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Ribonucleic acid metabolism during growth of excised root tips from normal and X-irradiated corn seeds
487
BIOCHIMICA ET BIOPHYSICAACTA
RIBONUCLEIC ACID METABOLISM DURING GROWTH OF ROOT TIPS FROM NORMAL AND X-IRRADIATED
EXCISED
CORN SEEDS
JOE H. CHERRY* Department of Agronomy, University o] Illinois, Urbana, Ill. (U.S.A.) (Received July 24th, I961)
SUMMARY Growth of excised corn root tips was found to involve a degradation and loss of protein and ribonucleic acid. During growth (mostly cell elongation) of the normal excised root tips there is a maj or redistribution of ribonucleic acid among the subcellular components. The microsomal and soluble ribonucleic acids rapidly decline with a concomitant increase in the nuclear and mitochondrial fractions. There was little redistribution of ribonucleic acid in the root tips from X-irradiated seeds, since the transformation had already occurred as a result of X-irradiation prior to incubation of the root tips. Incorporation of uracil into the subcellular ribonucleic acid fractions b y excised root tips revealed that the greatest incorporation was in the nuclei, followed in order by mitochondria, microsomes and the soluble fraction. X-irradiation greatly enhanced the incorporation of uracil into ribonucleic acid. Growth of root tips from normal and X-irradiated seeds increased the ribonuclease activity as much as 2 to 4-fold. Root tips grown from X-irradiated seed had an initial ribonuclease activity 2 to 3-fold greater than normal tissue.
INTRODUCTION Previous results show that X-irradiation of dry corn seeds inhibits cell division in the root tip of the growing seedlingI. Growth of seedlings from X-irradiated seeds appears to be due to elongation of cells pre-existing in the embryo at the time of irradiation. Available evidence2-~ indicates that the RNA associated with the subcellular particulates of plants undergoes major changes during cell growth and maturation. These results show that more of the RNA and protein of mature plant cell homogenates sediment with the nuclear and mitochondrial fractions than in the case of meristematic cells, where the microsomal fraction contains most of the RNA. Recent evidence8 shows that X-irradiation decreases the content of microsomal RNA in the meristematic region of germinating seedlings. Apparently, X-irradiation prevents the synthesis of microsomal RNA as ordinarily occurs with cell division, but allows normal catabolism of the ribonucleoprotein granules in expanding cells. Abbreviation: TCA, trichloroacetic acid. " Present address: Southern Research Laboratory, USDA, ARS, ~New Orleans I9, La. ~U.S.A.). Biochim. Biophys. Aota, 55 (1962) 487-494
488
j . H . CHERRY
X-irradiation enhances the incorporation of precursors into the nucleic acids of the subcellular components of growing roots s. X-irradiation greatly enhances the RNAase activity in root tips, which normally is low in activity 9. The data reported here are on the metabolism of RNA during growth of excised root tips from normal and X-irradiated seeds. The incorporation of uracil into RNA of the subcellular components was examined. Studies were made on the level of RNAase as affected b y cell elongation and X-irradiation of dry seed. MATERIALS AND METHODS Corn seeds were X-irradiated with 250 k R and germinated in a dark, humid atmosphere at 29 ° as previously described 1°.
Growth o/ excised root tips Root tips, i cm long, were sectioned from primary roots of 2~-day-old seedlings grown from normal and X-irradiated seeds. The tissue, about o.5 g, was rinsed in deionized water, blotted, and rapidly weighed before use. Root tips were placed in petri dishes and incubated at room temperature with gentle shaking for I to 16 h. Each dish contained IO ml of a solution containing 1 % sucrose, lO -8 M CaC12, IO 3 M KHzPO4 (pH 6.3 with NH4OI-I) and 1.6 #C of [2-14C]uracil (0.2/~ mole). After various incubation periods the root tips were washed, weighed and frozen for analysis. The experiments designed to determine the level of RNAase activity were made on root tips, 0.5 cm long, sectioned from primary roots of 2~-day-old seedlings from normal and X-irradiated seeds. As above, the tissue was incubated with a solution containing I °/o sucrose, lO -3 M CaC12, IO-s M KI-I~PO4 (pH 6.3 with N H 4 0 H ). Atteivarious incubation periods the root tips were washed, weighed and frozen for RNAase assay.
Isolation o/ cytoplasmic particulates Sections of the primary root after incubation with uracil were homogenized in 5 ml of an ice-cold medium containing o.5 M sucrose, 30 % glycerol, o.oi M Tris (pH 7.4) and o.oi M CaCla in an ice-jacketed glass homogenizer with a power-driven Teflon pestle for 2 rain. The homogenates were strained through a layer of Dacron fabric (openings of 3O-lOO/~). An aliquot of the debris-free homogenate was subjected to differential centrifugation in a Spinco Model L ultracentrifuge, each successive particulate fraction being obtained from the supernatant of the preceding fraction. The entire isolation procedure was performed at o to 4 °. The centrifugal forces, time, and the arbitrary designation of the isolated fractions were as follows: nuclei, 15oo × g for I2 rain; h e a v y mitochondria, 20 ooo × g for 15 rain; light mitochondria, 40 ooo × g for 20 min; microsomes, IOO ooo × g for i h; soluble, particulate-free supernatant. Each of the particulate fractions were washed once by resuspending in o.5 M sucrose and resedimenting at the same centrifugal force and time. The terms used to designate the cellular fractions are operational, as listed above, and do not necessarily mean the presence of pure morphological entities.
Biochim. Biophys. Acta, 55 (1962) 487-494
RNA
METABOLISM
DURING
GROWTH
OF EXCISED
ROOT TIPS
489
Analysis o/ subcellular /factions The RNA of the total debris-free homogenate and of the cytoplasmic fractions was extracted and measured b y a perchloric acid method previously described 1°. The RNA content of the debris-free homogenate is reported as total R N A in Table I.
Determination o/ radioactivity Samples of the RNA or total nucleic acid hydrolysates were neutralized with K O H , and the KC10, was removed b y centrifugation in the cold. Samples of these neutralized hydrolysates were then dried and counted with a Picker gas-flow proportional counter (window thickness less than 0.5 mg/cm*).
Assays /or RNAase activity Sections of the o.5-cm root tip after incubation were homogenized in 5 ml of o.5 M sucrose with an ice-jacketed glass homogenizer with a power-driven Teflon pestle. The homogenates were cleared of cell debris and nuclei b y centrifugation at 500 x g for IO rain. These cleared homogenates were filtered through glass wool and were used directly as a source of enzyme. RNAase activity was determined b y incubating I ml of the cleared homogenate with the additivies described b y HANSON11 for root tips. Each determination was made in the presence of 0.25 M sucrose, 5.1o -4 M MgSO4, 5 . I o - 3 M KC1, and I mg/ml of yeast RNA (pH 6.5). Final volume was made to 2 ml; incubation was for 30 min at 3 °0 with gentle shaking. The p H of the incubation medium was about 6.0. One set of tubes of each determination was held in ice to establish the initial level of RNA. Experimentation showed no RNA degradation at this temperature. At the termination of incubation, the tubes were placed in ice, and HC104 and uranium acetate were added to 0.2 N and 6. lO -3 M, respectively. The precipitates were washed twice with 0.2 N HC10,. The supernatant solution and the two washes were combined, made to volume and the absorbancy determined at 260 and 290 m/z. The relative increase in absorbancy difference at 260-290 m# of the acid-soluble fraction was used to compute the RNAase activity. Protein was determined b y the method of LOWRY et al. 12 on aliquots precipitated and washed in 5 % TCA. RESULTS
Growth and metabolism o[ R N A in elongating excised root tips Control root tips grew much faster (4 times) than root tips from X-irradiated seed (Fig. I). There was a slight lag period in the growth of control root tips, followed b y a linear rate. Root tips from X-irradiated seed lost weight immediately on incubation and did not begin to grow until 4 h later. Since growth of these excised root tips is essentially b y cell elongation, these data agree with the histological observations t which indicate that cells in the X-irradiated root tips are more mature, and therefore would have a smaller capacity for cell expansion. Control root tips took up slightly more uracil (counts/min/g initial fresh wt.) than those from X-irradiated seeds (Fig. 2). The uptake was linear with time for the
Biochim. Biophys. Acta, 55 (1962) 487-494
49 °
J.H.
CHERRY
first 8 h, but afterwards uracil was lost from the roots. Previous work s indicated a leakage of nucleotides from excised roots during incubation which agrees with these data. As previously discussed ~, very young actively dividing cells are characterized by a high level bf microsomal RNA, whereas mature cells have few ribonucleo.,..; 50
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Fig. I (left top). A comparison of growth of excised root tips (I cm) from normal and X-irradiated (25o kR) seeds. Fig. 2 (right top). A comparison of [2-14C]uracil uptake with growth of excised root rips (I cm) from normal and X-irradiated (25o kR) seeds. Fig. 3 (left middle). Changes in RNA content of the various subcellular particulates with growth of control 2 I-day-old excised root tips (I cm). Fig. 4 (right middle). Changes in RNA content of the various subcellular particulates with growth of excised root tips (i cm) from X-irradiated (25 ° kR) seeds. Fig. 5 (left bottom). Incorporation of [2-14C]uracil into RIgA of the various particulate fractions by 2 I-day-old excised control root rips (i cm). Fig. 6 (right bottom). Incorporation of [2-14C]uracil into RIgA of the various particulate fractions by 2 I-day-old excised root tips (I cm) from X-irradiated (25 ° kR)seeds. Biochim. Biophys. Acta, 55 (I962) 487-494
RNA
METABOLISM
DURING
GROWTH
O F ]~XCISED
ROOT TIPS
491
protein granules. Most of the nucleic acid of mature cells is found in the mitochondrial and nuclear fractions. Fig. 3 clearly shows a transformation of RNA in the subcellular fractions as the root cells elongate (see Fig. I for amount of growth). Microsomal and soluble RNA rapidly decline as the tissue grows; concomitantly there is an increase in heavy mitochondrial and nuclear RNA. No significant change in the amount of light mitochondrial RNA was observed. These analyses were based essentially on the same number of cells, but primarily at different stages of cell expansion, and therefore provide important information on the metabolism of RNA in relation to cell maturation. Growth had little effect on the intracellular distribution of RNA in excised root tips from X-irradiated seed (Fig. 4). However, there was a decline in microsomal RNA, even though the initial content was low. Light mitochondrial RNA increased, apparently at the expense of the microsomes. Table I shows the changes in total RNA content during growth of excised root tips. There was a decline in RNA of normal root tips during incubation except for the 2-h period. By 16 h, 24 % of the RNA was lost. Very little RNA was lost from the root tips grown from X-irradiated seed, The relative amount of uracil incorporated into RNA (assuming uracil is not converted to thymine) of the subceUular fractions of control root tips is presented in Fig. 5- The nuclei had a higher specific activity (counts/min [2-z~C]uracil/#g RNA) than the other fractions as was also demonstrated with other precursors z°. There was little difference in the specific activities of the mitochondrial and soluble fractions. The loss of uracil from the mitochondrial, nuclear, and soluble RNA after 8 h of incubation is believed to be due to the leakage of uracil from the root (Fig. 2). The microsomes incorporated uracil into RNA much more slowly than the other fractions, but at a linear rate. The time course of maximum labeling is instructive; nuclear nucleic acid is labeled first, followed in order b y mitochondrial and soluble fractions, and last the microsomal. Exposure of seed to X-irradiation altered the pattern of uracil incorporation (Fig. 6). The nuclei had a higher specific activity than did the other sub-cellular fractions, as in the case of control roots (note change in scale in comparison with Fig. 5). However, the amount incorporated was enhanced about 3-fold b y X-irradiation, suggesting an accelerated nucleic acid metabolism. The light and heavy mitochondrial fractions incorporated uracil into RNA throughout the growth period, but at a rate about one-half that of the nuclei. In comparison to the other subcellular fractions, the microsomes incorporated very little uracil. There is, of course, very little microsomal RNA in these roots. The X-irradiated root tips, unlike the controls, did not lose uracil from RNA after 8 h of incubation. The amount of soluble RNA was too small to measure any labeling (see Fig. 4). Growth and RNAase activity o/ excised root tips
Previous work 9 showed that RNAase activity was greater in root segments containing mostly maturing cells than in the case of meristematic tissue. X-irradiation of dry seeds greatly enhanced the RNAase activity of the growing seedlings, especially in the meristems. The previous work 9 suggested that RNAase activity was involved with cell growth and maturation. Biochim. Biophys. Acta, 55 (1962) 487-494
492
j.H.
CHERRY
TABLE I THE
CHANGE
IN
T O T A L l~l%~A C O N T E N T
DURING
GROWTH
OF E X C I S E D
ROOT
TIPS
The root tips, I cm long, were removed from seedlings grown from normal and X-irradiated seeds and incubated with 1 % sucrose, lO -3 M CaCI, and lO -8 M K H , P O 4 (pH 6. 3 with NHs} for the time periods indicated. The HC104-insoluble RNA was determined on the cleared homogenates. X-irradiation treatment Incubation time (h)
Control
250 kRtoseedapplied
#g nucleic aeid/g initial /resh weight
o I 2 4 7.5 16
1192 lO92 1287 lO86 962 907
TABLE C H A N G E S IN PROTEIN C O N T E N T A N D R N A A s E
627 638 627 589 597 593
II
ACTIVITY D U R I N G G R O W T H
OF EXCISED ROOT TIPg
ROOt tips, O.5 c m long, were removed from seedlings grown from normal and X-irradiated seeds and incubated with i ~o sucrose, lO -8 M CaCI~ and IO -8 M KI-12PO 4 (pFl 6.3 with N H 3 ) for the time periods indicated. R N A a s e activity was determined on the root homogenates in the presence of o.2 5 M sucrose, 5"IO-* M MgSO4, 5' lO-3 M KCII and I m g / m l of yeast R N A .
X-irradiation treatment
Incubation time (h)
Control Control Control Control 25o k R applied to seed 250 k R applied to seed 25o k R applied to seed 250 k R applied to seed
o 5 io I4 o
Percent gain in/resh weight
RNA ase Protein (#g/root tip) #g RNA degraded #g RNA degraded /hlper root tip /h/rag protein
29.4 53.8 71.8
5.9 5.5 4.3 4.2
2.I 4 .2 5 .o 7.2
364 803 1175 17°5
--
4.3
3.9
912
5
7 .2
3.4
3 .2
923
IO
16.6
3-2
4.o
1768
14
20.9
2.8
5.6
2oo8
T h e r e s u l t s p r e s e n t e d i n T a b l e I I o f f e r e v i d e n c e for a r e l a t i o n s h i p b e t w e e n g r o w t h ( m o s t l y cell e l o n g a t i o n ) of e x c i s e d r o o t t i p s a n d R N A a s e a c t i v i t y . G r o w t h of e x c i s e d r o o t t i p s i n t h e i n c u b a t i o n m e d i u m d e s c r i b e d i n v o l v e s a d e g r a d a t i o n a n d loss of p r o t e i n f r o m t h e r o o t t i p s as i n d i c a t e d e a r l i e r f r o m b o t h n o r m a l a n d X - i r r a d i a t e d seeds. G r o w t h of e x c i s e d r o o t s a l s o i n v o l v e s a loss of R N A ( T a b l e I ) a n d a m a j o r r e d i s t r i b u t i o n of R N A a m o n g t h e s u b c e l l u l a r c o m p o n e n t s (Fig. 3)- C o n c o m i t a n t l y w i t h t h e loss of p r o t e i n a n d R N A f r o m t h e n o r m a l e x c i s e d r o o t t i p s t h e r e is a l a r g e i n c r e a s e ( o v e r 3 - f o l d ) i n R N A a s e a c t i v i t y a f t e r 14 h of i n c u b a t i o n . T h e s p e c i f i c activity was enhanced over 4-fold in the same period. Biochim.
B i o p h y s . A c t a , 55 (1962) 487-494-
RNA
METABOLISM DURING GROWTH OF EXCISED ROOT TIPS
493
Root tips from X-irradiated seed grew less than normal root tips which was suggested previously1, s to be due to a large proportion of maturing cells not capable of much cell expansion under the described conditions. However, with tile small amount of growth with tile X-irradiated tissue, there was a degradation and loss (35 %) of protein from the root tips, and a doubling of specific RNAase activity.
DISCUSSION
The investigations presented in this paper show that growth of excised root tips involves a metabolism of protein and nucleic acids and the activity of RNAase. Incubation of normal excised root tips caused a rapid disappearance of microsomal and soluble RNA with a concomitant increase in the nuclear and mitochondrial fractions (Fig. 3)- This major redistribution of RNA among the subcellular fractions was accompanied by a loss of RNA from the root tip (Table I). The protein content also decreased with growth of both normal and X-irradiated tissue. Previous evidence is from this laboratory showed that there was a degradation and loss of protein and RNA from excised Cucumber and corn stems during incubation. As previously presentedS, 9 X-irradiation of dry corn seeds drastically affects the content and metabolism of nucleic acids of the growing seedling. It was shown that root tips from 3-day-old seedlings had an RNA distribution among the subcellular components typical of tissue possessing maturing cells. Since root tips from the X-irradiated seeds are atypical meristems containing little microso~al RNA, it is reasonable that little or no redistribution of RNA occurs with growth of excised X-irradiated tissue (Fig. 4). Tile incorporation of uracil into RNA of subcellular components b y excised root tips shows that the nuclear material incorporates more radioactivity than the other components (Figs. 3 and 4). Generally, there was little difference in incorporation of uracil into the RNA of the mitochondrial and soluble fractions. The microsomes incorporated the least amount of uracil into RNA, which is in agreement with previous work 9 and with the work of Ts'o AND SATO14. Previous workL2,s,s,9 and data presented in this paper indicates a metabolism of ribonucleoprotein granules is involved with cell growth and maturation in corn seedlings. Associated with cell maturation and disappearance of microsomal RNA is a large increase in RNAase activity (Table [I). It is to be noted that as the RNAase activity increases 2 to 4-fold the protein content decreases as much as 35 %. It is not known whether RNAase is responsible for tile RNA metabolism involving cell maturation. However, data presented suggest that the enzyme could serve an important physiological role in the metabolism of RNA during cell expansion. Evidently, the corn root must possess two kinds of RNA-metabolizing systems. The first system synthesizes large amount of soluble and microsomal RNA, and is susceptible to X-irradiation. The second system tentatively may be visualized as a "transferring" system, degrading the microsomal and soluble RNA produced by the first system. The second system then reforms the constituent nucleotides into RNA associated with the membranous fractions of the root during cell expansion. The "transferring" system appears not to be injured b y X-irradiation as judged by the incorporation of uracil into RNA and the level of RNAase activity. Biochim. Biophys. Acta, 55 (1962) 487-494
494
i . H . CHERRY ACKNOWLEDGEMENTS
The author is indebted to the U.S. Atomic Energy Commission (Grant No. 791) for its support. The author wishes to thank Mr. F. I. COLLINS for the X-irradiation of the seed and to Mrs. D. FLESHER for her excellent technical assistance. REFERENCES 1 2 3 4 5 6 8 9 10 11 x~ 19 ~
j . H. CHERRY, Radiation Research, in the press. H. A. LUND, A. E. MATTER AND J. B. HANSON, J. Biophys. Biochem. Cytol., 4 (1958) 87. G. SETTERFIELD, Can. J. Botany, 39 (1961) 469. p. O. P. T s ' o AND C. S. SATO, Exptl. Cell Research, 17 (1959) 227. j . L. KEY, J. B. HANSON, H. A. LUND AND A. E. MATTER,Crop Sci., I (1961) 5. j . H. CHERRY AND R. H. HAGEMAN, Plant Physiol., 36 (1961) 163. J. K. HEYES, Proc. Royal Soc. (London) B, 152 (196o) 218. j . H. CHERRY, R. H. HAGEMAN AND J. B. HANSON, Radiation Research, in the press. j. H. CHERRY, R. H. HAGEMAN AND J. B. HANSON, Radiation Research, in the press. j. H. CHERRY, R. H. HAGEMAN, F. I. CoLLINS AND DONNA FLESHER, Plant Physiol.,36 (1961) 566. j . B. HANSON, Plant Physiol., 35 (196o) 372. O. H. LowRY, N. J. ROSEBROUGH, A. L. FAIR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. S. H. WEST, J. B. HANSON AND J. L. KEY, Weeds, 8 (196o) 333. P. O. P. T s ' o AND C. S. SATO, Exptl. Cell Research, 17 (1958) 237.
Biochim. Biophys. Acta, 55 (1962) 487-494
BIOCHIMICA ET BIOPHYSICAACTA
RIBONUCLEIC ACID METABOLISM DURING GROWTH OF ROOT TIPS FROM NORMAL AND X-IRRADIATED
EXCISED
CORN SEEDS
JOE H. CHERRY* Department of Agronomy, University o] Illinois, Urbana, Ill. (U.S.A.) (Received July 24th, I961)
SUMMARY Growth of excised corn root tips was found to involve a degradation and loss of protein and ribonucleic acid. During growth (mostly cell elongation) of the normal excised root tips there is a maj or redistribution of ribonucleic acid among the subcellular components. The microsomal and soluble ribonucleic acids rapidly decline with a concomitant increase in the nuclear and mitochondrial fractions. There was little redistribution of ribonucleic acid in the root tips from X-irradiated seeds, since the transformation had already occurred as a result of X-irradiation prior to incubation of the root tips. Incorporation of uracil into the subcellular ribonucleic acid fractions b y excised root tips revealed that the greatest incorporation was in the nuclei, followed in order by mitochondria, microsomes and the soluble fraction. X-irradiation greatly enhanced the incorporation of uracil into ribonucleic acid. Growth of root tips from normal and X-irradiated seeds increased the ribonuclease activity as much as 2 to 4-fold. Root tips grown from X-irradiated seed had an initial ribonuclease activity 2 to 3-fold greater than normal tissue.
INTRODUCTION Previous results show that X-irradiation of dry corn seeds inhibits cell division in the root tip of the growing seedlingI. Growth of seedlings from X-irradiated seeds appears to be due to elongation of cells pre-existing in the embryo at the time of irradiation. Available evidence2-~ indicates that the RNA associated with the subcellular particulates of plants undergoes major changes during cell growth and maturation. These results show that more of the RNA and protein of mature plant cell homogenates sediment with the nuclear and mitochondrial fractions than in the case of meristematic cells, where the microsomal fraction contains most of the RNA. Recent evidence8 shows that X-irradiation decreases the content of microsomal RNA in the meristematic region of germinating seedlings. Apparently, X-irradiation prevents the synthesis of microsomal RNA as ordinarily occurs with cell division, but allows normal catabolism of the ribonucleoprotein granules in expanding cells. Abbreviation: TCA, trichloroacetic acid. " Present address: Southern Research Laboratory, USDA, ARS, ~New Orleans I9, La. ~U.S.A.). Biochim. Biophys. Aota, 55 (1962) 487-494
488
j . H . CHERRY
X-irradiation enhances the incorporation of precursors into the nucleic acids of the subcellular components of growing roots s. X-irradiation greatly enhances the RNAase activity in root tips, which normally is low in activity 9. The data reported here are on the metabolism of RNA during growth of excised root tips from normal and X-irradiated seeds. The incorporation of uracil into RNA of the subcellular components was examined. Studies were made on the level of RNAase as affected b y cell elongation and X-irradiation of dry seed. MATERIALS AND METHODS Corn seeds were X-irradiated with 250 k R and germinated in a dark, humid atmosphere at 29 ° as previously described 1°.
Growth o/ excised root tips Root tips, i cm long, were sectioned from primary roots of 2~-day-old seedlings grown from normal and X-irradiated seeds. The tissue, about o.5 g, was rinsed in deionized water, blotted, and rapidly weighed before use. Root tips were placed in petri dishes and incubated at room temperature with gentle shaking for I to 16 h. Each dish contained IO ml of a solution containing 1 % sucrose, lO -8 M CaC12, IO 3 M KHzPO4 (pH 6.3 with NH4OI-I) and 1.6 #C of [2-14C]uracil (0.2/~ mole). After various incubation periods the root tips were washed, weighed and frozen for analysis. The experiments designed to determine the level of RNAase activity were made on root tips, 0.5 cm long, sectioned from primary roots of 2~-day-old seedlings from normal and X-irradiated seeds. As above, the tissue was incubated with a solution containing I °/o sucrose, lO -3 M CaC12, IO-s M KI-I~PO4 (pH 6.3 with N H 4 0 H ). Atteivarious incubation periods the root tips were washed, weighed and frozen for RNAase assay.
Isolation o/ cytoplasmic particulates Sections of the primary root after incubation with uracil were homogenized in 5 ml of an ice-cold medium containing o.5 M sucrose, 30 % glycerol, o.oi M Tris (pH 7.4) and o.oi M CaCla in an ice-jacketed glass homogenizer with a power-driven Teflon pestle for 2 rain. The homogenates were strained through a layer of Dacron fabric (openings of 3O-lOO/~). An aliquot of the debris-free homogenate was subjected to differential centrifugation in a Spinco Model L ultracentrifuge, each successive particulate fraction being obtained from the supernatant of the preceding fraction. The entire isolation procedure was performed at o to 4 °. The centrifugal forces, time, and the arbitrary designation of the isolated fractions were as follows: nuclei, 15oo × g for I2 rain; h e a v y mitochondria, 20 ooo × g for 15 rain; light mitochondria, 40 ooo × g for 20 min; microsomes, IOO ooo × g for i h; soluble, particulate-free supernatant. Each of the particulate fractions were washed once by resuspending in o.5 M sucrose and resedimenting at the same centrifugal force and time. The terms used to designate the cellular fractions are operational, as listed above, and do not necessarily mean the presence of pure morphological entities.
Biochim. Biophys. Acta, 55 (1962) 487-494
RNA
METABOLISM
DURING
GROWTH
OF EXCISED
ROOT TIPS
489
Analysis o/ subcellular /factions The RNA of the total debris-free homogenate and of the cytoplasmic fractions was extracted and measured b y a perchloric acid method previously described 1°. The RNA content of the debris-free homogenate is reported as total R N A in Table I.
Determination o/ radioactivity Samples of the RNA or total nucleic acid hydrolysates were neutralized with K O H , and the KC10, was removed b y centrifugation in the cold. Samples of these neutralized hydrolysates were then dried and counted with a Picker gas-flow proportional counter (window thickness less than 0.5 mg/cm*).
Assays /or RNAase activity Sections of the o.5-cm root tip after incubation were homogenized in 5 ml of o.5 M sucrose with an ice-jacketed glass homogenizer with a power-driven Teflon pestle. The homogenates were cleared of cell debris and nuclei b y centrifugation at 500 x g for IO rain. These cleared homogenates were filtered through glass wool and were used directly as a source of enzyme. RNAase activity was determined b y incubating I ml of the cleared homogenate with the additivies described b y HANSON11 for root tips. Each determination was made in the presence of 0.25 M sucrose, 5.1o -4 M MgSO4, 5 . I o - 3 M KC1, and I mg/ml of yeast RNA (pH 6.5). Final volume was made to 2 ml; incubation was for 30 min at 3 °0 with gentle shaking. The p H of the incubation medium was about 6.0. One set of tubes of each determination was held in ice to establish the initial level of RNA. Experimentation showed no RNA degradation at this temperature. At the termination of incubation, the tubes were placed in ice, and HC104 and uranium acetate were added to 0.2 N and 6. lO -3 M, respectively. The precipitates were washed twice with 0.2 N HC10,. The supernatant solution and the two washes were combined, made to volume and the absorbancy determined at 260 and 290 m/z. The relative increase in absorbancy difference at 260-290 m# of the acid-soluble fraction was used to compute the RNAase activity. Protein was determined b y the method of LOWRY et al. 12 on aliquots precipitated and washed in 5 % TCA. RESULTS
Growth and metabolism o[ R N A in elongating excised root tips Control root tips grew much faster (4 times) than root tips from X-irradiated seed (Fig. I). There was a slight lag period in the growth of control root tips, followed b y a linear rate. Root tips from X-irradiated seed lost weight immediately on incubation and did not begin to grow until 4 h later. Since growth of these excised root tips is essentially b y cell elongation, these data agree with the histological observations t which indicate that cells in the X-irradiated root tips are more mature, and therefore would have a smaller capacity for cell expansion. Control root tips took up slightly more uracil (counts/min/g initial fresh wt.) than those from X-irradiated seeds (Fig. 2). The uptake was linear with time for the
Biochim. Biophys. Acta, 55 (1962) 487-494
49 °
J.H.
CHERRY
first 8 h, but afterwards uracil was lost from the roots. Previous work s indicated a leakage of nucleotides from excised roots during incubation which agrees with these data. As previously discussed ~, very young actively dividing cells are characterized by a high level bf microsomal RNA, whereas mature cells have few ribonucleo.,..; 50
"~ 40 .~ ~o 1200
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Fig. I (left top). A comparison of growth of excised root tips (I cm) from normal and X-irradiated (25o kR) seeds. Fig. 2 (right top). A comparison of [2-14C]uracil uptake with growth of excised root rips (I cm) from normal and X-irradiated (25o kR) seeds. Fig. 3 (left middle). Changes in RNA content of the various subcellular particulates with growth of control 2 I-day-old excised root tips (I cm). Fig. 4 (right middle). Changes in RNA content of the various subcellular particulates with growth of excised root tips (i cm) from X-irradiated (25 ° kR) seeds. Fig. 5 (left bottom). Incorporation of [2-14C]uracil into RIgA of the various particulate fractions by 2 I-day-old excised control root rips (i cm). Fig. 6 (right bottom). Incorporation of [2-14C]uracil into RIgA of the various particulate fractions by 2 I-day-old excised root tips (I cm) from X-irradiated (25 ° kR)seeds. Biochim. Biophys. Acta, 55 (I962) 487-494
RNA
METABOLISM
DURING
GROWTH
O F ]~XCISED
ROOT TIPS
491
protein granules. Most of the nucleic acid of mature cells is found in the mitochondrial and nuclear fractions. Fig. 3 clearly shows a transformation of RNA in the subcellular fractions as the root cells elongate (see Fig. I for amount of growth). Microsomal and soluble RNA rapidly decline as the tissue grows; concomitantly there is an increase in heavy mitochondrial and nuclear RNA. No significant change in the amount of light mitochondrial RNA was observed. These analyses were based essentially on the same number of cells, but primarily at different stages of cell expansion, and therefore provide important information on the metabolism of RNA in relation to cell maturation. Growth had little effect on the intracellular distribution of RNA in excised root tips from X-irradiated seed (Fig. 4). However, there was a decline in microsomal RNA, even though the initial content was low. Light mitochondrial RNA increased, apparently at the expense of the microsomes. Table I shows the changes in total RNA content during growth of excised root tips. There was a decline in RNA of normal root tips during incubation except for the 2-h period. By 16 h, 24 % of the RNA was lost. Very little RNA was lost from the root tips grown from X-irradiated seed, The relative amount of uracil incorporated into RNA (assuming uracil is not converted to thymine) of the subceUular fractions of control root tips is presented in Fig. 5- The nuclei had a higher specific activity (counts/min [2-z~C]uracil/#g RNA) than the other fractions as was also demonstrated with other precursors z°. There was little difference in the specific activities of the mitochondrial and soluble fractions. The loss of uracil from the mitochondrial, nuclear, and soluble RNA after 8 h of incubation is believed to be due to the leakage of uracil from the root (Fig. 2). The microsomes incorporated uracil into RNA much more slowly than the other fractions, but at a linear rate. The time course of maximum labeling is instructive; nuclear nucleic acid is labeled first, followed in order b y mitochondrial and soluble fractions, and last the microsomal. Exposure of seed to X-irradiation altered the pattern of uracil incorporation (Fig. 6). The nuclei had a higher specific activity than did the other sub-cellular fractions, as in the case of control roots (note change in scale in comparison with Fig. 5). However, the amount incorporated was enhanced about 3-fold b y X-irradiation, suggesting an accelerated nucleic acid metabolism. The light and heavy mitochondrial fractions incorporated uracil into RNA throughout the growth period, but at a rate about one-half that of the nuclei. In comparison to the other subcellular fractions, the microsomes incorporated very little uracil. There is, of course, very little microsomal RNA in these roots. The X-irradiated root tips, unlike the controls, did not lose uracil from RNA after 8 h of incubation. The amount of soluble RNA was too small to measure any labeling (see Fig. 4). Growth and RNAase activity o/ excised root tips
Previous work 9 showed that RNAase activity was greater in root segments containing mostly maturing cells than in the case of meristematic tissue. X-irradiation of dry seeds greatly enhanced the RNAase activity of the growing seedlings, especially in the meristems. The previous work 9 suggested that RNAase activity was involved with cell growth and maturation. Biochim. Biophys. Acta, 55 (1962) 487-494
492
j.H.
CHERRY
TABLE I THE
CHANGE
IN
T O T A L l~l%~A C O N T E N T
DURING
GROWTH
OF E X C I S E D
ROOT
TIPS
The root tips, I cm long, were removed from seedlings grown from normal and X-irradiated seeds and incubated with 1 % sucrose, lO -3 M CaCI, and lO -8 M K H , P O 4 (pH 6. 3 with NHs} for the time periods indicated. The HC104-insoluble RNA was determined on the cleared homogenates. X-irradiation treatment Incubation time (h)
Control
250 kRtoseedapplied
#g nucleic aeid/g initial /resh weight
o I 2 4 7.5 16
1192 lO92 1287 lO86 962 907
TABLE C H A N G E S IN PROTEIN C O N T E N T A N D R N A A s E
627 638 627 589 597 593
II
ACTIVITY D U R I N G G R O W T H
OF EXCISED ROOT TIPg
ROOt tips, O.5 c m long, were removed from seedlings grown from normal and X-irradiated seeds and incubated with i ~o sucrose, lO -8 M CaCI~ and IO -8 M KI-12PO 4 (pFl 6.3 with N H 3 ) for the time periods indicated. R N A a s e activity was determined on the root homogenates in the presence of o.2 5 M sucrose, 5"IO-* M MgSO4, 5' lO-3 M KCII and I m g / m l of yeast R N A .
X-irradiation treatment
Incubation time (h)
Control Control Control Control 25o k R applied to seed 250 k R applied to seed 25o k R applied to seed 250 k R applied to seed
o 5 io I4 o
Percent gain in/resh weight
RNA ase Protein (#g/root tip) #g RNA degraded #g RNA degraded /hlper root tip /h/rag protein
29.4 53.8 71.8
5.9 5.5 4.3 4.2
2.I 4 .2 5 .o 7.2
364 803 1175 17°5
--
4.3
3.9
912
5
7 .2
3.4
3 .2
923
IO
16.6
3-2
4.o
1768
14
20.9
2.8
5.6
2oo8
T h e r e s u l t s p r e s e n t e d i n T a b l e I I o f f e r e v i d e n c e for a r e l a t i o n s h i p b e t w e e n g r o w t h ( m o s t l y cell e l o n g a t i o n ) of e x c i s e d r o o t t i p s a n d R N A a s e a c t i v i t y . G r o w t h of e x c i s e d r o o t t i p s i n t h e i n c u b a t i o n m e d i u m d e s c r i b e d i n v o l v e s a d e g r a d a t i o n a n d loss of p r o t e i n f r o m t h e r o o t t i p s as i n d i c a t e d e a r l i e r f r o m b o t h n o r m a l a n d X - i r r a d i a t e d seeds. G r o w t h of e x c i s e d r o o t s a l s o i n v o l v e s a loss of R N A ( T a b l e I ) a n d a m a j o r r e d i s t r i b u t i o n of R N A a m o n g t h e s u b c e l l u l a r c o m p o n e n t s (Fig. 3)- C o n c o m i t a n t l y w i t h t h e loss of p r o t e i n a n d R N A f r o m t h e n o r m a l e x c i s e d r o o t t i p s t h e r e is a l a r g e i n c r e a s e ( o v e r 3 - f o l d ) i n R N A a s e a c t i v i t y a f t e r 14 h of i n c u b a t i o n . T h e s p e c i f i c activity was enhanced over 4-fold in the same period. Biochim.
B i o p h y s . A c t a , 55 (1962) 487-494-
RNA
METABOLISM DURING GROWTH OF EXCISED ROOT TIPS
493
Root tips from X-irradiated seed grew less than normal root tips which was suggested previously1, s to be due to a large proportion of maturing cells not capable of much cell expansion under the described conditions. However, with tile small amount of growth with tile X-irradiated tissue, there was a degradation and loss (35 %) of protein from the root tips, and a doubling of specific RNAase activity.
DISCUSSION
The investigations presented in this paper show that growth of excised root tips involves a metabolism of protein and nucleic acids and the activity of RNAase. Incubation of normal excised root tips caused a rapid disappearance of microsomal and soluble RNA with a concomitant increase in the nuclear and mitochondrial fractions (Fig. 3)- This major redistribution of RNA among the subcellular fractions was accompanied by a loss of RNA from the root tip (Table I). The protein content also decreased with growth of both normal and X-irradiated tissue. Previous evidence is from this laboratory showed that there was a degradation and loss of protein and RNA from excised Cucumber and corn stems during incubation. As previously presentedS, 9 X-irradiation of dry corn seeds drastically affects the content and metabolism of nucleic acids of the growing seedling. It was shown that root tips from 3-day-old seedlings had an RNA distribution among the subcellular components typical of tissue possessing maturing cells. Since root tips from the X-irradiated seeds are atypical meristems containing little microso~al RNA, it is reasonable that little or no redistribution of RNA occurs with growth of excised X-irradiated tissue (Fig. 4). Tile incorporation of uracil into RNA of subcellular components b y excised root tips shows that the nuclear material incorporates more radioactivity than the other components (Figs. 3 and 4). Generally, there was little difference in incorporation of uracil into the RNA of the mitochondrial and soluble fractions. The microsomes incorporated the least amount of uracil into RNA, which is in agreement with previous work 9 and with the work of Ts'o AND SATO14. Previous workL2,s,s,9 and data presented in this paper indicates a metabolism of ribonucleoprotein granules is involved with cell growth and maturation in corn seedlings. Associated with cell maturation and disappearance of microsomal RNA is a large increase in RNAase activity (Table [I). It is to be noted that as the RNAase activity increases 2 to 4-fold the protein content decreases as much as 35 %. It is not known whether RNAase is responsible for tile RNA metabolism involving cell maturation. However, data presented suggest that the enzyme could serve an important physiological role in the metabolism of RNA during cell expansion. Evidently, the corn root must possess two kinds of RNA-metabolizing systems. The first system synthesizes large amount of soluble and microsomal RNA, and is susceptible to X-irradiation. The second system tentatively may be visualized as a "transferring" system, degrading the microsomal and soluble RNA produced by the first system. The second system then reforms the constituent nucleotides into RNA associated with the membranous fractions of the root during cell expansion. The "transferring" system appears not to be injured b y X-irradiation as judged by the incorporation of uracil into RNA and the level of RNAase activity. Biochim. Biophys. Acta, 55 (1962) 487-494
494
i . H . CHERRY ACKNOWLEDGEMENTS
The author is indebted to the U.S. Atomic Energy Commission (Grant No. 791) for its support. The author wishes to thank Mr. F. I. COLLINS for the X-irradiation of the seed and to Mrs. D. FLESHER for her excellent technical assistance. REFERENCES 1 2 3 4 5 6 8 9 10 11 x~ 19 ~
j . H. CHERRY, Radiation Research, in the press. H. A. LUND, A. E. MATTER AND J. B. HANSON, J. Biophys. Biochem. Cytol., 4 (1958) 87. G. SETTERFIELD, Can. J. Botany, 39 (1961) 469. p. O. P. T s ' o AND C. S. SATO, Exptl. Cell Research, 17 (1959) 227. j . L. KEY, J. B. HANSON, H. A. LUND AND A. E. MATTER,Crop Sci., I (1961) 5. j . H. CHERRY AND R. H. HAGEMAN, Plant Physiol., 36 (1961) 163. J. K. HEYES, Proc. Royal Soc. (London) B, 152 (196o) 218. j . H. CHERRY, R. H. HAGEMAN AND J. B. HANSON, Radiation Research, in the press. j. H. CHERRY, R. H. HAGEMAN AND J. B. HANSON, Radiation Research, in the press. j. H. CHERRY, R. H. HAGEMAN, F. I. CoLLINS AND DONNA FLESHER, Plant Physiol.,36 (1961) 566. j . B. HANSON, Plant Physiol., 35 (196o) 372. O. H. LowRY, N. J. ROSEBROUGH, A. L. FAIR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. S. H. WEST, J. B. HANSON AND J. L. KEY, Weeds, 8 (196o) 333. P. O. P. T s ' o AND C. S. SATO, Exptl. Cell Research, 17 (1958) 237.
Biochim. Biophys. Acta, 55 (1962) 487-494