Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O₂ evolving activity ranging from 290 (for the wild type) to 1100 mol O₂ x mg chlorophyll^-1 x h^-1 (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochrome_b559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochrome_b559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochrome_b559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O₂ evolving photosystem II reaction center core complexes.Abbreviations PS photosystem - chl chlorophyll - LHCP light-harvesting chlorophyll a/b protein complex     

Electron transport activities of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants with impaired chloroplastic NAD(P)H dehydrogenase

The activities of electron transport are compared between wild-type Arabidopsis and two Arabidopsis mutants deficient for the chloroplastic NAD(P)H dehydrogenase (NDH) which catalyzes cyclic electron transport around photosystem I. The quantum yield of photosystem II and the degree of non-photochemical quenching of chlorophyll fluorescence were of similar levels in the two NDH-deficient mutants and the wild type under non-stressed standard growth conditions. Stromal over-reduction was induced in Arabidopsis NDH mutants with high light treatment, as is the case in tobacco NDH mutants. However, unlike tobacco mutants, photoinhibition was not observed in the Arabidopsis NDH mutants.     

Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803

We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 micromol x m(-2) x s(-1)), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light. (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII. These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.     

Characterization of Three Photosystem II Mutants in Zea mays L. Lacking a 32,000 Dalton Lamellar Polypeptide

Two fully blocked and one partially blocked photosystem II nuclear mutants have been selected in Zea mays. The fully blocked mutants lack photosystem II activity, variable fluorescence, the light-inducible C-550 signal, the high potential form of cytochrome b-559, and most or all of the low potential form of the cytochrome. The block in these mutants may primarily affect the reducing side of photosystem II, inasmuch as chloroplasts isolated from both mutants exhibit an elevated F695 fluorescence emission peak. The partially blocked mutant exhibits partial photosystem II activity and a reduction, but not the total loss of the variable fluorescence yield, the C-550 signal, and the high potential form of cytochrome b-559. Lamellae isolated from the fully blocked mutants are greatly deficient for a major lamellar polypeptide with an apparent molecular weight of 32,000 daltons, whereas lamellae from the partially blocked mutant show the partial loss of this same polypeptide, suggesting that the 32,000 dalton polypeptide is necessary for the proper function of photosystem II.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

温州蜜柑叶片光系统反应中心光能分配的变化

为深入了解果树光化学反应中心光能分配的状况,以柑橘为试材,采用调制荧光法对叶片光系统在高光强和低光强下的状态转换进行了研究．结果表明,光系统在100μmol·m^-2·s^-1的低光强下,由于QA的还原使PQ库处于还原状态,导致光能由PSⅡ转向PSⅠ分配,光系统处于状态2;在1000μmol·m^-2·s^-1的高光强下,PQ库无法得到电子而处于氧化状态,导致光能分配由PSⅠ转向PSⅡ,光系统处于状态1,叶片经磷酸酯酶抑制剂NaF处理后,光系统从高光强下状态2到状态1的转换受到抑制,高光强下过多的光能由PSⅠ向PSⅡ分配是导致PSⅡ光破坏的重要原因．     

Small Cab-like proteins retard degradation of photosystem II-associated chlorophyll in Synechocystis sp. PCC 6803: kinetic analysis of pigment labeling with 15N and 13C

Isotope (Na(15)NO(3), ((15)NH(4))SO(4) or [(13)C]glucose) labeling was used to analyze chlorophyll synthesis and degradation rates in a set of Synechocystis mutants that lacked single or multiple small Cab-like proteins (SCPs), as well as photosystem I or II. When all five small Cab-like proteins were inactivated in the wild-type background, chlorophyll stability was not affected unless the scpABCDE(-) strain was grown at a moderately high light intensity of 100-300 micromol photons m(-2) s(-1). However, the half-life time of chlorophyll was 5-fold shorter in the photosystem I-less/scpABCDE(-) strain than in the photosystem I-less strain even when grown at low light intensity (~3 micromol photons m(-2) s(-1)) (32 +/- 5 and 161 +/- 25 h, respectively). In other photosystem I-less mutants that lacked one to four of the scp genes the chlorophyll lifetime was in between these two values, with the chlorophyll lifetime generally decreasing with an increasing number of inactivated scps. In contrast, the chlorophyll biosynthesis rate was only marginally affected by inactivation of scps except when all five scp genes were deleted. Small Cab-like protein deficiency did not significantly affect photoinhibition or turnover of photosystem II-associated beta-carotene. It is concluded that SCPs do not alter the stability of functional photosystem II complexes but retard the degradation of photosystem II-associated chlorophyll, consistent with the proposed involvement of SCPs in photosystem II re-assembly or/and repair processes by temporarily binding chlorophyll while photosystem II protein components are being replaced.     

Loss of phylloquinone in Chlamydomonas affects plastoquinone pool size and photosystem II synthesis

Phylloquinone functions as the electron transfer cofactor at the A(1) site of photosystem I. We have isolated and characterized a mutant of Chlamydomonas reinhardtii, menD1, that is deficient in MenD, which encodes 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, an enzyme that catalyzes the first specific step of the phylloquinone biosynthetic pathway. The mutant is photosynthetically active but light-sensitive. Analysis of total pigments by mass spectrometry reveals that phylloquinone is absent in menD1, but plastoquinone levels are not affected. This is further confirmed by the rescue of menD1 by addition of phylloquinone to the growth medium. Analysis of electron transfer by absorption spectroscopy indicates that plastoquinone replaces phylloquinone in photosystem I and that electron transfer from A(1) to the iron-sulfur centers is slowed down at least 40-fold. Consistent with a replacement of phylloquinone by plastoquinone, the size of the free plastoquinone pool of menD1 is reduced by 20-30%. In contrast to cyanobacterial MenD-deficient mutants, photosystem I accumulates normally in menD1, whereas the level of photosystem II declines. This decrease is because of reduced synthesis of the photosystem II core subunits. The relationship between plastoquinone occupancy of the A(1) site in photosystem I and the reduced accumulation of photosystem II is discussed.     

Elimination of a group II intron from a plastid gene causes a mutant phenotype

Group II introns are found in bacteria and cell organelles (plastids, mitochondria) and are thought to represent the evolutionary ancestors of spliceosomal introns. It is generally believed that group II introns are selfish genetic elements that do not have any function. Here, we have scrutinized this assumption by analyzing two group II introns that interrupt a plastid gene (ycf3) involved in photosystem assembly. Using stable transformation of the plastid genome, we have generated mutant plants that lack either intron 1 or intron 2 or both. Interestingly, the deletion of intron 1 caused a strong mutant phenotype. We show that the mutants are deficient in photosystem I and that this deficiency is directly related to impaired ycf3 function. We further show that, upon deletion of intron 1, the splicing of intron 2 is strongly inhibited. Our data demonstrate that (i) the loss of a group II intron is not necessarily phenotypically neutral and (ii) the splicing of one intron can depend on the presence of another.     

Functional characteristics of green alga Scenedesmus obliquus (Chlorophyceae): 276–6 wild type and its two photosystems deficient mutants cultured under photoautotrophic,mixotrophic and heterotrophic conditions

Functional features of Scenedesmus obliquus: wild type 276–6 strain (WT) and its two mutants reported as photosystem I‐deficient (mutant 56.80) and photosystem II‐deficient (mutant 57.80) were characterized. Algae were cultured aseptically under continuous light or in darkness on mineral bold basal medium (BBM), yeast extract‐enriched BBM and yeast extract to evaluate the physiology of algal cells under photoautotrophic, mixotrophic and heterotrophic conditions. Growth, superoxide dismutase activity and photosynthetic parameters, including polyphasic fluorescence rise during the first seconds of chlorophyll a illumination (OJIP), were analyzed to find relationships between the photosynthetic/respiratory activity of the cells, occurrence of oxidative stress and trophic conditions applied to PSs‐deficient algae. Despite the highest superoxide dismutase activity, indicating the presence of oxidative stress, mixotrophic conditions appeared to be optimal for S. obliquus WT and mutant strains kept in non‐aerated cultures. OJIP analysis indicated that in mutant 56.80 part of photosystem (PS) I was functional and in mutant 57.80 residual PS II activity was found.     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

The PQ/PQH2 ratio and occupancy of photosystem II-QB site by plastoquinone control the degradation of D1 protein during photoinhibition in vivo

Photoinactivation of photosystem II (PSII) and light-dependent degradation of the reaction center II (RCII) protein D1 have been investigated in Chlamydomonas reinhardtii mutants D6, AC208, and B4 deficient in cytochrome b6/f, plastocyanin, and photosystem I (PSI) activity, respectively. These mutants possess active PSII and reduce plastoquinone (PQ) but cannot oxidize plastoquinol (PQH2) via light-dependent reduction of NADP. In light-exposed cells a high ratio PQH2/PQ and a low turnover of PQ/PQH2 at the RCII-QB site are maintained. In all mutants photoinactivation of RCII was slower as compared to the wild-type (wt) cells, and D1 degradation was drastically decreased. The degradation of D1 was also lower in the wt cells under anaerobic conditions and presence of ascorbate, while raising the concentration of dissolved oxygen increased the degradation of the D1 protein in the AC208 mutant. Photoinactivation and light-dependent degradation of the D1 protein were drastically increased in the Scenedesmus obliquus LF-1 mutant cells altered in its PSII manganese binding and thus unable to reduce PQ using water as an electron donor. Diuron inhibited the light-dependent degradation of D1 protein in both the LF-1 mutant and wt cells. Based on these results we propose that availability of PQ at the QB site is required for (i) the photoinactivation process of the RCII acceptor side followed by inactivation of the donor side leading to the generation of harmful cation radicals (Z+, P680+, chlz+) which damage the D1 protein, and (ii) the accessibility of the cleavage site of the damaged D1 protein to proteolytic degradation.     

A novel protein involved in the functional assembly of the oxygen-evolving complex of photosystem II in Synechocystis sp. PCC 6803

Mutation of Glu69 to Gln in the D2 protein of photosystem II is known to lead to a loss of photoautotrophic growth in Synechocystis sp. PCC 6803. However, second-site mutants (pseudorevertants) with restored photoautotrophic growth but still maintaining the E69Q mutation in D2 are easily obtained. Using a genomic mapping technique involving functional complementation, the secondary mutation was mapped to slr0286 in two independent mutants. The mutations in Slr0286 were R42M or R394H. To study the function of Slr0286, mutants of E69Q and of the wild-type strain were made that lacked slr0286. Deletion of slr0286 did not affect photoautotrophic capacity in wild type but led to a marked decrease in the apparent affinity of Ca(2+) to its binding site at the water-splitting system of photosystem II and to a reduced heat tolerance of the oxygen-evolving system, particularly in E69Q. Moreover, a small increase in the half-time for photoactivation of the oxygen-evolving complex of photosystem II for both wild type and the E69Q mutant was observed in the absence of Slr0286. The accumulation of photosystem II reaction centers, dark stability of the oxygen-evolving apparatus, stability of oxygen evolution, and the kinetics of charge recombination between Q(A)(-) and the donor side were not affected by deletion of slr0286. Slr0286 lacks clear functional motifs, and no homologues are apparent in other organisms, even not in other cyanobacteria. In any case, Slr0286 appears to help the functional assembly and stability of the water-splitting system of photosystem II.     

In vivo chlorophyll fluorescence screening allows the isolation of a Chlamydomonas mutant defective for NDUFAF3, an assembly factor involved in mitochondrial complex I assembly

The qualitative screening method used to select complex I mutants in the microalga Chlamydomonas, based on reduced growth under heterotrophic conditions, is not suitable for high‐throughput screening. In order to develop a fast screening method based on measurements of chlorophyll fluorescence, we first demonstrated that complex I mutants displayed decreased photosystem II efficiency in the genetic background of a photosynthetic mutation leading to reduced formation of the electrochemical proton gradient in the chloroplast (pgrl1 mutation). In contrast, single mutants (complex I and pgrl1 mutants) could not be distinguished from the wild type by their photosystem II efficiency under the conditions tested. We next performed insertional mutagenesis on the pgrl1 mutant. Out of about 3000 hygromycin‐resistant insertional transformants, 46 had decreased photosystem II efficiency and three were complex I mutants. One of the mutants was tagged and whole genome sequencing identified the resistance cassette in NDUFAF3, a homolog of the human NDUFAF3 gene, encoding for an assembly factor involved in complex I assembly. Complemented strains showed restored complex I activity and assembly. Overall, we describe here a screening method which is fast and particularly suited for the identification of Chlamydomonas complex I mutants.     

Photoinhibition of photosynthesis and growth responses at different light levels in psbA gene mutants of the cyanobacterium Synechococcus

Photoinhibition of photosynthesis and growth responses at diffrent light levels (10, 120 and 250 μmol m⁻² s⁻¹) were studied in psbA gene mutants R2S2C3 ( psbAI gene present) and R2K1 ( psbAIIIpsbAIII genes present) of the cyanobacterium Synechococcus sp . PCC 7942 ( Anacystis nidulans R2). Mutant R2K1 (possessing form II of the D1 protein of photosystem II) was much more resistant to photoinhibition than the mutant R2S2C3 (possessing form I of the D1 protein). At moderate inhibitory light levels (100 to 300 μmol m⁻² s⁻¹) this was largely ascribed to an increased rsistance of the photosystem II reaction cetres possessing form II of the D1 protein. However, at higher light levels the higher resistance mutant R2K1 was assigned to a higher rate of photosystem II repair, i.e. turnover of the D1 protein. Moreover, our results support the hypothesis that photoinhibition of photosystem II and photoinhibitory induced quenching are due to separate processes. Results from growth experiments show that the R2K1 mutant has a slower growth rate than the R2S2C3 mutant but shows an increased survival under high light stress conditions. It is hypothesized that high resistance to photoinhibition, though allowing a better survival under high light, is not advantageous for optimal growth.     

Different roles of alpha- and beta-branch xanthophylls in photosystem assembly and photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.     

Cytosolic APX knockdown rice plants sustain photosynthesis by regulation of protein expression related to photochemistry,Calvin cycle and photorespiration

The biochemical mechanisms underlying the involvement of cytosolic ascorbate peroxidases (cAPXs) in photosynthesis are still unknown. In this study, rice plants doubly silenced in these genes (APX1/2) were exposed to moderate light (ML) and high light (HL) to assess the role of cAPXs in photosynthetic efficiency. APX1/2 mutants that were exposed to ML overexpressed seven and five proteins involved in photochemical activity and photorespiration, respectively. These plants also increased the pheophytin and chlorophyll levels, but the amount of five proteins that are important for Calvin cycle did not change. These responses in mutants were associated with Rubisco carboxylation rate, photosystem II (PSII) activity and potential photosynthesis, which were similar to non‐transformed plants. The upregulation of photochemical proteins may be part of a compensatory mechanism for APX1/2 deficiency but apparently the finer‐control for photosynthesis efficiency is dependent on Calvin cycle proteins. Conversely, under HL the mutants employed a different strategy, triggering downregulation of proteins related to photochemical activity, Calvin cycle and decreasing the levels of photosynthetic pigments. These changes were associated to strong impairment in PSII activity and Rubisco carboxylation. The upregulation of some photorespiratory proteins was maintained under that stressful condition and this response may have contributed to photoprotection in rice plants deficient in cAPXs. The data reveal that the two cAPXs are not essential for photosynthesis in rice or, alternatively, the deficient plants are able to trigger compensatory mechanisms to photosynthetic acclimation under ML and HL conditions. These mechanisms involve differential regulation in protein expression related to photochemistry, Calvin cycle and photorespiration.