Planta (Bcrl.) 71, 107--112 (1966)
Short
Communication
EARLY FLOWERING IN ARABIDOPSIS INDUCED BY D N A BASE A N A L O G S * ** Y. HmoNo*** and G.P. R]~DEI Department of Field Crops, Curtis Hall University of Missouri, Columbia, Missouri Received June 30, 1966
Summary. Various isogenic lines of Arabidopsis differing in a single factor controlling flower initiation were cultured aseptically on media containing bromodeoxycytidine and bromodeoxyuridine (10-~ M). The wild type under short-day illumination (8 hours daily) and the late mutant gi2, in both continuous light and under short day, responded with dramatically earlier flower initiation. Another late mutant (/d) failed to respond. The effect of the DNA base analogs was nullified by the corresponding normal deoxyribonucleosides but not by the corresponding normal ribonucleosides. B~ow~ (1962) observed slight flower-promoting effects of iododeoxyuridine on vegetative plants of Arabidopsis thaliana. More recently it was found t h a t 8-azaadenine accelerated the flowering in Arabidopsis ( H m o ~ o and R]~DEI, 1966). I n the present communication it will be demonstrated that, in addition to the above-mentioned antimetabolites, several pyrimidine analogs promote floral development. I n the Columbia wild type of Arabidopsis and in some m u t a n t s these compounds appear to be the most effective chemicals known so far to enhance flowering. Gibberellic acid does not promote flowering in this material to an extent comparable to t h a t of some substituted pyrimidine deoxyribonucleosides. The Columbia wild type of Arabidopsis thaliana and several recessive, monogenie mutants of isogenic background (R]~DEI, 1962) were used in all experiments. The pyrimidines, the nucleosides and the analogs were obtained from Nutritional Biochemicals (Cleveland, Ohio, USA), Sigma (St. Louis, Missouri, USA), Calbiochem (Los Angeles, California, USA) companies. Each treatment involved about 20 plants. The experiments were repeated several times using several samples of the chemicals * Contribution from the Missouri Agricultural Experiment Station. Journal Series No 4005. Approved by the Director. ** Supported by National Science Foundation Grant GB-1174. *** Present address: Biology Department, Brookhaven National Laboratory, Upton, L.I., New York.
108
Y. Hrao~o and G.P. R~D]~I:
obtained from more than one source. The results were in remarkable agreement. The vurious compounds were Seitz-filtered and incorporated in u simple mineral nutrient solution fortified with 2 % dextrose and solidified with agar (0.8--1.2 % ). The plants were grown in a temperature-controlled greenhouse (200C at night, somewhat higher during sunny days) under natural light (500--1000 foot candles). Supplemental illumination during the night was provided b y incandescent lamps (100 foot-candies at the level of the culture tubes). Short-day conditions were secured by transferring the test-tube racks into a light-tight dark cabinet (20~ for 16 hours a day. Details of the aseptic culture technique were reported elsewhere (R]~D~I, 1962, 1965; HmoNo and l%s 1966). The Columbia wild type flowers very early when grown under continuous illumination (Table 1). Neither 5-bromodeoxycytidine (BDC) nor 5-bromodeoxyuridine (BDU) shortened the time to flowering in this genotype to any considerable extent under this light regime. I t appears that the shortest possible period for flowering is closely approached by long-day treatment alone. The lute mutant, gi 2 responded dramatically and about equally to both analogs. In the BDC- and BDU-treated series the number of days required from germination until the appearance of maeroscopically visible flower buds was reduced to less than haLf of that in plants grown on basal medium (Table 1). In an experiment conducted under lower, winter daylight intensities, the BDC- and BDU-treated gi 2 mutant flowered within less than 40 % of the time required for the control. In this Arabidopsis material, as in most other plants, the number of rosette and stem leaves is a reliable criterion for the extent of the vegetative development preceding the appearance of flower buds. The early wild type initiated flowers when it had one leaf less than the controls if either BDC or BDU was supplied to the medium. The control mutant gi 2 produced about three times as many leaves as the comparably handled wild type under continuous illumination. In the base-analog-treated series of the mutant, however, only a half or a third of the plants produced one leaf more than the wild type. The pyrimidine analogs practically eliminated the phenotypic difference between the mutant and the wild type. Dry weight data also reflect the extent of the vegetative development: the later the flowering the greater the weight of the plants. In soil culture under long-day conditions the gi 2 mutant may produce 20--25 times as much dry matter as the wild type (R~nwI, 1962). In test tubes, where the nutrients are limited, the difference is much less but still about 300--400 %. Both BDC and BDU shortened the vegetative development b y promoting flower differentiation and thus reduced dry weight. In the concentration indicated in the tables neither of these compounds
0
BDC 1' 10 5 BDC 1' 10 -5 4- DC 1" 10 -a DC 1- l0 s BDC 1" 10 5 -~C 1-10 u C 1-10 -a
gi 2
ffi2 ffi2
gi 2
ffi~
gi 2
gi 2 gi 2 gi 2 gi 2
B D U 1" l0 5 B D U 1' 10 ~ ~ T 1"10 -3 T 1" 10 -~ ]~DU 1" 10 5 + U 1 "10 -'~ U 1" 10 -a
0
0 BDC 1' 10 5 B D U 1" 10 5
wild wild wild
gi 2 gi s
Supplement (Mol.)
Genotype
• 0.40 ~ 0.55 4= 0.21 ~ 0.77
24.6 • 0.68
24.5 ~- 0.63 13.1 ~ 0.32
22.2 23.8 11.0 22.8
23.3 • 0.73 11.8 • 0.35
8,8 ~- 0.14 7,4 ~ 0.11 8.1 • 0.23 22.1 ~= 0,50 10.4 J: 0.22 22.4 i 0,97
:[VI=1=S.E.
103.4
102,9 55.0
100.5 100.0 46.2 95.8
105.4 53.4
100.0 84.1 92.1 100.0 47.1 101,4
%
Days from germination until visible fl0ral induction
0.05 0.10 0.12 0.18 0.18 1.05
~ 0.79 4_ 0.70 • 0.17 i 0.86
20.3 • 0.75
21,0 • 0.82 8.7 • 0.17
19.8 18.1 6.6 18.1
19.2 • 0.89 8.2 ~ 0.12
6.1 • 5.1 • 5.2 • 17,9 • 6,4 • 18,3 i
M=t=S.E.
112.2
116.0 48.1
110.6 100.0 36.5 100.0
107,3 45,8
100,0 83,6 85,3 100.0 35,8 102.2
%
Number of leaves developed beiore first flower on main stem
~ 0.07 ::t: 0.06 • 0.12 • 0.42 • 0,19 • 0,29
:[_ 0.57 • 0.34 =[_0,29 4- 0.27
13.7 :L 0.36
14.0 ~ 0.16 7.7 ::[_0,45
15.5 14.8 4.8 14.4
12.5 =[=0.43 8.0 • 0.39
4.1 2.0 1.5 14.7 4.5 11.8
M::t= S.~.
Dry weight (rag)
oxyuridine ( B D U ) , deoxyeytidine (DC), eytidine (C), thymidine ( T ) and uridine ( U ) under continuous illumination. ~u counted and dry weight determined at the termination o/the experiment
92,6
94.6 52.0
105.4 100.0 32.4 97.3
85,0 54.4
100.0 48.8 36,6 100,0 30.6 80.3
%
o] leaves
Table 1,_h'lowerinff o~ ~t~,ewild type and a late mutant (qi'~) of Arabidopsis thaliana ~n the, presence of 5-bromodeoxycgti&~ne ( BDC), 5-bromode-
%
r
o
5"
110
u Hrao~o and G.P. R~DEI:
h a d a seriously adverse effect on g r o w t h of the plants. Their effect is primarily developmental. I n a n o t h e r series of e x p e r i m e n t s b o t h the wild t y p e a n d t h e gi ~ m u t a n t were t e s t e d u n d e r 8 hours of d a i l y illumination. U n d e r these s h o r t - d a y conditions flowering was m u c h d e l a y e d in either g e n o t y p e . BDC a n d B D U , however, again r e d u c e d the t i m e r e q u i r e d to floral initiation (Table 2). I n these e x p e r i m e n t s the response of t h e wild t y p e was
Fig. 1. Typical response of Arabidopsis grown under short day conditions to bromodeoxycytidine. Left, controls; right, treated plants
Table 2. Acceleration o/ /lowering o/ the wild type and a late mutant (gi 2) o/Arabidopsis thaliana by bromodeoxycytidine ( BDC) under short-day conditions Genotype
BDC eoneentration (~ol.)
Days until visible floralinduction
M~: S.E.
wild wild gi 2 gi 2
0 1' 10-5 0 1" 10-5
49.3 :J: 1.06 16.3~ 0.39 60.0 ~ 0.98 19.5:L 0.35
%
i00.0 33.1 100.0 32.5
Number of leaves developed before first flower on main stem ~i
26.8 ~ 8.1~ 32.7 • 8.6 •
S.g.
0.43 0.17 0.47 0.15
%
100.0 30.2 100.0 26.3
Dry weight (rag)
N•
S.E.
15.1 • 0.46 5.5:j: 0.20 12.7 i 0.62 5.2 Jr 0.37
%
100.0 36.4 100.0 40.9
c o m p a r a b l e to t h a t of t h e gi~ m u t a n t . N e i t h e r of these two g e n o t y p e s flowered, however, as e a r l y in the analog series as t h e y d i d u n d e r continuous illumination. I n a n o t h e r series of e x p e r i m e n t s (to be p u b l i s h e d
Early Flowering in Arabidopsis Induced by DNA Base Analogs
111
in detail later) another late mutant, ld (I~gDEI, 1962) failed to react to BDU treatment under 8 hours daily illumination within a 110 days long culture period. Neither the controls nor the analog-treated plants homozygous for this recessive gene flowered. Under continuous illumination, however, the ld mutant is not much different from the wild type in flowering time. Both gi 2 and lcl mutants differ from the standard type in only one genetic factor. Under short-day conditions the wild type and gi 2 respond similarly to the analogs while ld is apparently quite insensitive to BDU. I t appears therefore that the control of flowering is quite different in the two non-allelie mutants, gi 2 and ld. Since the wild type and the gi 2 mutant, in contrast to Id, responded to the analogs very similarly under conditions unfavorable for flowering, it appears that there is only a quantitative difference between the flowering control of gi+ and gi ~ while ld, which does not flower at all under 8 hours daily illumination, has a qualitatively different mechanism of the photoperiodic response, insensitive also to base analogs. In addition to BDC and BDU several other base analogs were tested for their effects on flowering on the wild type and the gi 2 mutant under short- and long-day conditions. Iododeoxyuridine promoted flowering in these genotypes to about the same extent as BDC or BDU. Azauracil inhibited flowering and 5-bromouraeil had little if any effect on flower initiation. In contrast to some observations reported for other species (CHAILAKHYAN and KHLOPENKOV& 1961) the normal nueleosides alone did not affect flowering in our Arabidopsis material. I t was quite obvious, however, that the BDC effect could be nullified by deoxyeytidine and the BDU effect by thymidine (Table 1). Neither eytidine nor uridine, the corresponding ribonucleosides, interfered with the effect of the DNA-base analogs within a concentration range of 10.5 M to 10-a M. Thus it appears that these base analogs in some way affected DNA rather than RNA synthesis or function. CHA~P~. and BE~ZE~ (1962) reported that 5-fluorouraeil reversed certain mutant phcnotypes, apparently by being incorporated into mI~NA and presumably produeing normal (non-mutant) polypeptide chains by errors in base pairing. A similar mechanism of action of the deoxyribonueleoside analogs is not conceivable. Incorporation of these analogs into DNA is also unlikely since BDU, though extensively tried, failed to produce mutations in Arabidopsis (RI~D]~I, unpublished). Fluorodeoxyuridine and fluorouraeil repressed flowering in the shortday plant Pharbitis, perhaps by inhibition of DNA synthesis (ZEEvAA~T, 1962). In view of the normal cell divisions in our plants and the genotypic difference in response, it is not very likely that detectable inhibition of DNA synthesis in the treated Arabidopsis material would be the cause of the early flowering. If current concepts of gene function are
112
Y. HIt~ONO and G.P. R~DEI: Early Flowering in Arabidopsis
g e n e r a l l y applicable i t is difficult to u n d e r s t a n d w h y ribonueleoside analogs do n o t affect flowering in this Arabidopsis material. The mechanism of action of the deoxyribonueleoside analogs on flowering is n o t understood.
Acknowledgement. We are much indebted to Prof. A~ToN LA~G for the numerous suggestions concerning the manuscript.
Bibliography BROWn, J.A.M. : Effect of thymidine analogs on reproductive morphogenesis in Arabidopsis thaliana (L.) Heyn. Nature (Lond.) 196, 51--53 (1962). CIIAILAKnYAN,~V~.K., and L.P. KtILOPENKOVA: Influence of growth substances and nucleic acid metabolites on growth and flowering of photoperiodically induced plants [in Russian]. Dok!. Akad. Nauk SSSR l l l , 1497--1500 (1961). C~AI~Pv., S.P., and S. BENZER: Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequences in messenger-l~NA. Proc. Natl. Acad. Sci. (Wash.) 48, 532--546 (1962). HmoNo, u and G.P. RI~DEI: Acceleration of flowering of the long-day plant Arabidopsis by 8-azaadenine. Planta (Berh) 68, 88--93 (1966). RI~DEI, G.P.: Supervital mutants of Arabidopsis. Genetics 47, 443--460 (1962). - - Genetic blocks in the thiamine synthesis of the angiosperm Arabidopsis. Amer. J. Bot. 52, 834--841 (1965). ZEEVAART,J. A.D. : DNA multiplication as a requirement for expression of floral stimulus in Pharbitis nil. Plant Physiol. 117, 296--30~ (1962). u HnzoNo and G.P. R]h)EI Department of Field Crops, Curtis Hall University of Missouri Columbia, Missouri 65201, USA
Short
Communication
EARLY FLOWERING IN ARABIDOPSIS INDUCED BY D N A BASE A N A L O G S * ** Y. HmoNo*** and G.P. R]~DEI Department of Field Crops, Curtis Hall University of Missouri, Columbia, Missouri Received June 30, 1966
Summary. Various isogenic lines of Arabidopsis differing in a single factor controlling flower initiation were cultured aseptically on media containing bromodeoxycytidine and bromodeoxyuridine (10-~ M). The wild type under short-day illumination (8 hours daily) and the late mutant gi2, in both continuous light and under short day, responded with dramatically earlier flower initiation. Another late mutant (/d) failed to respond. The effect of the DNA base analogs was nullified by the corresponding normal deoxyribonucleosides but not by the corresponding normal ribonucleosides. B~ow~ (1962) observed slight flower-promoting effects of iododeoxyuridine on vegetative plants of Arabidopsis thaliana. More recently it was found t h a t 8-azaadenine accelerated the flowering in Arabidopsis ( H m o ~ o and R]~DEI, 1966). I n the present communication it will be demonstrated that, in addition to the above-mentioned antimetabolites, several pyrimidine analogs promote floral development. I n the Columbia wild type of Arabidopsis and in some m u t a n t s these compounds appear to be the most effective chemicals known so far to enhance flowering. Gibberellic acid does not promote flowering in this material to an extent comparable to t h a t of some substituted pyrimidine deoxyribonucleosides. The Columbia wild type of Arabidopsis thaliana and several recessive, monogenie mutants of isogenic background (R]~DEI, 1962) were used in all experiments. The pyrimidines, the nucleosides and the analogs were obtained from Nutritional Biochemicals (Cleveland, Ohio, USA), Sigma (St. Louis, Missouri, USA), Calbiochem (Los Angeles, California, USA) companies. Each treatment involved about 20 plants. The experiments were repeated several times using several samples of the chemicals * Contribution from the Missouri Agricultural Experiment Station. Journal Series No 4005. Approved by the Director. ** Supported by National Science Foundation Grant GB-1174. *** Present address: Biology Department, Brookhaven National Laboratory, Upton, L.I., New York.
108
Y. Hrao~o and G.P. R~D]~I:
obtained from more than one source. The results were in remarkable agreement. The vurious compounds were Seitz-filtered and incorporated in u simple mineral nutrient solution fortified with 2 % dextrose and solidified with agar (0.8--1.2 % ). The plants were grown in a temperature-controlled greenhouse (200C at night, somewhat higher during sunny days) under natural light (500--1000 foot candles). Supplemental illumination during the night was provided b y incandescent lamps (100 foot-candies at the level of the culture tubes). Short-day conditions were secured by transferring the test-tube racks into a light-tight dark cabinet (20~ for 16 hours a day. Details of the aseptic culture technique were reported elsewhere (R]~D~I, 1962, 1965; HmoNo and l%s 1966). The Columbia wild type flowers very early when grown under continuous illumination (Table 1). Neither 5-bromodeoxycytidine (BDC) nor 5-bromodeoxyuridine (BDU) shortened the time to flowering in this genotype to any considerable extent under this light regime. I t appears that the shortest possible period for flowering is closely approached by long-day treatment alone. The lute mutant, gi 2 responded dramatically and about equally to both analogs. In the BDC- and BDU-treated series the number of days required from germination until the appearance of maeroscopically visible flower buds was reduced to less than haLf of that in plants grown on basal medium (Table 1). In an experiment conducted under lower, winter daylight intensities, the BDC- and BDU-treated gi 2 mutant flowered within less than 40 % of the time required for the control. In this Arabidopsis material, as in most other plants, the number of rosette and stem leaves is a reliable criterion for the extent of the vegetative development preceding the appearance of flower buds. The early wild type initiated flowers when it had one leaf less than the controls if either BDC or BDU was supplied to the medium. The control mutant gi 2 produced about three times as many leaves as the comparably handled wild type under continuous illumination. In the base-analog-treated series of the mutant, however, only a half or a third of the plants produced one leaf more than the wild type. The pyrimidine analogs practically eliminated the phenotypic difference between the mutant and the wild type. Dry weight data also reflect the extent of the vegetative development: the later the flowering the greater the weight of the plants. In soil culture under long-day conditions the gi 2 mutant may produce 20--25 times as much dry matter as the wild type (R~nwI, 1962). In test tubes, where the nutrients are limited, the difference is much less but still about 300--400 %. Both BDC and BDU shortened the vegetative development b y promoting flower differentiation and thus reduced dry weight. In the concentration indicated in the tables neither of these compounds
0
BDC 1' 10 5 BDC 1' 10 -5 4- DC 1" 10 -a DC 1- l0 s BDC 1" 10 5 -~C 1-10 u C 1-10 -a
gi 2
ffi2 ffi2
gi 2
ffi~
gi 2
gi 2 gi 2 gi 2 gi 2
B D U 1" l0 5 B D U 1' 10 ~ ~ T 1"10 -3 T 1" 10 -~ ]~DU 1" 10 5 + U 1 "10 -'~ U 1" 10 -a
0
0 BDC 1' 10 5 B D U 1" 10 5
wild wild wild
gi 2 gi s
Supplement (Mol.)
Genotype
• 0.40 ~ 0.55 4= 0.21 ~ 0.77
24.6 • 0.68
24.5 ~- 0.63 13.1 ~ 0.32
22.2 23.8 11.0 22.8
23.3 • 0.73 11.8 • 0.35
8,8 ~- 0.14 7,4 ~ 0.11 8.1 • 0.23 22.1 ~= 0,50 10.4 J: 0.22 22.4 i 0,97
:[VI=1=S.E.
103.4
102,9 55.0
100.5 100.0 46.2 95.8
105.4 53.4
100.0 84.1 92.1 100.0 47.1 101,4
%
Days from germination until visible fl0ral induction
0.05 0.10 0.12 0.18 0.18 1.05
~ 0.79 4_ 0.70 • 0.17 i 0.86
20.3 • 0.75
21,0 • 0.82 8.7 • 0.17
19.8 18.1 6.6 18.1
19.2 • 0.89 8.2 ~ 0.12
6.1 • 5.1 • 5.2 • 17,9 • 6,4 • 18,3 i
M=t=S.E.
112.2
116.0 48.1
110.6 100.0 36.5 100.0
107,3 45,8
100,0 83,6 85,3 100.0 35,8 102.2
%
Number of leaves developed beiore first flower on main stem
~ 0.07 ::t: 0.06 • 0.12 • 0.42 • 0,19 • 0,29
:[_ 0.57 • 0.34 =[_0,29 4- 0.27
13.7 :L 0.36
14.0 ~ 0.16 7.7 ::[_0,45
15.5 14.8 4.8 14.4
12.5 =[=0.43 8.0 • 0.39
4.1 2.0 1.5 14.7 4.5 11.8
M::t= S.~.
Dry weight (rag)
oxyuridine ( B D U ) , deoxyeytidine (DC), eytidine (C), thymidine ( T ) and uridine ( U ) under continuous illumination. ~u counted and dry weight determined at the termination o/the experiment
92,6
94.6 52.0
105.4 100.0 32.4 97.3
85,0 54.4
100.0 48.8 36,6 100,0 30.6 80.3
%
o] leaves
Table 1,_h'lowerinff o~ ~t~,ewild type and a late mutant (qi'~) of Arabidopsis thaliana ~n the, presence of 5-bromodeoxycgti&~ne ( BDC), 5-bromode-
%
r
o
5"
110
u Hrao~o and G.P. R~DEI:
h a d a seriously adverse effect on g r o w t h of the plants. Their effect is primarily developmental. I n a n o t h e r series of e x p e r i m e n t s b o t h the wild t y p e a n d t h e gi ~ m u t a n t were t e s t e d u n d e r 8 hours of d a i l y illumination. U n d e r these s h o r t - d a y conditions flowering was m u c h d e l a y e d in either g e n o t y p e . BDC a n d B D U , however, again r e d u c e d the t i m e r e q u i r e d to floral initiation (Table 2). I n these e x p e r i m e n t s the response of t h e wild t y p e was
Fig. 1. Typical response of Arabidopsis grown under short day conditions to bromodeoxycytidine. Left, controls; right, treated plants
Table 2. Acceleration o/ /lowering o/ the wild type and a late mutant (gi 2) o/Arabidopsis thaliana by bromodeoxycytidine ( BDC) under short-day conditions Genotype
BDC eoneentration (~ol.)
Days until visible floralinduction
M~: S.E.
wild wild gi 2 gi 2
0 1' 10-5 0 1" 10-5
49.3 :J: 1.06 16.3~ 0.39 60.0 ~ 0.98 19.5:L 0.35
%
i00.0 33.1 100.0 32.5
Number of leaves developed before first flower on main stem ~i
26.8 ~ 8.1~ 32.7 • 8.6 •
S.g.
0.43 0.17 0.47 0.15
%
100.0 30.2 100.0 26.3
Dry weight (rag)
N•
S.E.
15.1 • 0.46 5.5:j: 0.20 12.7 i 0.62 5.2 Jr 0.37
%
100.0 36.4 100.0 40.9
c o m p a r a b l e to t h a t of t h e gi~ m u t a n t . N e i t h e r of these two g e n o t y p e s flowered, however, as e a r l y in the analog series as t h e y d i d u n d e r continuous illumination. I n a n o t h e r series of e x p e r i m e n t s (to be p u b l i s h e d
Early Flowering in Arabidopsis Induced by DNA Base Analogs
111
in detail later) another late mutant, ld (I~gDEI, 1962) failed to react to BDU treatment under 8 hours daily illumination within a 110 days long culture period. Neither the controls nor the analog-treated plants homozygous for this recessive gene flowered. Under continuous illumination, however, the ld mutant is not much different from the wild type in flowering time. Both gi 2 and lcl mutants differ from the standard type in only one genetic factor. Under short-day conditions the wild type and gi 2 respond similarly to the analogs while ld is apparently quite insensitive to BDU. I t appears therefore that the control of flowering is quite different in the two non-allelie mutants, gi 2 and ld. Since the wild type and the gi 2 mutant, in contrast to Id, responded to the analogs very similarly under conditions unfavorable for flowering, it appears that there is only a quantitative difference between the flowering control of gi+ and gi ~ while ld, which does not flower at all under 8 hours daily illumination, has a qualitatively different mechanism of the photoperiodic response, insensitive also to base analogs. In addition to BDC and BDU several other base analogs were tested for their effects on flowering on the wild type and the gi 2 mutant under short- and long-day conditions. Iododeoxyuridine promoted flowering in these genotypes to about the same extent as BDC or BDU. Azauracil inhibited flowering and 5-bromouraeil had little if any effect on flower initiation. In contrast to some observations reported for other species (CHAILAKHYAN and KHLOPENKOV& 1961) the normal nueleosides alone did not affect flowering in our Arabidopsis material. I t was quite obvious, however, that the BDC effect could be nullified by deoxyeytidine and the BDU effect by thymidine (Table 1). Neither eytidine nor uridine, the corresponding ribonucleosides, interfered with the effect of the DNA-base analogs within a concentration range of 10.5 M to 10-a M. Thus it appears that these base analogs in some way affected DNA rather than RNA synthesis or function. CHA~P~. and BE~ZE~ (1962) reported that 5-fluorouraeil reversed certain mutant phcnotypes, apparently by being incorporated into mI~NA and presumably produeing normal (non-mutant) polypeptide chains by errors in base pairing. A similar mechanism of action of the deoxyribonueleoside analogs is not conceivable. Incorporation of these analogs into DNA is also unlikely since BDU, though extensively tried, failed to produce mutations in Arabidopsis (RI~D]~I, unpublished). Fluorodeoxyuridine and fluorouraeil repressed flowering in the shortday plant Pharbitis, perhaps by inhibition of DNA synthesis (ZEEvAA~T, 1962). In view of the normal cell divisions in our plants and the genotypic difference in response, it is not very likely that detectable inhibition of DNA synthesis in the treated Arabidopsis material would be the cause of the early flowering. If current concepts of gene function are
112
Y. HIt~ONO and G.P. R~DEI: Early Flowering in Arabidopsis
g e n e r a l l y applicable i t is difficult to u n d e r s t a n d w h y ribonueleoside analogs do n o t affect flowering in this Arabidopsis material. The mechanism of action of the deoxyribonueleoside analogs on flowering is n o t understood.
Acknowledgement. We are much indebted to Prof. A~ToN LA~G for the numerous suggestions concerning the manuscript.
Bibliography BROWn, J.A.M. : Effect of thymidine analogs on reproductive morphogenesis in Arabidopsis thaliana (L.) Heyn. Nature (Lond.) 196, 51--53 (1962). CIIAILAKnYAN,~V~.K., and L.P. KtILOPENKOVA: Influence of growth substances and nucleic acid metabolites on growth and flowering of photoperiodically induced plants [in Russian]. Dok!. Akad. Nauk SSSR l l l , 1497--1500 (1961). C~AI~Pv., S.P., and S. BENZER: Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequences in messenger-l~NA. Proc. Natl. Acad. Sci. (Wash.) 48, 532--546 (1962). HmoNo, u and G.P. RI~DEI: Acceleration of flowering of the long-day plant Arabidopsis by 8-azaadenine. Planta (Berh) 68, 88--93 (1966). RI~DEI, G.P.: Supervital mutants of Arabidopsis. Genetics 47, 443--460 (1962). - - Genetic blocks in the thiamine synthesis of the angiosperm Arabidopsis. Amer. J. Bot. 52, 834--841 (1965). ZEEVAART,J. A.D. : DNA multiplication as a requirement for expression of floral stimulus in Pharbitis nil. Plant Physiol. 117, 296--30~ (1962). u HnzoNo and G.P. R]h)EI Department of Field Crops, Curtis Hall University of Missouri Columbia, Missouri 65201, USA
