential photosynthesis impairment in response to salinity in rice plants

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2 O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2 O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.

Bread making with Saccharomyces cerevisiae CEN.PK113‐5D expressing lipase A from Bacillus subtilis: leavening characterisation and aroma enhancement

SummaryLipase A from Bacillus subtilis was expressed in the yeast Saccharomyces cerevisiae CEN.PK113‐5D strain as a cell wall‐immobilised enzyme. The recombinant yeast was used in bread making to test the effect of lipase A on the bread properties such as rheological and aromatic properties. The results were compared to the not transformed strain and the commercial baker's yeast. The recombinant strain resulted a good leavening agent comparable to the commercial baker's yeasts provided 1% sucrose was added to the dough. Whereas, during the leavening, the trend of the rheological properties (cohesivness, gumminess and rigidity) differed from the commercial and the nontransformed (NT) strains. Moreover, using of the recombinant strain enhanced total aroma which achieved in the baked product a value of 9 μg per gproduct vs the 4 and 5 μg per gproduct of the NT strain and the commercial baker's yeast, respectively. To our knowledge, this is the first time that a bacterial lipase is used in bread making.

Introduction of the Arabidopsis PHYB gene increases resistance of photosynthetic apparatus in transgenic Solanum tuberosum plants to UV-B radiation

The influence of phytochrome B overproduction in 50- to 60-day-old transgenic potato plants (Solanum tuberosum L., lines Dara 5 and Dara 12 with moderate and intense expression of PHYB, respectively) on the resistance of photosynthetic apparatus to UV-B irradiation was investigated. In plants unexposed to UV-B, there was no significant difference in photosynthetic rates (P n) and fluorescence parameters (F v/F m, qN, qP) between the nontransformed (NT) line and Dara-5 and Dara-12 lines, whereas the content of photosynthetic pigments per 1 cm2 leaf area was higher in the transgenic plants. Irradiation with UV-B resulted in the decrease in photosynthetic rate in NT plants by 35–45%, whereas in Dara-12 line this rate was lowered by only 20–25%. Exposure to UV-B reduced the amplitudes of both fast and slow components of delayed fluorescence (DF), which indicated the diminished efficiency of photosystem II (PSII). The decrease in the maximum amplitude of the slow DF component was markedly lower in Dara-12 (19%) than in NT line (33%). The maximal photochemical quantum yield of PSII (F v/F m ratio) in plants exposed to UV-B was also suppressed stronger in NT line than in Dara-12. The line Dara-5 had intermediate position among other lines in terms of UV-B resistance of photosynthesis and PSII activity, but it was closer to NT than to Dara-12. Thus, the potato plants actively expressing the gene of Arabidopsis phytochrome B apoprotein (PHYB) demonstrated a higher resistance of photosynthetic apparatus to UV-B radiation compared to nontransformed plants. The elevated UV-B tolerance is most likely related to the increased leaf content of chlorophyll, carotenoids, and flavonoids.