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*