Differential Detergent Stability of the Major Light-Harvesting Complex II in Thylakoids Isolated from Monocotyledonous and Dicotyledonous Plants

A survey of isolated thylakoids from 11 different higher plant species (Spinacia oleracea L., Pisum sativum L., Vicia faba L., Brassica napus L., Vigna sinensis L., Vinca minor L., Secale cereale L., Triticum aestivum L., Triticosecale Wittn., Hordeum vulgare L., Zea mays L.) indicated that the ratio of the oligomeric:monomeric form of the light-harvesting complex II was twofold higher for the dicots (3.16 ± 0.35) than the monocots (1.64 ± 0.25) examined under identical separation procedures. Under conditions specifically designed to stabilize the oligomeric form in vitro, we show that the oligomeric form of dicot light-harvesting complex II is twice as stable to solubilization in the presence of sodium dodecyl sulfate (SDS) than that observed for monocots. This decreased stability of monocot light-harvesting complex II is associated with a twofold increase in the trienoic fatty acid level of thylakoid phosphatidylglycerol but with no significant changes in the trienoic fatty acid levels in the major galactolipids. In addition, SDS polyacrylamide gel electrophoresis and western blot analyses with monoclonal antibodies indicated that monocots exhibited greater heterogeneity in the polypeptide complements associated with subfractions of light-harvesting complex II than the dicots examined. The data indicate that the oligomeric form of the light-harvesting complex II is not the result of a simple oligomerization of a common monomeric unit. We suggest that the difference in stability of the oligomeric form of light-harvesting complex II in isolated thylakoids of monocots and dicots is probably due to a differential accessibility to SDS. The differential SDS accessibility may be due to differences in thylakoid protein-protein and/or protein-lipid interactions.     

Effects of 5-Aminolevulinic Acid on the Accumulation of Chlorophyll b and Apoproteins of the Light-Harvesting Chlorophyll a/b-Protein Complex of Photosystem II

The effects were examined of 5-aminolevulinic acid (ALA) onthe accumulation of Chl and apoproteins of light-harvestingChl a/b-protein complex of photosystem II (LHCII) in cucumbercotyledons under intermittent light. A supply of ALA preferentiallyincreased the accumulation of Chl a during intermittent illumination.However, when cotyledons were pretreated with a brief exposureto light or benzyladenine (BA), the stimulatory effect of ALAon the increase in the level of Chl b was greater than thatin the level of Chl a, resulting in decreased ratios of Chla/b. Time-course experiments with preilluminated cotyledonsrevealed that LHCII apoproteins accumulated rapidly within thefirst 30 min of intermittent illumination with a decline duringsubsequent incubation in darkness. A supply of ALA did not affectthe accumulation of LHCII apoproteins during the intermittentlight period, but it efficiently inhibited the decline in theirlevels during the subsequent darkness. After exposure to a singlepulse of light of BA-treated cotyledons, the prompt increasein levels of LHCII apoproteins was not accompanied by the formationof Ch b, which began to accumulate later. The pattern of changesin levels of LHCII apoproteins was quite similar to that inlevels of Chl a. These results suggest that LHCII apoproteinsare first stabilized by binding with Chl a and that an increasedsupply of Chl a and the accumulation of LHCII apoproteins areprerequisites for the formation of Chl b. ¹Present address: Department of Chemistry, Faculty of Scienceand Technology, Meijo University, Aichi, 468 Japan.     

Thermostability and Photostability of Photosystem II in Leaves of the Chlorina-f2 Barley Mutant Deficient in Light-Harvesting Chlorophyll a/b Protein Complexes

The chlorophyll-b-less chlorina-f2 barley mutant is deficient in the major as well as some minor light-harvesting chlorophyll-protein complexes of photosystem II (LHCII). Although the LHCII deficiency had relatively minor repercussions on the leaf photosynthetic performances, the responses of photosystem II (PSII) to elevated temperatures and to bright light were markedly modified. The chlorina-f2 mutation noticeably reduced the thermostability of PSII, with thermal denaturation of PSII starting at about 35[deg]C and 38.5[deg]C in chlorina-f2 and in the wild type, respectively. The increased susceptibility of PSII to heat stress in chlorina-f2 leaves was due to the weakness of its electron donor side, with moderate heat stress causing detachment of the 33-kD extrinsic PSII protein from the oxygen-evolving complex. Prolonged dark adaptation of chlorina-f2 leaves was also observed to inhibit the PSII donor side. However, weak illumination slowly reversed the dark-induced inhibition of PSII in chlorina-f2 and cancelled the difference in PSII thermostability observed between chlorina-f2 and wild-type leaves. The mutant was more sensitive to photoinhibition than the wild type, with strong light stress impairing the PSII donor side in chlorina-f2 but not in the wild type. This difference was not observed in anaerobiosis or in the presence of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea, diuron. The acceptor side of PSII was only slightly affected by the mutation and/or the aforementioned stress conditions. Taken together, our results indicate that LHCII stabilize the PSII complexes and maintain the water-oxidizing system in a functional state under varying environmental conditions.     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

Synthesis and Breakdown of the Apoproteins of Light-Harvesting Chlorophyll a/b Proteins in Chlorophyll b-deficient Mutants of Rice

Turnover, in the light, of apoproteins of light-harvesting chlorophylla/6-proteins for Photo-system I and II (LHC-I and LHC-II, respectively)was studied with the wild-type and three chlorophyll 6-deficientmutants of rice. (1) Synthesis of the 24 and 25 kDa apoproteinsof LHC-II and the 20 and 21 kDa apoproteins of LHC-I was examinedby incubating leaf segments with [³⁵S]-methionine. The threerice mutants, chlorina 2, which totally lacks chlorophyll b,and chlorina 11 and 14, which are partially deficient in chlorophyllb, synthesized the apoproteins as rapidly as did the wild typerice. (2) Pulse-chase experiments showed that breakdown of theapoproteins proceeded slowly, such that only a small proportionof the newly synthesized apoproteins was lost during the 48h of the chase in normal rice leaves. By contrast, large fractionsof the labelled apoproteins were rapidly degraded within thefirst several hours of the chase period in the chlorina mutants.The greater the deficiency in chlorophyll b of the mutant, thelarger were the rate and extent of the protein breakdown. Thisresult indicates that chlorophyll b is needed to stabilize theapoproteins of LHC-II and LHC-I. (3) However, even in chlorina2, there were small fractions of the apoproteins with lifetimesas long as those of apoproteins in the wild-type rice, suggestingthat the newly synthesized apoproteins are partially protectedby a factor(s) other than chlorophyll b. (4) The rate of turnoverof the apoproteins was significantly reduced in the dark andstrongly inhibited by prior treatment of leaf segments withchloramphenicol. (Received November 24, 1988; Accepted March 17, 1989)     

Organization and Stability of Polypeptides Associated with the Chlorophyll a-b Light-Harvesting Complex of Photosystem-II

In the oxygen-evolving photosystem-II (PSII) of higher plantchioroplasts and green algae, most of the light-harvesting functionis performed by the chlorophyll (Chl) a-b-protein complex (LHC-II).On the average, the LHC-II contains about 210 Chl (a+b) moleculesper PSII reaction center. The polypeptide composition, copynumber and organization of assembly in the LHC-II complex arenot fully understood at present. This work utilized the chlorinaf2 mutant of barley (lacking Chl b and having a LHC-II antennaof only 13 Chl a molecules) to determine the organization andstability of assembly of proteins in the LHC-II. High-resolutionSDS-PAGE and immunoblot analysis showed the presence of fourmain constitutive polypeptides in the wild-type LHC-II (termedhere subunits a, b, c and d) with molecular masses in the range30–25 kDa. Of those, only subunit d (a 25 kDa polypeptide)was found to occur at an equal copy number per PSII reactioncenter in both wild-type and in the Chl b-less chlorina f2 mutant.All other subunits were either absent or existed in much loweramounts in the mutant. Subunit d is a polypeptide constituentof the major Chl-protein subcomplex (CPII) of the LHC-II. Itis stably incorporated in the thylakoid membrane in the absenceof Chl b and probably binds the 13 Chl a molecules in the residualLHC-II antenna of the chlorina f2 mutant. We propose that, ofall LHC-II polypeptides, subunit d is most proximal to the PSIIcore and may serve as a linker in the process of excitationenergy transfer from the bulk LHC-II to the PSII reaction centerin chloroplasts. (Received February 25, 1992; Accepted May 12, 1992)     

Relation between the Light-Harvesting Chlorophyll a-Protein Complex LHCPa and Photosystem I in the Alga Chlamydobotrys stellata

The light-harvesting chlorophyll protein system of the alga Chlamydobotrys stellata consists of an as yet uncharacterized algal chlorophyll a-protein, called LHCPa, and a common photosystem II-related chlorophyll a/b-protein, called LHCPb (Brandt, Kaiser-Jarry, Wiessner 1982 Biochim Biophys Acta 679: 404-409). For further characterization, this LHCPa was isolated from the organism by polyacrylamide isoelectrofocusing and reelectrophoresis. It contains only chlorophyll a and has only one apoprotein (32,000 daltons). When separated from autotrophically grown cells, its absorption peak is at 674 nm and its isoelectric point at 5.3. Photoheterotrophic cultivation of the algae shifts the absorption maximum of LHCPa to 679 nm and its isoelectric point to 4.8. This LHCPa is a component of photosystem I particles. In relation to the total chlorophyll a content, the amount of LHCPa is low in autotrophic algae, but increases under photoheterotrophic growth conditions, where the organisms do not have the ability to assimilate CO₂ photosynthetically.     

Photoregulation of the Light-Harvesting Chlorophyll Protein Complex Associated with Photosystem II in Dunaliella tertiolecta: Evidence that Apoprotein Abundance but Not Stability Requires Chlorophyll Synthesis

The marine chlorophyte Dunaliella tertiolecta Butcher responds to a one-step transition from a high growth irradiance level (700 micromoles quanta per square meter per second) to a low growth irradiance level (70 micromoles quanta per square meter per second) by increasing the total amount of light-harvesting chlorophyll (Chl) a/b binding protein associated with photosystem II (LHC II), and by modifying the relative abundance of individual LHC II apoproteins. When high light-adapted cells were incubated with gabaculine, which inhibits Chl synthesis, and transferred to low light, the LHC II apoproteins were still synthesized and the ³⁵S-labeled LHC II apoproteins remained stable after a 24 hour chase. These results suggest that Chl synthesis is not required for stability of the LHC II apoproteins in this alga. However, when the control cells are transferred from high light to low light, the amount of the four LHC II apoproteins per cell increases, whereas it does not in the presence of gabaculine. These results suggest that Chl synthesis is required for a photoadaptive increase in the cellular level of LHC II.     

Effects of High Temperature Exposure of Spinach Intact Plants and Isolated Thylakoids on Light-Harvesting Complex 2 Protein Phosphorylation

After a 6 min exposure of isolated thylakoids to 43 °C, the extent of phosphorylation of light-harvesting complex of photosystem 2 (LHC2) was higher than in control thylakoids kept at 25 °C. Similarly, the exposure of intact spinach plants to 43 °C in dark for 11 h induced higher extent of thylakoid LHC2 phosphorylation than in control plants kept at 25 °C. The induced ability of LHC2 for enhanced phosphorylation may enable better energy distribution in favour of photosystem 1.     

Reorganization of the Photosystem II Unit in Developing Thylakoids of Higher Plants after Transfer to Darkness : Changes in Chlorophyll b, Light-Harvesting Chlorophyll Protein Content, and Grana Stacking

A light-dependent reversible grana stacking-unstacking process, paralleled by a reorganization of thylakoid components, has been noticed in greening etiolated bean (Phaseolus vulgaris, var. red kidney) leaves upon transfer to darkness. The reorganization, based on biochemical and biophysical criteria, involves mainly the photosystem II (PSII) unit components: upon transfer to darkness, the light-harvesting chlorophyll protein (LHCP), its 25 kilodalton polypeptide and chlorophyll b are decreased, while the CPa and its 42 kilodalton polypeptide are increased and new PSII units of smaller size are formed. This reorganization of components occurs only in thylakoids still in the process of development and not in those present in steady state conditions.

It is proposed that this process does not reflect the turnover of the LHCP component per se, but a regulatory process operating during development, by which the ratio of light-harvesting to PSII reaction center components, determined by the environmental conditions, controls the photosynthetic rate.

     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

Isolation and Partial Characterization of the NADH Dehydrogenase Complex from Barley Chloroplast Thylakoids

Using chloroplasts from barley leaves we attempt to purify andpartially characterize the NADH dehydrogenase complex. The enzymaticactivity was assayed as NADH-ferricyanide and NADH-nitro bluetetrazolium oxidoreductase. Analyzed by SDS-polyacrylamide gelelectrophoresis and subsequent enzymatic renaturation, the chloroplastsoluble fraction contains principally a 66 kDa enzyme. The membranousfraction solubilized with deoxycholate and analyzed by nativeelectrophoresis and NADH-nitro blue tetrazolium staining revealedthree enzymes: one with similar electrophoretic mobility tothat described for the soluble enzyme, another one which isa complex separated in 3% polyacrylamide gel and a third one,another complex separated in the top of the 5–22% linearpolyacrylamide gel gradient. The complex polypeptidic patternswere similar but different to those found for any thylakoidalproteinic complex known. Nine major polypeptides were detectedin the complex polypeptidic patterns, four of them constituentsof the small size thylakoid enzyme. The molecular masses ofsix polypeptides agreed with those indicated as encoded by 6chloroplast ndh genes. All the enzymes, including the 66 kDasoluble enzyme, contained a 53 kDa polypeptide, which is probablythe NADH-binding complex subunit. Isoelectric focusing of thethylakoidal enzyme points to a basic isoelectric point. Ion-exchangeor hydroxylapatite column chromatography followed by nativeelectrophoresis of the active fractions only separated the smallsize enzyme, which showed complex inactivation. (Received March 16, 1995; Accepted September 18, 1996)     

Supramolecular Assembly and Function of Subunits Associated with the Chlorophyll a-b Light-Harvesting Complex II (LHC II) in Soybean Chloroplasts

The chlorophyll (Chl) a-b light harvesting complex II (LHC II)contains more than 80% of the light-harvesting pigments of photosystemII (PS II) in chloroplasts. The supramolecular assembly andfunction of this auxiliary antenna system was investigated inChi b-deficient and Chi b-less mutant chloroplasts from soybeanand barley plants, and in their wild-type counterparts. Fourdistinct LHC II polypeptides were resolved by SDS-PAGE (subunitsa, b, c and d), having apparent molecular masses of 29, 28,27.2 and 26.8 kDa, respectively. The analysis of LHC II subunitcomposition in different developmental stages of the PS II unitin soybean (3>Chla/Chlbb>6), indicated the associationof specific subunits with the LHC H-inner and LHC II-peripheralin the chloroplast. The amount of subunit a in PS II was constantover a broad range of Chl a/Chl b ratios, suggesting that thissubunit is closely associated with the PS II-core complex. Subunitd also appeared to be constant over a wide range of Chl a/Chlb ratios, suggesting close association with the LHC II-inner.The PS II content in subunits b and c increased with the PSII antenna development in soybean but the ratio of b/c remainedconstant in all developmental stages and equal to 2 :1. Subunita was present in the Chl b-less chlorina f2 mutant of barleygrown under continuous illumination but was absent under intermittentillumination. The results suggest that each subunit binds 13-15Chl molecules. A working hypothesis is presented on the PS IIantenna development and LHC II subunit composition in soybeanchloroplasts. (Received October 11, 1988; Accepted January 19, 1989)     

叶绿素缺乏大麦突变体光系统I色素蛋白复合物的研究

同野生型大麦相比,突变大麦光系统ＩＩ捕光色素蛋白复合物的含量明显降低,其多肽组分也发生了变化：２６ｋＤ的多肽缺失,２４ｋＤ、２７ｋＤ和３０ｋＤ多肽含量减少。ＲＮＡ印迹杂交结果表明突变大麦中ｃａｂ基因的表达与野生大麦基本一致,说明突变大麦中ＬＨＣＩ多肽的缺乏不是在转录水平引起的障碍,而很可能是受转录后水平的调节。突变大麦叶绿体的内膜系统处于发育的初级阶段,基粒较少,类囊体膜的垛叠也受到很大的限制,这可能与２６ｋＤ多肽的缺失有关。     

叶绿素缺乏的大麦突变体的光合作用和叶绿素荧光

和早熟３号大麦（野生型）（ＤＧ）相比,黄化大麦（突变体）（ＬＧ）叶片Ｃｈｌ含量低,而Ｃｈｌａ／ｂ较高;光合速率低,表现量子效率低,而饱和光强较高,气孔导度较高;荧光参数Ｆｏ低,而ＰＳⅡ的光化学效率（Ｆｖ／Ｆｍ）以及Ｔ１／２、Ｆｍ／Ｆｏ、Φｅ均高。以单位叶绿素计算,黄化大麦的ＰＳⅡ电子传递活性和全链电子传递活性较高,而ＰＳⅠ电子传递活性较低。推测黄化大麦ＰＳⅡ的光化学效率增高可能是由于其ＰＳⅡ向ＰＳⅠ传递的激发能较少,是对Ｃｈｌ含量低的一种补偿。     

Organization of the Light-Harvesting Complex of Photosystem I and Its Assembly during Plastid Development

Photosystem I (PSI) holocomplexes were fractionated to study the organization of the light-harvesting complex I (LHC I) pigment-proteins in barley (Hordeum vulgare) plastids. LHC Ia and LHC Ib can be isolated as oligomeric, presumably trimeric, pigment-protein complexes. The LHC Ia oligomeric complex contains both the 24- and the 21.5-kD apoproteins encoded by the Lhca3 and Lhca2 genes and is slightly larger than the oligomeric LHC Ib complex containing the Lhca1 and Lhca4 gene products of 21 and 20 kD. The synthesis and assembly of LHC I during light-driven development of intermittent light-grown plants occurs rapidly upon exposure to continuous illumination. Complete PSI complexes are detected by nondenaturing Deriphat (disodium N-dodecyl-[beta]-iminodipropionate-160)-PAGE after 2 h of illumination, and their appearance correlates with that of the 730- to 740-nm emission characteristic of assembled LHC I. However, the majority of the newly synthesized LHC I apoproteins are present as monomeric complexes in the thylakoids during the early hours of greening. We propose that during development of the protochloroplast the LHC I apoproteins are first assembled into monomeric pigmented complexes that then aggregate into trimers before becoming attached to the pre-existing core complex to form a complete PSI holocomplex.     

Stacking Capacity and Chlorophyll Forms of Thylakoids in Normal and Mutant Maize Chloroplasts of Different Granum Content

Stacking of chloroplast lamellae, isolated from normal and carotenoid mutant chloroplasts of maize (Zea mays L.), was determined after a high-salt treatment. Stacking of isolated lamellae under favourable ionic conditions was almost identical with that occurring in intact chloroplasts; thus, differences in granum content could be attributed to the architectural properties of lamellae. Gaussian analyses, performed on the red band of room temperature absorption spectra, have shown that chloroplasts with lamellae of high stacking capacity contain relatively more Chl a₆₆₂ than chloroplasts containing lamellae of low stacking capacity. The presence of Chl a_705–708 was characteristic of preparations containing considerable amounts of stroma lamellae.     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

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 immuno-localization 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.     

Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)