共查询到20条相似文献,搜索用时 31 毫秒
1.
Janewit Wongratana Thanate Juntadech Chutima Sereeruk Chanan Angsuthanasombat Kittisak Yokthongwattana 《Journal of applied phycology》2013,25(2):445-452
Histidine tags attached to subunits of photosystem II (PSII) have proven to be very useful tools for isolation and purification of the complex for investigation of its components. However, it has been reported that Chlamydomonas reinhardtii transformants carrying N-terminal histidine-tagged version of the PSII D1 reaction center protein could not grow photoautotrophically. We report here a successful generation of C. reinhardtii transformants expressing histidine-tagged version of the PsbA protein that are capable of photoautotrophic growth. Biochemical and physiological analyses revealed that the histidine tag present at the N terminus of the D1 subunit did not cause total instability to the PSII complex as assessed by their phototrophic growth and oxygen evolution capability. Simple one-step affinity column chromatography also revealed that the histidine-tagged D1 subunit as well as its associated proteins could be effectively purified. These transformants could potentially serve as very good tools for the study of the PSII complex particularly the D1 protein. 相似文献
2.
Yuichiro Takahashi 《Journal of plant research》1998,111(1):101-111
The photosystem I, photosystem II, and cytochromeb
6
f complexes that are involved in electron transport of oxygenic photosynthesis consist of a number of subunits encoded by either
the chloroplast or nuclear genomes. In addition to the major subunits that carry redox components or photosynthetic pigments,
these complexes contain several to more than ten subunits with molecular masses of less than 10 kDa. Directed mutagenesis
has served as a powerful tool for investigation of the roles of these small subunits in the organization or function of the
complexes. Various chloroplast transformants of the green algaChlamydomonas reinhardtii and mutants of cyanobacteria in which a gene encoding a small subunit was deleted or altered have been constructed. Evidence
has accumulated suggesting that these small subunits function in the assembly, stabilization, or protection from photoinhibition
of the complexes or in the modulation or regulation of electron transport. This article presents an overview of the properties
and functions of the chloroplast-encoded small subunits of the three multiprotein complexes of photosynthetic electron transport
that have been mainly analyzed with chloroplast transformants ofC. reinhardtii and the corresponding cyanobacterial transformants.
Recipient of the Botanical Society Award for Young Scientists, 1995. 相似文献
3.
Heinz D. Osiewacz 《Archives of microbiology》1992,157(4):336-342
The gene encoding subunit IV of the cytochrome b6/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostos sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella protothecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.Abbreviations
aph
aminoglycoside 3-phosphotransferase
-
cpDNA
chloroplast DNA
-
km
kanamycin
-
PSI
photosystem I
-
PSII
photosystem II 相似文献
4.
One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1. 相似文献
5.
Gudynaite-Savitch L Gretes M Morgan-Kiss RM Savitch LV Simmonds J Kohalmi SE Hüner NP 《Molecular genetics and genomics : MGG》2006,275(4):387-398
Although cytochrome f from the Antarctic psychrophile, Chlamydomonas raudensis UWO 241, exhibits a lower apparent molecular mass (34 kD) than that of the mesophile C. reinhardtii (41 kD) based on SDS-PAGE, both proteins are comparable in calculated molecular mass and show 79% identity in amino acid
sequence. The difference in apparent molecular mass was maintained after expression of petA from both Chlamydomonas species in either E. coli or a C. reinhardtii ΔpetA mutant and after substitution of a unique third cysteine-292 to phenylalanine in the psychrophilic cytochrome f. Moreover, the heme of the psychrophilic form of cytochrome f was less stable upon heating than that of the mesophile. In contrast to C. raudensis, a C. reinhardtii ΔpetA mutant transformed with petA from C. raudensis exhibited the ability to undergo state transitions and a capacity for intersystem electron transport comparable to that of
C. reinhardtii wild type. However, the C. reinhardtii
petA transformants accumulated lower levels of cytochrome b
6
/f complexes and exhibited lower light saturated rates of O2 evolution than C. reinhardtii wild type. We show that the presence of an altered form of cytochrome f in C. raudensis does not account for its inability to undergo state transitions or its impaired capacity for intersystem electron transport
as previously suggested. A combined survey of the apparent molecular mass, thermal stability and amino acid sequences of cytochrome
f from a broad range of mesophilic species shows unequivocally that the observed differences in cytochrome f structure are
not related to psychrophilly. Thus, caution must be exercised in relating differences in amino acid sequence and thermal stability
to adaptation to cold environments.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
6.
The unicellular green alga Chlamydomonas reinhardtii possesses a [FeFe]-hydrogenase HydA1 (EC 1.12.7.2), which is coupled to the photosynthetic electron transport chain. Large
amounts of H2 are produced in a light-dependent reaction for several days when C. reinhardtii cells are deprived of sulfur. Under these conditions, the cells drastically change their physiology from aerobic photosynthetic
growth to an anaerobic resting state. The understanding of the underlying physiological processes is not only important for
getting further insights into the adaptability of photosynthesis, but will help to optimize the biotechnological application
of algae as H2 producers. Two of the still most disputed questions regarding H2 generation by C. reinhardtii concern the electron source for H2 evolution and the competition of the hydrogenase with alternative electron sinks. We analyzed the H2 metabolism of S-depleted C. reinhardtii cultures utilizing a special mass spectrometer setup and investigated the influence of photosystem II (PSII)- or ribulosebisphosphate-carboxylase/oxygenase
(Rubisco)-deficiency. We show that electrons for H2-production are provided both by PSII activity and by a non-photochemical plastoquinone reduction pathway, which is dependent
on previous PSII activity. In a Rubisco-deficient strain, which produces H2 also in the presence of sulfur, H2 generation seems to be the only significant electron sink for PSII activity and rescues this strain at least partially from
a light-sensitive phenotype. The latter indicates that the down-regulation of assimilatory pathways in S-deprived C. reinhardtii cells is one of the important prerequisites for a sustained H2 evolution. 相似文献
7.
《BBA》2013,1827(10):1183-1190
Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris–acetate–phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris–acetate–phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less 1O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of 1O2 originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (Em) of the quinone electron acceptors. Thermoluminescence indicated that the Em of the primary quinone acceptor (QA/QA−) of mixotrophic cells was stabilised while the Em of the secondary quinone acceptor (QB/QB−) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to 1O2 production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting photosystem II charge recombination events and photoinhibition. 相似文献
8.
PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I. 相似文献
9.
Growth of a cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be impaired under photoautrotrophic, but not under mixotrophic conditions. As compared to wild-type cells, oxygen evolution by the photoautotrophically grown mutant was considerably decreased and higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The capability for oxygen production was decreased in Chlamydomonas reinhardtii cells when grown in the presence of acetate without aeration. Wild-type cells grown under these conditions showed a rather low but significant oxygen evolution immediately after transfer to photoautotrophic conditions. This residual oxygen production was completely suppressed in the presence of acetate, obviously due to acetate inhibition of the water-splitting complex. In the case of our cadmium-tolerant mutant strain, however, residual oxygen production was measured even in the presence of acetate. After removal of acetate, oxygen evolution by the cadmium-tolerant mutant strain was increased to higher rates than measured for wild-type cells, but considerably higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The conclusion that the donor side of photosystem II is affected in our cadmium-tolerant mutant strain was further corroborated by a stronger decrease of the fluorescence level caused by hydroxylamine. 相似文献
10.
Anastasios Melis Akio Murakami Jeff A. Nemson Katsunori Aizawa Kaori Ohki Yoshihiko Fujita 《Photosynthesis research》1996,47(3):253-265
The work addressed the adjustment of the photosystem ratio in the green algaChlamydomonas reinhardtii. It is shown that green algae, much like cyanophytes and higher plants, adjust and optimize the ratio of the two photosystems in chloroplasts in response to the quality of irradiance during growth. Such adjustments are compensation reactions and helpC. reinhardtii to retain a quantum efficiency of oxygen evolution near the theoretical maximum. Results show variable amounts of PS I and a fairly constant amount of PS II in chloroplasts and suggest that photosystem stoichiometry adjustments, occurring in response to the quality of irradiance during plant growth, are mainly an adjustment in the concentration of PS I. The work delineates chromatic effects on chlorophyll accumulation in the chloroplast ofC. reinhardtii from those pertaining to the regulation of the PS I/PS II ratio. The detection of the operation of a molecular feedback mechanism for the PS I/PS II ratio adjustment in green algae strengthens the notion of the highly conserved nature of this mechanism among probably all oxygen evolving photosynthetic organisms. Findings in this work are expected to serve as the basis of future biochemical and mutagenesis experiments for the elucidation of the photosystem ratio adjustment in oxygenic photosynthesis. 相似文献
11.
Site-directed mutations were introduced into PsbO protein of photosystem 2 to study the role of two lysine residues, 223 and
226 (LGAKPPK), in the green alga Chlamydomonas reinhardtii. Lysines 223 and 226 homologous to His228 and His231 from cyanobacteria are located on the protein side facing the lumen
and can participate in formation of a channel connecting the Mn cluster with the intrathylakoid space. The K223E and K226E
mutants were generated on the basis of the ΔpsbO strain of C. reinhardtii with the substitution of glutamic acid for the lysine residues. The K226E mutation leads to a decrease in stability of the
protein and development of the ΔpsbO phenotype (the absence of both photosynthetic activity of photosystem 2 and photoautotrophic growth), with substantially
decreased PsbO content in the cells. In the case of K223E, the mutant strain accumulated the normal level of PsbO protein
and was able to grow photoautotrophically and to evolve oxygen. However, the rate of oxygen evolution and the F
v/F
m ratio were reduced by 15–20% compared to the control. Also, the time of the dark decay of F
v in the presence of DCMU in the cells of the K223E mutant was increased, indicating impairment in the water-oxidizing complex.
In general, our study shows the importance of amino acids K223 and K226 located at the lumenal surface of PsbO protein for
the activity of the water-oxidizing complex. 相似文献
12.
13.
This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function
of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted
transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding
protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated
to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the
stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane
domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin
genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying
slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the
Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of
sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and
H2-evolution related metabolism in this green alga. 相似文献
14.
Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum 总被引:10,自引:0,他引:10
Johanna Gehlen Ralph Panstruga Helga Smets Sabine Merkelbach Michael Kleines Petra Porsch Matthias Fladung Irmgard Becker Thomas Rademacher Rainer E. Häusler Heinz-Josef Hirsch 《Plant molecular biology》1996,32(5):831-848
Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K
m(PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing and antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.Abbreviations
CO2 compensation point
- CO2
quantum efficiency of CO2 assimilation
- PSII
quantum efficiency of photosystem II electron transport
- A
CO2 assimilation rate
- Ci
intercellular CO2 concentration; e, electron
- PFD
photon flux density
- QA
primary quinone electron acceptor of photosystem II
- QN
non-photochemical chlorophyll a fluorescence quenching
- qP
photochemical chlorophyll a fluorescence quenching 相似文献
15.
The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions. 相似文献
16.
17.
James V. Moroney N. E. Tolbert Barbara B. Sears 《Molecular & general genetics : MGG》1986,204(2):199-203
Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO2 for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO2 fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS
high salt medium which has 13 mM phosphate at pH 6.8
- HSA
high salt plus 36 mM acetate medium
- YA
high salt medium with 4 g yeast extract per L and 36mM acetate
- Arg
arginine
- cia-
CO2 accumulation mutants that cannot grow on low CO2
- Ci
inorganic carbon (CO2+HCO
-
3
)
- CA
carbonic anhydrase
- mt
mating type
Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station 相似文献
18.
The fluorescence kinetics of both intact needles and isolated chloroplasts of summer active and winter stressed Pinus sylvestris were measured at both room temperature and 77 K. It was confirmed that winter stress inhibited the photochemical capacity of photosystem II but also that winter stress caused the strongest inhibition of the electron transport at the site where the plastoquinone pool is reduced. Parallel analyses of the fluorescence characteristics of photosystem II (F693) and photosystem I (F729) during photosystem II trap closure furthermore revealed that the yield of spillover of excitation energy from photosystem II to photosystem I decreased upon winter stress. We suggest that this is because of an increased radiationless decay of excitation energy both at the reaction center and antennae levels of photosystem II. There is, however, also a possibility that the decreased yield of spill-over is accentuated by a partial detachment of the light harvesting chlorophyll a/b complex from photosystem II upon winter stress.Paper presented at the FESPP meeting in Strasbourg (1984). 相似文献
19.
Cong-Ping Tan Fang-Qing Zhao Zhong-Liang Su Cheng-Wei Liang Song Qin 《Journal of applied phycology》2007,19(4):347-355
A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding β-carotene hydroxylase that was able to catalyze the conversion of β-carotene to zeaxanthin and canthaxanthin to
astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast
genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RT-PCR assays showed that the H. pluvialis
crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred
from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible
mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation. 相似文献
20.
In order to investigate the possible impacts of increased atmospheric CO2 levels on algal growth and photosynthesis, the influence of CO2 concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO2 concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 M CO2 in C. reinhardtii, C. pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO2 was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N inC. pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p < 0.05) in high-CO2-grown cells. Elevation of the CO2 levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C. pyrenoidosa and S. obliquus when exposed to high photon flux density. The photoinhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO2-grownC. pyrenoidosa and S. obliquus. Although pH and pCO2 effects cannot be distinguished from this study, it can be concluded that increased CO2 concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus. 相似文献