Daucus carota tissues were grown on Murashige-Skoog medium (MS) at different concentrations with abscisic acid (ABA). Seven bands of chloroplast fractions were obtained on a sucrose gradient. At 10−5M, ABA highly increased chlorophyll and protein nitrogen content of medium density chloroplasts. With increasing age of the tissues, the most active chloroplasts according to their 14CO2 fixation were found in smaller numbers. When treated with 10−5 M ABA, 34 day-old tissues cultivated in vitro showed the chloroplast pattern of 110 day-old tissues. The effect of ABA—given to the tissues during a short pretreatment or continuously present in the culture medium—on the ribulose diphosphate carboxylase activity was analysed. It was found that ABA at 10−5 M strongly inhibited 14CO2 fixatio

Maillard-Sevhonkian, Sonia

Pilet, Paul-Emile

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Plant & CellPkysiol. 19(5): 811-817 (1978)Effect of abscisic acid on differentiation and ribulosediphosphate carboxylase of chloroplastsSonia Maillard-Sevhonkian and Paul-Emile PiletInstitute of Plant Biology and Physiology of the University1005 Lausanne, PI. de la Riponne, Switzerland(Received December 20, 1977)Daucus carota tissues were grown on Murashige-Skoog medium (MS) at differentconcentrations with abscisic acid (ABA). Seven bands of chloroplast fractions wereobtained on a sucrose gradient. At 10~5 M, ABA highly increased chlorophyll and proteinnitrogen content of medium density chloroplasts. With increasing age of the tissues,the most active chloroplasts according to their 14CC>2 fixation were found in smallernumbers. When treated with 10~5 M ABA, 34 day-old tissues cultivated in vitro showedthe chloroplast pattern of 110 day-old tissues. The effect of ABA—given to the tissuesduring a short pretreatment or continuously present in the culture medium—on theribulose diphosphate carboxylase activity was analysed. It was found that ABA at 10~5M strongly inhibited l4CO2 fixation.Key words: ABA — Chloroplasts — Differentiation enzyme — RuDP-carboxylase —Tissue culture.Analyses of the ABA effects on several tissues cultivated in vitro have given riseto a number of publications (12). In our Institute the changes in the ultrastruc-ture—induced by ABA—have been most particularly studied in the tissues of Rubushispidns cultivated in vitro (11, 13) especially in relation to their growth. Somechanges in plastid structures when these tissues were exposed to light for several dayshave been briefly reported. That is why experiments, using carrot tissues (rich inchloroplasts), were conducted to test the direct action of ABA on the chloroplastsand their chlorophyll content.Materials and methodsMaterialsDaucus carota tissue cultures (4, 8-10) were maintained on Murashige-Skoog(MS) medium containing 1.7X10~6M IAA and 6 X 1 0 - 9 M kinetin. Experimentsusing different concentrations of abscisic acid (ABA) were conducted with 34 day-oldcultures of tissues cultivated in vitro. The activity of ribulose diphosphate (Ru-DP) carboxylase was analysed using isolated chloroplasts from 48 day-old cultureswith or without ABA (10~5 M). Similar analyses were done on isolated chloroplastsfrom 110 day-old cultures pretreated with or without ABA (10~5 M).MethodsEach step of the extraction and isolation of chloroplasts until the osmotic shock811812 S. Maillard-Sevhonkian and P. E. Piletwas checked, on an untreated sample, by electron microscopy (Dr. R. M. Hofer, in afurther paper). After this check showed that the technique used yielded mainlystructurally intact chloroplasts, as routine electron microscopy examination ofevery chloroplast preparation would be extremely time-consuming, we checked thepellet placed on a discontinuous sucrose gradient under light microscope and phasecontrast. Chloroplasts in this pellet were of several sizes, smaller than those ofhigher plants, and predominantly of either class I or class II as described by Spencerand Unt (16), and Spencer and Wildman (15). Some of them appeared opaque,the grana difficult to differentiate, the margin of the chloroplasts well defined asdescribed by Leech (6) and Bourdu et al. (2) for membrane-bound chloroplasts;others appeared with a very granular appearance, diffused boundaries, presumablychloroplasts in which the external membrane was not complete, not refractive.Extraction: Tissues were ground with a mortar and pestle in a semi-frozenextraction buffer (0.02 M glycyl-glycine+5 mM MgCl2-6 H2O + I mM mercapto-ethanol+0.33 M saccharose) (5), and filtered through 4 layers of muslin and 2 layersof nylon gauze (pore size: 50 /xm) to remove cellular debris. The muslin waspressed and the nitrate collected and centrifuged (12,500 X g, 2 min). The pellet,corresponding to nearly all the chloroplasts, after light microscope examination,was placed on a discontinuous sucrose gradient (33^12-51-57—60-63-66-70% w/w)and centrifuged (23,000 X^, 90 min, 5°C) using the ultracentrifuge Christ Omega 2.Isolation of chloroplasts: Different types of chloroplasts were separated accord-ing to their density. The fractions were successively collected and their index ofrefraction at 25°C measured by refractometry. These fractions were diluted threetimes with the extraction buffer, without sucrose, and centrifuged (2 min, 7000 X g).The pellets—containing isolated chloroplasts—were taken up in the extraction bufferwithout sucrose in order to burst (osmotically) the chloroplasts, as Ru-DP carboxy-lase is found inside the chloroplast (14, 17), and to allow substrates to react withactive enzyme sites (2).Ru-DP carboxylase activity: 0.3 ml of the above fractions were mixed with10/xCi NaH14CO3 (50/xl), 0.5 /miole Ru-DP (disodium salt), 0.4 ml incubationbuffer (0.25 mM glycyl-glycine, 2.5 mM MgCl2-6H2O, 75 HIM NaHCOa, mercapto-ethanol 5 ^ 1/250 ml; pH 7.8) (5) in scintillation vials, which were then incubated(1 hr, 25°C) in white light (Philips: TL 40 w/33; blanc super; 1.5 X 103 lux).The reaction was stopped by adding 0.2 ml of 6 N acetic acid. The vials werethen flushed for 4 hr with air to remove the mineral-labelled CO2 in excess, and thesamples dried. Then 2.5 ml of H2O and 10 ml of Bray's scintillation fluid wereadded to the dry residues and the radioactivity of each sample was measured in aliquid scintillation counter.Analytical methods: Chlorophyll content was estimated according to a methodpreviously described (1, 3). An aliquot of the aqueous extract was diluted withacetone to adjust the water content to 20% (v/v). The absorbance was measuredwith a recording spectrophotometer at 663, 645 and 652 nm.Protein nitrogen content was estimated according to the Kjeldahl method. To1 ml of each fraction, 5 ml of 10% TCA were added. Proteins were precipitatedat 4°C for 18 hr and the tubes were then centrifuged (4,200 Xg, 30 min). The super-natant containing the non-protein nitrogen was discarded. To the pellets wereadded respectively 1 ml concentrated H2SO4, a small amount of selenium containingABA and the RuDP-carboxylase in chloroplasts 813catalyst and a few glass beads. The pellets were then hydrolyzed for 18 hr at 330°Cin a thermobloc (Lievisch, Planta Jenny). Ammonia was distilled according tothe Parnas-Wagner method and collected in 0.05 N H2SO4. The protein nitrogenwas determined by a back titration of the H2SO4 with 0.02 N NaOH.Results and discussionFirst, experiments were performed in order to study changes in the chloroplastdistribution with the increasing age of the tissues.Seven bands were observed in the sucrose gradient (Fig. 1A); they can beclassified into three groups, according to their density p:—Group I : light chloroplasts, density 1.141-1.213 (zones 33 to 42% sucrose)—Group II : medium chloroplasts, density 1.257-1.280 (zones 51 to 57%sucrose)—Group III : heavy chloroplasts, density 1.304-1.322 (zones 60 and 63 to 66%sucrose).In young tissues (27 days), there were more light chloroplasts (group I). Thoseof group II were almost nonexistent, while those of group III were of few importance.When the tissues became older (48 days), the number of chloroplasts in groups Iand III increased. When the tissues were old (110 days), chloroplasts from groupsI and III were at about the same level as in the 48 day-old tissues; in contrast,chloroplasts of group II became very abundant. There were a great many middleand high density chloroplasts.The distribution patterns of chloroplasts, obtained from tissues treated withABA, were changed in the following way (Fig. IB):—with 10~6 M ABA, the chlorophyll (Fig. 2) and protein (Fig. 3) content of group I3351£ 60ul(/Io5 6627z mmaI0"6 ID"5 10'ABA INMCHLOROPHYLL DENSITYLOW ->. HIGHFig. 1. Changes in chloroplast distribution patterns (separation in sucrose gradient). (A) With increasing ageof the tissues (in days) cultivated in vitro. (B) Effect of ABA (at several concentrations). Age ofcultures: 34 days.814 S. Maillard-Sevhonkian and P. E. Pilet2 0 -51 57 60SUCROSE IN o / oFig. 2. Chlorophyll content (in fig ml zone) of the seven isolated chloroplast sucrose zones. Chloroplasts wereprepared from carrot tissues cultivated on Murashige and Skoog medium in the presence of ABA atseveral concentrations. Age of cultures: 34 days.chloroplasts increased and became higher than that of group III. Group IIchloroplasts were still very few.—10~5 M ABA, the chlorophyll and protein levels of group I and group II chloro-plasts was clearly enhanced. As can be seen in Fig. 2, chlorophyll concentration(in /Ag/ml) rose from 5.81 to 13.00 in the 42% zone. In the 57% zone (a verythin band almost undistinguishable in the control), the chlorophyll content rosefrom 1.44 to 13.49/ig/ml. The 51% zone was stimulated as well as the 60%2000 - ,51 57 60SUCROSE IN °/oFig. 3. Protein content (in fig ml zone) of the seven isolated chloroplast sucrose zones. Chloroplasts were pre-pared from carrot tissues cultivated on Murashige and Skoog medium in the presence of ABA at severalconcentrations. See Fig. 2.ABA and the RuDP-carboxylase in chloroplasts 815one, in which chlorophyll content was increased from 5.78/xg/ml to 13.78 /*g/ml.In contrast the 63% and 66% zones "degenerated". The protein content(Fig. 3) was found to change in a similar way.—with 10~4 M ABA, stimulation of the light chloroplasts of group I was reduced,compared to the effect of 10~5 M ABA while group III zones—60, 63 and 66%sucrose—were destroyed. The chlorophyll (Fig. 2) and protein (Fig. 3) contentfor these zones was found to be decreased. Such results were expected, sincetissues showed necrosis in the presence of ABA at a concentration of 10~4 M.Next, changes in the chloroplast Ru-DP carboxylases were tested (Tables 1and 2). As more significant results were obtained with 10~5M ABA, a largenumber of tissue explants were cultivated (fresh weight=300 g) in Erlenmeyerflasks normal MS medium with or without ABA at 10~5 M. The Results are reportedin Table 1, as nanomoles of CO2 incorporated per mg of Chi, per ml and per hr,versus sucrose density. As can be seen, ABA has an inhibitory effect on all thezones except on the high density group of chloroplasts.Such an inhibition of Ru-DP carboxylase activity by ABA was abo obtainedfor the 110 day-old tissues, pretreated 17 hr in a phosphate buffer pH 6.1 (Table 2)containing 10~5 M ABA.When comparing Ru-DP carboxylase activity of untreated 48 day-old and 110day-old tissues, it may be noticed that CO2 fixation characterised mainly the chloro-plasts of 33, 42, 51, 57% zones. As a matter of fact, in old tissues, the larger numberof heavy chloroplasts (57, 60, 63 and 66% sucrose), less active in CO2 fixation, leadsthe tissues towards necrosis.Table I CO2 incorporated in organic compounds expressed in HM for two parameters {per mg ofCHL and per mgof NP) in zones of isolated chloroplasts from carrot tissues (cultivated 48 days on the Murashige andSkoog medium,with or without l-Kh5 M ABA)Average index ofrefraction "±Density D42° (/>)No ABA abCO2<»CO2<2>+ A B A abCO2<»CO2<2>331.38650.00301.14135.5189071613.5127.0438047513.8421.41390.00471.21392.0406081518.543.512402689.3Concentration of sucrose in %511.43100.00281.2574.65078217.58.26223114.1571.43900.00141.2802.85996262.924.012902815.2601.44710.00411.30428.617102434.028.418402433.7631.45260.00361.31614.212903013.311.015904495.3661.45590.00171.32216.512902573.38.312403562.4a: /*gof CHL/ml b:/igofNP/mlCO2 («: CO2 per mg of CHL/ml/hr CO2 <2>: CO2 per mg of NP/ml/hr" Evaluation for 9 experimentsRelative standard error in the determination of chlorophylls and protein nitrogen respectively: 6%and 0.25%.816 S. Maillard-Sevhonkian and P. E. PiletTable 2 CO% incorporated in organic compounds expressed in mi for two parameters {per mg ofCHL and per tngof NP) in zones of isolated chloroplastsfrom carrot tissues (cultivated 110 days on the Murashige and Skoog medium)and pretreated in a phosphate buffer, pH 6.1, with or without l-lCh5 M ABA for 17 hrNo ABA+ABAabCO2 <»CO2(»abCO2WCO2<2>3379.02000107042.0103.0175060235.44259.0107034419.098.683028233.5Concentration of sucrose in %5144.52120248052.291.220302059.25738.01520885.022.124.974033211.26024.4783237.27.418.47403779.56355.21570149.05.215.911104656.76642.81610155.64.114.09004156.5For a, b, COz*1' and CO2;2>, see Table 1.The chloroplast pattern of ABA-treated tissues is similar to that of old tissues;groups I and III remain. After treatment with ABA, two important populationsof chloroplasts appear in the 51 and 57% sucrose zones, in an identical pattern as for110 day-old tissues. ABA accelerates explant aging and produces the typicalchloroplast pattern of old tissues. With 10~4 M, "senescence" is set, almost allgroups of chloroplasts are touched. Those of group III are destroyed, and thetissues become necrotic.ConclusionTo explain the present data, two complementary hypotheses must be brieflypresented. ABA may first have a direct action on chloroplast differentiation—aspreviously reported (7)—in carrot tissues cultivated in vitro, causing thereby anacceleration of tissue maturation. But ABA could also control the metabolism—asalready discussed (72)—of the carrot tissues cultured in vitro, inducing some agingprocesses which could very well explain a rapid increase in the chloroplast content,usually higher in senescent tissues.On the other hand, whichever type of treatment is applied to the tissues (pre-treatment or continuous application of ABA), it always results in an inhibition ofthe Ru-DP carboxylase of chloroplasts isolated from the carrot tissues cultivatedin vitro and a strong decrease in the 14CC>2 fixation as well.We express our thanks to Dr. Rose-Marie Hofer, from our Institute, for her helpful participationin the present work.References( / ) Amon, D. I.: Copper enzymes in isolated chloroplasts in Beta vulgaris. Plant Physiol. 24: 1—15(1949).(2) Bourdu, R., Y. Mathieu, M. Miginiac-Maslow, R. Remy and A. Moyse: Structure granaire,reduction du NADP et photophosphorylation des chloroplastes isoles de feuilles d'orge. Planta80: 191-210 (1968).ABA and the RuDP-carboxylase in chloroplasts 817( 3) Bruinsma, J.: The quantitative analysis of chlorophylls a and b in plant extracts. Photochem.Photobiol. 2: 241-249 (1963).(4) Gautheret, R. J.: La culture des tissus vig'etaux. Masson Ed., Paris, 1959.( 5) Hanson, A. D. and J. Edelman: Photosynthesis by carrot tissue cultures. Planta 102: 11-25(1972).( 6) Leech, R. M.: The isolation of structurally intact chloroplasts. Biochitn. Biophys. Ada 79:637-639 (1964).( 7) Milborrow, B. V.: The chemistry and physiology of abscisic acid. Ann. Rev. Plant Physiol.25:259-307(1974).( 8) Pilet, P. E. and M. Fragata: Prot£ines et gradients auxines-oxydasiques dans les racines decarotte cultivees in vitro. Rev. gin. Bot. 70: 572-587 (1963).(9) Pilet, P. E.: Auxines et polarity morphologique des tissus de Carotte cultives in vitro, ibid.70: 572-587 (1963).(10) Pilet, P. E. and M. Fragata: A propos de l'extraction et de l'absorption du tryptophane dans lesracines de Carotte cultivees in vitro. C. R. Acad. Sci. 261: 513-516 (1965).(//) Pilet, P. E. and J. Cl. Roland: Effects of abscisic acid on the growdi and ultrastructure of tissuescultivated in vitro. Cytobiologie 4: 41-61 (1971).(12) Pilet, P. E.: ABA effects on growth in relation to auxin, RNA and ultrastructure. In Hormo-nal Regulation in Plant Growth and Development. Edited by H. Kaldewey and Y. Varder. p. 297-315.Verlag Chemie, Weinheim, 1972.(13) Roland, J. Cl. and P. E. Pilet: Modifications ultrastructurales des parois de cellules de Roncesous l'effect de l'acide abscissique. C. R. Acad. Sci. 272: 72-75 (1971).(14) Shumway, L. K., T. E. Weier and C. R. Stocking: Crystalline structures in Viciafaba chloro-plasts. Planta 76: 182-189 (1967).(15) Spencer, D. and S. G. Wildman: Observations on the structure of grana-containing chloroplastsand a proposed model of chloroplast structure. Aust. J. Biol. Sci. 15: 599-610 (1962).(16) Spencer, D. and H. Unt: Biochemical and structural correlations in isolated spinach chloro-plasts under Uotonic and hypotonic conditions, ibid. 18: 197-210 (1965).(17) Wildman, S. G., K. Chen, J. C. Gray, S. D. Kung, P. Kwanyuen and K. Sakano: Evolutionof ferredoxin and fraction I protein in the genus Nicotiana. In Genetics and Biogenesis ofChloroplasts and Mitochondria. Edited by P. S. Perlman, C. W. Birky and T. J. Byers. StateUniv. Press, Columbus. Chap. 9, p. 309-329. 1975.

Effect of abscisic acid on differentiation and ribulose diphosphate carboxylase of chloroplasts

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