Maize Genetics Cooperation Newsletter
vol 84 2010
Please Note: Notes submitted to the Maize Genetics
Cooperation Newsletter may be cited only with consent of authors.
DEFIANCE
OHIO, Defiance College
Mikula,
B.C., Studer, Anthony
Using
the R paramutation system, over the
past 50 years our reports to the CNL aimed to develop the evidence for photo
thermal (PT) control of heritable changes in a Mendelian gene. The challenge of the 20th
Century was to find a gene whose epigenetic variation could be induced and the
variation was heritable. Since the
environment was dismissed as Lamarckian in 20th Century efforts
needed to be directed at a specific Mendelian gene.
In
1956 R paramutation provided an
example in which every R allele from
the R/Rst heterozygote was heritably silenced
(reduced in level of pigmentation) thus violating the first law of Mendelian
genetics. Because R silencing
occurred in 100% of the R alleles
segregating from the heterozygote, there was a high probability that if the environment
could perturb the paramutation process, it was likely that a phenotypic change
could be detected. A further
advantage of R paramutation was that
a continuum of expression-states was available for monitoring changes in R on a test cross ear where as many as
200 kernels can exhibit changes in expression-states. Another advantage was paramutated R alleles showed incremental change (�memory�) from year to year
which meant small changes in expression one year could be amplified the
following year. The continuum of expression-states in testcrosses of
paramutated R alleles suggested that
progression of epigenetic variation in pollen from different tassel branches
needed to be examined carefully.
Thus
conditions were available to test whether environmental conditions (photo
therma, PT)
could influence paramutation, that is, cause heritable change in
a Mendelian gene expression.
Plants
grown under field conditions showed no clear variation between pollen tested
from the upper and lower tassel branches so effort was directed at timing how and when tassels formed in
the W22 inbred. The Kettering
Foundation provided controlled environment chambers where the maize seedling was
germinated under defined conditions of light (LD) and temperature.
Seedlings
germinated and maintained under continuous light (LL) developed quickly during
the first week after which necrotic lesions were observed at the leaf
tips. When light/dark (LD)
conditions were then applied, seedlings continued developing and necrotic
tissues were reduced. With the
appearance of necrotic lesions, following LL conditions, the seedling was ready
for tassel induction with the application of the LD conditions. Under LL seedling continued to initiate
leaf nodes until LD conditions are applied.
At maturity, pollen from upper tassel branches of
plants that as seedlings were subjected to controlled conditions showed more
silencing than pollen test crossed from lower branches of the same plant. Results shown in Fig.1 and were
reported to the Genetics Congress in Australia in 2003. The pooled means of columns three and
four, each representing several plants,
obscured the epigenetic changes in individual plants.
Early pollen from upper branches showed less pigment (more silencing) than
later pollen from lower branches among seedlings started at LL 22o. The epigenetic dynamics that was taking
place in the tassel pollen of individual plants was obscured in the pooled mean
scores.
A better graphic was needed to show the epigenetic
changes taking place in each tassel.
Figures 1-3 show the changes in 75 individual plants as pigment
expression profiles for the 75 plants whose pigment expression was presented as
pooled means in Table 1.
The mean values of 50 kernel-pigment scores from the
early and 50 from late pollen collections determined the linear profiles of the
75 plants in the three figures.
Pigment scores were determined by visually matching
individual kernels against a set o standard kernels that ranged from 1 (near
colorless) to 20 (full pigment).
Individual plant pigment-score profiles are distributed across the spread sheet in the three figures. In all 75 plants the earliest pollen sampled from the upper tassel
branches showed the greatest silencing expressions when compared with late
pollen from the same plant.
Fig. 1 represents plants that as seedlings received
mostly 32o conditions with pigment scores falling largely in the
lower half of the pigment scoring range (most silencing).
Fig. 3. Shows plants that received 22o
conditions as seedlings gave pigment scores represented in the lower half of
the scoring range (least silencing).
Fig. 2. Plants that received combinations of 32o
and 22o for various cycles over a six-day period show scores that
range across the upper and lower half of the pigment range.
The ovals in the three figures show how the pigment
scores were biased by the early conditions experienced by the seedlings.
The dynamics of paramutation responses, in single
plants, to PT conditions becomes more apparent in the spread sheet display of pigment
profiles figures 1-3. This dynamic was lost in the pooled means of Table 1,.
Within the second and third week a critical period
of development was identified by pigment responses from pollen test crosses of the mature plants exposed to earlier
temperature and LD cycles. In Fig.
2 temperature shifts for as few as two cycles into and out of 22o
and 32o can cause differences in the level of R silencing. The
variation in the profiles of the three figures helps to understand why the
continuum of pigment expression was difficult to interpret from field grown
material when, for 50 years, only single pollinations from single plants were
examined. The variation in a
single tassel was interpreted as stochastic when reported as pooled means from
single pollen samples from single plants.
The superscript numbers over each profile indicates
the interval in days between the early and late pollen samples from the same
plant. The number indicates that
the expression gradient between upper and lower tassel branches from plant to
plant could be quite variable suggesting that the rate of silencing in the
tassel was determined by early conditions but at the same time variable from
plant to plant.
The three figures provide 7500 epialleles, 100 from
each plant, 50 from early pollen and 50 from late. Only two days of pollen are represented in each profile. Since pollen is shed over a period of
eight days there is at least six times more variation available from the 75
plants sampled. Thus under the
conditions of paramutation an immense amount and range of R variation is
available for natural selection for a single generation, variation that can be
incremented the following generation.
The epigenetic nature of silencing means that the
expression-states can be incremented each generation at the lower temperature
or achieved within one or two LD cycles under the higher temperature. This means that temperature variation
early in plant development can have a significant role in determining variable
heritable phenotypes. What is
remarkable is that the variation is PT regulated. This means that pigment will be more intense at the lower
temperature and more UV will be filtered by the red pigment. 75 profiles of epigenetic R expression-states (epialleles) are
determined by PT conditions early in seedling development but expressed at
maturity in pollen testcrosses following gametogenesis. It is remarkable that
the silencing is orchestrated by a TE fragment in the promoter of an R inverted repeat and amplified by R
duplications on the homologous chromosome in the heterozygote R/Rst.
The TE silencing mechanism has taken on the role of
modulating gene expression in response to PT (environmental conditions) at a
critical stage of development. The
heritable epigenetic nature of the change in expression permits the inference
that TEs can have a role providing diverse expression-state for natural
selection.
References in support of our interpretation (see
also the accompanying note):
Kermicle, in a review of paramutation, suspected
involvement of doppia in
paramutation. The reports on tandem r repeats in the Rst haplotype made it possible to
consider that the role of doppia on
silencing was amplified by duplications on the homologous chromosome. Kermicle, J., 1996 Paragenetic
Modifications in Maize. In Epigenetic Mechanisms of Gene Regulation (ed. V. E.
A. Russo, R. A. Martiensses, and A. D. Riggs.) Cold Spr. Hbr. Lab. Pr
That R paramutation silencing
involved the silencing machinery was inferred from:
Sidorenko, Lyudmila and Vicki Chandler. 2008 RNA-Dependent RNA Polymerase Is
Required for Enahancer-Mediated Transcriptional Silencing Associated With
Paramutation. Genetics . 180: 1983-1993.
Chandler, V.
and Maike Stam, 2004 Chromatin
Conversations: Mechanisms and Implications of Paramutation. Nature
Reviews, GENETICS. 5:
534-543.
The interpretation of paramutation/silencing has
been based on three extensive reviews of epigenetic silencing associated with
transposable elements (TEs).
Fedoroff, Nina. 2000 Transposons and genome evolution in plants. PNAS
Vol. 97, No. 13,7002-7007.
Feschotte, C. and Ellen J. Pritham, 2007 DNA Transposons and the Evolution of the
Eukaryotic Genomes. Ann. Rev. Genet. 41: 331-368.
Slotkin, R. Keith, Robert Martienssen,
2007. Transposable elements and the epigenetic
regulation of the genome. Nature Reviews GENETICS Vol.
8, 272-285.
We are grateful to the Charles F. Kettering
Foundation for providing growth chambers and to former students Beth Besaw and
Tammie Rettig for technical assistance.
Comparison of Pigment Scores
of Earliest and Latest Pollen Samples
______________________________________________
Early
Late
Growth
Line n Pooled Means
Day 1-15 Day 16-21
48 8 8.0 �3.4 9.3 �5.0 LL 22o 2 LD 22o-4 LL32o
47 7 10.3 �2.0 13.5 �3.2 LL 22o 2 LD 22o-4 LL 22o
46 6 8.7 �3.1 10.9 �2.9 LL 22o 6 LL 22o
49
9 9.3 �1.7 12.5 �1.1 LL 22o 4 LD 22o-2 LL 22o
50 6 9.9 �4.0 14.4 �3.3 LL 22o 4 LD 22o-2 LL 32o
45 8 9.8 �2.6 13.4 �2.1 LL 22o 6 LD 22o
_________________________________________________________________
Part B
Day 1-10 Day 11-15
30 6 3.3 �.6 3.2 �1.5 LL 32o 5
LL 32o
31 7 8.2 �2.3 9.6 �3.8 LL 32o 5 LL 22o
26 5 8.7 �3.5 9.2 �3.2 LL 32o 5 LD 32o
27 7 9.3 �3.2 10.7 �2.5 LL 32o 5 LD 22o
28 7 9.2 �3.8 9.2 �3.6 LL 32o 2 LD 32o-3 LL 22o
29 4
8.8 �1.2 10.8 �3.2 LL 32o 2 LD 22o-3 LL 22o
_________________________________________________________________
Table 1 correlates the effect of
seedling photo thermal environments with phenotypic R paramutation pigment scores from pollen test crosses made from
upper (early) and lower (later) tassel branches at maturity. Multiple pollen test crosses
were made from the same plant within a period of eight days.
Part A. Seedlings were started in continuous light (LL) at 22o,
Days 1-15, as indicated in column five.
On days 16-21, seedlings were treated to a variety of LD (Light/Dark) LL
(continuous light) and temperature cycles as indicated in column six.
Part B. Seedlings were started in continuous light (LL) at 32o,
days 1-10, as indicated in column five.
On Days 11-15 seedlings were treated to a variety of LD, LL and
temperature cycles as indicated in column six. The lines of column one represent siblings whose treatments
differed as indicated in column siz.
The number of plants tested in each line is given in the second
column. The range of pigment
scores from single plants is presented in Figures 1-3 as pigment score profiles
for each of the 75 plants sampled.
1 2 3 4 5 6
7 8 9
10 11 12 13 14 15 16 17 18 19 20
. No. Earliest and Latest Pigment Scores
Environment
3
o 7 x LL32o
4 o 4 x
5LD32o
5
o 6 x
�������������������������������.
2 o 5 x
LL32o-
3 o4x 5LL32o
4 o3x
5 o 3 x
6 o 4 x
�������������������������������..
28-1
o3x
2
o 5
x
3 o 6 x
LL32o
5 o 4 x 2LD32o
6 o 6 x 3LL22o
7 o5x
8
o 6 x
�������������������������������..
Figure
1. Three
lines of sibling plants, 26, 30 and 28, were started in 32o LL
conditions, days 1-10, then shifted to conditions, in
bold type, days11-15, the critical period. Symbols o and
x represent means of 50 kernels
from early and late pollen test cross scores, respectively. The superscript over each profiele
indicates the interval in days between early and later pollen collections
1 2 3
4 5 6
7 8 9
10 11 12 13 14 15 16 17 18 19 20
2 o 6 x
4 o 5
x 5LL22o
5 o
5 x
6 o 4 x
�������������������������������.
27-1 o
5
x
2 o 6 x
3
o 6 x
LL32o
4
o 8 x
5LD22o
5
o 4
x
6 o 7 x
7
o 6 x
�������������������������������..
48-1
o 4 x
2
o 7 x
3
o 3 x
LL22o-
4 o2x
2LD22o
5
o4x
4LL32o
6
o4x
7 o4x
8 o 4 x
�������������������������������
50-1 o 6 x
2 o 7 x
3
o
7
x
4 o
5 x
LL22o
5 o 6
x 4LD22o
6
o 4 x 2LL32o
��������������������������������.
Figure
2. Lines 31 and 27 were started in 32oLL
conditions, days 1-10, then transferred to 5LL22o and
5LD,respectively, days 11-15.
Lines 48 and 50 were started in 22oLL conditions, days 16-17,
line 48 received two LD22o cycles, then was
transferred to 4LL32o cycles.
Days 16-19 line 50 received 4LD22o cycles then was transferred to 2LL32o
cycles, days 20-21. Symbols o and
x represent means of 50 kernels from early and later pollinations,
respectively. The superscript
indicates the interval in days between early and late pollen collections.
Sampled from single plants over an eight day period
1 2 3 4 5 6
7 8 9
10 11 12 13 14 15 16 17 18 19 20
2
o
7
x
3
o4x LL22o
4 o 5
x 2LD22o
5 o 5
x
4LL22o
6 o 4 x
8
o 5
x
�������������������������������
49-1
o 5 x
LL22o
2
o 4 x 4LD22o
3
o 5
x 2LL22o
4
o 8 x
5
o3x
6
o 5 x
7
o 6
x
8
o 5 x
�������������������������������..
45-1
o 2 x
2 o 7
x
3 o 6 x
LL22o
4 o
8 x
6LD22o
5 o
6
x
6 o
5 x
7 o
8
x
8
o5x
�������������������������������
46-1
o 5 x
2
o 4 x
LL22o
3
o 3 x
4
o 7
x
5
o 5
x
7
o 2 x
�������������������������������.
Figure 3. All four lines 47, 49, 45, and 46 were
started in 22oLL conditions, days 1-15. During the critical period, days 16-21 seedlings were
shifted to the conditions and numbers of cycles listed under column Early
Environment. Symbols o and x represent means of 50 kernels from early and late pollinations,
respectively. The superscript
indicates the interval in days between early and late pollen collections.