Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reduc-tase, type C （NTRC） in the light. Here we show overexpression of NTRC in Arabidopsis （NTRC°E） resulting in...Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reduc-tase, type C （NTRC） in the light. Here we show overexpression of NTRC in Arabidopsis （NTRC°E） resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants （ntrcl） exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the protein＇s biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure.. Complexes of higher molecular weight （HMW） showed stronger activity as a holdase chaperone, while low molecular weight （LMW） species exhibited weaker holdase chaperone activity but stronger disulfide reductase and fol-dase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser （C217/454S-NTRC） led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°E plants. However, after prolonged incubation under heat shock, NTRC°E plants tolerated the stress to a higher degree than C217/454S-NTRC°E plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.展开更多
To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially nfrC-deleted mutants of A. faecalis have been generated. To start w...To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially nfrC-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 pSUML The nfrCgene in pSUM1 was then replaced by a /acZ-Kmr fragment resulted in the generation of a plasmid pSUM2. The lacZfragment in pSUM2 was further removed and a plasmid pSUMS 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 nfrC-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%展开更多
Sunlight represents the energy source for photosynthesis and plant growth. When growing in the field, plant photosynthesis has to manage strong fluctuations in light intensities. Regulation based on the thio- redoxin ...Sunlight represents the energy source for photosynthesis and plant growth. When growing in the field, plant photosynthesis has to manage strong fluctuations in light intensities. Regulation based on the thio- redoxin （Trx） system is believed to ensure light-responsive control of photosynthetic reactions in the chlo- roplast. However, direct evidence for a role of this system in regulating dynamic acclimation of photosyn- thesis in fluctuating conditions is largely lacking. In this report we show that the ferredoxin-dependent Trxs ml and m2 as well as the NADPH-dependent NTRC are both indispensable for photosynthetic acclimation in fluctuating light intensities. Arabidopsis mutants with combined deficiency in Trxs ml and m2 show wild- type growth and photosynthesis under constant light condition, while photosynthetic parameters are strongly modified in rapidly alternating high and low light. Two independent trxmlm2 mutants show lower photosynthetic efficiency in high light, but surprisingly significantly higher photosynthetic efficiency in low light. Our data suggest that a main target of Trx ml and m2 is the NADP-malate dehydrogenase involved in export of excess reductive power from the chloroplast. The decreased photosynthetic efficiency in the high-light peaks may thus be explained by a reduced capacity of the trxm lm2 mutants in the rapid light acti-vation of this enzyme. In the ntrc mutant, dynamic responses of non-photochemical quenching of excita- tion energy and plastoquinone reduction state both were strongly attenuated in fluctuating light intensities, leading to a massive decrease in PSII quantum efficiency and a specific decrease in plant growth under these conditions. This is likely due to the decreased ability of the ntrc mutant to control the stromal NADP（H） redox poise. Taken together, our results indicate that NTRC is indispensable in ensuring the full range of dynamic responses of photosynthesis to optimize photosynthesis and maintain growth in fluctu- ating light, while Trxs ml and m2 are indispensable for full activation of photosynthesis in the high-light pe- riods but negatively affect photosynthetic efficiency in the low-light periods of fluctuating light.展开更多
文摘Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reduc-tase, type C （NTRC） in the light. Here we show overexpression of NTRC in Arabidopsis （NTRC°E） resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants （ntrcl） exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the protein＇s biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure.. Complexes of higher molecular weight （HMW） showed stronger activity as a holdase chaperone, while low molecular weight （LMW） species exhibited weaker holdase chaperone activity but stronger disulfide reductase and fol-dase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser （C217/454S-NTRC） led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°E plants. However, after prolonged incubation under heat shock, NTRC°E plants tolerated the stress to a higher degree than C217/454S-NTRC°E plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.
文摘To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially nfrC-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 pSUML The nfrCgene in pSUM1 was then replaced by a /acZ-Kmr fragment resulted in the generation of a plasmid pSUM2. The lacZfragment in pSUM2 was further removed and a plasmid pSUMS 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 nfrC-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%
文摘Sunlight represents the energy source for photosynthesis and plant growth. When growing in the field, plant photosynthesis has to manage strong fluctuations in light intensities. Regulation based on the thio- redoxin （Trx） system is believed to ensure light-responsive control of photosynthetic reactions in the chlo- roplast. However, direct evidence for a role of this system in regulating dynamic acclimation of photosyn- thesis in fluctuating conditions is largely lacking. In this report we show that the ferredoxin-dependent Trxs ml and m2 as well as the NADPH-dependent NTRC are both indispensable for photosynthetic acclimation in fluctuating light intensities. Arabidopsis mutants with combined deficiency in Trxs ml and m2 show wild- type growth and photosynthesis under constant light condition, while photosynthetic parameters are strongly modified in rapidly alternating high and low light. Two independent trxmlm2 mutants show lower photosynthetic efficiency in high light, but surprisingly significantly higher photosynthetic efficiency in low light. Our data suggest that a main target of Trx ml and m2 is the NADP-malate dehydrogenase involved in export of excess reductive power from the chloroplast. The decreased photosynthetic efficiency in the high-light peaks may thus be explained by a reduced capacity of the trxm lm2 mutants in the rapid light acti-vation of this enzyme. In the ntrc mutant, dynamic responses of non-photochemical quenching of excita- tion energy and plastoquinone reduction state both were strongly attenuated in fluctuating light intensities, leading to a massive decrease in PSII quantum efficiency and a specific decrease in plant growth under these conditions. This is likely due to the decreased ability of the ntrc mutant to control the stromal NADP（H） redox poise. Taken together, our results indicate that NTRC is indispensable in ensuring the full range of dynamic responses of photosynthesis to optimize photosynthesis and maintain growth in fluctu- ating light, while Trxs ml and m2 are indispensable for full activation of photosynthesis in the high-light pe- riods but negatively affect photosynthetic efficiency in the low-light periods of fluctuating light.
