Broquedis, M.

Cherrad, Monique

Colin, Lydia

Gény, Laurence

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 38 (3), 139-140 (1999) Research Note Activities of polyamine oxidases and diaminopropane contents during flowering of Vitis vinifera L. LYDIA CoLIN, LAURENCE GE.Nv, MoNIQUE CHERRAD and M. BROQUEDIS K e y · w o r d s : polyamine, diamine oxidases, polyamine oxidases, grapevine, spectrophotometric assay. Introduction: Polyamines (PA) are the newest class of compounds considered to function as possible plant growth regulators. Within their biosynthetic pathways the first PA formed is the diamine pulreseine (PUT) which may be converted into spermidine (SPD) and spennine (SPM) (MORRIS 1978, GALSTON 1983). PAs appear to be good biochemical markers for orga-nogenesis, especially with respect to the induction of flow-ering (MARTIN-T ANGuv 1985). In Doug1as fir trees, DAOUDI et al. (1994) observed that the PA Ievels are Jow during flowering, while arginine accumulates. This suggests the presence of diamine oxidase (DAO) and polyamine oxi-dase (PAO), which are responsible for the degradation of PAs. In grapevines GE.Nv et al. ( 1997) observed consider-able variation in the PA composition after anthesis. In par-ticular, diaminopropane (DAP), a product of PA Os, accu-mulated in young berries and was the major PA during the onset of fruit development. In view of the presumptive im-portance of these oxidases in berry development, we mea-sured, for the first time, their activities in floral buds, flow-ers and berries of cvs Merlot and Cabernet-Sauvignon at different developmental stages. The present paper describes the protein extraction to determine DAO and PAO activities and shows first results underlining the importance of PAO activities during flow-ering. Material and Methods: At different stages of devel-opment flowers and fruits of Merlot and Cabernet-Sauvignon were collected in a vineyard at U:ognan (Bor-deaux, France). Each sample, consisting of 10 grapes, was frozen in liquid nitrogen and stored at -50 °C. One gram fresh weight was suspended in 5 ml ofO.l M Tris-HCI buffer, pH 7.5. The extraction medium contained 5.7 mM ascorbic acid, 0.1% (w/v) Triton X-100 and 8% (w/v) polyethylene glycol4000 (Carbowax 4000). The ex-tracts were powdered in an ice-cold mortar. Polyvinyl-polypyrrolidone 5 % (w/v) was added. The samples were Correspondence to: Dr. LYDIA CoLIN, Laboratoire des Seiences de Ia Vigne, Universite de Bordeaux I , Avenue des Facultes, F-33405 Talence Cedex, France. Fax: +33-5-56842624. E-mail: lydiacolin@free.fr then centrifuged at 8,500 x g for 30 min at 4 °C and the supernatants adjusted to pH 7.5 with a saturated solution of Tris, were used immediately for the PAO assays. PAO activities were determined according to ÜKADA et al. ( 1983 ). The procedure involves the assessment of the rate of peroxidative oxidation of ortho-dianisidine by H20 2, released in enzyme assays containing SPD and SPM. The reaction mixture consisted of 8 ml of 0.1 M Tris-HCJ buffer (pH 7.5), 0.3 ml each of 1 % ortho-dianisidine and 60 U·mi-1 ofperoxidase (Horseradish Type li, Sigma). Af-ter preincubation of the components at 40 °C, the mixture was filtered; 0.4 ml ofthe crude enzyme extract and 0.1 ml of 10 mM SPD or SPM were added to 0.5 ml oftbis solution. The enzymatic activity was measured spectrophotometri-cally; absorbance at 460 nm was read after 30 min at 40 °C. DAO activities were detennined the same way, with 0.1 ml of 10 mM PUT in the assay instead ofSPM or SPD. Non-specific reactions were deterrnined in the absence of substrate. Free, conjugated and wall-bound PA were extracted ac-cording to the method of FLORES and GALSTON ( 1982) and analysed by HPLC according to SMJTH and DAVIES ( 1985), adapted for plant material by GENY et al. (1997). Results and Discussion: In both varieties, PAO ac-tivities were deterrnined in floral buds with a maximum in the inflorescences just before the onset of tlowering (June, 2 (Merlot) and June, 6 (Cabernet-Sauvignon)). At the end of tlowering and during fruit set, PAO activities decreased sharply and tended to zero (Figure, A). After anthesis, in both varieties considerable variation in the PA composition and content was observed. In flow-ers DAP was present at low Ievels and accumulated (prin-cipally in its conjugated form) predominantly in young ber-ries (Merlot: June, 15, Cabernet-Sauvignon: June, 23). Thereafter, DAP Ievels decreased and were very low in berries before and after veraison (Figure, B). During flowering and fruit development changes in free and conjugated PA contents were similar to-those reported for various other plants (MARTJN-T ANGUY 1997~. However, to our knowledge, this.is the first report on the occurrence and possible implication ofPAO activities and DAP in flow-ering and the pollination processes. In flowers and young berries, the PAO activities and the DAP content were inversely correlated. This correla-tion was similar in both varieties. lt was independent ofthe date but depended on the stage of development. The maxi-mum PAO activities occurred before DAP accumulated, i.e. flowering was characterized bfmaximum PAO activi-ties and pollination by maximum DAP contents. These com-pounds can be considered to be good indicators of these two developmental stages. These data raise the question of the function of bound and conjugated PAs and especially the function of DAP. DAP was the major PA observed in young berries at the onset of fruit development (GE:NY et a/. 1997). In plants, amine oxidases are involved in the regulation of PA con-centrations and transport at the subcellular Ievel, depend-ing on the physiological stage ofthe tissues (ARIBAUD et al. 140 LYDIA CoLrN, LAURENCE GENY, MoNtQUE CHERRAD and M. BROQUEDIS ~ Mertot -<l-Cabemet-Sauvignon 400 <: 300 ·~ g !. ~ 200 {A) PAO actlvities "' g ::> 100 0 250 200 ~ ... !50 (BI DAP content. "' ! 100 50 t0/8 919 Figure: PAO activities (A) and DAP contents (B) in floral buds and in young benies of grapevine. Values are means ± SD oftwo deterrninations ofsamples consisting of 10 grapes each. 1994). Data presented by SHIH et al. (1982) demoostrate that DAP is a potent inhibitor of dark-induced senescence inhibiting ethylene production. DAP may bind polyribosomes and affect protein synthesis, which may account for the decrease of protease activity. The antago-nism to Ca2+, however, suggests that the initia1 step ofthe retardation of senescence by DAP probably involves its attachment to membranes (SHJH et al. 1982). We believe that the study of the relationship between PA catabolism and pollination will Iead to further experi-mental efforts. One possible approach would be to utilize specific precursors or inhibitors of PA synthesis and to examine the resulting physiologica1 properties of the tis-sues. On the other hand, cloning of genes for amine oxi-dases in Vitis vinifera L. would allow to investigate the mechanisms controlling PA catabolism. This includes the knowledge of molecular mechanisms in which these PAs play a roJe in the processes of grapevine growth and devel-opment. This research was supported by the Conseil Interprofessionnel du Vin de Bordeaux (Bordeaux, France). The authors are grateful for financial help. We also thank Mrs. RosE-MARIE GuJLBAUD and Mrs. JosETTE LETOUZEY for their technical assistance, Professor BERNARD DoNECHE for helpful discussions and Mrs. JosETIE MAR.rJN-T ANGUY for her interest in our work and critical reading of the manu-script. ARIBAUD, M.; CARR.E, M.; MARTIN-TANGUY, J.; 1994: Polyamine metabo-lism andin vitro cell multiplication and ditferenciation in leaf explants ofChrysanthemum moritifolium Ramat. Plant Growth ReguL 15, 143-145. DAOUDI, E. H.; DouMAS, P.; BoNNn-MAstMBERT, M.; 1994: Changes in amino acids and polyamines in shoots and buds of Douglas-fir trees in-duced to flower by nitrogen and gibberellin treatments. Canad. J. Res. 24,1854-1863. FLORES, H. E.; GALSTON, A. W.; 1982: Analysis ofpolyamines in higher plants by high performance liquid chromatography. Plant PhysioL 69, 701-706. GALSTON, A. W.; 1983: Polyamines as modulators ofplant development. BioScience 33 (6), 382-387. GtNY, L.; BROQUEDIS, M .; MARTIN-T ANGUY, J .; BOtiARD, J .; 1997: Free, conju-gated and wall-bound polyamines in various organs offruiting cut-tings of Vitis vini[era L. cv. Cabemet Sauvignon. 1\rner. J. EnoL Viticult. 48(1),80-84. MARTIN-TANGUY, J .; 1985: The occurrence and possible function of hydroxycinnamoyl acid amides in plants. Plant Growth ReguL 3, 381-399. - -; 1997: Conjugated polyamines and reproductive development: Biochemi-cal, molecular and physiological approaches. PhysioL Plant. 100, 675-688. MoRRJs, D.; 1978: Biosynthesis of di-, tri- and polyamines in rat Ii ver. Mam-malian Rev. 13 (5), 651-663. ÜKADA, M.; KAWASHIMA, S.; IMAHORJ~K.; 1983: Purification ofputrescine oxydase from Micrococcus rubens by affinity chromatography. MethodEnzymoL 94,301-308. SHJH, L.; KAuR-SAWHNEY, K.; FuRHER, J.; SAMANTHA, S.; GALSTON, A. W.; 1982: Effects of exogenous I ,3-diaminopropane and spermidine on senescence of oat leaves. I. Inhibition of prolease activity, ethylene production and chlorophyll loss as related to poly-amine content. PlantPhysioL 15, 1592-1596. SMJTH, T. A.; DAVIES, J. P.; 1985: Separation and quantification ofpolyamine in plant tissue by high performance liquid chromatography of their dansyl derivatives. Plant PhysioL 79, 89-91 . 

Activities of polyamine oxidases and diaminopropane contents during flowering of Vinis vinifera L.

https://ojs.openagrar.de/index.php/VITIS/article/download/4784/4593

Activities of polyamine oxidases and diaminopropane contents during flowering of Vinis vinifera L.

Abstract

Similar works

Full text

Available Versions

JKI Open Journal Systems (Julius Kühn-Institut)