Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

Differential phosphorylation of thylakoid proteins in mesophyll and bundle sheath chloroplasts from maize plants grown under low or high light

In C4 plants, such as maize, the photosynthetic apparatus is partitioned over two cell types called mesophyll (M) and bundle sheath (BS), which have different structure and specialization of the photosynthetic thylakoid membranes. We characterized protein phosphorylation in thylakoids of the two cell types from maize grown under either low or high light. Western blotting with phosphothreonine antibodies and ProQ phosphostaining detected light-dependent changes in the protein phosphorylation patterns. LC-MS/MS with alternating CID and electron transfer dissociation sequencing of peptide ions mapped 15 protein phosphorylation sites. Phosphorylated D2, CP29, CP26, Lhcb2 proteins, and ATPsynthase were found only in M membranes. A previously unknown phosphorylation site was mapped in phosphoenolpyruvate carboxykinase from the BS cells. Phosphorylation stoichiometry was calculated from the ratios of normalized ion currents for phosphorylated to nonphosphorylated peptide pairs from the D1, D2, CP43, and PbsH proteins of photosystem II (PSII). Every PSII in M thylakoids contained on average 1.5 ± 0.1 or 2.3 ± 0.2 phosphoryl groups in plants grown under either low or high light, while in BS membranes the corresponding numbers were 0.25 ± 0.1 or 0.7 ± 0.2, respectively. It is suggested that the phosphorylation level, as well as turnover of PSII depend on the structure of thylakoids.     

Organization and activity of photosystems in the mesophyll and bundle sheath chloroplasts of maize

Photosystem I and Photosystem II activities, as well as polypeptide content of chlorophyll (Chl)-protein complexes were analyzed in mesophyll (M) and bundle sheath (BS) chloroplasts of maize (Zea mays L.) growing under moderate and very low irradiance. This paper discusses the application of two techniques: mechanical and enzymatic, for separation of M and BS chloroplasts. The enzymatic isolation method resulted in depletion of polypeptides of oxygen evolving complex (OEC) and alphaCF1 subunit of coupling factor; D1 and D2 polypeptides of PSII were reduced by 50%, whereas light harvesting complex of photosystem II (LHCII) proteins were still detectable. Loss of PSII polypeptides correlated with the decreasing of Chl fluorescence measured at room temperature. Using mechanical isolation of chloroplasts from BS cells, all tested polypeptides could be detected. We found a total lack of O2 evolution in BS chloroplasts, but dichlorophenolindophenol (DCPIP) was photoreduced. PSI activity of chloroplasts isolated from 14- and 28-day-old plants was similar in BS chloroplasts in moderate light (ML), but in low light (LL) it was reduced by about 20%. PSI and PSII activities in M chloroplasts of plants growing in ML decreased with aging of plants. In older LL-grown plants, activities of both photosystems were higher than those observed in chloroplasts from ML-grown plants. We suggest that in BS chloroplasts of maize, PSII complex is assembled typically for the agranal membranes (containing mainly stroma thylakoids) and is able to perform very limited electron transport activity. This in turn suggests the role of PSII for poising the redox state of PSI.     

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.     

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.     

Acclimation of mesophyll and bundle sheath chloroplasts of maize to different irradiances during growth

The regulation by light of the photosynthetic apparatus, and composition of light-harvesting complexes in mesophyll and bundle sheath chloroplasts was investigated in maize. Leaf chlorophyll content, level of plastoquinone, PSI and PSII activities and Lhc polypeptide compositions were determined in plants grown under high, moderate and low irradiances. Photochemical efficiency of PSII, photochemical fluorescence quenching and non-photochemical fluorescence quenching over a range of actinic irradiances were also determined, using chlorophyll a fluorescence analysis. Acclimation of plants to different light conditions caused marked changes in light-harvesting complexes, LHCI and LHCII, and antenna complexes were also reorganized in these types of chloroplasts. The level of LHCII increased in plants grown in low light, even in agranal bundle sheath chloroplasts where the amount of PSII was strongly reduced. Irradiance also affected LHCI complex and the number of structural polypeptides, in this complex, generally decreased in chloroplasts from plants grown under lower light. Surprisingly moderate and low irradiances during growth do not affect the light reaction and fluorescence parameters of plants but generated differences in composition of light-harvesting complexes in chloroplasts. On the other hand, the changes in photosynthetic apparatus in plants acclimated to high light, resulted in a higher efficiency of photosynthesis. Based on these observations we propose that light acclimation to high light in maize is tightly coordinated adjustment of light reaction components/activity in both mesophyll and bundle sheath chloroplasts. Acclimation is concerned with balancing light utilization and level of the content of LHC complexes differently in both types of chloroplasts.     

Acclimation of mesophyll and bundle sheath chloroplasts of maize to different irradiances during growth

The regulation by light of the photosynthetic apparatus, and composition of light-harvesting complexes in mesophyll and bundle sheath chloroplasts was investigated in maize. Leaf chlorophyll content, level of plastoquinone, PSI and PSII activities and Lhc polypeptide compositions were determined in plants grown under high, moderate and low irradiances. Photochemical efficiency of PSII, photochemical fluorescence quenching and non-photochemical fluorescence quenching over a range of actinic irradiances were also determined, using chlorophyll a fluorescence analysis. Acclimation of plants to different light conditions caused marked changes in light-harvesting complexes, LHCI and LHCII, and antenna complexes were also reorganized in these types of chloroplasts. The level of LHCII increased in plants grown in low light, even in agranal bundle sheath chloroplasts where the amount of PSII was strongly reduced. Irradiance also affected LHCI complex and the number of structural polypeptides, in this complex, generally decreased in chloroplasts from plants grown under lower light. Surprisingly moderate and low irradiances during growth do not affect the light reaction and fluorescence parameters of plants but generated differences in composition of light-harvesting complexes in chloroplasts. On the other hand, the changes in photosynthetic apparatus in plants acclimated to high light, resulted in a higher efficiency of photosynthesis. Based on these observations we propose that light acclimation to high light in maize is tightly coordinated adjustment of light reaction components/activity in both mesophyll and bundle sheath chloroplasts. Acclimation is concerned with balancing light utilization and level of the content of LHC complexes differently in both types of chloroplasts.     

Flash-induced 515 nm absorbance change in chloroplasts with various granum contents

The 515 nm absorbance change was studied in mesophyll and bundle sheath chloroplasts of maize, which contain different amounts of grana. The amplitude of the 515 nm signal (induced by 3 μs flashes repeated at 4 s intervals) has shown a correlation with the granum content of the samples. However, upon addition of N-methylphenazonium methosulphate the 515 nm signal became independent of the amount of grana: in agranal thylakoids a large pool of silent Photosystem I was activated and, as a result, the amplitude of the 515 nm signal of agranal chloroplasts increased to the level exhibited by granal chloroplasts.These data show that the 515 nm absorbance change is not limited to small closed vesicles like grana, but in the presence of suitable electron donors single lamellae of bundle sheath chloroplasts can also be active.     

Chloroplast thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology

Tissue-specific effects of low growth temperature on maize chloroplast thylakoid protein accumulation were analysed using immunocytology. Sections of leaves from plants grown at 25 and 14°C were probed with antibodies to specific chloroplast thylakoid proteins from the four major protein multisubunit complexes of the thylakoid membrane followed by fluorescein-conjugated goat anti-rabbit antibodies. At a normal growth temperature of 25°C, the 32 kDa D1 protein of the photosystem II reaction centre and the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II are both accumulated to a greater degree in the mesophyll than in the bundle sheath chloroplasts. In contrast, subunit II of photosystem I, cytochrome f and the α- and β-subunits of ATP synthetase are predominant in the bundle sheath thylakoids at 25°C. A striking difference between the 25°C-grown and the 14°C-grown leaf tissue was the presence in the latter of (20–30%) cells whose chloroplasts apparently completely lack several of the thylakoid proteins. In plants grown at 14°C, the accumulation of the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II was apparently unchanged, but other thylakoid proteins showed a significant reduction. The uneven distribution of proteins between the bundle sheath and mesophyll chloroplasts observed at 25°C was also maintained at 14°C. Reduction in the fluorescence at 14°C was manifested either as an overall reduction in the diffuse fluorescence across the chloroplast profiles or less frequently as a reduction to small discrete bodies of intense fluorescence. The significance of these results to low-temperature-induced reduction in the photosynthetic productivity of maize is discussed.     

Comparative analysis of biochemical properties of mesophyll and bundle sheath chloroplasts from various subtypes of C4 plants grown at moderate irradiance

The photochemical characteristics of mesophyll and bundle sheath chloroplasts isolated from the leaves of C4 species were investigated in Zea mays (NADP-ME type), Panicum miliaceum (NAD-ME type) and Panicum maximum (PEP-CK type) plants. The aim of this work was to gain information about selected photochemical properties of mesophyll and bundle sheath chloroplasts isolated from C4 plants grown in the same moderate light conditions. Enzymatic as well as mechanical methods were applied for the isolation of bundle sheath chloroplasts. In the case of Z. mays and P. maximum the enzymatic isolation resulted in the loss of some thylakoid polypeptides. It was found that the PSI and PSII activities of mesophyll and bundle sheath chloroplasts of all species studied differed significantly and the differences correlated with the composition of pigment-protein complexes, photophosphorylation efficiency and fluorescence emission characteristic of these chloroplasts. This is the first report showing differences in the photochemical activities between mesophyll chloroplasts of C4 subtypes. Our results also demonstrate that mesophyll and bundle sheath chloroplasts of C4 plants grown in identical light conditions differ significantly with respect to the activity of main thylakoid complexes, suggesting a role of factor(s) other than light in the development of photochemical activity in C4 subtypes.     

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.     

Lincomycin-induced alteration in the contents of chlorophyll-protein complexes of dimorphic maize chloroplasts and its effect on the temperature-induced spectral changes

The difference spectroscopy technique has been utilized to investigate the temperature-induced spectral changes in mesophyll and bundle sheath chloroplasts of maize ( Zea mays L. cv. Ganga-5) in order to assess the role of different pigment-protein complexes in the manifestation of temperature effect on the chloroplast membranes. Cooling and heating of both mesophyll and bundle sheath chloroplasts resulted in absorbance difference (AA) bands at similar wavelengths but the degree of absorb-ance changes were significantly higher in bundle sheath chloroplasts. For example, upon cooling to 7-8°C, positive AA bands were observed at 440, 490 and 680 nm in mesophyll chloroplasts and at 440, 495–500 and 680 nm in bundle sheath chloroplasts but the absorbance change at 680 nm was ca 2% in mesophyll chloroplasts, whereas it was ca 5% in bundle sheath chloroplasts, which have a lower content of light-harvesting pigment-protein complex. The role of chlorophyll-protein complexes was further investigated by monitoring the temperature-induced spectral changes of mesophyll and bundle sheath chloroplasts isolated from lincomycin-treated maize plants where lincomycin selectively inhibits the biosynthesis of specific chlorophyll-protein complexes. Results indicated that depletion of certain pigment-protein complexes in mesophyll chloroplasts made them more susceptible (a ca 4% vs ca 2% absorbance change upon cooling and a ca 6% vs ca 4% absorbance change upon heating) and less tolerant to temperature variation (a 76% vs 39% reversibility during ambient→Cooling→ambient temperature cycle). The data indicate that pigment-protein complexes play a significant role in protecting the chloroplast membranes against temperature variation.     

Ultrastructure and Dimensions of Chloroplasts in Leaves of Three Maize (Zea mays L.) Inbred Lines and their F1 Hybrids Grown Under Moderate Chilling Stress

Influence of moderate chilling stress on vascular bundle sheath cell (BSC) and especially mesophyll cell (MC) chloroplasts of mature maize leaves was studied by electron microscopy and stereology. Plants of two inbred lines of maize, differing in their photosynthetic activity, and their F₁ hybrids were cultivated during autumn in heated or unheated glasshouse. Generally, chilling temperatures resulted mainly in the decrease in stereological volume density (VD) of both granal and intergranal thylakoids of MC chloroplasts, while the ratio of granal to all thylakoids (granality) was less affected. The VD of peripheral reticulum and plastoglobuli usually increased after cold treatment of plants. The volume of MC chloroplasts usually increased under chilling stress, the shape of the chloroplasts changed only slightly. The ultra-structure of chloroplasts differed between individual genotypes; chilling-stressed hybrid plants showed positive heterosis particularly in the granal thylakoids' VD of MC chloroplasts.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment–protein complexes. Here, the thylakoids were isolated from light‐acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light‐harvesting chlorophyll a/b‐binding complexes (LHCII). Because the majority of plant light‐harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L‐LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L‐LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light‐dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D‐lpBN‐lpBN‐sodium dodecyl sulfate–polyacrylamide gel electrophoresis system demonstrated that PSII–LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L‐LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light‐dependent supramolecular organization of the photosystems into megacomplexes.     

Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Four types of differently phosphorylated hylakoids isolated from field grown spinach ( Spinacia oleracea L.) were tested for the sensitivity of photosystem II (PSII) to photoinactivation. Phosphorylation of light-harvesting II complexes (LHCII) protected PSII electron transfer from photoinhibitory damage, while the phosphorylation of the PSII core polypeptides slightly accelerated the decline of electron transfer during high irradiance treatment. Dephosphorylation of the CP43 apoprotein and PsbH protein by an alkaline phosphatase resulted in an extreme sensitivity of the thylakoids to strong illumination. The PSII photoinactivation of thylakoids with the impaired oxygen-evolving complex was found to be independent of phosphorylation.
The thylakoids of the thermophilic cyanobacterium Synechococcus elongates were used in order to compare the plants with an organism where LHCII complexes are missing and the PSII core proteins are not phosphorylated.     

Lead induced changes in phosphorylation of PSII proteins in low light grown pea plants

Light-intensity and redox-state induced thylakoid proteins phosphorylation involved in structural changes and in regulation of protein turnover. The presence of heavy metal ions triggers a wide range of cellular responses including changes in plant growth and photosynthesis. Plants have evolved a number of mechanisms to protect photosynthetic apparatus. We have characterized the effect of lead on PSII protein phosphorylation in pea (Pisum sativum L.) plants grown in low light conditions. Pb ions affected only slightly photochemical efficiency of PSII and had no effect on organization of thylakoid complexes. Lead activated strongly phosphorylation of PSII core D1 protein and dephosphorylation of this protein did not proceed in far red light. D1 protein was also not degraded in this conditions. However, phosphorylation of LHCII proteins was not affected by lead. These results indicate that Pb²⁺ stimulate the phosphorylation of PSII core proteins and by disturbing the disassembly of supercomplexes play a role in PSII repair mechanism. LHCII phosphorylation could control the distribution of energy between the photosystems in low light conditions. This demonstrates that plants may respond to heavy metals by induction different pathways responsible for protein protection under stress conditions.     

The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.     

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 1. The green tissues.

Exposure to continuous illumination causes the appearance of numerous plastoglobuli in the stroma of both the mesophyll and bundle sheath chloroplasts of the green tissues of the leaves of the japonica-2 mutant of maize. In the pale green tissues the thylakoids have markedly swollen membranes. Another feature of the plastids exposed to continuous illumination is the heavy accumulation of starch. The japonica-2 chloroplasts show a different sensitivity to light, the chloroplasts of the pale green tissues being affected more markedly than the ones of the dark green tissues, and the bundle sheath chloroplasts more than those of the mesophyll. The effects of continuous illumination may be interpreted as an acceleration of chloroplast ontogenesis.