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     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

The relative absorption cross-sections of Photosystem I and Photosystem II in chloroplasts from three types of Nicotiana tabacum

In the present study we used three types of Nicotiana tabacum, cv John William's Broad Leaf (the wild type and two mutants, the yellow-green Su/su and the yellow Su/su var. Aurea) in order to correlate functional properties of Photosystem II and Photosystem I with the structural organization of their chloroplasts. The effective absorption cross-section of Photosystem II and Photosystem I centers was measured by means of the rate constant of their photoconversion under light-limiting conditions. In agreement with earlier results (Okabe, K., Schmid, G.H. and Straub, J. (1977) Plant Physiol. 60, 150–156) the photosynthetic unit size for both System II and System I in the two mutants was considerably smaller as compared to the wild type. We observed biphasic kinetics in the photoconversion of System II in all three types of N. tabacum. However, the photoconversion of System I occurred with monophasic and exponential kinetics. Under our experimental conditions, the effective cross-section of Photosystem I was comparable to that of the fast System II component (α centers). The relative amplitude of the slow System II component (β centers) varied between 30% in the wild type to 70% in the Su/su var. Aurea mutant. The increased fraction of β centers is correlated with the decreased fraction of appressed photosynthetic membranes in the chloroplasts of the two mutants. As a working hypothesis, it is suggested that β centers are located on photosynthetic membranes directly exposed to the stroma medium.     

The effects of nuclear mutation on chloroplast development

Summary Under greenhouse conditions the dark green wild type (su/su) tobacco grows 2–3 times faster than the yellow mutant (Su/su) and contains five-fold more chlorophyll. On a fresh weight basis, however, both genotypes contain similar amounts of RuBPCase and fraction 11 protein in approximately equal proportion and have similar levels of 70s and 80s ribosomes. When seedlings are cultured on agar medium supplemented with sucrose and equal concentrations of IAA and kinetin or kinetin alone, a drastic reduction of RuBPCase and free 70s ribosomes, but not of chlorophyll content, were observed. Moreover, albino (Su/Su) seedlings developed on supplemented media still contain appreciable amounts of RuBPCase and free 70s ribosomes although chlorophyll levels are extremely low indicating no correlation between RuBPCase and chlorophyll content. RuBPCase crystallized from both wild type and yellow mutant plants seem to have identical composition and structure when examined by isoelectric focusing, amino acid analysis or peptide mapping techniques. The slow-growing yellow mutant is apparently deficient only in chlorophyll of the light harvesting chlorophyll-protein complex but with no alteration of the protein moiety or chlorophyll a/b ratio.     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.     

Culture and morphogenetic response of a lethal,chlorophyll-deficient mutant of tobacco to hormones,amino acid and sucrose

Summary This report describes the culture of Su/Su, Su/su and su/su tissue in vitro. High levels of auxin and low levels of cytokinin increase growth of the cells. The cells do not need exogenous amino acids for rapid growth and the chlorophyll deficiency cannot be overcome by amino acids. Reduced levels of auxin and sucrose enhance differentiation, whereas cysteine in the autoclaved medium inhibit differentiation. The Su chlorophyll mutant of tobacco provides a marker for in vitro studies on photosynthesis and photorespiration, chloroplast genetics and cell fusion techniques.     

Fluorescence properties of chloroplasts from manganese deficient and mutant tobacco

1. The kinetics of the fluorescence induction are described for chloroplasts from normal green tobacco, from the aurea tobacco mutant Su/su, from the photosynthetically inactive yellow patches of a variegated tobacco, and from tobacco plants grown in absence of manganese. The first two types display the well-known biphasic induction, but the Su/su chloroplasts have a distinctly slower rise time. Manganese deficient chloroplasts show a significantly higher fluorescence yield than any other type of chloroplasts studied. The kinetics of their fluorescence, on the other hand, are similar to those observed with the inactive chloroplasts from the variegated tobacco: the fluorescence rise is small, and the fluorescence yield is not changed very much by the addition of a reducing agent like hydrosulfite, or by addition of an oxidant like ferricyanide, or by an inhibition of the electron flow in Photosystem II with 3(3,4-dichlorophenyl)-1,1-dimethylurea.

2. Determinations of the amount of the primary electron acceptors associated with Photosystem II point to a 2- to 3-fold larger electron acceptor pool in chloroplasts of young Su/su plants than in chloroplasts of old Su/su plants and of various green leaves, including those from green tobacco. This finding agrees with recently published data on the size of the photosynthetic unit in tobacco mutants and normal green plants.

3. The different fluorescence characteristics of all four types of chloroplasts under study are discussed on the basis of their structure and their activity in photosynthetic O₂ evolution.     

Quantum efficiency and antenna size of Photosystems IIα, IIβ and I in tobacco chloroplasts

Reaction center concentrations were determined in chloroplasts of tobacco, cv John William's Broadleaf, and its mutants Su/su and Su/su var. Aurea. Quantum yields of the primary reactions of Photosystems I, II_α and II_β (Melis, A. and Homann, P.H. (1975) Photochem. Photobiol. 21, 431–437) were obtained by measurement of their rate constants and the absorbed energy, under conditions where all three photosystems operated simultaneously and produced almost irreversibly a single charge separation.The concentrations and reaction rates of the photosystems were different in chloroplasts from the wild type and the mutants, but in chloroplasts of each type of plant used essentially all quanta absorbed by chlorophyll caused a charge separation in PS I, PS II_α or PS II_β. Since the quantum efficiency of each photosystem was close to one, kinetic differences between the photosystems and between different kinds of chloroplasts were only due to differences in antenna size. From the rate constants the number of chlorophyll molecules in the antenna of each photosystem could be calculated. It is argued that PS II_α and PS II_β must be different, independent structures.     

Photoacoustic studies on the dependence of state transitions on grana stacking

Photoacoustic detection of oxygen evolution and Emerson enhancement in state 1 and state 2 were compared in a tobacco wild type and mutant (Su/su) deficient in chlorophyll. The mutant shows smaller changes in the distribution of excitation energy between the two photosystems than the wild type. Analysis of Emerson enhancement saturation curves indicates that in the mutant which is deficient in grana partitions and shows less stacking, state 1-state 2 transitions reflect changes in the yield of energy transfer from PS II to PS I (spillover). On the other hand, the wild type containing large grana shows changes in absorption cross-sections of the two photosystems upon state transitions. NaF, a specific phosphatase inhibitor, blocks the transition to state 1, indicating that LHC II phosphorylation has a role in excitation energy regulation in both the mutant as well as the wild type. It is demonstrated that N-ethylmaleimide, a specific sulfhydryl reagent, blocks the transition to state 2, suggesting that a disulfide-sulfhydryl redox couple activates the LHC II kinase in vivo.Abbreviations LHC II light harvesting chlorophyll a/b pigment protein complex of PS II - LHC II-P phosphorylated complex - NEM N-ethylmaleimide     

Evidence for Predominant Participation of Photosystem II{beta}-Units in Nitrogen Formation by Hydroxylamine-Treated Tobacco Chloroplasts

Relative optical cross sections for flash-induced O₂- and N₂-productionwith water and 1 mM NH₂OH as electron donors to PSII, respectively,as well as that for PSI-mediated O²-uptake have been measuredin tobacco chloroplasts by mass spectrometry. In the wild typetobacco these three reactions are driven by three populationsof photosynthetic units different with respect to the antennasize. The antenna size of O₂-evolving units is twice as largecompared to the N₂-evolving one, but both of these have thesame spectral characteristics in the far red region. In contrastto the wild type, the antenna sizes of O₂- and N₂-evolving unitsin the chlorophyll b-deficient tobacco mutant Su/su var. Aureaare the same as the sizes of the N₂-evolying units in the wildtype chloroplasts. Taking into account the data of Thielen andVan Gorkom [Biochim. Biophys. Acta (1981) 635:111] on a strongdifference in the relative amounts of PSII_ß and PSII_ßin the mutant compared with the wild type (the ratio about 1: 3 and 3 : 1 , respectively) it is concluded that mainly thestroma-exposed PSII_ß units are competent to N₂-evolution. (Received November 11, 1993; Accepted April 25, 1994)     

Characterization of the thylakoid membranes of the tobacco aurea mutant Su/su and of three green revertant plants

Phenotypic characterizations of the semidominant aurea tobacco (Nicotiana tabacum L.) mutant Su/su, the homozygous mutant Su/Su and three green revertants (R1, R2, and R3) are presented. The leaf color of Su/su plants varies from yellow to light-green when grown under high and low energy fluence rates (33.0 and 3.3 W m^–2), respectively. The change in visual phenotype under high-light conditions is correlated with decreased content of chlorophyll per leaf area, agranal chloroplast ultrastructure, changes in the number of chlorophyll-protein complexes, and absence of two or more of the light harvesting chlorophyll-polypeptides of 25,000–29,000 dalton. The homozygous mutant grown under low light was shown to be completely lacking in grana stacks and to be deficient in chlorophyll-protein complexes. Revertant R1 was found to be identical to wild-type plants in all parameters examined (leaf color, chloroplast ultrastructure, chlorophyll-protein complexes, chlorophyll-protein complex polypeptides) except in chlorophyll content. It did not show an increased chlorophyll and carotenoid content as did the wild-type plants when exposed to high light. Revertants R2 and R3 were similar to the heterozygous mutant Su/su in most of the parameters examined. They yellowed because of a loss of chlorophyll and an increase in the amount of carotenoids, had agranal chloroplasts, and had variant chlorophyll-protein complexes when grown under high light intensities. However, each appeared to contain some of the light-harvesting pigment-protein complex polypeptides found to be absent in Su/su when grown under high-light conditions.Abbreviations HL high light - LL low light - SDS sodium dodecyl sulfate This paper is part of a Ph.D. thesis by P.J.K. in the Program in Genetics, Michigan State University     

Quantum Yields of Photosystem II and Photosynthesis in an Aurea Mutant of Tobacco (C3) and an Oil Yellow Mutant of Maize (C4) Which Have High Capacities for Photosynthesis Despite Low Chlorophyll Contents

The quantum yields of O₂ evolution (_o2), CO₂ fixation (_co2)and of photosystem II measured by fluorescence analysis (_PSII),were determined in a mutant of tobacco (C₃) and a mutant ofmaize (C₄) having large deficiencies in light harvesting chlorophyll,but having similar capacities for photosynthesis on a leaf areabasis as the wild-type plants. The heterozygous dominant mutantof tobacco (aurea, Su/+) having 30% chlorophyll content of thewild-type, and of maize (oil yellow, Oy/+) having 60% chlorophyllcontent of the wild-type, maintain high leaf absorptances. Themaximum _o2 on an absorptance basis, measured under limitingred light and saturating CO₂ were the same in the mutant versuswild-type plants. _PSII and _co2 decreased in a similar mannerwith increasing light intensity, while the ratio of _PSII/_co2under varying light remained reasonably constant, in mutantversus wild-type seedlings. The results indicate that theseC₃ and C₄ mutants which have high capacities for photosynthesisdespite large deficiencies in chlorophyll content, maintaina high leaf absorptance, and a balanced utilization of absorbedquanta in photochemistry. ¹Present address: Botany Dept., University of California, Davis,CA, 95616, U.S.A.     

黄瓜矮生突变体C1056的茎解剖结构及其生理特性分析

该研究以黄瓜矮生突变体C1056和野生型CCMC为材料,对其主要生理特性、叶绿体超微结构以及茎显微结构进行了观察、测定和比较分析,以探讨黄瓜株高调控机理并挖掘新的矮化种质,为黄瓜的矮化育种提供依据。结果显示:(1)突变体C1056的株高较野生型极显著变矮,且叶色加深、叶脉加粗、叶尖内卷、叶片皱缩,但茎粗、节间数与野生型无显著差异,而节间长度极显著低于野生型。(2)茎横切显微结构显示,突变体的维管束数量与野生型无显著差异,但导管直径缩小;纵切结果显示,突变体茎节间细胞长度变短,细胞变小,细胞数目略有补偿。(3)与野生型相比,突变体的叶绿素和类胡萝卜素含量均有不同程度的下降,叶绿素/类胡萝卜素和叶绿素a/b的比值明显增高。(4)突变体叶绿素荧光各参数与野生型相比无明显变化;突变体的净光合速率较野生型降低8%,气孔导度、蒸腾速率较野生型分别提高15%和10%,但差异均不显著,而胞间CO2浓度显著高于野生型。(5)透射电镜观察结果发现,与野生型相比,突变体的叶肉细胞比较小,叶绿体所占细胞面积明增大,且叶绿体形状为半圆形和纺锤形,部分非正常结构的叶绿体的大部分基质、基粒片层未完全分化且不清晰,垛叠不整齐。研究表明,黄瓜矮生突变体C1056的矮化主要因其节间长度缩短以及细胞变小所致,且突变体的叶绿体结构受到一定程度的影响,但并未明显影响其光合能力。     

Chlorophyll-deficient Lespedeza procumbens

Summary A native plant of Lespedeza procumbens with yellow, chlorophyll-deficient leaves was discovered near Tallahassee, Florida. When selfed this plant yielded only yellow offspring. Normal, green plants of L. procumbens contained about 5.5 times more chlorophyll (dry weight basis) than yellow plants. At high light intensities photosynthetic rates on a chlorophyll basis in yellow plants were 3 to 8 times greater than in green plants. Chloroplasts in yellow plants did not possess high-stacked grana, and the thylakoids displayed unusual cross channels reported previously only in Cassia [7] and Rhodopseudomonas [15]. Chlorophyll deficiency in Lespedeza closely parallels that reported in yellow (Su/su) tobacco [11]. The present paper is the most detailed report on chlorophyll deficiency in a wild plant, all comparable work having been done on cultivars.These studies were partially supported by contract No. AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

Pigment and quinone content of two photosynthetic barley mutants

The pigment and quinone content of wild-type barley ( Hordeum vulgare L., cv. Svalöfs Bonus) and of two photosynthetic mutants was assayed. Wild type plants and the photosystem Hacking mutant viridis zb63 contained chlorophyll a and b. whereas chlorina-f2 contained only chlorophyll a The inability of the mutant chlorina-f2 to convert chlorophyll a into chlorophyll a appears to he the primary effect of the mutation. In both mutants, the carotenoid composition was virtually identical to that of the wild type. As compared to the wild type. chlorina-f2 contained less lutein and neoxanthin. The mutant viridis-zb63 contained less β-carotene but more antheraxanthin and xeaxanthin than the wild type. The quinone content and composition of the wild type and the photosynthetic mutants was similar, and both mutants biosynthesized plastid quinones and chromanols starting from [14C]-labeled tyrosine. The data indicate that carotenoid and quinone biosynthesis are not altered in the two mutants as compared to the wild type.     

Functional organization of thylakoid membranes in viable pea mutants with low chlorophyll content

The functional organizations of thylakoid membranes from wild type pea ( Pisum sativum L. cv. Kapital) and two viable mutants with low chlorophyll (Chl) contents were compared. Nuclear mutations in mutants 7 and 42 led to two- and three-fold decrease in total chlorophyll content, respectively. In spite of low Chl content mutants showed 80% photosynthetic activity, biological productivity, and seed production. It has been shown that mutant membranes differed from that of wild type by Chl distribution between the pigment-protein complexes and by stoichiometry of the main electrontransport complexes. The ratio photosystem I (PSI): photosystem II (PSII): cytochrome (Cyt) bjf complex: Chl was 1:1.1:1.2:650 in wild type chloroplasts, 1:1.8:1.7:600 in mutant 7 , and 1:1.5:1.9:350 in mutant 42 . PSI- and PSII-dependent electron-transport activities were enhanced in the mutants per mg Chl in proportion to number of reaction centers. The activity of the non-cyclic electron-transport chain increased in proportion to PSII and Cyt bjf complexes. The amount of ATP synthetase per unit of Chl as estimated by H⁻ATPase activity was much greater in mutant thylakoids, which is favorable for photosynthetic energy transduction. The low content of the light-harvesting complexes (LHC) in mutants is compensated by an increase of the number of PSII and Cyt bjf complexes, which eliminates the bottleneck at the site of plastoquinone oxidation.     

Control of carbohydrate metabolism in a starchless mutant of Arbidopsis thaliana

The biochemical regulation of photosynthate partitioning was investigated in a starchless mutant (TC7) of Arabidopsts thaliana (L.) Henyh, that was deficient in chloroplast phosphoglucomutase (Caspar et al. 1985. Plant Physiol. 79: 11–17). Plants were raised at 20°C with a 20 h light and 4 h dark period, so that the growth rates of the mutant and wild type were similar. Two or 3 isoforms of phosphoglucomutase were separated by ion-exchange chromatography using mutant and wild type leaf preparations, respectively. Initial rate kinetics of all isoforms were similar. Light-saturated photosynthetic oxygen evolution rates of the mutant and wild type were 224 and 302 nmol g^-1 chlorophyll h^-1, respectively. Starch, sucrose and hexose concentrations were unchanged in wild type leaves after a dark to light transition, whereas sucrose and hexose increased in mutant leaves. Hexose-phosphates accumulated in both genotypes in the light, although the steady-state leaf concentrations of glucose 6-phosphate were 3-fold higher in mutant than in wild type samples. Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate were lower in the mutant than in the wild type at the end of the dark period when mutant leaves were depleted of carbohydrates. Levels of UTP were lower in the mutant than in the wild type, possibly indicating that growth conditions had induced phosphate limited photosynthesis. These results are discussed in relation to the regulation of photosynthetic carbon metabolism.