of Etiolated Avena sativa Coleoptilest. Received for publication October 9, 1973 and in revised form February 9, 1974. A. W. BOWN AND T. AUNG2. Department ...
Plant Physiol. (1974) 54, 19-22
ThelInfluence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptilest Received for publication October 9, 1973 and in revised form February 9, 1974 A. W. BOWN AND T. AUNG2
Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada ABSTRACT The influence of indoleacetic acid, 0.03 % C02, and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein,
and the level of soluble protein. The combination of indoleacetic acid and C02 stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and C02. Evidence is presented that cycloheximide inhibited the stimulation of protein synthesis by C02, and that indoleacetic acid increased the incorporation of '4Cbicarbonate into protein. These data are discussed in the context of CO2-stimulated growth of etiolated tissue, and proposals that CO2-stimulated growth involves dark CO2 fixation.
The synergistic relationship between IAA and 0.03% CO2 in stimulating the growth of etiolated Avena coleoptile sections has been previously described (1), and the involvement of protein synthesis in auxin-stimulated growth is well documented (4, 10, 13). Experiments reported here were designed to study the separate and combined influence of IAA and CO2 on coleoptile protein metabolism, and to test the proposal that CO2stimulated growth is mediated by a process involving both dark CO2 fixation and CO2-stimulated protein synthesis (16). The results suggest that both IAA and CO2 stimulate protein synthesis, that CO2-stimulated protein synthesis involves dark CO2 fixation, and that elevated rates of protein synthesis result in increased incorporation of labeled bicarbonate into protein. It is proposed that CO2-stimulated growth is dependent on C02stimulated protein synthesis. MATERIALS AND METHODS "4C-sodium bicarbonate (59 mc/mmole) and L-"C(U)leucine (331 mc/mmole) were purchased from Amersham Searle; cycloheximide and IAA were purchased from the Sigma Chemical Company. Seeds of Avena sativa var. 'Victory' were grown and harvested as described previously (1). Except where mentioned, the 20-mm coleoptile section below the 3-mm tip was used.
Incubation Conditions. Weighed batches of tissue in 50-ml light-tight incubation tubes were submerged in 10 ml of phosphate buffer (25 mm, pH 7.5) and incubated at 25 C in the presence or absence of various test substances. In experiments involving 14C-leucine, the buffer was aerated with air or CO2free air at a rate of 10 I/hr batch of tissue; incubations involving "4C-bicarbonate were in closed tubes without aeration. The CO2 concentration used had no measurable influence on the pH of the incubation medium, and was measured with an infrared gas analyzer. Protein Determination. After incubation, tissue was washed thoroughly with water, frozen for 3 to 4 hr, thawed, and then ground vigorously in a Potter-Elvehjem tissue homogenizer for 5 min with 5 ml of phosphate buffer (1 mM, pH 7.5). The homogenate was centrifuged at 13,000g for 15 min, the supernatant fluid was collected, and the extraction procedure was repeated two more times with pH 7.5 phosphate buffer at 25 mm and 0.10 M. The three supernatant fluids were then combined. An equal volume of saturated ammonium sulfate was added to the combined extracts and, after 12 hr at 4 C, the precipitate was collected by centrifugation at 25,000g for 30 min. The pellet was washed with 5 ml of saturated ammonium sulfate containing nonradioactive leucine (0.1% w/v), centrifuged at 25,000g for 30 min, and then dissolved in phosphate buffer (25 mm, pH 7.5). Aliquots of this solution were used for soluble protein determination by the standard method of Lowry et al. (12), and for radioactivity determination. Radioactivity Determination. Duplicate 0.2-ml aliquots of the protein preparations were dissolved in 10 ml of a scintillation fluid containing in 1 liter, 333 ml of ethanol, 667 ml of toluene, 3.5 g of 2,5-diphenyloxazole, and 200 mg of 1,4bis(2, 4-methyl-5-phenyloxazolyl)benzene. Radioactivity was measured using a Packard Tri-Carb liquid scintillation spectrometer, Model 3310. RESULTS Figure 1 shows that in the presence of IAA and 0.03% CO2, the rate of radioactive leucine incorporation into soluble protein was linear for up to 3 hr of incubation. Figure 2 shows that the soluble protein level rose linearly for nearly 2 hr and then continued to rise more slowly. Similar but smaller changes were found in the absence of both factors. IAA and 0.03% CO2 stimulated the soluble protein levels by 35 % and 21 %, respectively, and increased radioactive leucine incorporation by 27% and 17%. In the presence of both factors, the rate of leucine incorporation was increased 59%, a value greater than the sum of the stimuli when both factors were used separately. This synergistic relationship between IAA and CO2 is not reflected in the soluble protein levels where the combined effects of these agents seems to be roughly additive (Table I).
'This study was supported by a National Research Council of Canada grant to A.W.B., and a Colombo Plan Scholarship to T.A. 2 Present address: Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada. 19
20
BOWN AND AUNG
synergistic stimulation of protein levels, and an approximately additive influence on the level of leucine incorporated into protein. In many respects, the presence of the tip of the coleoptile exerts an influence on protein metabolism similar to that of IAA (Table I). The results of investigations into the influence of malate and CO, on protein metabolism are shown in Table III. Both malate and CO2 stimulated the level of protein and the level of leucine incorporation to approximately the same extent; but while elevating these values further, the combined influence of both factors did not result in an additive influence on protein
200 0 x
120[
-C Q)
E 40 1 U-
0
0
Plant Physiol. Vol. 54, 1974
60
120
180
TIME (m;n) FIG. 1. Kinetics of the incorporation of radioactive leucine into soluble protein. Batches of 0.5 g of coleoptile tissue were incubated with 0.03% C02 and 20 AM IAA for 30 min before the addition of 8 X 105 dpm of "4C(U)-leucine. The incubations were terminated at
the times indicated.
3.6 3.2
levels or leucine incorporation. Thus the relationship between CO2 and malate seems to be different from that between CO2 and IAA. Table IV shows that cycloheximide reduced protein levels by approximately 5% and the rate of leucine incorporation into protein by 80%. Furthermore, in the presence of cycloheximide there was no significant stimulation by CO2 of soluble protein levels or leucine incorporation. A comparison of the data in Tables I to IV indicates that there was variability between experiments in the quantitative influence of test conditions on protein metabolism. Nevertheless, consistent qualitative results were obtained, and Tables I to IV show that in separate experiments CO. stimulated leucine incorporation into protein by 17, 43, 50, and 31%, respectively. Figure 3 indicates that the kinetics of incorporation of radioactive bicarbonate into soluble protein were linear for 60 min.
x 2.8 c
m 2.4
E
2.2 . 0
60
120
180
240
TIME (min) 2. of Kinetics FIG. changes in levels of soluble protein. Batches of 0.5 g of coleoptile tissue were incubated with 0.03% CO2 and 20 ,uM IAA for the times indicated.
Table 1. Inifliuenice of IAA anid CO! Onl Protei,i Metabolislmi Tissue (0.5 g) was incubated in buffer with or without 0.03'' CO2 in the presence or absence of 20 .m IAA for 120 min. After 30-min incubation, 8 X 106 dpm of 14C(U)-leucine was added. Results are expressed as the mean ± the standard deviation, and represent the average of duplicate determinations from four experiments. Figures in parentheses are percentages of values obtained in the absence of CO2 and IAA. Per Gram Fresh WN't Treatment
Treatment
AMg protein
-IAA -IAA +IAA +IAA
and and and and
-Co2 +CO2 -CO2 +CO2
2.66 3.22 3.60 4.04
i 0.10 + 0.37 + 0.44. ±+i 0.33
_____________
Dpm
(100)! 54,312 4 783 (100) (121) 63,536 i 821 (117) (135), 69,293 + 888 (127) (152) 86,318 + 1011 (159)
Table IL. Inifluenice of Coleoptile Tips and CO. Onl Preoteini Metabolism Coleoptiles (0.5 g), with or without the 3-mm tip, were incubated with or without 0.03% CO2 for 120 min. After 30-min incubation, 5 X 106 dpm of '4C(U)-leucine was added. Results are expressed as the mean + the standard deviation and represent the average of duplicate determinations from two experiments. Figures in parentheses are percentages of values obtained without tips or CO,. Per Gram Fresh WN't
Treatment
?sI,z protein
-Tips and -CO2 -Tips and +CO2 +Tips and -CO2 +Tips and +CO2
2.98 3.24 3.28 3.88
DTl)m
± 0.12 (100) 28,783 ± 718 (100)
± 0.05 (109) 41,088 + 745 (143) ± 0.07 (113) 47,443 + 841 + 0.14 (130) 57,762 + 870
(165) (206)
Table III. Ilnfluentce of Malc/te aiid C02 onl Proteini Metabolismn Tissue (0.5 g) was incubated with or without 0.03% , CO2 in the presence or absence of 0.5 mNi malate for 120 min. After 30-min incubation, 2 X 106 dpm of '4C(U)-leucine was added. Results are expressed as the mean ± the standard deviation, and represent the average of duplicate determinations from two experiments. Figures in parentheses are percentages of values obtained in the absence of CO2 and malate. Per Gram Fresh XV't Treatment
Table II shows the results of experiments to determine the influence of CO2 on the protein metabolism of coleoptiles with or without the 3-mm coleoptile tip present. The data indicate that both CO2 and the tips stimulated a significant increase in soluble protein and in the incorporation of labeled leucine into protein, and that the presence of both factors resulted in a
MIg protein
-Malate and -CO, -Malate and +CO2 +Malate and -CO2 +Malate and +CO2
3.03 i 0.11 3.53 + 0.07 3.59 ± 0.08 3.79 + 0.09
Dpm
(100) 25,888 + 552 (117)' 38,756 4 604 (118) 41,874 + 636 (125) 47,520 ±i 648
(100) (150) (162) (183)
Plant Physiol. Vol.
54, 1974
C02-STIMULATED PROTEIN SYNTHESIS
Table V demonstrates that during a 30-min incubation IAA stimulated by approximately 50% the incorporation of labeled bicarbonate into soluble protein. DISCUSSION Figure 2 shows that in the presence of CO2 and IAA the soluble protein level rose linearly for nearly 2 hr and then more slowly, demonstrating that, under the conditions used, the rate of protein synthesis exceeded the rate of degradation. The incorporation of radioactive leucine into soluble protein was linear for up to 3 hours (Fig. 1); thus, different levels of radioactivity after a 90-min incubation (Tables I to IV) indicate relative rates of incorporation. Given reported half-lives for proteins of 2 to 3 days (8), it is not likely that protein degradation would result in a significant loss of label during a 90-min incubation period, and different levels of incorporation are interpreted as indicating relative rates of protein synthesis, not changes in the rate of degradation. Thus the data in Tables I, II, and III indicates that IAA, CO, coleoptile tips, and malate all stimulate the rate of protein synthesis. Cleland (4) Table IV. In2fluence of Cycloheximide antd CO2 oni Proteinz Metabolism Tissue (0.5 g) was incubated with or without 0.03%c0 CO2 in the presence or absence of 6 ,ug/ml cycloheximide for 120 min. After 30-min incubation, 2 X 106 dpm of '4C(U)-leucine was added. Results are expressed as the mean i the standard deviation and represent the average of duplicate determinations from two experiments. Figures in parentheses are percentages of values obained in the absence of CO2 and cycloheximide. Per Gram Fresh Wt Treatment
Mg protein
-Cycloheximide and
Dpm
2.28 + 0.09
(100)1 15,922
2.67
(117) 20,827 ± 486 (131)
4
396 (100)
-Co2
-Cycloheximide and +C02 +Cycloheximide and -C02 +Cycloheximide and +C02
4
0.12
2.17 ± 0.07 (95)
2,834 i 188 (18)
2.19 + 0.05'(96)
2,924 i 190 (18)
100 2 x
60 cn Ul
E 2.0 0
TIME (m in) FIG. 3. Kinetics of the incorporation of "C-bicarbonate into soluble protein. Batches of 0.5 g of coleoptile tissue were incubated in closed containers in the presence of 20 AM LAA for 30 min before the addition of 2 X 10' dpm of "C-bicarbonate. The incubations were terminated at the times indicated.
Table V. Influence of IAA on the Inicorporationi of Radioactive Bicarbonate inito Soluble Proteini Twelve 0.5-g batches of coleoptile tissue from the same harvesting were incubated in closed containers for 60 min, six in the absence and six in the presence of 20 ,tM IAA. After 30-min incubation, 8 X 106 dpm of '4C bicarbonate was added. Results are expressed as the mean ±t the standard deviation and represent the average of duplicate determinations from the samples. Figures in parentheses are percentages of values obtained in the absence of IAA. Per Gram Fresh Wt
Treatment
-IAA
+IAA
Mg protein
Dpm
2.45 + 0.05 (100) 3.12 ± 0.10 (127)
4180 i 244 (100) 6333 ± 263 (152)
has shown that cycloheximide does not inhibit the uptake of labeled leucine into coleoptile tissue, and consequently, Table IV indicates that cycloheximide inhibited protein synthesis. There is substantial evidence that atmospheric levels of CO. can stimulate the growth of nonphotosynthetic plant tissue (1, 5, 7, 11, 17). Splittstoesser (16) proposed that CO2-stimulated growth involved dark CO2 fixation and the consequent synthesis of 4-carbon acids which can replace Krebs acid lost to the Krebs cycle during the biosynthesis of amino acids and other metabolites. He suggested that in the absence of CO2 the biosynthesis of amino acids and protein essential for growth would be reduced. This hypothesis is consistent with reports of dark fixation in which malate and aspartate are the first detectable labeled products (2, 15, 16), and with data indicating that in maize roots the rate of fixation of CO2 into a water soluble fraction is correlated with the rate of protein synthesis (16). In addition, atmospheric CO2 levels have been shown to stimulate the incorporation of labeled leucine into an ethanol insoluble residue from carrot and tomato roots (16), and it has been reported that malate can replace CO2 in stimulating the growth of etiolated coleoptiles (1). Despite this work, the dependency of C02-stimulated growth on CO2-stimulated protein synthesis has not been investigated. The data in Table IV demonstrate that cycloheximide inhibited C02-stimulated protein synthesis, and under similar conditions, it has been shown that cycloheximide inhibited CO2-stimulated growth of coleoptiles (1). It seems that CO2 not only stimulates growth and protein synthesis but also that C02-stimulated growth is dependent on protein synthesis. Further evidence supporting the involvement of CO2 fixation and protein synthesis in C02-stimulated growth is presented. Table III indicates that malate, a product of dark fixation, stimulated leucine incorporation into protein by 62% in the absence of CO2 and 33% in its presence. Similarly, the stimulation of leucine incorporation by CO2 was greater in the absence of malate. These data are consistent with malate and CO2 fulfilling a common function in protein synthesis, and reflect data showing that malate and CO2 are interchangeable in stimulating coleoptile growth (1). The hypothesis predicts that fixed CO2 would be incorporated into protein at rates dependent on the rate of protein synthesis. The data in Table V demonstrate that "C bicarbonate was incorporated into protein, and that the rate of incorporation was stimulated by IAA, which also stimulates the synthesis of protein (Table I). It cannot be concluded, however, that CO2-stimulated protein synthesis results in a subsequent stimulation of growth;
22
BOWN AND AUNG
the possibility remains that C02-stimulated growth occurs first, resulting in a later stimulation of protein synthesis that is necessary to maintain increased growth rates. Similarly, the data presented do not indicate the sequence by which IAA stimulates CO2 fixation, protein synthesis, and growth. It is interesting to compare the stimulation of protein synthesis by CO2 and IAA (Table I) to the reported synergistic growth stimulation by these two agents (1). The data demonstrate that the combination of CO2 and IAA stimulated incorporation of radioactive leucine by 59%, whereas the sum of the separate stimuli by CO2 and IAA amounted to 44%. Despite the synergistic stimulation of growth and protein synthesis, there is no reason to expect a close parallel between the rates of these two processes. Patterson and Trewavas (14) have indicated that the synthesis of many proteins is not affected by IAA, and it has been suggested that rapid cell elongation is limited by an unidentified growth-limiting protein whose synthesis or activation is stimulated by IAA (4). The somewhat similar stimulation of protein synthesis by IAA and coleoptile tips (Tables I and II) is not surprising in the light of reports that the coleoptile tip is a source of endogenous IAA (6). Nevertheless, since many experiments are performed using exogenous IAA, it is reassuring to find that applied IAA has a qualitatively similar influence on protein synthesis to that of the coleoptile tip. If, as appears probable, 0.03% CO2-stimulated growth results from dark fixation, tracing the molecular events from fixation to increased growth should result in relevant questions and insights concerning the growth process and the influence of growth regulators on it. For example, we have demonstrated ethylene inhibition of coleoptile growth in approximately 10 to 15 min (unpublished data), and in view of the antagonism between CO2 and ethylene in their influence on growth (3, 9), we are investigating the possibility that ethylene inhibition of growth is mediated by an inhibition of dark CO2 fixation.
Plant Physiol. Vol. 54, 1974 LITERATURE CITED
1. BOWN, A. W., I. J. DYMOCE, AND T. AUNG. 1974. A synergistic stimulation of Avena sativa coleoptile elongation by IAA and carbon dioxide. Plant Physiology. 54: 15-18. 2. BowN, A. W. AND W. W. LAMPMAN. 1971. The presence and role of phosphopyruvate carboxylase in etiolated coleoptiles of Avena sativa. Can. J. Bot. 49: 321-326. 3. BL-RG, S. P., AND E. A. BIJRG. 1967. Molecular requirements for the biological activity of ethylene. Plant Physiol. 42: 144-152. 4. CLELAND, R. 1971. Instability of the growth limiting proteins of the Avenia coleoptile and their pool size in relation to auxin. Planta 99: 1-11. 5. COCESHULL, K. E. AND 0. V. S. HEATH. 1964. Carbon dioxide effects on auxin responses of coleoptile sections. J. Exp. Bot. 15: 331-346. 6. GREENWOOD, M. S., S. SHAw, J. R. HILLMAN, A. RITCHIE, AND A\I. B. WILKINS. 1972. Identification of auxin from Zea coleoptile tips by mass spectrometry. Planta 108: 179-183. 7. HARRISON, A. 1965. Auxanometer experiments on extension growth of Arena coleoptiles in different carbon dioxide concentrations. Physiol. Plant. 18: 321-328. 8. HocK, B. AND H. BEEVERS. 1966. Development and decline of the glyoxylatecycle enzymes in watermelon seedlings. Z. Pflanzenphysiol. 55: 405-414. 9. KANG, B. G., C. S. YOCUm, S. P. BURG, AND P. M. RAY. 1967. Ethylene andl carbon dioxide. Mediation of hypocotyl hook opening response. Science 156: 958-959. 10. KEY, J. L. 1964. Ribonucleic acid and protein synthesis as essential process for cell elongation. Plant Physiol. 39: 365-370. 11. LEONARD, 0. A. AND J. A. PINUEARD. 1946. Effect of various oxygen and carbon dioxide concentrations on cotton root development. Plant Physiol. 21: 18-36.
12. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 13. NOODnN, L. D. AND K. V. THIm.ANN. 1966. Action of inhibitors of RNA and protein synthesis on cell enlargement. Plant Physiol. 41: 157-164. 14. PATTERSON, B. D. AND A. J. TREWAVAS. 1967. Changes in the pattern of protein synthesis induced by 3-indolylacetic acid. Plant Physiol. 42: 10811086. 15. SALTMAN, P., V. H. LYNCH, G. H. KuN\ITAXE, C. STITT, AND H. SPOLTER. 1957. The dark fixation of carbon dioxide by succulent leaves; metabolic changes subsequent to the initial fixation. Plant Physiol. 32: 197-200. 16. SPLITTSTOESSER, W. E. 1966. Dark carbon dioxide fixation and its role in the growtlh of plant tissue. Plant Physiol. 41: 755-759. 17. STOLWIJK, J. A. AND K. V. THIMANN. 1957. On the uptake of carbon dioxide and bicarbonate by roots and its influence on growth. Plant Physiol. 32: 513-520.
