Genome changes induced by auxin-herbicides in seedlings and calli of Zea mays L.

Environmentaland ExperimentalBotany, Vol. 28, No. 3, pp. 197 206, 1988.

0098-8472/88 $3.00 + 0.00 © 1988. Pergamon Press pie

Printed in Great Britain.

G E N O M E C H A N G E S I N D U C E D BY A U X I N - H E R B I C I D E S IN S E E D L I N G S AND CALLI OF ZEA M A Y S L. W. N A G L

Department of Biology, and Biotechnology Program, The University, P.O. Box 3049, 6750 Kaiserslautern, F.R.G. (Received9 November 1987; acceptedin revisedform 11 January 1988) NAGLW. Genomechangesinducedby auxin-herbiciagsin seedlingsand calli 0fZea mays L. ENVIRONMENTAL 28, 197-206, 1988.--Seedlings and calli derived from them were grown under the influence of the auxin-related herbicides 2,4-D, 2,4,5-T, MCPA and Picloram. Nuclear DNA was isolated and analyzed by CsC1 ultracentrifugation, thermal denaturation, renaturation and restriction digestion. Polyploidy levels were determined by cytophotometry, and chromosomes were counted in DAPI-stained squash preparations. The results indicated that the herbicides caused changes in the buoyant density of DNA in CsC1 gradients, the melting profile (and hence GC content), the reassociation kinetics (and hence the proportion of repetitive DNA fractions), the restriction pattern, and the average distribution of polyploidy. The findings are discussed as further evidence for the plasticity of the plant genome. AND EXPERIMENTAL BOTANY

INTRODUCTION

known to undergo rapid genomic diversifiHERBICIDES are widely used in agriculture with- cation/1°'~5/and hence may also strongly respond out precise knowledge of their multiple effects on to environmental and herbicide stress. cell structure(s) and metabolism. Some of their potential deleterious side-effects are seen in possMATERIALS AND METHODS ible toxic effects in animals and humans, mutagenie and other genomic effects in crops, and Whole plants possible selection for resistance in plants. ThereFor each test, five palettes each with 75 seeds fore, herbicide treatment can be compared with of Zea mays L., cv. Tombrid (a multiple hybrid; other environmental stress factors that are known Raiffeisen Inc., F.R.G.) were germinated at 27°C to cause changes in genome structure ~5'6'7/ and and grown at 22°C for 18 days. Then the seedlings gene expression/1'29/due to a "genomic shock"./16) were grown for a further 9 days and sprayed daily Herbicides are here envisaged as factors of with 50 ml (per palette), and watered every other environmental stress, but many of them also rep- day with solutions (0.2 mg/ml) of either 2,4-diresent growth substances and are indispensable for chlorophenoxyacetic acid (2,4-D), 2,4,5-tricell and tissue cultures. However, they may be chlorophenoxyacetic acid (2,4,5-T), 4-chlororesponsible for at least some of the genome insta- 2-methylphenoxyacetic acid (MCPA) or 4bilities at the DNA and chromosome levels, and amino-3,5,6-trichloropicolinic acid (Picloram or hence cause sornaclonal variation. C8/ Therefore, Tordon). T h e herbicides (synthetic auxins) were the effects of four commonly used herbicidal sub- purchased from Riedel-de-Haen, Hannover, stances, i.e. 2,4-D, 2,4,5-T, M C P A and Picloram, F.R.G., except Picloram, which was a gift of were investigated on seedlings and callus cultures Dow Chemicals, Indianapolis, U.S.A. Controls ofZea mays at the level of nuclear DNA. Maize is were sprayed and watered with tap water. 197

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Callus cultures Calli were induced from central stem parts of embryos from mature caryopses of both the cv. Tombrid and the inbred line B (Kleinwanzlebener Saatzucht, F.R.G.) on MS medium./18/ About 250 embryos were isolated, surface sterilized with a 2% solution of sodium hypochlorite for 15 min, and explanted to Petri dishes, five embryos per dish. The medium was supplemented with either 2,4-D, M C P A or Picloram at a concentration of 2.0 mg/1, or 2,4,5-T at various concentrations. A fight : dark regimen of 16 : 8 hr was chosen. Reinoculation onto the same media occurred every 2 weeks. After 6 months about 900 calli were used for the DNA and chromosome studies; the remaining calli were continued for other purposes. Isolation of D NA Nuclei were isolated from 20-40 g of leaves and roots, respectively, of the cv. Tombrid, and of callus cultures of both the genotypes cv. Tombrid and the inbred line B, according to the method ofLIMA-DE-FARIA et al. (13) and RIVlN et al. 1281DNA was isolated from the purified nuclei of seedling and calli by the technique described by MURRAY and THOMPSON, (19) using cetyltrimethylammonium bromide (CTAB) as a detergent. C3/ For most experiments, DNA was purified by ethidium bromide/CsC1 density gradient centrifugation (27'32/ and netropsin/CsCl gradients./9/ Between six and 12 replications were made of each isolation from the differently treated materials. D NA denaturation and reassociation Purified D N A showed extinction quotients of E260/E280 between 1.77 and 1.94, and of E260/E230 between 1.96 and 2.39. Denaturation and renaturation of DNA in 0.12 M phosphate buffer was monitored with a Gilford 250 spectrophotometer equipped with a thermocuvette; there were three to 12 experiments for each treatment. Restriction and gel electrophoresis The restriction enzymes Hae 3, Taq 1 and Sau 3 (Boehringer, Mannheim, F.R.G.) were used to cut total genomic DNA. Lamda-DNA, alternatively digested with Hind 3, Hind 3 + EcoR 1, Pst

1 and Bgl 1 were used as a marker for the molecular weight (size of fragments). Polyacrylamide gel electrophoresis was performed using gradient gels (2-16%). The gels were stained with ethidium bromide.

Cytophotometry Portions of plant and callus samples were fixed with ethanol: acetic acid (3 : 1) and Feulgenstained after hydrolysis with 5 N HC1 at 23°C for 30 min. DNA measurements were made in 2,4,5T treated specimens with the aid o f a Leitz scanning cytophotometer interfaced to a PDP-8 computer as described earlier./23/ Telophases were used to determine the 2C DNA content, and root tip nuclei of Allium cepa, cv. Stuttgarter Riesen (2C = 33.5 pg) were used to calibrate the absolute DNA values from the relative extinction values obtained. In total, 1200 maize and 300 onion nuclei were measured. Chromosome analysis Pieces of calli were treated with 0.5% colchicine for 3 hr, and fixed with ethanol:acetic acid (3: 1). After washing, the material was macerated with 40% pectinase for 2 hr, squashed in 45°/0 acetic acid, and air-dried after freezing and ethanol treatment. The slides were stained with 10 #g/ml DAPI (4',6-diamidino-2-phenylindole, chloride) and counterstained with 15 ~g/ml sulphorhodamine 101 in Mcllvaine buffer, pH 7, for 20 min. Chromosome counts were made in randomly scored appropriate metaphases using a Leitz fluorescence microscope. Statistics Statistical analyses were undertaken with an Apple I I a computer, employing the Kolmogorov-Smirnov test, the F test, and the t-test. RESULTS

Morphology The plantlets sprayed with the herbicides developed twisted stems and involucred or undulated leaves, as generally observed in plants treated with auxin-like substances. The roots developed thickened, sometimes bulb-like, tips after 3-4 days of treatment. The seedlings treated with Picloram yellowed rather quickly and their

G E N O M E C H A N G E S I N D U C E D BY A U X I N - H E R B I C I D E S

Examples of tissue grown fi'om embryonic axes under the influcncc of 2.0 mg/l M C P A {a) and 2,4,5-T (b). Magnifications (a) x 9.0, (b) x 1.5.

199

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FIO. 4. Examples of fragment size patterns of maize leaf DNA, as obtained by gradient polyacrylamide gel electrophoresis after digestion with the restriction enzymes Hae 3, Sau 3a and Taq 1 (from left to right).

GENOME CHANGES INDUCED BY AUXIN-HERBICIDES leaves were excluded from most of the additional experiments. The calli differed in their morphology, depending on the substances within the medium. Tissue grown under the influence of 2 mg/1 MCPA developed into a homogeneous mass of cells as typical for calli, tissue grown with 2 mg[1 2,4,5-T exhibited a strong tendency to develop root-like structures, much thicker than normal roots, and calli treated with 2,4-D showed intermediate responses. At a lower concentration of 2,4,5-T (0.2 mg/1), a mass of root-like structures evolved, while at a higher concentration (10 mg/1) compact calli originated (Fig. 1). Explants on the medium supplemented with Picloram grew extremely slowly, so that enough material was not obtained to undertake the same number of experiments as with the other treatments. In general, higher concentrations of all herbicides inhibited root growth and led to a more calluslike appearance./~7/

Buoyant density, satellite D NA Our experiments with DNA from young leaves of untreated plantlets, using ethidiumbromide/ CsC1 and netropsin/CsC1 density gradients, revealed just one, rather broad band. Root DNA differentiated into a main band and a satellite fraction. The treated seedlings did not yield enough nuclei and DNA to obtain reproducible data.

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The DNA from calli displayed a different behavior, depending on the herbicides in the culture medium and the morphology of the calli. Those grown in the presence of 2,4-D displayed one broad DNA band, while the DNA of those grown in the presence of 2,4,5-T separated into a main band and a satellite band. These cultures were morphologically characterized by the development of many root-like structures. The calli grown under the influence of MCPA also displayed satellite DNA, but of a higher density than the former. In some gradients the main band showed a non-distinct separation into two bands. The mean buoyant densities are given in Table 1.

Melting profiles, GC content of D NA The DNA from untreated plants showed a mean Tm (melting temperature) of 71.75°C, corresponding to an average GC content of 43.38%; DNA extracted from leaflets and roots was not significantly different. Under the influence of herbicides, small but not significant shifts in the Tm of the main fraction and the GC-rich thermal satellite fractions (as seen in derivative melting profiles) took place (Table 2). Callus DNA displayed a clear trend towards higher thermal stability, especially under the influence of 2,4-D. The differences between root and leaf DNA were, except for Picloram supplemented calli, statistically significant (Table 2). Among the diff-

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70 80 Temperature ('C)

90

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Fie,. 2. Examples of derivative melting profiles of DNA from maize roots, treated with different herbicides (D = 2,4-D, T = 2,4,5-T, M = MCPA; the broken line shows a directly obtained melting curve).

0.01

0.02 ~,

0.1

1 Cot

10

0.002 100

Fie,. 3. Examples for reassociation kinetics ( ) and derivative cot profiles ( ) of DNA from maize roots, treated with Picloram (P) and untreated roots (C).

202

W. NAGL Table 1. Buoyant densities of DNA extracted from maize tissues and callus cultures grown in the presence of various herbicides, compared to D NA of untreated roots and leaves DNA Main band Satellite band

Untreated Roots Leaves

Callus, treated with 2.0 mg]l 2,4-D 2,4,5-T MCPA

1.701 1.702

1.701 1.702

1.701 --

erently grown calli, all the Tm differences were statistically significant at the 1% level. Figure 2 shows examples of e x t r e m e l y large differences in melting profiles o f D N A e x t r a c t e d from maize calli. D N A reassociation kinetics Cot curves were established from p l a n t a n d callus D N A in o r d e r to d e t e r m i n e w h e t h e r a certain fraction o f repetitive D N A is the basis for changes in the G C content as revealed by t h e r m a l d e n a t u r a t i o n . I n general, the reassociation kinetics can be i n t e r p r e t e d to m e a n t h a t a b o u t 20% of the D N A represents highly repetitive sequences, while some 3 0 % represents modestly repetitive sequences. T a b l e 3 gives the pro-

1.701 1.702

1.703 1.705-6

portions of D N A reassociating with different c o t - 1 / 2 values. I t can be seen that several changes in the distribution o f highly a n d interm e d i a t e l y repetitive fractions occurred u n d e r the influence of herbicides in vivo a n d in vitro. Figure 3 shows representative cot curves and their first d e v i a t i o n for D N A of u n t r e a t e d a n d P i c l o r a m treated roots. Restriction analysis T o t a l D N A from roots, leaves a n d calli (from u n t r e a t e d a n d treated specimens) was digested with various restriction enzymes, a n d the fragments s e p a r a t e d by p o l y a c r y l gel electrophoresis using L a m d a - D N A as a m a r k e r for m o l e c u l a r weights. Digestion with H a e 3 led to the a p p e a r -

Table 2. Melting temperature and GG content of nuclear D NA isolated from different tissues ofZea mays (controls, and after treatment with various herbicides); means of 3 12 repeats Total DNA

Main fraction

Satellite fraction

% GC

Tm

°/o GC

Tm

% GC

71.8 71.8 71.8 71.2 71.0

43.4 43.4 43.4 42.0 41.5

70.5 70.5 70.6 70.1 69.9

40.9 40.3 40.6 39.4 38.9

79.3 79.5 79.5 79.5 79.1

61.7 62.3 62.5 62.3 61.3

Leaves, control 2,4-D 2,4,5-T MCPA Picloram

71.8 70.9 71.6 70.6 71.9

43.4 41.3 43.0 40.6 43.8

70.5 70.0 70.8 70.6 71.0

40.3 39.1 41.1 40.5 41.6

79.6 79.1 80.3 80.1 80.9

62.5 61.3 64.2 63.8 65.7

Calli, 2,4-D 2,4,5-T MCPA Picloram

85.7 74.0 87.9 71.3

77.35 48.80 82.72 42.21

Source and treatment

Tm

Roots, control 2,4-D 2,4,5-T MCPA Picloram

distribution too variable

GENOME CHANGES INDUCED BY AUXIN-HERBICIDES

203

Table 3. Cot-1/2 values of highly and moderately repetitivefractions of maize D NA isolatedfrom roots, leaves and calli treated with herbicides. Determinations were made in derivative reassociationprofiles (means of 3-6 repeats; significance tests were not possible, due to overlapping of several distributions) Highly rep.

Middle repetitive

Source and treatment

cot 1/2

%

I

II

III

IV

V

VI

VII

Roots, control 2,4-D 2,4,5-T MCPA Picloram

0.03 0.02 0.02 0.03 0.02

18.8 22.2 19.3 20.0 20.8

0.3 0.3 2.0 0.9 1.2

0.6 0.7 4.6 3.5 8.5

2.8 3.5 9.5 7.0

7.5 4.0 14.0 10.0

10.0 8.5 22.0 20.0

16.5 12.0

34.5

Leaves, control 2,4-D 2,4,5-T MCPA Picloram

0.03 0.03 0.03 0.04 0.04

17.11 26.5 21.7 21.8 19.3

0.4 0.5 0.5 0.8 1.0

0.6 3.0 1.7 1.8 10.0

3.3 8.0 7.8 4.3 24.0

5.5 20.5 11.0 19.0

14,0 -30.0 --

24.1

27.8

Callus, 2,4,-D Callus, MCPA

0.01 0.01

33.4 40.1

0.6 0.3

4.7 5.0

8.9 7.4

12.3 9.5

1,1 1.0

ance of one p r o m i n e n t b a n d o f 185 b p length, a n d a n u m b e r o f less distinct b a n d s between 800 a n d 10 b p f r a g m e n t lengths (Fig. 4). Restriction with Sau 3a g e n e r a t e d distinct b a n d s at 55 a n d 130 bp, a n d a d o u b l e b a n d at 180 b p lengths, as well as some a d d i t i o n a l very weak bands. T a q 1 cut the D N A to a p a t t e r n with distinct b a n d s corr e s p o n d i n g to f r a g m e n t lengths of 195, 180 a n d 155 bp, a n d some m o r e b a n d s of less intensity. U n d e r the influence o f herbicides, only the digestion of D N A with Sau 3a led to some q u a n titative differences. T h e d o u b l e b a n d at 180 b p a p p e a r e d less intensive, while the b a n d at 130 b p a p p a r e n t l y increased in intensity. As no densitometric analyses could be m a d e , these results represent p r e l i m i n a r y d e t e r m i n a t i o n s .

D NA content, ploidy level T h e 2C n u c l e a r D N A content a n d the freq u e n c y d i s t r i b u t i o n were a n a l y z e d in the i n b r e d line B, p a r t i c u l a r l y u n d e r the influence o f 2,4,5T. T h e 2C value was c a l c u l a t e d using 300 Allium cepa root meristem cell telophases as a reference to be 5.1 + 0.2 pg. S o m a t i c p o l y p l o i d was found in various tissues o f the y o u n g plants ( T a b l e 4). No c h a n g e in the frequency o f p o l y p l o i d nuclei could be d e t e c t e d u n d e r the influence of herbi-

-

cides in vivo, except in root tips (early differe n t i a t i n g region). A more detailed screening was u n d e r t a k e n in callus tissues. H e r e the effects of herbicides clearly d e p e n d e d on their concentration, exhibiting a m a x i m u m effect at an i n t e r m e d i a t e level ( T a b l e 4). A t 2.0 mg/l, a few nuclei reached 32-ploidy, while at lower a n d higher concentrations only octoploid nuclei were detected.

Karyotype T h e chromosome numbers as counted in mitoses after colchicine t r e a t m e n t o f calli roughly corresponded to the ploidy levels as d e t e r m i n e d by D N A measurements, b u t there was no absolute correlation. A m o n g 130 slides scored, which were m a d e from various cultures, the chromosomes could be c o u n t e d in only 82 mitotic figures. Fiftyeight displayed the diploid k a r y o t y p e (2n = 20), a n d only a few were polyploid. A n e u p l o i d mitoses (mainly hypoploid) a n d some structural a b n o r malities a p p a r e n t l y c o n t r i b u t e d to the v a r i a t i o n in D N A amounts. General aspects A l t h o u g h not all experiments could be m a d e with the same a m o u n t of m a t e r i a l a n d the same

204

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Table 4. Frequency distribution of ploidy levels in maize callus cultures under the effect of three concentrations of 2,4,5-T, in comparison to untreated and sprayed plant organs; the results of each test are based upon the measurement of 100 randomly scored nuclei

Tissue and treatment

2C

Per cent ploidy 4C 8C 16C

32C

Callus 0.2 mg/l 2.0 mg/1 10.0 mg/1

24 20 48

63 51 51

13 24 1

4 --

--

Root tip* untreated sprayed

38 22

56 36

4 22

1 14

1 6

Mesophyll untreated sprayed

78 74

22 24

.... 2

Scutellar internodium untreated

14

39

26

19

- 2

1

* Early differentiating region.

n u m b e r o f replications, certain conclusions seem reasonable. I n general, auxin-like herbicides affect maize plants and callus cultures at the D N A (genome) level. M C P A and Picloram exert stronger effects on genome composition in plantlets in vivo, but 2,4-D and 2,4,5-T affect nuclear D N A in in vitro cultures more than the former. DISCUSSION

T h e effect of herbicides on crop plants is of increasing interest to basic and applied plant science. It allows some insight into the function of natural growth substances in the regulation of gene expression, environmental stress, resistance phenomena, somaclonal variation in vitro, and genome and chromosome evolution in general. However, very little is known of herbicide effects at the genome level. T h e present results indicate a n u m b e r of effects of the auxin-related herbicides 2,4-D, 2,4,5-T, M C P A and Picloram on nuclear DNA. Changes induced by herbicides in the gross organization and composition of the maize genome were shown by several techniques. U p o n

CsCI density gradient ultracentrithgation, the existence and variation of satellite D N A were demonstrated for the first time, results not revealed in the present literature. Ill'31) There is good evidence that this satellite D N A is not identical to ribosomal DNA, as the latter is known to have its equilibrium at a b u o y a n t density of 1.710-1711 g/cm3, Ill'31/ i.e. clearly higher than that of the satellite band described in this paper. Also a plastidal contamination of the nuclei can be excluded, as we found a satellite band in gradients of untreated roots, but not leaves, in which chloroplast D N A m a y contribute as much as the nucleus to total cell DNA. Bacterial contamination can also be excluded, as satellite D N A could be detected in apparently sterile callus cultures as well. Even if there were some bacteria within the tissue, their D N A a m o u n t could not be responsible for a prominent band in the gradient. T h e satellite D N A is, moreover, not identical to that located in the knob heterochromatin of maize as described by PEACOCK et al. (zS) O n the basis of its different sedimentation in an ethidium bromide/CsC1 gradient it can be speculated, however, that it is circular D N A , a property suggested for amplified D N A sequences. Changes in genome composition could also be verified by D N A denaturation and reassociation. More than half of the maize genome is composed of repetitive sequences, with some clear variation between different cultivars and inbred lines, but also between tissues of an individual plant, and plants (organs) of different age. (1°'1523) Maize D N A exhibits a plurimodal distribution of base composition in derivative thermal melting profiles, indicating high heterogeneity. (26) T h e effects of herbicides on D N A composition (i.e. changes in certain fractions) are clearly expressed in the melting behavior, the total nuclear D N A content, and to some lesser extent in the restriction patterns. Very distinct effects of growth substances on nuclear D N A have been reported from protocorrn cultures of the orchid Cymbidium. In this plant, 2,4-D causes m a y differentiating cells to undergo increased amplification of an AT-rich D N A fraction, (22'~4) that is located in certain areas of chromocenters (heterochromatin). (~°) Somatic polyploidization is known to occur in

GENOME CHANGES INDUCED BY AUXIN-HERBICIDES several tissues of maize plantsJ 2) In callus cultures m a n y cells undergo aneuploidization and polyploidization in nearly all species, hence contributing to somaclonal variation./s'12) T h e present study indicates a stimulation towards higher levels of polyploidy by an intermediate concentration of synthetic auxins. I f the present and earlier results are joined together, there is strong support for the concept of rapid genomic changes in higher plants under stress conditions./33/ T h e mechanisms of action of herbicides are not yet known, but the simplest way to explain the described effects is by differential D N A replication, particularly the amplification of certain " a d a p t i v e " genes and "control" sequences (for details see some recently published reviews). 14'5'2°'2~ O n e of the most conspicuous consequences of gene amplification can be resistance to the stress factor. Additionally, a transposable element mechanism m a y be involved in genomic changes in maize./s'~4) Although the occurrence of differential D N A replication is evident and strongly supported by studies of tissue cultures, one has to be careful in the interpretation of D N A variation in this sense, because karyotype changes m a y also contribute to changes in base composition, and/or the proportion of repetitive DNA./2~) Therefore, a precise knowledge of chromosome numbers is a prerequisite for a definite interpretation of molecular data. T h e present study supports the suggestion that cell and tissue cultures m a y very well serve as model systems to study environmentally induced changes at the D N A and chromosome levels. Concomitant with these studies m a y come a better understanding of microevolution, origin of resistance, somaclonal variation, etc., and also the development of new strategies in the genetic manipulation and improvement of crop plants. I thank Mrs Silvia Moskopp, Mr F.-J. Busch, Mr U. Johann and Mr K. Schumann for providing some of the data.

Acknowledgements"

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