The Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
* Author for correspondence (e-mail: meins{at}fmi.ch)
Accepted 9 September 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cell-division factor, Cytokinin, Epigenetic changes, Epimutation, Habituation, Pseudodirected variation, Tobacco
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
More recently, it was recognized that some epigenetic changes could even be
transmitted meiotically. Examples of this phenomenon, called
epimutation (Jorgensen,
1993) include paramutation
(Chandler et al., 2000
),
presetting of transposable elements
(Fedoroff et al., 1989
),
transcriptional and post-transcriptional gene silencing
(Matzke et al., 2001
;
Plasterk, 2002
;
Bird, 2002
), and genomic
imprinting (Reik, 2001
;
Baroux et al., 2003
).
Epimutation is of particular interest because it raises the possibility that
some post-zygotic developmental events can be transmitted by sexual
reproduction and, hence, could play a role in evolution
(Jablonka and Lamb, 1995
).
The present study deals with the nature of heritable changes associated
with cytokinin habituation, i.e., the epigenetic, cell-heritable loss in the
requirement of plant cells for cell-division factors in culture
(Meins, 1989). Tobacco cells
cultured from explants of leaf exhibit a cytokinin-requiring
(C) phenotype; they show an absolute requirement for a cell
division factor such as cytokinin for continuous growth on an otherwise
complete culture medium containing auxin. In contrast, cells cultured from
explants of stem cortex exhibit a constitutive cytokinin autotrophic
(C+) phenotype, i.e., they can grow continuously in the absence of
added cytokinin. Cultures established from pith consist of a mixture of two
types of C cells. Inducible C cells
rapidly habituate, i.e., they shift to the C+ state in response to
cytokinin treatment or when cultured at elevated temperatures. Noninducible
C cells remain C under these conditions.
Cloning experiments have shown that both the C and
C+ states can be inherited at the cellular level. Nevertheless,
tissues of plants regenerated from C and C+
clones exhibit the cytokinin requirement of comparable tissues from seed-grown
plants indicating that the two mitotically transmissible states are not
permanent. This observation and the finding that the rates of induction and
reversion are high102- to 103-fold faster than
gametic mutationand developmentally regulated provide strong evidence
that tissue-specific states of cytokinin requirement result from epigenetic
changes.
Stable C+ variants can also be recovered from populations of
noninducible C cells serially propagated on media containing
reduced concentrations of cytokinin (Meins
and Foster, 1985; Meins and
Foster, 1986
). This form of variation has several surprising
features (Meins and Seldran,
1994
): First, leaf tissues of plants regenerated from these
variants, unlike those from inducible C cells, exhibit the
constitutive C+ phenotype in culture. This new phenotype, called
habituated leaf (Hl), is inherited meiotically as a dominant trait at the
Habituated leaf-2 (Hl-2) locus
(Meins and Foster, 1986
).
Second, although C+ cells arise by a random rather than by a
directed process, the rate at which they arise is extremely
highapproximately 102 per cell generation. Third,
cultured cells alternate between the C+ and C
states at this high rate in both the forward and back direction. This
phenomenon, called pseudodirected variation, results from phenotypic
changes so rapid that the classical distinction between random and induced
events is blurred. Because C and C+ cells differ
in growth rate in response to cytokinin, cytokinin can act by selection on the
alternating population of cells to give changes that appear to be induced when
examined at the tissue level.
We have combined cell cloning and plant regeneration experiments to show that cell-heritable states of cytokinin requirement generated by pseudodirected variation persist in regenerated plants and can be meiotically transmitted. Unlike most classical mutations, these heritable states undergo rapid reversion in successive sexual generations indicating that pseudodirected variation is a novel form of epimutation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Culture of tissues, cell cloning and plant regeneration
Methods for isolating tissues, culturing tissues, cloning cells and
regenerating plants have been described in detail elsewhere
(Binns and Meins, 1973;
Meins et al., 1980
). In brief,
C+ tissues were grown on a basal medium containing agar, salts,
sucrose, myo-inositol and thiamine at the concentrations recommended
by Linsmaier and Skoog (Linsmaier and
Skoog, 1965
) supplemented with 2.0 mg/l of the auxin
-naphthaleneacetic acid, and 5 mg/l of the pH indicator chlorophenol
red. The C tissues were grown on a complete medium
consisting of basal medium supplemented with 0.3 mg/l of the cytokinin
kinetin. Tissue explants,
10 mg in weight, were incubated in the light for
21 days at 25°C in shell vials containing 10 ml of medium. Clones were
obtained by marking the position of single cells plated in soft agar. Plants
were regenerated from cloned lines by incubating tissues on kinetin medium
(i.e., complete medium without auxin), and transferring the resultant shoots
on a rooting medium. Plants were placed in soil and grown to maturity in a
greenhouse. The regenerated plants are referred to as the S0
generation. Plants obtained by selfing S0 plants are referred to as
S1, S2, etc. for each successive generation. Haploid
plants were regenerated from cultured anthers as described by Bourgin and
Nitsch (Bourgin and Nitsch,
1967
).
Measuring cytokinin requirement
Two sets of four replicate tissue explants were subcultured twice, one set
on +kinetin (complete) medium and one set onkinetin (basal) medium and
were then weighed. Tissues were classified as C or
C+ using as the criterion relative growth rate (R) onkinetin
and +kinetin media. R was calculated from the expression
ln(W/W0)kinetin/ln(W/W0)+kinetin,
where W0 and W are the fresh weights of the inoculum and the tissue
after 3 weeks, respectively. Tissues giving an average R value greater than
0.4 were judged to be C+ (Binns
and Meins, 1973). Sampling error in distributions of progeny was
estimated by the binomial proportions test
(Simpson et al., 1960
).
Selection for variants
C+ variants were obtained from cloned lines of
C cells by subculturing tissues on medium containing 1% of
the kinetin concentration in complete medium as described previously
(Meins and Seldran, 1994).
C variants were obtained from cloned lines of C+
cells by subculturing tissues sequentially on media containing 1%, 10% and
100% of the kinetin concentration in complete medium and selecting for rapidly
growing colonies after each transfer.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
Stability of the revertant C phenotype in
regenerated plants
The protocol for studying the stability of the revertant
C phenotype in S0, S1 and
S2 generation plants is illustrated for the Hl-2/Hl-2 line
A in Fig. 2. One to three
replicate plants were regenerated from individual revertant and non-revertant
clones. Leaf tissues cultured from these plants were then assayed for their
cytokinin requirement. Table 7
shows that the phenotype of the non-revertant C+ clones persisted
in the regenerates: the seven S0 plants regenerated from the three
non-revertant C+ clones A7, A29 and A43 expressed the C+
phenotype. In contrast, the seven S0 plants regenerated from the
three C revertant clones A9, A10 and A11 varied widely in
phenotype. For example, all plants regenerated from clones A9 and A11 showed
the C+ phenotype and only one of the two plants regenerated from
clone A10 retained the C phenotype. A similar range of R
values was obtained for plants regenerated from different clones and for
sister plants regenerated from the same clone.
|
|
Meiotic transmission of the revertant C
phenotype
S0 generation plants regenerated from clones of line A and B
origin were selfed and leaves of the progeny were assayed for their cytokinin
phenotype. Progeny showing the C leaf trait were recovered
from S0 plants derived from both C and
C+ clones of homozygous line A
(Fig. 2,
Table 7). Segregation of the
C leaf trait in the S1 generation was variable,
viz., 26:4, 10:5, 14:1. Moreover, the C+ progeny showed a
`weak' C+ phenotype, with R values in the range 0.4-0.5, which is
far lower than the values of about 0.8-1.0 that are typical of leaves from
homozygous and heterozygous Hl-2 plants. These results show that the
revertant C phenotype arising in culture from homozygous
Hl-2/Hl-2 cells can be transmitted meiotically, but that its
inheritance is irregular. Similar conclusions can be drawn from the results
obtained with the heterozygous B line. In this case, the S0 plants
would be expected to be heterozygous for Hl-2 and the
C leaf trait should segregate 1:3 in the S1
generation. Instead, two of the three S0 plants tested gave an
unexpectedly large proportion of C progeny (data not
shown).
Two S1 plants, A9-1.2 and A9-1.9, descended from plant A9-1 regenerated from C revertant clone A9, were selfed (Table 7). Even though the two parent plants exhibited the recessive C phenotype, plants exhibiting the dominant C+ trait were recovered at frequencies of about 67% and 82% in the S2 generation. In our standard assay, tissues from only one leaf of each plant were scored. Thus, the irregular segregations observed could reflect chimerism for the Hl trait within individual plants. We confirmed that plants descended from A9-1 were variegated by comparing the R value of two different leaves from the same plants. The results showed that 3/12 of S1 plants, 4/15 of S2 progeny from plant A9-1.2 and 8/13 of S2 progeny from plant A9-1.9 exhibited different cytokinin phenotypes in the two leaves tested.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the distinction between genetic and epigenetic changes has been
debated, it is generally accepted that epigenetic changes result from
cell-heritable, but potentially reversible alterations in gene expression
(Meins, 1996;
Wu and Morris, 2001
;
van de Vijver et al., 2002
).
As judged by these criteria, the present study provides strong evidence that
epimutations, i.e., meiotically transmissible epigenetic changes
(Jorgensen, 1993
), can occur
reversibly and at high rates in culture. Most C+ clones resulting
from pseudodirected variation gave rise to plants showing the Hl phenotype,
which then segregated as a monogenic trait when the plants were selfed.
Therefore, the conversion of C to C+ cells is
associated with a meiotically heritable modification of a wild-type
hl allele to give a dominant Hl allele. Moreover, two
independent Hl-2 and Hl-3 mutants derived from C+
variants arising in culture were unstable in planta and reverted gametically
at rates roughly comparable to pseudodirected variation in culture, indicating
that the meiotically heritable changes we observed are potentially
reversible.
The finding that shoots regenerated from genetically mosaic Su/su
callus tissue are usually homogeneous in phenotype strongly suggests that
regenerated tobacco plants are clonally derived from single cells
(Lörz and Scowcroft,
1983). While leaf tissue from most of our regenerates showed the
same cytokinin phenotype as the clone from which they were derived, some,
e.g., plants A10-1, A11-1, A11-2, and A11-3
(Table 7) showed the
alternative phenotype. We believe that these plants are derived from a
subpopulation of cells that arose by rapid variation in culture subsequent to
cloning. Our finding that some regenerated plants were variegated in cytokinin
phenotype suggests, moreover, that rapid variation also occurs during the
regeneration process and the later development of the plant. This could
account for the irregular segregation of the Hl trait in the progeny of selfed
plants as reported for stable somatic mutations in tobacco
(Dulieu, 1974
;
Dulieu, 1975
;
Lörz and Scowcroft,
1983
).
Gametic revertants from Hl-2 and Hl-3 plants exhibit a stable C phenotype indistinguishable from wild type. Selfing of these progeny consistently gave exclusively C progeny (Table 4) indicating that the meiotically heritable C+ state has an epigenetic basis. In striking contrast, revertant C plants obtained by high-cytokinin selection of homozygous Hl-2/Hl-2 cells are unstable: they show mosaicism, irregular segregation of cytokinin phenotypes and high rates of reversion to the C+ state in the S2 and S3 generations (Table 7). This suggests that C+ Hl-2 cells can also revert incompletely to a metastable C state, which is distinct from the wild-type C state.
Cytokinins play a key role in regulating growth, differentiation and
morphogenesis (Schmülling,
2002). For example, acting in concert with auxins, cytokinins
induce shoot formation and inhibit root formation in undifferentiated cultures
of tobacco tissue (Skoog and Miller,
1957
). Recent studies with cytokinin-deficient transgenic tobacco
suggest cytokinins have a similar function in planta
(Werner et al., 2001
).
Organized structures arising under inductive conditions in culture are derived
from a subpopulations of committed, competent cells
(Meins, 1986
;
Merkle et al., 1995
). The
incidence of these committed cells depends on the concentration of cytokinin
and other factors in the culture medium as well as the internal epigenetic and
genetic state of the cells. Tobacco cells competent to form shoots in response
to cytokinin appear to arise reversibly in culture at rates roughly comparable
to that of pseudodirected variation (Meins
et al., 1982
). Thus, in principle, cytokinins might promote
organogenesis by selecting for a subpopulation of committed,
cytokinin-responsive cells that arise and are lost by a continuous process of
pseudodirected variation. It is often claimed that plant regeneration from
species in which regeneration is difficult in culture depends on selection
over many transfer generations to produce special morphological types of
callus. We speculate that pseudodirected variation provides a general
explanation for this phenomenon.
The molecular basis for the rapid variation we observed is not known.
Possibilities include positive autoregulation
(Meins and Binns, 1978),
reversible recombination switches
(Silverman et al., 1980
), RNA
silencing (Matzke et al.,
2001
) and stable chromatin modification
(Li et al., 2002
). Another
possibility is DNA methylation, which is known to be the basis for
well-characterized epimutations affecting the Arabidopsis SUPERMAN
gene (Jacobsen and Meyerowitz,
1997
) and the Lcyc gene of Linaria vulgaris
(Cubas et al., 1999
). Increased
DNA methylation has been shown to decrease the capacity for
cytokinin-independent growth of T-DNA transformants
(Amasino et al., 1984
;
van Slogteren et al., 1984
;
Sinkar et al., 1988
) and
tissues of tumor-prone interspecific GGLL Nicotiana hybrids
(Durante et al., 1989
;
Ahuja, 1996
). Finally, changes
in DNA methylation frequently occur in cultured plant tissues and are believed
to be a major cause for genetic as well as epigenetic forms of variation that
are sometimes meiotically transmissible
(Kaeppler et al., 2000
).
The few cases studied in detail suggest that methylation of specific genes
decreases in cultured plant tissues
(Kaeppler et al., 2000). Our
working hypothesis is that cytokinin requirement is epigenetically regulated
at loci such as Hl-2 or Hl-3 that are methylated and
transcriptionally inactive in C leaf and pith cells.
According to this hypothesis, these loci are demethylated at low rates in
culture to generate C+ cells heterozygous for the methylated
epiallele. This results in a dynamic equilibrium between the unmethylated
C+, hemimethylated C+ and methylated C
states. The state of methylation can also change at low rates in planta; but
in this case it appears that transitions to the methylated state are favored
since we have never found Hl progeny of wild-type plants
(Table 4). DNA methylation can
gradually and reversibly spread from an initial site to other sites along the
DNA leading to gradual, progressive epigenetic modifications in gene
expression (Bird, 2002
). As
judged by changes in R value, cells can show different degrees of stable
alteration; and, during prolonged culture, cells progressively increase in
their capacity for cytokinin-independent growth
(Meins and Binns, 1977
).
Graded differences in cytokinin requirement were also evident in progeny
obtained by selfing revertant C plants regenerated from
Hl-2/Hl-2 cells (Table 7). We
speculate that these metastable C states might represent
intermediate states of partial DNA methylation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahuja, M. R. (1996). Genetic nature of a nontumour mutant isolated from tumour-prone amphidiploid Nicotiana glauca-langsdorffii (GGLL): a critical assessment. Heredity 76,335 -345.
Amasino, R. M., Powell, A. L. G. and Gordon, M. P. (1984). Changes in T DNA methylation and expression are associated with phenotypic variation and plant regeneration in a crown gall tumor line. Mol. Gen. Genet. 197,437 -446.[Medline]
Baroux, C., Spillane, C. and Grossniklaus, U. (2003). Genomic imprinting during seed development. Adv. Genet. 46,165 -214.
Binns, A. and Meins, F., Jr (1973). Habituation of tobacco pith cells for factors promoting cell division is heritable and potentially reversible. Proc. Natl. Acad. Sci. USA 70,2660 -2662.[Abstract]
Bird, A. (2002). DNA methylation patterns and
epigenetic memory. Genes Dev.
16, 6-21.
Bourgin, J. P. and Nitsch, J. P. (1967). Obtention de Nicotiana haploides à partir d'etamines cultivées in vitro. Ann. Physiol. Végétale 9,377 -382.
Chandler, V. L., Eggleston, W. B. and Dorweiler, J. E. (2000). Paramutation in maize. Plant Mol. Biol. 43,121 -145.[CrossRef][Medline]
Cubas, P., Vincent, C. and Coen, E. (1999). An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401,157 -161.[CrossRef][Medline]
Dulieu, H. L. (1974). Somatic variation on a yellow mutant in Nicotiana tabacum L. (a+1/a1 a+2/a2). I. Non-reciprocal genetic events occurring in leaf cells. Mut. Res. 25,289 -304.
Dulieu, H. L. (1975). Somatic variation on a yellow mutant in Nicotiana tabacum L. (a+1/a1 a+2/a2). II. Reciprocal genetic events occurring in leaf cells. Mut. Res. 28, 69-77.
Durante, M., Cecchini, E., Natali, L., Citti, L., Geri, C., Parenti, R. and Ronchi, V. N. (1989). 5-Azacytidine induced tumorous transformation and DNA hypomethylation in Nicotiana tissue cultures. Dev. Genet. 10,298 -303.[Medline]
Fedoroff, N., Masson, P. and Banks, J.-A. (1989). Mutations, epimutations, and the developmental programming of the maize suppressor-mutator transposable element. BioEssays 10,139 -144.[Medline]
Jablonka, E. and Lamb, M. J. (1995). Epigenetic Inheritance and Evolution. The Lamarckian Dimension. Oxford, UK: Oxford University Press.
Jacobsen, S. E. and Meyerowitz, E. M. (1997).
Hypermethylation of SUPERMAN epigenetic alleles in Arabidopsis.Science 277,1100
-1103.
Jorgensen, R. (1993). The germinal inheritance of epigenetic information in plants. Phil. Trans. R. Soc. London Ser. B 339,173 -181.
Kaeppler, S. M., Kaeppler, H. F. and Rhee, Y. (2000). Epigenetic aspects of somaclonal variation in plants. Plant Mol. Biol. 43,179 -188.[CrossRef][Medline]
Lee, M. and Phillips, R. L. (1988). The chromosomal basis of somaclonal variation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39,413 -437.[CrossRef]
Li, G., Hall, T. C. and Holmes-Davis, R. (2002). Plant chromatin: development and gene control. BioEssays 24,234 -243.[CrossRef][Medline]
Linsmaier, B. and Skoog, F. (1965). Organic growth factor requirements of tobacco tissue culture. Physiol. Plant. 18,100 -127.
Lörz, H. and Scowcroft, W. R. (1983). Variability among plants and their progeny regenerated from protoplasts of Su/su heterozygotes of Nicotiana tabacum. Theor. Appl. Genet. 66,67 -75.
Matzke, M., Matzke, A. J. M. and Kooter, J. M.
(2001). RNA: Guiding gene silencing.
Science 293,1080
-1083.
Meins, F., Jr (1983). Heritable variation in plant cell culture. Annu. Rev. Plant Physiol. 34,327 -346.[CrossRef]
Meins, F., Jr (1986). Determination and morphogenetic competence in plant tissue culture. Bot. Monogr. 23,7 -25.
Meins, F., Jr (1989). Habituation: Heritable variation in the requirement of cultured plant cells for hormones. Annu. Rev. Genet. 23,395 -408.[CrossRef][Medline]
Meins, F., Jr (1996). Epigenetic modifications and gene silencing in plants. In Epigenetic Mechanisms of Gene Regulation (ed. V. E. A. Russo, R. A. Martienssen, and A. D. Riggs), pp. 415-442. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Meins, F., Jr and Binns, A. N. (1977). Epigenetic variation of cultured somatic cells: Evidence for gradual changes in the requirement for factors promoting cell division. Proc. Natl. Acad. Sci. USA 74,2928 -2932.[Abstract]
Meins, F., Jr and Binns, A. N. (1978). Epigenetic clonal variation in the requirement of plant cells for cytokinins. In The Clonal Basis for Development (ed. S. Subtelny and I. M. Sussex), pp. 185-201. New York: Academic Press Inc.
Meins, F., Jr and Foster, R. (1985). Reversible, cell-heritable changes during the development of tobacco pith tissues. Dev. Biol. 108,1 -5.[Medline]
Meins, F., Jr and Foster, R. (1986). A cytokinin mutant derived from cultured tobacco cells. Dev. Genet. 7,159 -165.[Medline]
Meins, F., Jr, Foster, R. and Lutz, J. (1982). Quantitative studies of bud initiation in cultured tobacco tissues. Planta 155,473 -477.
Meins, F., Jr, Foster, R. and Lutz, J. D. (1983). Evidence for a Mendelian factor controlling the cytokinin requirement of cultured tobacco cells. Dev. Genet. 4, 129-141.
Meins, F., Jr, Lutz, J. and Binns, A. N. (1980). Variation in the competence of tobacco pith cells for cytokinin habituation in culture. Differentiation 16, 71-75.
Meins, F., Jr and Seldran, M. (1994).
Pseudodirected variation in the requirement of cultured plant cells for
cell-division factors. Development
120,1163
-1168.
Merkle, S. A., Parrott, W. A. and Flinn, B. S. (1995). Morphogenic aspects of somatic embryogenesis. In In Vitro Embryogenesis in Plants (ed. T. A. Thorpe), pp. 155-203. Dordrecht: Kluwer Academic.
Nanney, D. L. (1958). Epigenetic control systems. Proc. Natl. Acad. Sci. USA 44,712 -717.
Plasterk, R. H. A. (2002). RNA silencing: The
genome's immune system. Science
296,1263
-1265.
Reik, W. (2001). Genomic imprinting: parental influence on the genome. Nature Rev. Genet. 2, 21-32.[CrossRef][Medline]
Schmülling, T. (2002). New Insights into the functions of cytokinins in plant development. J. Plant Growth Regul. 21,40 -49.[Medline]
Scowcroft, W. R., Brettell, R. I. S., Ryan, S. A., Davies, P. A. and Pallotta, M. A. (1987). Somaclonal variation and genomic flux. In Plant Tissue and Cell Culture (ed. C. E. Green, D. A. Somers. W. P. Hackett and D. D. Biesboes), pp.275 -288. New York: Alan R. Liss.
Silverman, M., Zieg, J., Mandel, G. and Simon, M. (1980). Analysis of the functional components of the phage variation system. Cold Spring Harb. Symp. Quant. Biol. 45, 17-26.
Simpson, G. G., Roe, A. and Lewontin, R. C. (1960). Quantatative Zoology. New York: Harcourt, Brace and World.
Sinkar, V. P., White, F. F., Furner, I. J., Abrahamsen, M., Pythoud, F. and Gordon, M. P. (1988). Reversion of aberrant plants transformed with Agrobacterium rhizogenes is associated with the transcriptional inactivation of the TL-DNA genes. Plant Physiol. 86,584 -590.
Skoog, F. and Miller, C. O. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol. 11,118 -131.
van de Vijver, G. E. R. T., van Spybroeck, L .I. N. D. amd De
Waele, D. A. N. I. (2002). Epigenetics: A Challenge for
Genetics, Evolution, and Development? Ann. NY Acad.
Sci. 981,1
-6.
van Slogteren, G. M. S., Hooykaas, P. J. J. and Schilperoort, R. A. (1984). Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment. Plant Mol. Biol. 3, 333-336.
Werner, T., Motyka, V., Strnad, M. and Schmülling, T.
(2001). Regulation of plant growth by cytokinin. Proc.
Natl. Acad. Sci. USA 98,10487
-10492.
Wu, C.-T. and Morris, J. R. (2001). Genes,
genetics, and epigenetics: A correspondence. Science
293,1103
-1105.
Related articles in Development: