Post on 02-Jun-2020
transcript
Supplemental Figure 1. DEX-Inducible Overexpression of At LOG Genes and Change in Cytokinin Levels.
Transgenic Arabidopsis seedlings harboring the empty vector pTA-7001 (Control), DEXpro:LOG1, DEXpro:LOG3,
DEXpro:LOG4, DEXpro:LOG5, and DEXpro:LOG8 were grown for 17 days on MGRL-agar medium and then transplanted
to MGRL-agar medium with or without 10 �M dexamethasone (DEX). After 5 days, seedling roots were harvested for
measuring cytokinin concentrations. gFW, g fresh weight. Error bars represent the SD (n = 3). Data sets marked with
an asterisk are significantly different from control as assessed by the Student’s t-test: P < 0.05. iP, N6-(�2-
isopentenyl)adenine; iPRPs, iP riboside 5’-phosphates; iP7G, iP-7-N-glucoside; iP9G, iP-9-N-glucoside.
Cy
tok
inin
co
nc
en
tra
tio
ns
(p
mo
l/g
FW
)
iP iPRPs
iP7G iP9G
- DEX+ DEX
0
5
10
0
50
100
150
0
0.5
1
* **
*
**
*
*
*
*
*
*
*
*
*
*
0
4
8
*
Supplemental Data. Kuroha et al. (2009). Functional Analyses of LONELY GUY Cytokinin-
Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis
1 2 3
A
B C D
Supplemental Figure 2. Complementation of the log3 log4 log7 Mutant by Introduction of the At LOG7 Gene.
(A) Four-week-old T1 transgenic plants of the log3 log4 log7 mutant (1 and 2) transformed with the empty vector pBG (1)
and pBG-LOG7 (2). For the control, LOG3pro:GUS plants (3) were grown in parallel. These T1 plants were selected on MS
medium and transplanted to rockwool blocks.
(B) to (D) Top of inflorescences of the T1 transgenic plants [(B) for 1, (C) for 2, (D) for 3] shown in (A). All opened flowers
were removed from the inflorescences.
Bars = 5 cm for (A), 1 mm for (B) to (D).
Supplemental Figure 3. Cytokinin Levels in the log3 log4 log7 Mutant.
Wild type (WT) and the log3 log4 log7 mutant were grown for 3 weeks on MGRL-agar medium, seedlings were
harvested, and cytokinin concentrations were measured. Error bars represent the SD (n = 3). gFW, g fresh
weight. Data sets marked with an asterisk are significantly different from WT as assessed by the Student’s t-test:
P < 0.05. iP, N6-(�2-isopentenyl)adenine; iPRPs, iP riboside 5’-phosphates.
Cy
tok
inin
co
nc
en
tra
tio
ns
(p
mo
l/g
FW
)
0
0.2
0.4
0.6
0.8
0
10
20
iP iPRPs
*
*
500 bp
LOG1pro:GUS
2002 bp 1024 bp
GUS
At LOG1
105 bp
GUS
105 bp
2113 bp
507 bp
LOG2pro:GUS
At LOG2
1832 bp
312 bp 123 bp
GUSLOG3pro:GUS
At LOG3
2125 bp
387 bp 123 bp
GUSLOG4pro:GUS
At LOG4
2036 bp
89 bp 108 bp
GUSLOG5pro:GUS
At LOG5
2000 bp
108 bp
GUS
1081 bp
LOG7pro:GUS
At LOG7
2051 bp
111 bp
GUS
350 bp
LOG8pro:GUS
At LOG8
Supplemental Figure 4. Schematic Representation of LOGpro:GUS Fusion Constructs.
The LOGpro:GUS fusion constructs (upper part) and coding regions of At LOG genes with their 5’ promoter regions (lower
part) are shown. Black and white rectangles indicate protein coding regions and untranslated regions, respectively. Lengths
of 5’ promoter regions, first exons, and first introns used for the fusion constructs are indicated.
Supplemental Figure 5. GUS Expression in Seedlings and Inflorescences of LOGpro:GUS Transgenic Plants.
GUS expression in seedlings [(A) to (E)] or inflorescences [(F) to (K)] of LOG1pro:GUS [(A) and (F)], LOG3pro:GUS
[(B) and (G)], LOG4pro:GUS [(C) and (H)], LOG5pro:GUS [(D) and (I)], LOG7pro:GUS (J), and LOG8pro:GUS [(E) and
(K)] plants 14 days [(A), (B) and (E)], 17 days [(C) and (D)], or 1.5 months [(F) to (K)] after germination are shown.
Bars = 1 mm for (A) to (D), 2 mm for (E) to (J).
A
C D E
F G H
I J K
B
ACT2
At LOG2
ACT2
At LOG4
ACT2
At LOG5
ACT2
At LOG7
ACT2
At LOG8
Supplemental Figure 6. Overexpression of At LOG Genes in 35Spro:LOG Transgenic Lines.
RT-PCR analysis of 14-day-old shoots in 35Spro:LOG transgenic lines. ACT2 is the extraction and loading
control. PCR reactions were performed with 25 temperature cycles except for At LOG4 gene (35 cycles).
Lines marked with an asterisk were used for further analyses.
Supplemental Figure 7. Seed Length of the 35Spro:LOG Transgenic Plants.
Seed length for wild-type (WT) and the 35Spro:LOG plants. Error bars represent the SD (n > 20). Data sets marked with an asterisk are significantly different from WT as assessed by the
Student’s t-test: P < 0.001.
* *** *
0
0.2
0.4
0.6
0.8
Se
ed
le
ng
th (
mm
)
C
0
1
2
3
Cell
are
a (
x10
3�
m2
)
0
2
4
6
8
Le
af
are
a (
mm
2)
D
E
A
0
1
2
3
4
5
Cell
nu
mb
er
(x10
3 c
ell
s/l
eaf)
Supplemental Figure 8. Phenotypes of 35Spro:LOG Cotyledons and Leaves.
(A) Shoots (left) and cotyledons and rosette leaves (right) of 3-week-old wild-type (WT) and 35Spro:LOG
seedlings. Arrows indicate cotyledons. Bar = 1 cm.
(B) Shoot phenotypes of WT (left) and 35Spro:LOG4 (right) seedlings 7 days after germination. Bar = 1 mm.
(C) to (E) Leaf area (C), cell area (D) or cell number (E) of WT and 35Spro:LOG4 cotyledons 7 days after
germination. Error bars represent the SD (n = 3 for leaf area, n = 12 for cell number, n = 30 for cell area).
Data sets marked with an asterisk are significantly different from WT as assessed by the Student’s t-test: P <
0.05.
*
*
WT
35Spro:LOG2
35Spro:LOG4
35Spro:LOG5
35Spro:LOG7
35Spro:LOG8
WT 35Spro:LOG4
B
Supplemental Figure 9. Cytokinin Levels in Roots and Shoots of 35Spro:LOG4 Transgenic Plants.
Cytokinin concentrations in roots and shoots of 3-week-old wild-type (WT) and 35Spro:LOG4 seedlings grown on
MGRL-agar plates were measured. gFW, g fresh weight. Error bars represent the SD (n > 3). Data sets marked with
an asterisk are significantly different from WT as assessed by the Student’s t-test: P < 0.05. iP, N6-(�2-
isopentenyl)adenine; iPRPs, iP riboside 5’-phosphates; iP7G, iP-7-N-glucoside; iP9G, iP-9-N-glucoside; tZ, trans-
zeatin; tZRPs, tZ riboside 5’-phosphates; tZ7G, tZ-7-N-glucoside; tZOG, tZ-O-glucoside.
Root
Shoot
Cyto
kin
in c
on
cen
trati
on
(p
mo
l/g
FW
)
0
0.4
0.8
0
4
8
** * 0
10
20
**
0
60
120
0
2
4
0
10
20
*
**
**
*
*
*0
100
200
300
0
100
200
300
*
WT 35Spro:LOG4
Supplemental Table 1. Structural Features of At LOGs and Deduced Proteins
Gene AGI Code LengthaMolecular
massb (kDa)
% identity to
LOGcChromosome
At LOG1 At2g28305 213 23.2 76.1 II
At LOG2 At2g35990 213 23.3 70.6 II
At LOG3 At2g37210 215 23.6 79.9 II
At LOG4 At3g53450 215 23.5 78.2 III
At LOG5 At4g35190 228 25.2 67.4 IV
At LOG6 At5g03270 229 25.0 67.0 V
At LOG7 At5g06300 217 23.9 72.2 V
At LOG8 At5g11950 216 23.8 65.6 V
At LOG9 At5g26140 143 16.1 63.7 V
aLengths are given in amino acids derived from predicted cDNA sequences in TAIR database.bCalculated molecular mass.cCalculated by TAIR BLAST 2.2.8 (http://www.arabidopsis.org/Blast/index.jsp)
Supplemental Table 2. Change of iP-type Cytokinin Concentrations in Roots of DEXpro:LOG2 Transgenic Plants
Cytokinins
iP 0.310 ± 0.143 0.173 ± 0.048 0.189 ± 0.027 0.665* ± 0.149
iPR 0.136 ± 0.060 0.129 ± 0.041 0.185 ± 0.054 0.178 ± 0.097
iPRPs 3.929 ± 0.530 4.214 ± 0.718 6.013 ± 0.317 3.888* ± 0.858
iP7G 10.346 ± 0.463 11.089 ± 0.677 12.159 ± 1.329 121.873* ± 14.261
iP9G 0.733 ± 0.031 0.722 ± 0.075 0.844 ± 0.087 4.749* ± 0.800
Transgenic Arabidopsis seedlings harboring empty pTA-7001 construct (Control) or DEXpro:LOG2 were grown for 17
days on MGRL-agar medium and then transplanted to MGRL-agar medium with or without 10 �M dexamethasone
(DEX). After 5 days, roots of seedlings were harvested and cytokinin concentration (pmol g-1 fresh weight) were
measured. Data are means ± SD (n = 3). Data sets marked with asterisk are significantly different from data without
DEX as assesed by Student's t-test: P < 0.02. iP, N6-(�2-isopentenyl)adenine; iPR, iP riboside; iPRPs, iPR
phosphate; iP7G, iP-7-N-glucoside; iP9G, iP-9-N-glucoside.
DEXpro:LOG2
- DEX
Control
+DEX + DEX- DEX
Supplemental Table 3. Summary of GUS Expression Patterns in LOGpro:GUS Transgenic Plants.
Organ At LOG3 At LOG8
Procambium - - +++ +++ - - -
Quiscent center - - - - - - +++
Other cells - - - ++ - - -
Elongation zone - - - ++ - +++ -
Vascular +++ - - +++ ++ - +++
Other cells ++ ++ - - - ++ +
Lateral root primordia - - - +++ - - -
Vascular + - - + - - +
Other cells - - - - - - -
Vascular +++ - - ++ - - +++
Other cells - - - - - + +++
Apical meristem +++ - - +++ - - -
Vascular +++ - + +++ +++ - +++
Trichome ++ - - ++ - +++ -
Other cells ++ ++ - ++ - ++ +++
Vascular ++ - - ++ - - +++
Other cells - - - - - - +++
Vascular ++ - - ++ - - +
Other cells - - - - + - +
Vascular ++ - - ++ - - ++
Other cells - - - - - - ++
Axillary bud - - ++ ++ ++ - ++
Petal - - - - ++ - +
Sepal - - - - ++ - ++
Immature + - - + +++ - -
Anther - - - - - - -
Filament - - - - ++ - ++
Stigma - - + + ++ - -
Style ++ - +++ - +++ - ++
Ovary ++ - + + + - ++
Immature + - - + - + -
Mature - - - - - ++ -
Endosperm - - - - - - -
Fruit abscission zone + - - +++ ++ - ++
Other cells - - - - - - +
Relative expression levels: +++, strong; ++, medium; +, weak; -, no.
Silique
Shoot
Cauline leaf
Pistil
Stamen
Hypocotyl
Stem
Premature rosette leaf
At LOG5
Cotylerdon
Mature rosette leaf
Pollen
Flower
Root
Meristem
Tissue name At LOG7
Root hair zone
At LOG1 At LOG2 At LOG4
Supplemental Table 4. Genetic Regulation of Seed Size by the 35Spro:LOG4 Transgene.
F1 seed genotype
� � SP EN EM
-/- -/- -/- -/-/- -/- 0.51 (0.02)a
-/- +/+ -/- -/-/+ -/+ 0.52 (0.02)a
+/+ -/- +/+ +/+/- +/- 0.65 (0.03)b
+/+ +/+ +/+ +/+/+ +/+ 0.63 (0.03)b
+/- +/+/- +/-
+/- -/-/- -/-0.60 (0.02)c
Average seed length of F1
Parental genotypes
+/- -/-
SP, EN, and EM refer to the maternal sporophyte, endosperm, and embryo genotypes, respectively.
The + and - designations indicate the presence and absence of the 35Spro:LOG4 transgene,
respectively. SD (n > 20) values are shown in parentheses. Different letters (a, b, and c) indicate
statistically significant differences between treatments (Student's t-test, P < 0.001).
Supplemental Table 5. T-DNA Insertion Sites and Primers Used for this Study.
Forward Reverse Forward Reverse
log1-1 SALK_027495 NT LOG1-RT-F LOG1-G-R LOG1-RT-F SALK Lba1
log1-2 SALK_143462 897 LOG1-RT-F LOG1-G-R LOG1-RT-F SALK Lba1
log2-1 SALK_052856 2319 LOG2-G-F LOG2-G-R LOG2-G-F SALK Lba1
log2-2 SALK_147463 2415 LOG2-G-F LOG2-G-R SALK Lba1 LOG2-G-R
log3-1 SALK_056659 950 LOG3-1-G-F LOG3-G-R LOG3-1-G-F SALK Lba1
log3-2 SALK_066969 1998 LOG3-2-G-F LOG3-RT-R SALK Lba1 LOG3-RT-R
log4-1 SALK_045912 1252 LOG4-12-G-F LOG4-12-G-R SALK Lba1 LOG4-12-G-R
log4-2 SALK_137667 1452 LOG4-12-G-F LOG4-12-G-R SALK Lba1 LOG4-12-G-R
log4-3 SALK_092241 2194 LOG4-3-G-F LOG4-RT-R SALK Lba1 LOG4-RT-R
At LOG5 log5-1 GABI_002G02 861 LOG5-RT-F LOG5-RT-R LOG5-RT-F GABI-Kat LB
At LOG7 log7-1 SALK_113173 2430 LOG7-G-F LOG7-G-R SALK Lba1 LOG7-G-R
log8-1 SALK_090077 144 LOG8-1-G-F LOG8-G-R LOG8-1-G-F SALK Lba1
log8-2 SALK_093520 NT LOG8-2-G-F LOG8-G-R LOG8-2-G-F SALK Lba1
*based on genomic sequences with translation start site (A of ATG = 1).
NT, Not tested.
Primer sequences are presented in Supplemental Data Set 2.
Primer
Gene specific T-DNA insertionGene Allele Line name
At LOG4
At LOG8
At LOG1
At LOG2
At LOG3
Site of T-DNA
insertion*