Stimulation of Malate Synthesis by Glycolate in Tomato Leaves in Relation to CO2 Concentration

The mechanism by which malate synthesis from CO2 is increasedunder low concentrations of CO₂ was investigated in C₃ plants.A number of metabolites were administered to illuminated tomatoleaves, and their effects on the incorporation of ¹⁴CO₂ intomalate were determined. Compared with water as a control, glycolate,glyoxylate, D,L-glycerate, glycine, phosphoglycolate and L-serineincreased malate synthesis by factors of 6.8, 3.8, 3.3, 2.5,2.3 and 2.2, respectively. The effect of exogenous glycolateon malate synthesis from CO₂ was dependent on its concentrationup to 100 mu, but was independent of ambient CO₂ concentration.The feeding of l-¹⁴C-glycolate in the light indicated that glycolatestimulated the carbon flow from CO₂ to malate. The analysis of the products of ¹⁴CO₂ fixation in illuminatedleaves supplied with glycolate showed increases in malate andsugar and decreases in serine and phosphate esters. However,this stimulated malate synthesis ceased when malonate was suppliedsimultaneously with glycolate. Treatment with glycolate didnot affect the dark ¹⁴CO₂-fixation, but increased the ¹⁴C-malatesynthesis, with a corresponding decrease in ¹⁴C-aspartate and14^C-glutamate. These results suggest that exogenous glycolateactivates malate dehydrogenase in leaves, and that the increasedglycolate formation at low CO₂ concentrations is associatedwith the increased malate synthesis from CO₂. (Received January 12, 1981; Accepted May 20, 1981)     

Organic Acid Metabolism in Cellular Organelles of Vigna cylindrica (Mitorisasage) Cotyledons

In starchy cotyledons of Vigna cylindrica (L.) Skeels (Mitorisasage)during seed germination, the enzymes of the glyoxylate cyclewere located in the matrix of mitochondria. Glyoxysomes wereabsent. The glyoxylate cycle in the mitochondria supplies organicacids to the tricarboxylic acid cycle. In mitochondria, isocitratelyase activity was much higher than malate synthase activity.Part of the glyoxylate thus produced in mitochondria may benonenzymatically converted to formate by H₂O₂ and the formatethen converted to CO₂ by peroxidase or by formic dehydrogenase.The activity of superoxide dismutase, which supplies H₂O₂, washigher in mitochondria than in peroxisomes. The remaining glyoxylatein mitochondria is possibly converted to glycine by alanine-glyoxylateaminotransferase or transported to peroxisomes which lackedisocitrate lyase activity but had high malate synthase activity.In peroxisomes, glyoxylate may be also produced from urate,as is suggested by the fairly high activities of uricase, allantoinaseand allantoicase. Judging from the enzyme distribution, Vignaperoxisomes should be capable of producing malate, oxalacetate,citrate, isocitrate and a-ketoglutarate. ¹Present address: Department of Horticulture, College of Agricultureand Animal Science, Yeugnam University, Gyeongsan 632, Korea. (Received May 27, 1987; Accepted October 7, 1987)     

Photosynthetic Changes in the Inducible CAM Plant Sedum telephium L. Following the Imposition of Water Stress. 1. General Characteristics

The effects of water stress (drought) on the pattern of photosynthesisin Sedum telephium have been determined. Well-watered plantsexhibit a weak-CAM pattern, with substantial CO₂ fixation inthe day, a low level of CO₂ fixation at night, high daytimestomatal conductance with a lower conductance at night, andno diurnal fluctuation in acid content. Imposition of water-stress causes a switch from weak-CAM toa full-CAM mode of photosynthesis, as indicated by cessationof daytime CO₂ fixation, a marked increase in night-time CO₂fixation, very low daytime stomatal conductance, increased night-timeconductance and significant diurnal fluctuations in acid content. Sedum telephium, CAM, CO₂ fixation, drought, malate, photosynthesis, water stress     

PHOTOSYNTHESIS OF GLYCINE AND SERINE IN GREEN PLANTS

1. The effects of "carbonyl" reagents on the photosyntheticin-corporation of ¹⁴CO₂ into the assimilation products of tobaccoand spinach leaves were studied. The presence of "carbonyl"reagents causes an increase in the ratio of ¹⁴CO₂ incorporatedin glycine and a decrease in serine. The incorporation of ¹⁴Cfrom glycolate-1-¹⁴C and glycolaldehyde-2-¹⁴C into glycine andserine was also affected by "carbonyl" reagents, as in the caseof ¹⁴CO₂-experiment. 2. The feeding experiments of glycine-1-¹⁴C and serine-1-¹⁴Cin the presence and in the absence of "carbonyl" reagents revealedthat these reagents inhibit the conversion of glycine to serine. 3. The results obtained above, together with the effects ofthiols on ¹⁴CO₂ incorporation presented in this paper, supportthe assumption that glycine and serine are formed via glycolateand glyoxylate during photosynthesis in green plants. 4. Comparison of ¹⁴C incorporation in malate from ¹⁴CO₂, glycolate-1-¹⁴C,glycine-1-¹⁴C and serine-1-¹⁴C in the presence and in the absenceof "carbonyl" reagents suggested the occurrence of the pathwayof the malate formation via glycolate and glyoxylate, not passingthrough glycine and serine, during photosynthesis. ¹ A part of this paper was presented at the Symposium on "Nitrogenand Plant" by the Japanese Society of Plant Physiologists, inOctober, 1963 ² Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka     

14CO2 Pulse-Chase Labelling in Clusia minor L.

Conditions and maintenance of growth were chosen so that plantsof Clusia minor L. were obtained which showed the C₃- and CAM-modes of CO₂-exchange, respectively. C. minor is known to accumulateconsiderable amounts of citric acid in addition to malic acidduring the dark-phase of CAM. ¹⁴CO₂-pulse-chase experiments were performed with these plants.Patterns of labelling during the pulse and redistribution oflabel during the chase in the C₃-mode were as expected for C₃-photosynthesis.Pulse-labelling in the CAM-mode during the last hour of thelight period, during the first part of the dark period and duringthe last hour of the dark period always led to an almost exclusiveincorporation of label into malate. Redistribution of labelfrom malate after the pulse at the end of the dark period duringthe chase in the subsequent light period followed the patternexpected for light-dependent reassimilation of CO₂ remobilizedfrom malate in CAM during the light period. During the chasesin the dark period, label was transferred from ^l4C-malate tocitrate. This suggests that during accumulation of citric acidin the dark period of CAM in C. minor, citrate is synthesizedin the mitochondria from malate or oxaloacetate after formationof malate via phosphoenolpyruvate carboxylase. The experiment also showed that no labelled compounds are exportedfrom leaves in the CAM-mode during the dark period. In plantsof the C₃-mode the roots proved to be strong sinks. Key words: Clusia minor, labelling, pulse-chase, ¹⁴CO₂     

The Organic Acid Metabolism of Bramley's Seedling Apple Peel1

1. The effect of various Krebs cycle acids on the respirationofdisks of apple peel at various stages of maturity was measuredin a Warburg respirometer.
2. Peel tissue from apples at thepre-climacteric and early post-climactericstages apparentlycontain sufficient of the Krebs cycle acidsused, with the exceptionof succinate, to maintain oxidativeprocesses at a maximum.
3. The addition of malate causes a large increase in the CO₂-outputof peel from post-climacteric and senescent fruit but not frompre-climacteric fruit, and a close correlation exists betweenthe climacteric and this decarboxylation of malate. The decarboxylationof malate does not affect the rate of O₂-uptake of peel tissue.The possible part played by the decarboxylation of malate inthe increased CO₂-output at the climacteric is discussed.
4. Addedpyruvate is decarboxylated by the tissue at all stagesof storagelife.
5. The decarboxylation of added malate is an aerobic fermentation,resulting in the quantitative production of acetaldehyde. Althoughthe presence of oxygen is necessary, the rate of O₂-uptake isnot affected by the reaction. Pyruvate decar boxylation doesnot require the presence of oxygen.
6. The O₂-uptake of peelfrom senescent apples can be stimulatedby addition of malate,succinate, and a-ketoglutarate. No evidencewas obtained, however,of oxidation of fumarate, citrate, orpyruvate. The additionof malate to senescent tissue restoresthe lower endogenousrate of O₂-uptake to that of early postclimacteric tissue.
7. Succinate and fumarate are toxic to peel tissue at concentrationabove 0.02M.

     

Antagonism Between Malate and Ethylene in the Regulation of Avena sativa Coleoptile Elongation

Etiolated Avena sativa L. coleoptile sections were used to determinethe influence of C₂H₄ on in vivo and in vitro rates of CO₂ fixation,and to measure the influence of various permutations of C₂H₄,CO₂, and malate on growth. Whereas 1 mM malate or 320 µI^-1 CO₂ stimulated growth by approximately 100 per cent, inhibitionof growth by 10-8 µ I_-1 C₂H₄ was substantial only in thepresence of malate or CO₂ The increase in growth rate in responseto these two agents was eliminated by the simultaneous applicationof C₂H₄. The in vivo rate of dark [¹⁴C]bicarbonate fixationand in vitro enzymic assays of fixation were not measurablyinhibited by C₂H₄. These results are discussed in the lightof evidence which indicates that CO₂-stimulated growth is mediatedby dark fixation. The data do not support the view that C₂H₄inhibition of growth results from an inhibition of fixation,but suggests that C₂H₄ may inhibit some step in the processby which malate stimulates growth.     

The Effect of Subjecting Peas to Air Enriched with Carbon Dioxide: II. RESPIRATION AND THE METABOLISM OF THE MAJOR ACIDS

Whole peas at about 75 per cent of their maximum fresh weightwere subjected to 5–30 per cent CO₂ in air for periodsof from 1–6 d, then returned to air for a further 1 d.Samples were withdrawn at intervals and organic acids, ^TCO₂and ethanol estimated as well as the rate of respiration. Slicesof cotyledons suspended in water were also subjected to highconcentrations of CO₂ in air for 3 h. The rate of respiration was inhibited progressively by increasein CO₂ content of the tissue. The high internal CO₂ contentof the intact pea causes an inhibition of its rate of respirationby about 25 per cent. Alcohol production commenced at between10 and 15 per cent CO₂ in the ambient gas and slowly increasedin rate up to 37 per cent. The CO₂-air mixtures reduced the content of malate, pyruvateand -oxoglutarate, increased that of succinate and left citrateunaffected. On return to air malate rose rapidly and succinatefell slowly to their original concentrations. During the sameperiod the concentration of PEP fell sharply and after about1 h rose again, whereas oxalacetate showed a reverse response.It is argued that the rapid re-synthesis of malate was by carboxylationof PEP to oxalacetate and that this reaction was stimulatedby a change in pH rather than by the direct effect of the changein concentration of CO₂. In one experiment ¹⁴CO₂ was supplied for 2 h before return toair and the movement of ¹⁴C followed for 6 h. The results supportthe method of re-synthesis of malate proposed.     

Differential Stimulation of PEP-carboxylation in Guard Cells and Mesophyll Cells by Ammonium or Fusicoccin

Dark CO₂-fixation in guard cells of Vicia faba was much moresensitive to ammonium than in mesophyll cells. Addition of ammonium(5.0 mol m^–3; pH₀ 7.6) caused up to a 7-fold increasein dark CO₂-fixation rates in guard cell protoplasts (GCP),whereas in leaf slices, mesophyll cells, and mesophyll protoplaststhe increase was only about 1.4-fold. In both cell or tissuetypes, total CO₂-fixation rates were higher in the light (2–12-foldhigher in GCP and 28-fold in mesophyll); these rates were onlyslightly changed by ammonium treatment. However, separationof ¹⁴C-labelled products after fixation of CO₂ in the lightby GCP revealed a large ammonium-induced shift in carbon flowfrom starch and sugars to typical products of C₄-metabolism(mainly malate and aspartate). In contrast, in mesophyll cellsamino acid and malate labelling was only moderately increasedby ammonium at the expense of sucrose. The data suggest thatin vivo ammonium might facilitate stomatal opening and/or delaystomatal closing through an increased production of organicacids. Key words: PEP-carboxylation, guard cell protoplasts, ammonium, fusicoccin     

Mitochondrial Changes in Harvested Carnation Flowers (Dianthus caryophyllus L.) during Senescence

Harvested carnation (Dianthus caryophyllus L.) flowers wereplaced in either a preservative solution or deionized waterand monitored through senescence during which time flower freshweight was measured as well as production of ethylene and CO₂.Flower fresh weight, ethylene, and CO₂ levels increased as theflowers aged, but fresh weight and CO₂ levels fell once flowersbegan to senesce regardless of holding solution. Preservative-treatedflowers senesced at a slower rate than deionized water-treatedflowers. The amount of ADP phosphorylated to ATP per oxygenatom consumed, using mitochondria isolated from petal tissueprovided with either succinate or malate as substrates, wasfound to increase as flowers senesced and then to decrease inthe later stages of senescence. Respiratory control ratios withsuccinate as the substrate did not change appreciably untilthe final stages of senescence white respiratory control valuesusing malate showed greater variation but no consistent patternrelative to the progress of senescence. Cyanide-resistant respirationwas noted with isolated mitochondria oxidizing either substrate,but no correlation between cyanide-resistant respiration andsenescence could be found. (Received July 10, 1984; Accepted April 16, 1985)     

The Influence of Nitrogen, Light and Water Stress on CO2 Exchange and Organic Acid Accumulation in the Tropical C3-CAM Tree, Clusia minor

The carbon balance and changes in leaf structure in Clusia minorL., were investigated in controlled conditions with regardto nitrogen supply and responses to low and high photosyntheticallyactive radiation (PAR). Nitrogen deficiency and high PAR ledto the production of smaller leaves with higher specific leafdry weight (SLDW) and higher leaf water content, but with lowerchlorophyll content. Nitrogen and PAR levels at growth alsoaffected CO₂ exchange and leaf area. In – N conditions,total daily net CO₂ uptake and leaf area accumulation were slightlyless for high-PAR-grown plants. In contrast, high-PAR-grownplants supplied with nitrogen showed about a 4-fold higher totaldaily CO₂ uptake and about twice the total leaf area of low-PAR-grownplants. Although total daily net CO₂ uptake of +N plants wasonly slightly higher than –N plants under the low PARlevel, –N plants produced almost three times more leafarea but with lower SLDW. Under well-watered conditions, low-PAR-grownplants showed only CO₂ evolution during the night and malicacid levels decreased. However, there was considerable night-timeaccumulation of titratable protons due to day/night changesin citric acid levels. High-PAR-grown plants showed net CO₂uptake, malate and citrate accumulation during the dark period.However, most of the CO₂ fixed at night probably came from respiratoryCO₂. Positive night-time CO₂ exchange was readily observed forlow-PAR-grown plants when they were transferred to high PARconditions or when they were submitted to water stress. In plantsgrown in high and low PAR, CAM leads to a substantial increasein daily water use efficiency for water-stressed plants, althoughtotal net CO₂ uptake decreased.     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Diurnal changes in malate-decarboxylation activity in the leaves of a CAM plant, Bryophyllum daigremontianum

Bryophyllum diagremontianum plants grown under light-dark regimeswere exposed to one more cycle of the regime or to continuousdarkness for 24 hr. Photosynthetic O₂ evolution by leaf segmentsfrom these plants was investigated in the presence of 15 mMNaHCO₃ (CO₂-dependent O₂ evolution) or in the absence of CO₂(malate-dependent O₂ evolution). The malate-dependent O₂ evolutionserved as an index of the activity of malate decarboxylation.Malate content was respectively 67, 64 and 85 µmoles/g.fwin leaves measured at 7 hr 30 min in light and 6 hr 26 min inthe dark from plants under the light-dark regime (light 12 hr/dark12 hr) and those measured at 6 hr 26 min in the dark from plantsunder the continuous dark regime. The malate- and CO₂-dependentphotosynthetic O₂ evolutions in the same leaves were 9.7 and22, 0.2 and 17, and 16 and 26 µmoles/g.fw.hr, respectively.Thus, the diurnal change in capacity for malate-dependent O₂evolution was relieved by continuous dark treatment. These results suggest that the diurnal change in malate decarboxylationin this crassulacean acid metabolism plant does not occur byan endogenous rhythm. This further indicates lack of an endogenousrhythm for the influx-efflux of malate across the vacuole andin malate decarboxylation enzyme activity. (Received August 1, 1979; )     

INHIBITION OF THE PHOTOSYNTHETIC CARBON DIOXIDE FIXATION OF GREEN PLANTS BY {alpha}-HYDROXYSULFONATES, AND ITS EFFECTS ON THE ASSIMILATION PRODUCTS

1. Several kinds of a-hydroxysulfonates, the bisulfite additioncompounds of aldehydes and ketones, were found to inhibit thephotosynthetic carbon dioxide fixation of the barley and wheatseedlings, tobacco leaf and Chlorella cells. Bisulfite additioncompounds of glyoxal, glyoxylate and benzaldehyde were moreeffective in this respect than those of formaldehyde and acetaldehyde.
2. The presence of -hydroxysulfonate causes an increase in ratiosof :¹⁴CO₂ incorporated in glycolate and alanine, and a decreasein incorporation in serine, malate, isocitrate and citrate.It was inferred that these changes are caused by the blockingof the formation of glyoxylate through inhibition of glycolicacid oxidase by the poison.
3. A reaction scheme was proposedto account for the above-statedresults, and the bearing ofthese findings on the possible roleof glycolic acid oxidasein the photosynthetic carbon dioxidefixation and in the formationof amino and organic acids wasdiscussed.

(Received December 8, 1961; )     

The Damping and Reinitiation of the Circadian Rhythm of CO2 Output in Bryophyllum Leaves in Relation to their Malate Content

The circadian rhythm of CO₂ output in leaves of Bryophyllumfedtschenkoi damps out after 3–4 d in continuous darknessand a CO₂-free air stream at 15°C. The rhythm is reinitiatedafter a single exposure to white light of 2, 4, 6 or 8 h duration,damps out again after a further 3–4 d and can be reinitiatedfor a second time by a further exposure to light. During the exposure to light there is a burst of CO₂ outputconsistent with the decarboxylation of malate, and the rhythmbegins afterwards with an initial high rate of CO₂ fixation.Malate gradually accumulates in the leaves in continuous darknessto attain a maximum value (35 mol m^–3) at the time whenthe circadian rhythm disappears, and decreases to a low value(19 mol m^–3) after a 4 h exposure to light which reinitiatesrhythmicity. These results support the hypothesis that damping of the rhythmof CO₂ output in continuous darkness is due to the accumulationof malate in the leaf cells, eventually reaching such a levelthat its removal from the cytoplasm into the vacuole cannottake place, with the result that PEPc activity, upon which therhythm of CO₂ output depends, remains allosterically inhibited. Key words: CAM, circadian rhythm, Bryophyllum, CO₂-fixation, malate metabolism     

The Effect of Subjecting Peas to Air Enriched with Carbon Dioxide: I. THE PATH OF GASEOUS DIFFUSION, THE CONTENT OF CO2 AND THE BUFFERING OF THE TISSUE

The intake of oxygen by an isolated pea was shown to be entirelythrough the micropyle; the loss of CO₂ was about two-thirdsvia the micropyle and the rest through the cuticle. The internalatmosphere of a pea in air contains about 11 per cent CO₂ v/v. Peas transferred from air to CO₂ in air mixtures required nearly24 h for their internal CO₂ content to come to equilibrium.The increase in internal CO₂ content was found to be balancedby a decrease in the content of malic acid and the pH of thetissue changed little. On return to air the changes were reversed.Slices of cotyledons in water apparently maintained their pHby loss of cations rather than change in malate content.     

Glyoxylate Inhibition of Ribulosebisphosphate Carboxylase/Oxygenase Activation State in vivo

Photosynthetic CO₂ exchange in photorespiration mutants of Arabidopsisthaliana showed a time-dependent inhibition at 350 µl/literCO₂ in 50% O₂ but not in 2% O₂. In a glycolate-P phos-phatasedeficient mutant, inhibition of photosynthesis was due to adepletion of ribulosebisphosphate. In the remaining mutants,which have defects in photorespiratory enzymes which metabolizeamino acids, reduced photosynthesis was accompanied by a declinein the activation level of ribulosebisphosphate carboxylase/oxygenase(Chastain and Ogren 1985), a decline in ribulosebisphosphateconcentration, and an accumulation of glyoxylate. Addition ofglyoxylate at submillimolar concentrations to intact spinach(Spinacea oleracea L.) chloroplasts inhibited light activationof ribulosebisphosphate carboxylase/oxygenase (rubisco) andCO₂ fixation. Similar concentrations of glyoxylate had no effecton A. thaliana rubisco activity in vitro. These results suggestthat glyoxylate accumulation indirectly inhibited rubisco activationstate in vivo. The inhibition of photosynthesis in mutants whichaccumulate glyoxylate may be attributed to a decline in ribulosebisphosphateconcentration, a reduction in rubisco activation state, or acombination of both phenomena. ³Present address: CSIRO, Division of Plant Industry, GPO Box1600, Canberra, ACT 2601, Australia. (Received May 12, 1989; Accepted July 8, 1989)     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

Photosynthesis and Diffusion Conductance of the Valencia Orange Fruit under Field Conditions

CO₂ uptake and diffusion conductance of Valencia orange fruits(Citrus sinensis L. Osbeck) were measured in the field duringthe growing season of 1977/78 to ascertain if, as in the leaf,stomata control photosynthesis and transpiration under changingenvironmental conditions. Measurements were made on 15 yearold trees grown in a sandy loam soil and receiving either adry or a wet treatment. Fruit diffusive conductance was measuredwith a modified water vapour diffusion conductance meter andgross photosynthesis was measured with a ¹⁴CO₂ uptake meter.Photosynthetically active radiation (PAR) was measured witha quantum sensor. Fruits exposed to light assimilated CO₂ ata rate which was 25–50% of that assimilated by leaves.The uptake was dependent on fruit size, PAR, chlorophyll content,and on diffusive conductance of the fruit epidermis. Epidermalconductance showed a diurnal trend which was similar in shapeto that of the leaf except in the late afternoon. Cuticularconductance of the fruit was calculated and ranged between 0.22and 0.30 mm s^–1. It was speculated that the CO₂ uptakeby the fruit could support the growth of flavedo cell layerswhen exposed to light. Dry soil caused an increase in the ¹⁴CO₂uptake by fruit possibly caused by the increased potential areaof the stomatal opening per unit of fruit surface area.     

Levels of endogenous ethylene, carbon dioxide, and soluble pectin, and activities of pectin methylesterase and polygalacturonase in ripening tomato fruits

In 4 cultivars of tomato (Lycopersicon esculentum Mill.), theearly detachment of fruits advanced ripening and considerablyreduced the threshold value of endogenous C₂H₄. This indicatesa supply from the vegetative parts of (a) labile ripening-inhibitingsubstance(s) antagonizing the action of C₂H₄. The endogenous level of CO₂ increased shortly after the risein C₂H₄, and maximum levels of C₂H₄ and CO₂ occurred almostsimultaneously. The activity of PE showed no connection with ripening, but PGactivity did not occur until the onset of ripening. However,this activity increased at considerably higher C₂H₄ concentrationsthan the rise in WSP, and was independent of the possible presenceof ripening inhibitor(s). Hence PG is considered not to be involvedin the primary events leading to fruit ripening. Exposure of fruits to different C₂H₄ concentrations in the ambientatmosphere also showed PG activity to increase only after therise in WSP had started. Other pectin degrading or synthesizingenzymes may be involved. In the non-ripening Rin mutant of cv. Rutgers, no rise occurredin C₂H₄, CO₂, WSP, and PG activity. ¹ Present address: Department of Agricultural Chemistry, Facultyof Agriculture, Kochi University, Otsu 200 Monobe, Nangoku City,Kochi Prefecture 783, Japan. (Received February 16, 1978; )