Planta
Planta 143, 1-4(1978)
9 by Springer-Verlag 1978
The Influence of Gibberellic Acid on Reassociation Kinetics of D N A of Daucus carota L. A. Schfifer and K.-H. Neumann Institut ffir Pflanzenern/ihrung der Universit/it, Abteilung Gewebekultur, Eichgfirtenallee 3, D-6300 Giegen, Federal Republic of G e r m a n y
Abstract. Carrot DNA, extracted from the tap root
of untreated and gibberellic acid (GA3)-treated plants and of different varieties, was analyzed by reassociation kinetics. Differences due to GA3 treatment appear mainly in the intermediate repeated D N A region. Differences in approximately the same region are found using carrot D N A of different varieties, which also show differences in the slow reassociating sequences. By hybridizing a " f a m i l y " of the " u n i q u e " D N A range with D N A obtained from GA3-treated plants and the controls, respectively, it could be shown that changes in the composition of total D N A are the result of GA3 treatment. Key words: sociation.
Daucus
-
D N A - Gibberellin - Reas-
opment of carrot plants (Schwab and Neumann, 1975), the effect of GA3 application on the reassociation behavior of DNA was investigated. Carrot roots of different origins were chosen for comparable studies. Furthermore, various fractions of D N A were described to characterize these fractions by means of reassociation kinetics and melting profiles.
Materials and Methods Growth of Plant Material Carrot plants (Daucus carota L., var. Rote Riesen) were grown as described by N e u m a n n and Schwab (1975). Plants were sprayed with 10 ml of a solution containing 1.73 x 10 -4 tool GA 3 twice a week from the appearance of the first normal leaves until harvest. In the first experiment (Fig. 1) plants were grown for 14 weeks and in the second experiment (Fig. 4) for 29 weeks. Carrots of Dutch and Italian origin were purchased from the market.
Introduction D N A Extraction
During the past few years problems of correlation between differentiation of higher plants and composition of D N A have been investigated (for review see Nagl, 1976). In previous papers the occurrence of several satellite D N A species of in vitro and in vivo differentiating tissues of the carrot (Schiller, 1976; Schfifer et al., 1978) implied differences in the qualitative composition of total DNA. The question of correlation between somatic differentiation and qualitative composition of total D N A was further examined by reassociation kinetic studies. Gibberellic acid (GA3) has been found to exert a strong influence on differentiation and development in higher plants (for review see Jones, 1973; Neumann, 1975). Since GA3 treatments influence the devel-
D N A was extracted and purified according to Jeannin et al. (1972).
Kinetics of D N A Reassociation Reassociation of sheared (48,000 psi) total D N A (after Nze-Ekekang et al., 1974) was carried out in 0.12 M sodium phosphate buffer (pH 6.8) at 60 ~ C. Reassociation was ascertained by digesting unreassociated D N A with nuclease S. Experimental values were renormalized to 100% and Cot values (the product of initial D N A concentration in moles nucleotide per liter and time in seconds according to Britten and Kohne, 1968) were corrected respectively. Each C0t curve is the result of 50 or more experimental points with the exception of the curves of + or - GA3, in which fraction 7 was added, consisting of 15 or more experimental points (Fig.
4). Fractionation o f D N A Based on Reassociation Rates
Abbreviations: Co t = p r o d u c t of D N A concentration (mol nucleotide I - i) x time (s) ; G A 3 = gibberellic acid; Tr, = temperature for 50% denaturation
The procedure of Nze-Ekekang et al. (1974) was used. Details of the fractionation are given in Figure 2.
0032-0935/78/0143/0001/$01.00
2
A. Schfifer and K.-H. N e u m a n n : Influence of G A on Reassociation of D N A
observed in the Cot region of 1.0-15. Differences in approximately the same Cot range are found when comparing total carrot D N A of the Dutch and Italian varieties, implying differences in the slowly reassociating sequences (Co t 80 and higher). For further analysis, total D N A of the Italian variety was fractionated into eight "families" according to reassociation rates as described in Figure 2. These eight fractions form three groups. The first group with the lowest Cot consists of only fraction 1 and amounts to ca. 23% of the total DNA. The second group (fractious 2, 3, 4, 5), reassociating at slower rates, amounts to 25% of the total DNA. The third group (44% of total D N A for fractions 6, 7, 8, or 52% if fraction 9 is included) reassociates at higher Cot values and could be classified as " u n i q u e " D N A according to Britten and Kohne (1968). The first and second group are " r e p e a t e d " DNA. T m determinations of various D N A fractions suggest a difference in base composition of these "families" (Sch/ifer, 1976). For further characterization of the influence of GAa treatments on reassociation kinetics, one of the fractions of the " u n i q u e " D N A group (fraction 7) was added to the total D N A of carrots treated with GA3 and to the total D N A of the control (Fig. 4). Since radioactive carrot D N A was not available at the time in sufficient amounts, a high proportion of the D N A of fraction 7 (45% of the D N A in the reaction mixture) was used for hybridization. Renormalized Cot curves are plotted in Figure 4a. The addition of fraction 7 resulted in remarkable differ-
Results
The reassociation curve of total D N A of the tap root o f D a u c u s carota is depicted in Figure 1. The reassociation of D N A sequences ranging fl'om Co t 0.1 to 1.0 and from Cot 15 to 1500 are similar in GA 3treated and control plants (Fig. la). However, in GA3-treated plants, a faster reassociation rate can be
z E)
s0 0
?, CD
a
I00 0.1
1
10 Cot (mot • s / I )
< -7 c3
100
1000
~, 50
"-
._.9.
r'~ 10C
b
r
t IFII
).1
II
I 1 IIllltl
1
[
I [iiillr
10 Cot (tool • s / t )
i
t'~
100
1000
Fig. 1. a The influence of G A 3 sprays on reassociation kinetics of the D N A of carrot roots. ( control, - - . - G A 3 treated). b Reassociation curve of the D N A of carrot roots of Italian and D u t c h origin. ( - Dutch variety, - - Italian variety)
100 mg D N A Cot 595
I
I (250-600) 17.9% fraction 7
I
1
Cotl259
Cot 2927 ]
S
D S (100 250) 12% fraction 6
[ D (600-3000) 14.2% fraction 8
I Cot 120 I
D
I Cot 50.6
s r (50-100) 6% fraction 5
I
D
S
~] (30008.6% fraction 9
t Co t19.5
L
--s
(10 50) 6.2% fraction 4
D I S (1-10) 7.2% fraction 3
I Cot 1.38 L
D
I Cot 0.1125
i (0-0.1) 22.7% fraction 1
S
[
D
I (0.1-1) 5.2% fraction 2
Fig. 2. Diagram of the fractionation of carrot D N A according to reassociation rates. Total D N A was incubated to a Cot of 595 (60~ 0.12 M PO~). Single stranded D N A (S) was separated from double-stranded D N A (D) by passing over a hydroxyapatite column. After denaturation of the double-stranded fractions, these were again incubated until the next lower Cot. Fractions obtained are designated with margin Cot-values in parentheses. The quantity of each final fraction is' expressed as the percentage of the total quantity of D N A recovered from all fractions (s. Fig. 3)
A. Schfifer and K.-H. N e u m a n n : Influence of GA on Reassociation of D N A 0
Planta 143, 1-4(1978)
9 by Springer-Verlag 1978
The Influence of Gibberellic Acid on Reassociation Kinetics of D N A of Daucus carota L. A. Schfifer and K.-H. Neumann Institut ffir Pflanzenern/ihrung der Universit/it, Abteilung Gewebekultur, Eichgfirtenallee 3, D-6300 Giegen, Federal Republic of G e r m a n y
Abstract. Carrot DNA, extracted from the tap root
of untreated and gibberellic acid (GA3)-treated plants and of different varieties, was analyzed by reassociation kinetics. Differences due to GA3 treatment appear mainly in the intermediate repeated D N A region. Differences in approximately the same region are found using carrot D N A of different varieties, which also show differences in the slow reassociating sequences. By hybridizing a " f a m i l y " of the " u n i q u e " D N A range with D N A obtained from GA3-treated plants and the controls, respectively, it could be shown that changes in the composition of total D N A are the result of GA3 treatment. Key words: sociation.
Daucus
-
D N A - Gibberellin - Reas-
opment of carrot plants (Schwab and Neumann, 1975), the effect of GA3 application on the reassociation behavior of DNA was investigated. Carrot roots of different origins were chosen for comparable studies. Furthermore, various fractions of D N A were described to characterize these fractions by means of reassociation kinetics and melting profiles.
Materials and Methods Growth of Plant Material Carrot plants (Daucus carota L., var. Rote Riesen) were grown as described by N e u m a n n and Schwab (1975). Plants were sprayed with 10 ml of a solution containing 1.73 x 10 -4 tool GA 3 twice a week from the appearance of the first normal leaves until harvest. In the first experiment (Fig. 1) plants were grown for 14 weeks and in the second experiment (Fig. 4) for 29 weeks. Carrots of Dutch and Italian origin were purchased from the market.
Introduction D N A Extraction
During the past few years problems of correlation between differentiation of higher plants and composition of D N A have been investigated (for review see Nagl, 1976). In previous papers the occurrence of several satellite D N A species of in vitro and in vivo differentiating tissues of the carrot (Schiller, 1976; Schfifer et al., 1978) implied differences in the qualitative composition of total DNA. The question of correlation between somatic differentiation and qualitative composition of total D N A was further examined by reassociation kinetic studies. Gibberellic acid (GA3) has been found to exert a strong influence on differentiation and development in higher plants (for review see Jones, 1973; Neumann, 1975). Since GA3 treatments influence the devel-
D N A was extracted and purified according to Jeannin et al. (1972).
Kinetics of D N A Reassociation Reassociation of sheared (48,000 psi) total D N A (after Nze-Ekekang et al., 1974) was carried out in 0.12 M sodium phosphate buffer (pH 6.8) at 60 ~ C. Reassociation was ascertained by digesting unreassociated D N A with nuclease S. Experimental values were renormalized to 100% and Cot values (the product of initial D N A concentration in moles nucleotide per liter and time in seconds according to Britten and Kohne, 1968) were corrected respectively. Each C0t curve is the result of 50 or more experimental points with the exception of the curves of + or - GA3, in which fraction 7 was added, consisting of 15 or more experimental points (Fig.
4). Fractionation o f D N A Based on Reassociation Rates
Abbreviations: Co t = p r o d u c t of D N A concentration (mol nucleotide I - i) x time (s) ; G A 3 = gibberellic acid; Tr, = temperature for 50% denaturation
The procedure of Nze-Ekekang et al. (1974) was used. Details of the fractionation are given in Figure 2.
0032-0935/78/0143/0001/$01.00
2
A. Schfifer and K.-H. N e u m a n n : Influence of G A on Reassociation of D N A
observed in the Cot region of 1.0-15. Differences in approximately the same Cot range are found when comparing total carrot D N A of the Dutch and Italian varieties, implying differences in the slowly reassociating sequences (Co t 80 and higher). For further analysis, total D N A of the Italian variety was fractionated into eight "families" according to reassociation rates as described in Figure 2. These eight fractions form three groups. The first group with the lowest Cot consists of only fraction 1 and amounts to ca. 23% of the total DNA. The second group (fractious 2, 3, 4, 5), reassociating at slower rates, amounts to 25% of the total DNA. The third group (44% of total D N A for fractions 6, 7, 8, or 52% if fraction 9 is included) reassociates at higher Cot values and could be classified as " u n i q u e " D N A according to Britten and Kohne (1968). The first and second group are " r e p e a t e d " DNA. T m determinations of various D N A fractions suggest a difference in base composition of these "families" (Sch/ifer, 1976). For further characterization of the influence of GAa treatments on reassociation kinetics, one of the fractions of the " u n i q u e " D N A group (fraction 7) was added to the total D N A of carrots treated with GA3 and to the total D N A of the control (Fig. 4). Since radioactive carrot D N A was not available at the time in sufficient amounts, a high proportion of the D N A of fraction 7 (45% of the D N A in the reaction mixture) was used for hybridization. Renormalized Cot curves are plotted in Figure 4a. The addition of fraction 7 resulted in remarkable differ-
Results
The reassociation curve of total D N A of the tap root o f D a u c u s carota is depicted in Figure 1. The reassociation of D N A sequences ranging fl'om Co t 0.1 to 1.0 and from Cot 15 to 1500 are similar in GA 3treated and control plants (Fig. la). However, in GA3-treated plants, a faster reassociation rate can be
z E)
s0 0
?, CD
a
I00 0.1
1
10 Cot (mot • s / I )
< -7 c3
100
1000
~, 50
"-
._.9.
r'~ 10C
b
r
t IFII
).1
II
I 1 IIllltl
1
[
I [iiillr
10 Cot (tool • s / t )
i
t'~
100
1000
Fig. 1. a The influence of G A 3 sprays on reassociation kinetics of the D N A of carrot roots. ( control, - - . - G A 3 treated). b Reassociation curve of the D N A of carrot roots of Italian and D u t c h origin. ( - Dutch variety, - - Italian variety)
100 mg D N A Cot 595
I
I (250-600) 17.9% fraction 7
I
1
Cotl259
Cot 2927 ]
S
D S (100 250) 12% fraction 6
[ D (600-3000) 14.2% fraction 8
I Cot 120 I
D
I Cot 50.6
s r (50-100) 6% fraction 5
I
D
S
~] (30008.6% fraction 9
t Co t19.5
L
--s
(10 50) 6.2% fraction 4
D I S (1-10) 7.2% fraction 3
I Cot 1.38 L
D
I Cot 0.1125
i (0-0.1) 22.7% fraction 1
S
[
D
I (0.1-1) 5.2% fraction 2
Fig. 2. Diagram of the fractionation of carrot D N A according to reassociation rates. Total D N A was incubated to a Cot of 595 (60~ 0.12 M PO~). Single stranded D N A (S) was separated from double-stranded D N A (D) by passing over a hydroxyapatite column. After denaturation of the double-stranded fractions, these were again incubated until the next lower Cot. Fractions obtained are designated with margin Cot-values in parentheses. The quantity of each final fraction is' expressed as the percentage of the total quantity of D N A recovered from all fractions (s. Fig. 3)
A. Schfifer and K.-H. N e u m a n n : Influence of GA on Reassociation of D N A 0
