The StBEL family of transcription factors in potato
KNOX and BEL1-like proteins are two large families of transcription factors in the three
amino- acid loop extension (TALE) superclass (Burglin, et al., 1997). POTH1 (potato
homeobox 1) is a member of the knotted1-like family of homeobox genes. During tuber
formation, POTH1 plays a positive role by repressing the level of GA in stolon tips
(Rosin, et al., 2003). Using the yeast two-hybrid system with POTH1 as the bait, seven
distinct BEL1-like proteins (Fig. 1) were isolated from cDNA libraries of potato (Chen, et
al., 2003).
StBEL genes (13 genes, two are inactive). Based on the whole genome sequence of
Solanum phureja in Potato Genome Sequencing Consortium Data, 11 BAC clones were
identified with especially low E-values (a good match), using 269 nucleotides from the
conserved BELL domain and homeodomain (Fig. 1) of StBEL5 as the query. By HMMBEL1-Like Proteins of Potato
based gene structure prediction on SoftBerry website, five new StBEL genes were
identified. These are designated StBEL6, -15, -31, -32, -33 and -34. (Fig. 2, StBEL6 is
not shown here).
Fig. 1 Common structure of BEL1-like proteins
Figure 3. A, Schematic of the amino acid sequence of the BEL1-like proteins of potato. Boxed regions represent conserved
sequences identified by aligning all seven BELs. Helices I to III of the homeodomain are designated. The Pro-Tyr-Pro (PYP)
loop extension is located between helices I and II. For clarity in labeling, the sequence is not drawn to scale. B, Predicted
helices of the putative protein-binding region (BELL domain) of the BEL1 protein StBEL5. The bold letters represent amino
acids conserved in other plant BEL1 proteins based on a BLAST analysis of StBEL5. The underlined portion of the sequence
represents a predicted "-helix. A consensus for the prediction of the sequence structure was derived by using three software
programs for amino acid sequence analysis: sspal, ssp, and nnssp (http://www.softberry.com/berry.phtml?topic"protein).
Four deletion constructs from Figure 5B are designated with arrows. Construct pAD5-1 contains amino acids 230 through
653 of pAD-05 (interaction with POTH1), and pAD5-2 contains amino acids 257 through 653 of pAD-05 (no interaction).
Fig. 2 Phylogenetic
tree of
the acids
BEL1-like
proteins
Solanum
POTH1
is aconsists
member
of the acids
Construct
pAD5-11 consists
of all
amino
1 through
286 ofinpAD-05
(notuberosum.
interaction), and
pAD5-9
of amino
I KNOTTED1-like
homeobox
1Class
through
315 (interaction with
POTH1).genes. POTH1 gives the tree a root. No ESTs were identified for BEL15
and -31.
1. StBEL15
(on St.AC.001.Scaffold000095):
Determining
the Protein-Binding
Regions in
the pGAD vector, no interaction was observed with
POTH1 and
the
BEL1-Like
Proteins
in pBridge.
StBEL15 is phylogenetically close to StBEL14, StBEL5
with 49.5%
similarity.
Fusion constructs of StBEL5 that dissected the 120Interaction
with is
StBEL5
wasfrom
observed
all de-StBELs
StBEL15
different
otherwith
identified
in the
conserved
domains.
amino
acid
domain (pAD5-2,
-3,First,
-4, -9, and -11) were
letions outside the KNOX domain, with pBHD2
tested
because
thisatisthe
one
of the regions
that is conStBEL15
does
not
have
the
“SKY”
box
(Fig.1),
with
an
arginine
position
for lysine.
(missing the amino terminus and the first 48 amino
served
in
BEL
TFs
from
other
plant
species
(Bellaoui
StBEL15
lost
thewith
thirdpBHD6
helix in the homeodomain. The third helix of the
acids of Second,
the KNOX
domain,has
Fig.
5A),
et
al.,
2001;
Fig.
3B).
Interaction
with
POTH1
was
contacts
DNA,
andofis highly conserved among all the homeobox
(missinghomeodomain
the carboxy terminus
andtarget
52 amino
acids
observed with all constructs that had deletions excluthe carboxy end of the KNOX domain), and with
sively outside of the conserved 120-amino acid box
pBHD-9 (first amino-terminal 114 amino acids but no
(Fig. 5B). The only exception to this was with
KNOX domain sequence). No interaction was obpAD5-9, which demonstrated an interaction and inserved with pBHD3 (missing all of the KNOX docluded a 43-amino acid deletion from the carboxy
main and the first 114 amino acids). Control experiend of the 120-amino acid domain. Even with as little
ments identified the first 114 amino acids of the N
as a 27-amino acid deletion from the amino end of the
terminus (pBHD9) as a transcriptional activator. This
120-amino acid domain, interaction did not occur
construct transformed alone into AH109 exhibited
proteins. StBEL15 does not contain the VSLTLGL box either, normally present in the Ctermini of other StBELs.
Besides these changes in the sequence of StBEL15, there are no EST matches with it in
the SolEST database. These observations suggest that StBEL15 has lost its function
during genome evolution.
2. StBEL31 and StBEL32 (on St.AC.001.Scaffold000399):
StBEL31 and StBEL32 are spatially close to each other on the same chromosome, with
only 2.2 kb between their respective coding sequences. StBEL31 and StBEL32 are
phylogenetically close to StBEL30, with 61.6% and 65.2% similarity respectively.
StBEL32 has a similar primary structure to other StBELs, conserved in all the 4
important domains. Blasted in EST database, StBEL32 gets identical matches, indicating
that StBEL32 is expressed in potato.
Although the protein sequence of StBEL31 is 60% similar to that of StBEL32, StBEL31 is
a truncated form of a StBEL gene, missing the VSLTLGL sequence just downstream of
Proteins of Potato
the homeobox (Fig. 3). Since there is no match for StBEL31 in the EST database,BEL1-Like
it is
possible that StBEL31 is either nonfunctional or that its mRNA is present at very low
levels.
Fig. 3 Protein structure of StBEL31
In the genome sequence, StBEL31 is contained in an ORF of a 12.6 kb region, including
23 exons. In the 23 exons, the first five of them encode StBEL31, while the following 14
encode a putative GTP-binding protein, and the last four of them encode a putative
peroxidase (Fig. 4). These last two parts have perfect matches respectively in the EST
database, meaning that there are two separate proteins encoded by this ORF expressed
in potato. The structure of this ORF is like an operon in prokaryotes, which is a cluster of
genes operated by a single promoter. However, the three genes are not expressed in
the same way, since StBEL31 is not expressed. It’s still unknown how these three genes
are controlled separately.
Figure 3. A, Schematic of the amino acid sequence of the BEL1-like proteins of potato. Boxed regions represent conserved
sequences identified by aligning all seven BELs. Helices I to III of the homeodomain are designated. The Pro-Tyr-Pro (PYP)
loop extension is located between helices I and II. For clarity in labeling, the sequence is not drawn to scale. B, Predicted
helices of the putative protein-binding region (BELL domain) of the BEL1 protein StBEL5. The bold letters represent amino
acids conserved in other plant BEL1 proteins based on a BLAST analysis of StBEL5. The underlined portion of the sequence
represents a predicted "-helix. A consensus for the prediction of the sequence structure was derived by using three software
programs for amino acid sequence analysis: sspal, ssp, and nnssp (http://www.softberry.com/berry.phtml?topic"protein).
Four deletion constructs from Figure 5B are designated with arrows. Construct pAD5-1 contains amino acids 230 through
653 of pAD-05 (interaction with POTH1), and pAD5-2 contains amino acids 257 through 653 of pAD-05 (no interaction).
Construct pAD5-11 consists
of
amino
acids
1
through
286
of
pAD-05
(no
interaction),
and
pAD5-9
consists
of
amino
acids
Fig.4 Structure of the ORF which contains StBEL31
1 through 315 (interaction with POTH1).
StBEL33 (on St.AC.001.Scaffold000522) and StBEL34 (on St.AC.001.Scaffold000116):
- StBEL33 the
andProtein-Binding
StBEL34 haveRegions
much in
lower similarity tothe
StBEL5
StBEL15,
31 andwas
32. observed with
Determining
pGADthan
vector,
no interaction
They are
a BEL1-Like
separate Proteins
branch in the phylogenetic treeStBEL5
of all the
StBELs.
POTH1
andin
the
in pBridge.
- StBEL33 and StBEL34 are very conserved in the homeobox
97.1% that
anddissected the 120Fusion domain,
constructswith
of StBEL5
Interaction with StBEL5 was observed with all deamino
acidhave
domain
(pAD5-2,
-3,
-4, -9, and -11) were
95.7%
similarity
to
StBEL5.
While,
both
StBEL33
and
StBEL34
lost
the
SKY
box.
letions outside the KNOX domain, with pBHD2
(missing the amino terminus and the first 48 amino
acids of the KNOX domain, Fig. 5A), with pBHD6
(missing the carboxy terminus and 52 amino acids of
the carboxy end of the KNOX domain), and with
pBHD-9 (first amino-terminal 114 amino acids but no
KNOX domain sequence). No interaction was observed with pBHD3 (missing all of the KNOX do-
tested because this is one of the regions that is conserved in BEL TFs from other plant species (Bellaoui
et al., 2001; Fig. 3B). Interaction with POTH1 was
observed with all constructs that had deletions exclusively outside of the conserved 120-amino acid box
(Fig. 5B). The only exception to this was with
pAD5-9, which demonstrated an interaction and in-
- StBEL34 has extra amino acids at the end of each helix of the BELL domain compared
to StBEL5. Especially at the end of the first helix, there are 23 additional amino acids.
The similarity between BELL domains in BEL5, -33 and -34: 5 vs. 33 = 72.7%; 5 vs. 34 =
60%; 33 vs. 34 = 59.6%.
EST matches have been found for both StBEL33 and StBEL34, indicating that both
StBEL33 and StBEL34 can be expressed.
100 AtBEL10
100
AtBLH5
OFP partners
AtBLH3
80
StBEL32
StBEL30
72
AtBLH6
98
AtBLH7
99
AtBLH11
63
StBEL31
99
AtBLH8 PNF
StBEL34
Shoot Development
100
21
AtBLH9
PNY BLR
40
StBEL33
100
100
StBEL11
33
StBEL5
Tuberization
StBEL29
60
AtBLH1
77
StBEL6
Shoot Development
AtATH1
100
100
StBEL14
Ovule morphogenesis
AtBEL1
StBEL15
63
100
StBEL22
99
StBEL13
Leaf Morphogenesis
AtBLH2 SAW1
76
AtBLH4 SAW2
100
AtSTM
POTH1
100
99
0.5
Figure 5. Revised phylogenetic analysis of StBEL family in potato reflecting all thirteen genes
that have been identified. The amino acid sequence of the thirteen potato BEL-like proteins was
analyzed and compared with the thirteen BEL proteins in Arabidopsis. StBELs are represented in
bold letters. Putative functions are listed for each group. Because of their presence in the phloem sieve tube system (Fig. 6), their unique
UTR sequences and their diverse RNA accumulation patterns, the family of
BEL1-like RNAs from potato represents a valuable model for studying the longdistance transport of full-length mRNAs and their role in development. This
research will focus on two projects: 1) auto-regulation of the StBEL5 gene and 2)
long-distance transport of RNAs of StBEL13, -14 and -30.
Fig. 6. RT-PCR products from RNA extracted from either stem sap or stolon tips for several BEL1- like
RNAs (designated 5, 13, 14, 22, 29, 30 and 11). Total RNA from Phloem-enriched exudate was extracted
and used as the RT-PCR template for detecting tissue-specific transcripts. G2 is RNA for a phloem-specific
transcription factor (Zhao et al. 2005) and NT2 is a positive control for root xylem cells (Nazoa et al. 2003).
(Campbell et al., 2008).
In Arabidopsis thaliana, there are thirteen BEL1-like family members, all of which
can form heterodimers with Class 1 KNOX proteins (Hackbusch, et al., 2005;
Kumar, et al., 2007). These BEL1-like proteins have significant roles in meristem
activities and flower development, and their functions are often overlapping and
redundant. BEL1-like proteins in Solanum tuberosum have high homologous
relationship with these AtBELs (Fig. 5). StBEL13 matches to SAW1 and SAW2
best. StBEL14 is close to AtBEL1. StBEL5 is close to AtBLH1. StBEL30 is close
to AtBLH6 and AtBLH7.
ATH1 (ARABIDOPSIS THALIANA HOMEOBOX 1), PNY (PENNYWISE), and
PNF (POUNDFOOLISH) are three BEL1-like proteins in Arabidopsis. They are
critical for the initiation, maintenance and development of the shoot apical
meristem. Triple mutant of these three genes have a more severe phenotype
than the single mutants. While in flower formation, ATH1 is a negative regulator,
opposite to the role of PNY and PNF (Rutjens, et al., 2009).
SAW1 (BLH2) and SAW2 (BLH4) are negative regulators of BP
(BREVIPEDICELLUS), an important Class I KNOX gene and positive regulator
for growth. In saw1 saw2 double mutant, BP can be expressed on the margin of
leaf, and the leaf will grow into a serrated and revolute shape. Since single
mutant saw1 and saw2 have no obvious phenotype, the function of SAW1 and
SAW2 are redundant in Arabidopsis (Kumar, et al., 2007).
AtOFPs are a family of proteins that control the development of ovule in
Arabidopsis. Yeast two-hybrid screen revealed complex interaction relationships
between AtOFPs proteins and TALE family proteins. Especially, BEL1, BLH1,
SAW1, BLH3, SAW2, BLH6 and BLH10 can bind with several of the AtOFPs
proteins (Hackbusch, et al., 2005). Indeed, misexpression of BLH1 in embryo sac
will switch one of the synergid cells into an egg cell (Pagnussat, et al., 2007), and
lose-of-function of BEL1 gene will block the development of integument of ovule
(Brambilla, et al., 2007). Do the StBEL proteins also interact with OFPs in
potato?
Figure 7. Northern-blot analysis of the accumulation of mRNA for four BEL1-like cDNAs (StBEL5,
-13, -14, and -30) in potato organs. Ten micrograms of total RNA from flowers, shoot tips (SAM),
leaves, stems, roots, unswollen stolons (U stolon), swollen stolons (S stolon), and tubers was
loaded per lane. Swollen stolons represent an early stage of tuber formation. A probe for the 18S
ribosomal RNA was used to verify equal loading of RNA samples (bottom). A gene-specific probe
for each BEL cDNA was used. The length of both 5ʹ′ and 3ʹ′ untranslated sequences are indicated
for each BEL RNA.
The RNA accumulation patterns of StBEL5, -13, -14 and -30 in different organs has been
characterized by Northern blot (Fig. 7). Consistent with the function of AtBEL1-like proteins in
ovule development, mRNAs of these four genes accumulate to high levels in flowers,
indicating a potential function of StBELs in flower. StBEL5, 13, and 30 also have high mRNA
concentrations in SAM, along with the significant role of AtBELs in meristem. StBEL30 has
higher accumulation in unswollen stolons, but much lower in swollen stolons and tuber. It is
feasible to suggest that StBEL30 may be functional in the tuber induction process.
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