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The Mediator Complex in Plants: Structure, Phylogeny, and Expression Profiling of Representative Genes in a Dicot (Arabidopsis) and a Monocot (Rice) during Reproduction and Abiotic Stress

Department of Plant Molecular Biology, University of Delhi, Old Delhi, NCT, India
Plant physiology (Impact Factor: 6.84). 12/2011; 157(4):1609-27. DOI: 10.1104/pp.111.188300
Source: PubMed
ABSTRACT
The Mediator (Med) complex relays regulatory information from DNA-bound transcription factors to the RNA polymerase II in eukaryotes. This macromolecular unit is composed of three core subcomplexes in addition to a separable kinase module. In this study, conservation of Meds has been investigated in 16 plant species representing seven diverse groups across the plant kingdom. Using Hidden Markov Model-based conserved motif searches, we have identified all the known yeast/metazoan Med components in one or more plant groups, including the Med26 subunits, which have not been reported so far for any plant species. We also detected orthologs for the Arabidopsis (Arabidopsis thaliana) Med32, -33, -34, -35, -36, and -37 in all the plant groups, and in silico analysis identified the Med32 and Med33 subunits as apparent orthologs of yeast/metazoan Med2/29 and Med5/24, respectively. Consequently, the plant Med complex appears to be composed of one or more members of 34 subunits, as opposed to 25 and 30 members in yeast and metazoans, respectively. Despite low similarity in primary Med sequences between the plants and their fungal/metazoan partners, secondary structure modeling of these proteins revealed a remarkable similarity between them, supporting the conservation of Med organization across kingdoms. Phylogenetic analysis between plant, human, and yeast revealed single clade relatedness for 29 Med genes families in plants, plant Meds being closer to human than to yeast counterparts. Expression profiling of rice (Oryza sativa) and Arabidopsis Med genes reveals that Meds not only act as a basal regulator of gene expression but may also have specific roles in plant development and under abiotic stress conditions.

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Available from: Saloni Mathur, May 19, 2014
Genome Analysis
The Mediator Complex in Plants: Structure, Phylogeny,
and Expression Profiling of Representative Genes in a
Dicot (Arabidopsis) and a Monocot (Rice) during
Reproduction and Abiotic Stress
1[W]
Saloni Mathur, Shailendra Vyas, Sanjay Kapoor, and Akhilesh Kumar Tyagi
2
*
Interdisciplinary Centre for Plant Genomics and Department of Plant Mol ecular Biology, University of Delhi
South Campus, New Delhi 110021, India
The Mediator (Med) complex relays regulatory information from DNA-bound transcription factors to the RNA polymerase II
in eukaryotes. This macromolecular unit is composed of three core subcomplexes in addition to a separable kinase module. In
this study, conservation of Meds has been investigated in 16 plant species representing seven diverse groups across the plant
kingdom. Using Hidden Markov Model-based conserved motif searches, we have identified all the known yeast/metazoan
Med components in one or more plant groups, including the Med26 subunits, which have not been reported so far for any
plant species. We also detected orthologs for the Arabidopsis (Arabidopsis thaliana) Med32, -33, -34, -35, -36, and -37 in all the
plant groups, and in silico analysis identified the Med32 and Med33 subunits as apparent orthologs of yeast/metazoan Med2/
29 and Med5/24, respectively. Consequently, the plant Med complex appears to be composed of one or more members of 34
subunits, as opposed to 25 and 30 members in yeast and metazoans, respectively. Despite low similarity in primary Med
sequences between the plants and their fungal/metazoan partners, secondary structure modeling of these proteins revealed a
remarkable similarity between them, supporting the conservation of Med organization across kingdoms. Phylogenetic analysis
between plant, human, and yeast revealed single clade relatedness for 29 Med genes families in plants, plant Meds being closer
to human than to yeast counterparts. Expression profiling of rice (Oryza sativa) and Arabidopsis Med genes reveals that Meds
not only act as a basal regulator of gene expression but may also have specific roles in plant development and under abiotic
stress conditions.
The proper functioning and development of an
organism is orchestrated by a large number of tran-
scription factors (TFs), which in turn regulate the
expression of several downst ream genes affecting
various regulatory and metabolic pat hways. Mediator
(Med) is a multiprotein complex that acts as an inter-
face to pass on the message from the TFs to the basal
transcriptional unit assem bled at the core promoter,
bringing about either transcriptional activation or
repression (Bjo
¨
rklund and Gustafsson, 2005; Conaway
et al., 2005b; Kornberg, 2005). Discovered in the bud-
ding yeast Saccharomyces cerevisiae, for its ability to
respond to a transcriptional activator (Kelleher et al.,
1990; Flanagan et al., 1991), Med was later shown to
participate in the transcription of a majority of yeast
genes (Holstege et al., 1998).
Electron microscopy studies have shown Med struc-
ture to form an unfolded arc around the RNA poly-
merase II (Pol II; Asturias et al., 1999), with the dense
regions corresponding to three distinct units, namely,
the head, the middle, and the tail modules. Yeast Med
is a complex of 25 subunits, where eight subunits form
the head module, seven constitute the middle module,
and six constitute the tail. In addition, a fourth regu-
latory module comprising two Med subunits along
with a cyclin-dependent kinase, Cdk8, and its associ-
ated cyclin, CycC, collectively called the kinase or
Cdk8-cyclin C module, is also part of the Med com-
plex. In contrast to the above model, Baidoobonso et al.
(2007) reported an intact and stable comp lex compris-
ing only the tail and head modules without the middle
module. Until recently, the metazoan Med complexes
were known to share 22 Med subunits with S. cerevisiae
(Bourbon et al., 2004), having eight subunits (Med23 to
-30) unique to them. Recent in silico studies, however,
have established that orthologs of all yeast Med
components are indeed represented in metazoans
(Bourbon, 2008).
Mediators facilitate transcription by increasing the
efficiency/rate of Pol II preinitiation complex forma-
tion at the promoters (Cantin et al., 2003) and activat-
ing transcription from promoters with stalled Pol II
(Lee et al., 2010). The recruitment of Pol II is proposed
to be achieved by direct contacts between the head and
1
This work was supported by the Department of Biotechnology
and the Department of Science and Technology, Government of
India.
2
Present address: National Institute of Plant Genome Research,
Aruna Asaf Ali Marg, New Delhi 110067, India.
* Corresponding author; e-mail akhilesh@genomeindia.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Akhilesh Kumar Tyagi (akhilesh@genomeindia.org).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.188300
Plant Physiology
Ò
, December 2011, Vol. 157, pp. 1609–1627, www.plantphysiol.org Ó 2011 American Society of Plant Biologists. All Rights Reserved. 1609
Page 1
the middle Med modules and the C-terminal domain
of the Rpb1 subunit (Asturias et al., 1999; Davis et al.,
2002). Pol II C-terminal domain phosphorylation has
been shown to be established in a mediator-dependent
fashion (Boeing et al., 2010). The kinase module phos-
phorylates subunits of the general transcription factor
(GTF) TFIID and Med2 (Hallberg et al., 2004; Liu et al.,
2004) and facilitates reinitiation of the preinitiation
complex (Yudkovsky et al., 2000). The Med complex
has the flexibility to acquire different structures upon
binding of different activators to different/same sub-
units (Taatjes et al., 2002, 2004). These distinct activa-
tor-Med structures differentially affect Pol II activity
(Meyer et al., 2010) and regulate Med function in gene-
specific ways (Ebmeier and Taatjes, 2010). Although
the function of Med as a GTF has been widely accepted
(Takagi and Kornberg, 2006), its role as a global reg-
ulator of tran scription has been questioned in some
recent reports (Deato et al., 2008; Thiaville et al., 2008).
However, Ansari et al. (2009) have shown Med to be a
direct requirement for Pol II association at constitu-
tively transcribed genes in yeast, justifying Med as a
GTF. Convincing evidence has also been provided for
the role of the Med complex in recruiting the cohesin
protein complex, which in turn promotes a nd/or
stabilizes the physical proximity between enhancers
and promoters (Kagey et al., 2010). Kim and coworkers
(2011) have expanded the role of the Med complex in
the Pol II-mediated intergenic transcription of small
and long noncoding RNAs.
Meds have been biochemically identified in several
fungi like S. cerevisiae (Kim et al., 1994; Li et al., 1995;
Myers et al., 1998) and Schizosaccharomyces pombe
(Spa
˚
hr et al., 2000), metazoans including mammals
(Fondell et al., 1996; Jiang et al., 1998; Malik and
Roeder, 2000; Sato et al., 2003), Drosophila melanogaster,
an insect (Park et al., 2001), and worms like Caeno-
rhabditis elegans (Kwon et al., 1999). Med homologs
have also been identified in several eukaryotes by
homology-based methods (Bourbon, 2008). The iden-
tification of Med subunits in various organisms has
also resulted in different nomenclatures for homologs
of the same subunit. To bring uniformity, Bourbon and
coworkers (2004) proposed a common unified nomen-
clature for different Med subunits across species.
Biochemical purification of the first plant Med com-
plex in Arabidopsis (Arabidopsis thaliana) identified 21
conserved and six Arabi dopsis-specific Med subunits
(Ba
¨
ckstro
¨
m et al., 2007). The study identified STRUW-
WELPETER (SWP) as Med14 and PHYTOCHROME
AND FLOWERING TIME1 (PF T1) as Med25 subunits.
Later, Med21 was shown to interact with HISTONE
MONOUBIQUITINATION1 (Dhawan et al., 2009) and
the Med12 -Med13 pair to regulate pattern formation
timing during embryogenesis in Arabidopsis (Gillmor
et al., 2010).
Although a wealth of information on fungal and
metazoan Med structure and function is available, a
similar understanding of this complex in plants is still
in its infancy. The past decade has witnessed a rapid
increase in the number of genome sequences for
several plant species. This study expands the search
for Med genes across different groups of the plant
kingdom, from algae to higher angiosperms, using in
silico approaches. We find that all the reported Med
subunits are present in one or the other plant group.
Our study also establishes that despite the low
sequence similarity between plant, fungal, and meta-
zoan homologs of the same Med subunits, these pro-
teins exhibit considerable similarity in their secondary
structures. Expression profiling supports the funda-
mental role for some Med genes in transcriptional
regulation and also highlights the role of other Meds in
regulating development- and stress-specific expres-
sion in rice (Oryza sativa) and Arabidopsis.
RESULTS AND DISCUSSION
Identification of Med Proteins in Pl ants
Med subunits have been biochemically identified in
several fungi and metazoans but only for one plant,
Arabidopsis (Ba
¨
ckstro
¨
m et al., 2007). In this study,
putative Meds have been identified in seven groups of
the plant kingdom by Hidden Marko v Model (HMM)
methods. Using a dual approach, both full-length (FL)
and high-homolgy (HH) HMM profiles were gener-
ated using complete sequences and highly conserved
regions across metazoan, fungi, and plant kingdoms.
This exercise identified 67 HH regions across all Meds,
with more than one HH region in 22 Med subunits
(Fig. 1). This intensive methodology enabled us to
identify novel Meds possessing one or more HH
regions. Furthermore, we singled out HH regions
that define highly conserved regions in plants (marked
as black boxes in Fig. 1) for each Med subunit owing to
their presence in all/most of the predicted plant
sequences (Supplemental Table S1). Cdk8 and CycC
counterparts were inferred using only the FL-HMMs
with very high exp ect values, to segregate them from
various other Cdk and cyclin family members. Bio-
informatics analysis has previously established yeast
Med2, -3, and -5 as orthologs of the metazoan Med29,
-27, and -24 subunits, respectively (Bourbon, 2008).
Therefore, the yeast nomenclature has been retained in
this study. Ba
¨
ckstro
¨
m et al. (2007) had identified six
new plant-specific Meds through biochemical anal-
ysis, which were numbered Med32 to -37. To find
orthologs of these Meds, Position-Specific Iterative
(PSI)-BLAST, HMM search, and the presence of com-
mon Pfam protein domains with the bona fide Arabi-
dopsis Meds helped us to identify putative Med32 to -37
in all the plant groups. Our in silico search revealed
that all the predicted plant Med33s were the same as
those we predicted as Med5 (Suppl emental Table S1).
In addition, several putative Med32 proteins were
found to have a conserved Med29 Pfam domain (Sup-
plemental Table S1), and all of the 18 Med32 predic-
tions were supported by PSI-BLAST analyses that
Mathur et al.
1610 Plant Ph ysiol. Vol. 157 , 2011
Page 2
were jump-started using Med2/29 alignment as quer y
sequences (Supp lemental Table S2) . These results cor-
roborated earlier predictions of plant Med32 and
Med33 to be apparent homologs of Med2/29 and
Med5/24, respectively (Bourbon, 2008). Consequently,
all Med32 and Med33 hits in this study were annotated
as Med2 and Med5, respectively. After removing all
the redundancy in various Med subunits, the Med
complex was finally found to have 32 unique Med
subunits (Med1 to -23, -25, -26, -28, -30, -31, and Med34
to -37) along with Cdk8 and CycC components of the
kinase module. All the gene s of the Med complex will
henceforth be collectively called MCC (for Med, Cdk8,
and CycC) genes.
Of the 739 MCC genes identified in the study (Fig.
2), 699 were Meds, 26 were Cdk8 orthologs, and 14
were CycC components. Our study establishes, to our
knowledge for the first time, that at least one homolog
for all the animal/fungal Med subu nits is represented
in the plant kingdom. We have predicted 52 Arabi-
dopsis MCC proteins (Fig. 2). These include more
paralogs for six Meds (Med15, -20, -19, and -35 to -37)
as well as orthologs for Med26, Med30, and the kinase
module subunits not identified earlier in the biochem-
ical screen (Ba
¨
ckstro
¨
m et al., 2007). We have identified
SENSITIVE TO FREEZING6 (SFR6) as Med16, a
nucleus-localized protein with roles in cold acclimation
(Knight et al., 2009) as well as photoperiodic and clock
gene expression (Knight et al., 2008). In addition, two
of the Arabidopsis Med33 subunits (AT2G48110 and
AT3G23590; Ba
¨
ckstro
¨
m et al., 2007) are annotated as
REDUCED EPIDERMAL FLUORESCENCE4 (REF4)
and REF4-RELATED1, respectively (The Arabidopsis
Information Resource [TAIR]). The dominant muta-
tions in REF4 lead to reduced accumulation of phenyl-
propanoid end products and affect plant growth
Figure 1. The distribution of the conserved HH regions of the Med complex proteins among various eukaryotes. The HH regions
(boxes) are numbered from the N terminus onward for each Med protein, represented as horizontal lines. The HH regions
defining the most conserved regions in plants are shown as black boxes. The number below each HH region shows its start
position relative to human or Arabidopsis (marked in the shaded area) Med proteins.
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1611
Page 3
(Stout et al., 2008). Although the exact function of REF4
has not been ascertained, the authors argue against the
function of REF4 as a TF, owing to putative membrane-
spanning domains in the protein. However, REF4 is
not a part of any membrane proteome (Stout et al.,
2008; TAIR) and has been isolated as a component of
the Med complex in a biochemical screen (Ba
¨
ckstro
¨
m
et al., 2007), making it a legitimate transcriptional
regulator. On the other hand, the rice counterparts of
Med5/33 have been annotated as “structural constit-
uent of ribosome” in the Rice Genome Annotation
Project (RGAP) database. This seems to be an anomaly
in the annotatio n. Domain identification for these
proteins in the Pfam, SMART, as well as InterProScan
databases did not provide information on any specific
protein signatures for the three rice Med5 paralogs. In
fact, in Pfam, there was no information on any con-
served protein domains for any of the 37 putative
Med5 proteins. Our results further contribute toward
enriching the current annotation of several plant
proteomes by annotating several genes of undefined
functions as Meds. These include Arabidopsis (8 of
Figure 2. The distribution of putative Med proteins across the plant kingdom. The Med subunits are grouped as head, middle,
tail, and kinase module according to Bourbon (2008). The unknown group has members whose positions in the complex are
unassigned. The left panel lists the number of biochemically purified Med proteins in S. cerevisiae (Sc), human (Hs), and
Arabidopsis (At). The right panel reports the number of homologs predicted in the study for diverse plants belonging to various
groups (marked at the top of each respective group) of the plant kingdom. The total numbers of Med proteins identified for an
organism and for an individual Med subunit are represented in shaded boxes at the ends of the columns and rows, respectively.
Organisms are as follows: G. max (Gm), P. trichocarpa (Pot), V. vinifera (Vv), C. papaya (Cap), S. lycopersicum (Sl), B. distachyon
(Bd), Z. mays (Zm), S. bicolor (Sb), O. sativa (Os), P. taeda (Pit), S. moellendorffii (Sem), P. patens (Pp), V. carteri (Vc), O.
lucimarinus (Ol), and C. merolae (Cm).
Mathur et al.
1612 Plant Ph ysiol. Vol. 157 , 2011
Page 4
52 = 15%), Cyanidioschyzon merolae (8 of 11 = 73%), rice
(20 of 55 = 36%), and Vitis vinifera (100%; Supplemental
Table S3).
Evidence of Animal Med26 in Plants
Orthologs of metazoan Med26 have not been re-
ported for plants in any study so far. Med26, a subunit
of the Cofactor Required for SP1 Activation complex
(Ryu et al., 1999), is a target of a zinc finger TF and is
involved in epigenetic silencing of neuronal gene
expression along with Med19 (Ding et al., 2009). Our
intensive HH study helped us to identify novel Med26
in all land plants; however, they were not detected in
the algal group. Of the six Med26 rice and Arabidopsis
proteins, four (two from each species) are annotated as
transcriptional elongation factors and one each as a
transcriptional regulator (in Arabidopsis) and a hypo-
thetical protein (in rice) in TAIR and RGAP, respec-
tively. Moreover, these six Med26 proteins showed
more than 90% probability of localizing to the nucleus
(data not shown). In addition, all the 40 predicted
Med26 subunits showed the Pfam Med26 domain
having the conserved TFIIS helical bundle domain, a
conserved N-terminal region found in the transcrip-
tion elongation factors TFIIS and Elongin A (Booth
et al., 2000). Although the role of Med26 in plants
remains to be established, it appears that Med26 may
play a role in transcription elongation, as has been
reported for the Cdk8 submodule in the serum re-
sponse network (Donner et al., 2010) and in p53-
directed Pol II elongation via the Med complex in
humans (Meye r et al., 2010).
Conservati on, Loss, and Duplication of Fungal/Animal
Med Subunits in Plants
For each core module of the Med (head, middle, and
tail), a high conservation except for one or two sub-
units was observed across kingdoms. All the 34 MCCs
could be identified in one or the other species studied
here (Fig. 2), with the fungal/metazoan Med7, -18, -21,
and -31 being represented in all seven groups. In yeast,
Med7 and -21 are important for cell viability and
constitute the core along with eight other subunits
upon which other Med subunits assemble (Bjo
¨
rklund
et al., 2001; Kang et al., 2001; Guglielmi et al., 2004).
High diversity in Med subunits was observed for the
plant-specific Me ds and those with unknown func-
tions (Fig. 2). Algal Meds (including plant-specific
subunits) were predominantly divergent from those in
the other classes of plants; however, as in yeast, algae
also lacked Med23, -25, -26, and -30 subunits. Except
for Volvox carteri, all the algal members also lacked the
kinase module counterparts. Functional Med com-
plexes lacking the kinase module have been well
documented in various cell types (Sato et al., 2004).
Despite a rigorous search, only the red alga C. merolae
had a detectab le Med1 subunit in the plant kingdom.
Med1 is the target for the aryl-hydrocarbon receptor
(Wang et al., 2004) and several liganded nuclear re-
ceptors via its LxxLL (Leu-X-X-Leu-Leu) motif (Yuan
et al., 1998; Malik et al., 2002). CmMed1_1 has two
LxxLL motifs at positions 219 and 384. In mammals,
Med1 exists only in a subpopulation of the total Med
complexes (Zhang et al., 2005), and in yeast , it is not
critical for cell survival (Balciunas et al., 1999). It
appears that Med1 functions have either been lost in
higher plants or the roles have been acquired by other
Med subunits/transcription regulators during the
course of evolution.
The yeast Med complex has single homologs of all
the MCC components, while the more complex meta-
zoans possess additional Med subunits as well as more
than one member in the kinase module. However,
wherever detected, only Med21 and -23 showed a
single homolog across all plant species studied. And
within the submodules, only Carica papaya exhibited a
single homolog in the head and the middle modules
while Brachypodium distachyon , Physcomitrella patens,
Ostreococcus lucimarinus, and C. merolae had a single
homolog only in the middle module. In this collection
of plant Meds, Populus trichocarpa had the highest
number of paralogs (nine for Med15) among all the
yeast/metazoan Meds. In addition, several other plant
species showed high numbers of Med15 paralogs.
Med15 physically interacts with several disparate TFs
both in metazoans and S. cerevisiae (Kato et al., 2002;
Yang et al., 2006; Thakur et al., 2009). Consistent with
this, an increased number of plant Med15 paralogs
could bestow additional regulatory potential to the
Med compl ex to interact with diverse signals.
The largest numbers of MCC homologs were iden-
tified in Glyc ine max (91 proteins). Interestingly, G. max
had one or more additional paralogs for each MCC
family than Arabidopsis, except Med6 to -8, -10, -13,
-15, -20 to -23, -25, -34, and CycC, where the homolog
numbers for both the organisms were the same. G. max
has a highly duplicated genome (Walling et al., 2006),
with nearly 75% of the genes present in multiple
copies (Schmutz et al., 2010). The expansion in the
genome size in G. max and also P. trichocarpa (Tuskan
et al., 2006), which has the second highest number of
predicted MCC paralogs (77) in our repertoire of
Meds, may account for the increased number of de-
tectable MCC paralogs in these dicots as compared
with Arabidopsis. Likewise, C. papaya , reported to
have fewer genes than any other sequenced angio-
sperm (Ming et al., 2008), had the least number of
MCC paralogs among dicots. Similarly, in monocots,
the reported similar gene numbers across a broad
diversity of grasses (International Brachypodium Ini-
tiative, 2010) was reflected in the number of MCC
paralogs identified. Dicots showed a wider variation
in the total numbers of MCC proteins (32–91) for
various subunits than monocots (45–55).
Detection of all known metazoan/fungal Meds in
plants (except Med1 in higher plants) suggests a
conservation of Med function across kingdoms. In
addition, the presence of novel plant-specific Meds
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1613
Page 5
along with an enhanced number of paralogs for sev-
eral Meds suggests that these subunits may have been
acquired to perform a specialized role in plants. La ck
of several Meds and the presence of Med1 in algae
suggest a variable composition of the Med complex
in certain groups. However, it is prudent to say that
with the availability of more sequence (genome/
transcriptome) data on the species studied and com-
plete genome sequences of more organisms, further
insights can be obtained in the future.
Evolutionary Relatedness between Plant, Fungal, and
Metazoan Mediators
To assess the evolutionary relationship and conser-
vation between homologs of various Med families,
phylogenetic analysis was performed on sequences
from the yeast, human, and one representative mem-
ber from the seven plant groups selected in the study
(Arabidopsis, rice, Pinus taeda, Selagine lla moellendorffii,
P. patens, V. carteri, and C. merolae). These sequences
were divided according to the different Med modules
followed by generating respective phylogenetic trees
(Fig. 3A; Supplemental Figs. S1 and S2). Considering a
bootstrap cutoff of at least 50% as significant, the
cumulative depiction of different Med homologs is
provided in Figure 3B. A single clade representing all
the putative plant homologs as well as their fungal and
metazoan counterparts (category A) was seen for three
subunits: one belonging to the middle module and two
to the kinase module. “All plan ts” refers to a group
consisting of all the terrestrial plants (angiosperms,
gymnosperms, pteridophyte s, and bryophytes) in a
single clade that also included all or at least one
member of the algal taxa with high confidence within
the same clade. In addition, four other subunits (cat-
egory B) formed a single clade comp rising all the plant
homologs but lacking the yeast and human members.
There were 22 subunits (categories C, D, and E) where
all the terrestrial plants exhibited a high degree of
conservation but their algal partners did not. Of note,
these also included 12 Med subunits for which we did
not find any detectable Meds in the algal group (see
above). While both human and yeast Meds shared the
clade with terrestrial plants in category C, both were
absent in category E. Category D had the human Meds
grouping with the terrestrial plant clade, while yeast
did not. If the terrestrial plants themselves either
formed more than one clade for a Med class (Med9)
or had less than 50% bootstrap values (Med10, -31,
and -2), they were put in category F. Med1, with no
members in the land plants, was also assigned to
category F.
The phylogenetic analysis highlighted that higher
plants grouped together in a single clade for 29 MCC
families (groups A–E). Apparently, the plant Meds
were found to be closer to human Meds than their
yeast counterparts. The former shared the plant clade
for eight MCC families (groups A, C, and D) as
opposed to just five (groups A and C) for yeast (Fig.
3B). Investigations on the relationship between rice
and Arabidopsis revealed that they grouped together
to form an angiosperm-specific clade with high confi-
dence of 50% or greater in seven MCC families (black
asterisks in Fig. 3 and Supplemental Figs. S1 and S2).
Furthermore, lineage-specific clades were formed for
both dicots (Med4, -18, -22, and -25) and monocots
(Med11, -16, -20, and -30), pointing to independent
expansion of Med gene families in the angiosperms.
Structural Conservation of Mediator Submodules
in Plants
This study, along with a previous work (Bourbon,
2008), shows that Med sequences are only weakly
conserved between different organisms. We reason
that if the function of a Med has been conserved across
kingdoms, then during evolution, the selection pres-
sure would favor sustaining struct ural determinants
that support intersubunit contacts. Therefore, we com-
pared the protein secondary structures between yeast,
human, and a plant (Arabidopsis) Meds for the de-
fined submodules as well as those identified through
yeast two-hybrid screens.
Med7 is the key architectural subunit of the middle
module and forms subcomplexes with Med21 and -31;
the Med7/21 dimer in turn binds to the Med10 C
terminus (C) in yeast (Koschubs et al., 2009, 2010).
Secondary structure mod eling of these four Meds
showed high conservation, with the exception of an
extended Arabidopsis Med21 N terminus (N; Fig. 4).
The two yeast Med7N poly-Pro stretches, which aid in
wrapping around Med31 helices (Koschubs et al.,
2009), were conserved in plants too (Supplemental
Fig. S3A). Notably, like human Med7, there were fewer
amino acids intervening in these Pro regions in plan ts
than in yeast. The only plant lacking this region was P.
patens, which appeared to be a partial sequence. We
noted that the plant Med31C sequences exhibited
poly-Pro regions followed by a nuclear localization
signal domain (Supplemental Fig. S3B). Interestingly,
these sequences were absent in yeast, human, as well
as algae, highlighting that these sequences were very
similar early in evolution, when lower plants diversi-
fied from the fungal/animal group, and the higher
plants probably acquired them later.
The middle module Med4/9 also forms a dimer
(Koschubs et al., 2010). Secondary structure compari-
son of Med4 highlighted the high level of homology
these proteins shared, including in the regions that
have been shown t o interact with Med7 and -21
(Guglielmi et al., 2004; Fig. 4). The yeast Med4C region
required for cell via bility (amino acid residues
194–250; Fig. 4) showed considerable structural simi-
larity with plants; however, its human counterpart
formed more sheets in this region. The Med9C region
showed much more structural conservation between
the three organisms as compared with highly diver-
gent N termini (Fi g. 4). This is not surprising, as in
yeast, Med9N is not stably bound to the core complex
Mathur et al.
1614 Plant Ph ysiol. Vol. 157 , 2011
Page 6
and the predicted Med9 loop comprising amino acid
residues 19 to 63 is not required for Med4/9 formation
(Koschubs et al., 2010).
The yeast Med14N (amino acid residues 1–259)
connects the tail to the middle module through
Med10 (Guglielmi et al., 2004); moreover, in Arabi-
dopsis, Med14N (amino acids 1–959) interacts with a
transcription regulator (Gonzalez et al., 2007). The
yeast and Arabidopsis Med14 sequences aligned only
for 171 amino acids in the N-terminal region, showing
only 21% identity and 41% similarity (data not shown).
However, within this region, all three proteins showed
the same number of helices (fi ve) and sheets (three;
Fig. 4). We note that Ar abidopsis and human Med14C
shared higher structural correlation than their yeast
counterpart.
Figure 3. Phylogenetic relationship of Med subunits among various members of the yeast, metazoan, and plant kingdoms. Med
proteins from one representative member of the seven plant groups, human (Hs), and S. cerevisiae (Sc) were assigned into the
different modules of the Med complex. A, An unrooted tree of the head module constructed using the PHYLIP program by the
neighbor-joining method. Numbers at the nodes represent bootstrap values from 1,000 replicates. A bootstrap value of at least
500 was used to define the Med subunits into groups A to G (see B). All the terrestrial plants (angiosperms, gymnosperms,
pteridophytes, and bryophytes) are shown in green, algal members in blue, and Hs and Sc sequences in purple and black,
respectively. Angiosperm-specific clades are marked by black asterisks and mixed clades by red asterisks. The scale bar
represents amino acid substitutions per site. For the phylogenetic trees of the middle, tail, unknown, and kinase modules, see
Supplemental Figures S1 and S2. B, Summary of the phylogenetic grouping of plants with Sc and Hs Med subunits. Groups A and
B represent a single clade of all the putative Med proteins in terrestrial plants and algal members (black boxes) or at least one
member of the algal group (red boxes) at 500 or greater bootstrap value. Groups C to E represent only the terrestrial plant
members (either the algal members have not been predicted or do not group together with the land plants). Groups A and C
include both the Sc and Hs members, while group D has only Hs members in the same clade as the plants. In groups B and E, the
Sc and Hs members do not group together with plants. When the land plants did not group together to form a single clade, they
were assigned to group F. Med subunits having angiosperm-specific clades are marked by asterisks.
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1615
Page 7
Figure 4. Secondary structure comparison among human, yeast, and Arabidopsis mediator sequences. The protein secondary
structures were predicted using PSIPRED and superimposed on the alignments generated using MAFFT. The purple rectangles
and green arrowheads denote the predicted protein helices and sheets, respectively. A solid black line indicates no secondary
structure, and a dotted line denotes a gap in the alignment. Each red bar represents a length equivalent to 20 amino acids. Blue
lines indicate the extent of interaction of Med4 with Med7 and Med21. Hs, Human; Sc, S. cerevisiae; At, Arabidopsis.
Mathur et al.
1616 Plant Ph ysiol. Vol. 157 , 2011
Page 8
Med8N in S. cerevisiae binds to a TATA box-binding
protein, and Med8C attaches the Med18/20 submod-
ule to the mediator (Larivie
`
re et al., 2006, 2008). The
domains of the Med8/18/20 triad are interdependent
on each other for fo lding and proper complex forma-
tion (Shaikhibrahim et al., 2009). Surprisingly, the
conserved Med8 domain could not be detected in
any of the plant species (Supplemental Table S1),
including the one identified via biochemical screening
in Arabidopsis (Ba
¨
ckstro
¨
m et al., 2007). Despite this,
comparative modeling indicated that Arabidopsis
Med8 as well as its interacting Med18/20 partners
adopt spatial conformations significantly similar to
those determined for the yeast proteins (Fig. 4). We
observed that the putative plant Med8 sequences were
nearly double in size than their yeast and human
counterparts.
Med17 is the most important scaffolding subunit of
the head module; its conditional knockout mutants in
yeast are incapable of expressing most of the protein-
coding genes (Thompson and Young, 1995). Compar-
ative modeling confirmed the importance of Med17 in
showing high structural similarity between the three
organisms (Fig. 4). Yeast two-hybrid screens support
the interaction of Med17 with Med8C and -22 as well
as Med6 (Guglielmi et al., 2004). That study also
identified interactions between Med22 and -11 as
well as Med16 and -5. Structural comparisons revealed
similar topology for Med5, -6, -11, -16, and -22 in the
three organisms modeled (Fig. 4). Of note, the Arabi-
dopsis Med5C formed more helices, owing to longer
protein length.
The Med2/3 pair interacts with Med15 to form a
triad in yeast (Zhang et al., 2004). Notably, the human
and plant Med2 and -3 sequences are closer than their
fungal counterpart, whereas, apart from Med 15N and
parts of its mi ddle region, there was less apparent
structural similarity between the three organisms
for Med15 (Fig. 4). The yeast Med15N KIX domain
(Novatchkova and Eisenhaber, 2004) is a bundle of
three helices that forms the interface for transcriptional
regulator binding (Thakur et al., 2009). The metazoan
Med15N has an ARC105 domain, a three-helix struc-
ture with marked similarity to the KIX domain (Yang
et al., 2006). The canonical Pfam KIX domain was not
apparent in the bona fide Arabidopsis Med15 (Sup-
plemental Table S1) but was represented in some
dicots (C. papaya, G. max, and P. trichocarpa) and
monocots (rice and B. distachyon). The structural com-
parison showed that several plant Med15s shared the
three-helix structure; moreover, amino acid residues
implicated in the interaction of the KIX domain
(Thakur et al., 2009) were also conserved/similar
between plants and yeast (Supplemental Fig. S4).
The ARM domain shared by elongation factor TFIIS
and Med26 enables yeast TFIIS to interact with SAGA
and Med13 (Diebold et al., 2010). Secondary structure
analysis showed a similar pattern of conservation
between the Arabidopsis and human Med26 ARM
domains (Fig. 4). Moreover, the conserved “LFG”
(Leu-Phe-Gly) motif between yeast Med13 and TFIIS
had similar “P(Pro)FG” and “A(Ala)FG” motifs in
human and Arabidopsis seque nces, respectively (data
not shown).
This extensive secondary structure analysis sup-
ports a conserved structural organization for the Med
submodules across kingdoms. However, the presence
of divergent regions in Meds may have evolved to
facilitate interactions with different host-specific TFs
in an organism-specific manner.
Differential Regulation of Meds during
Plant Development
Out of the 52 rice and 43 Arabidopsis MCC genes
represented on the microarray chips, 50 and 42 genes
were differentially expressing at a statistically signif-
icant value of P # 0.05 in at least one of the develop-
mental stages analyzed in the monocot and the dicot,
respectively (Supplemental Tables S4 and S5). Further-
more, we kept the criteria for significant differential
expression to be 2-fold change with respect to the
control. A total of 39 rice (Fig. 5) and 33 Arabidopsis
(Fig. 6) genes showed more than 2-fold change in at
least one reproductive substage in comparison with
either of the vegetative stages. These gene numbers
stood at 22 and 13 in rice and Arabidopsis (Fig. 7, A
and B), respectively, when both the vegetative controls
were assessed vis-a-vis the reproductive stages. Fur-
thermore, OsMed5_1 showed opposite regulation in
P1, S1, and S5 stages with respect to leaf and root
controls. Detailed assessment between the various
reproductive stages in comparison with both the veg-
etative controls revealed that the P1 stage in panicle
and the S3 and S5 stages in seed for rice as well as the
F15 stage in flower and the S9 and S10 stages in seed
development for Arabidopsis had the most differen-
tially regulated genes. The expression of none of the
genes changed significantly in all flower/panicle sub-
stages with respect to the controls. However,
OsMed37_1 and OsMed37_6 showed more than 2-fold
induction, while the expression of AtMed36_1 was
depressed in all the seed stages. The seed stages
exhibited much more pronounced expression, high-
lighting that more MCC genes are directly involv ed
during embryo development and seed maturation
stages. A search for cis-elements in the promoter
regions of these Med genes revealed several motifs
found in the promoters of seed storage protein genes
as well as those involved in embryo and/or endo-
sperm development (Supplemental Table S6). In addi-
tion, elements involved in pollen development, the
binding site of LEAFY (important for the transition
from the vegetative to the reproductive phase) in
OsMed31_2, as well as the CArG consensus sequence
found in the promoter of the flowering pathway
integrator gene SUPPRESSOR OF OVEREXPRES-
SION OF CONSTANS1 were also identified in regula-
tory regions of some of the genes that had high
expression in panicle/flower development.
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1617
Page 9
The normalized expression values of developmental
stages for rice and Arabidopsis are provided in Sup-
plemental Tables S7 and S8, respectively. A compari-
son of the expression patterns obtained for selected
genes using quantitative real-time PCR (QPCR) ex-
hibited a similar trend of expression to that observed
for the microarray data, having correlation coefficients
of more than 0.9 (Fig. 8).
Limited Regulation of Plant Meds in R esponse to
Abiotic Stress
Mi croarray analysis in response t o thre e stress
conditions (desiccation, cold, and salt) was also per-
formed. When the expression profiles were compared,
29 rice genes (Supplemental Table S4) and only four
Arabidopsis genes (Supplemental Table S5) were dif-
ferentially expressed in at least one of the stress phases
as compared with the unstressed control. Further-
more, expression levels for 10 rice genes (Fig. 9) and
none of the Arabidopsis MCC genes were significantly
affected in response to any of the stress treatments. We
noted that the exp ression profiles for all these 10 rice
genes during desiccation and salt stress were compa-
rable. This is in accordance with the similar responses
these stresses are known to trigger in affected plants
(Bartels and Sunkar, 2005). Only OsMed37_6 showed
more than 2-fold change in transcript abundance in all
three stress conditions tested. An in silico analysis
Figure 5. Microarray-based expression analysis of selected Med genes
in rice development stages. Expression profiles of at least 2-fold
differentially regulated Med genes at P # 0.05, with respect to the
vegetative controls (mature leaf [ML] and root [R]), are shown. Devel-
opmental stages are listed at the top of each column in the temporal
order of development. Reproductive stages comprise panicle (P1–P6)
and five stages of seed (S1–S5) development. Hierarchical clustering of
the expression profile was done with log-transformed average values,
taking mature leaf as the baseline. The color scale at the bottom of the
heat map is given in log
2
intensity value, whereby green represents low-
level expression, black shows medium-level expression, and magenta
signifies high-level expression.
Figure 6. Microarray-based expression analysis of selected Med genes
in Arabidopsis development stages. Expression profiles of at least 2-fold
differentially regulated Med genes at P # 0.05, with respect to the
vegetative controls (leaf [L] and root [R]), are shown. Developmental
stages are listed at the top of each column in the temporal order of
development. Reproductive stages comprise flower (F9–F28) and seed
(S3–S10) stages. Hierarchical clustering of the expression profile was
done with log-transformed average values, taking leaf as the baseline.
The color scale at the bottom of the heat map is given in log
2
intensity
value, whereby green represents low-level expression, black shows
medium-level expression, and magenta signifies high-level expression.
Mathur et al.
1618 Plant Ph ysiol. Vol. 157 , 2011
Page 10
showed the presence of abiotic stress recognition mo-
tifs in the promoters of some of these genes (Supple-
mental Table S6). We note that the expression of more
rice genes (seven) decreased in response to at least one
of the stresses than those that were augm ented (three;
Fig. 7C). It appears that thousands of years of domes-
tication of rice have rendered it less tolerant to stresses
and probably explains why it down-regulates its tran-
scription mach inery during stress. The expression
profiles of three genes under stress conditions were
also verified by QPCR (Fig. 8).
Med genes have been widely accepted as GTFs. The
ubiquitous expression of many MCC genes at similar
levels during stress and development (Supplemental
Tables S9 and S10) supports the basic role these genes
play as components of the transcription machinery.
However, differential expression of some Med genes
in specific states provides ample possibilities for the
differential regulation of plant processes as well.
Expression Profile of the Core Module Gene s during
Reproductive Development
In order to determine a role of the Med complex in
reproductive devel opment, we compared the expres-
sion patterns of various Med genes in a module. In the
head module, Med17 (the most important scaffolding
gene of this module), -6,-20,-22, -28, and -30 did not
show significant differential expression in the repro-
ductive stages vis-a-vis both the vegetative controls in
both the angiosperms. The same was true for rice
Med18 and Arabid opsis Med8 and -19; however,
AtMed18_1 showed 4-fold down-regulation in the S9
stage with respect to the vegetative controls, and
OsMed8_1 was up to 3-fold induced in the early
panicle stages P1 and P2. Also, while OsMed19_1
showed down-regulation in the P4 stage, the expres-
sion rose in the P1 stage of OsMed19_2. In addition,
OsMed11_1 showed more than 2-fold enhanced ex-
pression in the S2 and S3 stages of seed development,
while the Ar abidopsis counterpart could not be as-
sessed, as it was not represented on the chip.
Expression profiles for Med7, the key architectural
unit of the middle module, and Med4 showed that
their expression was not significantly altered in repro-
ductive stages in the two angiosperms. The expression
data for AtMed21_1 showed its specific differential
regulation in seed stages (i.e. approximately 2-fold
from S7 to S9 and then staying high in S10; Supple-
mental Table S10). These seed stages in Arabidopsis
correspond to embryo development and cotyledon
expansion stages (Schmid et al., 2005), providing cre-
dence to the portrayed role of Med21 in embryo
development (Dhawan et al., 2009). In rice, on the
other hand, the expression of Med21 was 2-fold up-
regulated in only the P1 stage of panicle development
with respect to the vegeta tive controls, while in seeds,
its expression levels remained sim ilar to those in
vegetative tissues (Supplemental Tables S4 and S9).
Thus, it appears that Med21 plays a distinctive role in
Arabidopsis seed development; however, in rice , its
function might be more pronounced during early
Figure 7. Expression analysis of rice and Arabidopsis Med genes using a microarray. A, Expression profiles for genes that are
more than 2-fold differentially regulated at P # 0.05 in rice panicle (P1–P6) and seed (S1–S5) stages with respect to both the
vegetative controls, leaf and root. Red and green boxes represent up- and down-regulated genes, respectively. A box marked in
blue defines an opposite regulation in that substage with respect to leaf and root controls. B, Expression profiles for Arabidopsis
flower (F9–F28) and seed (S3–S10) stages. C, Differentially expressing Med genes in rice in abiotic stress stages. CS, Cold stress;
DS, desiccation stress; SS, salt stress.
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1619
Page 11
Figure 8. QPCR results for the expression of selected genes during development and stress and their correlation with microarray
data. Three biological replicates were taken for both QPCR and microarrays. Three technical replicates were employed for each
QPCR biological replicate. Error bars show
SE values for data obtained using both techniques. QPCR data were normalized to
ease profile matching with each microarray’s data. Pearson correlation coefficients between QPCR and microarray data are
Mathur et al.
1620 Plant Ph ysiol. Vol. 157 , 2011
Page 12
panicle development. The same study also suggested
Med21 to be activated by microbial infection and also
by factors involved in stress signaling; however, we
did not find any positive correlation when assaying for
the three abiotic stress responses in both the angio-
sperms (Supplemental Tables S4 and S5). It is possible,
however, that Med21 is involved in stress signaling
during reproductive stages and not in younger vege-
tative tissues. In rice , another Med (OsMed31_2) was
up-regulated in the P1 and P2 stages of panicle devel-
opment, while its paralog OsMed31_1 showed 3-fold
enhanced regulation in leaf tissue as compared with
root. However, in Arabidopsis, Med31 did not show
any significant differential expression across the stages
analyzed. Likewise, while the expression remained
unchanged for Med9 in the flower stages of Arabidop-
sis with respect to the vegetative controls, OsMed9_2
was 2-fold down- regulated in the P6 stage. Of note, the
expression of AtMed9_1 was augmented in the S9 and
S10 stages, and the expression in OsMed9_1 declined in
the equivalent substages. Of the remaining middle
module gene, Med10, the lone rice homolog did not
show differential expression in reproductive stages
when compared with the vegetative stages; however,
the Arabidopsis homolog AtMed10_1 exhibited up-
regulation in the S9 and S10 stages. AtMed10_2 could
not be assessed, as it was not represented on the chip.
Four of the seven Med genes in the tail module
showed appreciable differential transcript abundance
in at least one stage of reproductive devel opment in
either Arabidopsis or rice with respect to the vegeta-
tive controls. These were Med3,-5,-14,and-15 (Fig. 7,
AandB).ForMed3, only the Arabidopsis homolog
showed up-regulation in th e S9 and S10 stages of seed
development. In Arabidopsis, SWP (Med14) has been
shown to interact with the corepressor LEUING
(Gonzalez et al., 2007) and its expression levels are
important in defining the duration of cell proliferation
during leaf development; thus, it plays a role in pat-
tern formation at the meristem (Autran et al., 2002) as
well as in the regulation of root elongation by repres-
sing the root-specific gene Lateral Root Primordium1 via
histone deacetylation (Krichevsky et al., 2009). We
identified two Med14 paralogs in rice, of which only
OsMed14_1 had significant differential exp ression in
some reproductive substages (P1, P4, S1, and S3) with
respect to both the vegetative controls. Notably, there
was an 11-fold up-surge in its transcript abundance
soon after fertilization in the S1 stage, and in the
subsequent developmental stage, the expression levels
came down to levels similar to vegetative stages.
OsMed14_2, on the other hand, did not show any
differential transcript accumulation in any of the
reproductive stages vis-a-vis the vegetative controls.
AtMed14_1 showed specific enhanced expression only
in the S9 and S10 stages. Among the three rice Med5
homologs and two members in Arabidopsis, only
OsMed5_2 showed significant change in its mRNA
levels in a reproductive stage (S5; down-regulation) in
comparison with both the vegetative controls. Of the
two Med15 homologs in rice, only OsMed15_1 showed
high-level expression in some seed stages, the expres-
sion being up-regulated by 75-, 60-, and 5-fold in the
S2, S3, and S4 stages, respectively, signifying a possible
role in rice embryo development. Of the three Arabi-
dopsis Med15 paralogs, AtMed15_1 is not represented
on the chip and the other two did not show apprecia-
ble differential expression in reproductive stages with
respect to leaf and root. Med16 is a coactivator of
lipopolysaccharide- and heat shock-induced transcrip-
tional activator in Drosophila (Kim et al., 2004) and is
known as SFR6 in Arabi dopsis. SFR6 is part of a
complex network regulating the photoperiodic path-
way and circadian clock gene expression in response
to external and internal changes such as light, meta-
bolic status, and temperature (Knight et al., 2008). The
rice as well as Arabidopsis Med16 genes did not
display much variation in expression patterns across
all the developmental stages. The sfr6 mutant was
isolated for its inability to cold acclimate to freezing
temperatures (Warren et al., 1996). Surprisingly, we
did not see any differential response in both plant
species even under cold stress (Supplemental Tables
S4 and S5). However, this is in accordance with the
Figure 8. (Continued.)
indicated in parentheses. The y axis represents raw expression values obtained from microarray analysis; the x axis depicts
various developmental stages or abiotic stress conditions. ML, Mature leaf; R, root; S1 to S5, seed development stages; CS, cold
stress; SS, salt stress; DS, desiccation stress.
Figure 9. Microarray-based expression analysis of selected Med genes
in rice during abiotic stress conditions. Expression profiles of at least
2-fold differentially regulated Med genes at P # 0.05, with respect to
the control (untreated 7-d-old-seedling), are shown. Hierarchical clus-
tering of the expression profile was done with log-transformed average
values taking an untreated 7-d-old-seedling as the baseline. The color
scale at the bottom of the heat map is given in log
2
intensity value,
whereby green represents low-level expression, black shows medium-
level expression, and magenta signifies high-level expression.
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1621
Page 13
reported limited transcriptional regulation of the gene
by cold; SFR6, in fact, has been proposed to act
posttranslational ly on the C box-binding factor TFs
to modulate their activity to provide cold tolerance
(Knight et al., 2009).
Expression analysis between comparable reproduc-
tive stages of the dicots and the monocots highlights
that not only do some members of the same gene
family exhibit variable expression patterns between
the two angiosperms but also among its paralogs. This
not only provides evidence for tissue- or cell-specific
dynamics in the constitution of Med complexes, as has
been well documented in mammals (Conaway et al.,
2005a; Taatjes, 2010), but also differential regulation of
Med in a species-specific manner.
Expression Profile of the Kinase Module and Other
Associated Med Genes during
Reproductive Development
The kinase module of the Med complex can repress
as well as activate transcription (Taatjes, 2010), where
Med12 and Med13 subunits play a critical role
for the subcomplex-dependent repression (Knuesel
et al., 2009). Med12 promotes the epigenetic silencing
of target genes by recruiting a histone methyltransfer-
ase and methylating chromatin (Ding et al., 2008). In
Arabidopsis, Med12 (CENTER CITY [CCT]) and Med13
(GRAND CENTRAL [GCT]) exert a transient repres-
sion on the transcriptional machinery that interferes
with embryo development (Gillmor et al., 2010). Mu-
tations in these genes delay the specification of cell
identity and the globular-to-heart transition but have
minimal effects on the initial growth rate of the em-
bryo. In our microarray analysis, this expected en-
hancement in transcript levels was clearly visible
for the Arabidopsis homolog and one rice paralog
(OsMed12_2). The expression levels were several-fold
higher in the equivalent Arabidopsis S5 and rice S2
stages as compared with the preceding seed develop-
ment stages (Suppl emental Tables S9 and S10). It is
noteworthy that these stages represent the late globu-
lar embryo stage of seed development (Schmid et al.,
2005; Agarwal et al., 2007). While the expression
continued to be high in the S3 and S4 stages in rice,
dipping only in the S5 stage, the Arabidopsis homolog
exhibited highest expression in the S9 and S10 stages,
which are maturation stages of embryo development.
Apparently, the other rice paralog does not seem to
participate in this activity. In contrast to Med12, the
Med13 genes of both the angiosperms did not show
any significant differential expression dur ing stages
of reproductive development; however, OsMed13_1
showed approximately 3-fold up-regulation in leaf
with respect to root. Between the CyclinC-Cdk8 genes,
all the CycC homologs in Arabidopsis and rice as well
as OsCdk8_1 were not significantly differentially reg-
ulated in reproductive stages when compared with
leaf as well as root. However, the expression increased
in S9 and S10 for AtCdk8_1, while OsCdk8_1 was up-
regulated by more than 3-fold in only leaf in compar-
ison with root.
There are several Meds whose positions in the
complex are uncertain. One of them is Med25, which
is absent in yeast but plays a role in the regulation of
xenobiotic metabolism and lipid homeostasis in hu-
man liver (Rana et al., 2011). A biochemical screen
identified Arabidopsis PFT1 as Med25 (Ba
¨
ckstro
¨
m
et al., 2007). The PFT1 negatively regulates the phyto-
chrome signaling pathway (Wollenberg et al., 2008),
regulating flowering in response to light quality
(Cerda
´
n and Chory, 2003) and by acting as a positive
regulator of jasmonic acid signaling, which regulates
plant defense responses during fungal pathogen in-
fection (Kidd et al., 2009). The expression profile of
AtMed25_1 revealed very similar transcript abundance
in flower as compared with vegetative tissues, aug-
menting by approximately 2-fold in the S9 and S10
seed stages. The rice homolog had comparable tran-
script levels in the reproductive and vegetative stages.
This is not surprising, since PFT1 is known to exert its
effect on floweri ng only in response to a suboptimal
light environment (Cerda
´
n and Chory, 2003), specific
conditions not analyzed in this study. Moreover, the
role of PFT1 in chromatin remodeling via histone
acetylases like human Med25 (Lee et al., 2007) cannot
be underestimated.
The other animal-specific Med in this category is
Med26. Of the three rice Med26 paralogs, OsMed26_1
did not show any significant differential expression
(Supplemental Table S9), OsMed26_2
transcripts in-
creased in the S3, S4, and S5 stages, and OsMed26_3
transcripts dipped by more than 4-fold in leaf tissue in
comparison with roots (Supplemental Table S4) . Arab-
idopsis also has three Med26 members, of which
AtMed26_1 was not represented on the chip, while
AtMed26_2 and AtMed2 6 _ 3 were not significantly dif-
ferentially regulated in reproductive stages in com-
parison with both the vegetative stages. However, like
OsMed26_3, the expression of these genes was down-
regulated in leaf in compari son with roots. Several of
the plant-specific Med34, -35, -36, and -37 homologs
showed high expression in both the reproductive
stages as compared with the vegetative stages, signi-
fying the important roles these genes may play in
regulating plant-specific functions.
CONCLUSION
Among eukaryotes, plants are unique in being ses-
sile, enduring the vagaries of their surrounding envi-
ronment, both biotic and abiotic. In addition, several
processes like flowering and response to the quality as
well as the quantity of light in several metabolic as
well as developmental processes are specific to plants.
Thus, it is not surprising that several plant-specific TF
families have emerged during the course of evolution
(Riechmann et al., 2000). Consequently, one would
expect a similar expansion in the Med components
Mathur et al.
1622 Plant Ph ysiol. Vol. 157 , 2011
Page 14
that act as a link between these TFs and the Pol II core
complex. Our in-depth in silico prediction for these
subunits revealed more than one homolog for various
Med subunit s in different plants. This expands the
possibility of the number of potential Med complexes
that might form and, in turn, regulate myriad func-
tions during different developmental stages as well as
in response to external environmental cues. In this
regard, the identification of several Med subunits as
components of the plant machinery that participate in
cell proliferation (SWP), embryo development (CCT
and GCT), chromatin modification (Med21), and re-
sponse to external stimuli like light quality, which in
turn regulates genes involved in flowering (PFT1) and
circadian rhythms (SFR6), is beginning to unravel.
This view is strengthened by the genome-wide ex-
pression analysis using microarrays of Med genes in
two angi osperms, a dicot and a monocot, in various
plant developmental stages and during abiotic stress
conditions in this study. The differential transcript
abundance of several Med genes (or their paralogs) in
both/either angiosperms highlights their importance
in regulating plant development in a redundant as
well as organism-specific manner. These observations
also present the possibility that alternative forms of
Med complexes may be functional in various cell types
during different developmental stages. We identify 34
unique Med components, including their kinase and
cyclin counterparts in plants. The data show that
mediators are conserved across the plant kingdom
and suggest that all the yeast/metazoan Med subunits
may have been incorporated within the Med complex
before the plants diverged from fungus/animal
groups; however, it appears that as the plants evolved
and attained specialized functions and structures,
some of these Meds were lost (Med1) while others
were gained (Med34 to -37). Secondary structure
modeling of Arabidopsis proteins and its comparison
with yeast and human counterparts revealed that
despite high sequence variatio n, the functions of
Meds may have been maintained across eukaryotes
by adopting similar structures. However, some sub-
units may have gained additional regulatory power
either by acquiring new regions or by forming new
conformations to interact with TFs or chromatin-
modifying proteins. Future studies will be required
not only to confirm the existence of t hese predicted
Med homologs as structural components of the
Med com plex but also to asc erta in th eir b iolog ical
functions.
MATERIALS AND METHODS
Sequence Retrieval
In this study, putative Med subunits have been identified from 16 plant
species belonging to various groups. Protein sequences were downloaded for
the angiosperms Arabidopsis (Arabidopsis thaliana), Brachypodium distachyon,
Carica papaya, Glycine max, rice (Oryza sativa), Populus trichocarpa, Sorghum
bicolor, Vitis vinifera,andZea mays, the pteridophyte Selaginella moellendorffii,
the bryophyte Physcomitrella patens, the green algae Volvox carteri f. sp.
nagariensis and Ostreococcus lucimarinus, and the red alga Cyanidioschyzon
merolae strain 10D. EST clusters were downloaded for Solanum lycopersicum (an
angiosperm) and Pinus taeda (a gymnosperm) and translated with ESTScan-
3.0.2 using plant-specific matrices (Iseli et al., 1999). The details of the source
databases are provided in Supplemental Table S11. For promoter analysis,
2-kb sequences upstream of the translational initiation sites of some rice and
Arabidopsis Med genes were downloaded from the RGAP and TAIR data-
bases, respectively.
In Silico Identification of Mediator Subunits in Plants
The sequences obtained from published literature covering nearly 70
organisms (Bourbon, 2008) and Med sequences downloaded from the Na-
tional Centre for Biotechnology Information (NCBI) by name search (Supple-
mental Table S12) and cross-verified to be “mediator specific” at a cutoff E
value of less than e-20 in the SwissProt database (Boeckmann et al., 2003) were
used to build HMM profiles. An abbreviation was specified for every
organism (Supplemental Table S13). The sequences were categorized into
different Med families, and HMM profiles were generated on alignments
generated using ClustalW version 1.83 (Thompson et al., 1994) followed by
identification of Meds using HMMER (http://hmmer.org/) in all the plant
species. In the preliminary screening, protein hits having a cutoff E value of
e
22
or less were selected as putative Meds; furthermore, Med sequences of
metazoan, fungal, and plant origin were selected to construct FL-HMM. In
order to pick distantly related Med homologs, HH regions for each Med
subunit were defined. For this purpose, continuous stretches of at least seven
amino acids having a quality score of 60 or more each were delineated over the
entire length of an alignment, and the mean score for each block was
calculated. The highest scoring amino acid stretch was singled out follow ed
by the identification of other blocks having an average score within 3%
(to account for a kingdom-specific bias [viz. metazoan, fungi, and plants]) of
the highest HH score. These were collectively selected as HH regions and
numbered from the N terminus onward; an example is shown in Supple-
mental Figure S5. Furthermore, the alignments corresponding to these
HH regions were extracted using the multiple alignment editor Jalview
(Waterhouse et al., 2009), HMM profiles (HH-HMMs) for Med1 to -31 were
generated, and fresh HMMER searches were performed. Hits with E values of
e
23
or less using FL-HMMs and E values of e
21
or less using HH-HMMs
(expect values determined on bona fide Arabidopsis Med hits) were further
screened on the basis of the most prevalent HH regions among hits and/or the
presence of Med-specific Pfam domains (Finn et al., 2010; Supplemental Table
S1). For identifying Cdk8 and CycC homologs, a stringent E value cutoff of
e
290
or less was used after cross-referencing the hits using the Pfam database.
For identifying Med32 to -37, reported only in Arabidopsis, initially, PSI-
BLAST (Altschul et al., 1997) searches were performed on the protein
sequences of the plant species selected for the study using the BLOSUM 62
substitution matrix and an iteration threshold of 0.001. The best hit from each
plant species was selected and used to generate the FL- and HH-HMMs using
the criteria described above. Med proteins with comparable expect values to
bona fide Arabidopsis Med proteins and the presence of common Pfam
domains were selected. The predicted plant Meds were named as follows:
a rice Med11 gene having two paralogs were designated OsMed11_1 and
OsMed11_2, respectively, numbered according to the increasing order of locus
identifiers. To remove redundancy, all the splice variants of a locus were
considered as a singular entry. All the putative plant Meds and an identity
converter are listed in Supplemental Table S14.
Other Bioinformatics Tools Used
Protein domain identification was done using the Pfam, SMART (Letunic
et al., 2009), and InterProScan (Quevillon et al., 2005) databases. Subcellular
localization of proteins was performed using MultiLoc2 (Blum et al., 2009),
and nucleus-localizing signals were identified using NLStradamus (Nguyen
Ba et al., 2009). Protein secondary structures were predicted using PSIPRED
(Bryson et al., 2005). The cis-elements in the promoter sequences were
identified using the PLACE database (Higo et al., 1999). All these databases
were queried at the default parameters.
Phylogenetic Analysis
Multiple alignments for Med proteins were generated on full-length amino
acid sequences using the MAFFT iterative refinement method (1,000 iterations)
Genome-Wide Investigations on the Plant Mediator Complex
Plant Physiol. Vol. 157, 2011 1623
Page 15
incorporating local pairwise alignment (Katoh et al., 2005). The alignments
were corrected manually, and a PHYLIP neighbor tree with 1,000 replicates
defined as the bootstrap value was built using an online tool (http://bioweb2.
pasteur.fr/phylogeny/). The tree was viewed in FigTree version 1.2.3 (avail-
able at http://tree.bio.ed.ac.uk/software/figtree/).
Microarray Data Analysis
Microarray analysis was performed on expression data obtained in our
laboratory previously (Agarwal et al., 2007; Arora et al., 2007) on Affymetrix
GeneChip Rice Genome Arrays representing 49,824 rice transcripts for three
vegetative stages (mature leaf, 7-d-old seedling, and its root), six panicle
development stages (all stages collected at a particular length [in cm] of
panicles as follows: P1, 0–3; P2, 3–5; P3, 5–10; P4, 10–15; P5, 15–22; P6, 22–30),
five seed development stages (S1, 0–2 d after pollination [DAP]; S2, 3–4 DAP;
S3, 5–10 DAP; S4, 11–20 DAP; S5, 21–29 DAP), and three types of stress (cold,
salt, and desiccation) in rice. The raw data (*.cel) files (platform accession no.
GPL2025 under the series accession nos. GSE6893 and GSE6901, deposited at
the Gene Expression Omnibus database of the NCBI) were imported to
GeneSpring GX 10 software (Agilent Technologies) for detailed analysis. The
redundancy in the probe sets was taken care of by preferring the 3#-most
probe set for each gene (R. Sharma, P. Agarwal, S. Ray, P. Deveshwar, P.
Sharma, N. Sharma, A. Nijhawan, M. Jain, A.K. Singh, V.P. Singh, J.P. Khurana,
A.K. Tyagi, and S. Kapoor, unpublished data). Normalization of the raw data
was performed using the Gene Chip Robust Multiarray Analysis algorithm
(Wu et al., 2003), data were baseline transformed, following which signal
intensity values were log transformed and averages of the three biological
replicates for each sample were calculated. One-way ANOVA statistical
analysis was used to perform differential expression analysis by taking
vegetative controls as reference to identify genes expressing at greater than
2-fold in different stages of reproductive development (panicle and seed),
with P # 0.05. Similarly, for identifying differentially expressing stress-
induced genes, analysis was performed by taking the untreated 7-d-old
seedling as the reference.
The expression data for Arabidopsis was obtained from the Gene Expres-
sion Omnibus under the series accession numbers GSE5620, GSE5621,
GSE5623, GSE5624, GSE5629, GSE5630, GSE5631, GSE5632, and GSE5634.
These series represent Affymetrix GeneChip ATH1 Genome Arrays of stages
comparable to those used for rice. A total of 55 *.cel files representing 21 stages
of development as well as stress treatments were analyzed as described above
for rice followed by generating heat maps for all/selected genes.
QPCR Analysis
QPCR was performed using three biological replicates. Each biological
replicate in turn was assessed using three technical replicates. Primers were
designed from the 3# end of the selected genes using Primer Exp ress (Applied
Biosystems) at its default settings and checked for specificity by performing
BLAST at the NCBI with other regions of the genome. DNase (Fermentas Life
Sciences)-treated RNA was reverse transcribed to synthesize the first strand-
cDNA (cDNA Archive kit; Applied Biosystems). SYBR Green PCR Master Mix
(Applied Biosystems) was used to estimate the expression levels for the
selected genes using the ABI Prism 7000 Sequence Detection System (Step One
Plus; Applied Biosystems). The expression obtained using the ACTIN gene
was used as the endogenous control to normalize the variation among
different samples using cycle threshold (Ct value) as a discrimina ting criteria.
The data were further normalized against the maximum average expression
values obtained from the microarray.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogenetic relationship of Med subunits for
the middle and tail modules.
Supplemental Figure S2. Phylogenetic relationship of Med subunits for
the kinase and unknown modules.
Supplemental Figure S3. Amino acid alignments of Med7N and Med31C
for plant, yeast, and human Med sequences.
Supplemental Figure S4. The conserved Med15 KIX region.
Supplemental Figure S5. Defining the HH regions for a Med subunit.
Supplemental Table S1. Occurrence of the HH regions and Pfam domains
in various Med gene families.
Supplemental Table S2. PSI-BLAST result of plant Med32, jump-started
using Med2/Med29 aligned sequences.
Supplemental Table S3. List of Med genes with no definitive annota tion in
respective databases.
Supplemental Table S4. Differential expression analysis between vegeta-
tive stages, vegetative and reproductive stages, and stress conditions in
rice.
Supplemental Table S5. Differential expression analysis between vegeta-
tive stages, vegetative and reproductive stages, and stress conditions in
Arabidopsis.
Supplemental Table S6. Identification of cis-elements in promoter regions
of selected genes differentially expressing in reproductive stages in rice
and Arabidopsis as well as under stress conditions in rice.
Supplemental Table S7. Normalized expression values obtained for all
rice Med genes as revealed by microarray analysis.
Supplemental Table S8. Normalized expression values obtained for all
Arabidopsis Med genes as revealed by microarray analysis.
Supplemental Table S9. Differential expression analysis between all the
reproductive stages in rice.
Supplemental Table S10. Differential expression analysis between all the
reproductive stages in Arabidopsis.
Supplemental Table S11. Database sources of proteins for different
organisms.
Supplemental Table S12. List of mediators from the NCBI used to build
HMM profiles.
Supplemental Table S13. List of organisms and their abbreviations
belonging to different groups.
Supplemental Table S14. List of putative plant mediators identified in this
study.
ACKNOWLEDGMENTS
Dr. Ramesh Hariharan of Strand Life Sciences, India, and Dr. Sanjeev
Singh of Institute of the Informatics and Communication, University of Delhi
South Campus, India, are thanked for useful discussions.
Received October 1, 2011; accepted October 20, 2011; published October 21,
2011.
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Plant Physiol. Vol. 157, 2011 1627
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    • "Apart from this, 115 RNA helicases and 31 DNA helicases have also been identified (). Rice genome has been found to code for 51 Mediator complex protein coding genes, which include all subunits identified so far in various organisms (Mathur et al. 2011 ). Eleven regulators belonging to various families have the KIX domain, which is responsible for protein-protein interactions (Thakur et al. 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Rice is one of the main pillars of food security in India. Its improvement for higher yield in sustainable agriculture system is also vital to provide energy and nutritional needs of growing world population, expected to reach more than 9 billion by 2050. The high quality genome sequence of rice has provided a rich resource to mine information about diversity of genes and alleles which can contribute to improvement of useful agronomic traits. Defining the function of each gene and regulatory element of rice remains a challenge for the rice community in the coming years. Subsequent to participation in IRGSP, India has continued to contribute in the areas of diversity analysis, transcriptomics, functional genomics, marker development, QTL mapping and molecular breeding, through national and multi-national research programs. These efforts have helped generate resources for rice improvement, some of which have already been deployed to mitigate loss due to environmental stress and pathogens. With renewed efforts, Indian researchers are making new strides, along with the international scientific community, in both basic research and realization of its translational impact.
    Full-text · Article · Dec 2016 · Rice
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    • "The same observations apply to the increased weight, not always observed in s1 progeny grown in control conditions, as reported in the present work; also in such a case, subtle differences among experimental conditions which might override parental effects. Arabidopsis thaliana AtTFIIS-like is one of the putative proteins of subunit 26 of the Mediator complex, i.e., MED26_3 (Mathur et al., 2011); although its expression is only slightly induced by Fe deficiency, nevertheless the expression of MED16, another subunit of Mediator which is undoubtedly involved in the regulation of the Fe deficiency response (Yang et al., 2014; Zhang et al., 2014), does not change at all under Fe deficiency, making the observed change in AtTFIIS-like gene expression, profoundly relevant. The reported data, highlighting the involvement of the TFIIS-like gene in the plant response to Fe-deficiency stress, provide for the first time evidence of the role played in this context by the TC-NER sub-pathway. "
    [Show abstract] [Hide abstract] ABSTRACT: We investigated the existence of the transgenerational memory of iron (Fe) deficiency stress, in Arabidopsis thaliana. Plants were grown under Fe deficiency/sufficiency, and so were their offspring. The frequency of somatic homologous recombination (SHR) events, of DNA strand breaks as well as the expression of the transcription elongation factor TFIIS-like gene increase when plants are grown under Fe deficiency. However, SHR frequency, DNA strand break events, and TFIIS-like gene expression do not increase further when plants are grown for more than one generation under the same stress, and furthermore, they decrease back to control values within two succeeding generations grown under control conditions, regardless of the Fe deficiency stress history of the mother plants. Seedlings produced from plants grown under Fe deficiency evolve more oxygen than control seedlings, when grown under Fe sufficiency: however, this trait is not associated with any change in the protein profile of the photosynthetic apparatus and is not transmitted to more than one generation. Lastly, plants grown for multiple generations under Fe deficiency produce seeds with greater longevity: however, this trait is not inherited in offspring generations unexposed to stress. These findings suggest the existence of multiple-step control of mechanisms to prevent a genuine and stable transgenerational transmission of Fe deficiency stress memory, with the tightest control on DNA integrity.
    Full-text · Article · Sep 2015 · Frontiers in Plant Science
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    • "This proves unequivocally that Mediator constitutes important part of the basal transcriptional machinery. However, drastic morphological changes in mutants of individual Mediator subunits suggest that Mediator could also act as selective gene regulator both in metazoans and plants (Malik and Roeder, 2010; Taatjes, 2010; Kidd et al., 2011; Mathur et al., 2011; An and Mou, 2013; Poss et al., 2013; Allen and Taatjes, 2015). As the present review is plant specific, the following is an account and critical analyses of important functions of Mediator subunits reported from different plant species through mutational and genome-wide transcriptom analyses (Tables 1 "
    [Show abstract] [Hide abstract] ABSTRACT: Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.
    Full-text · Article · Sep 2015 · Frontiers in Plant Science
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