1. Free IAA (nmol g-1 DW)
IAA ｈ 1 24 72 - ０ h + 10 20 30 40 50 60 70 80
0.01
0.02
0.03
0.04
0.05
Supplemental Figure S1. Quantification of the IAA concentration of K. nitens in the absence or presence of exogenous 100 µM IAA.
K. nitens was cultured for 0, 1, 24, and 72 h in the absence or presence of exogenous 100 µM IAA. The cells were harvested by filtration and washed with water. The intracellular IAA concentration of K. nitens was quantified by liquid chromatography-tandem mass spectrometry. Error bars represent SD of values for three replicates. Inner graph shows a magnified view of the y axis. DW, dry weight.

2. Duration of culture (h)
day 0
day 1
day 3
day 7
Control
100 μM NPA B
0.2
0.4
0.6
0.8
1.0
1.2 20 40 60 80
100
120
140
160
180
Control
100 μM NPA
Optical density (OD)=550
Duration of culture (h)
Supplemental Figure S2. Growth of K. nitens in the presence of NPA.
K. nitens was cultured in the presence of 100 μM NPA. A, Cell cultures were photographed at 0, 1, 3, and 7 d. B, Growth curves for K. nitens in the absence or presence of NPA. Error bars represent SD of values for three replicates.

3. Duration of culture (h)
day 0
day 1
day 3
day 7
Control
1 μM　BBo
10 μM BBo
100 μM BBo B
0.2
0.4
0.6
0.8
1.0
1.2 20 40 60 80
100
120
140
160
180
Control
1 µM BBo
10 µM BBo
100 μM BBo
Optical density (OD)=550
Duration of culture (h)
Supplemental Figure S3. Growth of K. nitens in the presence of BBo.
K. nitens was cultured in the presence of 0, 1, 10, or 100 μM BBo. A, Cell cultures were photographed at 0, 1, 3, and 7 d. B, Growth curves for K. nitens for each concentration of BBo. Error bars represent SD of values for three replicates.

4. Duration of culture (h)
day 0
day 1
day 3
day 7
Control
100 μM IAA
Control + C
100 μM IAA + C B
1.6
Control
1.4
100 μM IAA
1.2
Control + C
Optical density (OD)=550
100 μM IAA + C
1.0
0.8
0.6
0.4
0.2 20 40 60 80
100
120
140
160
180
Duration of culture (h)
Supplemental Figure S4. Growth of K. nitens in the presence of calcofluor white.
K. nitens was transiently stained with calcofluor white (+ C) and cultured in the absence or presence of IAA. A, Cell cultures were photographed at 0, 1, 3, and 7 d. B, Growth curves for transiently stained or unstained K. nitens in the absence or presence of IAA. Error bars represent SD of values for three replicates.

5. Ratio of total cells (%)B
day 0
day 0
day 1
day 1
day 2
day 3 C 20 40 60 80
100
First cell division
Second cell division Ⅰ Ⅱ
n=355
n=378
day 2
Ratio of total cells (%)
day 3
Supplemental Figure S5. Measurement of the frequency of cell division in K. nitens by calcofluor white staining.
A, K. nitens transiently stained with calcofluor white was traced from 0 to 3 d. Red and blue fluorescence indicate chloroplasts (chlorophyll autofluorescence) and cell wall (calcofluor white), respectively. The numerals indicate borders of the cells at day 0. Yellow arrows indicate borders strongly stained with calcofluor white at day 0. Fluorescence from cell borders produced by incomplete cell division makes it difficult to accurately measure the frequency of cell division. Scale bars = 25 μm. B, Enlarged views of A. Scale bars = 5 μm. C, The borders formed by the first and second cell division were counted in K. nitens transiently stained with calcofluor white. The black bar (I) represents the ratio of new borders strongly stained after cell division, and the gray bar (II) represents the ratio of new borders weakly or not stained after cell division. The number of cell borders counted was 355 for the first cell division and 378 for the second cell division. This experiment was performed one time.

6. A B Frequency of cell division 20 40 60 80 100 3 2.75 2.5 2.25 2 1.5 120 40 60 80
100 3
2.75
2.5
2.25 2
1.5 1
Ratio of total cells (%)
IAA - - - + + +
Day 1 2 3 1 2 3 B
Frequency of cell division 20 40 60 80
100 3
2.75
2.5
2.25 2
1.5 1
Ratio of total cells (%)
IAA - - - + + +
Day 1 2 3 1 2 3
Supplemental Figure S6. Measurement of the frequency of cell division in K. nitens by calcofluor white staining in the presence of IAA.
The reproducible experiments of Figure 5B. K. nitens transiently stained with calcofluor white was traced from 0 to 3 d in the absence or presence of 100 μM IAA on solid medium. The frequency of cell division was measured for 1, 2, and 3 d in the absence or presence of 100 μM IAA on solid medium. Bar graphs represent the percentage of each frequency class of cell division. A, Analysis of 128, 210, and 283 samples for the control cultured for 1, 2, and 3 d, respectively, and 143, 139, and 138 samples for the IAA treatment cultured for 1, 2, and 3 d, respectively. B, The number of samples analyzed was 229, 258, and 443 for the control cultured for 1, 2, and 3 d, respectively, and 250, 317, and 347 for the IAA treatment cultured for 1, 2, and 3 d, respectively.

7. A 5 10 15 20 25 30 35 40 2 4 6 8 12 14 16 18 22 24 26 28 32 34 36 38 42
day 0
Control day 5
IAA day 5
Ratio of total cells (%)
Cell length
(μm) B 5 10 15 20 25 30 35 2 4 6 8 12 14 16 18 22 24 26 28 32 34 36 38 40 42
day 0
Control day 5
IAA day 5
Ratio of total cells (%)
Cell length
(μm)
Supplemental Figure S7. Distribution of cell length at 5 d in the absence or presence of 100 µM IAA.
The reproducible experiments of Figure 6B. K. nitens was cultured for 5 d in the absence or presence of 100 μM IAA on solid medium. A, The number of samples measured was 264 (control, 0 d), 310 (control, 5 d), and 273 (IAA treatment, 5 d). B, The number of samples measured was 408 (control, 0 d), 351 (control, 5 d), and 412 (IAA treatment, 5 d).

8. A
0.5
1.0
1.5
2.0
2.5
Control
100 µM IAA
2.5 *
2.0
1.5
Ratio of cell length (day 1/day 0)
1.0
Contol
0.5
100 µM IAA 5 10 15 20 25 30
Cell length at day 0 (µm) B
0.5
1.0
1.5
2.0
2.5 5 10 15 20 25 30
Control
100 μM IAA
0.5
1.0
1.5
2.0
2.5 *
Ratio of cell length (day 1/day 0)
Control
100 µM IAA
Cell length at day 0 (µm)
Supplemental Figure S8. Cell elongation ratio as calculated in the absence or presence of 100 µM IAA.
The reproducible experiments of Figure 6C. The cell elongation ratios are represented by dot (left) and box (right) plots. A, The number of samples measured was 147 (control) and 172 (IAA treatment). B, The number of samples measured was 79 (control) and 107 (IAA treatment). *P < 0.01 (Student’s t-test).

9. A
0.5
1.0
1.5
2.0
2.5
3.0 5 10 15 20 25
Control
100 μM TIBA
3.0 *
2.5
2.0
Ratio of cell length (day 1/day 0)
1.5
1.0
0.5
Control
100 µM TIBA
Cell length at day 0 (µm) B
Cell length at day 0 (µm)
0.5
1.0
1.5
2.0
2.5
3.0 5 10 15 20 25
Control
100 µM TIBA
0.5
1.0
1.5
2.0
2.5
3.0 *
Ratio of cell length (day 1/day 0)
Control
100 µM TIBA C
3.0
3.0 *
2.5
2.5
2.0
2.0
Ratio of cell length (day 1/day 0)
1.5
1.5
1.0
1.0
Control
0.5
0.5
100 μM TIBA 5 10 15 20 25
Control
100 µM TIBA
Cell length at day 0 (µm)
Supplemental Figure S9. Cell elongation ratio as calculated in the absence or presence of 100 µM TIBA.
The reproducible experiments of Figure 7B. The cell elongation ratios are represented by dot (left) and box (right) plots. A, The number of samples measured was 166 (control) and 110 (TIBA treatment). B, The number of samples measured was 156 (control) and 159 (TIBA treatment). C, The number of samples measured was 169 (control) and 135 (TIBA treatment). *P < 0.01 (Student’s t-test).

10. A 20 40 60 80
100 1
1.5 2
2.25
2.5
2.75 3
Control day 3
100 µM NPA day 3
Ratio of total cells (%)
Number of cell divisions B
0.5
1.0
1.5
2.0
2.5
3.0 5 10 15 20 25
control
100 μM NPA
Ratio of cell length (day 1/day 0)
0.5
1.0
1.5
2.0
2.5
3.0
Cell length at day 0 (µm)
Control
100 µM NPA C
0.5
1.0
1.5
2.0
2.5
3.0 5 10 15 20 25
0.5
1.0
1.5
2.0
2.5
3.0
Ratio of cell length (day 1/day 0)
control
100 μM NPA
Cell length at day 0 (µm)
Control
100 µM NPA D
0.5
1.0
1.5
2.0
2.5
3.0 5 10 15 20 25
0.5
1.0
1.5
2.0
2.5
3.0
Ratio of cell length (day 1/day 0)
control
100 μM NPA
Cell length at day 0 (µm)
Control
100 µM NPA
Supplemental Figure S10. Cell division and cell elongation of K. nitens grown in the presence of NPA.
A, Frequency of cell division for 3 d in the absence or presence of 100 μM NPA. Error bars represent SD of values for three replicates. The number of samples measured for the three replicates was 198, 196, and 201 (control) and 160, 223, and 237 (NPA treatment). B, Cell elongation ratio as represented in dot (left) and box (right) plots. The number of samples was 181 (control) and 156 (NPA treatment). This experiment was independently performed three times (C, D). C and D, The reproducible experiments of B. C, The number of samples measured was 167 (control) and 163 (NPA treatment). D, The number of samples measured was 154 (control) and 178 (NPA treatment).

11. A 5 10 15 20 25 30 35 2 4 6 8 12 14 16 18 22 24 26 28 32 34 36 38 40 42
0 day
Control day 5
BBo day 5
Ratio of total cells (%)
Cell length
(μm) B 5 10 15 20 25 30 35 40 45 2 4 6 8 12 14 16 18 22 24 26 28 32 34 36 38 42
0 day
Control day 5
BBo day 5
Ratio of total cells (%)
Cell length
(μm)
Supplemental Figure S11. Distribution of cell length at 5 d in the absence or presence of 30 µM BBo.
The reproducible experiments of Figure 8B. K. nitens was cultured for 5 d in the absence or presence of 30 μM BBo on solid medium. A, The number of samples measured was 307 (control, 0 d), 313 (control, 5 d), and 310 (BBo treatment, 5 d). B, The number of samples measured was 243 (control, 0 d), 276 (control, 5 d), and 265 (BBo treatment, 5 d).

12. A
2.5
0.5
1.0
1.5
2.0
2.5 *
2.0
1.5
Ratio of cell length (day 1/day 0)
1.0
Control
0.5
30 µM BBo 5 10 15 20 25
Control
30 µM BBo
Cell length at day 0 (µm) B
3.0
3.0 *
2.5
2.5
2.0
2.0
Ratio of cell length (day 1/day 0)
1.5
1.5
1.0
1.0
Control
0.5
0.5
30 µM BBo 5 10 15 20 25
Control
30 µM BBo
Cell length at day 0 (µm)
Supplemental Figure S12. Cell elongation ratio as calculated in the absence or presence of 30 µM BBo.
The reproducible experiments of Figure 8C. The cell elongation ratios are represented by dot (left) and box (right) plots. A, The number of samples measured was 127 (control) and 123 (BBo treatment). B, The number of samples measured was 227 (control) and 172 (BBo treatment). *P < 0.01 (Student’s t-test).

13. A B 1 2 3 4 5 6 7
Control
100 μM IAA 1 2 3 4 5 6 7
***
Control * **
100 μM IAA
Relative expression level
Relative expression level **
Hours
Hours C
0.5
1.0
1.5
2.0
2.5
3.0 1 2 3 4 5 6 7
Control
100 μM IAA
Relative expression level ** **
Hours
Supplemental Figure S13. The initial expression changes of certain mRNAs in the downregulated Cluster8 and upregulated Cluster11.
The initial expression changes of certain mRNAs in the downregulated Cluster8 and upregulated Cluster11 were analyzed by real-time PCR of samples from cells incubated for 1, 3, and 6 h in the absence or presence of 100 μM IAA. (A) The expression of kfl00295_0090 and (B) the expression of kfl00812_0030 in upregulated Cluster11. C, The expression of kfl00396_0100 in downregulated Cluster8. All error bars represent SD of values for four replicates. A, B and C, *P < 0.1, **P < 0.05, ***P < 0.01 (Student’s t-test).

14. Supplemental Figure S14. Phylogenetic tree analysis of LBD genes. 75 92 74 51 80 31 38 33 22 46 81 66 98 52 63 97 99 29 93 26 58 94 9 79 14 18 25 59 48 43 39 73 53 87 35 21 19 56 44 71 69 8 7 12 78 65 86 15 5 28 6 23 2 1
Potri.005G
Potri.005G
AT1G16530
AT1G31320
49084
Pp1s66 56V6.1
59179
Potri.014G
Potri.008G
LOC Os03g
AT2G28500
LOC Os03g
LOC Os05g
Potri.001G
AT2G30130
Potri.012G
AT3G26620
LOC Os03g
Pp1s9 186V6.1
37169
Potri.013G
AT2G40470
LOC Os01g
Potri.013G
AT2G30340
AT3G50510
59642
AT3G03760
Pp1s157 25V6.1
AT2G45420
AT4G00220
Potri.002G
LOC Os03g
Potri.002G
AT2G45410
AT4G00210
Potri.010G
AT5G06080
LOC Os03g
LOC Os02g
Potri.002G
AT2G42430
AT2G31310
LOC Os03g
Potri.002G
AT2G42440
AT3G58190
Potri.008G
AT3G11090
Potri.008G
AT1G65620
LOC Os05g
LOC Os03g
AT2G23660
AT5G66870
Pp1s71 7V6.1
68988
LOC Os01g07480
AT5G63090
Potri.001G
AT3G27650
27657
Pp1s73 7V6.1
Potri.003G
AT3G13850
AT1G72980
LOC Os08g
Potri.012G
AT3G47870
Potri.018G
AT5G15060
LOC Os01g
AT2G19820
LOC Os01g
Potri.013G
AT1G06280
kfl00147_0060_v1.1
kfl00115_0010_v1.1
kfl00255_0040_v1.1
kfl00500_0060_v1.1
kfl00209_0160_v1.1
Pp1s69 130V6.1
69267
LBD18
LBD16
LBD29
LBD33
KnLBD1
A. thaliana
O. sativa
P. trichocarpa
P. patens
S. moellendorffii
K. nitens
0.2
Supplemental Figure S14. Phylogenetic tree analysis of LBD genes.
Protein sequences of LBD genes (Supplemental Table S2) were collected from datasets by PFAM (Finn et al., 2016). After removing sequences that were inadequate for phylogenetic analysis (short sequence length, low quality, large deletion, high similarity sequences in each species, etc.), sequences were aligned using MAFFT (Katoh and Standley, 2013) v trimAl (Capella-Gutiérrez et al., 2009) v1.2 was used to remove any poorly conserved regions, and the amino acid substitution model was calculated with Aminosan52 (Tanabe, 2011). Phylogenetic tree analysis of LBD using the maximum likelihood method in MEGA6 (Tamura et al., 2013) based on the LG model +G (8 categories) with 500 bootstrap replicates. Each symbol represents a species. Red indicates auxin-inducible LBD genes in A. thaliana. The thick branches were supported by bootstrap values ≥70. The scale bar represents 0.2 amino acid substitutions per site.

15. Supplemental Figure S15. Multiple sequence alignment of ABP1 homologs.
A. thaliana (NP_192207)
P. trichocarpa (XP_ )
O. sativa (XP_ )
S. moellendorffii (XP_ )
P. patens (XP_ )
K. nitens (GAQ82792)
C. reinhardtii (XP_ )
C. variabilis (XP_ )
C. variabilis (XP_ )
O. sativa (ABA99317)
A. thaliana (NP_192207)
P. trichocarpa (XP_ )
O. sativa (XP_ )
S. moellendorffii (XP_ )
P. patens (XP_ )
K. nitens (GAQ82792)
C. reinhardtii (XP_ )
C. variabilis (XP_ )
C. variabilis (XP_ )
O. sativa (ABA99317)
A. thaliana (NP_192207)
P. trichocarpa (XP_ )
O. sativa (XP_ )
S. moellendorffii (XP_ )
P. patens (XP_ )
K. nitens (GAQ82792)
C. reinhardtii (XP_ )
C. variabilis (XP_ )
C. variabilis (XP_ )
O. sativa (ABA99317)
Supplemental Figure S15. Multiple sequence alignment of ABP1 homologs.
Amino acid sequences of ABP1 homologs were aligned using MUSCLE (Edgar, 2004). Red, blue, and green boxes represent metal-core sites, hydrophobic sites, and the endoplasmic reticulum retention motif, respectively. The GenBank accession number of each protein is shown in parentheses.

16. Supplemental References
Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses.　Bioinformatics 25: 1972–1973
Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797
Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, et al (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44(D1): D279–D285
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol 30: 772–780
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 2725–2729
Tanabe AS (2011) Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data. Mol Ecol Resour 11: 914–921