Donald Miles

Use of a nuclear mutant of maize to identity components of Photosystem II

Abstract Previous analysis of a high fluorescent, nuclear mutant of maize, designated hcf-3 , indicated that the primary alterations of the photosynthetic membranes were associated with Photosystem (PS) II (Leto, K. and Miles, C.D. (1980) Plant Physiol. 66, 18–24). Further investigation reveals that the mutant thylakoids contain less than 20% of the wild-type manganese level and are missing most or all of at least six major polypeptides with apparent molecular masses of 49, 45, 34, 32, 16 and 10 kDa. The data provide evidence for the regulated production of a physiological PS II unit made up of these polypeptides and in terms of electron transport, extending from the water-splitting apparatus up to but not including the plastoquinone pool. This mutant may aid in the identification of membrane components associated with PS II as well as provide information concerning nuclear control of production of thylakoid complexes.

Analysis of Leaf Sectors in the NCS6 Mitochondrial Mutant of Maize.

The nonchromosomal stripe (NCS6) mutation of maize is a partial deletion of the mitochondrial cytochrome oxidase subunit 2 (Cox2) gene. The Cox2 deletion and a narrow yellow striping phenotype are inherited together in a maternal fashion. The striped plants are heteroplasmic for mutant and normal Cox2 genes. Only the mutant Cox2 gene is detected within the yellow stripes, whereas both normal and mutant forms of the gene are present in the green sectors of the NCS6 plants. In the green leaves of nonstriped relatives, only the normal Cox2 gene is found. Both the structure and functioning of the chloroplasts in the yellow leaf sectors of NCS6 plants are altered. The pleiotropic effects of the NCS6 mutation suggest that mitochondrial function is required for the development of photosynthetically competent chloroplasts.

Biochemical characterization of a highly active O2-evolving Photosystem II preparation from maize

Abstract An O2-evolving Photosystem (PS) II preparation was isolated from maize by a Triton X-100 procedure (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539). A highly active O2-evolving preparation was obtained which evolved O2 at 76% the rate of fresh chloroplasts (H2O → 2,6-dichloro-p-benzoquinone) and was very sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. There was no detectable PS I activity in the preparation (2,3,5,6-tetramethyl-p-phenylenediamine → methyl viologen). When analyzed by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis the O2-evolving preparation was shown to be highly depleted in CP I, CF1, and devoid of cytochromes f and b-563 (the absence of which was confirmed by difference spectroscopy). The preparation was enriched in the PS II reaction center polypeptides I and II, the 34 kDa polypeptide (Metz, J., Wong, J. and Bishop, N.I. (1980) FEBS Lett. 114, 61–66), the Coomassie blue-stainable 32 kDa polypeptide (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys. Acta 581, 228–236), LHCP-associated polypeptides and cytochrome b-559. Polypeptides of unknown function at 40.5, 25, 24, 22, 16.6 and 14 kDa were also present in the O2-evolving preparation. Triton X-114 phase partitioning (Bricker, T.M. and Sherman, L.A. (1982) FEBS Lett. 149, 197–202) indicated that the majority of these polypeptides were intrinsic. Only the polypeptides at 32, 25, 24 and 14 kDa were extrinsic. When examined by the octylglucoside procedure of Camm and Green (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) the PS II O2-evolving preparation was shown to contain the chlorophyll-proteins CP 27, CP 29, CP II∗, D, and CP a-1 and CP a-2. Chlorophyll-proteins associated with PS I were highly depleted. The visible absorption spectra indicated an enrichment of chlorophyll b and carotenoids in the preparation. The 77 K fluorescence emission spectrum (excitation wavelength = 435 nm) exhibits a strong F-686 with little F-695 shoulder and a broad, low-intensity F-735 emission.

Nuclear mutations affecting plastoquinone accumulation in maize.

We have isolated and characterized two nuclear mutations which affect plastoquinone accumulation in maize. The mutations, hcf103 and hcf114, modify the same genetic locus. Plants homozygous for either mutant allele exhibit reduced PS II electron transport activity, reduced variable chlorophyll fluorescence and reduced delayed fluorescence yield. In these ways, hcf103 and hcf114 resemble previously described PS II mutants which lack stably assembled PS II reaction center complexes. However, unlike most previously described PS II mutants, hcf103 and hcf114 possess stable membrane-associated PS II complexes. Plastoquinone (PQ-9), which performs a variety of redox functions essential to normal non-cyclic electron transport, is severely depleted in the mutants. The lack of PS II electron transport activity is attributed to the absence of PQ-9. This is the first report of mutants deficient in PQ which do not also suffer serious pleiotropic defects.

Purification of cytochrome b-559 from oxygen-evolving Photosystem II preparations of spinach and maize

A rapid and simple procedure is presented for the purification of chloroplast cytochrome b-559. The method is based on the protocol devised by Garewal and Wasserman (Garewal, H.S. and Wasserman, A.R. (1974) Biochemistry 13, 4063–4071), which we have modified to eliminate the requirement for a lengthy electrophoretic step. Novel features of our method include: the use of oxygen-evolving Photosystem II preparations (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539) as the starting material; isocratic elution of cytochrome b-559 from a DEAE-cellulose column (yielding the protein in a pure state); and a simple column procedure for removal of excess Triton X-100. The procedure has been applied to both spinach and maize (Zea mays L.). Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. Lithium dodecyl sulfate polyacrylamide gel electrophoresis of cytochrome b-559 from both spinach and maize reveals a major polypeptide band (apparent molecular mass = 9 kDa), and two minor bands (apparent molecular masses = 10 kDa and 6 kDa).

Transposon mutagenesis of nuclear photosynthetic genes in Zea mays.

The discovery of a new maize (Zea mays L.) transposon system, Mutator, and the cloning of the 1.4 kilobase transposon, Mul, have made feasible the isolation of nuclear photosynthetic genes which are recognized only by their mutant phenotype. Mutant maize plants which express a high chlorophyll fluorescent (hcf) phenotype due to a defect in the electron transport or photophosphorylation apparatus have been isolated following mutagenesis with an active Mutator stock. The affected genes and their products in these mutants are inaccessible to classical methods of analysis. However, mutagenesis with the Mutator transposon makes it possible to isolate these genes.Although the PSII-deficient mutant hcf3 has been thoroughly studied by classical photo-biological methods, the nature of the lesion which results in the observed phenotype has not been established. A Mutator-induced allele of hcf3 has been isolated. A fragment of genomic DNA has been identified which is homologous to Mul and co-segregates with the mutant phenotype. This fragment is expected to contain a portion of the hcf3 locus which will be used to clone the normal gene. Direct study of the gene can provide insight into the nature and function of its polypeptide product.This approach can be used to study any photosynthetic gene which has been interrupted by a transposon. The isolation of more than 100 different chemically-induced hcf mutants, most of which can not be fully characterized using classical means, indicates the wealth of information which can be obtained using a transposon tagging technique.

Anomalous electron transport activity in a photosystem I-deficient maize mutant

Photosynthesis mutations were induced in maize lines bearing the transposable DNA element system, Mutator. Two Photosystem I mutants (hcf101 and hcf104) which were isolated are described here. Maize plants homozygous for the hcf104 mutation are seedling lethal and exhibit a high in vivo chlorophyll fluorescence yield. They lack ∼60% of CP1, P700 and PSI-specific electron transport activity relative to normal sibling plants. The comparable depletion of these three measures of PS I content conforms to the pattern reported for many other PS I-deficient mutants. Maize plants homozygous for hcf101 are seedling lethal and also exhibit high in vivo chlorophyll fluorescence yield. They lack 80–90% of CP1 and P700 but sustain steady state levels of PS I-specific electron transport activity at 70% of normal. Previous reports of similar apparent PS I hyperactivity are discussed and an explanation for the elevated steady state level of PS I electron transport activity in hcf101 is proposed.

Energy transfer for low temperature fluorescence in PS II mutant thylakoids.

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.

Activation of the Maize Chloroplast Coupling Factor ATPase

The chloroplast coupling factor (CF1) is part of the proton-translocating ATPase (H+-ATPase) complex which phosphorylates ADP in the process of photophosphorylation. CF1 consists of 5 different kinds of subunits, denoted α -e in order of decreasing molecular weight.

PHOTOSYNTHETIC CHARACTERIZATION OF MUTANTS OF ZEA MAYS INDUCED BY THE DNA TRANSPOSABLE ELEMENT, MUTATOR

Fourteen new photosynthesis mutants were isolated from Zea mays L. (maize) stocks which were outcrossed to lines containing active Mutator elements. This outcrossing causes replication and translocation of the DNA elements leading to new insertion into genes at random and mutation (transposon mutagenesis). After two generations of self-pollinations (M2 generation) new mutant genes which were induced segregate in a 1∶3 ratio. Photosynthesis mutants were screened and isolated by monitoring the increased level of chlorophyll fluorescence in the intact plant. Mutant genes which cause a visibly high chlorophyll fluorescence are termed hcf. Upon standard analysis of the state of photosynthetic electron transport, several different types of photosynthesis mutants were discovered which cause sufficient limitation in photosynthesis to increase whole plant visual fluorescence yield.