Jul 8, 1986 - the lower temperature while that of sucrose synthase was increased at the higher .... was terminated by the addition of 1 ml of Sumner reagent.
Plant Physiol. (1987) 84, 447-449
0032-0889/87/84/0447/03/$0 1.00/0
Effect of Night Temperature on the Activity of Sucrose Phosphate Synthase, Acid Invertase, and Sucrose Synthase in Source and Sink Tissues of Rosa hybrida cv Golden Times Received for publication July 8, 1986 and in revised form February 2, 1987
ELI KHAYAT* AND NAFTALY ZIESLIN Department of Ornamental Horticulture, The Hebrew University of Jerusalem, Rehovot 76100, Israel (HTR). During the day, temperatures were maintained in both treatments at a minimum of 18°C and a maximum of 30°C (21). Sucrose phosphate synthase and acid invertase activities in the mature In plants grow at both night temperature regimes branches leaves of roses (Rosa hybrida cv Golden Times) were greater in plants bearing flower buds with unopened sepals were decapitated above grown under a higher night temperature than under a lower temperature the third 5-leaflet leaf from the top. The activity of SS (UDPregime. In young shoots, the activity of acid invertase was promoted by glucose:i-fructose 2-glucosyl transferase, EC 2.4.2.13) was measthe lower temperature while that of sucrose synthase was increased at ured in the newly growing, second shoot from the top of the the higher temperature. At both temperatures benzyladenine when ap- branch. The activity of SPS (UDP-glucose:-fructose-6-P 2 gluplied to the axillary bud stimulated sucrose phosphate synthase activity cosyl transferase, EC 2.4.1.14) was determined in the leaf adjaand advancement of its peak of activity in the leaf subtending to the bud, cent to this shoot at 5- or 10-d intervals following decapitation. and also stimulated sucrose synthase activity in the young shoot. At the Acid invertase (f3-D-fructofuranoside fructohydrolase, EC lower temperature, application of benzyladenine to the axillary bud 3.2.1.26) activity was measured in both tissues. All samples were stimulated acid invertase activity in the young shoot but not in the leaves. harvested and extracted at 0800 (about 2 h after the transition of dark to light periods). Enzyme Extraction. Young shoots, 10 to 15 mm long, or their subtending leaves, were ground by hand in liquid N2 and homogenized (3 g) in 25 ml of extraction medium containing: 50 mM Hepes-NaOH buffer (pH 7.5), 0.5 mM Mg C12-H20, lm M Flower bud atrophy in rose plants is promoted by a decline in Na2 EDTA, 2 mM diethyldithiocarbamic acid (DIECA), 2.5 mM solar radiation and a drop in temperature (21, 22). The effects DTT, 1% BSA, and 2% PVPP, employing a modification of a of these two environmental factors are more pronounced in the method described previously by Schaffer (19, 20). The extract lower shoots on the stem than in the upper ones (23). Atrophy was filtered through six layers of cheesecloth and centrifuged for of the flower buds has been attributed to a reduced transport of 20 min at 20,000g. The supernatant was passed through a assimilates to the apices at the early stages of shoot development Sephadex G-25 column (4 x 350 mm) and assayed for enzymatic (10, 14) and to alterations in the distribution of assimilates activity. The results of a series of control experiments show 90% among the various parts of the plant (10). The application of cytokinins to axillary buds or sprouting shoots resulted in in- recovery of the activity of all three enzymes examined. SPS and SS Assay. Aliquots (70 Al) of tissue extract were creased mobilization of carbohydrates at the treated apex (15) incubated for 30 min at 37°C with an equal volume of reaction and reduced flower bud abortion (24, 25). Sucrose is the main carbohydrate transported in plants. SPS' mixture according to Rufty and Huber (18), with minor modiplays a key role in the conversion of triose-P to sucrose in source fications: for SPS assay, it contained 15 mm UDP-glucose, 15 leaves and may be subject to coarse control by the demand of mM fructose 6-P, 5 mM MgCl2.6H20, 5 mM NaF, 5 mm Na2 the sink tissues for assimilates (18). SS and acid invertase are MoO4. 2H20, and 50 mM Hepes-NaOH buffer (pH 7.5), and for present in source and sink tissues (7). Both sucrose synthase and sucrose synthase assay the fructose 6-P was replaced by fructose. acid invertase activities in the sink tissue can be taken as a Reaction was terminated by the addition of 70 ,l of 1 N NaOH, and sucrose formation was determined by the resorcinol colorireflection of the mobilization strength of the sink (3, 4). The purpose of the present study was to investigate the effect metric method (18). Enzymatic activity was determined by refof night temperature and cytokinin application on the activities erence to a blank reaction mixture from which the UDP glucose of SPS, SS, and acid invertase in source and sink tissues extracts was excluded. The activities of SPS and SS were also examined of rose plants. by HPLC using a strong anion exchange column (Partisil SAX, Whatman) and measuring UDP release (9). Differences between the results obtained by the two methods were negligible and their MATERIALS AND METHODS average is presented here. Plant Material. Rose plants (Rosa hybrida cv Golden Times) Acid Invertase Assay. Acid invertase activity was determined grown under greenhouse conditions (21) were exposed to two according to Schafer (19). Aliquots (200 ul) ofplant extracts were different night temperature regimes: (a) 12°C (LTR) and (b) 18°C incubated for 30 min at 37°C with an equal quantity of 1 M sucrose and 600 ul phosphate citrate buffer (pH 5.0). Reaction ' Abbreviations: SPS, sucrose phosphate synthase; SS, sucrose synwas terminated by the addition of 1 ml of Sumner reagent. thase; HTR, high temperature regimen; LTR, low temperature regime; Enzyme blanks were incubated in Sumner reagent. Activity was PVPP, polyvinylpolypryolidine. expressed as the quantities of reducing sugars formed by sucrose ABSTRACT
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KHAYAT AND ZIESLIN 448448
Plant Physiol. Vol. 84, 1987
both temperature treatments a peak of SPS activity was observed in the leaves on the 10th d following decapitation, even though in LTR plants shoot elongation was retarded by more than 10 d relative to HTR plants (Fig. 2). Under both temperature regimes application of BA to the bud resulted in more rapid shoot elongation and earlier bud sprouting, (Fig. 2); moreover, SPS activity peaked 5 d earlier (on the 5th d following decapitation) RESULTS than in untreated plants (Fig. 1). Treatment with BA prior to Prior to decapitation, the activity of SPS was higher in leaf decapitation also resulted in a marked increase of SPS activity extracts of plants exposed to HTR than to LTR (Fig. 1). For in the leaves, which was more pronounced under HTR than under LTR. By the 20th d following decapitation, SPS activity under both temperature regimes had declined below starting levels. Acid invertase activity in leaf extracts of plants was also higher 30 under HTR than under LTR (Table I). Application of BA to the bud did not effect the activity. The activity of acid invertase measured in the extracts of shoots 1 cm in length was significantly greater than in the mature leaf tissue; it was also significantly greater in young shoot tissue of plants exposed to LTR than in HTR (Table I). Acid invertase activity in the shoot extracts 7ri increased by the application of BA to the bud only in plants exposed to LTR (Table I). The activity of SS detected in the extracts of young shoots was greater in plants grown under HTR than under LTR (Table I). Application of BA caused an increase in SS activity of 90% at HTR and of 140% at LTR.
hydrolysis (10). Cytokinin Treatment. The lateral bud second from the top of the decapitated branch was treated with a lanolin paste containing 0.75% (w/w) BA immediately after decapitation. Lateral shoot elongation was measured in the various treatments.
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DISCUSSION The floral apex of the young rose shoot during the first few days after sprouting is extremely sensitive to changes in environmental conditions such as light and temperature (8, 12, 22). At this stage of development the photosynthetic capacity of the shoots is low (1); their growth and normal flower bud develop15 5 10 20 ment therefore depend on their ability to mobilize assimilates DAYS AFTER DECAPITATION from the leaves of the previous growth cycle (13). The activity of FIG. 1. Effect of hiight temperature and the application of BA on the SPS, which plays a major role in the regulation of sucrose activity of SPS in the second leaf from the top at 5- to I0-d intervals synthesis (7), can serve as an indicator of the availability of following decapitation. Values are means of 3 samples SD. *, 12C; 0, carbohydrates for export from the source leaves (1 1). 12C + BA; A, 18C; A, 18C + BA. No SD bars are shown where the The activity of SPS was lower in the extracts from the leaves values are smaller than the symbol sign. of plants grown under the lower than under the 4igher night temperature regime (Fig. 1). However, sucrose concentrations in 401 1 A I the leaves were higher at the lower temperature (10). Thus, a higher concentration of sucrose is not necessarily indicative of a higher rate of synthesis, but may also result from the accumulation of sucrose compartmented in the vacuoles (5). The attainment of the maximum level of SPS activity defected in the extracts of the source leaf (Fig. 1) precedes the phase of rapid shoot elongation by a few days (Fig. 2). Application of BA to the axillary bud (sink tissue) was accompanied by a sharp rise in the J activity within the leaf (source tissue) and an advancement of the peak of its activity by 5 d. This may reflect an increased X- 201demand for carbohydrates by the sink as a result of BA application (13). It may also suggest that increased sink activity precedes the acceleration of growth. More SPS activity was defected by 4( the application of BA to the bud after decapitation occurred at both temperature regimes, but was more pronounced in plants 10h grown under HTR. The gradual decline in SPS activity may indicate a gradual shift of the growing lateral shoots to a state of self-supply of carbohydrates; however, it may also be attributable to senescence of the source leaves (2). VMI Acid invertase activity is higher in the extracts of sink tissue 15 25 10 20 30 of young lateral shoots than in the mature source leaves (Table DAYS FROM DECAPITATION I). Similar observations have been reported for in other plants FIG. 2. Effect of night temperature and the application of BA on the (6, 19). Surprisingly the activity of acid invertase in the extracts growth of the second axillary shoot from the top, following its decapita- of young shoots was higher in the plants exposed to LTR (Table tion. Values are means of 20 branches SD. *, 12C; 0, 12C + BA; A, I) which showed slower growth rate than plants under HTR (Fig. 1). Perhaps this increase of acid invertase activity by low tem18C; A, 18C + BA. 0
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Plant Physiol. (1986) 81, 577-583 0032-0889/86/8 1/0577/07/$0 1.00/0
Effects of Orthophosphate and Adenosine 5'-Phosphate on Threonine Synthase and Cystathionine ey-Synthase of Lemna paucicostata Hegelm. 6746 Received for publication November 19, 1985 and in revised form February 24, 1986
JOHN GIOVANELLI*, S. HARVEY MUDD, ANNE H. DATKO, AND GREGORY A. THOMPSONI Laboratory of General and Comparative Biochemistry, National Institute ofMental Health, Building 32, Room 101, 9000 Rockville Pike, Bethesda, Maryland 20892 ABSTRACT Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine y-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine 'y-synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts.
nine synthase and cystathionine y-synthase of Lemna, and examines the physiological significance ofthese effects on threonine and methionine biosynthesis. MATERIALS AND METHODS
General Methods. Methods have been described previously for paper electrophoresis and paper chromatography (7), for determination of protein in crude enzyme extracts (30), and for determination of total plant protein (9). 32P was determined with an efficiency of at least 90% in a Beckman model LS 6800 liquid scintillation counter. Solvents. Solvents for chromatography include: solvent A, isobutyric acid:H20:15 M NHI,OH:0. 1 M Na2EDTA (100:55.8: 4.2:1.6, v/v); solvent B, propyl acetate: HCOOH: H20 (11:5:3, v/v). Chemicals. Preparation of [U-'4C]OPH has been described (14). Other compounds were obtained commercially. Plants. Lemna paucicostata Hegelm. 6746 was grown in medium 4 with 20 gM inorganic sulfate (9), except that the standard concentration of Pi in the medium (400 uM) was varied as noted for individual experiments. For preparation of media containing 25 or 50 mm Pi, required amounts of solid KH2PO4 were dissolved in standard medium, the pH adjusted with KOH to 5.85, and the solution filter sterilized. Values for frond- d equivalent to a frond * doubling were derived from an expression given by Datko and Mudd (3). Determination of Pi and AMP. Lemna, pre-grown for at least 4.5 doublings with unlabeled Pi at concentrations of 1 FM, 400 ,M, or 25 mm, was cultured to isotopic equilibrium (4.4-5.2 doublings) in medium containing 32Pi at corresponding concenIn plants, threonine synthase and cystathionine -y-synthase trations, and specific radioactivities ranging from 60 to 10 cpm/ catalyze the first committing steps in the threonine and methio- nmol at the times of inoculation. Plants were rinsed with nonnine biosynthetic branches of the aspartate family of amino acids radioactive medium of the same chemical composition as used ( 11) (Fig. 1). These two enzymes would therefore be expected to for growth, frozen in liquid N2, and homogenized in 10% TCA play important roles in regulating the entry of 4-carbon units containing [3H]AMP (500 nmol, and known amount of radiofrom OPH2 into the two biosynthetic branches. While it is activity) and 0. 15% (w/v) 8-hydroxyquinoline to prevent adsorpestablished that cystathionine y-synthase is a major site for tion of phosphate esters to the TCA-insoluble fraction (18). regulation of methionine biosynthesis (13, 30, 31), studies of Radioactivity in the TCA-insoluble and -soluble fractions was threonine synthase have failed to reveal any regulatory property determined (4). The TCA-soluble fraction was extracted with that would allow threonine to regulate specifically its own syn- ether, and electrophoresed in 0.35 M acetic acid:0.47 M HCOOH thesis (14). Recently it was reported in preliminary form (33) (pH 1.9) at 2000 V for 2 h. The proportion of 32p that migrated that Pi and AMP each inhibits threonine synthase. Pi inhibits as a peak between 8 and 14 cm toward the anode provided a also cystathionine y-synthase, but less efflciently than threonine measure of 32Pi. The radiopurity of this material was demonsynthase. This work details the effects of Pi and AMP on threo- strated by its comigration with authentic 32Pi during paper chromatography with solvents A and B. 32p that remained with [3H]'Present address: Calgene, Inc., Davis CA 95616. AMP at the origin of the electrophoretogram was eluted and 2Abbreviations: OPH, O-phospho-L-homoserine; AdoMet, S-adeno- further purified by successive paper chromatography with solsylmethionine. vents A and B. After chromatography with the latter solvent, a 577
