Developmental Studies on Glyoxysomes in R I C I N U S Endosperm
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DEVELOPMENTAL STUDIES ON GLYOXYSOMES IN R I C I N U S ENDOSPERM B. P. GERHARDT and HARRY BEEVERS From the Department of Biological Sciences, Purdue University, Lafayette, Indiana 47906 . Dr. Gerhardt's present address is the Botanisches Institut der Universitat Koln, 5 Koln-Lindenthal, Germany. Dr. Beevers' present address is the Division of Natural Sciences, University of California, Santa Cruz, California 95060 ABSTRACT The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient . It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized . We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat break- down correspond to the net production and destruction of this organelle . INTRODUCTION During the germination of fatty seeds such as those early in germination is now known to be due to a of castor bean, the stored fat is converted to de novo synthesis of the enzymes (6, 8, 11, 15) that carbohydrate (1) . One stage in the reaction are present in the plants only during the period sequence from fat to carbohydrate is the glyoxylate when fat is being utilized . cycle (9) which converts acetyl CoA, arising from In castor bean seeds, the fat is localized in the the breakdown of fatty acids, to succinate. During endosperm, and the carbohydrate produced, the germination period the key enzymes of the principally sucrose, is absorbed by the cotyledons glyoxylate cycle, isocitrate lyase and malate syn- of the growing embryo (10) . The rate of fat thetase, undergo striking changes in activity (4, conversion reaches its maximum at the 5th day of 18). From extremely low levels in the unger- germination. From the endosperm of 5-day-old minated seeds, the activities of both enzymes seedlings Breidenbach and Beevers (2, 3) isolated increase rapidly until the time when the rate of a new kind of organelle carrying the enzymes of conversion of fat to carbohydrate in the endosperm the glyoxylate cycle and therefore named the reaches its maximum at day 5 . Subsequently the glyoxysome . This finding raised the possibility that activities decline . The increase of these activities the striking changes observed in the activities of 94 isocitrate lyase and malate synthetase during Inc ., Lincoln, Nebraska) . All operations were carried germination could be related to the formation and out at 0-4 °C. disappearance of this organelle. Biochemical Assays MATERIALS AND METHODS All enzymes were assayed spectrophotometrically . Isocitrate lyase and malate synthetase were assayed Preparative Procedures essentially as described by Hock and Beevers (8) . Succinic dehydrogenase was assayed by the method of Seeds of castor bean, Ricinus communis L . var. Baker Hiatt (7) with the use of an extinction coefficient 296 or Hale, were soaked for 12-16 hr in tap water at 1 .56 X 10 7 cm2/mole for dichlorophenol indophenol room temperature and then placed in moist (16) . Catalase was assayed by a slight modification of vermiculite at 30 °C and constant humidity in the the method of Luck (12) with the use of an extinction dark (day 0) . Seedlings were harvested at daily in- coefficient 6 .7 X 10 4cm2/mole for H2O2 (13) . Protein tervals, the testa and the embryo (root, hypocotyl, was determined by a modification of the Lowry pro- and cotyledons) were removed, and only the endo- cedure (5) . Sucrose concentrations (w/v) were deter- sperm tissue was used as experimental material . This mined with a Bausch & Lomb Abbe Refractometer tissue was homogenized in a solution of 0 .4 M sucrose, (Bausch & Lomb, Inc., Rochester, N.Y.) . 0.167 M Tris buffer pH 7 .5 or 0 .167 M Tricine buffer (California Biochemical Corp ., Los Angeles, Calif.) Labeling Experiments pH 7.8, 0 .01 M dithiothreitol, 0.01 M KCl, 0.001 M MgCls, and 0.01 M ethylenediaminetetracetic acid 0.25 µc of leucine- 14C (8 .0 X 10-4 µ.moles in 5 µl) (EDTA), pH 7.5. Usually 2 ml of medium was used or 2 .5 µc of leucine-3H (6.25 X 10-5 moles in 5 µ1) for each gram of tissue . After the tissue was treated were applied directly to each of 10-20 endosperm in an onion chopper, it was carefully ground in a halves (17) . The tissue was incubated at 30 °C in mortar, and the resulting crude homogenate was moist atmosphere in Petri dishes for a definite time of filtered through two layers of cheesecloth. After treatment (2-6 hr) and then homogenized together centrifugation at 270 g for 10 min, the supernatant with unlabeled endosperm. Organelle fractions were solution (supernatant I) was centrifuged at 10,800 g isolated as described above . The incorporation of for 30 min . The resulting pellet (the crude particu- radioactivity into these fractions was determined by late fraction) was resuspended in a few ml of 32% the method of Mans and Novelli (14) and the pre- sucrose (w/v) and layered on the top of a sucrose cipitated protein samples were assayed on a Beckman density gradient . Discontinuous gradients were pre- DPM Scintillation counter (Beckman Instruments, pared by adding 5 ml of 60% sucrose, and 10 ml of Inc ., Fullerton, Calif.). For the double labeling exper- 57% sucrose, 15 ml of 51% sucrose, 15 ml of 44% iments, the window of the 3H-channel was adjusted sucrose, and 5 ml of 32% sucrose, in sequence in so that the overspill of 14C was about 15% (internal cellulose nitrate tubes . Usually, the crude particulate standard). No 3H was detected in the 14C-channel . fraction obtained from 40 g of tissue was applied to such gradients . Linear continuous gradients were pre- RESULTS pared from 19 .5 ml of 32% sucrose and 19.5 ml of 60% sucrose over a cushion of 11 ml of 60% sucrose, Protein and on these gradients the particulate fraction from During the first 3 days of germination of the not more than 10 g of tissue was applied . For the ex- castor bean seeds the protein content of the whole periments in which mitochondria were purified by flotation, the mitochondrial fraction from a discon- endosperm remains constant, but, as shown in Fig . tinuous gradient was concentrated from 43 to 57% 1, the protein content of the crude particulate sucrose by adding 70% sucrose . This suspension was fraction increases rapidly until the third day . used as the cushion for a linear continuous gradient After the third day, when the total protein of the grading from 57 to 35% sucrose . All sucrose solutions endosperm begins to decrease, the protein of the for gradients contained 0 .001 M EDTA pH 7.5. The crude particulate fraction accounts constantly for gradients were centrifuged at 64,300 g in a Spinco about 13 % of the total protein . Thus, early in rotor SW 25 .2 (Beckman Instruments Inc ., Spinco germination a portion of the protein of the seed is Div., Palo Alto, Calif.) for 5 hr ; flotation gradients transformed into newly synthesized organelles, were spun for 8 hr. The gradients were fractionated from the bottom of the tubes . From discontinuous and at later stages, when a net loss of protein gradients the bands and interphases were usually col- occurs, total protein and particulate protein are lected as whole fractions ; from continuous gradients 1 degraded at the same rate (Fig. 1) . Eventually the ml fractions were collected by an Isco density gradient products are completely absorbed by the growing fractionator (Instrumentation Specialties Company, embryo (17) . B . P. GERHARDT AND HARRY BEEVERs Glyoxysomes in Ricinus Endosperm 95 H Y) 800 8000 F Z Q 0U F F Q 120 a 600 6000 W 0 100 0U \ 400 4000 Z Z W LIA 80 F I- 0 0 M a R 2000 E D: 200 a. CD E CP 60 E 0 2 4 6 8 40 DAYS OF GERMINATION FIGURE 1 Changes in the protein content of the 20 endosperm (supernatant I and of the crude particulate fraction during the germination of castor bean seeds . The results are expressed as mg protein/100 g fresh I I weight of endosperm and as mg protein/crude par- 2 4 6 ticulate fraction from 100 g fresh weight of endo- DAYS OF GERMINATION sperm . FIGURE 2 Changes in the protein content (mg pro- tein/100 g fresh weight of endosperm) of the mito- Centrifugation of the crude particulate fraction chondrial, proplastid, and glyoxysome bands isolated on a sucrose density gradient results in three on discontinuous sucrose density gradients during the major protein bands-the mitochondria, the pro- germination of castor bean seeds . plastids and the glyoxysomes (2) . From the 5th day onward, the protein present in these bands accounted for about 60% of that added as crude incorporated into trichloroacetic acid-precipitable particulate protein .