Ethylene production in rice bronzing leaves induced by ferrous iron

Bronzing, a nutritional disorder of rice plants which is widely distributed in tropical lowlands, was induced by dipping the cut end of rice leaves into FeSO₄ solution (pH 3.5). Ethylene production; the activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase; and the effects of Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, and 1-aminocyclopropane-1-carboxylate, were investigated in the course of bronzing development. It was found that ethylene production could be stimulated up to about 20 times that of the control by Fe²⁺, and a peak could be reached at about 24 h after incubation. The Fe²⁺-treated leaves also had 10-fold higher peroxidase activity than the control, whereas in vitro enzyme activity was inhibited by Fe²⁺. Cycloheximide retarded in vivo stimulation of peroxidase, indicating that in vivo stimulation resulted from inducing de novo synthesis of the enzyme. No changes in the activities of phenylalanine ammonia-lyase and polyphenol oxidase were observed. The results, obtained from the incubation of leaves with Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, or 1-aminocyclopropane-1-carboxylate, showed that ethylene production was the effect of Fe²⁺ stress and that it was not involved in the process of bronzing development, which is probably an acclimation process to enable plants to cope with stress. The accelerated peroxidase activity may be associated with bronzing development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - EFE ethylene forming enzyme - PAL phenylalanine ammonia-lyase - POD peroxidase - PPO polyphenol oxidase - SE standard error     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Plant enzyme synthesis: Hormonal regulation of invertase and peroxidase synthesis in sugar cane

Summary Using sugar-cane internodal tissue in which RNA synthesis was ratelimiting for invertase of peroxidase synthesis, measurements were made of enzymeforming-capacity after blocking further RNA synthesis with actinomycin D or 6-methylpurine. In this way it was possible to determine whether added auxin (naphthaleneacetic acid) or gibberellic acid (GA₃) affected steps prior or subsequent to synthesis of the RNA fractions specifically required for synthesis of either enzyme. Both auxin and GA₃ increased the enzyme-forming-capacity for invertase but not for peroxidase. The effects of the two hormones are interpreted as causing stabilization of mRNA for invertase.Abscisic acid (ABA) increased the rate of synthesis of invertase but not peroxidase. ABA did not change the rate of loss of invertase when peptide-bond formation was blocked with cycloheximide, but stimulated its synthesis when RNA synthesis was blocked with 6-methyl purine. Hence, the site of action of ABA is subsequent to invertase-mRNA formation and prior to invertase destruction.Kinetin had no short-term effects when RNA synthesis was limiting for invertase production, and does not appear to directly modulate mRNA synthesis or stabilization, or amino-acid-polymerization steps. In treatments longer than 5 hours, kinetin inhibited synthesis of all three enzymes studied, so that its effect on enzyme synthesis in this tissue appears to be unspecific.Abbreviations used throughout text ABA (±)-abscisic acid (abscisin II, dormin) - GA₃ gibberellic acid - NAA -naphthaleneacetic acid     

Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction.     

Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants

Application of regurgitant from Leptinotarsa decemlineata Say on wound surfaces of one wounded leaf of intact bean (Phaseolus vulgaris L.) plants resulted in activation of ethylene biosynthesis followed by an increase of both peroxidase and polyphenol oxidase activity. The aim of the present investigation was to study the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants. As the regurgitant contained relative high activities of both peroxidase and polyphenol oxidase, there is a possibility that increased enzyme activities in bean leaves following regurgitant treatment is an artifact of insect-derived enzymes. Localisation experiments and electrophoretic analysis revealed that only part of the increased enzyme activities could be attributed to regurgitant-derived enzymes. Both increase of ethylene production and oxidative enzyme activities depended on protein synthesis. To demonstrate if the increase of oxidative metabolism was ethylene-dependent, seedlings were pretreated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, and 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. Increase of both peroxidase and polyphenol oxidase activity in wounded and subsequently regurgitant-treated leaf was abolished by both aminooxyacetic acid and 1-MCP. Inhibitor studies indicated that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for regurgitant-induced increase of ethylene production and oxidative enzyme activities.     

模拟氮沉降对太岳山油松林土壤酶活性的影响

为研究土壤酶活性对氮沉降增加的响应,以山西太岳山油松人工林和天然林为研究对象,于2009年8月开始实施模拟氮沉实验,试验设置对照(CK,0 kg N hm-2a-1);低氮(LN,50 kg N hm-2a-1);中氮(MN,100 kg N hm-2a-1);高氮(HN,150 kg N hm-2a-1)4种氮处理,自2012年起每年5、7、9月在各处理样方采集表层0—20 cm土壤,测定土壤酶活性(过氧化物酶、多酚氧化酶、纤维素酶、蔗糖酶、脲酶、中性磷酸酶)。研究结果表明:施氮处理下的脲酶与中性磷酸酶活性均有所提高,而低氮处理下天然林中的多酚氧化酶与人工林中的蔗糖酶显著低于对照,中氮、高氮处理下过氧化物酶、多酚氧化酶、天然林中的纤维素酶以及人工林中的蔗糖酶显著降低。总的来说,人工模拟氮沉降促进了土壤中脲酶和中性磷酸酶的活性,抑制了过氧化物酶和多酚氧化酶的活性,并降低了天然林土壤中的纤维素酶活性和人工林中的蔗糖酶活性,但对天然林中蔗糖酶和人工林中的纤维素酶无影响。主导木质素降解的多酚氧化酶活性与纤维素酶、蔗糖酶活性显著相关,纤维素酶与蔗糖酶活性的下降可能是由木质素降解受到抑制,土壤微生物可利用碳源减少所引起。另外,受到天然林土壤含氮量较高的影响,与人工林相比,天然林的多酚氧化酶活性对模拟氮沉降更敏感。由于被抑制的酶均与土壤有机质降解密切相关,氮沉降增加将减缓山西油松林土壤有机质的降解,有利于有机质在土壤中的积累。     

Effect of Gibberellic Acid and Ethylene on Peroxidase in Pea Internodes

Ethylene at 5–80 µl l^–1 inhibited elongationand induced swelling in internodes of light-grown normal anddwarf pea plants; GA₃ did not prevent swelling in response toethylene. GA₃ neither inhibited nor enhanced the activity of isoperoxidasesin the internodes, regardless of its effect on their elongation.Ethylene at 80 µl l^–1 enhanced peroxidase in GA₃-untreatedand treated normal and dwarf plants. At 5 µl l^–1,ethylene had only a weak effect on peroxidase activity or none.The enzyme enhancement by ethylene was not related to its effecton cell expansion and seems do be due, at least in part, tochemical injury. Electron microscopy revealed peroxidase activity in the roughER and cell walls, including intercellular spaces. Stainingof walls in ethylene-treated tissues was more pronounced thanin untreated ones. Golgi vesicles did not seem to be involvedin the assembly of the enzyme carbohydrate moiety in ethylene-treatedcells. The peroxidase fraction extracted with 20 mM phosphate buffer,pH 6, and that extracted from wall debris with 1 M NaCl accountedfor 98% of total enzyme activity. Both fractions contained thesame six cathodic isoforms which comprised 85–90% of theiractivity. Electrophoresis did not reveal differences in thequalitative isoenzyme patterns in relation to variety, age,GA₃, or ethylene. The only observed quantitative differenceswere age-dependent. Procedural artefacts during separation of protoplast and wallionically bound peroxidase fractions are discussed.     

三峡库区不同林龄马尾松土壤养分与酶活性的关系

基于对三峡库区不同林龄马尾松林下土壤养分和酶活性的测定及典范对应分析,探讨了不同林龄马尾松林土壤养分、酶活性特征及其相互关系.结果表明:马尾松林0 ～ 20 cm土壤有机质、总氮、铵态氮和有效磷含量均表现出成熟林＞中龄林＞近熟林;随着林龄的增加,土壤转化酶活性先降低后增加,纤维素酶、多酚氧化酶活性逐渐降低,而脲酶和过氧化物酶活性先增加后降低.经典范对应分析,不同林分中主要土壤养分对酶活性的影响顺序为总氮＞有机质＞pH＞容重＞铵态氮＞有效磷,转化酶与土壤有机质、总氮、总磷呈显著正相关,过氧化物酶与有机质、总氮、总磷、容重呈显著负相关,土壤主要养分含量高,转化酶活性较高,过氧化物酶活性相对较低.转化酶、纤维素酶和过氧化物酶是评价土壤质量及肥力较好的生物学指标.     

咖啡酸和氯化钴对茉莉酸甲酯诱导抗病相关酶活性的影响

烟草愈伤组织在含咖啡酸（5 mmol/L）和/或CoCl2(10 mmol/L,乙烯合成抑制剂)的MS培养基上暗培养,同时用茉莉酸甲酯（1 mg/ml,简称MJ）处理愈伤组织。处理后测定乙烯、水杨酸和病程相关蛋白（PR）含量及一些抗病相关酶活性。MJ明显促进乙烯产生、增加水杨酸和PR蛋白含量,提高苯丙氨酸解氨酶（PAL）、β1,3-葡聚糖苷酶和几丁酶的活性。咖啡酸降低MJ对乙烯和水杨酸诱导,CoCl2明显降低MJ对乙烯的诱导,但没有明显影响MJ对水杨酸的诱导,两者都促进MJ诱导PAL活性而抑制MJ诱导β1,3-葡聚糖苷酶活性。咖啡酸明显影响MJ诱导内切几丁酶,几乎完全抑制对外切几丁酶的诱导;CoCl2对MJ诱导内切几丁酶没有影响,促进对外切几丁酶的诱导。实验结果表明,不同的抗病相关酶活性诱导有不同的信号传递途径,在所测几种酶的诱导中,水杨酸起主要作用,乙烯作用较小,MJ的诱导作用主要是由水杨酸所转导。     

Changes in the forms of invertase during germination of mung bean seeds

High levels of ‘alkaline’ invertase activity occur in dormant mung bean seeds. During germination this activity decreases rapidly and is replaced by high ‘acid’ invertase activity. Cycloheximide prevented the formation of the latter activity and also inhibited germination. It is suggested that de novo synthesis of ‘acid’ invertase occurs during germination. Both enzymes bind to concanavalin A and, hence, are presumed to be glycoproteins. Affinity-purified enzyme samples show similar ratios of ‘acid’ and ‘alkaline’ invertase activities to the crude preparations indicating that specific enzyme inhibitors or activators are probably not involved in controlling the activities in vivo.     

Protein Dysfunction and Inhibition of Amino Acid Incorporation in Root Segments from Wheat and Mung Bean Seedlings caused by some Amino Acid Analogues

The effects of para-fluorophenylalanine (p-FPA) and azetidine-2-carboxylicacid (AZ) on acid phosphatase and peroxidase activity, and onL-leucine incorporation, in root segments of Triticum aestivumL. cv. Fenman and Vigna radial a (L.) Wilczek, were studied.Incubation with 50 mmol^–3 AZ significantly reduced phosphataseand peroxidase activities in wheat roots, but with 20 mol m^–3p-FPA, only the peroxidase activity was reduced. In mung beanroots, phosphatase activity was inhibited by both AZ and p-FPA.Effects of the ortho- and meta- isomers of FPA on wheat rootphosphatase and peroxidase, and on mung bean phosphatase, werealso examined. Leucine uptake and incorporation were not inhibitedby 5 h pre-incubation with either p-FPA or AZ, but were inhibitedafter 24 h of pre-incubation. The results support the view that,in the shorter term, the analogues inhibit enzyme activity bybecoming incorporated to produce non-functional protein and,in the longer term, metabolism is further affected by inhibitionof protein synthesis. Key words: Fluorophenylalanin, Azetidine-2-carboxylic acid, Triticum aestivum, Vigna radiata     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Effect of auxin and other hormones on the metabolic response to wounding in sweet potato roots

In roots of sweet potato (Ipomoea batatas Lam. cv. Kokei 14),the metabolic response to wounding was remarkable only in theproximal side. We assumed that the polarity resulted from apolar movement of indole-3-acetic acid (IAA) produced in thecut surface (8). As the metabolic response was slight in thedistal side, the effect of IAA and the other plant hormoneson the development of various enzyme activities was examinedin this side. Increases in activities of L-phenylalanine ammonia-lyase,acid invertase, NADPHa₂ : cytochrome c oxidoreductase, peroxidase,cytochrome c : O₂ oxidoreductase and o-diphenol oxidase, whichdeveloped in response to wounding, were stimulated by the treatmentwith IAA. Gibberellic acid had a stimulative effect on the developmentof only acid invertase activity. Abscisic acid and kinetin hadlittle effect. The results strongly support our hypothesis thatIAA plays an important role in the metabolic response to wounding. (Received September 29, 1979; )     

Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess

Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed.     

Rapid Ethylene Production in Soybean in Response to the Cupric Ion

Very rapid accumulation of free 1-aminocyclopropane-1-carboxylicacid (ACC), followed by stimulation of ethylene production wereinduced by a Cu²⁺ in soybean cuttings. The accumulation mustbe attributed to an increase in ACC synthesis, because: (1)it was completely inhibited by aminoethoxyvinylglycine (AVG);and (2) the ethylene stimulation was inhibited by AVG, indicatingthat free ACC cannot be released from its conjugated form. Theactivity of the ethylene-forming enzyme slightly decreased followingthe Cu²⁺ pulse, and this event was accompanied by a slight increasein electrolyte leakage from the treated soybean tissues. Glycine max L., soybean, ethylene, cupric ion     

模拟氮沉降增加条件下土壤团聚体对酶活性的影响

氮沉降增加改变了森林土壤生态系统物质输入,影响土壤生物及酶活性,而土壤团聚体内相对稳定的微域生境可能减弱或延缓土壤生物和酶对氮沉降增加的响应强度。以广东省东莞大岭山森林公园荷木人工林为研究对象,用模拟N沉降方法,分析了2011年12月到2012年11月一年内氮沉降增加条件下表层混合土壤和土壤团聚体内脲酶、蔗糖酶和酸性磷酸酶活性的变化及影响因素,旨在理解氮沉降增加条件下土壤团聚体对酶活性的影响。结果表明:氮沉降增加对表层混合土壤中脲酶和蔗糖酶的抑制作用不显著,而酸性磷酸酶受氮沉降显著影响,表现为低氮(50 kg N hm-2a-1)促进,高氮(300 kg N hm-2a-1)抑制的规律。表层土壤团聚体内脲酶活性随氮沉降增加而降低,N300处理显著低于对照;蔗糖酶和酸性磷酸酶活性随氮沉降增加先降低后增加,N100处理最低,分别比其他处理降低了6.46%—25.53%和42.33%—68.25%。试验区内各粒径土壤团聚体内酶活性高于混合土壤,且随团聚体粒径增加酶活性均为先增加后降低。不同粒径土壤团聚体的3种酶活性均以2—5 mm最高,但脲酶、酸性磷酸酶在各团聚体粒径间差异不显著,蔗糖酶活性2—5 mm显著高于5—8 mm。土壤酶相对活性指数和相对活性综合指数结果显示,超过85%的团聚体粒径内的相对酶活性指数大于1,而土壤酶相对活性综合指数均大于1。以上结果表明,氮沉降增加条件下土壤团聚体对其团聚体内的土壤酶活性有隔离保护作用,但其隔离保护效果与酶的种类和土壤团聚体粒径有关。     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.     

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO₂, 6% O₂, and 88% N₂ for 14 weeks at 12°C, exhibited a temporal separation of certain biochemical events associated with ripening.

The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.

When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.

To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22°C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.