Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

Detection and Evaluation of Serine Enzymes by [3H]-DFP Affinity Labeling in Spinach Plants

Serine enzymes were detected in spinach plants by affinity labelingwith [³H]-di-isopropyl phosphorofluoridate (DFP) and SDS-polyacrylamidegel electrophoresis. Two serine enzymes were detected in thedry seeds, and another 4 major and 3 minor serine enzymes weredetected in 48-hr soaked seeds, especially in the cotyledons.Fourteen serine enzymes, including 9 enzymes in the cotyledons,were detected in mature leaves. [³H]-DFP binding with some serineenzymes in mature leaves was inhibited by a prior treatmentwith phenylmethylsulfonyl fluoride, a more specific probe ofserine proteases. Other affinity labeling reagents for serineproteases, L-1-tosylamide-2-phenylethyl chloromethyl ketoneand N-p-tosyl-L-lysine chloromethyl ketone also decreased DFP-bindingto some serine enzymes. These results are evidence that the enzymes found are serineproteases. Natural inhibitors for serine proteases, leupeptin,aprotinin and soybean trypsin inhibitor had no effect on [³H]-DFPbinding. DFP-binding with all the serine enzymes detected inthe mature leaves was decreased by p-chloromercuric benzoatebut not by EDTA. (Received June 12, 1982; Accepted September 28, 1982)     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 2. The Incorporation of 13C into Carbohydrates, Organic Acids, Amino Acids and some Storage Compounds

Nodulated soya bean (Glycine max L.) plants at the early floweringstage were allowed to assimilate ¹³CO₂ under steady-state conditions,with a constant ¹³C abundance, for 8 h in the light. The plantswere either harvested immediately or 2 d after the end of the¹³CO₂ feeding, divided into young leaves (including flower buds),mature leaves, stems+petioles, roots and nodules; the ¹³C abundancein soluble carbohydrates, organic acids, amino acids, starchand poly-ß-hydroxybutyric acid was determined witha gas chromatography-mass spectrometry. The rapid turnover of ¹³C in the sucrose pools observed in allorgans of the plants showed that sucrose was the principal materialin the translocation stream of primary products of photosynthesis.At the end of the ¹³CO₂ exposure, sucrose in the mature leavesas the major source organs and in the stems+petioles was labelledwith currently assimilated carbon to about 75 per cent, whereasa much higher labelling of sucrose was found in the roots andin the nodules. This suggests the existence of two or more compartmentedpools of sucrose in mature leaves and also in stems+petioles. The relative labelling patterns of individual organic acidsand amino acids were similar in various plant organs. However,the rapid turnover of succinate and glycine was characteristicof nodules. Treatment with a high concentration of nitrate inthe nutrient media increased the turnover rate of amino acidcarbon in shoot organs and roots, while it markedly decreasedthe labelling of amino acids in nodules. The cyclitols, exceptfor D-pinitol, were significantly labelled with assimilated¹³C in mature leaves, but in nodules, the labelling was verymuch less. In the nodules, which were actively fixing atmospheric nitrogen,a large proportion (80–90 per cent) of currently assimilatedcarbon was found as sucrose and starch at the end of the ¹³CO₂feeding. This was also true of the roots. On the other hand,in young growing leaves, the distribution of currently assimilatedcarbon into sucrose, starch and other soluble compounds wasmuch less. This suggests that a large amount of carbon assimilatedby and translocated to young leaves was used to make up structuralmaterials, mainly protein and cell wall polymers synthesis,during the light period. Glycine max L., soya bean, ¹³CO₂ assimilation, carbon metabolism in nodules     

Photosynthesis of White Clover Leaves as Influenced by Canopy Position, Leaf Age, and Temperature

Microswards of white clover (Trifolium repens L.) were grownin controlled environments at 10/7, 18/13 and 26/21 °C day/nighttemperatures. The vertical distribution of leaves of differentages and their rates of ¹⁴CO₂-uptake in situ were studied. Extending petioles carried the laminae of young leaves throughthe existing foliage. A final position was reached within 1/4to 1/3 of the time between unfolding and death. Newly unfoldedleaves had higher rates of ¹⁴CO₂-uptake per leaf area than olderones at the same height in the canopy. At higher temperatures,the decrease with age was faster. However, the light-photosynthesisresponse of leaves which were removed from different heightsin the canopy varied much less with leaf age than did the ratesof ¹⁴CO₂-uptake in situ. The comparison of the rates of ¹⁴CO₂-uptake in situ with thelight-photosynthesis response curves suggests that young leavesreceive more light than older ones at the same height in thecanopy. This would imply that young white clover leaves havethe ability to reach canopy positions having a favourable lightenvironment. This ability may improve the chances of survivalof white clover in competition with other species. Trifolium repens L., white clover, photosynthesis, canopy, leaf age, ¹⁴CO₂-uptake, ecotypes, temperature     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine     

Nitrate Nutrition and Temperature Effects on Wheat: Enzyme Composition, Nitrate and Total Amino Acid Content of Leaves

Lawlor, D. W., Boyle, F. A., Kendall, A. C. and Keys, A. J.1987. Nitrate nutrition and temperature effects on wheat: Enzymecomposition, nitrate and total amino acid content of leaves.—J.exp. Bot. 38: 378–392. Wheat plants were grown in controlled environments in two temperatureregimes with two rates of nitrate fertilization. In some experimentstwo light intensities were combined with the nitrogen and temperaturetreatments. The composition of the third leaf was studied fromsoon after emergence until early senescence. The amounts ofchlorophyll, soluble protein, ribulose bisphosphate carboxylase-oxygenase(RuBPc-o) protein, nitrate, and total amino acids were measuredtogether with the activities of RuBPc-o, fructose- 1,6-bisphosphatase,glycolate oxidase, carbonic anhydrase, nitrate reductase, glutaminesynthetase and serine- and glutamate-glyoxylate aminotransferases.Additional nitrate supply increased the amounts, per unit leafarea, of chlorophyll, total soluble protein and RuBPc-o proteinand the activities of all the enzymes. The ratio of RuBP carboxylaseto RuBP oxygenase activity, when measured at constant CO²/O²ratio and temperature, was unaffected by growth conditions orleaf age. Leaves grown at the lower temperature, especiallywith more nitrate, contained much more soluble protein, nitratereductase, fructose bisphosphatase and free amino acids perunit area than the plants grown in the warmer conditions. However,young leaves grown in the warm contained more nitrate than thosegrown in the cool. Amounts of protein, amino acids and chlorophylland most enzyme activities reached maxima near full leaf expansionand decreased with age; additional nitrate slowed the decreaseand senescence was delayed. Nitrate content and nitrate reductaseactivities were highest in leaves before full expansion andthen fell rapidly after full expansion. Increased light intensityincreased the content of RuBPc-o protein at the higher rateof nitrate supply. Chloroplast components and, to a lesser extent,peroxisomal enzymes associated with photosynthetic nitrogenassimilation changed in proportion with different treatmentsbut nitrate reductase activity was not closely related to chloroplastenzymes. Control of tissue composition in relation to environmentalconditions is discussed. Key words: Nitrate nutrition, temperature, wheat, enzyme, amino acid, leaves, ribulose bisphosphate carboxylase oxygenase, nitrate reductase     

Effects of Gibberellic Acid on the Activation, Synthesis, and Release of Acid Phosphatase in Barley Seed

Acid phosphatase activity was present in unimbibed barley seed,but rose during incubation of embryoless half-seeds and isolatedaleurone layers, and was further increased by 10^–6 M gibberellicacid (GA₃). Release of total acid phosphatase activity fromhalf-seeds and aleurone layers was markedly enhanced by GA₃.Inhibitor studies with cycloheximide and actinomycin D suggestedthat de novo synthesis of acid phosphatase occurred followingimbibition. Gel nitration, electrophoresis, and [¹⁴C]leucineincorporation studies revealed that a single molecular formof acid phosphatase was present in dry seed, whereas on incubationtwo further forms arose. A proportion of the three molecularforms of the enzyme was synthesized de novo. Gibberellic acidstimulated activation, but not de novo synthesis, of all threemolecular forms of acid phosphatase. Although a small amountof one of the molecular forms was secreted in the absence ofGA₃, the presence of gibberellin greatly increased secretionof the same form of acid phosphatase.     

Two-Dimensional Protein Patterns of Arabidopsis Wild-Type and Auxin Insensitive Mutants, axr1, axr2, Reveal Interactions between Drought and Hormonal Responses

In order to detect gene products involved in Arabidopsis droughtadaptive strategy, 2D-PAGE protein patterns of two auxin-insensitivemutants, axr1, axr2, differentially affected in specific droughtresponses, were compared to the wild-type Columbia ecotype,in well-watered and drought-stressed conditions. Coupled tocomputer analysis of polypeptide amounts, 2D-electrophoresisrevealed subtle changes in protein expression induced by progressivedrought stress and/or mutations affecting the auxin responsepathway. The differential protein patterns of axr1 and axr2 were consistentwith their contrasting drought responses. The specific leafand root protein patterns of axr1 showed that this mutationdisrupts drought responses related to auxin regulation. In particular,the near absence of drought rhizogenesis in axr1 was associatedwith a root protein pattern closer to the well-watered thanto the water-stressed axr2 and Columbia wild-type root proteinpatterns. Also, the largely different effects of axr1 and axr2mutations suggest that they affect different pathways in auxinresponse. Several sets of polypeptides, whose regulation wasaffected by drought and/or mutation, were thus detected. Thesepolypeptides could play a role both in the auxin and the droughtresponse pathways. Their identification, through microsequencing,should be most informative. ⁴Current address: Laboratoire de BioénergétiqueCellulaire, Département d'Ecophysiologie Végétaleet de Microbiologie, Centre d'Etudes de Cadarache, 13108 SaintPaul lez Durance, France     

Effects of cycloheximide and kinetin pretreatments on responses of susceptible and resistant Avena leaf protoplasts to the phytotoxin victorin

Avena leaf protoplasts from varieties susceptible (cv. Park)and resistant (cv. Victory) to victorin incorporate the labelfrom ³H-L-leucine, ³H-uridine and ³H-thymidine into trichloroaceticacid-insoluble materials at comparable rates. When incubatedin the presence of the toxin, susceptible protoplasts beginto lyse after a short period of time; the effect on resistantprotoplasts is minimal. Victorin also significantly reducesincorporation of label from leucine and to a lesser extent fromuridine and thymidine into susceptible protoplasts but has anegligible effect on resistant protoplasts. Pretreatment ofsusceptible leaves with cycloheximide but not kinetin protectsagainst the subsequent action of victorin on apparent proteinsynthesis and lysis of protoplasts. ¹Permanent address: Institut National de la Recherche Agronomique,Station de Physiologie et Biochemie Végétales,"La Grande Ferrade", Centre de Recherches de Bordeaux, 33 Pont-de-la-Maye,Bordeaux, France. ²School of Forestry and Environmental Studies, Yale University. ³Supported by grant GI-32958 of the RANN program of the NationalScience Foundation to A. W. Galston. We are indebted to WhitneyAdams, jr. for expert assistance. (Received April 10, 1976; )     

Comparison of Photosynthetic and Photorespiratory Enzyme Activities between Green Leaves and Colorless Parts of Variegated Leaves of a C4 Plant, Stenotaphrum secundatum (Walt.) Kuntze

The activities of enzymes involved in C⁴ photosynthesis andphotorespiration in colorless parts of variegated leaves ofStenotaphrum secundatum (Walt.) Kuntze were compared with thosein green leaves. Chlorophyll content of the colorless part wasonly about 0.3–3% of that of the green leaves. The activities of chloroplastic enzymes, pyruvate, P_i dikinase,NADP⁺-malic enzyme and NADP⁺-glyceraldehyde 3-phosphate dehydrogenasewere considerably lower in colorless tissue on a fresh weightor protein basis (the ratios of the activities in the green/colorlesstissues ranging from 5 to 20). A cytoplasmic enzyme, UDP-glucosepyrophosphorylase as well as aspartate and alanine aminotransferasesshowed comparable activities in the two types of tissue, whereasPEP carboxylase in the colorless tissue had only the one-thirdactivity of that in green tissue. Differences in activitieswere also observed for the glycolate pathway enzymes (the ratiosranging from 2 to 7 for glycolate oxidase, hydroxypyruvate reductaseand serine hydroxymethyltransferase, and 7 to 15 for catalase),while cytochrome c oxidase showed comparable activity in thetwo types of tissue. The results suggest that the deficiency of thylakoid developmentin the colorless tissue influences enzyme activities not onlyin plastids but also in other cellular compartments. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 26, 1986; Accepted June 17, 1986)     

Sur une nouvelle applicaton de la theorie de P information a la description des series chronologiques planctoniques

Corrélations simples (r) et pondérées (r_p)entre S. setosa (trois stades cumulés), S. minima etS. inflata (stade 1), S. bipunctata, et les quatre paramétreshydrologiques retenus. ^aSeuil de signification 5%. ^bSeuil designification 1%     

FEEDING ACTIVITY OF LIMAX VALENTIANUS FERUSSAC: NOCTURNAL RHYTHM AND ALIMENTARY COMPETITION

Feeding activity of the slug Limax valentianus Férus-sacthroughout a 24-hour period began with the decrease in light.Feeding began 41 minutes after the start of locomotor activityand showed a maximum level in the beginning of the night L.valentianus had two to five meals each night, each meal lasting8.5 to 14 minutes. The overcrowding of the slugs induced a shorterduration of individual feeding periods and acts of aggressionduring meals. ^*Present address: Centre de Neurochimie, Laboratoire de neurobiologiemolécuiaire des interactions cellulaires. 5 rue BlaisePascal, 67084 Strasbourg, France (Received 7 July 1996; accepted 21 April 1997)     

Action of Benzoic Acid and its o-, m- and p-Hydroxy-Derivatives on the Dark- and Light-Induced Leaflet Movements in Cassia fasciculata Michx.

Benzoic acid and its o-, m- and p-hydroxy derivatives appliedon excised leaves of Cassia fasciculata modified the dark-induced(scotonasty) and light-induced (photonasty) leaflet movements.Benzoic acid inhibited the scotonastic closure in a dose-dependentmanner from 10^–4M to 10^–3M and promoted the photonasticopening at optimum concentration of 5.10^–4M. These effectswere dependent upon the position of hydroxyl group on the benzoicring, the o-derivative inducing a stronger effect than the m-and p-derivatives. Experiments showed that treatment with o-hydroxybenzoicacid had not to exceed 30–60 min and that the maximumeffect was obtained at pH 5.5. (Received September 16, 1986; Accepted June 22, 1987)     

Age-related Changes in Stomatal Response to Cytokinins and Abscisic Acid

Kinetin and zeatin(100 mmol m^–3)reversald the ABA-mediated(100mmol ^m-2)closure of stomata of young maize leaves but did notaffect stomatal apertures of these leaves when applied alone.As leaves aged, kinetin or zeatin alone promoted increased stomatalapertures, while abscisic acid (ABA) applied alone had a reducedeffect on stomata. Even with older leaves, cytokinins reversadthe effect of ABA on stomata. Maize, stomata, abscisic acid, kineusc, zeatin, Zea mays     

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves ofCitrus sinensis L. Osbeck is affected by the presence of thefruitlet on the shoot. Previous work gave evidence that gibberellinsmay be involved in this 'fruit effect'. In the present workwe have studied the effect of gibberellic acid (GA₃) on ¹⁴C-sucroseuptake by leaf discs and whether its action could be due toa modulation of the plasma membrane ATPase, which maintainsthe H⁺ gradient that drives H⁺/sucrose co-transport. The effect of GA₃ on ¹⁴C-sucrose uptake depended on the osmolarityof the assay medium. At 300 mOsm a reduction in the uptake ratewas observed. The inhibitory effect of the hormone disappearedafter preincubating the leaf discs with para-chloromercuri-phenylsulphonicacid (PCMPS), a sulphydril binding inhibitor. ATPase activityof isolated plasma membrane vesicles was inhibited by IAA treatments,while GA₃ or ABA did not affect this enzyme, even after a 3h preincubation period. However, in the absence of a surfactantin the assay medium, GA₃, together with turgor pressure, modulatedplasma membrane ATPase activity, possibly through modificationsof membrane permeability. The hormone effect on ¹⁴ C-sucroseuptake may involve action on the sucrose carrier.Copyright 1994,1999 Academic Press Abscisic acid, Citrus sinensis, gibberellic acid, indoleacetic acid, orange, osmotic pressure, plasma membrane ATPase, ¹⁴C-sucrose uptake     

Partitioning and redistribution of sulphur during S-stress in Macroptilium atropurpureum cv. Siratro

During the first 7 d of sulphate-deprivation stored SO₄^2- wasredistributed and assimilated into organic forms in the tropicallegume Macroptilium atropurpu-reum cv. Siratro. However, whilstthe sulphate content of all tissues declined after removingthe external SO₄^2- supply this was slowest in mature leaves.By contrast, the total S content of mature leaves declined markedlyin the absence of external sulphate whilst that of both youngleaves and roots increased. Furthermore, when radiolabelledSO₄^2- was applied to abraded surfaces of mature leaves, mostof the translocated label was recovered in the root following2 d SO₄^2- deprivation. By contrast, radiolabelled SO₄^2-appliedto young leaves was mostly retained in these tissues and nottranslocated. Within 3 d of removing the SO₄^2- supply there was a large increasein extractable APS-sulphotransferase activity in roots accompaniedby a decline in nitrate reductase activity, but these effectswere not seen in leaves. Five days after the removal of SO₄^2-there was a large increase in the content of asparagine in roots. The results are discussed in relation to the co-ordination ofNO₃^- and SO₄^2- uptake and assimilation and the partitioningof sulphur during S-stress. Key words: Sulphate supply, stomatal conductance, ATP-sulphurylase, APS-sulphotransferase, nitrate reductase     

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)     

Formation of multiple forms of acid and alkaline phosphatases in relation to the culture age of Aspergillus niger

Acid phosphatase (EC 3.1.3.2 [EC] ) was extracted from mycelia ofAspergillus niger, then separated and purified into four fractions.These acid phosphatases, designated IA, IB, II and III, hadpH optima at 5.0, 4.5–5.0, 4.5 and 2.5, respectively.None required the presence of divalent cations, and all werestrongly inhibited by NaF. They were non-specific acid phosphatasesbut varied in their activities with various substrates. Thealkaline phosphatase (EG 3.1.3.1 [EC] ) of A. niger was also separatedinto two fractions, alkaline phosphatases I and II. Changes in the activity ratios of these acid and alkaline phosphataseswere studied during culture in a peptone medium. The activityof acid phosphatase II was higher than the others when the culturewas young. The activity of acid phosphatase III increased toa maximum in the actively growing phase, then decreased. Thatof acid phosphatase I became highest in the mature culture.In contrast, the activity of alkaline phosphatase I was higherthan the others in young cultures, while alkaline phosphataseII became dominant in the mature culture. Activities of the various acid and alkaline phosphatases indifferent regions of the growing colonies were also studied.The changing patterns of these enzymes in both liquid and surfacecultures were compared. When A. niger was cultured in a medium containing a low concentrationof phosphate, acid phosphatase activity greatly increased afterthe consumption of phosphate, but alkaline phosphatase activitydid not. ¹ The present experiments were carried out, for the most partat the Institute of Applied Microbiology of the University ofTokyo. (Received February 10, 1975; )