Zhipan Yang

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO2 assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H2O2 in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.

Heat Stress Induces an Aggregation of the Light-harvesting Complex of Photosystem II in Spinach Plants

Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25°C–50°C) in the dark for 30 min. At temperatures higher than 35°C, CO2 assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45°C and decreased only slightly at 50°C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25°C to 50°C in the dark for 10 min. At temperatures over 35°C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25°C to 50°C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35°C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.

Characterization of photosystem II in transgenic tobacco plants with decreased iron superoxide dismutase

Iron superoxide dismutases (FeSODs) play an important role in preventing the oxidative damage associated with photosynthesis. To investigate the mechanisms of FeSOD in protection against photooxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with severely decreased FeSOD by using a gene encoding tobacco chloroplastic FeSOD for the RNAi construct. Transgenic plants were highly sensitive to photooxidative stress and accumulated increased levels of O₂•⁻ under normal light conditions. Spectroscopic analysis and electron transport measurements showed that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses showed that PSII protein accumulation was also decreased in transgenic plants. PSII photodamage and D1 protein degradation under high light treatment was increased in transgenic plants, whereas the PSII repair was not affected, indicating that the stability of the PSII complex was decreased in transgenic plants. The results in this study suggest that FeSOD plays an important role in maintaining PSII function by stabilizing PSII complexes in tobacco plants.

Sucrose Regulates Elongation of Carrot Somatic Embryo Radicles as a Signal Molecule

Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5%(145 mM). This effect would not be released until the sucrose concentration was lowered again. Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds. Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect. It is thus clear that the regulatory effect is not a result of simple osmotic stress. Hexokinase inhibitors such as glucosamine and N-acetyl-glucosamine did not exert any influence on the regulation–deregulation effects of sucrose. Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating. It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase. As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene (cSUT) expressed in the embryos and roots specifically was isolated. Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity. Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription. This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

Decreased glutathione reductase2 leads to early leaf senescence in Arabidopsis.

Abstract Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) and participates in the ascorbate‐glutathione cycle, which scavenges H2O2. Here, we report that chloroplastic/mitochondrial GR2 is an important regulator of leaf senescence. Seed development of the homozygous gr2 knockout mutant was blocked at the globular stage. Therefore, to investigate the function of GR2 in leaf senescence, we generated transgenic Arabidopsis plants with decreased GR2 using RNAi. The GR2 RNAi plants displayed early onset of age‐dependent and dark‐ and H2O2‐induced leaf senescence, which was accompanied by the induction of the senescence‐related marker genes SAG12 and SAG13. Furthermore, transcriptome analysis revealed that genes related to leaf senescence, oxidative stress, and phytohormone pathways were upregulated directly before senescence in RNAi plants. In addition, H2O2 accumulated to higher levels in RNAi plants than in wild‐type plants and the levels of H2O2 peaked in RNAi plants directly before the early onset of leaf senescence. RNAi plants showed a greater decrease in GSH/GSSG levels than wild‐type plants during leaf development. Our results suggest that GR2 plays an important role in leaf senescence by modulating H2O2 and glutathione signaling in Arabidopsis.

Characterization of photosystem II photochemistry in transgenic tobacco plants with lowered Rubisco activase content.

Rubisco activase plays an important role in the regulation of CO(2) assimilation. However, it is unknown how activase regulates photosystem II (PSII) photochemistry. To investigate the effects of Rubisco activase on PSII photochemistry, we obtained transgenic tobacco (Nicotiana tabacum) plants with 50% (i7), 25% (i28), and 5% (i46) activase levels as compared to wild type plants by using a gene encoding tobacco activase for the RNAi construct. Both CO(2) assimilation and PSII activity were significantly reduced only in transgenic i28 and i46 plants, suggesting that activase deficiency led to decreased PSII activity. Flash-induced fluorescence kinetics indicated that activase deficiency resulted in a slow electron transfer between Q(A) (primary quinine electron acceptor of PSII) and Q(B) (secondary quinone electron acceptor of PSII). Thermoluminescence measurements revealed that activase deficiency induced a shift of S(2)Q(A)(-) and S(2)Q(B)(-) recombinations to higher temperatures in parallel, and a decrease in the intensities of the thermoluminescence emissions. Activase deficiency also dampened the period-four oscillation of the thermoluminescence B-band. Protein gel blot analysis showed that activase deficiency resulted in a significant decrease in the content of D1, D2, CP43, CP47, and PsbO proteins. Transmission electron microscopy analysis demonstrated that activase deficiency induced a significant decrease in the number of grana stacks per chloroplast and discs per grana stack. Our results suggest that activase plays an important role in maintaining PSII function and chloroplast development.

Functional analysis of the rice rubisco activase promoter in transgenic Arabidopsis

To gain a better understanding of the regulatory mechanism of the rice rubisco activase (Rca) gene, variants of the Rca gene promoter (one full-length and four deletion mutants) fused to the coding region of the bacterial reporter gene β-glucuronidase (GUS) were introduced into Arabidopsis via Agrobacterium-mediated transformation. Our results show that a 340 bp fragment spanning from -297 to +43 bp relative to the transcription initiation site is enough to promote tissue-specific and light-inducible expression of the rice Rca gene as done by the full-length promoter (-1428 to +43 bp). Further deletion analysis indicated that the region conferring tissue-specificity of Rca expression is localized within a 105 bp fragment from -58 to +43 bp, while light-inducible expression of Rca is mediated by the region from -297 to -58 bp. Gel shift assays and competition experiments demonstrated that rice nuclear proteins bind specifically with the fragment conferring light responsiveness at more than one binding site. This implies that multiple cis-elements may be involved in light-induced expression of the rice Rca gene. These works provide a useful reference for understanding transcriptional regulation mechanism of the rice Rca gene, and lay a strong foundation for further detection of related cis-elements and trans-factors.

The Phytol Phosphorylation Pathway Is Essential for the Biosynthesis of Phylloquinone, which Is Required for Photosystem I Stability in Arabidopsis

Phytyl-diphosphate, which provides phytyl moieties as a common substrate in both tocopherol and phylloquinone biosynthesis, derives from de novo isoprenoid biosynthesis or a salvage pathway via phytol phosphorylation. However, very little is known about the role and origin of the phytyl moiety for phylloquinone biosynthesis. Since VTE6, a phytyl-phosphate kinase, is a key enzyme for phytol phosphorylation, we characterized Arabidopsis vte6 mutants to gain insight into the roles of phytyl moieties in phylloquinone biosynthesis and of phylloquinone in photosystem I (PSI) biogenesis. The VTE6 knockout mutants vte6-1 and vte6-2 lacked detectable phylloquinone, whereas the phylloquinone content in the VTE6 knockdown mutant vte6-3 was 90% lower than that in wild-type. In vte6 mutants, PSI function was impaired and accumulation of the PSI complex was defective. The PSI core subunits PsaA/B were efficiently synthesized and assembled into the PSI complex in vte6-3. However, the degradation rate of PSI subunits in the assembled PSI complex was more rapid in vte6-3 than in wild-type. In vte6-3, PSI was more susceptible to high-light damage than in wild-type. Our results provide the first genetic evidence that the phytol phosphorylation pathway is essential for phylloquinone biosynthesis, and that phylloquinone is required for PSI complex stability.

Cloning and expression of DnaJ homolog in carrot somatic embryo

Abstract As the co-chaperone of DnaK/Hsp70 protein, DnaJ/Hsp40 protein influences the synthesis and assembly of the protein complex by regulating ATPase activity of DnaK/Hsp70 protein. By employing the modified method of cDNA representational difference analysis, a homologous fragment of DnaJ was isolated from the deregulated carrot somatic embryos and it was further used as the probe to screen the cDNA library of carrot somatic embryo deregulated for 12 h. As the result. DcJ1 gene, the homologous gene of DnaJ, was isolated from carrot. Sequence analysis showed that its coding region is 1257 bp, which codes 418 amino acids and comprises 3 highly-conserved characteristic domains. Southern blot analysis suggested that the DcJ1 gene seems to be a single copy in the genome, while Northern blot result indicated that DcJ1 expresses only in roots and its degree of expression changes obviously with the regulation-deregulation process. These results suggest that DcJ1 is correlated with the early development of car...