Interaction between chloroplast and cytoplasmic factors in the inhibition of l-phenylalanine ammonia-lyase activity

Components of the system inactivating cytoplasmic l -phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) in barley seedlings ( Hordeiim vulgare L. cv. Polon) were investigated. In vitro light-activated chloroplasts and two cytoplasmic factors participate in the enzyme inhibition. One role of the chloroplasts is to mediate the reaction between PAL and the cytoplasmic factors, resulting in inactivation of the enzyme. Uncoupling (carbonyl cyanide m-chlorophenyl hydrazone, valinomycin) or inhibiting [3-(3,4-dichlorophenyl)-l,l-dimethylurea] photosynthetic electron transport showed that electron transport is necessary to enable chloroplasts to mediate PAL inhibition. Preliminary results also indicate that mitochondria are able to mediate the inhibition. The role of this inhibiting system in the regulation of PAL activity in vivo is discussed.     

Differential regulation of phenylalanine ammonia lyase activity and protein level by light in tomato seedlings

Red light, acting via phytochrome, stimulates phenylalanine ammonia lyase (PAL) activity in cotyledons and hypocotyls of tomato seedlings. The time course of photoinduction of PAL activity has a peak level at 4 h after which activity declines significantly. In tomato seedlings PAL activity comprised of three isoforms and light stimulated activity of all three isoforms. A polyclonal antibody raised against PAL purified from tomato leaves recognized PAL protein belonging to PAL-II and PAL-III isoforms. The mode of increase in PAL activity was investigated by immunochemical techniques. The photostimulated increase in PAL activity appeared to be dependent on de novo synthesis of protein and nucleic acid. However, inhibition of protein phosphatase activity blocked increase in PAL activity without affecting the increase in PAL protein levels. The results indicate that in addition to de novo synthesis, the photostimulation of PAL activity likely requires dephosphorylation by a type 2C protein phosphatase.     

Diurnal oscillation of amylolytic activity in spinach chloroplasts

Chloroplasts isolated from spinach (Spinacia oleracea L., cv. vital^R) plants grown under controlled light/dark and temperature regimes, contained the phosphorolytic and amylolytic pathways for starch breakdown. The latter consists at least of α- and β-amylase and maltase. Only low amylolytic activity was observed in chloroplasts isolated during the light phase. In chloroplasts prepared during the dark phase, this activity was almost twice as high. These diurnal oscillations of the amylolytic activity were maintained when the plants were kept in prolonged darkness or continuous light. The amylolytic system exhibited a sharp pH optimum between 5.8 and 6.0. Phosphorylase activity, when assayed with saturating concentrations of inorganic phosphate, did not show diurnal fluctuations.     

Effect of Photosynthetic Inhibitors on Induction of Tryptamine Pathway-mediated Resistance in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

Rice cv. Sekiguchi‐asahi was identified as a lesion mimic mutant from cv. Asahi. This mutant showed light‐enhanced resistance to Magnaporthe grisea infection, inducing Sekiguchi lesion (sl) formation and tryptamine accumulation. To elucidate the role of chloroplasts on the activation of tryptamine pathway, the effect of some photosynthetic inhibitors on light‐dependant Sekiguchi lesion formation, tryptamine accumulation, and enzyme activities was investigated. When detached leaf blades which had been pretreated with Linuron, 1, 10‐Phenanthroline or Poly‐l ‐lysine were inoculated with M. grisea, respectively, Sekiguchi lesion formation and typtamine accumulation were suppressed even under light, suppressing gene expression of tryptophan decarboxylase and monoamine oxidase activity. On the other hand, catalase activity was significantly inhibited in control leaves infected with M. grisea under light, but kept at high level in chemical‐pretreated leaves. These results suggested that photosynthetic activity in chloroplasts was involved in inhibition of antioxidant sysytem and activation of tryptamine pathway responsible for light‐enhanced resistance in sl mutant to M. grisea infection.     

Inhibitory effect of p-nitrothiophenol in the light on the photosystem II activity of spinach chloroplasts.

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH'S caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiopehnol to causing the specific inhibition of Photosystem II.     

Reduction of 2,6-dichlorophenol-indophenol (Hill Reaction) by Chloroplasts with Different Chlorophyll a/b Ratios Under monochromatic Light

Chloroplasts with different chlorophyll a/b ratios were isolated from 7 to 8 days old wheat seedlings and the activities of reduction of 2,6-dichlorophenol-indophenol (DPIP) by these chloroplasts as function of the chlorophyll a/b ratios were studied under mono-chromatic light (650 m μ, 670 mμ, 680 mμ, 707 mμ). It was found that the DPIP reducing activities by these chloroplasts varied with their chlorophyll a/b ratios, and these variations are affected by the wavelengths of the illuminating light. Under 650 mμ, at the a/b ratios of 2.2 to 2.82, the activities of DPIP reduction in- creased with the a/b ratios, but decreased when the a/b ratios were higher than 2.82. Under 670 mμ, the DPIP reducing activities also varied with the a/b ratios of isolated chloroplasts. However, the variation was more gradual and steadier. Under 680 mμ, the DPIP reducing activities increased with the a/b ratios over 3.0, but decreased rather suddenly at a/b ratio of 3.30. Essentially the same relation held for 707 mμ, but the Hill reaction activities ceased to decline farther when a/b ratio rose to 3.40 at 707 mμ. When the results were analyzed in terms of the “relative activities” of the chloroplasts of the above mentioned wave lengths, it was found that the values of the "relative activity" (a/a+b) declined steadily from 1.47–1.29 at an a/b ratio range of 2.05–3.40, while the values of the "relative activity" (b/a+b)increased steadily from 3.10--4.40 at the same range of a/b ratios. But it is to be noted that, under 650 mμ, the (a/a+b) was 1.36 at the a/b ratio range of 2.63–2.82. Interesting enough, the DPIP reducing activities were the highest of all with these a/b ratios. Activities of DPIP reduction by isolated chloroplasts kept at 0 ℃, 20 ℃, 30 ℃, and 45 ℃ diminished with time when illuminated at all wavelengths. However, for those kept at 45 ℃ their activities were lost after 20 minutes, except those illuminated with the wave length of 680 mμ which still maintained 30 % of the initial activity. When kept at the above mentioned temperatures, the chlorophyll a/b ratios of all batches of chloroplasts declined steadily with time. The above results are interpreted as being indicative of the possibility that the pigment systems for the partial reaction (Hill reaction) of the over-all photosynthesis process consisted mainly of Chlb650 and Chla670. And the correlation of temperature and abolition of activity reduction of DPIP of chloroplasts varied under monochromatic light of different wave lengths.     

Induction of phenylalanine ammonia-lyase activity by tryptophan in Ustilago maydis.

To understand the regulation of phenylalanine ammonia-lyase (PAL) activity in the corn smut fungus, Ustilago maydis, we examined the effects of different media, metabolic effectors (including aromatic amino acids), and environmental factors on induction and repression of PAL activity. PAL was detected only in cell extracts and not in the culture medium. U. maydis PAL is constitutively produced at a low level in all media tested but its regulation can be influenced by aromatic amino acids. L-Tryptophan (0.3 mM) induces PAL activity 3- to 5-fold but tryptophan analogs and tryptophan-related metabolites do not. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. No induction or inhibition of PAL activity was observed as a function of culture temperature, pH or light. PAL induction was repressed by glucose but not by its reaction product, t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be involved. This study shows that the regulation of U. maydis PAL is very different from the patterns known for plants and other fungi.     

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m^-2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine-and tyrosine-ammonia-lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize ( Zea mays L. cv. Cross Bantam T51). Growth rate of dark-grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p -coumaric acid ( p -CA) increased by severalfold in cell walls of light-grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p -CA and the decrease in cell wall extensibility. Light decreased the wall-bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p -CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.     

水稻、大麦幼苗胚乳中的苯丙氨酸解氨酶调节因子(PAL-R)

从萌发的水稻、大麦胚乳中,通过85％硫酸铵盐析、DE52纤维素和Sephadex G100柱层析,获得部分纯化的苯丙氨酸解氨酶调节因子(PAL—R),研究其基本性质,及体外PAL-R对PAL和PAL-I的影响,表明它们之间存在着相互作用。     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

A direct light effect on maintaining photosynthetic activity of Nitella chloroplasts

The chloroplasts of internodal cells of Nitella are fixed to a stationary layer of cytoplasm whereas the nuclei and most of the cytoplasm stream along the longitudinal axis. Isolated internodal cells were maintained for several days with half the cell kept in the dark, the other half kept under continuous light. Photosynthetic activity of the cells was checked by placing the cell evenly illuminated in a ¹⁴CO₂ atmosphere. Chloroplasts of the previously dark half of the cell were found to fix only half as much CO₂ as the chloroplasts which were continuously illuminated. These results are discussed in relation to the possible direct effect of light on biosynthetic reactions of mature chloroplasts.     

Chloroplastenausbildung und Morphogenese der Gametophyten von Dryopteris filix-mas (L.) Schott nach Applikation von Chloramphenicol und Actidion (Cycloheximid)

Summary Morphogenesis and differentiation of the young gametophytes (=sporelings) of Dryopteris filix-mas are controlled by light. Blue light leads to the formation of normal two-dimensional prothallia; under red light, however, the gametophytes grow as cellular filaments. Morphogenesis in blue light is connected with an increase in protein synthesis; in red light the protein content of the sporelings is markedly lower. The size of the chloroplasts is correlated with the protein content of the sporelings.In the present paper the diverse effect of chloramphenicol (CAP) and actidione (cycloheximide, ACT) was studied in connection with the formation of chloroplasts. ACT blocks the growth of the gametophytes, while the chloroplasts remain functional. On the other hand, CAP does not influence morphogenesis of the gametophytes. In particular the activity of the dividing apical cells remains untouched. Even when the light quality is changed during the development the corresponding specific effect of the light quality on morphogenesis is normal. The chloroplasts, however, become smaller, probably by inhibition of synthesis of structural proteins. But their synthetic activity is not completely suppressed. The specific blue or red light dependent morphogenesis is not changed, when protein synthesis in the chloroplasts is inhibited.     

Thylakoid-bound proteolytic activity against LHC II apoprotein in bean

Triton X-100 solubilized thylakoids, isolated from Phaseolus vulgaris chloroplasts, degrade endogenous or exogenously added LHC II. The degradation, as monitored by immunodetection of the remaining LHC II after incubation at 37°C, is activated by Mg⁺⁺ and inhibited by pCMB, EDTA, PMSF and benzamidine; the activity under high light conditions parallels chlorophyll photooxidation. The thylakoid-bound proteolytic activity is under phytochrome control. Etiolated plants pretreated by a white light pulse, and kept in the dark thereafter, show enhanced proteolytic activity, which follows rhythmical oscillations. On the other hand, chloramphenicol pretreatment of etiolated plants, prior to their transfer to continuous light, reduces the proteolytic activity against LHC II. The results suggest that the degradation involves a serine type protease, which depends on SH group(s), coded by the plastid genome; the protease action on LHC II is regulated by Mg⁺⁺, phytochrome, the biological clock and chlorophyll accumulation in the thylakoid. The stroma lamellar fraction, separated from French press disrupted chloroplasts, exhibits higher activity towards exogenous LHC II than the grana fraction. The stroma of intact chloroplasts exhibits also high proteolytic activity, which is drastically reduced when the lysis medium is supplemented with cations. This suggests that the protease is bound mainly on stroma lamellae and peripheral granal membranes, its association to the membranes being possibly under cation control.Abbreviations CAP chloramphenicol - CL continuous light - LHC II light harvesting complex of Photosystem II     

Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Photoregulation of L-phenylalanine ammia-lyase: an immunochemical approach

The photoregulation of L-Phenylalanine ammonia-lyase (PAL) is studied by immunochemical methods. We used a partly purified L-Phenylalanine ammonia-lyase: F1 light fraction, the corresponding inactive one provided from dark-grown cotyledons: F1 dark fraction and the antisera specific of these two fractions. The complete absorption of PAL activity from F1 light fraction with the anti-F1 light immune serum shows the antigenicity of PAL and the specificity of this serum for all forms of PAL present in F1 light fraction. The presence of an inactive L-Phenylalanine ammonia-lyase in the 36 h dark-grown cotyledons suggested by preliminary results of absorption is conformed by showing that less PAL activity is precipitated from the fraction F1 light by a same amount of IgG anti-F1 light when F1 dark fraction is added. This result is explained by a competition between active and inactive forms of PAL for the IgG extracted from an immune serum specific for F1 light fraction. By measuring the absorption of PAL activity from FO fraction (crude extract) obtained from 18 h, 36 h and 48 h light-grown cotyledons when increasing amounts of IgG anti-F1 36 h light are added, we demonstrate the presence of at least two isozymes A and B, the synthesis of B being shifted in time in comparison to A.     

Regulation of phenylalanine ammonia-lyase activity and growth in lettuce by light and gibberellic acid

Abstract The effects of light and gibberellic acid (GA3) on growth and phenylalanine ammonia-lyase (PAL) activity were studied in seedlings of lettuce (Lactuca sativa L.). Using an in vivo assay for PAL it was shown that wounding caused by excising hypocotyls results in an increase in PAL activity with time that can mask the effect of light on the activity of this enzyme. When hypocotyl sections were excised from light-treated seedlings immediately prior to the in vivo assay of PAL, light was shown to cause a marked increase in PAL activity. Experiments with an inhibitor of PAL activity, α-aminooxy-β-phenylpropionic acid (AOPP), confirmed that the volatile radioactive products measured in the in vivo assay resulted from the activity of PAL. Gibberellic acid suppresses the light-induced increase in PAL activity and there is an inverse relationship between GA₃-induced growth and the activity of PAL. Over a wide range of GA₃ concentrations, the activity of PAL is also inversely correlated with growth rate along the length of the hypocotyl section; the upper halves of sections elongate more rapidly and have lower levels of PAL than the lower halves. Despite the strong correlation between growth and PAL activity, experiments with AOPP and t-cinnamic acid show that it is unlikely that elongation is regulated directly by products of PAL activity.     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.     

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.     

Induction of Phenylalanine Ammonia-lyase in Strawberry Leaf Disks: Action Spectra and Effects of Wounding, Sucrose, and Light

The increase in phenylalanine ammonia-lyase (PAL) activity in strawberry (Fragaria vesca var. WSU-1232) leaf disks required wounding, sucrose, and light and was cycloheximide-sensitive. In injured leaves and in leaf disks, the highest PAL activity was detected nearest the wounded tissues. Without wounding, no increase in activity was observed when leaves were cultured in sucrose and light.     

Chloroplast Response to Low Leaf Water Potentials: II. Role of Osmotic Potential

Electron transport in chloroplasts isolated from desiccated sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves was compared with electron transport in sunflower chloroplasts in sorbitol-containing media having various osmotic potentials. In media having low osmotic potentials and dichloroindophenol as electron acceptor, the activity for electron transport was inhibited, but the inhibition was much less than that due to comparable desiccation in vivo. The inhibition at low osmotic potentials was rapidly reversed by returning the chloroplasts to media having high osmotic potentials, but the activity of chloroplasts from desiccated tissue showed no reversal when the chloroplasts were placed in media having high osmotic potentials. Nevertheless, the inhibition of chloroplast activity due to desiccation in vivo was basically reversible, because chloroplasts recovered quickly when they were rehydrated in vivo. The large differences between desiccation in vivo and exposure to low osmotic potential in vivo indicate that osmotic solutions did not reproduce the effects of tissue desiccation. It is concluded that decreases in the Gibbs free energy of water due to decreased osmotic potentials probably have only a small effect on electron transport in chloroplasts from desiccated tissue and do not account for the major effects of leaf desiccation on electron transport.