Spermidine levels and its relationship to DNA synthesis in outgrowing spores and vegetative cells of Bacillus subtilis

It is now established that the light-harvesting chlorophyll—protein complex (LHCP) of chloroplasts becomes phosphorylated in the light. In this study subfractionation of phosphorylated intact chloroplasts has been carried out to compare the phosphorylation of LHCP in non-appressed and appressed thylakoid regions. The results show around 10-times higher relative phosphorylation in the non-appressed regions than in the appressed ones. Since the non-appressed thylakoids also contain almost all photosystem 1, this region is likely to be the site for energy transfer from LHCP to photosystem 1 under phosphorylated conditions.     

Polypeptide composition and spectral properties of light-harvesting chlorophyll ab-protein complexes from intact and trypsin-treated chloroplast thylakoid membranes

The polypeptide composition and spectral properties of isolated light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complexes from intact and trypsin-treated thylakoid membranes of Hordeum vulgare and Vicia faba are compared. The LHCP complexes consist of four distinct polypeptides with molecular weights between 21 000 and 25 000 occurring in equal relative amounts in the whole polypeptide spectra of thylakoid membranes. It is shown indirectly that the two major polypeptides very probably belong to different chlorophyll-proteins. The loss of a small segment from both polypeptides during trypsin digestion of thylakoids does not substantially alter the spectral properties and cation-mediated aggregation of isolated LHCP complexes.     

State 1-state 2 transition in leaves and its association with ATP-induced chlorophyll fluorescence quenching

By using chlorophyll fluorescence, a study has been made of changes in spillover of excitation energy from Photosystem (PS) II to PS I associated with the State 1–State 2 transition in intact pea and barley leaves and in isolated envelope-free chloroplasts treated with ATP. (1) In pea leaves, illumination with light preferentially absorbed by PS II (Light 2) led to a condition of maximum spillover (state 2) while light preferentially absorbed by PS I induced minimum spillover condition (State 1) as judged from the redox state of Q and low-temperature emission spectra. The State 1–State 2 transitions took several minutes to occur, with the time increasing when the temperature was lowered from 19 to 6°C. (2) In contrast to the wild type, leaves of a chlorophyll b-less mutant barley did not exhibit a State 1–State 2 transition, suggesting the involvement of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex in spillover changes in higher plants. (3) Spillover in isolated pea chloroplasts was increased by treatment with ATP either (a) in Light 2 in the absence of an electron acceptor or (b) in the dark in the presence of NADPH and ferredoxin. These observations can be interpreted in terms of the model that a more reduced state of plastoquinone activates the protein kinase which catalyzes phosphorylation of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981). Nature 291, 25–29). This process was found to be very temperature sensitive. (4) Pea chloroplasts illuminated in the presence of ATP seemed to exhibit a slight decrease in the degree of thylakoid stacking, and an increased intermixing of the two photosystems. (5) The possible mechanism by which protein phosphorylation regulates the State 1–State 2 changes in intact leaves is presented in terms of changes in the spatial relationship of two photosystems resulting from alteration in membrane organization.     

High light fluence impairs light-harvesting Chl a/b complex apoprotein syntheses and/or membrane uptake in the CD3 mutant of wheat

The CD3 mutant of wheat is a chlorophyll(Chlo-deficient mutant the phenotype of which depends upon the accumulation of the light-harvesting Chl a/b protein complex in leaves in response to the intensity of illumination. In the present studies, the rates of synthesis and/or uptake, and degradation of the light-harvesting Chl apoprotein in chloroplasts of wild-type wheat ( Triticum aestivum L. selection ND 496) and CD3 wheat leaf segments were examined in response to two different intensities of illumination. We were interested particularly in the 21. 23 kDa proteins of the light-harvesting Chl a/b complex of photosystem I (LHCI) and the 25. 27. 29 kDa proteins of the light-harvesting Chl a/b complex of photosystem II (LHCII). The accumulation of [³⁵S]-Met into the light-harvesting Chl protein of CD3 wheat chloroplasts was impaired by a high but not by a low light fluence. The levels of radiolabel in the supernatant fractions of leaf tissue homogenates from the wild-type and CD3 wheats were not significantly different over time, suggesting that the cellular uptake of [³⁵S]-Met was not limiting in the mutant. The high fluence did not enhance the degradation of light-harvesting Chl protein from CD3 wheat thylakoids. Our data indicate an impairment in the light-harvesting Chl protein synthesis/membrane uptake system in CD3 wheat leaves under high fluence. A recovery in levels of the inner LHCPII, but not of LHCPI, was observed in the Chl-deficient wheat mutant after a prolonged (4 days) exposure to high fluence. Under low fluence, LHCP was added to both photosystem II (PSH) and photosystem I (PSI) but only that added to PSI remained in thylakoids after seedlings were switched to high fluence.     

Integration of nuclear-encoded proteins into pea thylakoids with different pigment contents

The in vitro membrane integration of the light-harvesting protein of photosystem II (LHCP), the Rieske FeS protein of the cytochrome (Cyt) blf-complex, and the NADPH:protochlorophyllide oxidoreductase (Pchlide reductase) into pea thylakoids with different pigment composition was studied. Pea plants (Pisum sativum L. cv. Kelvedon Wonder) with different contents of chlorophyll (Chl) and carotenoids were obtained by growing the seedlings in a greenhouse or in weak red light with or without the herbicide Norflurazon, an inhibitor of carotenoid biosynthesis. Chloroplasts from untreated and Norflurazon-treated plants grown in weak red light contained approximately 29 and 14% of Chl compared to chloroplasts from untreated plants grown in the greenhouse. The corresponding carotenoid contents were 66 and 5%. Following an integration reaction using LHCP precursor protein and chloroplast lysate, thylakoids from untreated and Norflurazon-treated plants grown in weak red light contained approximately 30 and 5% of protease-protected LHCP, respectively, compared to thylakoids of untreated plants grown in a greenhouse. In contrast to LHCP, the in vitro assembly of the Pchlide reductase was only sligthly reduced in chloroplast lysates of plants grown in weak red light compared to greenhouse-grown plants. In chloroplast lysates of Norflurazon-treated plants, however, the amount of membrane associated, protease-protected Pchlide reductase was reduced to 32% of the amount in untreated plants grown under the same light conditions. In contrast, the integration of the Rieske FeS protein occurred to almost similar levels irrespective of light conditions and herbicide treatments. Reconstitution assays where stroma from Norflurazon-treated plants was added to thylakoids from untreated plants, showed that the herbicide did not affect any stromal component(s) vital for the insertion reaction. Removal of samples during the integration reaction of LHCP showed that no degradation of the protein occurred during the assay. Neither was the assembled protein degraded up to 24 h after the termination of the assay. This indicates that growing plants in weak red light, with or without Norflurazon treatment, mainly affected the primary step in thylakoid assembly of LHCP, i.e. the insertion reaction into the membrane. The results further indicate that proteins normally bound to pigments also require pigments for membrane recognition or integration.     

Oceanic picophytoplankton having a high abundance of chlorophyll b in the major light harvesting chlorophyll protein complex

Chl a-containing, very small unicellular, eukaryotic phytoplankton (picophytoplankton) often become the dominant organisms near the bottom of the euphotic zone in the ocean, where light is limited, not only in intensity (about 0.5% of the surface irradiance), but also in quality (dominant in blue to green wavelengths). We have isolated picophytoplankton from subsurface waters (from 75 to 150 m in depth) of the Kuroshio area near Japan. EM observations showed that a single chloroplast occupies a large part of the cytoplasm. Some of the isolates have a flagellum. The major photosynthetic pigments found in these isolates were chlorophyll a and b. The light-harvesting chlorophyll a/b complex (LHCP) was isolated from three clones of picophytoplankton, one flagellated form (NIBB8001) and two coccoid forms (94B8100A and 94B5100C) . More than 50% of the total chlorophylls were recovered in the major LHCP fraction. A common feature of the major LHCPs isolated from the three picophytoplankton clones was a high abundance of chlorophyll b: the ratios of chlorophyll a to b were about 0.8, 0.7 and 0.6 for the clones NIBB8001, 94B8100A and 94B5100C, respectively. These values were very low compared with those in chlorophyll a/b-binding LHCIIs in higher plants and in the major chlorophyll a/b-binding LHCPs in microalgae (higher than 1.0). The major LHCP apoproteins of NIBB8001 and 94B5100C contained one major polypeptide; the apparent molecular masses analyzed with SDS-PAGE were about 22 kDa and 27 kDa, respectively. The major LHCP apoprotein of 94B8100A had two major polypeptides having apparent molecular masses of about 23 and 25 kDa. None of the thylakoid proteins cross-reacted with an antibody raised against the LHC II apoprotein of spinach. It is suggested that the high abundance of chlorophyll b in picophytoplankton, together with a large chloroplast in a small cell, enable them to utilize the reduced light in their habitat.     

Assembly of chloroplast signal recognition particle involves structural rearrangement in cpSRP43

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54_pep). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54_pep in a 1:1 stoichiometry with an apparent K_d of ∼ 1.06 μM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54_pep interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54_pep shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.     

The Stay-Green Rice like (SGRL) gene regulates chlorophyll degradation in rice

The Stay-Green Rice (SGR) protein is encoded by the SGR gene and has been shown to affect chlorophyll (Chl) degradation during natural and dark-induced leaf senescence. An SGR homologue, SGR-like (SGRL), has been detected in many plant species. We show that SGRL is primarily expressed in green tissues, and is significantly downregulated in rice leaves undergoing natural and dark-induced senescence. As the light intensity increases during the natural photoperiod, the intensity of SGRL expression declines while that of SGR expression increases. Overexpression of SGRL reduces the levels of Chl and Chl-binding proteins in leaves, and accelerates their degradation in dark-induced senescence leaves in rice. Our results suggest that the SGRL protein is also involved in Chl degradation. The relationship between SGRL and SGR and their effects on the degradation of the light-harvesting Chl a/b-binding protein are also discussed.     

The influence of metabolic state on the level of phosphorylation of the light-harvesting chlorophyll-protein complex in chloroplasts isolated from maize mesophyll

The activity of the protein kinase that phosphorylates the light-harvesting chlorophyll-protein of Photosystem II (LHCP) has been investigated in intact chloroplasts isolated from maize mesophyll cells. Measurements of ³²P incorporation into LHCP, ATP concentration, $\text{ATP}\text{ADP}$ ratio, ΔpH, chlorophyll fluorescence and oxygen evolution were made in the presence of different metabolic substrates. Without added substrate a high level of LHCP phosphorylation was observed which was suppressed by addition of oxaloacetate or phosphoglycerate but stimulated by pyruvate. Whereas no correlation was observed between LHCP phosphorylation and adenylate status, a clear effect of redox state on protein kinase activity was observed. A correlation between a highly reduced electron-transfer chain (produced under conditions which favour cyclic electron flow) and the maximum level of protein phosphorylation was observed. The regulation of kinase activity and its dependence on electron transfer and carbon assimilation are discussed.