Involvement of DEG5 and DEG8 proteases in the turnover of the photosystem II reaction center D1 protein under heat stress in Arabidopsis thaliana

Deg5,deg8 and the double mutant,deg5deg8 of Arabidopsis thaliana were used to study the physiological role of the DEG proteases in the repair cycle of photosystem II (PSII) under heat stress. PSII activity in deg mutants showed increased sensitivity to heat stress, and the extent of this effect was greater in the double mutant, deg5deg8, than in the single mutants, deg5 and deg8. Degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, our results indicate that DEG5 and DEG8 may have synergistic function in degradation of D1 protein under heat stress.     

Recombinant PsbF from Synechococcus sp. PCC 7002 forms β:β homodimeric cytochrome b559

All organisms with oxygenic photosynthesis contain two photosystems:photosystem Ⅰ(PSⅠ)and photo-systemⅡ(PSⅡ),The minimal photosystem Ⅱ particles which are photochemically active contain three subunits:D1,D2 and cytochrome b559 (Cyt b559),The function of Cyt b559 remains unclear,We have successfully overxpressed the psbF gene,encoding the β subunit of Cyt b559,from a marine cyanobacterium Synechoccous sp.PCC 7002 as a fusion gene and obtained a redox-active from of Cyt b559,When the N-terminal GST protein of the fusion gene product was removed with thrombin ,the PsbF protein was still redox-active,suggesting that the recombinant PsbF can form dimer in Escherichia coli.The absorption spectra of either the oxidized from or the reduced form of both GST fusion protein and the purified PsbF dimer and the difference Spectra between the two forms are the same as that of the Cyt b559 isolated from the higher plants .Redox titration analysis of recombinant PsbF showed that the mid-point redox potential of the recombinant Cyt b559 was approximately 50 mV, which is close to the low potential of Cyt b559 ,The results are helpful to the understanding of locatlization and function of Cyt b559 on thylakoid membranes.Ⅰ     

Improving photosynthesis of microalgae by changing the ratio of light-harvesting pigments

Changing the ratio of light-harvesting pigments was regarded as an efficient way to improve the photosynthesis rate in microalgae, but the underlying mechanism is still unclear. In the present study, a mutant of Anabeana simensis （called SP） was selected from retrieved satellite cultures. Several parameters related with photosynthesis, such as the growth, photosynthesis rate, the content of photosynthetic pigment, low temperature fluorescence spectrum （77K） and electron transport rate, were compared with those of the wild type. It was found that the change in the ratio of light-harvesting pigments in the mutant led to more efficient light energy transfer and usage in mutant than in the wild type. This may be the reason why the mutant had higher photosynthesis and growth rates.     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.     

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coil When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.     

Role of two phosphohexomutase genes in glycogen synthesis in Synechocystis sp. PCC6803

Phosphohexomutases catalyze the interconversion between hexose-6-phosphate and hexose-l-phosphate and play important roles in polysaccharide synthesis. In Synechocystis sp. PCC 6803, sl10726 is predicted to encode PGM （phosphoglucomutase）, slr1334 is predicted to encode a PGM/PMM （phosphomannomutase） bifunction enzyme. In comparison to the wild type, a sllO726-null mutant showed 3.4% PGM activity but 45%-69% glycogen content. Down-regulation of slr1334, an essential gene, by using a copper regulated promoter further decreased the PGM activity in the sllO726：：Kmr PpetE-slr1334 double mutant to 0.3% of the wild type level. However, the glycogen content was not further decreased in parallel. In vitro, recombinant Sl10726 or S1r1334 showed predicted enzyme activities. Our results indicate that a relatively high level of glycogen can be maintained in Synechocystis mutants with low levels of PGM activity. The high PGM activity in the cyanobacterium may be required for turnover of glycogen or synthesis of other polysaccharides or oligosaccharides.     

Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

Low concentrations of NaHSO3 enhance NAD(P)H dehydrogenase-dependent cyclic photophosphorylation and alleviate the oxidative damage to improve photosynthesis in tobacco

Bisulfite at low concentrations(L-NaHSO3) increases cyclic electron transport around photosystem I(PSI) and photosynthesis.However,little is known regarding the detailed contribution of cyclic electron transport to the promoted photosynthesis by L-NaHSO3.In the present work,we used tobacco mutant defective in ndhC-ndhK-ndhJ(ndhCKJ) to investigate the role of NAD(P)H dehydrogenase(NDH)-dependent cyclic electron transport around PSI in an increase in photosynthesis by L-NaHSO3.After the treatment of tobacco leaves with L-NaHSO3(10 μmol L-1),the NDH-dependent cyclic electron transport,monitored by a transient post-illumination increase in Chl fluorescence and the amount of NDH,was notably up-regulated in wild type(WT).The NDH-dependent cyclic electron transport was severely impaired in ndhCKJ and was not significantly affected by treatment with L-NaHSO3.Accordingly,the NDH-dependent transthylakoid membrane proton gradient(pH),as reflected by the slow phase of millisecond-delayed light emission(ms-DLE),was increased by L-NaHSO3 in WT,but not in ndhCKJ;the enhancement of cyclic photophosphorylation(PSP) activity by L-NaHSO3 was more obvious in WT than ndhCKJ.The accumulation of both superoxide and hydrogen peroxide was reduced in WT when subjected to L-NaHSO3 treatment,but not in ndhCKJ.Furthermore,the increase of photosynthetic O 2 evolution rate by L-NaHSO3 was more significant in WT than in ndhCKJ.We therefore conclude that L-NaHSO3 alleviates the photo-oxidative damage by the enhancement of NDH-dependent cyclic PSP,thereby improving photosynthesis.     

Preliminary study on a gravity－insensitive rice mutant

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant‘s shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.     

An on-line multi-parameter analyzing optical biosensor for real-time and non-invasive monitoring of plant stress responses in vivo

Photosynthetic dysfunction and reactive oxygen species （ROS） production are the common features of plant stress responses. Based on quantitative measurement of ROS production and delayed fluorescence （DF） emission, which is an excellent marker for evaluating photosynthesis, an on-line multiparameter analyzing optical biosensor for detecting plant stress responses was developed. Performances of the proposed biosensor were tested in the wild type （WT） Arabidopsis and heat shock protein （Hsp） 101 T-DNA knockout mutant （hsp101） plants with different thermotolerance. Results demonstrated that DF intensity correlates with net photosynthesis rate （Pn） in response to elevated temperature in both the WT Arabidopsis and hsp101 mutant plants. The light response characteristics and the recovery dynamics of the DF intensity were also in line with those of Pn in both the WT Arabidopsis and hsp101 mutant plants after heat stress （HS, 40℃ for 30 min）, respectively. In all experiments discussed above, the hsp101 plant showed the worse photosynthetic performance than the WT plant. Moreover, after HS, more ROS production in the hsp101 mutant than in WT Arabidopsis, which was found to be mainly localized at chloroplasts, could be directly detected by using the proposed biosensor. In addition, the hsp101 mutant showed severer chloroplasts alterations than the WT plant within the first 1 h of recovery following HS. Nevertheless, pre-infiltration with catalase （CAT） reduced ROS production and prevented the declines of the DF intensity. Therefore, HS-caused declines of photosynthetic performance might be due to oxidative damage to photosynthetic organelle. To sum up, we conclude that Hsp101 plays an important role in preventing oxidative stress, and the proposed optical biosensor might be a powerful tool to determine plant stress responses and identify plant resistant difference.     

Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60A and F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b, E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration of the surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochrome bj, its variants and cytochrome c at different temperature and ionic strength exhibited an order of F35Y > wild type > E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggesting that the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rate constants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer process. The significant difference of Cyt b, F35Y and E44/48/56A/D60A from the wild type protein further confirmed the great importance of the electrostatic interaction in the protein electron transfer.     

Increase in electron transfer activity in photosystem Ⅱ of spinach thylakoids caused by conversion of phosphatidyl- glycerol to phosphatidic acid molecules

The techniques of oxygen electrode polarography, sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) were employed to investigate the effect of phospholipase D treatment on physiological function of spinach thylakoids. It was shown that the phospholipase D treatment on thylakoid resuited in the degradation of phosphatidylglycerol (PG) and occurrence of phosphatidic acid (PA). The changes of PG to PA molecules caused an increase in oxygen evolution in photosystem Ⅱ (PS Ⅱ), which was accompanied by an uncoupling effect on thylakoid membrane. It was revealed that the head-groups of PG molecules play an important role in themaintenance of the appropriate physiological activity of thylakoid membrane.     

Construction and characterization of partially ntrC-deleted mutants in Alcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Kmr fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC-) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifA-lacZ gene was introduced into the ntrC- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC- mutant was nif + , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

An Arabidopsis ctpA homologue is involved in the repair of photosystem II under high light

A T-DNA insertion mutant AtctpA1 was identified to study the physiological roles of a carboxyl-terminal processing protease (CtpA) homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.     

Sinorhizobium meliloti nifA gene exerts a pleiotropic effect on nodulation through the enhanced plant defense response

Sinorhizobium meliloti nifA gene is required for the expression of a bunch of nif and fix genes. Here, we report its pleiotropic effects on the nodule formation. Compared with wild type strain, nifA mutant sig- nificantly reduced nodule suppression rate in split-root system. The plants inoculated with mutant strain produced lower amount of daidzein and less necrotic cells on their roots. In addition, the defense genes failed to be evoked by nifA mutant at the early nodulation stage. These findings indicated that host defense response was one of the mechanisms mediated by nifA gene to regulate nodule formation during symbiosis. Even though nifA mutant could increase the number of nodules in host plant, it synthesized lower Nod factors than wild type. This suggested that nifA gene mediated multiple and diverse instances in nodulation formation.     

Protection of phosphatidylcholine to photosystem Ⅱ membrane during heat treatment

Photosystem Ⅱ membrane was reconstituted with phosphatidylcholine (PC) with different kinds of fatty acyl chains and the protection of PC to photosystem Ⅱ (PS Ⅱ)membrane during heat treatment was investigated using oxygen electrode, variable fluorescence and circular dichroism (CD) spectroscopy. Heat treatment decreased the oxygen evolution rate and the F′v/Fm′ ratio of PS Ⅱ membrane and influenced CD spectra of PS Ⅱ membrane, but PC inhibited the effect of heat treatment on the oxygen evolution rate, the F′v/F′m ratio and CD spectra of PS Ⅱ membrane. The results indicate that PC can protect PS Ⅱ membrane against heat treatment and the alterations in the unsaturated fatty acid extent in PC can cause the changes of the protection ability.     

Thermal stability of oxygen evolution in photosystem Ⅱ core complex in the presence of digalactosyl diacylglyceroli

The influence of digalactosyldiacylglycerol (DGDG), one of the photosynthetic membrane lipids, on heat inactivation of the process of oxygen evolution has been studied in vitro in photosystem Ⅱ (PSⅡ) core complex. It was found that the temperature of semi-inactivation of oxygen evolution in the complex increased from 40.0 to about 43.0℃ in the presence of DGDG with 5-min heat treatment in the dark. Furthermore, when PSⅡ core complex was incubated for 5 min at 45.0℃, the oxygen evolution in the complex was completely lost, whilst the DGDG-complexed PSⅡ core complex still retained a 16% of activity (100% for 25.0℃). In addition, a 1-h incubation at 38.0℃ inactivated absolutely the oxygen evolution for the PSⅡ core complex. By contrast, there remained about 20% of activity (zero time for 100%) for the complex in the presence of DGDG under the same condition. These results indicate a new role of DGDG in the protection of PSⅡ core complex against the deleterious effects of temperature. It was most likely that DGDG-mediated stability toward thermal denaturation of oxygen evolution in PSⅡ core complex is due to the protective effect of DGDG on the release of the 33 kD protein from PSⅡ core complex.     

ATPG is required for the accumulation and function of chloroplast ATP synthase in Arabidopsis

The subunit Ⅱ of chloroplast ATP synthase is one of the two peripheral stalks, which associates the catalytic CF1 with mem-brane-spanning CFo . Although the structural and functional roles of chloroplast ATP synthase have been extensively examined, the physiological significance of subunit Ⅱ in vivo is still unclear. In this work, we identified one Arabidopsis T-DNA insertion mutant of atpG gene encoding the subunit Ⅱ of chloroplast ATP synthase. The atpg null mutant displayed an albino lethal pheno-type, as it could not grow photoautotrophically. Transmission electron microscopy analysis showed that chloroplasts of atpg lacked the organized thylakoid membranes. Loss of subunit Ⅱ affected the accumulation of CF1-CFo complex, however, it did not seem to have an effect on the CF1 assembly. The light induced ATP formation of atpg was significantly reduced compared with the wild type. Based on these results, we suggested that ATPG was essential for the accumulation and function of chloroplast ATP synthase.     

The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain （DJ 194） of Azotobacter vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (△nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194).The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that △nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, △nifZ Avl was also similar to OP Av1 at the molybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (△ε) of circular dichroism (CD)at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the △nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H^ and N2)-reduction activity of △nifZ Av1 were 74% and 46%-50% of those of OP Av1, respectively. Furthermore, the △ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between △nifZ Avl and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in △nifZ Av1, but the composition or redoxstate of P-cluster in △nifZ Av1 were not changed. Thus it could propose that △nifZ Av1 is composed of two different αβsubunit pairs. One is a FeMoco-and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-containing pair. Since the deletion of nifZ gene leads to the deficiency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.