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Satoh et al. Ramosa 3 trehalose.
Supplementary Figures and Legends
Supplementary Figure 1. Positional cloning of RA3.
a, Markers for fine mapping of RA3 on BAC c0387K01 are shown as black dots, with
number of recombinants marked. The adjacent gene, SRA, is a homolog of RA3. b, RA3
gene structure with predicted transcription start (arrow) and stop (vertical line), introns
(horizontal lines) and exons (boxes) marked. Grey boxes = TPP domain, black boxes =
phosphatase boxes. c, RT-PCR of mRNAs from 1cm long ears of wild-type (1), ra3-ref
(2), ra3-fea1 (3), ra3-EV (4), ra3-NI (5), ra3-bre (6). The UBI control shows roughly
equal amplification in all samples.
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Satoh et al. Ramosa 3 trehalose.
Supplementary Figure 2. Phylogenetic analysis of selected TPP genes.
The best phylogenetic trees estimate that the duplication of RA3/SRA occurred near the
base of the major diversification of the grasses. None of the topologies in the 95%
credible set of trees satisfied the constraint of a RA3/SRA duplication at the base of the
panicoid clade, or at the base of the centothecoid + panicoid clade, allowing us to reject
these hypotheses. In contrast, 25 of the 554 topologies in the 95% credible set estimated
the RA3/SRA duplication occurred prior to the chloridoid + centothecoid + panicoid clade,
indicating that we are unable to reject the hypothesis that the RA3/SRA duplication
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Satoh et al. Ramosa 3 trehalose.
occurred near the base of the PACCAD clade29.
Numbers above or below braches represent percent clade credibility. Species
abbreviations are: Bacteria: Pca = Pelobacter cabinolicus, No = Nitrosococcus oceani, Rf
= Rhodoferax ferrireducens. Insect: Dm = Drosophila melanogaster. Eudicots: At =
Arabidopsis thaliana, Br = Brassica rapa, Nt = Nicotiana tabacum. Grasses: As = Avena
strigosa, Cl = Chasmanthium latifoium, Et = Eragrostis tef, Hv = Hordeum vulgare, Os
= Oryza sativa, Pc = Phalaris canariensis, Pg = Pennisetum glaucum, Sb = Sorghum
bicolor, Sv = Setaria viridis, Zm = Zea mays. Because the maize genes are not yet
annotated we named them “ZmRA3 Like” (“ZmRA3L”).
These
sequences
correspond
to
Genbank
numbers
except
for
AZM
from
http://maize.tigr.org/ RfTPP: NZ_AAJK01000029, PcaTPP: NC_007498, NoTPP:
NC_007484, DmTPS1: NM_134983, At1g22210: NM_102071, AtTPPB: NM_106458,
OsRA3L1: AC103551, ZmRA3L1: AZM4_101301, AsSRA: DQ436922, HvSRA:
DQ436929, OsSRA: AK109902, SbSRA: DQ436924, ZmSRA: DQ436921, ClSRA:
DQ436926, EtSRA: DQ436927, ZmRA3: DQ436920, SbRA3: ASB1339, SvRA3:
DQ436925, OsRA3L2: AP004727, OsRA3L3: XM_467838, PgRA3L3: DQ436923,
OsRA3L4: XM_450681, EtRA3L4: DQ436928, PcRA3L4: DQ436930, OsRA3L5:
XM_482349, At1g35910: NM_103289, At4g39770: BT005555, At2g22190: BT010648,
At5g65200: BT015352, BrTPP1: AC146875, BrTPP2: AC155344, At5g10100:
NM_121048, AtTPPA: BT008467, At4g22590: NM_118385, At4g12430: BT002566,
NtTPP:
AY570725,
OsTPP1:
NM_197683,
XM_473444.
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OsTPP2:
XM_467162,
OsTPP3:
Satoh et al. Ramosa 3 trehalose.
Supplementary Figure 3. Expression of the rice SRA gene measured by RT-PCR.
RT-PCR of mRNAs from leaf blade (B), root (R), leaf sheath containing vegetative apex
(V) and young inflorescence (I). The rice SRA gene is expressed strongly in root and
young inflorescence and it is also detected in vegetative tissues (upper panel). The rice
UBI positive control (lower panel) shows roughly equal amplification in all samples.
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Satoh et al. Ramosa 3 trehalose.
4
3.5
3
2.5
2
1.5
1
0.5
0
T6P
G6P
S6P
F6P
Ser/Thr
-0.5
Supplementary Figure 4a. RA3 has trehalose phosphate phosphatase activity.
The chart shows phosphate release,
following treatment of various phosphorylated
substrates. RA3 full length protein or the TPP domain alone, and Mycobacterium TPP30,
specifically catalyzed dephosphorylation of T6P (trehalose-6-phosphate), with no activity
on G6P, S6P or F6P (glucose, sucrose or fructose-6-phosphates), nor on a Ser/Thr
(serine/threonine phosphopeptide) as a reporter of protein phosphatase activity (Promega).
The N-terminal region of RA3 had no enzyme activity and the positive control SAP
(shrimp alkaline phosphatase) showed phosphatase activity against all substrates.
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Satoh et al. Ramosa 3 trehalose.
Supplementary Figure 4b. RA3 complements a yeast TPP mutant.
RA3 full length protein or TPP domain of RA3 rescued the growth of a yeast TPP mutant
at the non-permissive temperature in the presence of 1M NaCl.
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Satoh et al. Ramosa 3 trehalose.
Supplementary methods
Phylogenetic analysis
Total RNA was extracted from 1-10 mm long developing inflorescences of barley
(Hordeum vulgare), green millet (Setaria viridis), inland sea oats (Chasmanthium
latifolium), oats (Avena strigosa), tef (Eragrostis tef) and sorghum (Sorghum bicolor)
using RNAwiz (Ambion, Austin, TX). RA3/SRA genes were amplified using one-step
RT-PCR
(Invitrogen,
Carlsbad,
CA)
using
either
(ATGACGAASCACGCCGSCTWCKCC)
and
(STCWCKCAGMGWGTAGGATGC)
or
(AGCAGATCGTCATGTTCCTGGACTA)
and
the
primers
RA3-1F
RA3-1015R
SRA-293F
SRA-1042R
(CCAACTTGCGCAGGAACT) designed using Primaclade31. Amplified fragments were
cleaned, sub-cloned and sequenced as described32. 43 TPP gene nucleotide sequences
were aligned using MacClade 4.033 and ClustalX34 using the amino acid translation as a
guide. Bayesian phylogenetic estimates were produced using the General Time Reversal
model with some invariant sites and gamma distributed rates (GTR + I + G) based on two
separate runs of 10 million Markov Chain Monte Carlo (MCMC) generations using
MrBayes 3.121. Trees were sampled every 500 generations and burn-in was determined
empirically by plotting likelihood score against generation number. After burn-in trees
had been removed, clade credibility values (CC) and the 95% credible set of trees were
estimated using MrBayes 3.121. The 95% set of credible trees contained 554 different
topologies. Constraint trees reflecting different SRA/RA3 duplication scenarios were
generated in MacClade34. The set of 95% credible trees were then filtered using these
constraint trees within PAUP*4.035.
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Satoh et al. Ramosa 3 trehalose.
Molecular biology
For
RT-PCR
in
Supplementary
figure
3,
the
primers
NS466
(GGTTGATTTTGGGGGATGAC) and NS469 (TACTTGCCAATCAGCCCTTTG), UBI
RTF1
(CTGATCTTCGCTGGCAAGCAG)
and
UBI
RTR1
(CATATCATACGGACTCGATGGTCCA) were used to amplify the rice SRA and UBI
genes respectively.
Protein expression and phosphatase asssay
RA3 full length cDNA or a cDNA of the TPP domain were ampified using primers NS487
(CGAGCCATGACGAAGCAC) and NS429 (ATAAGCGCCTCTTTGCTGTTG or
NS483 (GGCGACTGGATGGAGAAGCA) and NS429, and cloned into the vector pCR
T7/NT-TOPO containing an N-terminal histidine tag (Invitrogen). Recombinant proteins
were expressed in E. coli and purified by a batch purification method (Qiagen). The
proteins were used at a concentration of 70ng/
phosphopeptide
as
described23.
Phosphate
release
was
measured
as
OD600
(serine/threonine phosphatase assay system, Promega).
Yeast complementation test
RA3 full length cDNA or a cDNA of the TPP domain were amplified using primers
NS489 (AAGGAAAAAAGCGGCCGCGCCATGACGAAGCACGCCGCCTACTC) and
NS490
NS490
(ACGAGGTCGTGCCTGCCGCTCATGGTTGGCGCGCCCCCTTCT)
and
(CGCGCCGCCGGCGGCCGCGACATGGACTGGATGGAGAAGCACCCGTC)
or
NS500
and
cloned into a yeast shuttle vector, pFL61, which drives strong expression from the
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Satoh et al. Ramosa 3 trehalose.
phosphoglycerate kinase promoter36. The Arabidopsis TPPB gene was used as a positive
control. The constructs were transformed into the yeast strain YSH6.106.-8C, which has a
deletion of the TPS2 gene and hence lacks TPP activity. This mutant strain is sensitive to
high temperature and salt concentrations24, so we assayed for growth on selective media
at 40.5oC, the non-permissive temperature, in the presence of 1M NaCl.
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Satoh et al. Ramosa 3 trehalose.
Supplementary Tables
Supplementary Table1. Molecular markers used for RA3 mapping (Fig. 2).
Marker
name
og.1
Nature of marker
Sequence
indel marker
cb. g1E
CAPS marker with Mse1
NS362
indel marker
NS364
indel marker
NS346
indel marker
cb. e
RFLP marker with BamH1
GAGCGGGAGGTGTAGAGACAAG and
CCAGCTTCACGAAGTTGAACAC
GTGCTGTGGTCCTTCAATTCTG and
GCGCAGATCACGGCTCCCAT
ACGTGTAGCCACACACAGTCG and
GCAACGACATACACCACGAGA
ATGCACGAGGTCGTCTTCATT and
GAGATTCGTCCGTCCATCAAG
AAGCAGCCCAAAGAACAACAA and
CTTTTGCATCGGGAAGAAGTG
Probe was made with primers
CCCAGAGCATCCATAACTCTGAT and
AGTCTCCTTTTTCGACGATGTTG
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Satoh et al. Ramosa 3 trehalose.
Supplementary Table2. Molecular lesions in ra3 alleles.
Allele
Source
ra3-ref
Maize Genetics Stock Center
ra3-fea1 Mu transposon stock (D.
Jackson)
ra3-EV
Ac-Ds stock (E. Vollbrecht)
ra3-NI
ra3-bre
EMS screen (N. Inada)
Unknown (E. Irish)
ra3-JL
Mu transposon screen
ra3-NS
Spm transposon screen
Lesion
4bp insertion (AGCT) in exon 7 after nt 743
~2kb ILS-like transposon37 between TATA
box and exon1 and 4bp insertion (CCGG)
into exon 7 after nt 769
4bp insertion (GCGC) into exon 6 after nt
663
~150bp insertion* into exon 10 after nt 995
10bp (ATATATATAT) insertion into exon 6
after nt 617
42bp deletion and 15bp insertion
(GCCATCGCCTGCGTCCACTTCCGCT)
in exon 6 after nt 649
2bp insertion (CA) into exon 6 after nt 662
Nucleotide (nt) numbers are counted from the predicted ATG.
*GTTTGTTTGGATAATCTCGGATTGAAATGGATTGAGGTGGTATGTGGTGT
ATTGAAGTGTAATATGAACTAATTTTTTTTCAATCCCCTTCAATACACCTTA
ATACACTTCAATCCCTCTGTACCCAAACAAAG; similar to repetitive sequence
associated with the B transcriptional activator b1-B' allele, (genbank # AY078063) and the
sucrose synthase (shrunken1) gene (genbank # AF544125).
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Satoh et al. Ramosa 3 trehalose.
Supplementary notes
29.
Grass Phylogeny Working Group. Phylogeny and subfamilial classification of the
grasses (Poaceae). Ann. Mo. Bot. Gard. 88, 373-457 (2001).
30.
Edavana, V. K. et al. Cloning and expression of the trehalose-phosphate
phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from
Mycobacterium smegmatis. Arch Biochem Biophys 426, 250-257 (2004).
31.
Gadberry, M. D., Malcomber, S. T., Doust, A. N. & Kellogg, E. A. Primaclade - a
flexible tool to find conserved PCR primers across multiple species.
Bioinformatics 21, 1263-1264 (2005).
32.
Malcomber, S. T. & Kellogg, E. A. Heterogeneous expression patterns and
separate roles of the SEPALLATA gene LEAFY HULL STERILE1 in grasses. Plant
Cell 16, 1692-706 (2004).
33.
Maddison, W. P. & Maddison, D. R. MacClade: Analysis of Phylogeny and
Character Evolution. (Sinauer Associates, Sunderland, 2003).
34.
Jeanmougin, F. J., Thompson, D., Gouy, M., Higgins, D. G. & Gibson, T. J.
Multiple sequence alignment with Clustal X. Trends in Biochem. Sciences 23,
403-405 (1998).
35.
Swofford, D. L. PAUP*: Phylogenetic Analysis Using Parsimony. (Sinauer
Associates, Sunderland, 2000).
36.
Minet, M., Dufour, M. E. & Lacroute, F. Complementation of Saccharomyces
cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J 2,
417-422 (1992).
37.
Alrefai,R., Orozco,B. and Rocheford,T. Detection and sequencing of the
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Satoh et al. Ramosa 3 trehalose.
transposable element ILS-1 in the Illinois long-term selection maize strains. Plant
Physiol. 106, 803-804 (1994).
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