Structure and Function of Chloroplast Membrane III. The Effects of Mg++ and K+ Ions Toward the Ultrastructure of Two Kinds of Chloroplast Thylakoid Membranes

Based upon our previous work on the relationship between structure and function of chloroplast of wheat in connection with PSⅡ reaction, we studied the effects of MgCl2 and KC1 toward two kinds of thylakoid membranes. After exposing etiolated wheat seedlings to intermittent light (cycle of 2 min. light, 118 min. dark) for 24 hr, we obtain ed an incompletely developed chleroplast membrane. Completely developed chloroplast membrane was obtained from wheat seedlings grown under normal light-dark regime. Thylakoid membranes of plants grown under intermittent light failed to form grana stacks they remained as single lamellae in the suspension containing Mg++ or K+ of high concentration although simple stackings not more than two thylakoids c.ould be found. However, thylakoids grown under normal light-dark regime showed well developed grand stacks. Isolated chloroplast samples from two kinds of seedlings were suspended in 5 mM MgCl2 and 100 mM KC1 solutions for a definite time, portions of each samples were processed for electron microscopic observations and their photosynthetic activities were measured at the same time (It will be dealt with in another article). When these two kinds of isolated plastids were suspended either in MgCl2 (5 mM) or KC1 (100), the normally developed grana thylakoids stacked closely but the incompletely developed thylakoid- membranes did not stack. The incompletely developed chloroplast thylakoid membranes,, in either Mg++ or K+ ions could not induce stacking of the scattered thylakoid membranes to form grana. Therefore, we presume that light- harvesting chlorophyll a/b protein complex is on internal factor to induce thylakoid- membranes stacking and a definite concentration of caionions is an important factor in maintaining the stacking of thylakoid membranes. These results further prove the close association between structure and function in our previous studies on the mesophyll cell of the winter wheat.     

Structure and Function of Chloroplast Membrane V.Ultrastructural Biogenesis of Chloroplasts from Different Ploids of Wheat

Based upon our previous studies on the ultrastructure and constituents of chloroplast membrane in relation to the function of photosystem Ⅱ, and the effects of intermittent light and cations toward the ultrastructurc and the absorption spectrum and function of photosystem Ⅱ of wheat chloroplast membranes. Then we studied the ultrastructure of the chloroplast membrane in different ploids of wheat during their biogenesis. Seedlings of diploid wheat (Tr. monococcum), tetraploid wheat (Tr. durum Desf), hexaploid wheat, "Nongda 139" (Tr. aestivum) and octoploid wheat (Triticale Hungarian) were grown in dark for 7 days and exposed to light (1500 lux) for greening at a temperature about 26℃. Samples were taken after exposing to light for 1hr, 3 hrs, 4 hrs, and 22 hrs for ultrastructural observation of the biogenesis of chloroplasts. We found that the chloroplastid membranes of different ploids of wheat appeared to have a similar stepwise biogenesis process. We also found that the development rate and degree of constitution and form of their membrances were distinctly different. Especially, it was remarkable and interesting that we found numerous "vesicles" in dfferent sizes and number within the stroma of the proplastid of the diploid, tetraploid and octoploid wheat after exposing 1 hr and 4 hrs of light. In so doing, gemmalike bodies appeared from the outer envelope membrane from the yellow proplastid of tetraploid and octoploid wheat. These bodies looked quite like the photosynthetic membranes of prokaryote. It is worth notice that this unusual ultrastructure has not been reported before. We wonder if the gemmalike bodies are probably another form of reproduction of chloroplast. How does this phenomenon be explained? Why do these bodies were found only in tetraploid and octoploid wheat? And what are the factors controlling? These processes are needed to study in the future.     

Structure and Function of Chloroplast Membrane XI. The Effects of Linolenic Acid on the Structure and the Absorption and Fluorescence Spectra of Wheat Chloroplast Membranes as well as Regulations by MgCl2

The effects of various concentrations of linolenic acid on the structure and the absorption and fluorescence spectra of wheat chloroplast membranes were studied. Linolenic acid increases the absorption peaks in both red and blue regions of the chloroplast membranes. The degree of increase in the absorption peaks is proportional to the concentration within a range of concentration. Linolenic acid increased the fluorescence yield of F685 and F738 of chloroplast membranes. Electron microscopical studies revealed that the increases were mainly due to the disappearance of grana stacks and the unfolding of thylakoid membranes. The causes of effects of linolenic acid on the absorption and fluorescence spectra and the structure of chloroplast membranes as well as the reversion and regulation by MgCl2 were discussed.     

Effect of Bovine Serum Albumin Incubation on Lettuce Chloroplasts Digested by Trypsin

1. Bovine serum albumin stimulates the DCIP photoreduction activity of lettuce chloroplasts which has been treated with trypsin. When these chloroplast preparations were washed by tricine buffer such "reversible action" can still be obtained. It is possible that bovine serum albumin may be incorporated into trypsin destroyed site of the membrane. 2. Trypsin-induced CCCP inhibitory effect on DCIP photoreduction activity is reversed by bovine serum albumin. 3. Bovine serum albumin partially reverses the trypsin-induced unstacking of lettuce chloroplast membranes. 4. After trypsin digestion, there are absorbance decreases around 500–640 nm. Bovine serum albumin has no effect on these absorbance decreases. It is concluded that the membrane-bound proteins responsible for different functions of chloroplast are heterogeneous. The results also show that there are gate and channel near the position of PSⅡ on chloroplast membrane.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

Fluorescence of Wheat with Incompletely Developed Chloroplast Membranes

Low temperature (77K) fluorscenee emission spectrum of the incompletely developecl chloroplast membranes of wheat, and the fluorescence induction transient of its intact leaf at room temperature were studied. The main peaks of the fluorescence emission spectrum of the incompletely developed chloroplast membranes were at 685 nm and 725 nm respectively. The positions of these two' peaks were almost the same as that in chlorophyll bless mutant barley Chlorina f2. This showed that the incompletely develop- ed chloroplast membranes of wheat did not develope peripheral antenna of PSI but only contained internal antenna of PSI as the case of Chlorina f2. The fluorescence induction transient of wheat leaf with incompletely developed chloroplast membranes did not show the typical time course of O→P→S→M→T and lacked second peak (M) and showed a slow decline as P decayed. This is the same as that of Chlorina f2 leaf. The fluorescence rise in induction period of the leaf with incompletely developed chloroplast membranes was much different from that of normal wheat leaf. These results can be explained by our previous assumption[3] that the occurence of typical fluorescence induction transient is based on the coexistence of LHCP of PSII and peripheral antenna of PSI and on their cooperation with each other.     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

亚硫酸对ＰＳII颗粒活性的伤害及Ca2+的影响

PSⅡ1)颗粒的荧光产值依SO32-浓度和处理时间的增加而减少,pH7.3以上受害严重;SO32-对新鲜叶绿体的光化学活性不产生伤害,对老化叶绿体伤害严重,其叶绿素的分解速度低于DCIP2)光还原的降低速度。Ca2+能减轻或消除SO32-对叶绿体的伤害;对于PSⅡ颗粒则有加剧SO32-伤害的作用,其规律可用Logistic方程表示。     

Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Damage of PSII by Sulfite

The photoreduction of DCIP by PSⅡ pasticles isolated from spinach leaves was inhibited by sulfite and the degree of inhibition was increased with the increase of sulfite concentration. The site of sulfite damage was on the oxidation side. In dark, electron flow from H2O to DCIP and from DPC to DCIP was not affected by sulfite. With certain concentrations of sulfite, the damage to PSⅡ particle varied with time of sulfite treatment and the mechanism of the damage might be related to the discretion of 33 kD polypeptide from thylakoid membrane and the leakage of Mn. Sulfite did not specifically damage the newly prepared thyla- koid, but this was the case with aged thylakoid. The rate of DCIP photoreduction decreased as the aging process was prolonged. Decrease in Mn content correlated with the decrease of DCIP photoreduction. Especially in the presence of EDTA, with the decrease of Mn, the rate of electron transport was severely reduced.     

Multi-temperature effects on Hill reaction activity of barley chloroplasts.

1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian). 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2--38degreesC. The apparent Arrhenius activation energy (Ea) for the reaction was 48.1 kJ/mol. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the Ea for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36degreesC. The Ea was highest below 9degreesC at 63.7 kJ/mol. Between 9 and 20degreesC the Ea decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29degreesC. Between 29 and 36degreesC there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29degreesC were reversible. At temperatures above 36degreesC (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred. 4. Temperature-induced changes in Ea were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in Ea in the absence of an uncoupler. 6. The temperature-induced changes in Hill activity at 9 and 29degreesC coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.     

Effects of water stress on photochemical function and protein metabolism of photosystem II in wheat leaves

The effects of osmotic dehydration in wheat leaves ( Triticum aestivum L. cv. Longchun No. 10) on the photochemical function and protein metabolism of PSII were studied with isolated thylakoid and PSII membranes. The results indicated that PSII was rather resistant to water stress as mild water deficit in leaves did nut significantly affect its activity. However, extreme stress conditions such as 40% decrease in relative water content (RWC) or 1.8 MPa in water potential (Ψ) caused ca 50% reduction in O₂ evolution and ca 25% inhibition of DCIP (2.6-dichlorophenol indophenol) photoreduction of PSII. In addition, it was found that the inhibited DCIP photoreduction of PSII could not be reversed by DPC (2.2-diphenylcarbazide), a typical electron donor to PSII, suggesting that water stress did not affect electron donation to PSII. Urea-SDS-PAGE and western blot analysis showed that the steady slate levels of major PSII proteins, including the D1 and D2 proteins in the PSII reaction center, declined on a chlorophyll basis with increasing water stress, possibly as a result of increased degradation. In vitro translation experiments and quantitative analysis of chloroplast RNAs indicated that the potential synthesis of chloroplast proteins from their mRNAs was impaired by water stress. From the results it is concluded that the effects of water stress on PSII protein metabolism, especially on the reaction center proteins, may account for the damage to PSII photochemistry.     

The Regulation and Distribution of LHC-I and LHCP2 between the Photosystem I and Photosystem II

Evidences were provided in this paper that the relative distribution of chl-protein complexes of PSⅠ and PSⅡ could be regulated by Mg2+. addition of Mg2+ led to decrease in the amount of chl-protein complexes of PSⅠ and increase in the amount of chl-protein in complexes of PSⅡ. There was no effect of Mg2+ on the spectral property of LHCP1, but the addition of Mg2+ could change the spectral property of LHCP2 so that it became similar to that of the LHC-Ⅰ. CPIa2 was a complex of reaction centre of PSⅠ and LHC-I. LHC-I might be contacted specially with LHCP2 in chloroplast membranes. Addition of Mg2+ probably cansed the motion of LHC-I from PSⅠ to PSⅡ and became more closely connected with LHCP2. The relative amount of CPIa2, CPIa1, LHCP1 and LHCP2 in chloroplast membranes could be regulated by different light intensity. There were more CPIa2, LHCP1 and less LHCP2 in chloroplast membranes from the shade plant Malaxis monophyllos and sunflower grown under weak light, both of them lacked equally CPIa1. There were less CPIa2, LHCP1 and more LHCP2 in the sun plant spinach and sunflower grown under strong light, and they possessed equally CPIa1 chl-protein complexes. It is suggested that LHCP1 and LHCP2 are different light-harvesting Chl-protein complexes. The LHC-I and LHCP2 are mobile light-harvesting chl-protein complexes and shuttle back and forth between PSⅠ and PSⅡ They play an important role in the regulation and distribution of excitation energy between the two photosystems.     

Mechanism of Change in the Cation-Induced Excitation Energy Distribution Between PSⅡ and PS Ⅰ in the Chloroplast Membrane from Zostera marina

The interrelations between thylakoid polypeptide components and Mg2+-induced Chl a fluorescence and thylakoid surface charge changes were investigated in Zostera marina chloroplasts treated with Ca2+ and trypsin. It was observed that: 1. The increase of Mg2+- induced PS Ⅱ fluorescence intensity was closely related to the decrease of Mg2+-induced surface charge density of the thylakoid membrane in the normal chloroplast; 2. Removal of the 32～34 kD polypeptides of the thylakoid surface by Ca2+ extraction of the chloroplast did not affect the Mg2+-induced phenomena; 3. If the Ca2+-treated chloroplast was further digested by trypsin to remove the 26 kD polypeptide of the membrane surface, the Mg2+-induced phenomena disappeared completely. These results clearly indicated that the 26 kD polypeptide of thylakoid surface is the specific acting site of the cation that induced these two correlated phenomena in the chloroplast from Zostera marina. The mechanism on the regulating effect of the cation on excitation energy distribution between PS Ⅱ and PS Ⅰ was discussed.     

Effects of trypsin and cations on chloroplast membranes

A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl₂) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl₂) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the “diffuse electrical layer” hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism.     

The Supramolecular Architecture in Chloroplast Thylakoid Membranes of Warigal Wheat

Thylakoid membranes of chloroplast from first leaf and flag leaf of wheat Warigal were examined by freeze-fracture and rotary shadowing etectron microscopy. The shape, size, density and size distribution of freeze-fracture partieles of their four faces were measured and plotted as three-dimensional histograms by a Hewlett-packard 9874 A digitizer with a HP 9845 B Computer and HP 9872 C plotter. When comparisons were made among different fracture faces and between the corresponding faces of the first leaf and the flag leaf, we found that the supramolecular architecture on the four fracture faces of the flag leaf differs from that on the corresponding faces of the chloroplast thytakoid membranes of the first leaf. The most significant difference was that the EFs particles contain the photosystem Ⅱ reaction centres associated with LHCP and the PFs particles were mostly light-harvesting complex. There was a 15% increase in EFs particle density, a 22% increase in PFs particle density and a 28% increase in EFu particle density. The large PFu particles contained the photosystem Ⅰ reaction centre and the flag lcaves contained 5% more than the first leaves. In addition, the stacking of thylakoid membranes in the flag leaf was 5% more than those in the first leaf.Thus, it provides theoretical basis for the fact that the flag leaf has higher photosynthetic rate.     

Reconstitution of the Light Harvesting Chlorophyll a/b Pigment-Protein Complex into Developing Chloroplast Membranes Using a Dialyzable Detergent

Conditions were developed to isolate the light-harvesting chlorophyll-protein complex serving photosystem II (LHC-II) using a dialyzable detergent, octylpolyoxyethylene. This LHC-II was successfully reconstituted into partially developed chloroplast thylakoids of Hordeum vulgare var Morex (barley) seedlings which were deficient in LHC-II. Functional association of LHC-II with the photosystem II (PSII) core complex was measured by two independent functional assays of PSII sensitization by LHC-II. A 3-fold excess of reconstituted LHC-II was required to equal the activity of LHC developing in vivo. We suggest that a linker component may be absent in the partially developed membranes which is required for specific association of the PSII core complex and LHC-II.     

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.