Characterization of HD2C and its interaction with novel partners in Arabidopsis thaliana This thesis is presented for the degree of Doctor of Philosophy of the University of Western Australia Michael Van der Kwast Biochemistry and Molecular Biology School of Chemistry and Biochemistry April 2013 Supervisors: Dr Thomas Martin and Dr Martha Ludwig
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Characterization of HD2C and its interaction with novel partners in
Arabidopsis thaliana
This thesis is presented for the degree of Doctor of Philosophy of the University of Western Australia
Michael Van der Kwast
Biochemistry and Molecular Biology
School of Chemistry and Biochemistry
April 2013
Supervisors: Dr Thomas Martin and Dr Martha Ludwig
Declaration
The work presented in this thesis is my own work except where stated. This work was
carried out in the School of Chemistry and Biochemistry, Faculty of Life and Physical
Sciences, at the University of Western Australia. The material presented in this thesis
has not been presented for any other degree.
Michael Van der Kwast
April 2013
1
Acknowledgment
I would like to express my sincere gratitude and appreciation for the hard work of my
principle supervisor Dr. Thomas Martin. It was only through his guidance and
determination that I achieved my goal of finishing my PhD and accomplishing my
successes in the lab. I gained many insights into the scientific way of thinking through
our frequent meetings (I also enjoyed our many discussions on the merits of various
cricket players).I would also like to thank my second supervisor, Dr. Martha Ludwig,
who offered alternative viewpoints so that I could take my research down unexpected
avenues. Special thanks as well to all of the present and former members of the labs of
Dr. Patrick Finnegan and Martha. I enjoyed our fortnight meetings despite the
occasional disheartened atmosphere as we would share a frustrating or failed
experiment. In particular I must mention the early help that I received from Hung Chi
Lui, who took me under his wing at the start of my PhD.
I have been privileged to work with some talented scientists in Thomas’ lab. In
particular I would like to thank Adrian Sheng Hao Tong, Ruo Han Li, Sally Grasso. A
super, special thanks to Julia Man, for her amazing help when I was writing-up; their
presence kept me motivated (and occasionally entertained) in the lab and I will be sad
to leave such an enriching environment.
I am grateful for the help of Mr John Murphy and Dr Paul Rigby in learning and
operating the confocal microscope. My project was dependent on their skills and their
time was greatly appreciated.
I would like to acknowledge and thank the help of my family and friends. Daniel
Pegdon, I promised to acknowledge you here, but now you have to read EVERY WORD
of this thesis. My parents were always willing to help, and their gift of cooking dinner
when I was stressed was something that I often joked about, but really appreciated.
Lastly I would like to acknowledge and thank my wife, Karen. I am truly grateful for her
unwavering support and help whether I felt that I needed it or not. This was an equally
difficult time for her (although I’m sure that she appreciates all of the new things that
she has learnt about histone deacetylases).
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Abstract
Histone deacetylases are known to bind a variety of targets and interacting proteins
which are responsible for determining its activity in a temporal and spatial context.
Here, Bimolecular Fluorescent Complementation (BiFC) assays were used to analyse
putative interactions of the plant specific histone deacetylase HD2C isoform from
Arabidopsis thaliana. Specifically, HD2C was shown to form homo- and hetero-dimers
through interaction with other HD2 isoforms; it was shown to interact with both
epsilon and non-epsilon 14-3-3 isoforms; and lastly it was shown to interact with the
transcription factor TGA6.
Localization of HD2C-GFP traced accumulation of HD2C to the nucleus and nucleolus of
Arabidopsis leaf cells. This was evidently dynamic, as salt stress induced the HD2C-GFP
to localize to the nucleolus. This was linked to HD2C dimers, which showed a similarly
nucleolar localization pattern. The nuclear localization was shown to be dependent on
an evolutionarily conserved KKAK motif.
14-3-3 interaction with HD2C was investigated using Bimolecular Fluorescent
Complementation. HD2C bound to both epsilon and non-epsilon classes of 14-3-3
proteins, while the other HD2 isoforms were able to bind to 14-3-3 epsilon. The region
required for this interaction was traced to three distinct areas of the HD2C protein.
Site directed mutagenesis of serine and threonine residues was used to identify critical
potentially phosphorylated binding sites within the C-terminal region of the HD2C. The
study revealed that substitutions of S284, T235 and S239 by alanine abolished 14-3-3
binding to a C-terminal peptide of HD2C.
Lastly, it was attempted to identify a link between HD2C and salicylic acid (SA) and
jasmonic acid (JA) signalling. Interaction between TGA6, a known SA and JA response
transcription factor with HD2C was demonstrated using BiFC. In addition two lines
over-expressing a HD2C-GFP construct in Arabidopsis plants were compared with Col-0
wild type plants to determine if there a SA or JA dependent phenotypes could be
linked to the enzyme. An increased sensitivity of the overexpressing lines to SA and JA
was found during germination. However, when analysing HD2C expression, exogenous
application of salicylic acid and jasmonic acid had no evident effect on HD2C
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expression. Finally, HD2C was tested for its ability to regulate as-1 like promoter cis-
element containing genes which are known to be regulated by SA and/or JA. A slight
decrease in the expression of PDF1.2, a JA responsive gene, as well as in the expression
of the SA and JA- RLK1 gene was observed, suggesting that HD2C may be somewhat
and TGA2 shared expression in the hypocotyl. Together these results suggest that
TGA6 interaction with HD2C should be biologically relevant given the shared sub-
cellular localization pattern and the overlapping gene expression.
5.2.6 Analysis of the expression of genes controlled by TGA6 in HD2C modified
plants
A model for HDAC action suggests that the enzymatic deacetylation activity is directed
to specific chromatin regions by facilitating proteins that recognize specific DNA cis-
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elements (Luo, Postigo et al. 1998; Doetzlhofer, Rotheneder et al. 1999). TGA6 is
known to bind to the as-1-like motif in the promoter in SA and JA related genes
(Niggeweg, Thurow et al. 2000) and to regulate expression of a number of SA and JA
regulated genes (Zhou, Trifa et al. 2000; Zander, La Camera et al. 2010). Based on the
validated interaction between HD2C and TGA6, it was next necessary to test the
hypothesis that HD2C regulates those same genes. A selection of genes related to the
plant defence response were chosen for an expression analysis using RT-PCR.
Arabidopsis lines Col-0, HD2C-L1 and HD2C L2 were grown for 2 weeks on 0.5MS plates
before RNA was extracted, converted to cDNA which was used to perform a RT-PCR.
Actin and ATPase were amplified in parallel as housekeeping gene controls. Results
indicated that there was no change in the intensity of PCR product in the actin and
ATPase housekeeping controls (figure 5.7) The expression of EDS5, ORA, SID2, EIGT,
UGT and GST6 were not affected by the overexpression of HD2C (figure 5.7). The most
obvious change in intensity indicating a difference in expression was observed for USP,
where expression was much higher in the two 35S:HD2C-GFP lines compared to Col-0.
There was also a visible increase in the expression of PR1 compared to Col-0, although
this was less obvious from the band intensities (figure 5.7). PDF1.2 and RFLK appeared
to be expressed in lower amounts in the 35S:HD2C-GFP lines compared to Col-0.
Together these results indicate that HD2C overexpression alters the expression of USP,
PR1, PDF1.2 and RFLK, but has no effect on EDS5, ORA, SID2, EIGT, UGT or GST6.
5.3 Discussion
5.3.1 Summary
Here, it was investigated what role HD2C may have in the salicylic acid and jasmonate
response pathway. Initially it was shown in Col-0 wild type plants that exogenous
application of salicylic acid was sufficient to induce expression of the salicylic response
gene PR1, but have no evident effect on HD2C expression. Similarly, exogenous
application of methyl-jasmonate was sufficient to induce expression of the jasmonate
response gene PDF1.2, but had no evident effect on HD2C expression. Next, two lines
over-expressing a HD2C-GFP construct in Arabidopsis plants were compared with Col-0
wild type plants to determine if there was any quantifiable phenotypic evidence that
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HD2C had a role in either SA or JA response. A root length analysis on either media did
not produce any significant difference between the Arabidopsis lines. However a
germination analysis showed that over-expression of HD2C-GFP yielded a sensitive
response phenotype compared to wild type at 0.25mM, 0.5mM and 0.75mM SA and
0.25mM and 0.5mM MeJA. Next, interaction between HD2C and TGA6, a transcription
factor involved in both SA and JA signalling, was shown using BiFC. Micro-array data
suggested that there was some overlap in expression between these two proteins in
seed tissue and in the developmental progression of senescence. To test the
hypothesis that TGA6 directed HD2C to its as-1 like promoter cis-elements, RT-PCR was
used as a semi-quantitative measure of various genes that were shown to be regulated
by TGA6. A slight decrease in the JA responsive PDF1.2 expression, as well as in the SA
and JA-responsive RLK1 expression was observed, suggesting that HD2C may be
involved in regulating their expression.
5.3.2 Plants expressing 35S:HD2C-GFP have altered development in response to SA
and MeJA
It was unknown at the onset of this analysis if HD2C had a role in either the SA or JA
signalling pathways, therefore germination and root length data were obtained for
transgenic lines exposed to SA and MeJA, a biological more active form of the JA
hormone. Despite some overlapping mechanisms with respect to the defence
pathway, each hormone elicits a distinct molecular response and hence the extent of
the response was further evaluated using gene expression studies.
Germination is the process where an embryonic plant contained within its seed
transitions from dormancy to growth. This is mediated by sensitive signal transduction
pathways which induce growth in response to critical conditions such as water
availability, temperature, light and oxygen. Imbibition, where the embryonic tissue
swells sufficiently to break the seed coat, leads to the release of the embryonic root
(radicle) and is an effective marker of germination. The link between HD2 expression
and germination was previously explored in relation to salt and ABA tolerance, where
it was found that overexpression of HD2C leads to an increase in the germination
frequency compared to wild type on 150mM salt as well as in the presence of 15mM
ABA (Sridha and Wu 2006). This is consistent with the very high HD2 expression in
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barley embryos which led to the suggestion that HD2C is involved in germination.
Germination is controlled principally through the balance between ABA and GA
hormones, whereby dormancy is maintained by ABA until activation of GA biosynthesis
and signalling occur. Aside from the well-defined balance between ABA and GA, other
plant hormones have been shown to impact on seed germination. For example, SA and
MeJA were shown to inhibit germination of Col-0 Arabidopsis seeds at concentrations
>0.1mM.
Given the established link between HD2C expression and germination, the role of SA
and MeJA in delaying germination and the possible role of HD2C in signalling, it was
not surprising to find that HD2C-GFP over-expressing seeds showed an altered
response when germinated on SA or MeJA containing media. Here, in contrast to the
ABA treatment where HD2C:GFP overexpression led to a resistance phenotype, MeJA
and SA treatment of 0.25mM, 0.5mM and 0.75mM resulted in sensitivity phenotype,
as germination was evidently inhibited when compared to the wild type. This
suggested that the germination pathway is affected in different ways by ABA and SA.
This is difficult to rationalize, as previous studies appear to suggest that SA uses an
identical pathway to suppress germination. In monocots, this occurs by suppressing
the GA activated alpha-amylase (Xie, Zhang et al. 2007). The effect on germination by
SA (250µM ) required more than 10 times the molar concentration compared to ABA
(20µM). In the literature, the higher sensitivity to ABA is commonly accounted for by
the fact that SA affects the mRNA accumulation of alpha-amylase through the action
of WRKY38 negative regulation of its gene expression whilst ABA in addition to
negative transcription regulation, it directly decreases amylase secretion and
enzymatic activity, thereby effectively blanketing the cue to initialize germination. In
dicots it appears that salicylic acid inhibition of root growth is mediated by the defence
response. This pathway transfers energy from growth and reallocates it towards
fighting off pathogen attack (Heil, Hilpert et al. 2000).
The question remains therefore as to why the germination data suggest that plants
over-expressing HD2C-GFP are more tolerant to ABA as shown by (Sridha and Wu
2006), yet less tolerant to SA and JA as demonstrated in the study here.
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The answer may lie in the antagonistic relationship that exists between ABA and SA. It
was shown earlier that pathogenesis by P. syringae is marked by pathogen induced
stimulation of ABA accumulation, leading to a decrease in the SA-mediated defence
(de Torres, Bennett et al. 2009). Similarly exogenous application of small amounts of
SA was shown to null the effect of abiotic stresses such as salt which are controlled by
the ABA pathway (Borsani, Valpuesta et al. 2001). Moreover, the molecular basis of
ABA response and SA response appears consistent with this. 35S:HD2C-GFP plants
showed decreased expression of ABA response genes, therefore the antagonistic
crosstalk with GA is reduced, leading to an increase in expression. Results from the
analysis presented here showed that the SA responsive genes PR1 and EDS5 were
increased in 35S:HD2C-GFP plants. Therefore repression of germination by the
aforementioned inhibition of alpha-amylase is more likely following the alpha-amylase
pathway. This hypothesis clearly requires further investigation, as the data presented
offers only a tentative link between any cross talk of ABA and SA pathways.
5.3.3 HD2C binds TGA6 transcription factor
To rationalize the biological link of SA and MeJA with HD2C, an analysis of putative
binding partners revealed that HD2C interacted with TGA6 in planta. TGA6 belongs to
the TGA family of transcription factors, so-named because they bind to promoter cis-
elements designated as-1 like sequences which are composed of two tandem repeats
of TGACG (Idrovo Espín, Peraza-Echeverria et al. 2012). TGA6 and its redundant
homologues TGA2 and TGA5 were shown to be involved in both the SA and JA induced
plant defence response (Fan and Dong 2002). In the salicylic acid pathway TGA6 binds
NPR1 in the nucleus and activates SA-response genes such as PR1. In the jasmonic acid
pathway, TGA6 activates the jasmonate responsive PDF1.2 and b-chi genes (Zander, La
Camera et al. 2010). However it is also involved in antagonistic crosstalk between the
two pathways, with co-stimulation of SA and MeJA resulting in clade II TGAs binding
ROXY19/GRX480 and repressing the JA-responsive genes, thereby blocking the JA-
defence pathway (Zander, Chen et al. 2012; Gatz 2013).
Sensitive phenotypes in the 35S:HD2C-GFP plants when exposed to SA or MeJA suggest
that HD2C has a role in both of the pathways. Given the binding of a transcription
factor to a negative regulator of gene expression, the possibility that TGA6 was
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mediating negative regulation of its target through interaction was explored. On the
surface, this hypothesis was not consistent with the data- TGA6 has been shown to
activate SA- and JA-responsive genes, how then did HD2C factor into this relationship?
Indeed, the RT-PCR data was not overwhelmingly supportive of this hypothesis. Of the
TGA targets measured, the most obvious difference in expression was a large induction
of expression for the VSP1 gene, although there was slight induction in expression of
PR1. This clearly suggested that HD2C was not catalytically active on these promoters.
This was surprising given that HD2C was shown to bind HDA19, and that HDA19 was
involved in repressing PR1 expression (Choi, Song et al. 2012). This suggests that HD2C
is involved in a different process than HDA19, and that binding probably mediates a
different process. Similarly there was no obvious change in the expression of ORA,
SID2, EIGT, UGT or GST6; this suggests that HD2C was similarly not active on these
promoters. Given the diverse range of target genes that were targeted by TGA6, this
suggested that HD2C was not binding TGA6 to negatively regulate these targets.
Rather than engaging in the regulation of SA- and JA- responsive genes singularly,
HD2C may be involved in the cross-talk between the two pathways. The mechanism
for this cross talk has not yet been established. Given the repressive role of HD2C on
gene expression, and this unknown negative regulation on JA-responsive genes when
challenged with SA, TGA6 may direct HD2C to JA-responsive genes when stimulated by
SA. Consistent with this, RT-PCR analysis of 35S:HD2C-GFP showed that PDF1.2 and
RLK1 expression was repressed compared to the Col-0 wild type. Although there was
no salicylic induction, this may demonstrate some element of SA-JA crosstalk. These
are JA-responsive factors, with PDF1.2 being the classic example of SA mediated JA-
responsive gene repression. However, this hypothesis is tempered by a study on the
repression of the core JA-responsive element PDF1.2, where it was revealed that its
repression by the SA pathway was not mediated directly by its chromatin acetylation
state (Koornneef, Rindermann et al. 2008). Assuming that HD2C is using TGA6 to
deacetylate the PDF1.2 promoter, this suggests that it is not involved in SA-JA
crosstalk, but is active on the promoter through interaction with another pathway.
A significant amount of effort for this study was directed towards establishing a link
between HD2C action and the plant defence response. Partly this was instigated by the
broad role of TGA19 salicylic and jasmonate plant defence (Zhou, Zhang et al. 2005;
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Choi, Song et al. 2012). Additionally the already established link between HD2C and the
ABA mediated abiotic stress response provided a tentative link which suggested that
there may be a role linking HD2C with the biotic stress response (Sridha and Wu 2006).
Lastly, there is a significant current interest in linking epigenetic pathways with various
stress responses in an effort to improve plant tolerance to various harsh
environmental conditions. From the data however there is not sufficient evidence to
lead to this conclusion. Although HD2C appears to be involved in SA and JA responses,
the molecular mechanism does not appear to involve binding AS-1 like cis-factors via
TGA interaction. This may partly be due to a confounding effect caused by HD2C
interaction with other factors involved in these pathways. Principally, HD2C interaction
with ERF1 was described in relation to Longan. Assuming that this is conserved in
Arabidopsis, it is likely that this interaction would also produce the SA and MeJA
phenotypes that were observed. Future studies into this interaction may instead look
to the expression data presented in figures 5.5 and 5.6. Interestingly, HD2C and TGA6
overlap most strongly in senescing tissue and seeds. This suggests that interaction is
most likely relevant during these developmental stages.
5.3.4 Conclusions and future work
The most significant aspect of this study was the identification of HD2C interaction
with TGA6, as it provides further evidence that there is significant cross-talk between
general transcriptional regulation elements and the epigenetic pathway. As previously
mentioned, this interaction is evolutionarily practical given the specificity of
transcription factors to significant subsets of genes and the passive but stable ability
for acetylation state to control gene expression. Given the inability to trace any
overwhelming evidence that HD2C is targeting as-1 like cis binding sites to act on,
further characterization into its role is required. The possibility of a role in senescence
was suggested from micro-array data. Both SA and JA have been shown to mediate this
developmental stage.
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TGA6-YC/ HD2C-YN
TGA6-YC/ Δ HD2C-
YN
YFP CHLOROPHYLL DAPI MERGE A
B
Mouse anti-cmyc
Mouse anti-HA
Figure 5.8: BiFC analysis to determine HD2C interaction with TGA6 IN N benthamiana leaf tissue (A) Interaction of HD2C with TGA6 was evident by fluorescence caused by reconstitution of the split YFP fused to each HD2 protein. The YFP emission of each dimer overlaps with the DAPI stained nucleus in a discrete nucleolar compartment. No signal could be detected in the YFP channel for interaction between TGA6 and the 60AA N-terminal region of HD2C. A negative control consisting of the N-terminal (1-60 amino acids) of HD2C was tested for interaction. It yielded no fluorescence. (B) Expression was measured using western blot against the c-myc epitope from the YN vector and HA epitope from the YC vector. Expression was present in both tissues. Scale bar represents 20μm.
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Chapter 6
Final Discussion
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6.1 Summary
In this thesis the original aims were to characterize putative interactions to develop
insight into possible regulatory pathways that had not previously been identified.
Initially, localization of HD2C was analysed to trace the accumulation of HD2C in the
cell. HD2C was present in the nucleus and nucleolus when fused to GFP. Next, HD2C
dimers were tested to determine their fluorescent localization pattern. This revealed a
predominantly nucleolar pattern, distinct from HD2C-GP. it appears that this
localization may be dynamic, as stably transformed 35S:HD2C-GFP plants showed
increased accumulation of nucleolar fluorescence after 24 hours of salt stress. The
mechanism of this accumulation remains unknown; however a hypothesis is that
dimerization of HD2s appears to be involved based on the similar localization between
HD2C dimers and the pattern observed in salt treatment. Next two domains of HD2C
were shown to be necessary for nuclear and nucleolar localization. The nuclear
localization was shown to be dependent on an evolutionarily conserved KKAK motif,
while this did not disrupt nucleolar localization.
The interaction of 14-3-3 and HD2C was also investigated using Bimolecular
Fluorescent Complementation. It was shown that HD2C bound to both epsilon and
non-epsilon classes of 14-3-3 proteins, while the other HD2 isoforms were able to bind
to 14-3-3 epsilon. To identify the region required for this interaction, a deletion
analysis of the HD2C protein mapped out the interaction of HD2C with 14-3-3 epsilon.
Three distinct regions were found which enable interaction. Site directed mutagenesis
of possible serine and threonine residues that could correspond to the critical
phosphorylated binding site on the C-terminal region of the protein revealed that
residue S284 and one of T235 and S239, when substituted to alanine, abolished 14-3-3
binding
Lastly, interaction was investigated between TGA6 and HD2C. A link between HD2C
and SA and JA mediated plant defence was tried to be established. However,
exogenous application of salicylic acid and jasmonic acid had no evident effect on
HD2C expression. In addition two lines over-expressing a HD2C-GFP construct in
Arabidopsis plants were compared with Col-0 wild type plants to determine if there
was any phenotypes that suggest that HD2C had a role in either SA or JA response.
There was no statistically significant change in root growth on either media, however a
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sensitivity phenotype was evident for both hormones in a germination assay. Finally,
HD2C was tested for its ability to regulate as-1 like promoter cis-elements related to
stress response. A slight decrease in the JA responsive PDF1.2 expression, as well as in
the SA and JA-responsive RLK1 expression was observed, suggesting that HD2C may be
involved in regulating their expression.
6. 2 Results suggest new model of HD2 action
Prior to this thesis a HD2 model describing its functionality had not been explicitly
stated, but could be derived by amalgamating several key studies from the past 20
years (figure 6.1A). HD2C is expressed in the cytoplasm as a protein containing an N-
terminal HDAC domain, central acidic domain and C-terminal zinc finger domain. The
functional protein is part of a large, 400kDa protein complex, phosphorylated at
multiple sites and capable of removing acetyl-groups from histone H3 which was
related to down-regulating gene expression. Accumulation in the nucleus and
nucleolus was driven by an unknown mechanism. The enzymatic activity was targeted
to various genes relating to ABA response, rRNA expression and development by an
unknown mechanism. It has been speculated that molecular interactions with different
factors of the transcriptional machinery may mediate interactions of HD2C with
specific histone areas. However only the ethylene response transcription factor ERF7
was experimentally shown to bind a Longan HD2 homologue.
The work of this thesis has expanded this model by focusing on the missing elements
of the regulatory pathway. As shown in figure 6.1B, the nuclear and nucleolar
accumulation were shown to be independent signalling events, where nuclear
accumulation was mediated by a bipartite NLS and the nucleolar accumulation was
driven by a further C-terminal region. Furthermore the nucleolar targeting appeared to
be stress responsive, as nucleolar accumulation was increased in response to salt
stress. It appears that formation of the complex may have a role here, as HD2C-HD2
dimers were shown to accumulate in the nucleolus compared to the nuclear and
nucleolar pattern of the HD2C-GFP. The role of 14-3-3 binding to the C-terminal end of
the protein was shown not be a critical determinant of nuclear or nucleolar
localization, as mutation of these sites resulted in the same localization pattern as the
non-modified version of the enzyme. Lastly, the role of HD2C towards SA and JA
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signalling was investigated. Results indicated that HD2C expression was linked to SA
and MeJA sensitivity in the context of root growth and germination with
overexpression causing greater sensitivity to these enzymes during germination.
Binding of TGA6 was initially hypothesised to be a critical determinant of this
phenotype; however semi-quantitative analysis of SA and JA-responsive factors that
were shown to be controlled by clade II TGA factors did not show any significant
repression despite heightened HD2C presence in the cell.
Together these results contribute new knowledge to HD2C regulation. Firstly, this
revealed and characterized differences in HD2C localization. Specifically, the difference
between HD2C:HD2 dimers and stress induced HD2C-GFP nucleolar fluorescent
patterns are distinct from the patterns observed in non-stressed HD2C-GFP, HD2C:14-
3-3 and HD2C:TGA6 interaction studies. Together this suggests that HD2C localization
is dynamic and can be manipulated by external conditions. Secondly, characterization
of three different classes of proteins interacting with HD2C extends our current
knowledge of the diversity of the HD2Cs interactome.
6.3 HD2C for use in genetically modified plants
A significant motivation for plant molecular biology research is the potential for its
insights to be harnessed towards manipulating crop plants to yield more food for less
human input. The potential for histone deacetylases to be modified to alter plant
growth and cropping has been documented. For example it was shown that the
acetylation state of the genome is dynamic, with acetylation of specific areas related
to growth and development shifting from acetylated to deacetylated in response to
various stages of plant growth (Kouzarides 1999; Tian, Fong et al. 2005). In Arabidopsis
non-specific blocking of histone deacetylation activity resulted in pleiotropic effects on
growth and development which could be traced to its inability to operate as an
acetylation antagonist (Tian and Chen 2001). Similarly knocking out various individual
HDAC genes results in a number of growth and developmental defects. From these
studies it is clear that acetylation per se is central to growth and development, which
necessitates understanding of the deacetylation component of this system. Precisely
how the HD2 family of histone deacetylases fits into this and how manipulation of their
pathways may lead to a genetically superior crop has not yet been established.
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Modified expression of HD2s by a constitutive promoter or by gene knockout does not
appear to be the answer. Plants over-expressing HD2C are resistant to salt and drought
stress during germination, survival rate and root growth assays. However the plants
inherent pay-back for this phenotype was evident in a higher proportion of sterility.
Similarly, it was reported that Arabidopsis plants over-expressing HD2A developed
pleiotropic growth defects manifested by altered leaf and flower morphology. Plants
with HD2 knockouts were similarly deficient; HD2C knockouts were correspondingly
sensitive to salt and drought stress and germinated poorly on sucrose media. Together
these results suggest that a simple over- or under-expression of HD2 is not suitable as
a crop modification tool. The results of this thesis provide a starting point for a non-
expression driven alteration in HD2C which has the potential to impact on plant
growth, development and stress response.
This thesis focused on identifying aspects of the enzyme’s regulation that may be
modified to produce a more pathway specific response. For example, this study
showed that it was the nucleolar retention rather than the nuclear translocation which
was the most relevant mechanism in localization control. This was evident both from
the apparent nucleolar targeting which took place following salt stress and the
nucleolar localization of the functional HD2 complex. The region required for nucleolar
retention was narrowed to a C-terminal portion of the protein which did not wholly
overlap with the nuclear targeting sequence. Further analysis of this region yielded the
identification of a 14-3-3 binding site; however mutation of this site did not yield any
evident shift in localization pattern when the mutated HD2C protein was tested as a
GFP-fusion. Together these data suggest that the regulation of nucleolar localization is
a possible site for manipulation. The next step is likely the identification of factors
which mediate this regulation so that the HD2 sequence dependent on these
interactions could be altered to only respond to specific factors. This would achieve an
imposed specificity for nucleolar localization that occurs only in response to
predetermined conditions. This is obviously an ambitious hypothesis given the number
of unknown regulatory factors and the potential diversity of the signal transduction
event. The impact of chemicals on targeting specific interactions in the context of a
whole cell were seen most exquisitely in the context of 14-3-3 binding to H+ATPase in
tomato by fusicoccin. Here fusicoccin locked together the subunits of the H+ATPase.
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This was shown to cause hyperpolarization of the cell membrane and lead to an
induced pathogen response.
An alternative approach is focussing on the interaction with transcription factors which
mediate contact between HD2 and DNA. This thesis determined that there was
interaction between TGA6 and HD2C. Although it appears that this is not significant in
the context of mediating regulation of SA or JA responsive factors, the developmental
implications of this interaction have yet to be determined. Whatever the biological
consequence of this interaction, it is now clear that interaction with transcription
factors is possible, given the interactions of HD2C with ERF1 and TGA6. The next step is
to determine the HD2C binding site that mediates this contact. Given this information,
modification of the HD2 sequence would enable a direct manipulation on the pathway
that involves the transcription factor. Given the inherent ability for HD2s to regulate
large subsets of genes via its chromatin condensation ability, this would enable a large
scale manipulation on genetic interactions with external stimuli which could be
harnessed to modify plant growth and response to the environment.
6.4 C-terminal domain appears to be necessary for protein binding
Throughout this thesis there were several lines of evidence to suggest that the C-
terminal domain of HD2C was significant in the context of protein-protein interaction.
Most overt was the direct mapping of a 14-3-3 epsilon binding sites, where two
different sites were mapped to the C-terminal end of HD2C. Similarly, the N-terminal
1-60AA fragment of HD2C was used both in dimerization experiments of HD2s and in
the interaction study with TGA6 and failed to interact with either of the two. This
suggests that interaction with other proteins does not reuiqre the N-termianl part of
HD2C. A spin-off of this discovery is the application of the N-terminal fragment as a
negative control for protein interaction experiments. . Furthermore, the tiling deletion
analysis that was performed on HD2C to map 14-3-3 binding sites was performed for
TGA6, mapping a region that it required for binding to a C-terminal site overlapping
the NLS (study not completed, data not shown). The significance of this is two-fold:
Firstly it hints at a fundamental domain structure within the protein, where the N-
terminus contains a conserved enzymatic domain and functional diversity is
135
manifested by the C-terminal end where various subsets of interactors bind. In The
RPD3-like superfamily in Arabidopsis contains a similar domain structure. There, the N-
terminal HDAC site is highly conserved and diversity is most evident in the C-terminal
end.
Secondly, it suggests that there is a co-operation between the two domains. It suggests
that the C-terminal domain is necessary for protein interaction which guides the HDAC
initially into the nucleus and nucleolus, and subsequently to its chromatin targets. Next
the N-terminal end performs its HDAC function. The fact that this relationship is both
synergistic and essential is clear from each study in this thesis. Firstly, the NLS and
nucleolar retention signal are located in the C-terminus which is essential for
colocalization of HD2 with its substrate. Secondly, specific chromatin regions are the
focus of the HDAC activity, which is probably provided via interaction with TGA and
ERF transcription factors and possibly others. Lastly, the enzyme requires
phosphorylation to form an active complex. Initially it was hypothesised that this
suggested N-terminal phosphorylation sites which would stabilize the active domain
and control enzyme activity. However a phosphorylation site was found in the N-
terminus by phosphoproteomics and two 14-3-3 sites which bind phosphorylation sites
were similarly found in this region (Durek, Schmidt et al. 2010). Therefore it is possible
that enzyme regulation is dependent upon secondary mechanisms. Although the
results do not provide clear insight, it may be speculated that phosphorylation
regulates formation of the active HD2 complex or mediates contact with substrate.
Moreover, the presence of the zinc-finger domain in HD2A and HD2C isoforms is a
feature of the C-terminus which remains uncharacterized. It was suggested that the
single zinc finger of HD2C cannot interact with DNA, instead it would provide a domain
for protein or RNA interactions. However, hetero- and Homo- dimers of HD2s raise the
possibility that multiple Zinc-fingers from HD2A and HD2C isoforms make direct DNA
contact possible in such complexes. . Its overlap with a 14-3-3 binding site similarly
suggests that its role may be dynamically regulated by phosphorylation. Whatever its
role, its evolutionary conservation suggest that this domain is a critical feature of this
C-terminus whose function is yet to be determined.
136
6.5 Future
HD2s principle role is to modify gene expression. So far this has been traced by testing
the expression of genes of interest in plants with over- or under-expressing HD2C
genes. For example this approach was used to confirm the impact of HD2C expression
on various ABA responsive genes to rationalize the observation that plants with
modified HD2C expression had different responses to salt, drought and ABA. These
small scope studies shed light on specific pathways that relate to HD2C expression and
function. The risk of this is that the holistic role of HD2C function is diluted by specific
research areas where time is more heavily invested. An alternative approach is the so-
called ‘-omic’ analyses which allow a large scale interpretation of protein activity by
offering a holistic monitoring of responses.
HD2C is an enzyme that affects transcription of genes, and does this by modifying the
chromatin structure of the genome. Two ‘-omic’ approaches are immediately relevent
here.
Micro-array measures mRNA amounts for large subsections of the coded genome (Ball,
Sherlock et al. 2002). In a case relevant to this thesis, it was used to identify novel
functions of a clade II TGA isoform in response to salicylic acid (Thibaud‐Nissen, Wu et
al. 2006). It is anticipated that a similar approach could be used in plants with modified
HD2C expression to identify novel pathways related to HD2C function.
Secondly, various chromatin immune-precipitation (ChIP) techniques such as chip
hybridization (ChIP-chip) (Buck and Lieb 2004; Park 2009) or next generation
sequencing (ChIP-seq) have been developed to screen for modified acetyl-chromatin
states in response to development, stress or mutation. The advantage of this over
micro-array analysis is that it allows the direct visualization of HD2C activity. Thus it
would provide direct evidence relating to the binding targets of HD2C enzyme and
enable an insight to be made into the core function of HD2C.
137
Chapter 7 References
138
Abràmoff, M. D., P. J. Magalhães, et al. (2004). "Image processing with ImageJ." Biophotonics international 11(7): 36-42.
Aitken, A. (2006). 14-3-3 proteins: a historic overview. Seminars in cancer biology, Elsevier. Aravind, L. and E. V. Koonin (1998). "Second Family of Histone Deacetylases." Science
280(5367): 1167. Atchley, W. R. and B. K. Hall (1991). "A model for development and evolution of complex
morphological structures." Biological Reviews 66(2): 101-157. Aufsatz, W., M. F. Mette, et al. (2002). "HDA6, a putative histone deacetylase needed to
enhance DNA methylation induced by double-stranded RNA." The EMBO journal 21(24): 6832.
Bader, J. S., A. Chaudhuri, et al. (2003). "Gaining confidence in high-throughput protein interaction networks." Nature biotechnology 22(1): 78-85.
Bae, M. S., E. J. Cho, et al. (2003). "Analysis of the Arabidopsis nuclear proteome and its response to cold stress." The Plant Journal 36(5): 652-663.
Ball, C. A., G. Sherlock, et al. (2002). "Standards for microarray data." Science (New York, NY) 298(5593): 539.
Berger, S. L. (2007). "The complex language of chromatin regulation during transcription." Nature 447(7143): 407-412.
Berger, S. L., T. Kouzarides, et al. (2009). "An operational definition of epigenetics." Genes & Development 23(7): 781-783.
Berman, M. and T. DeJong (1996). "Water stress and crop load effects on fruit fresh and dry weights in peach (Prunus persica)." Tree Physiology 16(10): 859-864.
Bogdanović, O. and G. J. C. Veenstra (2009). "DNA methylation and methyl-CpG binding proteins: developmental requirements and function." Chromosoma 118(5): 549-565.
Borsani, O., V. Valpuesta, et al. (2001). "Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings." Plant Physiology 126(3): 1024-1030.
Boulon, S., B. J. Westman, et al. (2010). "The nucleolus under stress." Molecular Cell 40(2): 216-227.
Bradford, K. J. and H. Nonogaki (2007). Seed development, dormancy and germination, Blackwell publishing.
Brooks, A. R., R. N. Harkins, et al. (2004). "Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle." The journal of gene medicine 6(4): 395-404.
Brosch, G., A. Lusser, et al. (1996). "Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos." Biochemistry 35(49): 15907-15914.
Buck, M. J. and J. D. Lieb (2004). "ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments." Genomics 83(3): 349-360.
Cameron, R. K., N. L. Paiva, et al. (1999). "Accumulation of salicylic acid and PR-1 gene transcripts in relation to the systemic acquired resistance (SAR) response induced by< i> Pseudomonas syringae</i> pv.< i> tomato</i> in< i> Arabidopsis</i>." Physiological and molecular plant pathology 55(2): 121-130.
Camoni, L., J. F. Harper, et al. (1998). "14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)." FEBS letters 430(3): 381.
Carmo-Fonseca, M. (2002). "The contribution of nuclear compartmentalization to gene regulation." Cell 108(4): 513-522.
Chen, L.-T., M. Luo, et al. (2010). "Involvement of Arabidopsis histone deacetylase HDA6 in ABA and salt stress response." Journal of experimental botany 61(12): 3345-3353.
Chen, W. and K. B. Singh (1999). "The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element." The Plant Journal 19(6): 667-677.
139
Chini, A., S. Fonseca, et al. (2007). "The JAZ family of repressors is the missing link in jasmonate signalling." Nature 448(7154): 666-671.
Chinnusamy, V., Z. Gong, et al. (2008). "Abscisic Acid‐mediated Epigenetic Processes in Plant Development and Stress Responses." Journal of Integrative Plant Biology 50(10): 1187-1195.
Choi, S. M., H. R. Song, et al. (2012). "HDA19 is required for the repression of salicylic acid biosynthesis and salicylic acid‐mediated defense responses in Arabidopsis." The Plant Journal 71(1): 135-146.
Cokol, M., R. Nair, et al. (2000). "Finding nuclear localization signals." EMBO reports 1(5): 411-415.
Collura, V. and G. Boissy (2007). From Protein—Protein Complexes to Interactomics. Subcellular Proteomics, Springer: 135-183.
Colville, A., R. Alhattab, et al. (2011). "Role of HD2 genes in seed germination and early seedling growth in <i>Arabidopsis</i>." Plant Cell Reports 30(10): 1969-1979.
Costa, F. F. (2008). "Non-coding RNAs, epigenetics and complexity." Gene 410(1): 9-17. Covello, P. S. and M. W. Gray (1989). "RNA editing in plant mitochondria." Creelman, R. A. and J. E. Mullet (1995). "Jasmonic acid distribution and action in plants:
regulation during development and response to biotic and abiotic stress." Proceedings of the National Academy of Sciences 92(10): 4114-4119.
Dangl, M., G. Brosch, et al. (2001). "Comparative analysis of HD2 type histone deacetylases in higher plants." Planta 213(2): 280-285.
Dathan, N., L. Zaccaro, et al. (2002). "The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2–His2 zinc finger motif." Nucleic Acids Research 30(22): 4945-4951.
Davies, P. J. (2010). Plant hormones: biosynthesis, signal transduction, action!, Springer. Davies, P. J. (2010). The plant hormones: their nature, occurrence, and functions. Plant
hormones, Springer: 1-15. de Ruijter, A. J., A. H. Van Gennip, et al. (2003). "Histone deacetylases (HDACs):
characterization of the classical HDAC family." Biochemical Journal 370(Pt 3): 737. de Torres, Z. M., M. Bennett, et al. (2009). "Antagonism between salicylic and abscisic acid
reflects early host-pathogen conflict and moulds plant defence responses." The Plant journal: for cell and molecular biology 59(3): 375.
DeLille, J. M., P. C. Sehnke, et al. (2001). "The Arabidopsis 14-3-3 family of signaling regulators." Plant Physiology 126(1): 35-38.
Demetriou, K., A. Kapazoglou, et al. (2009). "Epigenetic chromatin modifiers in barley: I. Cloning, mapping and expression analysis of the plant specific HD2 family of histone deacetylases from barley, during seed development and after hormonal treatment." Physiologia Plantarum 136(3): 358-368.
DeRisi, J. L., V. R. Iyer, et al. (1997). "Exploring the metabolic and genetic control of gene expression on a genomic scale." Science 278(5338): 680-686.
Després, C., C. DeLong, et al. (2000). "The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors." The Plant Cell Online 12(2): 279-290.
Dez, C., M. Dlakić, et al. (2007). "Roles of the HEAT repeat proteins Utp10 and Utp20 in 40S ribosome maturation." Rna 13(9): 1516-1527.
Díaz-Moreno, I., D. Hollingworth, et al. (2009). "Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14-3-3 binding." Nature Structural & Molecular Biology 16(3): 238-246.
Doetzlhofer, A., H. Rotheneder, et al. (1999). "Histone deacetylase 1 can repress transcription by binding to Sp1." Molecular and cellular biology 19(8): 5504-5511.
Dong, X. (2004). "NPR1, all things considered." Current opinion in plant biology 7(5): 547-552.
140
Durek, P., R. Schmidt, et al. (2010). "PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update." Nucleic Acids Research 38(suppl 1): D828-D834.
Eberharter, A. and P. B. Becker (2002). "Histone acetylation: a switch between repressive and permissive chromatin." EMBO reports 3(3): 224-229.
Eden, A., F. Gaudet, et al. (2003). "Chromosomal instability and tumors promoted by DNA hypomethylation." Science 300(5618): 455-455.
ERARD, M. S., P. Belenguer, et al. (1988). "A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone Hl." European Journal of Biochemistry 175(3): 525-530.
Eulgem, T. (2005). "Regulation of the< i> Arabidopsis</i> defense transcriptome." Trends in Plant Science 10(2): 71-78.
Eulgem, T. and I. E. Somssich (2007). "Networks of WRKY transcription factors in defense signaling." Current opinion in plant biology 10(4): 366-371.
Fahrenkrog, B. and U. Aebi (2003). "The nuclear pore complex: nucleocytoplasmic transport and beyond." Nature Reviews Molecular Cell Biology 4(10): 757-766.
Fan, W. and X. Dong (2002). "In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid–mediated gene activation in Arabidopsis." The Plant Cell Online 14(6): 1377-1389.
Fernández-Calvo, P., A. Chini, et al. (2011). "The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses." The Plant Cell Online 23(2): 701-715.
Fischle, W., F. Dequiedt, et al. (2002). "Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR." Molecular Cell 9(1): 45-57.
Fu, H., R. R. Subramanian, et al. (2000). "14-3-3 proteins: structure, function, and regulation." Science Signaling 40(1): 617.
Fuglsang, A. T., S. Visconti, et al. (1999). "Binding of 14-3-3 protein to the plasma membrane H+-ATPase AHA2 involves the three C-terminal residues Tyr946-Thr-Val and requires phosphorylation of Thr947." Journal of Biological Chemistry 274(51): 36774-36780.
Galis, I. and K. Matsuoka (2007). Transcriptomic analysis of salicylic acid-responsive genes in tobacco BY-2 cells. Salicylic Acid: A Plant Hormone, Springer: 371-396.
Gardino, A. K., S. J. Smerdon, et al. (2006). Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms. Seminars in cancer biology, Elsevier.
Gatz, C. (2013). "From Pioneers to Team Players: TGA Transcription Factors Provide a Molecular Link Between Different Stress Pathways." Molecular plant-microbe interactions 26(2): 151-159.
Gill, S. S. and N. Tuteja (2010). "Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants." Plant Physiology and Biochemistry 48(12): 909-930.
Ginisty, H., H. Sicard, et al. (1999). "Structure and functions of nucleolin." Journal of Cell Science 112(6): 761-772.
Glazebrook, J. (2005). "Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens." Annu. Rev. Phytopathol. 43: 205-227.
Goldberg, A. D., C. D. Allis, et al. (2007). "Epigenetics: a landscape takes shape." Cell 128(4): 635.
Görlich, D., S. Kostka, et al. (1995). "Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope." Current Biology 5(4): 383-392.
Graves, P. R., C. M. Lovly, et al. (2001). "Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding." Oncogene 20(15): 1839.
Grebenok, R. J., E. Pierson, et al. (1997). "Green‐fluorescent protein fusions for efficient characterization of nuclear targeting." The Plant Journal 11(3): 573-586.
141
Grozinger, C. M., C. A. Hassig, et al. (1999). "Three proteins define a class of human histone deacetylases related to yeast Hda1p." Proceedings of the National Academy of Sciences 96(9): 4868-4873.
Grozinger, C. M. and S. L. Schreiber (2000). "Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization." Science Signaling 97(14): 7835.
Grunstein, M. (1997). "Histone acetylation in chromatin structure and transcription." Nature 389(6649): 349-352.
Guo, M., A. Yang, et al. (2012). "The new understanding of Arabidopsis thaliana proteins associated with salinity." Journal of Plant Interactions 7(4): 348-355.
Hannon, G. J. (2002). "RNA interference." Nature 418(6894): 244-251. Harootunian, A., S. Adams, et al. (1993). "Movement of the free catalytic subunit of cAMP-
dependent protein kinase into and out of the nucleus can be explained by diffusion." Molecular biology of the cell 4(10): 993.
Heil, M., A. Hilpert, et al. (2000). "Reduced growth and seed set following chemical induction of pathogen defence: does systemic acquired resistance (SAR) incur allocation costs?" Journal of Ecology 88(4): 645-654.
Hinshaw, J. E., B. O. Carragher, et al. (1992). "Architecture and design of the nuclear pore complex." Cell 69(7): 1133-1141.
Hirayama, T. and K. Shinozaki (2007). "Perception and transduction of abscisic acid signals: keys to the function of the versatile plant hormone ABA." Trends in Plant Science 12(8): 343-351.
Hirota, K., T. Miyoshi, et al. (2008). "Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs." Nature 456(7218): 130-134.
Hollender, C. and Z. Liu (2008). "Histone deacetylase genes in Arabidopsis development." Journal of Integrative Plant Biology 50(7): 875-885.
Holliday, R. (1987). "The inheritance of epigenetic defects." Science 238(4824): 163-170. Holliday, R. (1990). "Mechanisms for the control of gene activity during development."
Biological Reviews 65(4): 431-471. Holliday, R. (2006). "Epigenetics: a historical overview." Epigenetics 1(2): 76-80. Idrovo Espín, F. M., S. Peraza-Echeverria, et al. (2012). "< i> In silico</i> cloning and
characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in< i> Carica papaya</i>." Plant Physiology and Biochemistry 54: 113-122.
Imamura, T., S. Yamamoto, et al. (2004). "Non-coding RNA directed DNA demethylation of< i> Sphk1</i> CpG island." Biochemical and Biophysical Research Communications 322(2): 593-600.
Ito, M., A. Koike, et al. (2003). "Methylated DNA-binding proteins from Arabidopsis." Plant Physiology 133(4): 1747-1754.
Iuchi, S. (2001). "Three classes of C2H2 zinc finger proteins." Cellular and Molecular Life Sciences CMLS 58(4): 625-635.
Jaenisch, R. and A. Bird (2003). "Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals." Nature genetics 33: 245-254.
Jans, D. A., C. Y. Xiao, et al. (2000). "Nuclear targeting signal recognition: a key control point in nuclear transport?" Bioessays 22(6): 532-544.
Johnson, C., S. Crowther, et al. (2010). "Bioinformatic and experimental survey of 14-3-3-binding sites." Biochemical Journal 427(Pt 1): 69.
Jönsson, H., M. Heisler, et al. (2005). "Modeling the organization of the WUSCHEL expression domain in the shoot apical meristem." Bioinformatics 21(suppl 1): i232-i240.
Kadonaga, J. T. (1998). "Eukaryotic transcription: an interlaced review network of transcription factors and chromatin-modifying machines." Cell 92: 307-313.
Kende, H. and J. Zeevaart (1997). "The Five" Classical" Plant Hormones." The Plant Cell 9(7): 1197.
142
Kim, C. A. and J. M. Berg (1996). "A 2.2 Å resolution crystal structure of a designed zinc finger protein bound to DNA." Nature Structural & Molecular Biology 3(11): 940-945.
Kim, J.-M., T. K. To, et al. (2008). "Alterations of lysine modifications on the histone H3 N-tail under drought stress conditions in Arabidopsis thaliana." Plant and cell physiology 49(10): 1580-1588.
Klug, A. and D. Rhodes (1987). "‘Zinc fingers’: a novel protein motif for nucleic acid recognition." Trends in Biochemical Sciences 12: 464-469.
Koltunow, A. M., J. Truettner, et al. (1990). "Different temporal and spatial gene expression patterns occur during anther development." The Plant Cell 2(12): 1201.
Koornneef, A., K. Rindermann, et al. (2008). "Histone modifications do not play a major role in salicylate-mediated suppression of jasmonate-induced PDF1. 2 gene expression." Communicative & integrative biology 1(2): 143-145.
Koroleva, O., G. Calder, et al. (2009). "Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia." The Plant Cell Online 21(5): 1592-1606.
Kouzarides, T. (1999). "Histone acetylases and deacetylases in cell proliferation." Current opinion in genetics & development 9(1): 40-48.
Kouzarides, T. (2007). "Chromatin modifications and their function." Cell 128(4): 693-705. Kuang, J.-f., J.-y. Chen, et al. (2012). "Histone deacetylase HD2 interacts with ERF1 and is
involved in longan fruit senescence." Journal of experimental botany 63(1): 441-454. Lagace, M., S. C. Chantha, et al. (2003). "Fertilization induces strong accumulation of a histone
deacetylase (HD2) and of other chromatin-remodeling proteins in restricted areas of the ovules." Plant Molecular Biology 53(6): 759-769.
Laity, J. H., B. M. Lee, et al. (2001). "Zinc finger proteins: new insights into structural and functional diversity." Current opinion in structural biology 11(1): 39-46.
Lange, A., R. E. Mills, et al. (2007). "Classical nuclear localization signals: definition, function, and interaction with importin α." Journal of Biological Chemistry 282(8): 5101-5105.
Larsen, F., G. Gundersen, et al. (1992). "CpG islands as gene markers in the human genome." Genomics 13(4): 1095-1107.
Lawrence, R. J., K. Earley, et al. (2004). "A Concerted DNA Methylation/Histone Methylation Switch Regulates rRNA Gene Dosage Control and Nucleolar Dominance." Molecular Cell 13(4): 599-609.
Lawton, K., K. Weymann, et al. (1995). "Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene." MPMI-Molecular Plant Microbe Interactions 8(6): 863-870.
Lee, B. J., A. E. Cansizoglu, et al. (2006). "Rules for nuclear localization sequence recognition by karyopherinβ2." Cell 126(3): 543-558.
Lee, M. S., G. P. Gippert, et al. (1989). "Three-dimensional solution structure of a single zinc finger DNA-binding domain." Science 245(4918): 635-637.
Li, J., G. Brader, et al. (2004). "The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense." Science Signaling 16(2): 319.
Lin, M., X. Shen, et al. (2011). "PAIR: the predicted Arabidopsis interactome resource." Nucleic Acids Research 39(suppl 1): D1134-D1140.
Lin, M., X. Zhou, et al. (2011). "The predicted Arabidopsis interactome resource and network topology-based systems biology analyses." The Plant Cell Online 23(3): 911-922.
Liu, D., J. Bienkowska, et al. (1995). "Crystal structure of the zeta isoform of the 14-3-3 protein."
Loidl, P. (2004). "A plant dialect of the histone language." Trends in Plant Science 9(2): 84-90. Luger, K. and T. J. Richmond (1998). "The histone tails of the nucleosome." Current opinion in
genetics & development 8(2): 140-146. Luo, M., Y.-Y. Wang, et al. (2012). "HD2C interacts with HDA6 and is involved in ABA and salt
stress response in Arabidopsis." Journal of experimental botany 63(8): 3297-3306.
143
Luo, M., Y.-Y. Wang, et al. (2012). "HD2 proteins interact with RPD3-type histone deacetylases." Plant Signaling & Behavior 7(6): 608-610.
Luo, R. X., A. A. Postigo, et al. (1998). "Rb interacts with histone deacetylase to repress transcription." Cell 92(4): 463-473.
Lusser, A., G. Brosch, et al. (1997). "Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein." Science 277(5322): 88-91.
Malamy, J. and D. F. Klessig (1992). "Salicylic acid and plant disease resistance." The Plant Journal 2(5): 643-654.
Mancini, D. N., S. M. Singh, et al. (1999). "Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors." Oncogene 18(28): 4108.
Manners, J. M., I. A. Penninckx, et al. (1998). "The promoter of the plant defensin gene PDF1. 2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate but not to salicylic acid." Plant Molecular Biology 38(6): 1071-1080.
Margueron, R. and D. Reinberg (2010). "Chromatin structure and the inheritance of epigenetic information." Nature Reviews Genetics 11(4): 285-296.
Margueron, R., P. Trojer, et al. (2005). "The key to development: interpreting the histone code?" Current opinion in genetics & development 15(2): 163-176.
Marras, E. and E. Capobianco (2008). "A Multiscale Tour in Protein Interactomics." Martin, W. (2010). "Evolutionary origins of metabolic compartmentalization in eukaryotes."
Philosophical Transactions of the Royal Society B: Biological Sciences 365(1541): 847-855.
Mattick, J. S. (2001). "Non-coding RNAs: the architects of eukaryotic complexity." EMBO reports 2(11): 986-991.
McGonigle, B., K. Bouhidel, et al. (1996). "Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression." Genes & Development 10(14): 1812-1821.
Métraux, J., H. Signer, et al. (1990). "Increase in salicylic acid at the onset of systemic acquired resistance in cucumber." Science 250(4983): 1004-1006.
Mitsuda, N. and M. Ohme-Takagi (2009). "Functional analysis of transcription factors in Arabidopsis." Plant and cell physiology 50(7): 1232-1248.
Moore, M. S. (1998). "Ran and nuclear transport." Journal of Biological Chemistry 273(36): 22857-22860.
Moore, T. C. (1979). Biochemistry and physiology of plant hormones, Springer-Verlag. Moorhead, G., P. Douglas, et al. (1999). "Plant proteins containing phosphopeptide motifs that
bind to 14-3-3 proteins." Plant J 18: 1-12. Morris, J. (2001). "Genes, genetics, and epigenetics: a correspondence." Science 293(5532):
1103-1105. Mou, Z., W. Fan, et al. (2003). "Inducers of plant systemic acquired resistance regulate NPR1
function through redox changes." Cell 113(7): 935-944. Muslin, A. J., J. W. Tanner, et al. (1996). "Interaction of 14-3-3 with signaling proteins is
mediated by the recognition of phosphoserine." Cell 84(6): 889-897. Muslin, A. J. and H. Xing (2000). "14-3-3 proteins: regulation of subcellular localization by
molecular interference." Cellular signalling 12(11): 703-709. Ndamukong, I., A. A. Abdallat, et al. (2007). "SA‐inducible Arabidopsis glutaredoxin interacts
with TGA factors and suppresses JA‐responsive PDF1. 2 transcription." The Plant Journal 50(1): 128-139.
Niggeweg, R., C. Thurow, et al. (2000). "Tobacco transcription factor TGA2. 2 is the main component of as-1-binding factor ASF-1 and is involved in salicylic acid-and auxin-inducible expression of as-1-containing target promoters." Journal of Biological Chemistry 275(26): 19897-19905.
Noll, M. (1974). "Subunit structure of chromatin." Nature 251(5472): 249-251.
144
Obsilova, V., J. Vecer, et al. (2005). "14-3-3 Protein interacts with nuclear localization sequence of forkhead transcription factor FoxO4." Biochemistry 44(34): 11608-11617.
Pabo, C. O. and R. T. Sauer (1992). "Transcription factors: structural families and principles of DNA recognition." Annual review of biochemistry 61(1): 1053-1095.
Panni, S., L. Montecchi‐Palazzi, et al. (2011). "Combining peptide recognition specificity and context information for the prediction of the 14‐3‐3‐mediated interactome in S. cerevisiae and H. sapiens." Proteomics 11(1): 128-143.
Park, P. J. (2009). "ChIP–seq: advantages and challenges of a maturing technology." Nature Reviews Genetics 10(10): 669-680.
Paul, A.-L., L. Liu, et al. (2009). "Comparative interactomics: analysis of Arabidopsis 14-3-3 complexes reveals highly conserved 14-3-3 interactions between humans and plants." Journal of proteome research 8(4): 1913-1924.
Pedone, P. V., R. Ghirlando, et al. (1996). "The single Cys2-His2 zinc finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding." Proceedings of the National Academy of Sciences 93(7): 2822-2826.
Penfield, S., Y. Li, et al. (2006). "Arabidopsis ABA INSENSITIVE4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm." The Plant Cell Online 18(8): 1887-1899.
Peng, S., J. Huang, et al. (2004). "Rice yields decline with higher night temperature from global warming." Proceedings of the National Academy of Sciences of the United States of America 101(27): 9971-9975.
Pérez-Núñez, M., R. Souza, et al. (2009). "Detection of a SERK-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos." Plant Cell Reports 28(1): 11-19.
Perez‐Terzic, C., M. Jaconi, et al. (1997). "Nuclear calcium and the regulation of the nuclear pore complex." Bioessays 19(9): 787-792.
Pieterse, C. M. and L. C. van Loon (1999). "Salicylic acid-independent plant defence pathways." Trends in Plant Science 4(2): 52-58.
Pollard, V. W., W. M. Michael, et al. (1996). "A novel receptor-mediated nuclear protein import pathway." Cell 86(6): 985.
Purwestri, Y. A., Y. Ogaki, et al. (2009). "The 14-3-3 protein GF14c acts as a negative regulator of flowering in rice by interacting with the florigen Hd3a." Plant and cell physiology 50(3): 429-438.
Razin, A. and A. D. Riggs (1980). "DNA methylation and gene function." Science 210(4470): 604-610.
Robert-Seilaniantz, A., M. Grant, et al. (2011). "Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism." Annual review of phytopathology 49: 317-343.
Sasai, N., M. Nakao, et al. (2010). "Sequence-specific recognition of methylated DNA by human zinc-finger proteins." Nucleic Acids Research 38(15): 5015-5022.
Saslowsky, D. E., U. Warek, et al. (2005). "Nuclear localization of flavonoid enzymes in Arabidopsis." Journal of Biological Chemistry 280(25): 23735-23740.
Saxonov, S., P. Berg, et al. (2006). "A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters." Proceedings of the National Academy of Sciences of the United States of America 103(5): 1412-1417.
Schenk, P. M., K. Kazan, et al. (2000). "Coordinated plant defense responses in Arabidopsis revealed by microarray analysis." Proceedings of the National Academy of Sciences 97(21): 11655-11660.
Schijlen, E. G., C. Ric de Vos, et al. (2004). "Modification of flavonoid biosynthesis in crop plants." Phytochemistry 65(19): 2631-2648.
Shaffer, K. L., A. Sharma, et al. (2005). "Regulation of protein compartmentalization expands the diversity of protein function." Developmental cell 9(4): 545-554.
145
Shah, J. (2003). "The salicylic acid loop in plant defense." Current opinion in plant biology 6(4): 365-371.
Shah, J., P. Kachroo, et al. (1999). "The Arabidopsis ssi1 mutation restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependent." The Plant Cell Online 11(2): 191-206.
Shah, J. and D. Klessig (1999). "Salicylic acid: signal perception and transduction." New Comprehensive Biochemistry 33: 513-541.
Shaw, P. (2013). The Plant Nucleolus. Plant Genome Diversity Volume 2, Springer: 65-76. Sittka, A., S. Lucchini, et al. (2008). "Deep sequencing analysis of small noncoding RNA and
mRNA targets of the global post-transcriptional regulator, Hfq." PLoS genetics 4(8): e1000163.
Song, Y., K. Wu, et al. (2010). "< i> Arabidopsis</i> DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity." Biochemical and Biophysical Research Communications 396(2): 187-192.
Song, Y. A., K. Q. Wu, et al. (2010). "Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity." Biochemical and Biophysical Research Communications 396(2): 187-192.
Spoel, S. H., A. Koornneef, et al. (2003). "NPR1 modulates cross-talk between salicylate-and jasmonate-dependent defense pathways through a novel function in the cytosol." The Plant Cell Online 15(3): 760-770.
Sridha, S. and K. Q. Wu (2006). "Identification of AtHD2C as a novel regulator of abscisic acid responses in Arabidopsis." Plant Journal 46(1): 124-133.
Staswick, P. E., I. Tiryaki, et al. (2002). "Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation." The Plant Cell Online 14(6): 1405-1415.
Swatek, K. N., K. Graham, et al. (2011). "The 14-3-3 isoforms chi and epsilon differentially bind client proteins from developing Arabidopsis seed." Journal of proteome research 10(9): 4076-4087.
Tada, Y., S. H. Spoel, et al. (2008). "Plant immunity requires conformational charges of NPR1 via S-nitrosylation and thioredoxins." Science 321(5891): 952-956.
Tajrishi, M. M., R. Tuteja, et al. (2011). "Nucleolin: The most abundant multifunctional phosphoprotein of nucleolus." Communicative & integrative biology 4(3): 267-275.
Thaler, J. S. (1999). "Jasmonate-inducible plant defences cause increased parasitism of herbivores." Nature 399(6737): 686-688.
Theunissen, O., F. Rudt, et al. (1992). "RNA and DNA binding zinc fingers in Xenopus TFIIIA." Cell 71(4): 679-690.
Thibaud‐Nissen, F., H. Wu, et al. (2006). "Development of Arabidopsis whole‐genome microarrays and their application to the discovery of binding sites for the TGA2 transcription factor in salicylic acid‐treated plants." The Plant Journal 47(1): 152-162.
Thines, B., L. Katsir, et al. (2007). "JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling." Nature 448(7154): 661-665.
Thomma, B. P., K. Eggermont, et al. (1998). "Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens." Proceedings of the National Academy of Sciences 95(25): 15107-15111.
Thoms, H. C., C. J. Loveridge, et al. (2010). "Nucleolar targeting of RelA (p65) is regulated by COMMD1-dependent ubiquitination." Cancer research 70(1): 139-149.
Tian, L. and Z. J. Chen (2001). "Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development." Proceedings of the National Academy of Sciences 98(1): 200-205.
146
Tian, L., M. P. Fong, et al. (2005). "Reversible histone acetylation and deacetylation mediate genome-wide, promoter-dependent and locus-specific changes in gene expression during plant development." Genetics 169(1): 337-345.
Toroser, D., G. S. Athwal, et al. (1998). "Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins." FEBS letters 435(1): 110-114.
Traw, M. B. and J. Bergelson (2003). "Interactive effects of jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis." Plant Physiology 133(3): 1367-1375.
Tseng, T.-S., C. Whippo, et al. (2012). "The role of a 14-3-3 protein in stomatal opening mediated by PHOT2 in Arabidopsis." The Plant Cell Online 24(3): 1114-1126.
Uquillas, C., I. Letelier, et al. (2004). "NPR1-independent activation of immediate early salicylic acid-responsive genes in Arabidopsis." Molecular plant-microbe interactions 17(1): 34-42.
Verdel, A., S. Curtet, et al. (2000). "Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm." Current Biology 10(12): 747-749.
Wang, A. H., M. J. Kruhlak, et al. (2000). "Regulation of histone deacetylase 4 by binding of 14-3-3 proteins." Molecular and cellular biology 20(18): 6904-6912.
Wang, R. and M. G. Brattain (2007). "The maximal size of protein to diffuse through the nuclear pore is larger than 60kDa." FEBS letters 581(17): 3164-3170.
Watt, F. and P. L. Molloy (1988). "Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter." Genes & Development 2(9): 1136-1143.
Weiner, H. and W. M. Kaiser (1999). "14-3-3 proteins control proteolysis of nitrate reductase in spinach leaves." FEBS letters 455(1): 75-78.
Wu, K. Q., L. N. Tian, et al. (2000). "Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana." Plant Journal 22(1): 19-27.
Wu, K. Q., L. N. Tian, et al. (2003). "Repression of gene expression by Arabidopsis HD2 histone deacetylases." Plant Journal 34(2): 241-247.
Xie, Z., Z.-L. Zhang, et al. (2007). "Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene." Plant Molecular Biology 64(3): 293-303.
Xiong, L., K. S. Schumaker, et al. (2002). "Cell signaling during cold, drought, and salt stress." The Plant Cell Online 14(suppl 1): S165-S183.
Xu, Y., P.-F. L. Chang, et al. (1994). "Plant defense genes are synergistically induced by ethylene and methyl jasmonate." The Plant Cell Online 6(8): 1077-1085.
Yaffe, M. B. (2002). "How do 14-3-3 proteins work?–Gatekeeper phosphorylation and the molecular anvil hypothesis." FEBS letters 513(1): 53-57.
Zander, M., S. Chen, et al. (2012). "Repression of the Arabidopsis thaliana jasmonic acid/ethylene-induced defense pathway by TGA-interacting glutaredoxins depends on their C-terminal ALWL motif." Molecular plant 5(4): 831-840.
Zander, M., S. La Camera, et al. (2010). "Arabidopsis thaliana class‐II TGA transcription factors are essential activators of jasmonic acid/ethylene‐induced defense responses." The Plant Journal 61(2): 200-210.
Zeng, L. and M.-M. Zhou (2002). "Bromodomain: an acetyl-lysine binding domain." FEBS letters 513(1): 124.
Zhang, D. (2011). "The structure of arabidopsis NPR1: its function as a salicylic acid receptor and a metal-binding protein."
Zhang, X. (2008). "The epigenetic landscape of plants." Science 320(5875): 489-492. Zhang, X., M. I. Francis, et al. (2010). "Over-expression of the Arabidopsis NPR1 gene in citrus
increases resistance to citrus canker." European journal of plant pathology 128(1): 91-100.
147
Zhang, Y., W. Fan, et al. (1999). "Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene." Proceedings of the National Academy of Sciences 96(11): 6523-6528.
Zhang, Y., M. J. Tessaro, et al. (2003). "Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance." The Plant Cell Online 15(11): 2647-2653.
Zhou, C., H. Labbe, et al. (2004). "Expression and function of HD2‐type histone deacetylases in Arabidopsis development." The Plant Journal 38(5): 715-724.
Zhou, C., L. Zhang, et al. (2005). "HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis." The Plant Cell Online 17(4): 1196-1204.
Zhou, C. H., H. Labbe, et al. (2004). "Expression and function of HD2-type histone deacetylases in Arabidopsis development." Plant Journal 38(5): 715-724.
Zhou, J.-M., Y. Trifa, et al. (2000). "NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid." Molecular plant-microbe interactions 13(2): 191-202.