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Cell arrest in G2 in root meristems: A control factor from the cotyledons
Preliminary notes
nitrocellulose filters have to be changed, since otherwise the duration of a pulse treatment may be longer than intended or, if the treatment is continuous, the local dactinomycin concentration may exceedthe bulk one. Furthermore, if diluted dactinomycin solutions are sterilized by filtration through nitrocellulose filters the final concentration may decreaseconsiderably. Grants: The Sigrid Juselius Foundation Foundation for Cancer Research, Finland.
and the
47 I
[2]. It is possible that fewer cells arrested in G2 in the older cultures becausea substance(s) was depleted in the root that is normally supplied by other plant organs. In the early stages of seedling growth the cotyledons are the chief source of most cell nutrients and consequently, they are most suspect as the tissue origin of the postulated substance(s) affecting cell arrest in G2. The experiments described in this paper were designed to test if the cotyledons influence cell arrest in G2 in root meristems of Pisum.
References 1. Grobstein, C, Exptl cell res 10 (1956) 424. 2. Jainchill, J, Saxen, L & Vainio, T, J embryo1 exptl morph01 12 (1964) 597. 3. Nordling, S, Miettinen, H, Wartiovaara, J & Saxen, L, J embryo1 exutl morph01 26 (1971) 231. 4. Sax& L & Sakseia, E, Exptl cell res 66 (1971) 349. 5. Wessells, N K, Devel biol 9 (1964) 92. 6. Wessells, N K & Wilt, F H, J mol biol 13 (1965) 767. Received June 25, 1973 Revised version received August 20, 1973
Cell arrest in G2 in root meristems:
A control factor from the cotyledons L. S. EVANS and J. VAN’T HOF, Biology Department, Brookhaven National Laboratory, Upton, N. Y. 11973, USA Summary. A substance promoting cell cycle arrest in G2 in the root meristem is demonstrated. This substance is produced in the cotyledons and is transported to the root.
Arrest in the mitotic cycle by meristematic cells in cultured primary root tips after protracted carbohydrate starvation is a nonrandom process [l]. Few, if any, cells stop during DNA synthesis and none arrest during mitosis. The probability of arrest in Gl and in G2 is not always equal. For example, in freshly excised carbohydrate-deficient meristems of Vicia faba the proportion of cells arrested, G 1 : G2, was approx. 1: 3 while that of older cultured meristems was about 1: 1
Materials and Methods The general procedures used in our experiments have been described previously [3]. Seeds of Pisum satiaum were surface sterilized in undiluted Clorox bleach containing 5.2 X sodium hypochlorite for 5 min, washed with water, and placed in sterile vermiculite. Three and 7 days after planting, l-l.5 cm primary root tips were placed into culture medium [6] with or without sucrose. Roots from seedlings were fixed after treatment in ethanol/acetic acid (3 : I, v/v). Relative amounts of DNA/nucleus were determined on Feulgen-stained nuclei from O-2 mm terminal meristems with a Leitz microspectrophotometer [2] with the use of the two-wavelength method of Ornstein [4] and Patau [5]. Some samples were examined with a Zeiss scanning microspectrophotometer at 560 nm. Both methods were comparable when DNA/ nucleus was normalized with readings of one-half telophase and prophase taken to be 2C and 4C values, respectively:
Results and Discussion
Specifically, we approached the problem of whether cotyledon presence and absence influenced cell arrest in three ways. The first experiments were performed to demonstrate reduced ceil arrest in G2 in older cultured primary root tips of Pisum satiuum. Excised l-l.5 cm root tips immediately starved of carbohydrate for 4 days, had terminal O-2 mm apices with 41 k3 % cells of a 2C nuclear amount and 59 & 3 % witha4Camount of DNA (fig. 1). In apices of root tips first cultured for 7 days in medium with 2% sucrose and then starved of carbohydrate for 5 days, 78 k 2 y; of the nuclei had a 2 C and 22 L 2 % had a 4C nuclear DNA content (fig. 2). The Exptl Ceil Res 82 (1973)
472
Preliminary notes
additional time in culture produced a 37 % decrease in the proportion of cells arrested in G2. In our second experiment we determined if the decrease in cell arrest in G2 in older cultures was due to the length of time the root tips were separatedfrom the cotyledons. This was done by removing the cotyledons from seedlings 4 days before the root tips were excised and cultured in medium deficient of carbohydrate. The controls in the experiments were seedlings in which the cotyledons remained attached up to the time of excision and subsequent starvation. If the cotyledons were supplying a substance to the primary root meristem, the root tips of seedlings from which the cotyledons were detached earlier should have fewer cells arrested in G2. Microspectrophotometric measurements of nuclei of cells in the control (fig. 3) showed 6222% with a 2C and 38+2% with a 4C Figs 1-8. Abscissa: relative amount of DNA; ordinate: no. of nuclei. amount of DNA. In root apices of seedlings Histograms of the relative amount of nuclear DNA from which the cotyledons were removed measured by Feulgen stain microspectrophotometry at 560 nm with either a Zeiss scanning photometer earlier 86+3 % of the nuclei had a 2C and or by the two wavelength method of Ornstein [4] and 14i3 % had a 4C amount of DNA (fig. 4). Patau 151.The 2C and 4C values were determined from Ii, -telophase and prophase mitotic figures and Detachment of the cotyledons 4 days prior were given relative values of 0.5 and 1.O, respectively. The estimate of the number of cells in DNA synthesis to starvation resulted in 24% fewer cells was made between values 0.6 and 0.8 and was checked arrested in G2. by autoradiography in meristems of 3-day-old seedOur third experiment tested if decreased lings. Both methods produced similar estimates. With microspectrophotometry 20% of the cells were esticell arrest in G2 in the root meristem was mated to be synthesizing DNA; after a 1 h pulse with associated only with the detachment of the SH-thymidine 19 % of the cells were fabled with radioactive tracer. N, number of nuclei measured. Fig. 1. cotyledons and not with the absence of the Nuclei selected randomly from squash preparations of O-2 mm tips of excised primary roots cultured for 3 leaves. In these experiments the cotyledons days in White’s medium [6] without carbohydrate; were removed from 3-day-old seedlings and fig. 2, nuclei of tips of roots cultured in medium with the entire remaining plantlet including the carbohydrate for 7 days and then starved for 5 days; fig. 3, nuclei of tips of the primary root of 7-day-old epicotyle was cultured in carbohydrateseedlings that were excised and starved for 4 days; deficient medium, in the dark, for 7, 10 and fig. 4, nuclei of root tips of 7-day-old seedlings from which the cotyledons were removed 4 days before 13 days. If the cotyledons were the major excision and starvation-the duration of starvation source of substance(s) affecting cell arrest, was 4 days; figs 5-7, nuclei of root tips of whole seedlings minus the cotyledons that were cultured in then a shift in the proportion of 2C and 4C carbohydrate-deficient medium, in the dark, for 7, nuclei should be observed. Measurements of 10, and 13 days, respectively; fig. 8, nuclei of tips of the primary root of complete S-day-old seedlings prior cells in the primary root tips are shown in to any experimental manipulation. figs 5, 6, and 7, respectively, for seedlings starved for 7, 10, and 13 days. I
(I)
’
I
0
I
’
I
N =200
Exptl Cell Res 82 (1973)
I
’
(5)
1
I 1 N=96
I
’
Preliminarjs
In each case the duration of starvation was insufficient to produce cell arrest in the mitotic cycle. Apparently, the seedlings without cotyledons had enough endogenous carbohydrate to maintain cell division for some time. In meristems of cultured seedlings without cotyledons that were starved for 7 days, the percentage of nuclei with a 2C and a 4C amount of DNA was 6Oi13 and 17i7, respectively, in plants starved 10 days the percentages were 8 I+ 6 and lOt_4, and in those starved 13 days they were 75 1-2 and 12*2. The percentage of nuclei with a 2C amount of DNA in meristems of 3-day-old seedlings with leaves and cotyledons attached, was 37.6, while 43.7 had a 4C amount (fig.8). Whether starved for 7, 10, or 13 days, meristems of seedlings without cotyledons always had fewer nuclei with a 4C amount of DNA than those grown with cotyledons and leaves attached. Pea root meristem cells respond to two sets of mitotic cycle controls; one in Gl and another in G2. These controls respectively determine if cells in G 1 will progress to S and those in G2 will advance to mitosis. Carbohydrate starvation results in cells that are deficient in requisite proteins for the G 1 + S and G2 + M transitions and consequently they arrest in G 1 and G2 [7]. Our present results show that to arrest in G2 many cells must not only be deficient in certain proteins but also must be under the influence of a transportable factor originating in the cotyledons. The importance of the G 1 + S and G 2 L M transitions in the control of cell division
notes
473
in complex animal tissue is demonstrated by the work of Gelfant [8, 9, 10, 1I] and others [12, 131. Gelfant proposed two discrete cell populations in the mouse epidermis, one which stops in G 1 and another that is blocked in G 2. Furthermore, in mammals, substances that effect cell arrest or blockage in G 1 or in G2, called chalones, have been reported [14-161. These compounds are nontransportable, tissue specific, and have a cellular effect similar to that which we have described for the pea cotyledonal substance. This research was partially supported by the USAEC and partially supported by USPHS (grant no. 1 F02-CA52766-01). References 1. Van? Hof, J & Kovacs, C J, Advan exptl med
biol 18 (1972) 15. 2. Van’t Hof, 3, Hoppin, D P & Yagi, S, Am j bot 60 (1973) 889. 3. Van? Hof, J, Am j bot 53 (1966) 970. 4. Ornstein, L, Lab invest 1 (1952) 250. 5. Patau, K, Chromosoma 5 (1952) 341. 6. White, P R, A handbook of plant tissue culture. J Cattell & Co., Lancaster, Pa (1943). 7. Webster, P L & Van’t Hof, J, Am j bot 57 (1970) 130. 8. Gelfant, S, Exptl cell res 26 (1962) 395. 9. - Ibid 32 (1963) 521. 10. - Methods in cell physiol (ed D M Prescott) vol. 2, p. 359. Academic Press, New York (1966). 11. Gelfant, S & Smith, J G, Science 178 (1972) 357. 12. Epifanova. 0 I & Tersikikh, V V, Cell tissue kinet 2<1969) 75. 13. Post, J, Sklarev, R J & Hoffman, J, J natl cancer inst 50 (1973) 403. 14. Bullough, W S & Johanna, U R, Symp sot exptl biol 25 (1971) 255. 15. Marks, F, Hoppe-Seyler’s Z physiol them 352 (1971) 1273. 16. Frankfurt, 0 S, Exptl cell res 64 (1971) 140. Received August 30, 1973
Exptl Cell Re., 82 (1973)
nitrocellulose filters have to be changed, since otherwise the duration of a pulse treatment may be longer than intended or, if the treatment is continuous, the local dactinomycin concentration may exceedthe bulk one. Furthermore, if diluted dactinomycin solutions are sterilized by filtration through nitrocellulose filters the final concentration may decreaseconsiderably. Grants: The Sigrid Juselius Foundation Foundation for Cancer Research, Finland.
and the
47 I
[2]. It is possible that fewer cells arrested in G2 in the older cultures becausea substance(s) was depleted in the root that is normally supplied by other plant organs. In the early stages of seedling growth the cotyledons are the chief source of most cell nutrients and consequently, they are most suspect as the tissue origin of the postulated substance(s) affecting cell arrest in G2. The experiments described in this paper were designed to test if the cotyledons influence cell arrest in G2 in root meristems of Pisum.
References 1. Grobstein, C, Exptl cell res 10 (1956) 424. 2. Jainchill, J, Saxen, L & Vainio, T, J embryo1 exptl morph01 12 (1964) 597. 3. Nordling, S, Miettinen, H, Wartiovaara, J & Saxen, L, J embryo1 exutl morph01 26 (1971) 231. 4. Sax& L & Sakseia, E, Exptl cell res 66 (1971) 349. 5. Wessells, N K, Devel biol 9 (1964) 92. 6. Wessells, N K & Wilt, F H, J mol biol 13 (1965) 767. Received June 25, 1973 Revised version received August 20, 1973
Cell arrest in G2 in root meristems:
A control factor from the cotyledons L. S. EVANS and J. VAN’T HOF, Biology Department, Brookhaven National Laboratory, Upton, N. Y. 11973, USA Summary. A substance promoting cell cycle arrest in G2 in the root meristem is demonstrated. This substance is produced in the cotyledons and is transported to the root.
Arrest in the mitotic cycle by meristematic cells in cultured primary root tips after protracted carbohydrate starvation is a nonrandom process [l]. Few, if any, cells stop during DNA synthesis and none arrest during mitosis. The probability of arrest in Gl and in G2 is not always equal. For example, in freshly excised carbohydrate-deficient meristems of Vicia faba the proportion of cells arrested, G 1 : G2, was approx. 1: 3 while that of older cultured meristems was about 1: 1
Materials and Methods The general procedures used in our experiments have been described previously [3]. Seeds of Pisum satiaum were surface sterilized in undiluted Clorox bleach containing 5.2 X sodium hypochlorite for 5 min, washed with water, and placed in sterile vermiculite. Three and 7 days after planting, l-l.5 cm primary root tips were placed into culture medium [6] with or without sucrose. Roots from seedlings were fixed after treatment in ethanol/acetic acid (3 : I, v/v). Relative amounts of DNA/nucleus were determined on Feulgen-stained nuclei from O-2 mm terminal meristems with a Leitz microspectrophotometer [2] with the use of the two-wavelength method of Ornstein [4] and Patau [5]. Some samples were examined with a Zeiss scanning microspectrophotometer at 560 nm. Both methods were comparable when DNA/ nucleus was normalized with readings of one-half telophase and prophase taken to be 2C and 4C values, respectively:
Results and Discussion
Specifically, we approached the problem of whether cotyledon presence and absence influenced cell arrest in three ways. The first experiments were performed to demonstrate reduced ceil arrest in G2 in older cultured primary root tips of Pisum satiuum. Excised l-l.5 cm root tips immediately starved of carbohydrate for 4 days, had terminal O-2 mm apices with 41 k3 % cells of a 2C nuclear amount and 59 & 3 % witha4Camount of DNA (fig. 1). In apices of root tips first cultured for 7 days in medium with 2% sucrose and then starved of carbohydrate for 5 days, 78 k 2 y; of the nuclei had a 2 C and 22 L 2 % had a 4C nuclear DNA content (fig. 2). The Exptl Ceil Res 82 (1973)
472
Preliminary notes
additional time in culture produced a 37 % decrease in the proportion of cells arrested in G2. In our second experiment we determined if the decrease in cell arrest in G2 in older cultures was due to the length of time the root tips were separatedfrom the cotyledons. This was done by removing the cotyledons from seedlings 4 days before the root tips were excised and cultured in medium deficient of carbohydrate. The controls in the experiments were seedlings in which the cotyledons remained attached up to the time of excision and subsequent starvation. If the cotyledons were supplying a substance to the primary root meristem, the root tips of seedlings from which the cotyledons were detached earlier should have fewer cells arrested in G2. Microspectrophotometric measurements of nuclei of cells in the control (fig. 3) showed 6222% with a 2C and 38+2% with a 4C Figs 1-8. Abscissa: relative amount of DNA; ordinate: no. of nuclei. amount of DNA. In root apices of seedlings Histograms of the relative amount of nuclear DNA from which the cotyledons were removed measured by Feulgen stain microspectrophotometry at 560 nm with either a Zeiss scanning photometer earlier 86+3 % of the nuclei had a 2C and or by the two wavelength method of Ornstein [4] and 14i3 % had a 4C amount of DNA (fig. 4). Patau 151.The 2C and 4C values were determined from Ii, -telophase and prophase mitotic figures and Detachment of the cotyledons 4 days prior were given relative values of 0.5 and 1.O, respectively. The estimate of the number of cells in DNA synthesis to starvation resulted in 24% fewer cells was made between values 0.6 and 0.8 and was checked arrested in G2. by autoradiography in meristems of 3-day-old seedOur third experiment tested if decreased lings. Both methods produced similar estimates. With microspectrophotometry 20% of the cells were esticell arrest in G2 in the root meristem was mated to be synthesizing DNA; after a 1 h pulse with associated only with the detachment of the SH-thymidine 19 % of the cells were fabled with radioactive tracer. N, number of nuclei measured. Fig. 1. cotyledons and not with the absence of the Nuclei selected randomly from squash preparations of O-2 mm tips of excised primary roots cultured for 3 leaves. In these experiments the cotyledons days in White’s medium [6] without carbohydrate; were removed from 3-day-old seedlings and fig. 2, nuclei of tips of roots cultured in medium with the entire remaining plantlet including the carbohydrate for 7 days and then starved for 5 days; fig. 3, nuclei of tips of the primary root of 7-day-old epicotyle was cultured in carbohydrateseedlings that were excised and starved for 4 days; deficient medium, in the dark, for 7, 10 and fig. 4, nuclei of root tips of 7-day-old seedlings from which the cotyledons were removed 4 days before 13 days. If the cotyledons were the major excision and starvation-the duration of starvation source of substance(s) affecting cell arrest, was 4 days; figs 5-7, nuclei of root tips of whole seedlings minus the cotyledons that were cultured in then a shift in the proportion of 2C and 4C carbohydrate-deficient medium, in the dark, for 7, nuclei should be observed. Measurements of 10, and 13 days, respectively; fig. 8, nuclei of tips of the primary root of complete S-day-old seedlings prior cells in the primary root tips are shown in to any experimental manipulation. figs 5, 6, and 7, respectively, for seedlings starved for 7, 10, and 13 days. I
(I)
’
I
0
I
’
I
N =200
Exptl Cell Res 82 (1973)
I
’
(5)
1
I 1 N=96
I
’
Preliminarjs
In each case the duration of starvation was insufficient to produce cell arrest in the mitotic cycle. Apparently, the seedlings without cotyledons had enough endogenous carbohydrate to maintain cell division for some time. In meristems of cultured seedlings without cotyledons that were starved for 7 days, the percentage of nuclei with a 2C and a 4C amount of DNA was 6Oi13 and 17i7, respectively, in plants starved 10 days the percentages were 8 I+ 6 and lOt_4, and in those starved 13 days they were 75 1-2 and 12*2. The percentage of nuclei with a 2C amount of DNA in meristems of 3-day-old seedlings with leaves and cotyledons attached, was 37.6, while 43.7 had a 4C amount (fig.8). Whether starved for 7, 10, or 13 days, meristems of seedlings without cotyledons always had fewer nuclei with a 4C amount of DNA than those grown with cotyledons and leaves attached. Pea root meristem cells respond to two sets of mitotic cycle controls; one in Gl and another in G2. These controls respectively determine if cells in G 1 will progress to S and those in G2 will advance to mitosis. Carbohydrate starvation results in cells that are deficient in requisite proteins for the G 1 + S and G2 + M transitions and consequently they arrest in G 1 and G2 [7]. Our present results show that to arrest in G2 many cells must not only be deficient in certain proteins but also must be under the influence of a transportable factor originating in the cotyledons. The importance of the G 1 + S and G 2 L M transitions in the control of cell division
notes
473
in complex animal tissue is demonstrated by the work of Gelfant [8, 9, 10, 1I] and others [12, 131. Gelfant proposed two discrete cell populations in the mouse epidermis, one which stops in G 1 and another that is blocked in G 2. Furthermore, in mammals, substances that effect cell arrest or blockage in G 1 or in G2, called chalones, have been reported [14-161. These compounds are nontransportable, tissue specific, and have a cellular effect similar to that which we have described for the pea cotyledonal substance. This research was partially supported by the USAEC and partially supported by USPHS (grant no. 1 F02-CA52766-01). References 1. Van? Hof, J & Kovacs, C J, Advan exptl med
biol 18 (1972) 15. 2. Van’t Hof, 3, Hoppin, D P & Yagi, S, Am j bot 60 (1973) 889. 3. Van? Hof, J, Am j bot 53 (1966) 970. 4. Ornstein, L, Lab invest 1 (1952) 250. 5. Patau, K, Chromosoma 5 (1952) 341. 6. White, P R, A handbook of plant tissue culture. J Cattell & Co., Lancaster, Pa (1943). 7. Webster, P L & Van’t Hof, J, Am j bot 57 (1970) 130. 8. Gelfant, S, Exptl cell res 26 (1962) 395. 9. - Ibid 32 (1963) 521. 10. - Methods in cell physiol (ed D M Prescott) vol. 2, p. 359. Academic Press, New York (1966). 11. Gelfant, S & Smith, J G, Science 178 (1972) 357. 12. Epifanova. 0 I & Tersikikh, V V, Cell tissue kinet 2<1969) 75. 13. Post, J, Sklarev, R J & Hoffman, J, J natl cancer inst 50 (1973) 403. 14. Bullough, W S & Johanna, U R, Symp sot exptl biol 25 (1971) 255. 15. Marks, F, Hoppe-Seyler’s Z physiol them 352 (1971) 1273. 16. Frankfurt, 0 S, Exptl cell res 64 (1971) 140. Received August 30, 1973
Exptl Cell Re., 82 (1973)