Seasonal Changes in Activities of Enzymes Responsible for the Formation of C6-aldehydes and C6-alcohols in Tea Leaves, and the Effects of Environmental Temperatures on the Enzyme Activities

When tea leaves were homogenized and incubated, the volatileC₆-compounds hexanal, cis-3-hexenal, cis-3-hexenol and trans-2-hexenalwere formed much more by summer leaves than by winter leavesof tea plants (Camellia sinensis). The enzymes lipolytic acylhydrolase (LAH), lipoxygenase, fatty acid hydroperoxide lyase(HPO lyase) and alcohol dehydrogenase (ADH) and an isomerizationfactor were responsible for the sequential reactions of C₆-compoundformation from linoleic and linolenic acids in tea leaf lipids,and there were seasonal changes in their activities. The tealeaf enzymes were of 3 types: LAH and lipoxygenase, which hadhigh activities in summer leaves and low activities in winterleaves; ADH, which had low activity in summer leaves and highactivity in winter ones; and HPO lyase and the isomerizationfactor, which did not seem to have any effect on the rate ofC₆-compound formation throughout the year. Changes in enzymeactivities were induced by shifts in the environmental air temperaturerather than by the age of the leaves. The combined activitiesof these enzymes determined the amounts and compositions ofthe volatile C₆-compounds formed, which are the factors thatcontrol the quality of the raw leaves processed for green tea. (Received October 6, 1983; Accepted December 20, 1983)     

Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

Effects of Anoxia on Phloem Loading in C3 and C4 Species

Changes in phloem loading rate were inferred from observationsof ¹¹C export from attached leaves of C₃ and C₄ monocots anddicots. Loading decreased under anoxia in C₃ leaves, but notin general in the leaves of either C₄ monocots or dicots inthe light. However, loading rate in the C₄ leaves did reduceif the leaf was also darkened or received no CO₂. We suggestthat insensitivity to anoxia in C₄ leaves is due to oxygen fromphotosynthesis which is retained in the bundle sheath at a concentrationsufficient to energize a phloem loading system. There may alsobe another system which is insensitive to anoxia since the effectsof shade and CO₂ deprivation were not always seen, and loadingwas not completely stopped by these treatments. Key words: Phloem loading, C₃, C₄     

Studies in the Respiratory and Carbohydrate Metabolism of Plant Tissues1: XVII. THE EFFECT OF IODOACETATE ON THE CO2 OUTPUT, PATHWAYS OF GLUCOSE RESPIRATION AND CONTENTS OF PHOSPHATE ESTERS IN STRAWBERRY LEAVES

When specifically labelled glucose was fed to strawberry leaves,the C₆/C₁, quotient (rate of release of ¹⁴CO₂ from glucose-6-¹⁴C/rateof release of ¹⁴CO₂ from glucose-1¹⁴C ranged from 0.27 to 0.35in leaves in water and from 0.46 to 0.96 in leaves fed withiodoacetate. These quotients indicate that both the glycolyticand the pentose phosphate pathways participate in the respirationof strawberry leaves, with a greater contribution from the formerin the iodoacetate increased CO₂ output. Concurrently with the increase of CO² output in iodoacetate,the contents of glucose-6-phosphate (G6P), fructose-6 (F6P)and fructose-1,6-diphosphate (FDP) increased greatly; therewas a smaller increase of phosphoenol-pyruvate (PEP). The increasein the CO₂ output in iodoacetate may be explained solely onthe basis that the increases of G6P and FDP accelerate the ratesrespectively of the pentose phosphate pathway and of glycolysisand traffic into the tricarboxylic acid cycle. The increasein content of G6P and FDP is attributed to an increase in theaccessibility of enzymes and substrates caused by iodoacetate.Alternatively the increased CO₂ output in iodoacetate may bepartly due to uncoupling of oxidative phosphorylation.     

Lipoxygenase, Hydroperoxide Lyase and Volatile C6-Aldehyde Formation from C18-Fatty Acids during Development of Phaseolus vulgaris L.

Kidney bean plants (Phaseolus vulgaris) were found to have thecapability to produce C₆-aldehydes (hexanal and hexenals) fromlinoleic and linolenic acids. The various organs tested hadlipoxygenase and hydroperoxide lyase activities responsiblefor the C₆-aldehyde formation. Young leaves showed relativelyhigh activities for C₆-aldehyde formation. However, the activitiesof the leaves decreased gradually with leaf expansion. Seedlingsand seeds containing cotyledons showed low activities for C₆-aldehydeformation, because of the occurrence of an inhibitory factorin the cotyledons. The substrate specificity of the enzymeswas essentially the same among the various developmental stagesof leaves tested. (Received February 5, 1982; Accepted March 19, 1982)     

Immunologically Distinct Forms of Adenylate Kinase in Leaves: Comparison of Subunit Size of Adenylate Kinase from C3 and C4 Plants

Kleczkowski, L. A. and Randall, D. D. 1987. Immunologicallydistinct forms of adenylate kinase in leaves: comparison ofsubunit size of adenylate kinase from C₃ and C₄ plants.—J.exp. Bot. 38: 1440–1445. Antibodies prepared against maize leaf adenylate kinase (E.C.2.7.4.3 [EC] ) cross-reacted with the enzyme isolated from leavesof both C₃ and C₄ plants. The immunoreaction was very specificas judged by the presence of a single band on Western immunoblotscontaining proteins from leaf extracts of several species. Themolecular weight (M₁) of adenylate kinase determined by meansof the immunoblotting was 29 kD and 27 kD for C₄ and C₃ species,respectively. For both C₃ and C₄ plants, the antibodies failedto precipitate all adenylate kinase activity in leaf extracts,while they were 100% effective in pelleting the enzyme frommaize mesophyll chloroplasts. This indicated the presence ofat least two immunologically distinct forms of adenylate kinasein leaves. It is suggested that the observed differences in molecular structure(M₁s) of adenylate kinase from C₃ and C₄ species might be responsiblefor distinct catalytic and functional properties of the enzymein these two groups of plants. The irrununologically-determinedoccurrence of distinct pools of adenylate kinase in leaves supportsprevious evidence obtained by means of subcellular fractionationstudies.     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Isoforms of NADP-Malic Enzyme from Mesembryanthemum crystallinum L. That are Involved in C3 Photosynthesis and Crassulacean Acid Metabolism

Exposure of the facultative halophyte Mesembryanthemum crystallinumL. to salt stress induces a shift from C₃ photosynthesis toCrassulacean acid metabolism (CAM). During induction of CAM,the activity of NADP-malic enzyme (EC 1.1.1.40 [EC] ) increased asmuch as 12-fold in leaves, while the enzymatic activity in rootsfell to half of the original level. These changes in the activityof the enzyme corresponded to changes in levels of the enzymeprotein. NADP-malic enzymes extracted from leaves in the C₃and CAM modes could be distinguished by differences in electrophoreticmobility during electrophoresis on a non-denaturing polyacrylamidegel. NADP-malic enzyme extracted from roots in the C₃-mode andin the CAM mode migrated as fast as the enzyme extracted fromleaves in the CAM mode on the same gel. Although the patternof peptide fragments from NADP-malic enzyme from CAM-mode leaveswas similar to that from C₃-mode leaves, as indicated by peptidemapping, both immunoprecipitation and an enzyme-linked immunosorbentassay revealed some antigenic differences between the enzymesextracted from leaves in the C₃ and the CAM modes. These resultssuggest the existence of at least two isoforms of NADPmalicenzyme that differ in their levels of expression during inductionof CAM. (Received April 21, 1994; Accepted September 5, 1994)     

The metabolism of L-[6-14C]ascorbic acid in detached grape leaves

Grape leaves (Vitis labrusca L.) that are removed from the positionopposite the flower cluster either 28 or 14 days before anthesiscleave L-ascorbic acid (AA) at the C4-C5 bond into a C₄ and,presumably, a C₂ fragment. Leaves taken from this position 14days after anthesis fail to cleave AA. The C₄ fragment is utilizedfor L(+)-tartaric acid (TA) biosynthesis while the C₂ fragmentis recycled into hexose and products of the hexose metabolism.When [6-₁₄C]AA is the source of the label, the sucrose-derivedglucose from labeled leaves has a distribution of ₁₄C in thecarbon skeleton as follows: Cl, 35%; C2, 14%; C3, 4%; C(4+5),13% and C6, 34%. The effect of inhibitors of the glycolate pathwayon [6-^l4C]AA metabolism is examined. ¹This work was supported by Grant No. GM-22427 from the NationalInstitute of General Medical Science, National Institute ofHealth, United States Public Health Service, Bethesda, Maryland.This is Scientific paper No. 5305, Project 0266, College ofAgricultural Research Center, Washington State University, Pullman,WA 99164, U.S.A. ²Present address: The Radioisotope Research Center, Kyoto University,Kyoto 606, Japan. (Received August 29, 1979; )     

Characterization of the Type of Photosynthetic Carbon Dioxide Fixation in Jute (Corchorus olitorius L.)

Activities of photosynthetic and photorespiratory enzymes viz.,ribulose bisphosphate carboxylase, phosphoenol pyruvate carboxylaseand glycolate oxidase from jute (Corchorus olitorius L.; cv.JRO 632) leaves were compared with those from maize (C₄) andsunflower (C₃) leaves. The photosynthetic CO₂ fixation products,the release of ¹⁴CO₂ in light and dark following photosynthesisin ¹⁴CO₂, chlorophyll a: b ratio, gross leaf photosyntheticrate and dry matter production rate were also studied. The resultsshow that jute is a C₃ plant. Key words: Jute, Corchorus olitorius, C₃ photosynthesis     

Preparation of Radioactive Starch, Glucose and Fructose from C14O2

Radioactive starch, glucose and fructose have been preparedfrom tobacco leaves after assimilation of C¹⁴O₂. The apparatusused for photosynthesis consisted of a shallow Perspex leafchamber connected to a closed gas system, in which C¹⁴O₂ wasgenerated from BaC¹⁴O₂. Six leaves, area 14 to 18 sq. dm. whenexposed to bright sunlight with an initial CO₂ concentrationof 8 to 10 per cent., assimilated 3.35 g. of C¹⁴O₂ in 8 to 10hours. At least 80 per cent. of the C¹⁴O₂ supplied appearedin the leaves as starch and sugar and over 80 per cent. of theradioactivity was accounted for in these carbohydrates. Thespecific activity per m. atom of carbon of the isolated productswas 85 to 90 per cent. of that of the C¹⁴O₂. Small amounts ofradioactive carbon were also incorporated in the leaf proteinand in the celluose, hemicellulose and polyuronides.     

Ethylene Evolution from Bracts and Leaves of Poinsettia, Euphorbia pulcherrima Willd

Woodrow, L. and Grodzinski, B. 1987. Ethylene evolution trombracts and leaves ol Poinsettia, Euphorbia pulcherrima Willd.—J.exp. Bot. 38: 2024–2032. Ethylene release from fully expanded, red and white bracts andleaves of poinsettia, Euphorbia pulcherrima Willd., was compared.On a laminar (area) basis leaves contained about 50 times morechlorophyll and demonstrated 10 times the photosynthetic rateof the bracts. Both tissues contained starch, however, solublecarbohydrate in the bracts consisted primarily of reducing hexoseswhile the leaves contained mainly sucrose for translocation.The total free alpha-amino nitrogen content of the bract tissuewas twice that of the leaf tissue. The leaves contained moreACC (1-aminocyclopropane-1-carboxylic acid) and produced proportionallymore endogenous C²H⁴ than either the red or white bracts. ACC-stimulated₂H₄ release was also greatest from the green tissue indicatingthat the EFE (ethylene forming enzyme) was most active in theleaves. The specific activity of the ¹⁴C₂H₄/¹²C₂H₄ releasedfrom [2,3-¹⁴C]ACC confirmed ACC as the primary precursor ofC₂H₄ in this tissue. Ethylene release from the non-photosynthetic,bract tissue was not markedly affected by alterations in CO₂or light conditions. In green leaf tissue endogeneous ethylenerelease increased from 1·5 to 6·0 pmol C₂H₄ cm^–2h^–1 while ACC-stimulated ethylene release increased from10 to 35 pmol C₂H₄ cm^2– h^1– as the CO₂ partial pressureincreased from 100 to 1 200 µbar. Key words: Poinsettia, ethylene, bracts     

The Mechanism of Photosynthesis in the Tea Plant (Camellia sinensis L.)

Leaves of the tea plant photosynthesizing in ¹⁴CO₂ incorporatedmuch radioactivity into intermediates of the glycolate pathwayand little into C₄ acids. Increased O₂ in the atmosphere decreasedphotosynthesis, stimulated photorespiration, and increased theCO₂ compensation point. In air the rate of photorespirationwas 19% of net photosynthesis. These observations indicate aC₃ rather than a C₄ mechanism of photosynthesis.     

The Influence of Leaf Development on the Expression of C4 Metabolism in Flaveria trinervia, a C4 Dicot

The initial products of ¹⁴CO₂ assimilation were determined understeady state illumination of leaves of Flaveria trinervia, aC₄ dicot of the NADP-mialic enzyme subgroup. Leaf age influencedthe partitioning of ¹⁴CO₂ between the C₄ cycle and the reductivepentose phosphate (RPP) pathway. An estimated 10 to 12%of theCO₂ entered the RPP pathway directly in leaves about 20% fullyexpanded, whereas CO₂ was apparently fixed entirely throughthe C₄ pathway in leaves 75% or more expanded. This partitioningpattern was attributed to the bundle sheath compartment in youngleaves having a relatively high conductance to CO₂ (i.e., beingsomewhat leaky). Of the initially labelled C₄ acids, the proportion that wasmalate, relative to aspartate, increased continuously duringleaf expansion (from 60 : 40 to 87 : 13 at full expansion).Concurrently, there was an increase in the whole leaf activityof NADP malate dehydrogenase and a decrease in the activitiesof aspartate and alanine aminotransferases. Low chlorophylla/b values were observed in young leaves, which may coincidewith an enhanced capacity for non-cyclic electron transportin the bundle sheath chloroplasts of such tissue. Both enhancedaspartate metabolism and direct fixation of CO₂ in the bundlesheath could provide a greater sink for utilization of photochemicallyderived NADPH in the bundle sheath of young leaves. Such metabolicchanges are discussed in relation to a possible decrease inCO₂ conductance of the bundle sheath during leaf development. (Received March 4, 1986; Accepted June 25, 1986)     

Distribution and Accumulation of Thiophenes in Plants and Calli of Different Tagetes Species

The diversity of thiophenes (natural biocides) and the differencesbetween the concentrations of these compounds in the leavesand roots of Tagetes erecta L., T. patula L. cv. Nana furia,and T. minuta L. (marigolds) indicated the presence of at leasttwo different sites of accumulation: leaves and roots. Leafexplants of Tagetes, however, are used by preference to obtaincallus cultures. Once subcultured, secondary (C₂) calli of T.patula obtained from leaves of 4 to 7-week-old plants, containedhigher amounts of accumulated thiophenes (up to 80% of the amountsin the leaves) than original (C₁) or twice subcultured calli(C₃). The concentrations of thiophenes in C₂ calli of T. minutawere about half those of C₁ calli, while the concentrationsof thiophenes of C₁ calli amounted to 1-2% of the leaf values.Most of the C₃ calli of T. minuta did not contain thiophenesat all. Although C₁ calli of T. erecta also contained considerableamounts of thiophenes, the C₂ calli died, most likely owingto high levels of accumulated polyphenolic compounds. The combinationof species effects and the physiological state of plants andcalli provides adequate information to decide whether Tagetescalli are able to produce thiophenes or not. It is concludedthat the ability to produce thiophenes does not depend on theorgan used, but on the genetic information present in the species,and on the physiological state of plants and calli, particularlytheir age. Key words: Callus, explant selection, Tagetes erecta, Tagetes minuta, Tagetes patula, thiophenes     

Prolonged Survival of CAM-Mode Mesembryanthemum crystallinum in Darkness and its Possible Dependence on Malate

A remarkable difference was found in the survival of leavesof Mesembryanthemum crystallinum with plants grown in the C₃versus the CAM mode. With excised leaves (petiole in solution)of C₃-mode plants subjected to 6 days of darkness, there wasa large reduction in the chlorophyll content of the leaf andleaf turgor had decreased. By day 9, the chlorophyll had disappeared,except at the major veins, and the leaf tip had dried and turnedbrown. In contrast, the leaf tissue in the CAM mode showed onlya partial loss of chlorophyll during the same period, and evenafter 17 days of darkness, the tissue at the base was stillalive. Similarly, intact plants grown in the C₃ mode deterioratedmuch faster during 20 days of darkness than did plants grownin the CAM mode. Chlorophyll content, chlorophyll a/b ratio,phosphoenolpyruvate carboxylase, NADP-malic enzyme, malate andstarch content were measured. In both C₃- and CAM-mode plants,the starch content decreased rapidly during the dark periodand was nearly depleted after two days. In the CAM-mode tissue,there was a relatively high level of malate during prolongeddarkness (up to 17 days), with a transitory rise early in thedark period. In contrast, the malate content was low and rapidlydepleted in the C₃-mode leaves kept in darkness. These findingssuggest that malate may be an important source of carbon forsustaining leaves of CAM-mode M. crystallinum during prolongeddarkness. (Received May 20, 1987; Accepted October 23, 1987)     

Panicum milioides, a Gramineae plant having Kranz leaf anatomy without C4-photosynthesis

Light and electron microscopic observations of the leaf tissueof Panicum milioides showed that the bundle sheath cells containeda substantial number of chloroplasts and other organelles. Theradial arrangement of chlorenchymatous bundle sheath cells,designated as Kranz leaf anatomy, has been considered to bespecific to C₄ plants. However, photosynthetic ¹⁴CO₂ fixationand ¹⁴CO₂ pulse-and-chase experiments revealed that the reductivepentosephosphate pathway was the main route operating in leavesof P. milioides. The interveinal distance of the leaves wasintermediate between C₃and C₄Gramineae species. These resultsindicate that P. milioides is a natural plant species havingchracteristics intermediate between C₃ and C₄ types. (Received March 6, 1975; )     

Glycolate synthesis in a C3 C4 and intermediate photosynthetic plant type

Excised leaves of a C₃-photosynthetic type, Hordeum vulgare,a C₄-type, Panicum miliaceum, and an intermediate-type, Panicummilioides, were allowed to take up through their cut ends a1 mM solution of butyl hydroxybutynoate (BHB), an irreversibleinactivator of glycolate oxidase. After 30 to 60 min in BHB,extractable glycolate oxidase activity could not be detectedin the distal quarter of the leaf blades. Following this pretreatment,recovery of ¹⁴C-glycolate from ¹⁴CO₂ incorporated in a 10 minperiod was nearly maximal for each of the three plant types.Labeled glycolate was 51% of the total ¹⁴CO₂ incorporated forthe C₃-species, 36% for the intermediate-species, and 27% forthe C₄-species Increased labeling of glycolate was compensatedfor primarily by decreased labeling of the neutral and basicfractions for the C₃ and intermediate-type species. In the C₄-type,label decreased primarily in the neutral and insoluble fractions,but increased in the basic fraction. A lower rate of glycolatesynthesis is indicative of a lower rate of photorespirationand consistent with a lower O₂/CO₂ ratio present in the bundle-sheathcells of C₄-plants. We conclude that both decreased glycolatesynthesis and the refixation of photorespiratory-released CO₂are important in maintaining a lower rate of photorespirationin C₄-plants compared to C₃ plants. Intermediate glycolate synthesisin Panicum milioldes is consistent with its intermediate levelof O₂ inhibition of photosynthesis and intermediate rate ofphotorespiration. (Received May 6, 1978; )     

Atmospheric CO2 as a Global Change Driver Influencing Plant-Animal Interactions

Plants respond to changes in atmospheric carbon dioxide. Toherbivores, the decreased leaf protein contents and increasedC/N ratios common to all leaves under elevated atmospheric carbondioxide imply a reduction in food quality. In addition to thesefine-scale adjustments, the abundance of C₃ and C₄ plants (particularlygrasses) are affected by atmospheric carbon dioxide. C₄ grassescurrently predominate over C₃ grasses in warmer climates andtheir distributions expand as atmospheric carbon dioxide levelsdecreased during glacial periods. C₄ grasses are a less nutritiousfood resource than C₃ grasses both in terms of reduced proteincontent and increased C/N ratios. There is an indication thatas C₄-dominated ecosystems expanded 6–8 Ma b.p., therewere significant species-level changes in mammalian grazers.Today there is evidence that mammalian herbivores differ intheir preference for C₃ versus C₄ food resources, although thefactors contributing to these patterns are not clear. Elevatedcarbon dioxide levels will likely alter food quality to grazersboth in terms of fine-scale (protein content, C/N ratio) andcoarse-scale (C₃ versus C₄) changes.