Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immunolocalization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.Leaves of C4 plants contain two distinct types of photosynthetic cells, mesophyll (M3) and bundle sheath (BS), that are quite distinctly organized both structurally and functionally. Since the discovery of C4-dicarboxylic acid photosynthesis, much effort has been directed to determining the sequence of reactions in that pathway (3,11,20,26,28,29). Accumulated data agree upon the existence in C4 plants of an active PSI in both cell types and of PSII in M cells; however, there is disagreement about the existence of PSII activity in BS cells of maize and even about the presence of some of the constituents of PSII in BS cells (2,3,10,20,26,28,29 (3,26,28). In all of these studies, BS cells were first separated from M cells and then examined for the presence of mRNA for LHCII, or for the polypeptide itself.The apoproteins of LHCII are encoded by a nuclear gene family and synthesized on cytoplasmic ribosomes as a watersoluble, higher mol wt precursor form(s), preLHCII. During its insertion into the thylakoid membranes it is processed to its mature, water-insoluble form, and photosynthetic pigments are added to it (7,14,24,25,30). The sequence of events that leads to its incorporation into the LHCII complex is, however, largely unknown. Uptake experiments in which in vitro synthesized preLHCII is incubated with a suspension of intact chloroplasts show that the efficiency with which the preLHCII is processed to its mature size and incorporated into the pigmented LHCII complex depends upon the stage of plastid development (5). Import ofthe precursor by plastids is energy dependent and requires a putative receptor in the chloroplast envelope (9,13,16). A stromal factor(s), probably proteinaceous, is required for insertion into the thylakoid membrane (5,8).In the present study we show qualitatively, using in situ hybridization and in situ immunolocalization techniques which avoid fractionation of the two cell types, that mRNA for LHCII and the LHCII gene translation product(s) accumulate to a detectable level i...
The photosystem I (PSI) complex of Lemna gibba, isolated by deriphat/polyacrylamide gel electrophoresis of thylakoids solubilized in glycosidic surfactants, has been fractionated into its two chlorophyll-protein complexes: a core component (CCI) and a light-harvesting component (LHCI), using either non-denaturing gel electrophoresis or ion-exchange chromatography/sucrose gradient centrifugation. Both methods yielded an LHCI component that contained only one apoprotein of approximately 20 kDa. All the chlorophyll b and lutein of the PSI complex is associated with this LHCI preparation. The chlorophyll a/b ratio of this chlorophyll-protein is 2.5, and lutein is essentially the only carotenoid present. While the purified LHCI from Lemna cross-reacts with antibodies raised against spinach LHCPIb of Lam et al. [FEBS Lett. 168, 10 -14 (1984)], no cross-reactivity occurred between it and the major light-harvesting chlorophyll-a/b -protein of PSII, LHCIIP. This and a comparison of the amino acid and pigment compositions of the apoproteins of the LHCI and LHCIIP chlorophyll-proteins indicate that these are two distinct but similar chlorophyll-proteins.Thylakoid membrane polypeptides are organized in situ into several discrete multiprotein complexes (e. g. the photosystem I, photosystem 11, cytochrome b/f and ATPase complexes). Only the photosystem I and 11 (PSI and PSII) complexes contain the photosynthetic pigments and thus these complexes function to harvest light and perform the primary photochemical event [I]. It is convenient, as well as being technically correct, to think of the pigments in both the PSI and PSII complexes as being distributed between two components: a light-harvesting component (LHC) and a core component (CC); in the latter both light-harvesting and primary photochemistry take place [I]. Mullet et al. [2] presented the seminal report on the CCI and LHCI components of PSI. PSI particles, isolated from pea thylakoids solubilized with Triton X-100, were shown to have a ratio of 110 chlorophylls per P700, to contain chlorophylls a and b and 11 polypeptide subunits, and to emit a 735-nm fluorescence at 77 K [2]. Those polypeptides, migrating between 22.5 kDa and 24.5 kDa, were proposed to be the apoproteins of LHCI. The putative chlorophyll-protein complex was believed to contain the site of the 735-nm fluorescence. Later, Haworth et al. [3] isolated the LHCI complex from peas and showed that indeed it contained four protein subunits. The isolated material had a chlorophyll a/b ratio of 3.7, and it emitted maximally at 730 nm at 77 K. The release of LHCI from the PSI complex yielded a residual CCI, which contained only seven polypeptide subunits: the reaction center apoprotein(s) of about 68 kDa and six smaller polypeptides A PSI complex (e.g. CCI + LHCI) can also be purified by mild SDS/polyacrylamide gel electrophoresis [7 -91. Using this technique, Kuang et al. [lo] and Argyroudi-Akoyunoglou [ll] observed an LHCI component containing only a 21-kDa polypeptide in pea PSI-1 10 and CPIa, respectivel...
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