Characterization of a family of chlorophyll-deficient wheat (Triticum) and barley (Hordeum vulgare) mutants with defects in the magnesium-insertion step of chlorophyll biosynthesis.

During thylakoid membrane biogenesis, chlorophyll (Chl) biosynthesis and the accumulation of Chl-binding proteins are tightly linked, light-regulated processes. We have investigated the consequences faced by mutant plants with defects in Chl biosynthesis by studying a series of five homeologous allelic chlorina mutants in wheat (Triticum) and one phenotypically related barley (Hordeum vulgare) mutant that express the same pleiotropic mutant phenotype but to different extents. These mutants accumulate Chl at different rates, with the most severely affected plants having the slowest rate of Chl accumulation. Analysis of precursor pools in the Chl synthesis pathway indicates they have a partial block in Chl synthesis and accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The affected plants with the most severe phenotypes accumulate the most Proto. Chloroplasts isolated from these mutants exhibit a lower activity of the enzyme Mg-chelatase, which catalyzes the first committed step in Chl synthesis. The most severely affected plants exhibit the greatest reduction in Mg-chelatase activity. Heme levels and protoporphyrinogen oxidase activity were the same for mutant and wild-type plants. We suggest that a block in Mg-chelatase activity in these mutants could account for the other traits of their pleiotropic phenotype previously described in the literature.     

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.     

High Irradiance Induced Pigment Degradation and Loss of Photochemical Activity of Wheat Chloroplasts

Loss of chlorophyll (Chl) and carotenoids (Car) of leaves and changes in Chl fluorescence emission and polarisation, malondialdehyde (MDA) accumulation, and 2,6-dichlorophenol indophenol (DCPIP) photoreduction in chloroplasts of wheat seedlings grown under different irradiance and subsequently exposed to high irradiance stress (HIS; 250 W m^–2) were studied in mature and senescent primary wheat leaves. Faster rate of pigment loss was observed in leaves of moderate irradiance (MI; 15 W m^–2) grown plants, compared to high irradiance (HI-1 and HI-2; 30 and 45 W m^–2) ones when exposed to HIS. A relatively lower loss of Car in the plants grown in HI-1 and HI-2 exposed to HIS suggests HI adaptation of these seedlings. The slower rate of increase in the ratio of Chl fluorescence emission (F₆₈₅/F₇₃₅) also may suggest photoprotective strategy of HI grown seedlings. There was a positive correlation between MDA accumulation and Chl fluorescence polarisation. The DCPIP photoreduction activity in chloroplasts isolated from HI-1 and HI-2 grown plants exposed to HIS showed slower loss of electron transport activity compared to MI grown plants. These observations suggest that plants grown under higher irradiance have capacity to manage the excess quanta better than those grown under lower irradiance.     

Partial blocks in the early steps of the chlorophyll synthesis pathway: A common feature of chlorophyll b-deficient mutants

We have analyzed precursor pools in the chlorophyll (Chi) synthesis pathway for a set of eighteen well studied Chl b -defident mutants in monocotyledonous (barley, maize and wheat) and dicotyledonous plants ( Antirrhinum, Arabidopsis , soybean, tobacco and tomato) that form abnormal thylakoid membrane systems. All of these mutants have a partial block in Chl synthesis and nearly all of them accumulate protoporphyrin IX (Proto), the last porphyrin compound common to both heme and Chl synthesis. The large number of mutants at several genetic loci affecting this critical branchpoint in tetrapyrrole biosynthesis suggests that the Mg-chelatase enzyme, catalyzing the first committed step of Chi biosynthesis, is a multimeric complex composed of the products of some of these genetic loci, and perhaps regulated by others. We hypothesize that these mutants are Chi b -deficient and have reduced amounts of light-harvesting antenna complexes (LHCs.) and develop abnormal thylakoid membranes as a direct result of limited Chl synthesis. The observed bottleneck in Chl synthesis can also explain the light-intensity-dependent and temperature-dependent expression of the mutant phenotype. This hypothesis offers a simple explanation for the wide variety of pbenotypes that have been reported for the many Chl-deficient mutants in the literature. Our findings are also consistent with the notion that Chl b is made from "left over" Chl a molecules and suggest that the Chi b -deficient mutants should be considered more appropriately as leaky Chl-deficient mutants.     

Inhibitory effect of hypergravity on photosynthetic carbon dioxide fixation in Euglena gracilis.

Photosynthesis, the conversion of light energy into chemical energy, is a critical biological process, whereby plants synthesize carbohydrates from light, carbon dioxide (CO2) and water. The influence of gravity on this biological process, however, is not well understood. Thus, centrifugation was used to alter the gravity environment of Euglena gracilis grown on nutritive agar plates illuminated with red and blue light emitting diodes. The results showed that hypergravity (up to 10xg) had an inhibitory effect on photosynthetic CO2 fixation. Chlorophyll accumulation per cell was essentially unaffected by treatment; however, Chl a/Chl b ratios decreased in hypergravity when compared to 1xg controls. Photosynthesis in Euglena appears to have limited tolerance for even moderate changes in gravitational acceleration.     

Inhibition of chlorophyll biosynthesis at the protochlorophyllide reduction step results in the parallel depletion of Photosystem I and Photosystem II in the cyanobacterium Synechocystis PCC 6803

In most oxygenic phototrophs, including cyanobacteria, two independent enzymes catalyze the reduction of protochlorophyllide to chlorophyllide, which is the penultimate step in chlorophyll (Chl) biosynthesis. One is light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) and the second type is dark-operative protochlorophyllide oxidoreductase (DPOR). To clarify the roles of both enzymes, we assessed synthesis and accumulation of Chl-binding proteins in mutants of cyanobacterium Synechocystis PCC 6803 that either completely lack LPOR or possess low levels of the active enzyme due to its ectopic regulatable expression. The LPOR-less mutant grew photoautotrophically in moderate light and contained a maximum of 20 % of the wild-type (WT) Chl level. Both Photosystem II (PSII) and Photosystem I (PSI) were reduced to the same degree. Accumulation of PSII was mostly limited by the synthesis of antennae CP43 and especially CP47 as indicated by the accumulation of reaction center assembly complexes. The phenotype of the LPOR-less mutant was comparable to the strain lacking DPOR that also contained <25 % of the wild-type level of PSII and PSI when cultivated under light-activated heterotrophic growth conditions. However, in the latter case, we detected no reaction center assembly complexes, indicating that synthesis was almost completely inhibited for all Chl-proteins, including the D1 and D2 proteins.     

Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors

Light induction of light-harvesting chlorophyll a/b-binding protein (LHCP) mRNA accumulation was studied in light-dark synchronized cultures of Chlamydomonas reinhardi. LHCP mRNA accumulation was prevented by the chlorophyll-synthesis inhibitor alpha,alpha-dipyridyl which blocks late steps in the chlorophyll biosynthetic pathway and leads to the accumulation of the porphyrin intermediate magnesium protoporphyrin methyl ester. LHCP mRNA accumulated normally, however, when chlorophyll synthesis was blocked by inhibitors such as hemin and levulinic acid which interfere with early steps in the chlorophyll biosynthesis pathway prior to the formation of magnesium protoporphyrin methyl ester. Similar effects were observed in the light induction of LHCP mRNA levels in protoporphyrin IX-accumulating mutants, brc-1 and brs-1. These mutants have low levels of LHCP mRNA when grown under heterotrophic conditions in the dark where they accumulate protoporphyrin IX. However, LHCP mRNA is light-induced in brc-1 which synthesizes chlorophyll in the light and presumably consumes porphyrin intermediates in doing so. These results suggest that the chlorophyll-synthesis intermediates, magnesium protoporphyrin methyl ester and its immediate precursors, inhibit by a feedback mechanism the light induction of LHCP mRNA accumulation. Low magnesium protoporphyrin methyl ester levels permit the light-induced accumulation of LHCP mRNA, whereas high magnesium protoporphyrin methyl ester levels destabilize LHCP mRNA regardless of the illumination conditions. Preliminary experiments show that LHCP mRNA accumulation in C. reinhardi is stimulated by blue light, and not by red light which stimulates LHCP mRNA accumulation in higher plants.     

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll <Emphasis Type="Italic">b</Emphasis>-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 μmol m⁻² s⁻¹). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (F_v/F_m), quantum efficiency of PS2 electron transport (Φ_PS2), photochemical quenching (q_P), as well as rate of photochemistry (P_rate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (D_rate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–q_P (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 μmol m⁻² s⁻¹) and high, HI (700 μmol m⁻² s⁻¹) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.     

Influence of clinorotation on some parameters of photosynthetic apparatus

This study aimed to examine the electron transport rates in the thylakoids, isolated from leaves of pea plants grown under clinorotation and in vertical control, to measure the chlorophyll a/b (Chl a/b) ratio in such thylakoids and in photosystem I (PSI) particles isolated from them, to elucidate if there are any differences in changes of PS II activity in thylakoids and Chl a/b ratio in PS I particles under phosphorylation of polypeptides of thylakoid pigment-protein complexes.     

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.     

Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655

Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A.     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

Chlorophyllide a Oxygenase mRNA and Protein Levels Correlate with the Chlorophyll a/b Ratio in Arabidopsis thaliana

Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.     

Limitation of nocturnal import of ATP into Arabidopsis chloroplasts leads to photooxidative damage

When grown in short day conditions and at low light, leaves of Arabidopsis plants with mutations in the genes encoding two plastidial ATP/ADP transporters (so-called null mutants) spontaneously develop necrotic lesions. Under these conditions, the mutants also display light-induced accumulation of H(2)O(2) and constitutive expression of genes for copper/zinc superoxide dismutase 2 and ascorbate peroxidase 1. In the light phase, null mutants accumulate high levels of phototoxic protoporphyrin IX but have only slightly reduced levels of Mg protoporphyrin IX. The physiological changes are associated with reduced magnesium-chelatase activity. Since the expression of genes encoding any of the three subunits of magnesium-chelatase is similar in wild type and null mutants, decreased enzyme activity is probably due to post-translational modification which might be due to limited availability of ATP in plastids during the night. Surprisingly, the formation of necrotic lesions was absent when null mutants were grown either in long days and low light intensity or in short days and high light intensity. We ascribe the lack of lesion phenotype to increased nocturnal ATP supply due to glycolytic degradation of starch which may lead to additional substrate-level phosphorylation in the stroma. Thus, nocturnal import of ATP into chloroplasts represents a crucial, previously unknown process that is required for controlled chlorophyll biosynthesis and for preventing photooxidative damage.     

Restriction of Chlorophyll Synthesis Due to Expression of Glutamate 1-Semialdehyde Aminotransferase Antisense RNA Does Not Reduce the Light-Harvesting Antenna Size in Tobacco

The formation of 5-aminolevulinate is a key regulatory step in tetrapyrrole biosynthesis. In higher plants, glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes the last step in the sequential conversion of glutamate to 5-aminolevulinate. Antisense RNA synthesis for GSA-AT leads to reduced GSA-AT protein levels in tobacco (Nicotiana tabacum L.) plants. We have used these transgenic plants for studying the significance of chlorophyll (Chl) availability for assembly of the light-harvesting apparatus. To avoid interfering photoinhibitory stress, plants were cultivated under a low photon flux density of 70 [mu]mol photons m-2 s-1. Decreased GSA-AT expression does not seem to suppress other enzymic steps in the Chl pathway, indicating that reduced Chl content in transgenic plants (down to 12% of the wild-type level) is a consequence of reduced GSA-AT activity. Chl deficiency correlated with a drastic reduction in the number of photosystem I and photosystem II reaction centers and their surrounding antenna on a leaf area basis. Different lines of evidence from the transgenic plants indicate that complete assembly of light-harvesting pigment-protein complexes is given preference over synthesis of new reaction center/core complexes, resulting in fully assembled photosynthetic units with no reduction in antenna size. Photosynthetic oxygen evolution rates and in vivo Chl fluorescence showed that GSA-AT antisense plants are photochemically competent. Thus, we suggest that under the growth conditions chosen during this study, plants tend to maintain their light-harvesting antenna size even under limited Chl supply.     

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.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress.     

Influence of Potassium Deficiency on Photosynthesis,Chlorophyll Content,and Chloroplast Ultrastructure of Cotton Plants

In cotton (Gossypium hirsutum L.) grown in controlled-environment growth chamber the effects of K deficiency during floral bud development on leaf photosynthesis, contents of chlorophyll (Chl) and nonstructural saccharides, leaf anatomy, chloroplast ultrastructure, and plant dry matter accumulation were studied. After cotton plants received 35-d K-free nutrient solution at the early square stage, net photosynthetic rate (P _N) of the uppermost fully expanded main-stem leaves was only 23 % of the control plants receiving a full K supply. Decreased leaf P _N of K-deficient cotton was mainly associated with dramatically low Chl content, poor chloroplast ultrastructure, and restricted saccharide translocation, rather than limited stomata conductance in K-deficient leaves. Accumulation of sucrose in leaves of K-deficient plants might be associated with reduced entry of sucrose into the transport pool or decreased phloem loading. K deficiency during squaring also dramatically reduced leaf area and dry matter accumulation, and affected assimilate partitioning among plant tissues.     

Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: I. Pigment Composition

The use of n-octyl-beta-d-glucopyranoside along with sodium dodecyl sulfate improves the retention of chlorophyll (Chl) by chlorophyll-protein complexes (CPs) prepared from thylakoids of Euglena gracilis Klebs var bacillaris Cori and yields several additional complexes. Thylakoids from wild-type (WT) cells, solubilized in these detergents and subjected to polyacrylamide gel electrophoresis at 0 degrees C, yield the following CPs, in order of relative molecular weight, containing the pigments shown in parentheses with their respective molar ratios where determined: CP Ia (Chl a, diadinoxanthin and beta-carotene; 100:12:5); CP I (Chl a and beta-carotene; 100:6-12); CPx (Chl and carotenoids); LHCP(2) (light-harvesting CP oligomer) (Chl a, Chl b, diadinoxanthin and neoxanthin; 12:4:3:1); CPy (Chl a, diadinoxanthin and beta-carotene; 100:14:8); CPa (Chl a and beta-carotene; 100:18-25) and LHCP (monomer) (Chl a, Chl b, diadinoxanthin and neoxanthin; 12:6:4:1). The LHCP complexes retain up to 40% of the total Chl and 80% of the Chl b in the thylakoids. CP Ia contains only a trace of Chl b (Chl a/b [mol/mol] = 62). The lower amount of Chl b in Euglena (about 10% of Chl a + b) compared to higher plants (about 30% of Chl a + b) is probably a consequence of the lower Chl b (relative to Chl a) in the LHCPs of Euglena rather than of fewer LHCPs being present. G(1)BU, Gr(1)BSL, and O(4)BSL, mutants of bacillaris low in Chl b (1-2% of Chl a + b), lack the CP Ia, LHCP, and LHCP(2) found in wildtype (WT); G(1) and O(4) also lack CPy. The mutants contain reduced amounts of Chl a (two-thirds of WT in Gr(1) and one-third in G(1) and O(4)) and neoxanthin (20-40% of WT) but retain levels of beta-carotene and diadinoxanthin close to those in cells of WT. The CPs remaining in the mutants have pigment compositions very similar to their counterparts from WT.