Photoconversion of Photochlorophyllide in the y-1 Mutant of Chlamydomonas reinhardtii

Dark-grown y-1 mutant cells of Chlamydomonas reinhardtii accumulate protochlorophyllide (Pchlide) in both 635 nanometers (P635) and 650 nanometers (P650) forms. Plastids in these cells lack the normal thylakoid membrane structure except some remnants of membrane vesicles. Using difference spectrophotometry, P635 is shown to be photoconverted to chlorophyllide at 672 nanometers (C672) and P650 is photoconverted to C688 followed by a rapid shift to C672 (Shibata shift) and regeneration of P650. Some of the Pchlide is not photoconverted despite repeated illumination. Although P650 is destroyed by freezing and thawing, it is not transformed into P635. Freezing and thawing treatment also made Pchlide no longer photoactive.     

Effect of Dim Light on the y-1 Mutant of Chlamydomonas reinhardtii

The y-1 mutant of Chlamydomonas reinhardtii tends to die or revert to wild type when grown in the dark for a long period of time. A small amount of white light (0.5 lux) enables the y-1 mutant to grow indefinitely in a “near dark” condition. Under this condition, the y-1 mutant is physiologically and ultrastructurally similar to the dark-grown y-1 yet remains genetically stable.     

Origin of Thylakoid Membranes in Chlamydomonas reinhardtii y-1 at 38 degrees C

The origin of thylakoid membranes was studied in Chlamydomonas reinhardtii y-1 cells during greening at 38°C. Previous studies showed that, when dark-grown cells are exposed to light under these conditions, the initial rates of accumulation of chlorophyll and the chlorophyll a/b-binding proteins in membranes are maximal (MA Maloney JK Hoober, DB Marks [1989] Plant Physiol 91: 1100-1106; JK Hoober MA Maloney, LR Asbury, DB Marks [1990] Plant Physiol 92: 419-426). As shown in this paper, photosystem II activity, which was nearly absent in dark-grown cells, also increased at a linear rate in parallel with chlorophyll. As compared with those made at 25°C, photosystem II units assembled during greening at 38°C were photochemically more efficient, as judged by saturation at a lower fluence of light and a negligible loss of excitation energy as fluorescence. Electron microscopy of cells in light for 5 or 15 minutes at 38°C showed that these initial, functional thylakoid membranes developed in association with the chloroplast envelope.     

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

Studies of in vitro processing of precursors of the major chlorophyll a/b-binding polypeptides of Chlamydomonas reinhardtii y-1 were undertaken to define the precursor-product relationships. Analysis of translates, prepared from C. reinhardtii poly(A)-rich RNA in a rabbit reticulocyte lysate system, which were incubated with the soluble fraction from C. reinhardtii cells, showed that the 31,500 relative molecular mass (M_r) precursor was converted to the M_r 29,500 thylakoid membrane polypeptide whereas the M_r 30,000 precursor was converted to the M_r 26,000 product. Furthermore, the M_r 31,500 polypeptide, when bound to antibodies, was not processed to the mature polypeptide of M_r 29,500, although the presence of antibodies did not prevent the precursor of M_r 30,000 from being converted to the mature M_r 26,000 polypeptide. The mature fraction of M_r 26,000, was separated into two bands corresponding to polypeptides 16 and 17 in the electrophoretic system of Chua and Bennoun (1975 Proc Natl Acad Sci USA 72: 2175-2179).

Processing activity was present in the soluble fraction obtained from cells grown in the light or in the dark. Therefore, processing of the precursor polypeptides does not appear to be involved in the regulation by light of the accumulation of these polypeptides in thylakoid membranes.

     

Chloroplast Development in the Chloroplast Ribosome Deficient Mutant (y-1 ac-20) of Chlamydomonas reinhardtii

To study the participation of chloroplast protein synthesisduring the three phases [Matsuda (1974) Biochim. Biophys. Acta366:45] of the greening process in Chlamydomonas reinhardtiiy-1, the greening characteristics in the low-chloroplast ribosomemutant y-1 ac-20 were compared with those in the y-1. In thedouble mutant cells Chl synthesis proceeded with an extendedlag and without a second transition point. The development ofpotential for rapid Chl synthesis (P-factor formation) was alsodelayed. Furthermore, PS I activity increased significantly,whereas PS II activity developed very little during greeningof the double mutant cells. The results indicate that greeningin double mutant cells occurs with no apparent late phase. (Received November 26, 1984; Accepted February 25, 1985)     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

Studies on chloroplast development in Chlamydomonas reinhardtii IV. Control of rapid chlorophyll formation in greening y-1 cells

Effects of protein synthesis inhibitors, CAP and CHI, on diegreening of Chlamydomonas reinhardtii y-1 cells, particularlyon die P-factor formation (19) in the early phase, were studied.Chlorophyll synthesis in the normal greening process, whichis divided into three phases, was strongly inhibited by bothantibiotics, although the inhibition by CAP was weaker in themiddle and late phases. The development of potential for rapidchlorophyll formation (P-factor formation) that takes placein dark-grown cells during dark incubation following brief illuminationwas completely blocked by CHI, but not by CAP. A "CHI-sensitive"period for the P-factor formation was restricted to the initial30 min during the dark incubation following brief illumination(10 min). This initial 30-min period appeared to correspondto the time of protochlorophyll(ide) formation which was inhibitedby CHI. Light-dependent conversion of protochlorophyll(ide) to chlorophylland also the subsequent protochlorophyll(ide) synthesis, whichis "CHI-sensitive" seem to be prerequisite for the inductionof P-factor synthesis. A possible control mechanism involvedin the early phase of the greening process in y-1 cells is discussed. (Received February 12, 1976; )     

A Secondary Processing Site in the Precursor of the Small Subunit of Ribulose Bisphosphate Carboxylase of Chlamydomonas reinhardtii y-1

When the precursor of ribulose bisphosphate carboxylase of Chlamydomonas reinhardtii y-1 is bound to antibodies and treated with the soluble cell fraction, it is cleaved to the mature form (M_r 16,500) via an intermediate of M_r 18,500. Although this intermediate has only been observed in vitro, it may be produced during processing of the precursor in vivo.     

Dual Role of Methionine in the Biosynthesis of Diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii

Biosynthesis of the polar group of diacylglyceryl-O-4′-(N,N,N-trimethyl)homoserine (DGTS) was studied in intact cells of Chlamydomonas reinhardtii Dangeard. Among the three C₄ amino acids tested, only l-methionine could specifically inhibit the photosynthetic incorporation of [¹⁴C]NaHCO₃ into the polar group of DGTS. The radioactivity in l-[¹⁴C]methionine, which was labeled at either the C3 + C4, the C1, or the methyl carbon, was efficiently incorporated into the polar group of DGTS. These results suggest that the C₄ backbone and the S-methyl group of l-methionine are precursors to the C₄ backbone and the N-methyl groups of DGTS, respectively.     

Kinetics of Chlorophyll Accumulation and Formation of Chlorophyll-Protein Complexes during Greening of Chlamydomonas reinhardtii y-1 at 38 degrees C

The initial kinetics of accumulation of chlorophylls (Chl) were analyzed during optimal greening of Chlamydomonas reinhardtii y-1 at 38°C. Acetate was required for maximal synthesis of Chl, which occurred at a linear rate when degreened cells were exposed to light. During the first hour Chl a and b accumulated predominantly as geranylgeraniol esters, with lesser amounts of the species with more reduced alcohol side chains. When Chl synthesis was blocked either by treatment with gabaculine or by transfer to the dark, the distribution shifted to the more reduced forms. Similar kinetic patterns indicated that a common pool of chlorophyllides a and b provided substrate for the enzymatic system that performs esterification and reduction of the sldechain for each group of Chl. Chl b was essentially quantitatively integrated into light-harvesting complexes as indicated by energy transfer to Chl a. In the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis, Chl b did not accumulate and Chl a production was reduced about one-half. The results demonstrate that Chl a/b-protein complexes assemble rapidly during greening and that reduction of the alcohol side chain of the Chl is not required for assembly of these complexes.     

Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control

The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38°C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38°C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells.     

缺氮介导的莱茵衣藻油脂合成过程的内参及标志物蛋白质的筛选鉴定

微藻被认为是最有潜力的生物能源原料之一,了解油脂合成机理、提升油脂合成的效率是重要的生物学问题.莱茵衣藻缺氮胁迫是油脂合成机理研究的模式系统,组学研究已经积累了大量的数据,但针对莱茵衣藻缺氮介导的油脂合成过程的内参及标志物蛋白质还鲜有报道.本研究对莱茵衣藻进行了对照和缺氮胁迫培养,比较了多个时间点(0、1、2、4和6d)在2种处理条件下的培养物表型、细胞密度、油脂含量以及总蛋白质含量的变化等特征.结果显示:莱茵衣藻细胞在受到缺氮胁迫后表现为培养物颜色由绿变黄;A750和细胞计数结果显示细胞生长趋于停滞;尼罗红染色定量实验鉴定到油脂含量的显著升高;考马斯亮蓝染色实验检测到总蛋白质含量降低.以20个莱茵衣藻蛋白质为候选,利用蛋白质印迹技术(Western blot,WB)检测了其在不同处理和不同时间点的表达特征变化,通过计算总蛋白质和候选蛋白质含量的皮尔森相关系数(Pearson’s correlation coefficient, PCC)筛选了莱茵衣藻缺氮胁迫的内参蛋白质,发现Histone H3、 RBCL(ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit)和BCR1(biotin carboxylase,ACCase complex 1)在对照和缺氮胁迫条件下均与总蛋白质含量变化呈现极显著或显著正相关,所以被选作内参蛋白质.进而通过比较候选蛋白质的平均相对倍率变化(average relative fold change, ARF),鉴定了莱茵衣藻缺氮胁迫的标志物蛋白质,发现ATPs-β(ATP synthase CF1beta subunit)、 GAP2(glyceraldehyde 3-phosphate dehydratase 2)和RMT1(rubisco large subunit N-methyltransferase 1)蛋白质的ARF值分别为180.59、52.90和12.48,明显高出其他蛋白质,由此把它们选做缺氮胁迫的标志物蛋白质.接下来,对缺氮胁迫早期(0、2、4、8、12、18、24和48 h)的样品进行蛋白质印迹法分析,发现可检测的ATPs-β、GAP2和RMT1的缺氮诱导条带出现的时间分别是8、18和12 h.综上可以认为,在所有候选蛋白质中,ATPs-β是出现最早且变化幅度最大的缺氮处理标志物蛋白质.本研究鉴定的内参和标志物蛋白质对了解缺氮应答及油脂合成机理会有所帮助,所积累的蛋白质表达信息可供研究同行参考.     

Blue-Light-Regulated Expression of Genes for Two Early Steps of Chlorophyll Biosynthesis in Chlamydomonas reinhardtii

In light:dark-synchronized cultures of Chlamydomonas reinhardtii, the genes encoding the enzymes for two early steps of chlorophyll biosynthesis, glutamate-1-semialdehyde aminotransferase (gsa) and [delta]-aminolevulinic acid dehydratase (alad), are expressed at high levels early in the light phase, just prior to a rapid burst of chlorophyll synthesis. Induction of gsa mRNA in synchronized cells is totally dependent on light, whereas induction of alad mRNA occurs to approximately one-half the light-induced level even in cells kept in the dark during the light phase and appears to be dependent on the cell cycle or a circadian rhythm. gsa mRNA and alad mRNA accumulation is induced by light that was passed through blue (400-480 nm) or green (490-590 nm) filters but not by light that was passed through orange (>560 nm) or red (>610 nm) filters, indicating the participation of a blue-light photoreceptor system rather than a protochlorophyllide- or rhodopsin-based photoreceptor. Light induction of gsa mRNA accumulation is absent in a carotenoid-deficient mutant, which suggests that a carotenoid-containing blue-light photoreceptor is involved. In contrast, pretreatment of wild-type cells with either of two flavin antagonists, phenylacetic acid and KI, does not prevent the light induction. In the later part of the light phase, the gsa mRNA level decreases more rapidly than that of alad mRNA. Turnover studies indicate that the half-life of alad mRNA is twice that of gsa mRNA. This difference in mRNA stability partially accounts for the more rapid decline in gsa mRNA levels after the peak of light induction is reached. Thus, differential blue-light induction and stability of mRNAs regulates the expression of these two chlorophyll biosynthetic genes.     

Development of Photosystem II Complex during Greening of Chlamydomonas reinhardi y-1

The relative content of organized pigment, active centers, and acceptor pools of photosystem II and their interconnection during the development of the photosynthetic membranes of Chlamydomonas reinhardi y-1 have been measured using the fluorescence induction technique. The degree of connectivity and efficiency of the developing system has been assessed also from measurements of maximal rates, quantum yield, and flash yield of 2,6-dichlorophenolindophenol photoreduction using H₂O as the electron donor. The results obtained indicate that the process of membrane development in this organism consists of two phases: an initial phase of reorganization and connection between pre-existing components, and a second phase of actual accumulation of newly formed, complete, and active units. The ratio of active centers to Chl remains practically constant throughout the process while the degree of connectivity between the active center and the plastoquinone pool was doubled during the early phase of the greening. In addition the degree of connectivity between the plastoquinone pool and the rest of the electron transport chain increases as demonstrated by a 10- to 20-fold rise in the quantum yield and a 10-fold rise in the maximal rate and the flash yield. The ratio of light harvesting Chl to active centers remains apparently constant during the second phase of the greening as indicated by light saturation experiments and by the constancy of the apparent photosynthetic unit size. Electron donation from H₂O seems to develop slower than the activity of the rest of the complex as demonstrated by measurements of 2,6-dichlorophenolindophenol photoreduction using 1,5-diphenylcarbazide as the electron donor. The value of all the above parameters which remain constant during the second phase of the greening are comparable to those obtained with membranes of light-grown cells.     

Flagellar Morphology in Stumpy-Flagella Mutants of Chlamydomonas reinhardtii1

Sixteen new mutants of the biflagellate green alga Chlamydomonas reinhardtii with either stumpy-flagella or no flagella at all were examined by electron microscopy. Four of the mutants were found to carry short bulbous flagella containing amorphous electron-dense material which may represent unassembled flagellar protein. Basal bodies of normal ultrastructure were present in all mutants. Dikaryon dominance tests indicated that the stumpy mutations were recessive to wild-type in all cases tested. Stumpy mutations also conferred a measure of detergent resistance to Chlamydomonas, apparently by affecting the detergent-solubility of the flagellar membrane.