Maize Thylakoids Research Articles

The short-term response of Arabidopsis thaliana (C3) and Zea mays (C4) chloroplasts to red and far red light.

Light quality has various effects on photochemistry and protein phosphorylation in Zea mays and Arabidopsis thaliana thylakoids due to different degrees of light penetration across leaves and redox status in chloroplasts. The effect of the spectral quality of light (red, R and far red, FR) on the function of thylakoid proteins in Zea mays and Arabidopsis thaliana was investigated. It was concluded that red light stimulates PSII activity in A. thaliana thylakoids and in maize bundle sheath (BS) thylakoids, but not in mesophyll (M) thylakoids. The light quality did not change PSI activity in M thylakoids of maize. FR used after a white light period increased PSI activity significantly in maize BS and only slightly in A. thaliana thylakoids. As shown by blue native (BN)-PAGE followed by SDS-PAGE, proteins were differently phosphorylated in the thylakoids, indicating their different functions. FR light increased dephosphorylation of LHCII proteins in A. thaliana thylakoids, whereas in maize, dephosphorylation did not occur at all. The rate of phosphorylation was higher in maize BS than in M thylakoids. D1 protein phosphorylation increased in maize and decreased in A. thaliana upon irradiation with both R and growth light (white light, W). Light variations did not change the level of proteins in thylakoids. Our data strongly suggest that response to light quality is a species-dependent phenomenon. We concluded that the maize chloroplasts were differently stimulated, probably due to different degrees of light penetration across the leaf and thereby the redox status in the chloroplasts. These acclimation changes induced by light quality are important in the regulation of chloroplast membrane flexibility and thus its function.

Phosphorylation of PSII proteins in maize thylakoids in the presence of Pb ions

Lead is potentially toxic to all organisms including plants. Many physiological studies suggest that plants have developed various mechanisms to contend with heavy metals, however the molecular mechanisms remain unclear. We studied maize plants in which lead was introduced into detached leaves through the transpiration stream. The photochemical efficiency of PSII, measured as an Fv/Fm ratio, in the maize leaves treated with Pb was only 10% lower than in control leaves. The PSII activity was not affected by Pb ions in mesophyll thylakoids, whereas in bundle sheath it was reduced. Protein phosphorylation in mesophyll and bundle sheath thylakoids was analyzed using mass spectrometry and protein blotting before and after lead treatment. Both methods clearly demonstrated increase in phosphorylation of the PSII proteins upon treatment with Pb2+, however, the extent of D1, D2 and CP43 phosphorylation in the mesophyll chloroplasts was clearly higher than in bundle sheath cells. We found that in the presence of Pb ions there was no detectable dephosphorylation of the strongly phosphorylated D1 and PsbH proteins of PSII complex in darkness or under far red light. These results suggest that Pb2+ stimulates phosphorylation of PSII core proteins, which can affect stability of the PSII complexes and the rate of D1 protein degradation. Increased phosphorylation of the PSII core proteins induced by Pb ions may be a crucial protection mechanism stabilizing optimal composition of the PSII complexes under metal stress conditions. Our results show that acclimation to Pb ions was achieved in both types of maize chloroplasts in the same way. However, these processes are obviously more complex because of different metabolic status in mesophyll and bundle sheath chloroplasts.

Photoinhibition and D1 protein degradation in mesophyll and agranal bundle sheath thylakoids of maize.

Susceptibility of photosystem II complex (PSII) to photoinhibition and degradation of D1 protein has been described in the chloroplasts of C3 plants but so far, the PSII turnover has not been characterised in any C4 plant, which contains two types of chloroplasts differing biochemically and structurally. In maize (Zea mays L. Oleńka), chloroplasts located in mesophyll (M) develop grana, while bundle sheath (BS) chloroplasts are agranal. In this paper, we report the D1 protein phosphorylation, damage and proteolysis in mesophyll as well as in agranal bundle sheath thylakoids of maize plants. Photoinhibitory treatment (1800 μmol photons m-2 s-1) of isolated thylakoids led to donor side inhibition of PSII electron transport and then to damage of reaction centre in both M and BS thylakoids. Rate of D1 degradation rate was faster in BS than in M thylakoids, and the addition of ATP to incubation medium delayed D1 degradation in both types of thylakoids. Furthermore, we demonstrated that the proteases belonging to FtsH and Deg families were present but their amounts significantly differed in M and BS thylakoids. Protease inhibitor studies revealed that serine- and metallo-proteases were involved in degradation of D1 protein. Apparent existence of D1 degradation cycle and the presence of proteolytic enzymes responsible for this process in BS thylakoids confirm that PSII plays an important role in agranal membranes, and when damaged, D1 can be rapidly degraded to enable PSII repair and restoration in these membranes.

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids

The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b 6f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b 6f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.

Extrinsic photosystem II carbonic anhydrase in maize mesophyll chloroplasts.

One form of carbonic anhydrase (CA) has been observed in maize (Zea mays) thylakoids and photosystem II (PSII)-enriched membranes. Here, we show that an antibody produced against a thylakoid lumen-targeted CA found in Chlamydomonas reinhardtii reacts with a single 33-kD polypeptide in maize thylakoids. With immunoblot analysis, we found that this single polypeptide could be identified only in mesophyll thylakoids and derived PSII membranes, but not in bundle sheath thylakoids. Likewise, a CA activity assay confirmed a large amount of activity in mesophyll, but not in bundle sheath membranes. Immunoblot analysis and CA activity assay showed that the maximum CA can be obtained in the supernatant of the PSII-enriched membranes washed with 1 M CaCl(2), the same procedure used to remove all extrinsic lumenal proteins from PSII. Because this CA reacts with an antibody to lumen-directed CA in C. reinhardtii, and because it can be removed with 1 M CaCl(2) wash, we refer to it tentatively as extrinsic CA. This is to distinguish it from another form of CA activity tightly bound to PSII membranes that remains after CaCl(2) wash, which has been described previously. The function of extrinsic CA is not clear. It is unlikely to have the same function as the cytoplasmic CA, which has been proposed to increase the HCO(-)(3) concentration for phosphoenolpyruvate carboxylase and the C(4) pathway. We suggest that because the extrinsic CA is associated only with thylakoids doing linear electron flow, it could function to produce the CO(2) or HCO(-)(3) needed for PSII activity.

In VitroReconstitution of the Recombinant Photosystem II Light-harvesting Complex CP24 and Its Spectroscopic Characterization

The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or beta-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions. The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a.

Reconstitution and pigment-binding properties of recombinant CP29.

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.

Excited state equilibration in the photosystem I-light-harvesting I complex: P700 is almost isoenergetic with its antenna.

Photosystem I with its full antenna complement (PSI-LHCI) has been prepared by mild detergent solubilization with octyl beta-D-glucopyranoside from maize thylakoids. A preliminary polypeptide analysis is presented. At room temperature, the steady-state fluorescence derives from an almost perfectly thermalized state, as demonstrated by a Stepanov analysis, in which about 90% of the excited states are associated with the red chlorophyll spectral forms absorbing above 700 nm. Equilibration is temperature-sensitive and is lost at T < 200 K. A careful analysis of fluorescence between 75 and 280 K clearly demonstrates the presence of at least three red chlorophyll spectral forms with emission maxima at 720, 730, and 742 nm, the absorption origin bands of which have been calculated at 714, 725, and 738 nm. On the basis of a minor deviation from thermal equilibration around 695 nm, it is suggested that at least 3-4 antenna chlorophylls, with an average absorption near 695 nm, are strongly coupled to P700. Thermodynamic analysis of absorption and fluorescence spectra indicates that the equilibrium, absorption-weighted excited state population of the P700 dimer is around 0.013 assuming that the low-energy exciton state possesses all the oscillator strength. The average free energy for excitation transfer from antenna to P700 is thus calculated to be -0.26 kT at room temperature. This indicates that P700 is almost isoenergetic with its antenna at room temperature when the red forms are taken fully into account. From the calculated excited state population of P700, we estimate that the primary charge separation rate in PSI is 1-2 ps-1.

Oxidation-reduction potential dependence of formate binding to Photosystem II in maize thylakoids

The affinity of Photosystem II for formate is less when thylakoid membranes are incubated with ferricyanide in complete darkness. A redox titration of this effect showed that the affinity for formate is not determined by the oxidation state of the non-heme iron (Q 400) associated with the reaction center complex, nor does formate influence the mid-point potential, E m, of Q 400. Rather, the formate affinity is determined by the oxidation state of a component with an E m of about +480 mV at pH 7.0. The E m of this single-electron redox mediator, here referred to as D 480, has a pH-dependence of approx. 60 mV/pH. D 480 is tentatively identified as an auxiliary electron donor ( E m = + 475 mV at pH 7.6), first described by Bearden and Malkin (Bearden, A.J. and Malkin, R. (1973) Biochim. Biophys. Acta 325, 266–274). When D 480 is oxidized in the dark by ferricyanide, the affinity of Photosystem II for other monovalent anions such as bicarbonate and chloride is low. A single saturating flash reverses the effect of ferricyanide, indicating that oxidized D 480 can be reduced in the light. The binding of bicarbonate ions to formate-inhibited thylakoids shows a period-of-two oscillation with flash number. Oxidation of D 480 by ferricyanide has a strong damping effect on these oscillations.

High rates of photosystem II electron flow occur in maize thylakoids when the high-affinity binding site for bicarbonate is empty of all monovalent anions or has bicarbonate bound

Monovalent anions, such as formate and particularly bicarbonate, can control the rate of electron flow between Photosystem I and II. There are two explanations for this effect that differ in the residency state of a high-affinity binding site for bicarbonate when normal (rapid) electron flow occurs. One explanation, the 'bound-bicarbonate' hypothesis, requires that the site be filled with bicarbonate for high rates of electron flow. A second explanation, the ‘inhibitory-anion’ hypothesis, requires that the site either be filled with bicarbonate or be entirely free of any inhibitory monovalent anion. Electron flow in maize thylakoids was monitored by rates of oxygen evolution with dichlorobenzoquinone as an electron acceptor. The amount of bicarbonate bound to the high-affinity site in maize thylakoids was determined by measuring, the amount of bicarbonate displaced by injection of formate. The following results were obtained. Thylakoids with high-affinity binding sites devoid of bicarbonate and other monovalent anions have high rates of electron flow. The dissociation constant for bicarbonate at the high-affinity site is 40 ± 11 μ M; there are 2.4 ± 0.6 bicarbonate bound and 1.8 ± 0.3 atrazine bound per Photosystem II reaction center active in charge separation; in maize thylakoids 94% of separated charge in Photosystem II is used in oxygen evolution. Uncharacterized respiratory processes occur in spinach and pea thylakoid preparations. It is concluded that the inhibitory-anion hypothesis is correct. Also, bicarbonate might be present for high rates of electron flow in vivo, in which case it binds to its high-affinity site and excludes other monovalent anions that are inhibitory.

Modifications to Thylakoid Composition during Development of Maize Leaves at Low Growth Temperatures

The effects of reductions in growth temperature on the development of thylakoids of maize (Zea mays var LG11) leaves are examined. Thylakoids isolated from mesophyll cells of leaves grown at 17 degrees and 14 degrees C, compared with 25 degrees C, exhibited a decreased accumulation of many polypeptides, which was accompanied by a loss of activity of photosystems (PS) I and II. Probing the polypeptide profiles with a range of antibodies specific for thylakoid proteins demonstrated that a number of polypeptides encoded by the chloroplast genome failed to accumulate at low temperatures. Although thylakoid protein synthesis was reduced severely at 14 degrees C compared with 25 degrees C, major synthesis of both chloroplast and nuclear encoded polypeptides was detected. It is suggested that the lack of accumulation of some thylakoid proteins at low temperatures may be due to an inability to stabilize the proteins in the membranes. A number of thylakoid polypeptides were found to appear as the growth temperature was decreased. Analyses of pigments and polypeptides demonstrated that decreases in the photosystem reaction center core complexes occur relative to the light harvesting complex associated with PS II at reduced growth temperatures. Differential effects on the development of PSI and PSII were also observed, with PSII activity being preferentially reduced. Reductions in PSII content and activity occurred in parallel with decreases in the quantum yield and light-saturated rate of CO(2) assimilation. Fractionation of thylakoid pigment-protein complexes showed that the ratio of monomeric:oligomeric form of the light harvesting complex associated with PSII increased at low growth temperature, which is consistent with a chill-induced modification of thylakoid organization. Many, but not all, of the characteristic changes in thylakoid protein metabolism, which were observed when leaves were grown at low temperatures in controlled environments, were identified in leaves of a field maize crop during the early growing season when low temperatures were experienced by the crop. Chill-induced perturbations of thylakoid development can occur in the field in temperate regions and may have implications for the photosynthetic productivity of the crop.

Characteristics of the Mg2+-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg(2+)-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na(2)SO(3)-dependent Mg(2+)-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg(2+)-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg(2+)-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg(2+)-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t((1/2)) of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.

Absence of a Formate-Induced Release of Bicarbonate from Photosystem II

Formate has been proposed to inhibit electron flow in photosystem II by replacing endogenous bound bicarbonate on the reaction center complex. A mass spectrometer was used to measure directly the CO(2)/HCO(3) (-) released when maize thylakoids, showing normal rates of electron flow, were treated with formate. Although the formate inhibited electron flow by 95%, no release (displacement) of CO(2)/HCO(3) (-) was detected. This is consistent with the concept that membrane-bound HCO(3) (-) is not a requirement for normal rates of electron flow through photosystem II. Moreover, formate and other monovalent anions do not inhibit electron flow by removing bound HCO(3) (-) but by binding to empty sites. The "bicarbonate effect" is a reversal, by high concentrations of exogenous bicarbonate, of anion inhibition of photosystem II.

The roles of low temperature and light in accumulation of a 31‐kDa polypeptide in the light‐harvesting apparatus of maize leaves

Abstract Previously, accumulation of a 31‐kDa polypeptide had been observed in the light‐harvesting apparatus of thylakoids of maize leaves exposed to 5°C and high light (Hayden et al., 1986). The accumulation and disappearance of this 31‐kDa polypeptide in thylakoids of maize leaves are examined as a function of photon flux density and temperature. The accumulation of large amounts of the polypeptide at 5°C was light‐dependent during a 6‐h chill period, with 50% of maximal accumulation occurring at a photon flux density of 60 μmol m−2 s−1.Some polypeptide accumulation did occur in leaves kept in the dark at 5°C for 6 h, i.e. ca. 18% of that accumulating at a photon flux density of 1500 μmol m−2 s−1. The temperature optimum for polypeptide accumulation was ca. 9°C with greater than 50% of maximal accumulation being achieved between 5 and 11°C. The breakdown of maximally accumulated polypeptide on returning leaves to 25°C was complete after 1 h, had a half‐time of ca. 20 min and was independent of light. Breakdown of the polypeptide was also observed when thylakoids isolated from chilled leaves were incubated at 25°C. Reductions of thylakoid incubation temperature between 13 and 5°C markedly reduced the rate of polypeptide disappearance. The accumulation of the polypeptide is discussed in relation to temperature and light effects on the rate of the polypeptide synthesis and of peptidase activities. The results are also discussed in the context of accumulation of the polypeptide in maize leaves in the field and consideration is given to the possible physiological significance.

Organization of the photosynthetic membrane in maize mesophyll and bundle sheath chloroplasts studied by two-dimensional gel electrophoresis

Polypeptide composition and the distribution of Photosystem II polypeptides were studied in mesophyll and bundle sheath chloroplasts of maize by using a modified two-dimensional gel electrophoresis technique of O'Farrell (J. Biol. Chem. (1975) 250, 4007–4021). The modifications were LiDS solubilization instead of SDS, reverse isofocusing and sensitive silver staining procedure. This high-resolution technique allowed us to separate 160 polypeptides in the two types of thylakoid membrane. We found that both types of lamella contained nearly equal amounts of polypeptides, but 73 polypeptides were different in the two preparations. In mesophyll thylakoids, 75 polypeptides out of 140 were found to be characteristic, and 54 of them were exclusively present in mesophyll preparations. In bundle sheath, 24 polypeptides out of 106 were characteristic and 19 of these polypeptides were exclusively present in bundle sheath lamellae. In functional Photosystem II prepared from maize thylakoids, 68 individual polypeptides could be distinguished. Most of these polypeptides were present in both mesophyll and bundle sheath lamellae, but 19 of them were more pronounced in mesophyll than in bundle sheath lamellae. On the other hand, 11 polypeptides of Photosystem II were distinctly different in mesophyll and bundle sheath lamellae. These differences may be in close connection with the Photosystem II heterogeneity existing in the two types of thylakoid.

Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0.

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].

Differential expression of six light-harvesting chlorophyll a/b binding protein genes in maize leaf cell types.

Bundle sheath chloroplasts of maize leaves contain about one-fourth as much light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP-II) as do mesophyll chloroplasts. We have determined that this difference is, in part, the result of differential expression of different LHCP-II genes. We have prepared and partially characterized cDNA clones specific for six LHCP-II genes of maize. Transcripts of these six LHCP-II genes are present at vastly different levels and account for about 95% of total LHCP-II mRNAs in bundle sheath and mesophyll cells of illuminated dark-grown maize leaves. Three genes are preferentially expressed in mesophyll cells, and their mRNAs constitute about 54% of the total LHCP-II transcripts in greening (24 hr) maize leaves. Two genes are expressed equally in bundle sheath and mesophyll cells. Most interestingly, the RNA of one gene that contributes about 8% of the total LHCP-II transcripts in leaves greening for 24 hr is present at a much higher level in bundle sheath than in mesophyll cells. Moreover, immunoblot analysis of maize thylakoids reveals at least five sizes of LHCP-II; these also differ from one another in their relative abundance in bundle sheath and mesophyll cells of developing maize leaves.

Protein PSII-G. An additional component of photosystem II identified through its plastid gene in maize.

An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.

Chlorophyll-proteins of two photosystem I preparations from maize

Two photosystem I (PSI) preparations were purified by non-denaturing SDS-PAGE or sucrose gradient ultracentrifugation and examined as to their chlorophyll-protein composition. In both preparations a minimum of two chlorophyll-proteins can be distinguished in addition to the 110 kDP-700 Chla-P1 complex. One (LHCI-730) is a chlorophyll-protein (Mr 40 kD) having a high chlorophylla/b ratio, and a major 77 K fluorescence peak at 730 nm. It consists of three polypeptides with apparent molecular weights of 21, 22.5 and 24 kD. Another chlorophyll-protein (LHCI-680) with a lower molecular weight (Mr 25 kD) fluoresces at 77 K with a maximum at 680 nm. This chlorophyll-protein has a high chlorophyllb content and two constituent polypeptides of 20 and 25 kD. Absorption, 77 K fluorescence and circular dichroism spectra of the PSI related chlorophyll-proteins are presented and compared with those of photosystem II chlorophyll-proteins from maize thylakoids. We propose a model for energy transfer in the photosystem I reaction centre with the following sequence: LHCI-730→LHCI-680 →Chla-P1. LHCI-680 acts as a connecting antenna which can also transfer energy from Chla/b-P2. This model was used to interpret the 77 K fluorescence emission from two barley mutants.

Functional discrimination between Photosystem-II associated chlorophyll a proteins in Zea mays

Three minor Chl a proteins were detected in electrophoretic profiles from wild-type maize thylakoids. The spectral characteristics of these Chl proteins and the apparent molecular weights of their constituent apoproteins suggested that they were associated with the Photosystem-II reaction center. One of these Chl a-proteins, CPa-1, was present in wild-type thylakoids and a photochemically active Photosystem-II particle, but was missing from thylakoids of a mutant-lacking Photosystem-II reaction center. CPa-2, on the other hand, was enriched in mutant thylakoids but was completely missing from the Photosystem-II particles. We conclude that CPa-1 is most likely to contain the photoactive chlorophyll of Photosystem II, while CPa-2 is not required for Photosystem-II activity. The apparent molecular weights of the major CPa-1 and CPa-2 apoproteins were 48 000 and 42 000, respectively. The third minor Chl protein seems most likely to be an electrophoretic variant of CPa-1 and has been designated CPa-1∗. Seven other Chl proteins were detected in wild-type profiles. Many of these Chl proteins appeared to be oligomers or highly order complexes of LHCP and CP-1.