Methods and Materials
Fourier transform infrared spectroscopic analysis
For Fourier Transform Infrared analysis (FT-IR), pre-cleaned by solvent extractions and
dried plant stems were pooled and homogenized by ball milling. The powder was dried at
30°C overnight, mixed with KBr, and pressed into 13-mm pellets. Fifteen FT-IR spectra
for each line were collected on a Thermo Nicolet Nexus 470 spectrometer
(ThermoElectric Corporation, Chicago, IL) over the range of 4,000–400 cm-1. For each
spectrum, 32 scans were averaged at a resolution of 8 cm-1 for Fourier transform
processing and absorbance spectrum calculation by using OMNIC software (Thermo
Nicolet, Madison, WI). Spectra were corrected for background by automatic subtraction
and saved in JCAMP.DX format for further analysis. Using Win-DAS software (Wiley,
New York), spectra were baseline-corrected and were normalized and analyzed by using
the principal component (PC) analysis covariance matrix method (McCann et al., 1997).
Allelism test between ugt80B1 and tt15
ugt80B1 was crossed to tt15 and 100 F1 heterozygous seeds were generated. None of the
doubly heterozygous seeds displayed a visible increase in seed color hue from either the
parent alleles. This suggests that, on the basis of seed coat hue, the ugt80A1 and tt15
mutations are allelic (confirmed based on a personal communication by Isabelle
Debeaujon).
Electrolyte leakage analysis was determined as the ratio of conductivity measured in the
water before and after boiling the samples, and was achieved using a HI8820N
conductivity meter (Hanna Instruments, Kehl, Germany) by the methods of Rohde et al.
(2004). Tissue used for electrolyte leakage was rosette leaves and 12-36 plants were
analyzed from both the mutant and wild-type plants.
Generation
of
UGT80B1
and
UGT80A2
promoter
GUS
fusions
and
complementation of ugt80B1. Transgenic plants containing the genomic DNA
sequences 5’ of the UGT80B1 and UGT80A2 coding sequences fused to the Escherichia
coli β-glucuronidase (GUS) coding sequence were obtained as follows. The promoters
were amplified from genomic DNA with Expand High Fidelity (Roche Molecular
Biochemicals) by using primer pairs GGCCGCCCCGGGtttggtcccgtaatgtagca and
GGCCGCGTCGACaaaactgaattcaactaaacagctctc
for
UGT80B1
(At1g43620)
GGCCGCGGATCCccaaggcaagcttcatcata
and
and
GGCCGCCTGCAGccagaagatgaaaagatcgaaaa for UGT80A2 (At3g07080). The 2,170-bp
amplified fragment for UGT80B1 was cloned into pGEMT-easy (Promega), sequenced
and subcloned into pCAMBIA1305.1 as a Xma1/Sal1 fragment. The purified 1,441-bp
fragment of UGT80A2 was cloned into pGEMT-easy (Promega) and subcloned as a
BamH1/Pst1 fragment into pCAMBIA 1305.1. Both constructs were introduced into
Agrobacterium tumefaciens (with gentamicin, rifampicin, and kanamycin selection) and
introduced into Col-0 Arabidopsis plants using a standard transformation protocol
(Clough and Bent, 1998). The open reading frame for UGT80B1 was amplified by
reverse transcription polymerase chain reaction from mRNA extracted from Arabidopsis
seedlings using a commercially available kit (QIAGEN) and Gateway compatible
primers: ggggacaagtttgtacaaaaaagcaggcttcATGGCTAGTAATGTATTTGATCATCC and
ggggaccactttgtacaagaaagctgggtCACGCCACCACATGGAAGACAACACT.
The
amplified purified product was cloned directly into the pDONR vector via BP clonase
reaction (Invitrogen, Carlsbad, CA) prior to being sub-cloned into the destination vector
pMDC32 (Curtis and Grossniklaus, 2003)(35S:UGT80B1). The construct was sequenced
using three nested primers CACTGTCCCGTCATTTTG, GTGCCATTCTTTGGGGAT,
CGATGTGCAGCCTTTTCT.
The
destination
construct
was
transformed
by
electroporation into Agrobacterium tumefaciens, which was used to transform ugt80B1
mutant plants for complementation analysis and protein localization studies. Transgenic
plants were identified by hygromycin resistance, rescue of transparent testa phenotype of
the ugt80B1, and PCR confirmation.
-Glucuronidase (GUS) staining
Arabidopsis seedlings and whole plants expressing the UGT80A2 or UGT80B1 GUS
reporter constructs were harvested and treated as described previously (Bergmann et al.,
2004). GUS activities were visualized after incubating the seedlings with the substrate XGluc for 3-13 hours.
Table S1. SG and ASG levels are reduced in ugt80A2 and ugt80B1 mutants.
Characterization of the sterol and sterol derivative profile in wild-type and ugt80A2,
ugt80B1 and ugt80A2,B1 mutants. SG and ASG are reduced in ugt80A2 and ugt80B1 and
highly reduced in the ugt80A2,B1 double mutant. Sterol profiles in wild-type and mutants
are similar except that the levels of cycloartenol and cholesterol appeared to decrease,
while isofucosterol appeared to increase in the ugt80A2, ugt80B1 and ugt80A2,B1
mutants. The data represent two independent experimental analyses.
WT
Rosette leaf
Stem
Inflorescence
WT
Rosette leaf
Stem
Inflorescence
(SG+ASG)/FS (%)
ugt80A2 ugt80B1
15
6
5
18
6
11
17
7
4
ugt80A2,B1
2
8
8
(ASG)/SE (%)
ugt80A2 ugt80B1
41
8
7
43
8
9
9
4
2
ugt80A2,B1
10
5
1
FS= free sterols, SE= steryl esters, SG= steryl glycosides, ASG= acyl steryl
glycosides
0.7
2.3
1.5
18
1
74
1
WT
S
4
4
16.5
1
trace
16
1
57
0.5
IN + SQ
3.5
3.5
7
1.5
2
14
trace
67
1.5
L
2
1.2
2
2
2
15
4
70
2
100
100
100
100
Sterol (%)
Pentacyclic triterpenes
Cycloartenol
Cholesterol
Brassicasterol
24-methylene cholesterol
Campesterol
Isofucosterol
Sitosterol
Stigmesterol
L
1.5
Total
ugt80A2
S
3
1
6
2.5
1
23
2
62
1
100
IN + SQ
3
0.5
4
1.5
2
18
4
68
1
L
1.5
0.1
1.6
1.6
1.7
16
4.5
72
1
100
100
ugt80B1
S
1
1
5
2
1
24
1
64
1
100
IN + SQ
1
0.5
2
1.5
2
18
4
70
1
L
1
trace
1
2
3
17
3.5
71
1.5
100
100
ugt80A2,B1
S
IN + SQ
0.5
0.3
1
trace
3
2
2.5
2
1
4
24
18
1
5
66
67
1
1.7
100
100
Supplemental Figure Legends
Fig. S1. Polymerase chain reaction genotyping of ugt80A2 and ugt80B1 mutant
alleles. (A) UGT80A2 allele-specific primers for ugt80A2 amplify 440 and 581 bp
products in mutant and wild-type, respectively. (B) UGT80B1 allele-specific primers for
ugt80B1 amplify 595 and 455 bp products in mutant and wild-type, respectively. Marker
(right lane) indicates 500 and 750 bp sizes.
Fig. S2. Growth defects of ugt80A2, ugt80B1 and ugt80A2,B1 mutant alleles. Root
lengths of ugt80A2 and ugt80B1 mutants and wild-type were measured daily while grown
on plates at 10, 15 and 22oC temperatures. The growth curves indicate that mutants, in
particular the ugt80A2,B1 double mutant exhibits a slower growth rate at all
temperatures.
Fig. S3. Electrolyte leakage in ugt80A2 and ugt80B1 mutant alleles. (A) and (B) show
results for non-acclimated plants grown in a long-day phytotron for six weeks. (A) The
ugt80A2,B1 double mutant appears slightly more cold resistant than wild-type controls
Col-0 and WS. However only three values represent a significant difference. (C) and (D)
show results for acclimated plants. Plants were grown in a long-day phytotron for six
weeks and following two more weeks in a 4°C chamber. Results indicate no apparent
difference in cold tolerance between the ugt80A2,B1 double mutant and Col-0 and WS,
respectively.
Fig.
S4.
proUGT80A2::GUS
reporter
gene.
Gene
expression
of
promotorUGT80A2::GUS. A) Pollen, scale bar = 100 m. B) Stamen, scale bar = 0.2
mm. C) Patchy distribution in around the bases of siliques, scale bar = 2 mm. D) Staining
observed around the base of floral organs, scale bar = 1 mm.
Fig.
S5.
proUGT80B1::GUS
reporter
gene.
Gene
expression
of
promotorUGT80B1::GUS was primarily in developing inflorescences, seeds and the root
size of seedlings. A and E) Stained untransformed seed and embryo. B and C) Stained
promotorUGT80B1::GUS seed after clearing with chloral hydrate solution made from an
8:3:1 mixture of chloral hydrate, H2O, and glycerol. D) Seed coat epidermal cell
boundaries stained with GUS. F, G and H) Characterization of the GUS staining pattern
in embryos revealed that expression is strongest around the apical tip of the cotyledons
and at the root apex in the promotorUGT80B1::GUS stained embryos (Scale bars = 1
mm). I) Staining pattern in the entire root and root hair of 7-day-old seedlings, scale bar =
4 mm. H) GUS expression was also apparent at the inflorescence in particular the stigma,
Scale bar = 1 mm.
Fig. S6. Seed weight experiment. Measurements of dry seed were performed in batches
of 25 seed from wild-type, ugt80A2 and ugt80B1, rescue and ugt80A2,B1 mutants and
recorded in micrograms. Standard error from the mean was calculated based on triplicate
analyses.
Fig. S7. DPBA and iodine staining of cotyledons showed altered hydathode
morphology in developing cotyledons from ugt80B1 and ugt80A2,B1 mutants. Dark
grown Arabidopsis seedlings were stained with a DPBA solution and imaged under UV
illumination. A) The staining pattern for wild-type, ugt80A2, ugt80B1 and ugt80A2,B1
seedlings showed that wild-type and ugt80A2 cotyledons display small zone of blue
coloration (sinapate and derivatives) proximal to the hydathode whereas ugt80B1 and
ugt80A2,B1 displayed a marked increase in the zone of blue coloration that was capable
of encompassing half the cotyledon. B) Starch accumulation was stained using iodine and
visualized as brown coloration using light microscopy. Iodine staining of cotyledons
showed no brown coloration of wild-type seedlings. In contrast, the hydathode region of
ugt80A2,B1 was stained by iodine. Scale bar = 1 mm unless indicated otherwise within
the panel.
References
Bergmann DC, Lukowitz W, Somerville CR (2004) Stomatal Development and Pattern
Controlled by a MAPKK Kinase Science 304, 1494-1497.
Clough SJ, Bent AF (1998) Simplified Arabidopsis Transformation Protocol Plant J. 16,
735-743.
Curtis MD, Grossniklaus U (2003) gateway cloning vector set for high-throughput
functional analysis of genes in planta. Plant Physiol. 133, 462-469.
McCann M, Chen L, Roberts K, Kemsley E, Sene C, Carpita N, Stacey N, Wilson R
(1997) Infrared microspectroscopy: Sampling heterogeneity in plant cell wall
composition and architecture. Physiol Plant. 100, 729-738.
Rohde P, Hincha DK, Heyer AG (2004) Heterosis in the freezing tolerance of crosses
between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show
differences in non-acclimated and acclimated freezing tolerance. Plant J. 38, 790799