Further studies of the thylakoid membrane surface charges by particle electrophoresis.

1. Above pH 4.3 the outer surface of thylakoid membranes isolated from pea chloroplasts is negatively charged but below this value it carries an excess of positive charge. 2. Previously the excess negative charge has been attributed to the carboxyl groups of glutamic and aspartic acid residues (Nakatani, H.Y., Barber, J. and Forrester, J.A. (1978), Biochim. Biophys. Acta 504, 215-225) and in this paper it is argued from experiments involving treatments with 1,2-cyclohexanedione that the positive charges are partly due to the guanidino group of arginine. 3. The electrophoretic mobility of granal (enriched in chlorophyll b and PS II activity) and stromal (enriched in PS I activity) lamellae isolated by the French Press technique were found to be the same. 4. Treatment of the pea thylakoids with trypsin or pronase, sufficient to inhibit the salt induced chlorophyll fluorescence changes, increased their electrophoretic mobility indicating that additional negative charges had been exposed at the surface. 5. Polylysine treatment also inhibited the salt induced chlorophyll fluorescence changes but unlike trypsin and pronase, decreased the net negative charge on the surface. 6. The isoelectric point defined as the pH which gave zero electrophoretic mobility (about 4.3) was independent of the nature of the cations in the suspending medium (monovalent vs. divalent).

Surface charges on chloroplast membranes as studied by particle electrophoresis.

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts. 2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge. 3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface. 4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues. 5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting chlorophyll a/chlorophyll b pigment . protein complex. 6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts. 7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory. 8. Calculations of the zeta-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500--2000 A2. 9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.

The influence of the thylakoid membrane surface properties on the distribution of ions in chloroplasts.

Thylakoid membranes isolated from peas have been subjected to ionic analyses using the technique of neutron activation. This has allowed the analyses of K+, Na+, Mg2+, Ca2+ and Cl- to be measured simultaneously on the same sample. By varying the ionic composition of the suspending medium it has been shown that these chloroplast membranes have no obvious chemical specificity for the inorganic cations studied and that the major controlling factor is the electrostatic neutralization of the surface negative charges. In agreement with the Gouy-Chapman theory and for the conditions used, divalent cations were preferentially attracted to the membrane surface. This finding, together with the ionic analysis of the unwashed thylakoids and of isolated intact chloroplasts, indicated that the major physiological surface cation is Mg2+ and that K+ is probably the main inorganic cation of the stroma. This conclusion is discussed in terms of counterion movement in response to light induced proton pumping at the thylakoid membrane.

Photosynthetic oxygen evolution does not require the participation of polypeptides of 16 and 24 kilodaltons.

Oxygen evolution from Triton X-100 prepared photosystem II particles from spinach was inhibited 70-80% after treatment of the particles with high concentrations of salt (0.5 or 1 M NaCl). The salt-washed preparations were depleted of 16 and 24 kilodalton polypeptides. Nearly complete restoration of herbicide-sensitive electron transport activity was observed upon addition of Cl-. Maintenance of the maximal rates required the presence of Ca2+. Restoration of the activity did not require the readdition (reconstitution) of the extracted polypeptides indicating that the 16 and 24 kilodalton polypeptides do not participate directly in the water-oxidation process. They may, however, play a regulatory role, e.g., in the sequestering of Cl- and/or Ca2+.

Inhibition of photosynthetic oxygen evolution by calmodulin-type inhibitors and other calcium-antagonists.

Several studies have recently implicated a role for Ca2+ in photosynthetic oxygen evolution (9-11). Our previous study indicated that Ca2+ was likely acting at the level of the Cl- cofactor requirement in photosystem II (9). We now demonstrate, through the use of calmodulin-type inhibitors ( calmidazolium and trifluoperazine) and metal Ca2+-antagonists (e.g., Tb+3 and La+3), the function of Ca2+ on the oxidizing side of photosystem II. In addition, the peroxide (H2O2) electron donation site was differentiated from the electron donation site of NH2OH, Mn2+ and diphenyl carbazide in the photosystem II complex.

A clover mutant lacking the chlorophyll a- and b-containing protein antenna complexes.

A chlorophyll deficient mutant of clover has been examined by SDS-PAGE, spectrophotometric and electron microscopic methods. By a comparison of the absorption and first derivative spectra of acetone extracts from the mutant and normal biotypes, we observed a deficiency in chlorophyll b for the mutant biotype. The calculated chlorophyll a/b ratios, using the method of Arnon (Plant Physiol. 24, 1-15, 1949), approached infinity for the mutant whereas it ranged from 3.0-3.3 in the wild-type. The low temperature (77 degrees K) fluorescence emission bands in the 685-695 nm region could not be differentiated for the biotypes; however, the long wavelength emission band (near 740 nm in the wild-type) was shifted to shorter wavelengths (ca 720-725 nm) in the mutant indicating loss of photosystem I antenna. The SDS-PAGE profile of the mutant biotype showed a dramatic decline in the Coomassie stained polypeptides of apparent molecular weights similar to those of LHC II. Transmission electron micrographs of the mutant and normal tissue exhibited similar extents of grana-stacking, indicating that a component(s) other than the LHC II may be responsible for membrane adhesion in this mutant.

Light-dependent redistribution of ions in suspensions of chloroplast thylakoid membranes.

Ion movements associated with the pH rise that is observed upon illumination of thylakoid suspensions at low pH have been studied by a multiparameter technique. Light-dependent, dark-reversible fluxes of H(+), Cl(-), Na(+), K(+) and divalent cations were monitored, together with simultaneous changes in the optical density of the suspension. Extensive uptake of Cl(-) and efflux of Mg(2+) accompany the apparent inward movement of H(+) in the light. Only minor efflux of K(+) is seen and Na(+) appears immobile. The Cl(-) and Mg(2+) fluxes together compensate for most of the charge transferred as H(+), contributing respectively about 49% and 43% on an equivalent basis. The ratio of Cl(-) influx to Mg(2+) efflux is variable, but usually >1.0. The Mg(2+) flux can be supplanted by (1) K(+) flux, if the K(+)/Mg(2+) activity ratio in the suspension is high, and (2) Ca(2+) flux, if the thylakoids are equilibrated with suspending media containing Ca(2+). The affinity of the divalentcation-binding sites, or carrier mechanism, is greater for Ca(2+) than for Mg(2+). Schemes can be drawn up to account for the observed ion movements on the basis of either a chemical or a chemiosmotic mechanism for energy transduction in chloroplasts. In intact chloroplasts, light-dependent control of Mg(2+) distribution between thylakoid and stroma could serve to regulate enzyme activities in the carbon fixation pathway, and hence photosynthesis.

Paraquat resistance in conyza.

A biotype of Conyza bonariensis (L.) Cronq. (identical to Conyza linefolia in other publications) originating in Egypt is resistant to the herbicide 1,1'-dimethyl-4,4'-bipyridinium ion (paraquat). Penetration of the cuticle by [(14)C]paraquat was greater in the resistant biotype than the susceptible (wild) biotype; therefore, resistance was not due to differences in uptake. The resistant and susceptible biotypes were indistinguishable by measuring in vitro photosystem I partial reactions using paraquat, 6,7-dihydrodipyrido [1,2-alpha:2',1'-c] pyrazinediium ion (diquat), or 7,8-dihydro-6H-dipyrido [1,2-alpha:2',1'-c] [1,4] diazepinediium ion (triquat) as electron acceptors. Therefore, alteration at the electron acceptor level of photosystem I is not the basis for resistance. Chlorophyll fluorescence measured in vivo was quenched in the susceptible biotype by leaf treatment with the bipyridinium herbicides. Resistance to quenching of in vivo chlorophyll fluorescence was observed in the resistant biotype, indicating that the herbicide was excluded from the chloroplasts. Movement of [(14)C] paraquat was restricted in the resistant biotype when excised leaves were supplied [(14)C]paraquat through the petiole. We propose that the mechanism of resistance to paraquat is exclusion of paraquat from its site of action in the chloroplast by a rapid sequestration mechanism. No differential binding of paraquat to cell walls isolated from susceptible and resistant biotypes could be detected. The exact site and mechanism of paraquat binding to sequester the herbicide remains to be determined.

The origin of the long-wavelength fluorescence emission band (77 degrees K) from photosystem I.

Isolated photosystem I (PSI)-110 particles, prepared using a minimal concentration of Triton X-100 [J. E. Mullet, J. J. Burke, and C. J. Arntzen (1980) Plant Physiol. 65, 814-822] and further subjected to short-term solubilization with sodium dodecyl sulfate (SDS), were resolved into four pigment-containing bands on polyacrylamide gel electrophoresis (PAGE). We have identified these in order of increasing electrophoretic mobility as being (a) CPIa, (b) CPI, (c) the light-harvesting complex of photosystem I (LHC-I), and (d) a free pigment-zone. LHC-I had an absorption maximum in the red at 668-669 nm and a shoulder at 650 nm, which was resolved by its first-derivative spectrum to indicate the presence of chlorophyll b. LHC-I exhibited a 77 degrees K fluorescence emission maximum at 729-730 nm. The 77 degrees K fluorescence emission maxima of CPIa and CPI, excised from the gel, were at 729 and 722 nm, respectively. The LHC-I band, excised from the gel and rerun on dissociating SDS-PAGE, was resolved into two polypeptide doublets of 24-22.5 and 21-20.5 kDa. The CPIa band under similar conditions was resolved into polypeptides of 68, 24, 22.5, 21, 20.5, 19, 15, and 14 kDa; on the contrary, CPI contained only the 68-kDa polypeptide. When intact thylakoids were subjected to "nondenaturing" SDS-PAGE, LHC-I comigrated with an oligomeric form (dimer) of the light-harvesting chlorophyll a/b pigment-protein that preferentially serves photosystem II (LHCP-II). When this combined LHC-I/LHCP-II pigment-protein band was prepared by SDS-PAGE from isolated stroma lamellae, it exhibited a long-wavelength fluorescence band near 730 nm at 77 degrees K. When a similar preparation was obtained from sucrose density gradients containing SDS [J. Argyroudi-Akoyunoglou and H. Thomou (1981) FEBS Lett. 135, 171-181], it was found to be enriched in a 21-kDa polypeptide. The data suggest that the 21-kDa polypeptide of LHC-I is the chlorophyll-containing polypeptide responsible for the long-wavelength fluorescence of LHC-I; other polypeptides in the complex (20.5, 22.5, and 24 kDa) presumably bind chlorophyll and also serve an antennae function.