1. PLANT EPIGENETIC MECHANISMS
Classical epigenetic systems
Gene silencing
Viral cross-protection
Epigenetics in plant development
Epigenetics is the analysis of genetic changes that are stable, often heritable, but not irreversible in the same sense as are base changes, insertions, deletions, and other rearrangements. Until just a few years ago -- somewhere between 5 and this field was considered slightly disreputable. Anyone interested in DNA methylation, an epigenetic modification, wasn’t quite taken seriously. But no longer. The analysis of epigenetic phenomena, particularly gene silencing, is not is suddenly one of the most exciting areas of contemporary biology -- both in plants and in animals. My purpose here is to give you a brief overview of epigenetic studies in plants, from their origins in the studies of McClintock and other maize geneticists, to contemporary applications in plant protection
I will start bybriefly describing two classicalmaize epigenetic systems and then touch on more recent observations. Development, of course, is an epigenetic phenomenon and we increasingly understand that setting up differential gene expression patterns and locking them in during development occur by different mechanisms and involve different genes. I’ve long thought that plants offer a unique opportunity to analyse epigenetic phenomena because they produce germcells repeatedly during development, making it possible occasionally to catch epigenetic mutations, or epimutations, in the germline for genetic analysis. There is some truth to this in the sense that there were well-develop genetic analyses of epigenetic phenomena in plants earlier than there were in other eukaryotic genetic systems. But I think that the differences are not fundamental. Recent progress in understanding position effect variegation and Polycomb gene function in Drosophila and of X- inactivation and imprinting in mammals has made it evident that the commonalities are greater than the differences.

2. CLASSICAL EPIGENETIC SYSTEMS
Transposons - change of phase
Paramutation in maize
The two classical epigenetic phenomena that were first characterized genetically in maize are changes in the activity phase of transposable genetic elements, identified and described by Barbara McClintock, and paramutation, described by Alexander Brink and his students. Although McClintock is widely credited with the discovery of transposition, it is less widely appreciated that she was also the first to describe and analyse an epimutations, although she certainly didn’t call them that.

3. Changes in Spm activity phase
Heritable, but reversible
Epimutants differ in their
developmental expression patterns
The transition from active to cryptic (and the
reverse) takes several plant generations
McClintock reported in the 1950s that transposons can undergo reversible, albeit heritable, changes in activity, behaving as if they’d disappeared, then reappearing. In fact, she believed that transposons were present in the genome in a silent form, which I later designated “cryptic,” and she set out to prove it by “evoking” if you will, a transposon that her friend Marcus Rhoades had identified, but which she’d never seen in her cultures. And of course she succeeded.
She invested a great deal of effort in studying the inactivation and reactivation of the Suppressor-mutator or Spm element. She understood that the Spm transposon could exist in a variety of heritable forms -- epimutants -- that differed in either the frequency, the timing, or the developmental pattern of transposon inactivation and reactivation. In one form, the transposon alternated between active and inactive phases at intervals of a few cell divisions, creating a somatic mosaic for transposon activity. The kernel in the photograph shows the effect of such a transposon on the activity of a gene in the anthocyanin biosynthetic pathway. When the transposon is on, the gene is off and vice versa. She identified other transposon epimutants which were active only in certain tissues or in certain parts of the plant, and still other epimutants that remain largely inactive, returning to an active form very occasionally.
Then, in a brilliant series of genetic experiments, she deduced that an active transposon can communicate with an inactive transposon to reactivate it when the two are brought together by a genetic cross. We subsequently showed that the interaction also heritably alters the inactive element. Exposure to an active element promotes the reactivation of an inactive element. But the process takes several plant generations before an inactive element is fully and heritably activated.
So McClintock not only discovered transposition, but also provided the first genetic evidence for the existence of an epigenetic modifier.

4. Genetic analysis of phase change
McClintock: an inactive transposon wakes up when an
active transposon is present, but segregates unchanged
Fedoroff: an active element can heritably wake up an
inactive or a cryptic element
The transition from active to cryptic (and the reverse)
takes several plant generations
McClintock reported in the 1950s that transposons can undergo reversible, albeit heritable, changes in activity, behaving as if they’d disappeared, then reappearing. In fact, she believed that transposons were present in the genome in a silent form, which I later designated “cryptic,” and she set out to prove it by “evoking” if you will, a transposon that her friend Marcus Rhoades had identified, but which she’d never seen in her cultures. And of course she succeeded.
She invested a great deal of effort in studying the inactivation and reactivation of the Suppressor-mutator or Spm element. She understood that the Spm transposon could exist in a variety of heritable forms -- epimutants -- that differed in either the frequency, the timing, or the developmental pattern of transposon inactivation and reactivation. In one form, the transposon alternated between active and inactive phases at intervals of a few cell divisions, creating a somatic mosaic for transposon activity. The kernel in the photograph shows the effect of such a transposon on the activity of a gene in the anthocyanin biosynthetic pathway. When the transposon is on, the gene is off and vice versa. She identified other transposon epimutants which were active only in certain tissues or in certain parts of the plant, and still other epimutants that remain largely inactive, returning to an active form very occasionally.
Then, in a brilliant series of genetic experiments, she deduced that an active transposon can communicate with an inactive transposon to reactivate it when the two are brought together by a genetic cross. We subsequently showed that the interaction also heritably alters the inactive element. Exposure to an active element promotes the reactivation of an inactive element. But the process takes several plant generations before an inactive element is fully and heritably activated.
So McClintock not only discovered transposition, but also provided the first genetic evidence for the existence of an epigenetic modifier.

5. Paramutation at the R locus in maize
A directed, heritable change in gene expression
r-st and r-mb termed PARAMUTAGENIC
R-r termed PARAMUTABLE
Altered expression is heritable
Partial reversion when homozygous
A paramutable allele can become paramutagenic
upon exposure to a paramutagenic allele
The second classical maize epigenetic system I’ll discuss is R locus paramutation. A tenet of classical genetics is that alleles of a gene segregate unaltered from a heterozygote. Paramutation is the name given by Alexander Brink to a phenomenon that he discoverd in maize in the 1950s that violates this most fundamental of genetic laws.
In R paramutation, one allele imposes a directed heritable change on the expression of another allele. The maize R locus, which we now know encodes transcription factors required for expression of anthocyanin biosynthetic genes, is subject to paramutation.
Brink reported that the expression of a wild-type R-r allele decreased in the next generation after exposure to either an allele called r-st (or another such allele, called r-mb, which was later identified). The alleles that do the job are termed PARAMUTAGENIC, and those which change are called PARAMUTABLE.
The alteration is heritable and can be maintained, but can also be gradually lost in certain genetic configurations.
More than that, after exposure to the paramutagenic allele, the paramutable allele can itself become paramutagenic.
Brink, R. A., Styles, E. D. and Axtell, J. D. (1968) Science, 159:

6. R gene paramutation in maize
This is illustrated here in a figure taken from a recent paper by Elsbeth Walker.
A heterozygote between the paramutable R-r allele and the paramutagenic R-mb allele gives rise to a strongly pigmented F1 heterozygote. When the heterozygote is used as the male parent in a backcross to a colorless allele, the resulting heterozygote is very much more weakly pigmented than its parent.
You will also note that the effect appears to be more pronounced when the paramutable R-r allele is transmitted through the male than through the female.
Now let’s look at what’s known about the molecular basis paramutagenicity and paramutability.
Walker, E. L. (1998), Genetics, 148:

7. Structure of a paramutagenic R allele
The R-st allele contains several highly homologous repeats
Paramutagenicity is directly proportional to the number of repeats
Transcription start sites are methylated
The structure of a paramutagenic allele of the R locus is shown here.
The key element is that this is a highly redundant locus that contains multiple copies of virtually identical coding sequences derived by unequal crossing-over.
Kermicle and his colleagues identified 80 derivaties of this locus that arose by unequal crossingover and correlated the structure of the allele with its paramutagenicity -- a prodigious amount of work.
These investigators found that the paramutagenicity of the derivatives was -- almost without exception -- proportional to the number of repeats.
It had long been hypothesized that the transposon in the paramutagenic allele was responsible for its paramutagenicity, but derivatives lacking the transposon remain paramutagenic.
Kermicle, J. L., Eggleston, W. B. and Alleman, M. (1995), Genetics, 141:

8. Structure of the paramutable R-r allele
The structure of the paramutable R locus is shown here and appears to have been derived by several steps of duplication. The functional genes are P, S1 and S2; q is a piece of a gene. The sigma component of this locus is the relic of an ancient transposon that belongs to the same family of transposons as Spm and it is now the promoter sequence for the S genes, which are divergently transcribed. The critical points are these:
1. Paramutation of this locus results in methylation of all of the promoter sequences.
2. This locus, when paramutated and methylated itself becomes paramutagenic.
3. If this central region containing the promoters is deleted, this locus loses its paramutability: it can neither be methylated nor can it become paramutagenic.
Walker, E. L. (1998), Genetics, 148:

9. transposon inactivation and paramutation
Common themes in
transposon inactivation and paramutation
Sequence duplication is central
Promoter sequences are methylated
Genes/TEs transcriptionally silenced
Silencing is heritable, but reversible
Both involve transposon sequences
So what is common to transposon inactivation and paramutation is the following:
1. Silencing is associated with sequence duplication. In the case of transposons, this generally occurs as a consequence of transposition.
2. Promoter sequences become methylated preferentially.
3. Both transposons and R genes are transcriptionally silenced
4. Silencing is heritable, but reversible. When silenced TEs or R genes are reactivated, methylation decreases or disappears.
5. There is evidence of communication between homologous sequences located at different chromosomal sites. In the case of paramutation, the silenced locus can impose silencing on allelic loci, even when they are located elsewhere in the genome. In the case of TEs, a transcriptionally active transposon can activate an inactive one, leading to its heritable reactivation and demethylation. As I mentioned earlier, this is mediated by an element-encoded protein in the case of Spm. Another maize transposon, Mu, undergoes concerted methylation and inactivation of copies throughout the genome.
6. Finally, both involve transposon sequences. In the case of the paramutable R locus, a transposon sequence is integral to the promoter. The difficulty in interpreting this observation is that the deletion simultaneously removes the promoter sequences and the transposon sequences, so it cannot be ascertained whether paramutability and the ability to become paramutagenic requires the transposon sequences or transcription of these genes -- or both.

10. Gene silencing (co-suppression) by trangenes
Transgenes can silence endogenous genes
More transgenes, more gene silencing
Inverted repeats are especially effective
Silenced genes are often methylated
Silencing can be heritable
Silenced genes can be “paramutagenic”
A phenomenon that resembles paramutation was discovered when people began to make transgenic plants. One of the earliest reports was from Rich Jorgensen, who introduced a chalcone synthase transgene into Petunia plants and found that both the transgene and the endogenous gene could be inactivated simultaneously. The gene was commonly inactivated in part of the floral tissue, as shown in this illustration, or it could be silenced throughout the plant.
Similar observations were subsequently made by many investigators for many different transgenes. It appears that a certain fraction of transformants derived by the commonly used Agrobacterium transformation technology exhibit what has come to be called gene silencing or co-suppression. What this means is that if an extra copy of a gene already present in the genome is introduced, both the endogenous and the introduced copies are silenced together, hence the name “co-suppression.” But the gene silencing is not confined to endogenous genes. A transgene can be silenced, as well, and the probability that it is silenced increases with the number of copies. The salient features of co-suppression are these:
1. The probability of inactivation increases with copy number
2. The copies can be tandem direct repeats or inverted repeats and inverted repeats are more effective than direct repeats.
3. Silencing can be heritable, but isn’t always.
4. A silenced multicopy locus can be “paramutagenic,” meaning that it can transfer its “silenced” state to another allele of the gene located elsewhere in the genome.
Que, Q, Want, H.-Y, and Jorgensen, R.A. (1998). Plant J. 13: 401-9

11. Transcriptional and post-transcription silencing
(TGS and PTGS)
Silencing can be transcriptional, post-transcriptional or both
TGS is associated with promoter methylation
PTGS is associated with coding sequence methylation
Promotor methylation is not required for initiation of silencing
Methylation is required for the maintenance of silencing
Now as I noted earlier, some genes silenced by the introduction of transgenes are re-expressed when segregated from the transgene and in other cases they are not.
1. Transgenes that are readily reactivated upon segregation are generally not methylated and are inactivated post-transcriptionally. This is called post-transcriptional gene silencing or PTGS.
2. Transcription from such genes, as detected by run-on transcription assays, is not inhibited. That is, the phenotypically silenced genes are transcribed, often at high levels, but there is little steady-state RNA accumulation. This means that the silencing occurs by a post-transcriptional mechanism that either destabilizes the RNA or results in its premature truncation.
3. Promoter methylation is generally detected when transgenes are transcriptionally silenced, while post-transcriptionally silenced genes are often methylated in the coding region.
4. There is evidence from Herve Vaucheret’s lab that promoter methylation is required for the mainenance of transcriptional gene silencing, but not its initiation.
Having made all these generalizations, I have to remind you that all generalizations, including the present ones, are suspect. Going back a moment to maize and paramutation, what I described for the R locus clearly falls into the category of TGS. But paramutation is likely to involve more than one epigenetic mechanism. Paramutation has also been studied at the maize B locus and found to have quite different properties. In this case, the paramutagenic allele exerts its influence in the F1 heterozygote and methylation has never been detected. What’s common to both is that the interaction between alleles results in a heritable change in gene expression of the paramutable allele.

12. Gene silencing and viral resistance
Over the past few years, it has become increasingly evident that there is a connection between the phenomenon of post-transcriptional gene silencing and the acquisition of viral immunity by plants. It has long been known that plants can recover from viral infection and become immune to further infection, as shown in the illustration taken from a recent publication from David Baulcombe’s lab. Resistance can be conferred on a plant by a coat protein transgene -- this observation was made quite some time ago by Roger Beachy and his colleagues, as well as other groups -- but the mechanism has remained elusive.
Dougherty’s lab, for example, showed that:
1. Potato virus X resistance could be conferred by expression of a coat gene whose transcript couldn’t be translated, as well as one that could be translated. This implies that it is the RNA and not the protein that is responsible for virus resistance.
2. The most resistant lines had methylated transgenes that were transcribed at high levels, but had a low steady-state transcript level. This is much the same as is observed in post-transcriptional gene silencing or PTGS.
Viral infection confers immunity to further infection
Transgenic plants expressing coat protein are resistant
Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276:

13. Viral resistance is RNA-mediated
PVX
W22
PVX. W22
Transgene-induced resistance resembles PTGS
Resistance is mediated by RNA
Virus infection can result in co-suppression
Cross-protection between viruses -- these have RNA genomes for the most part -- is a function of their sequence similarity.
1. Baulcombe’s lab showed that cross-protection between viruses that normally don’t cross protect could be transferred by introducing a sequence from one into the other in such a way that it does not affect expression of the viral proteins.
2. This implies that the viral RNA itself can trigger a process in which closely related sequences are targetted for destruction (or, perhaps, aborted replication).
3. When a plant is infected with a virus into whose genome a copy of either an endogenous plant gene or a resident transgene has been inserted, the resident genes are silenced, much as is observed with co-suppression.
All of these observations suggest a close relationship between the induction of viral immunity and PTGS.
Ratcliff, F., Harrison, B. D. and Baulcombe, D. C. (1997). Science 276:

14. Gene silencing: a systemic signal
Local infection confers systemic viral immunity, implying that an inducing signal is propagated throughout the plant. This isn’t too surprising, since viruses encode a movement protein that promotes their systemic propagation.
1. In 1997, Baulcombe’s and Vaucheret’s labs reported the remarkable finding that gene silencing could spread throughout a plant from a local site of DNA introduction. This is shown here for a GFP gene taken from a Baulcombe’s initial report. In this experiment, the GFP gene was introduced on an Agrobacterium T-DNA vector on a lower leaf, indicated by the arrow, and silencing of a GFP gene already in the plant was induced in other parts of the plant, as evidenced by the red fluorescent sectors in these leaves. On the right are control leaves and leaves taken from different parts of a plant in which gene silencing had been initiated at a distant site.
2. Vaucheret’s lab used different transgenes, an NIA and a GUS gene, with similar results. And they also reported that silencing is graft transmissible, more evidence for a transmitting signal.
3. There is evidence that the signal moves both symplastically through plasmodesmata, as well as systemically through the phloem.
4. These are curious observations, indeed. Here is evidence that some sort of signal originates from a cell that receives a foreign gene and replicates and spreads much like a virus!
Voinnet, O., and Baulcombe, D. C. (1997). Nature 389: 553

15. The systemic gene silencing signal is RNA
Non-overlapping gene fragments cross-silence
RNA moves between cells in plants
Plants encode RNA-dependent RNA polymerases
What might the systemic signal be? The evidence is still a bit indirect, but favors the idea that the signal is an RNA, be it double or single stranded.
1. Baulcombe’s lab reported that a promoter is not required to initiate gene silencing by a gene fragment, although there is data from other laboratories suggesting that transcription is necessary. Curiously enough, non-overlapping gene fragments can induce cross-silencing. That is, a gene fragment can confer silencing on a viral vector containing a non-overlapping gene fragment. This suggests that the silencing is mediated by an interaction between the introduced sequence and either the resident gene itself or its transcript. Since there is ample evidence that silencing is sequence specific, the logical intermediary is the gene transcript that overlaps the two fragments.
2. So it is currently hypothesized that the signal is either an RNA degradation product or double-stranded RNA. Peter Waterhouse’s lab recently reported that transgenes comprising inverted repeats of gene sequences are particularly efficient silencing signals, but only if they have a promoter. They therefore suggest that double-stranded RNA might be the trigger.
3. And there are other clues. It has long been known that plants encode RNA-dependent RNA polymerases and their presence has been a continuing mystery. A recent exciting discovery in Neurospora is that RNA “quelling”, which strongly resembles post-transcriptional gene silencing in plants, requires a gene with homology to RNA-dependent RNA polymerase.
4. Unfortunately, the most straightforward test of the hypothesis, the induction of systemic silencing with double-stranded RNA, has not yet been successful in plants, although double-stranded RNA induces gene-specific silencing in C. elegans and in Drosophila.

16. TGS and PTGS: is there a relationship?
Replication incompetent
Replication competent
P35S PSTVd cDNA pAnos
P35S PSTVd cDNA pAnos
Transcription only
No replication
No methylation
Transcription
Replication
Methylation
Are these two mechanisms wholly independent or is there a linkage?
A paper published in 1994 by Wassenegger et al. suggests that there is, indeed, a feed-back loop between transcript abundance and methylation of the cognate gene. The system they used is wholly artificial: these investigators introduced cDNA copies of a potato spindle tuber viroid genome into tobacco plants. Viroids do not normally give rise to DNA copies and integrate into the genome and viroids do not encode proteins. However, transcription of an artificial cDNA can give rise to an internal viroid infection.
1. The authors found that the viroid cDNA sequence was methylated if the integrated cDNA encoded a replication-competent form of the viroid, but was not methylated if the integrated cDNA encoded a replication-incompetent sequence. That is, if the viroid RNA accumulated to high levels as a result of replication, the gene was methylated. If it was just transcribed, the gene did not get methylated. Moreover, the replication incompetent viroid sequence became methylated if the transgenic line was infected with the viroid. Large amounts of viroid RNA were detected only when the viroid could replicate and the implication of these observations is that there is a feedback loop from the production of large amounts of RNA to methylation of the coding sequence.
2. Recently, this group has looked at the methylation patterns in detail, reporting that all of the C residues in both strands are methylated in the region corresponding to the cDNA. They detected increased methylation of adjacent sequences, but at somewhat lower levels.
3. Thus these investigators have tapped into an endogenous mechanism that shuts down genes that are expressed at high levels.
Wassenegger, M., Heimes, S., Reidel, L., and Sanger, H. L. (1994) Cell 76:

17. microRNAs and silencingRNAs in plants
Finally, do epigenetic mechnisms of the kind I’ve discussed play any role in normal plant development?
Well, the evidence is beginning to accumulate in bits and pieces that they do. First of all, several DNA methylase genes have been identified and cloned in Jean Finnegan’s lab over the past few years. Methylase antisense plants have been made and have substantially reduced DNA methylation levels. Although such plants are viable, they tend to have many developmental anomalies that become progressively more acute with each generation. Similarly, the ddm1 mutation identified by Eric Richards, which affects a putative chromatin protein, results in the general hypomethylation of DNA and a similar range of developmental anomalies that worsen as the homozygote is propagated.
I draw your attention to this interesting parallel: methylation antisense plants and ddm1 mutant plants mirrors what has been observed with transposon inactivation and reactivation, paramutation and PAI gene methylation. In all of these different cases, changes in methylation are slow, progressing over several plant generations. Recovery from hypomethylation is also slow: methylation levels recover slowly when the methylase transgene or the ddm mutation are segregated away. But these are gross effects, difficult to dissect.
Evidence is accumulating that methylation is involved in the transition to the reproductive phase of development. Flowering is controlled by a number of different signals, including day length, temperature, light and hormone levels. Finnegan and her co-workers have reported that vernalization, the requirement for a period of cold temperatures for the initiation of flowering, involves DNA methylation.
Mallory, A. C., and Vaucheret, H. (2004) Current Opinion in Plant Biology, 7:

18. microRNAs and silencingRNAs in animals
Finally, do epigenetic mechnisms of the kind I’ve discussed play any role in normal plant development?
Well, the evidence is beginning to accumulate in bits and pieces that they do. First of all, several DNA methylase genes have been identified and cloned in Jean Finnegan’s lab over the past few years. Methylase antisense plants have been made and have substantially reduced DNA methylation levels. Although such plants are viable, they tend to have many developmental anomalies that become progressively more acute with each generation. Similarly, the ddm1 mutation identified by Eric Richards, which affects a putative chromatin protein, results in the general hypomethylation of DNA and a similar range of developmental anomalies that worsen as the homozygote is propagated.
I draw your attention to this interesting parallel: methylation antisense plants and ddm1 mutant plants mirrors what has been observed with transposon inactivation and reactivation, paramutation and PAI gene methylation. In all of these different cases, changes in methylation are slow, progressing over several plant generations. Recovery from hypomethylation is also slow: methylation levels recover slowly when the methylase transgene or the ddm mutation are segregated away. But these are gross effects, difficult to dissect.
Evidence is accumulating that methylation is involved in the transition to the reproductive phase of development. Flowering is controlled by a number of different signals, including day length, temperature, light and hormone levels. Finnegan and her co-workers have reported that vernalization, the requirement for a period of cold temperatures for the initiation of flowering, involves DNA methylation.
Mallory, A. C., and Vaucheret, H. (2004) Current Opinion in Plant Biology, 7:

19. The Arabidopsis hyl1 mutation
No ABA
0.6 µM ABA
wildtype
hyl1

20. The hyl1 mutation affects miRNA levelswt
hyl1
hen1-1 1 3
35S::HYL1
miR167
miR171
tRNA +
5S rRNA
miR159 wt
hyl1
hen1-1 1 3
ARF8
SCL6-III
rRNA
MYB33
35S::HYL1
UBQ1
DCL1 wt
hyl1
hen1-1

21. The hyl1 mutation affects mRNA stability
100
hyl1
hyl1 wt
hyl1 wt wt wt 50
35S::HYL1
35S::HYL1
35S::HYL1
35S::HYL1 30
% initial value
MYB33
ANP1
SCL6-III
ARF8 10 4 8 12 4 8 12 4 8 12 4 8 12
Time (hrs)

22. HYL1 is in nuclear bodies

23. but codes for two proteins
Spm has one gene,
but codes for two proteins
TnpA and TnpD are required for transposition
TnpA is also a weak transcription factor
promoter
TnpA
mRNA
Our laboratory cloned the Spm element some years ago and we have explored the molecular basis of Spm inactivation and reactivation. We’ve done this genetically in maize and we’ve used reconstructed transgenic systems with introduced cloned element-encoded genes and Spm promoter-reporter gene fusions. The essential findings are these:
1. When the element is inactive, its promoter sequence is methylated.
2. The heritability of the inactive or silenced state is correlated with the methylation level of a downstream, very GC-rich seqence.
3. The promoter is rapidly methylated in transgenic plants, but only if it contains the GC-rich downstream sequence.
4. The element encodes two proteins that are necessary for transposition and one of these, TnpA, is both a transposition protein and a regulator.
5. TnpA binds to multiple sites present in both direct and inverted orientations at both element ends.
6. TnpA can reactivate a silenced, methylated element both transiently and heritably, identifying it a sequence-specific epigenetic regulatory protein.
TnpD
mRNA
TnpA
TnpD
active
Spm
TnpD
Transposition

24. Changes in Spm activity phase
Promoter methylated, element inactive
Methylation of GC-rich sequence confers heritability
Reversed by Spm-encoded TnpA
promoter
Our laboratory cloned the Spm element some years ago and we have explored the molecular basis of Spm inactivation and reactivation. We’ve done this genetically in maize and we’ve used reconstructed transgenic systems with introduced cloned element-encoded genes and Spm promoter-reporter gene fusions. The essential findings are these:
1. When the element is inactive, its promoter sequence is methylated.
2. The heritability of the inactive or silenced state is correlated with the methylation level of a downstream, very GC-rich seqence.
3. The promoter is rapidly methylated in transgenic plants, but only if it contains the GC-rich downstream sequence.
4. The element encodes two proteins that are necessary for transposition and one of these, TnpA, is both a transposition protein and a regulator.
5. TnpA binds to multiple sites present in both direct and inverted orientations at both element ends.
6. TnpA can reactivate a silenced, methylated element both transiently and heritably, identifying it a sequence-specific epigenetic regulatory protein.
GC-rich sequence
TnpA
cryptic
Spm
active
Spm
Methylated site
Unmethylated site

25. Molecular mechanism of Spm activation
TnpA is a weak transcription factor
TnpA binds unmethylated and hemimethylated DNA
TnpA promotes active demethylation
Molecular mechanism of Spm activation
Methyl group
promoter
replication
TnpA 1.
TnpA
TnpA

26. Transposon silencing: the chromatin connection
mRNA
siRNAs?
transposition
siRNAs
DNA methylase
histone deacetylase
chromatin remodeling proteins
These investigators found that overall DNA methylation levels were reduced by some 15% when plants were vernalized, recovering rapidly when plants were returned to normal growth temperatures. Experimental reduction of methylation by treatment with azaC, introduction of a methylase antisense gene or the ddm1 mutation all hastened flowering without a cold treatment. A gene encoding a repressor of flowering, designated FLC and FLF in different labs, may be either the or a key methylation-sensitive gene. FLF overexpression delays flowering, while reduced expression shortens the time to flowering. Vernalization down-regulates the FLF gene (and reduces methylation generally). Hypomethylation results in a reduction in the level of FLF transcripts. Thus FLF may be a central repressor of flowering and when the gene is hypomethylated, the transcript level is reduced, allowing earlier flowering.
The next question is what controls the methylation level, of course, and that remains to be discovered. Nonetheless, there is growing evidence that methylation is a component of the complex mechanism that links environmental signals to the initiation of flowering.
What other aspects of development might involve epigenetic mechanisms? I’d venture a guess that there will be many more, although not all will involve DNA methylation. The recent discovery that plants have genes resembling the Drosophila polycomb group genes and the emerging evidence that these are involved in determining developmental patterns of gene expression suggests that we are at just at the beginning of understanding developmental epigenetics.
In closing, I hope I have presented a reasonable case that epigenetic mechanisms are central to the survival, development, and evolution of plants. That related or overlapping mechanisms are involved should be no surprise. That’s pretty much how evolution works: duplication and diversification.

27. The story of papaya ringspot virus
So whats the relationship between transcriptional and post-transcriptional gene silencing?
The clear differences are these:
1. In TGS, a gene isn’t transcribed and all, while in PTGS, the gene is transcribed, but the transcripts are either truncated or degraded.
2. In TGS, the promoter is methylated (although it should be noted that some promoters are still transcribed even when methylated). In PTGS, the gene’s coding region tends to be methylated.
3. TGS is generally heritable, PTGS is generally not heritable.
4. And finally, there are Arabidopsis mutations that prevent PTGS without affecting TGS, uncoupling the two genetically.

28. Papaya ringspot virus
So whats the relationship between transcriptional and post-transcriptional gene silencing?
The clear differences are these:
1. In TGS, a gene isn’t transcribed and all, while in PTGS, the gene is transcribed, but the transcripts are either truncated or degraded.
2. In TGS, the promoter is methylated (although it should be noted that some promoters are still transcribed even when methylated). In PTGS, the gene’s coding region tends to be methylated.
3. TGS is generally heritable, PTGS is generally not heritable.
4. And finally, there are Arabidopsis mutations that prevent PTGS without affecting TGS, uncoupling the two genetically.

29.
Papaya ringspot virus
1960s: Papaya industry moves to Puna district
1940s: PRS virus discovered in Hawaii
So whats the relationship between transcriptional and post-transcriptional gene silencing?
The clear differences are these:
1. In TGS, a gene isn’t transcribed and all, while in PTGS, the gene is transcribed, but the transcripts are either truncated or degraded.
2. In TGS, the promoter is methylated (although it should be noted that some promoters are still transcribed even when methylated). In PTGS, the gene’s coding region tends to be methylated.
3. TGS is generally heritable, PTGS is generally not heritable.
4. And finally, there are Arabidopsis mutations that prevent PTGS without affecting TGS, uncoupling the two genetically.
1950s: Oahu’s papaya industry wiped out

30. Papaya ringspot virus TGS No
So whats the relationship between transcriptional and post-transcriptional gene silencing?
The clear differences are these:
1. In TGS, a gene isn’t transcribed and all, while in PTGS, the gene is transcribed, but the transcripts are either truncated or degraded.
2. In TGS, the promoter is methylated (although it should be noted that some promoters are still transcribed even when methylated). In PTGS, the gene’s coding region tends to be methylated.
3. TGS is generally heritable, PTGS is generally not heritable.
4. And finally, there are Arabidopsis mutations that prevent PTGS without affecting TGS, uncoupling the two genetically.

31. Papaya ringspot virus
1980s: PRSV-resistance project started under direction of Dennis Gonsalves
1991: First transgenic PRSV-resistant papaya plant
1992: PRSV discovered in Puna district
1992: First field trials PRSV-resistant papaya plants
1994: USDA granted permission for large scale field trials
So whats the relationship between transcriptional and post-transcriptional gene silencing?
The clear differences are these:
1. In TGS, a gene isn’t transcribed and all, while in PTGS, the gene is transcribed, but the transcripts are either truncated or degraded.
2. In TGS, the promoter is methylated (although it should be noted that some promoters are still transcribed even when methylated). In PTGS, the gene’s coding region tends to be methylated.
3. TGS is generally heritable, PTGS is generally not heritable.
4. And finally, there are Arabidopsis mutations that prevent PTGS without affecting TGS, uncoupling the two genetically.
: Approvals for release from USDA, EPA, FDA
: PRVS spread; many farmers went out of business
1998: Seeds released, free of charge, to growers
2000: Papaya industry bounced back; crop back to pre-1995 levels

32. Papaya ringspot virus
These investigators found that overall DNA methylation levels were reduced by some 15% when plants were vernalized, recovering rapidly when plants were returned to normal growth temperatures. Experimental reduction of methylation by treatment with azaC, introduction of a methylase antisense gene or the ddm1 mutation all hastened flowering without a cold treatment. A gene encoding a repressor of flowering, designated FLC and FLF in different labs, may be either the or a key methylation-sensitive gene. FLF overexpression delays flowering, while reduced expression shortens the time to flowering. Vernalization down-regulates the FLF gene (and reduces methylation generally). Hypomethylation results in a reduction in the level of FLF transcripts. Thus FLF may be a central repressor of flowering and when the gene is hypomethylated, the transcript level is reduced, allowing earlier flowering.
The next question is what controls the methylation level, of course, and that remains to be discovered. Nonetheless, there is growing evidence that methylation is a component of the complex mechanism that links environmental signals to the initiation of flowering.
What other aspects of development might involve epigenetic mechanisms? I’d venture a guess that there will be many more, although not all will involve DNA methylation. The recent discovery that plants have genes resembling the Drosophila polycomb group genes and the emerging evidence that these are involved in determining developmental patterns of gene expression suggests that we are at just at the beginning of understanding developmental epigenetics.
In closing, I hope I have presented a reasonable case that epigenetic mechanisms are central to the survival, development, and evolution of plants. That related or overlapping mechanisms are involved should be no surprise. That’s pretty much how evolution works: duplication and diversification.

33. Epigenetic mechanisms: plant evolution, defense and development
Gene silencing is a response to gene duplication
(evolution of duplicated genes; transposon control)
Gene silencing is a response to gene overexpression
(dosage compensation)
Gene silencing is a defense response
(viral cross protection; rapid environmental responses)
Epigenetic mechanisms are used in plant development
(JAW miRNA in leaf morphogenesis)
These investigators found that overall DNA methylation levels were reduced by some 15% when plants were vernalized, recovering rapidly when plants were returned to normal growth temperatures. Experimental reduction of methylation by treatment with azaC, introduction of a methylase antisense gene or the ddm1 mutation all hastened flowering without a cold treatment. A gene encoding a repressor of flowering, designated FLC and FLF in different labs, may be either the or a key methylation-sensitive gene. FLF overexpression delays flowering, while reduced expression shortens the time to flowering. Vernalization down-regulates the FLF gene (and reduces methylation generally). Hypomethylation results in a reduction in the level of FLF transcripts. Thus FLF may be a central repressor of flowering and when the gene is hypomethylated, the transcript level is reduced, allowing earlier flowering.
The next question is what controls the methylation level, of course, and that remains to be discovered. Nonetheless, there is growing evidence that methylation is a component of the complex mechanism that links environmental signals to the initiation of flowering.
What other aspects of development might involve epigenetic mechanisms? I’d venture a guess that there will be many more, although not all will involve DNA methylation. The recent discovery that plants have genes resembling the Drosophila polycomb group genes and the emerging evidence that these are involved in determining developmental patterns of gene expression suggests that we are at just at the beginning of understanding developmental epigenetics.
In closing, I hope I have presented a reasonable case that epigenetic mechanisms are central to the survival, development, and evolution of plants. That related or overlapping mechanisms are involved should be no surprise. That’s pretty much how evolution works: duplication and diversification.