The Accumulation of Protochlorophyllide in Cells of Synechocystis PCC 6714 with a Low PSI/PSII Stoichiometry

Changes in intracellular levels of Chl a precursors were examinedin relation to changes in the PSI/PSII stoichiometry in thecyanophyte Synechocystis PCC 6714. Protochlorophyllide (Pchlide)accumulated markedly in cells with a low PSI/PSII stoichiometrygrown under light that is absorbed by Chl a (PSI light) whereasno accumulation occurred in cells with a high PSI/PSII stoichiometrygrown under light absorbed by phycobilisomes (PSII light). Levelsof Pchlide in cells grown under PSI light decreased rapidlyupon a shift to PSII light. The rapid decrease in Pchlide accompanieda transient increase in chlorophyllide a, indicating that reductionof Pchlide was enhanced by shift to PSII light. The action spectrumindicated that the Pchlide decrease upon the shift to PSII lightdepended on excitation of Pchlide, suggesting that the accumulationof Pchllide was due to limited excitation of Pchlide, so thatPchlide photoreduction, under PSI light. However, comparisonof levels of Pchlide and the photosystem complexes in wild-typePlectonema boryanum with those in a mutant that lacked the darkPchlide reductase (YFC 1004) indicated that dark reduction compensatedfor the limited photoreduction under PSI light. Similar compensationby dark reduction was confirmed with Synechocystis PCC 6714.In cultures of Synechocystis under conditions where Pchlidecould not be photoreduced, accumulation of Pchlide and low PSI/PSIIstoichiometry occurred only when cells were illuminated withlight that preferentially excited PSI. The results indicatethat the low PSI/PSII stoichiometry in cells grown under PSIlight is not a result of inefficient synthesis of Chl a witha reduced rate of Pchlide photoreduction. They suggest furtherthat accumulation of Pchlide under PSI light results from retardationof the Chl a synthesis due to suppression of PSI synthesis. ¹Present address: Tsurukawa 5-15-11, Machida, Tokyo, 195 Japan.     

Identification of the Product of ndhA Gene as a Thylakoid Protein Synthesized in Response to Photooxidative Treatment

A 76 amino acid sequence of NDH-A (the protein encoded by plastidndhA gene) from barley (Hordeum vulgare L.) was expressed asa fusion protein with rß-galactosidase in E. coli.The corresponding antibody generated in rabbits was used toinvestigate localization, expression and synthesis in vitroof NDH-A. NDH-A was identified as a 35 kDa polypeptide localizedin thylakoid membrane. Western blots shows a large increasein NDH-A levels when barley leaves were incubated under photooxidativeconditions, which was more pronounced in mature-senescent leavesthan in young leaves. Immunoprecipitation of the [³⁵S]methioninelabelled proteins, synthesized in vitro by isolated chloroplasts,demonstrated the synthesis in chloroplasts of the NDH-A 35 kDapolypeptide when barley leaves had been incubated under photooxidativeconditions. The results indicate that ndh genes may be involvedin the protection of chloroplasts against photooxidative stress,particularly in mature-senescent leaves. (Received November 13, 1995; Accepted February 5, 1996)     

Regulation of the Size of Light-Harvesting Antennae in Response to Light Intensity in the Green Alga Chlorella pyrenoidosa

Changes in size of the light-harvesting Chl-protein complex(LHC) induced by changes in light intensity were studied withthe green alga Chlorella pyrenoidosa. The Chi a/b ratio, whichis correlated with the size of LHC, varied over a wide range(2.5–5.0) when the light intensity for autotrophic growthwas changed. Comparison of properties of LHC II isolated fromcells grown under light of high and low intensity indicatedthat the large difference in Ch1 a/b ratios in cells grown underlight of different intensities is due mainly to changes in levelsof LHC. Reduction in levels of LHC under light of high intensitydid not occur when proliferation of cells was suppressed. Thisresult indicates that reduction in levels of LHC is not attributableto acceleration of the degradation of LHC under light of highintensity. Stimulation of formation of LHC occurred even underlight of high intensity when formation of photosystems was suppressedby chloram-phenicol (CAP). Analysis of the CAP-induced formationof LHC indicated that (1) such formation of LHC was regulatedby light intensity, being less active under higher intensity,and (2) the suppressive effect of gabaculine, an inhibitor ofthe synthesis of porphyrin, and thus of Ch1, was greater underlight of high intensity while the suppressive effect of cycloheximide,an inhibitor of the synthesis of apoprotein, was slightly greaterunder light of low intensity. The results described in thisreport indicate that (1) intensity-induced changes in the sizeof LHC in Chlorella pyrenoidosa are due to regulation of theassembly of LHC and (2) the regulation occurs primarily at thelevel of the synthesis of Ch1. (Received June 1, 1989; Accepted August 21, 1989)     

The Relation Between Ion Absorption and Protein Synthesis in Beet Disks

Disks of red beet storage tissue were incubated under asepticconditions permitting the development of various metabolic processescommonly associated with aged disks, and the effects of chloramphenicoland puromycin on protein synthesis, on the development of invertaseactivity and ion absorption capacity, and on ion absorptionper se were determined. Low concentrations of chloramphenicoland puromycin inhibit the development of ion absorption capacitybut stimulate invertase development and protein synthesis, whilehigher concentrations inhibit all three processes. In contrastion absorption itself is unaffected by puromycin, but is sensitiveto quite low concentrations of chloramphenicol The D-threo andL-threo isomers of chloramphenicol have sharply contrasted effectson the development, as distinct from the utilization, of ionabsorption capacity. The D isomer inhibits the development ofion absorption capacity more effectively than the L isomer whichin turn inhibits absorption more effectively than the D isomer. A reappraisal is made of the hypothesis that ion absorptionis directly linked with protein turnover and to account forthe results a model is proposed in which D-threo-chloramphenicolis active both as an uncoupler of oxidative phosphoryalationand as an inhibitor of protein synthesis, while L-threo-chloramphenicolacts only in the former capacity and puromycin only in the latter.It is concluded that the inhibition of ion uptake by chloramphenicolcannot be attributed to a contemporaneous effect on proteinsynthesis. However, the results are consistent with the involvementof ATPase proteins in ion uptake.     

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Cytochrome systems in cells of a denitrifying bacterium, Pseudomonasstutzeri (VAN NIEL strain), grown under different atmosphericconditions were compared with reference to the effects of nitrateand nitrite on cytochrome synthesis. When a culture was sufficiently aerated (aerobic conditions),synthesis of all cytochrome components was repressed, regardlessof the presence or absence of nitrate and nitrite. When aeratedmoderately (semi-aerobic conditions), both soluble and paniculatecytochromes c-552 and cytochrome b-558 contents markedly increasedeven in the absence of nitrate and nitrite. Under anaerobic or semi-aerobic conditions, nitrite inducedcytochrome a₂–c synthesis. This inductive effect of nitritewas counteracted by nitrate. Nitrate also repressed particulatecytochrome c-552 synthesis to some extent but nitrite did not. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan (Received June 24, 1969; )     

Water-Stress-Induced Changes in the Pattern of Protein Synthesis in Maize Seedling Mesocotyls: A Comparison with the Effects of Heat Shock

Cut segments from the growing and non-growing regions of 4-d-oldmaize seedling mesocotyls exhibit reduced protein synthesiswhen subjected to water-stress imposed directly by incubationin solutions of polyethylene glycol. Growing mesocotyl tissuecan recover full protein synthesis upon return to a fully-hydratedcondition following stress to –0.5 MPa, while non-growingtissue recovers well from greater stresses (–0.75 to –1.0MPa), and in lesser time. Using two-dimensional electrophoresisand fluorography of de novo synthesized proteins in vivo itwas possible to follow changes in protein synthesis in the non-growingtissue under water-stress and upon subsequent recovery. Thesynthesis of several proteins was reduced by stress, althoughthat of others intensified. Those proteins exhibiting reducedsynthesis under stress were synthesized at control levels (i.e. the same levels as in unstressed tissue) when the stresswas relieved. Some proteins whose synthesis was intensifiedunder stress retained this intensification upon return to fullhydration. No unique proteins were synthesized in response towater-stress, in contrast to the ‘heat-shock’ proteinsinduced when the mesocotyls were subjected to high temperature.Hence water-stress induces quantitative rather than qualitativechanges in the pattern of protein synthesis. Key words: Zea mays, Water-stress, Pattern of protein synthesis     

Preferential inhibition by ethidium bromide of photosynthetic apparatus formation in Rhodopseudomonas spheroides cells

Ethidium bromide (EB) inhibited growth of cells of the non-sulfurpurple photosynthetic bacterium Rhodopseudomonas spheroides.The inhibitory action of EB on the light-anaerobic culturedcells was stronger than on dark-aerobic cultured ones. EB alsosuppressed the induced synthesis of bacteriochlorophyll (Bchl)and carotenoids in the dark-aerobically grown cells incubatedwith gentle aeration under no-growth conditions, suggestingthat the target of the inhibitory action of EB on the photosyntheticgrowth of R. spheroides cells is chromatophore formation. EBdepressed the incorporation of ³H- or ¹⁴C-uracil into both RNAand DNA fractions from cells incubated with gentle aeration.In contrast, inhibition by EB of ³H-uracil incorporation intothe DNA fraction was not observed under vigorous aeration. Ourfindings seemed to favor the hypothesis described previously(10) that lowering the intracellular oxidation-reduction potentialmight bring about a unique synthesis or turnover of DNA responsiblefor chromatophore formation. (Received December 22, 1977; )     

Regulation of Stoichiometry between PSI and PSII in Response to Light Regime for Photosynthesis Observed with Synechocystis PCC 6714: Relationship between Redox State of Cyt b6-f Complex and Regulation of PSI Formation

The effect of the Cyt b₆-f redox state on the PSI formationwas examined with the cyanophyte Synechocystis PCC 6714 by usinga Q-cycle inhibitor, HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide).HQNO inhibited the rapid reduction of flash-oxidized Cyt f,the reaction correlating with the stimulation of PSI formation,on one hand, and accumulated reduced Cyt b₆, on the other, indicatingthat the electron flow in the Q-cycle correlates with regulationof PSI synthesis. HQNO also inhibited the stimulation of PSIformation under PSII light, resulting in a low PSI/PSII ratioeven under PSII light, while the PSI formation under PSI lightwas not suppressed by HQNO. Simultaneous inhibition of Cyt b₆oxidation through the Q-cycle and the stimulated PSI formationby HQNO suggests that an HQNO-sensitive Cyt b₆ oxidation isinvolved in the mechanism of monitoring the state of electrontransport system for regulation of PSI formation. (Received March 3, 1993; Accepted August 9, 1993)     

Investigation of sucrose synthase from rice for the synthesis of various nucleotide sugars and saccharides

The unique character of the plant glucosyltransferase sucrosesynthase, to catalyse in vitro the synthesis and cleavage ofsucrose under appropriate conditions, can be exploited for theenzymatic synthesis of carbohydrates. The present paper describesthe potential utilization of sucrose synthase from rice forthe enzymatic synthesis of activated sugars and saccharides.In the cleavage reaction of sucrose, the nucleoside diphosphatescan be used in the order UDP > TDP > ADP > CDP >GDP to obtain the corresponding activated glucoses. In batchreactions, >90% conversion of UDP and TDP could be achieved.Substituting different di- and trisaccharides for sucrose inthe cleavage reaction with UDP 2-deoxysucrose was the most promisingsubstrate. Sucrose synthase was combined with UDP-galactose4'-epimerase and ß1–4 galactosyltransferaseto synthesize N-acetyllactosamine with in situ regenerationof UDP-glucose. In the synthesis reaction of sucrose synthase,different donor (UDP-sugars) and acceptor substrates were investigated.UDP-N-acetylglucosamine and UDP-xylose could be used in combinationwith fructose as acceptor. D-Xylulose, D-tagatose, D-lyxose,D-psicose, L-sorbose, D-mannose, L-arabinose, 1, 6 anhydroglucose,lactulose, raffinose and isomaltulose can serve as acceptorsfor UDP-glucose. N-acetyllactosamine nucleotide sugars saccharides sucrose synthase     

Photosynthetic carbon metabolism in wild, primitive and cultivated forms of wheat at three levels of ploidy: Role of the glycolate pathway

¹⁴CO₂ assimilation was studied with diploid, tetraploid, hexaploidspecies of the genera Triticum and their wild relatives Aegilops.Attached mature leaves of 3–4 weekold plants were allowedto undergo photosynthesis under air at ambient temperature.The pattern of distribution of ¹⁴C was notably similar in Triticumand Aegilops species whatever the level of ploidy. Sucrose wasthe sink for photosynthetic carbon. ¹⁴C for sucrose synthesis was supplied either through the glycolatepathway by glycolate, the product of the photorespiration orby the Calvin cycle intermediates exported into the cytoplasm.Depending on the species, the glycolate pathway provided 40to 75%of the sucrose ¹⁴C. The higher labeling of sucrose was associated with the greaterparticipation of the glycolate pathway in the wild diploid (DD)A. squarrosa and in the cultivated hexaploid (AABBDD) T. aestivum.The results suggest that the expression of the male D genomeis dominant over the female AB genome in T. aestivum. In T. aestivum under ambient conditions lowering (low temperature)or hindering (1% O₂ ) photorespiration, sucrose labeling decreased,but serine and glycine labeling was favoured. We propose thatin wheat leaves, the role of photorespiration is to drain artof the carbon exported from the chloroplast as glycolate, towardssucrose synthesis. (Received March 16, 1979; )     

Changes in Levels of Heme a, Protoheme and Protochlorophyll(ide) in Submerged Rice Seedlings after Exposure to Air

Rice (Oryza sativa L. cv. Yamabiko) seedlings germinated underwater for 5 days contained small amounts of heme a and protohemebut no protochlorophyll(ide) [Pchl(ide)]. Levels of hemes andPchl(ide) increased rapidly upon transfer to air. When expressedin terms of fresh weight of tissue, hemes reached the levelsin aerobic controls after 24 h of contact with air, but Pchl(ide)did not. A comparison of the increases during 24-h adaptationto air in levels of heme a and Pchl(ide), which are specificto mitochondria and plastids, respectively, suggested that thedevelopment of mitochondria preceded that of plastids. The rateof synthesis of 5-aminolevulinic acid (ALA) was low in submergedseedlings, as compared to the rate in aerobic controls, butit increased during air adaptation. The sum of the amounts ofheme a, protoheme and Pchl(ide) increased in parallel with theamount of porphyrins, equivalent to the amount of ALA synthesizedduring the experimental period. When submerged seedlings thathad been pretreated with levulinic acid were exposed to air,no Pchl(ide) was formed. In contrast, Pchl(ide) accumulatedunder water when submerged seedlings were fed with ALA. Theseresults indicate that the synthesis of ALA, the limiting stepin the synthesis of Pchl(ide), is repressed under hypoxic conditions. ¹ Present address: KRI International, Inc., Kyoto Research Park17, Chudoji Minami-machi, Shimogyo-ku, Kyoto, 600 Japan. ² Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

Content and De Novo Synthesis of Cocaine in Embryos and Endosperms from Fruit of Erythroxylum coca Lam

There is controversy as to whether the mature fruit of Erythroxylumcoca var. coca Lam. contains the cocaine alkaloid (benzoylmethylecgonine).In the present study, cocaine was monitored to determine ifit was present in embryos and endosperms of mature fruit ofE. coca var. coca Lam., and if present, the time required forde novo synthesis in imbibing seed. Seeds from mature fruitof E. coca were dissected to separate the embryos from the endosperms.The separated embryos and endosperms were analysed for cocaine.Subsequently, endosperms and embryos from seed imbibed. undera light and dark treatment were separated on days 3, 6, 9, 12and 15 and analysed for cocaine. Cocaine was present in embryos(0.005% of d. wt) and endosperms (0–001% of d. wt) ofmature fruit of E. coca. De novo synthesis of cocaine occurredonly in embryos of seed imbibed under light after day 9 of imbibition. Erythroxylum coca, alkaloid, benzoylmethylecgonine, cocaine, embryo, endosperm, seed imbibition     

Limitation to Photosynthesis in Water-stressed Leaves: Stomata vs. Metabolism and the Role of ATP

Decreasing relative water content (RWC) of leaves progressivelydecreases stomatal conductance (g_s), slowing CO₂ assimilation(A) which eventually stops, after which CO₂ is evolved. In somestudies, photosynthetic potential (A_pot), measured under saturatingCO₂, is unaffected by a small loss of RWC but becomes progressivelymore inhibited, and less stimulated by elevated CO₂, below athreshold RWC (Type 1 response). In other studies, A_pot andthe stimulation of A by elevated CO₂ decreases progressivelyas RWC falls (Type 2 response). Decreased A_pot is caused byimpaired metabolism. Consequently, as RWC declines, the relativelimitation of A by g_s decreases, and metabolic limitation increases.Causes of decreased A_pot are considered. Limitation of ribulosebisphosphate (RuBP) synthesis is the likely cause of decreasedA_pot at low RWC, not inhibition or loss of photosynthetic carbonreduction cycle enzymes, including RuBP carboxylase/oxygenase(Rubisco). Limitation of RuBP synthesis is probably caused byinhibition of ATP synthesis, due to progressive inactivationor loss of Coupling Factor resulting from increasing ionic (Mg²⁺)concentration, not to reduced capacity for electron or protontransport, or inadequate trans-thylakoid proton gradient (pH).Inhibition of A_pot by accumulation of assimilates or inadequateinorganic phosphate is not considered significant. DecreasedATP content and imbalance with reductant status affect cellmetabolism substantially: possible consequences are discussedwith reference to accumulation of amino acids and alterationsin protein complement under water stress.     

Accumulation of Betacyanin in Phytolacca americana Cells and of Anthocyanin in Vitis sp. Cells in Relation to Cell Division in Suspension Cultures

In suspension cultures of Vitis sp., maximal accumulation ofanthocyanin was observed during the stationary phase. Accumulationof anthocyanin occurred in parallel with the cessation of celldivision under conditions such as a reduction of the concentrationof phosphate in the medium, or the presence of aphidicolin,an inhibitor of DNA synthesis. By contrast, in suspension culturesof Phytolacca americana, aphidicolin inhibited the accumulationof betacyanin and cell division. When aphidicolin was removedfrom cells by washing, partially synchronized division of cellswas induced and the accumulation of betacyanin also occurred,in conjunction with cell division. In the absence of phosphatefrom the medium, cell division did not occur and accumulationof betacyanin also ceased. Readdition of phosphate to cellsstarved for phosphate induced both cell division and the accumulationof betacyanin. These results indicate a positive correlationbetween the accumulation of betacyanin and cell division inPhytolacca which contrasts with a negative correlation betweenthe accumulation of anthocyanin and cell division in Vitis. (Received April 17, 1989; Accepted December 23, 1989)     

The Effect of Growth-regulating Substances and Light on Olive Callus Growth in vitro

In vitro olive callus cultures were established; auxin was notobligatory for the early development of the callus. However,after a number of subcultures, both an auxin and a cytokininwere needed for growth. IAA was the most efficient auxin ininducing growth, although 2,4-D was also very active. Very shortperiods of red-light illumination were sufficient to effectgreening of the tissue. A particularly high ratio of chlorophyllb/a was found under all light conditions tested. All light treatmentsstimulating chlorophyll synthesis caused a reduction in growthafter four subcultures.     

Genetic and Hormonal Regulation of Vitellogenesis in Drosophila

In Drosophila the female-specific yolk protein, or vitellogenin,is synthesized in the fat body. In D. melanogaster, vitellogenin,is first detected in the female hemolymph at the time of adulteclosion and in the ovaries 20 hours later, suggesting differentregulatory mechanisms for the processes of synthesis and uptake.Transplantations of pupal or immature adult ovaries into D.melanogaster adult males induce vitellogenin synthesis, implicatingan ovarian agent in the control of synthesis. Larval ovariesfail to stimulate synthesis. Female-sterile mutants with rudimentaryor previtellogenic ovaries synthesize and accumulate large quantitiesof vitellogenin in the hemolymph, but not in the ovaries. Transplantationof these rudimentary ovaries into males induces vitellogeninsynthesis, suggesting that the ovarian inducing agent does notoriginate from the germ cells. Treatment of the homozygous female-sterilemutants with juvenile hormone stimulates the uptake of vitellogeninby the ovary in some strains. This shows that juvenile hormoneplays a role in vitellogenin uptake. The potential importanceof Drosophila vitellogenesis for studies of gene regulationis discussed.     

Photoregulation of Phytochrome Synthesis in Germinating Embryos of Avena sativa L.

Hilton, J. R. and Thomas, B. 1987. Photoregulation of phytochromesynthesis in germinating embryos of Avena sativa L.—J.exp. Bot. 38: 1704–1712. The effect of light on the accumulation of phytochrome in germinatingAvena embryos was determined. A quantitative ELISA using monoclonalantibody AFRC MAC 56 was used to measure specifically type 1(or dark) phytochrome. A pulse of red light given after 14 himbibition but prior to the onset of type 1 phytochrome synthesis,strongly inhibited subsequent type 1 phytochrome accumulation.This effect of red light at 14 h was reversible by far-red lightindicating the involvement of phytochrome. Red light also inhibitedphytochrome synthesis after 18 h and 24 h imbibition but after24 h, far-red light did not reverse the effect. The effect ofred light treatment at 18 h was reversed by giving a pulse offar-red light at any time up to 30 h. Seed germination was notinfluenced by light under the conditions of these experiments.It is proposed that type 2 (or light) phytochrome may be responsiblefor photoregulation of type 1 phytochrome synthesis in germinatingAvena embryos. Key words: Photoregulation, phytochrome, seed.     

Development of Lipid Synthesis from CO2 in Avena Leaves during Greening of Etiolated Seedlings

The development of the lipid synthesizing system in Avena leafsections was examined in connection with carbon fixation duringthe greening of etiolated seedlings under light. During theinitial 2 h illumination there was a low level of CO₂ fixationby PEP carboxylation, but its products, malate and citrate,did not serve as a carbon source for lipid synthesis, althoughlipid synthesis from acetate had already been established. Withthe initiation of Calvin cycle activity after the initial 2h illumination, lipid synthesis began, with CO₂ fixed by RuBPcarboxylation serving exclusively as the carbon source. Fattyacid synthesis in the leaves during the initial 3 h illumination,unlike the fatty acid synthesis thereafter, was insensitiveto thiolactomycin, an inhibitor of type II fatty acid synthetasecontained in the plastids, and was not dependent on light, incontrast to light-dependent activity in greened leaves. The distribution of ¹⁴C incorporated into lipid molecules fromNaH¹⁴CO₃ showed an equal ratio of ¹⁴C in fatty acid, glyceroland choline moieties of labeled phosphatidylcholine, but a denserradioactivity in the galactose moiety than in the residual moietyof mono- and di-galactosyldiacylglycerols. This suggests a regulatedsupply of glycerol, choline and fatty acid moieties for phosphatidylcholinesynthesis, and an excess supply of galactose to diacylglycerolmoiety for galactosyldiacylglycerol synthesis in Avena leaves. (Received October 31, 1984; Accepted January 25, 1985)