Supplemental Information Side-Chain Modification of ... · PDF fileSide-Chain Modification of Cytokinins Controls Shoot ... which is essential for P450 ... Transverse sections of Col-0

Developmental Cell, Volume 27

Supplemental Information

Side-Chain Modification of Cytokinins

Controls Shoot Growth in Arabidopsis

Takatoshi Kiba, Kentaro Takei, Mikiko Kojima, and Hitoshi Sakakibara

Inventory of Supplemental Information Figure S1 related to Figure 1 Figure S2 related to Figure 2 Figure S3 related to Figure 3 Figure S4 related to Figure 5 Figure S5 related to Figure 7 Table S1 related to Figure 1 (see separate Excel file) Table S2 related to Figure 2 Table S3 related to Figure 3 Table S4 related to Figure 5 Table S5 related to Figure 7 Supplemental Experimental Procedures Supplemental References

Hor

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CYP735A1

CYP735A2

ACT2

Col-0 a1-1 a1-2 a2-1 a2-2 a1-1a2-1

a1-2a2-1

a1-1a2-2

a1-2a2-2

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a2-1

a2-2

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A1-1Col-

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ADP/ATP

DMAPP+

iPRPs

iP

tZRPs

tZ

CYP735AIPT

LOGLOG

Degradation/Inactivation

0

300 tZ-typecZ-typeiP-type

**

CYP735A1

CYP735A2

ATG

ATG

TGA

TGA

cyp735a1-1 cyp735a1-2

cyp735a2-1 cyp735a2-2

H

H

B

Figure S1. Hormone concentrations in cyp735a1 and cyp735a2 mutants, and complemented lines, related to Figure 1. (A) A simplified model of cytokinin metabolism. Further details can be found in Sakakibara (2006) and Kamada-Nobusada and Sakakibara (2009). cZ, cis-zeatin; DMAPP, dimethylallyl diphosphate; iP, N6-(∆2-isopentenyl)adenine; iPRPs, iP-riboside 5’-phosphates ; IPT, adenosine phosphate-isopentenyltransferase; LOG, LONELY GUY; tZ, trans-zeatin: tZRPs. tZ-riboside 5’-phosphates. Blue arrows indicate enzymatic reactions. (B) Expression of the CYP735A1 and CYP735A2 genes in cyp735a1 and cyp735a2 single and double mutants. (top) Schematic representation of cyp735a1 and cyp735a2 T-DNA insertion alleles. Boxes represent exons; horizontal bars, introns; triangles, T-DNA insertion sites; ATG, initiation codon; TGA, termination codon; arrows, primers used in RT-PCR analyses shown in the lower panel and (E). The zone labelled “H” in the fifth exon represents the region encoding a heme binding signature, which is essential for P450 catalytic activity. (bottom) RT-PCR analyses of cyp735a1 and cyp735a2 single and double mutants. Analyses were conducted with cDNA generated from the plants indicated and specific primers at saturating PCR cycle numbers. ACT2 was used as an internal control. (C, D) Abscisic acid (ABA) and indole-3-acetic acid (IAA) concentrations in the shoot (C) and root (D) of the wild type (Col-0), single, and double mutants of cyp735a1 and cyp735a2. Seedlings were grown as described in Figure 1. Asterisks indicate statistically significant differences compared with Col-0 (P < 0.01, one-way ANOVA follwed by Dunnett’s test). gFW, gram fresh weight. Concentrations of each hormone are given in Table S1. (E, F) Complementation of the cyp735a1 cyp735a2 mutant by CYP735A1 and CYP735A2 genomic fragments. (E) Expression of the CYP735A1 and CYP735A2 genes in wild type (Col-0), a1-1 a2-1 (cypDM), and in independent lines of cypDM carrying a genomic fragment of CYP735A1 (Comp. A1-1 and Comp. A1-2) or CYP735A2 (Comp. A2-1 and Comp. A2-2). Semi-quantitative RT-PCR analyses were conducted with cDNA generated from the plants indicated and specific primers shown in (B). ACT2 served as an internal control. (F) Cytokinin concentrations in the shoots of 21-day-old plants grown on soil. Error bars represent standard deviation of four biological replicates.

Col-0 a1-1 a1-2 a2-1 a2-2

a1-1 a2-1 a1-1 a2-2 a1-2 a2-1

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Figure S2. Shoot phenotype of all double mutant combinations for cyp735a1 and cyp735a2, and complemented lines, related to Figure 2. (A, B) Appearance (A) and rosette diameter (B) of 21-day-old wild type (Col-0), a1-1, a1-2, a2-1, a2-2, a1-1 a2-1, a1-2 a2-1, a1-1 a2-2, and a1-2 a2-2 grown on soil. Error bars represent standard deviation of four biological replicates. Asterisks indicate statistically significant differences compared with Col-0 (*, P < 0.05; **, P < 0.01 one-way ANOVA followed by Dunnett’s test). (C) Stature of 49-day-old Col-0 and mutants. (D, E) Leaf senescence is not altered in cyp735a1 cyp735a2 mutants. (D) Dak-induced senescence of Col-0 and cyp735a1-2 cyp735a2-2 (cypDM) detached leaves. Appearance (left) and chlorophyll content (right) of Col-0 and cypDM. Fifth leaves of plants grown on MS plates were floated on distilled water for the indicated periods. The chlorophyll content before dark incubation (1.77 ± 0.11 and 1.68 ± 0.12 µg/g fresh weight for Col-0 and cypDM, respectively) was set to 100% for each genotype. (E) Age-dependent leaf senescence of Col-0 and cypDM. Appearance (left) and chlorophyll content (right) of Col-0 and cypDM. The chlorophyll contents of the first rosette leaf of soil-grown plants were measured at 21, 26, and 31 days after germination. The chlorophyll content at 21 days after germination (1.51 ± 0.13 and 1.32 ± 0.09 µg/g fresh weight for Col-0 and cypDM, respectively) was set to 100% for each genotype. Error bars represent standard deviations of five biological replicates. (F) Shoot branching phenotype of cyp735a1-2 cyp735a2-2 (cypDM) at 40 days after germination. Leaves were lined up in the order of age starting from the left. CB indicates a primary cauline branch. The primary inflorescence stem is shown at the top of each picture. (G) Complementation of cyp735a1-1 cyp735a2-1 by CYP735A1 and CYP735A2 genomic fragments. Overall appearance of 27-day-old Col-0, cyp735a1-1 cyp735a2-1 (a1-1 a2-1), and representative complemented plants (Comp. A1-1 and Comp. A2-1). Scale bars: 1 cm for (A) and 5 cm for (C), (F) and (G).

Col-0 cypDMA

B Col-0 cypDM

Col-0 cypDMD

CCol-0 cypDM

Figure S3. Cellular organization around columella meristem and in the vasculature of the cyp735a1 cyp735a2 primary root, related to Figure 3. (A, B) Root tip of Col-0 and cyp735a1-2 cyp735a2-2 (cypDM) stained with propidium iodide (A) and Lugol’s solution (B). (C, D) Transverse sections of Col-0 and cypDM primary roots stained with toluidine blue. Sections were made in the young (C) and old (D) part of the root. Seedlings were grown on 1/2MS vertical agar plates without sucrose for 5 days. White and black arrowheads, quiescent center cells; black arrows, columella initial cells; red arrows, xylem pole. Scale bar, 25 µm

line 9

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Figure S4. Overexpression of CYP735A2 does not reduce root growth, related to Figure 5. (A, B) Primary root length (A) and lateral root number (B) of wild type (Col-0) and two independent lines of CYP735A2-ox (line 1 and line 9). (C) Cytokinin concentrations in Col-0, CYP735A2-ox line 1 and line 9 roots. Seedlings were grown for 2 weeks on 1/2MS vertical agar plates. Concentrations of each cytokinin species are shown in Table S4. Error bars represent standard deviations of at least five biological replicates. Asterisks indicate statistically significant differences in total cytokinin concentration compared with Col-0 (P < 0.05, one-way ANOVA followed by Dunnett’s test).

A B

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Figure S5. Expression of CYP735A2 in the shoot and root as detected by GUS staining, related to Figure 7. Histochemical localization of GUS activity in ProCYP735A2:GUS transgenic plants grown on MGRL agar plates. Representative GUS staining in (A) 5-day-old and (B-H) 10-day-old seedlings; (B) is a composite of two pictures taken from the same plant. Letters with black arrows in (B) indicate the positions where pictures (C) to (G) were taken. Scale bar: 5 mm for (A) and (B), 1 mm for (C), 100 µm for (D)-(G), and 10 µm for (H).

Genotype aBolting time (DAG) Leaf number at bolting b1st leaf with abaxial trichome cNumber of CI cNumber of RI cNumber of node with ABLCol-0 26.1 ± 0.8 13.9 ± 0.8 6.5 ± 0.5 3.5 ± 0.7 5.4 ± 0.8 5.8 ± 0.9a1-1 a2-1 27.4 ± 1.3* 13.9 ± 0.7 8.4 ± 0.7* 2.3 ± 0.4* 9.4 ± 1.0* 10.6 ± 0.5*a1-2 a2-2 28.9 ± 1.3* 13.8 ± 0.8 9.4 ± 0.7* 2.3 ± 0.5* 9.2 ±1.2* 11.2 ±0.4*

cMeasurement was done on 40th day after germination (16 h-light/8 h-dark cycles)

aBolting time was defined as the time when the inflorescence stem reached 5 cm length.

Table S2. Flowering and shoot branching phenotype in cyp735a1 cyp735a2 double mutants, related to Figure 2

Data shown are mean ± standard deviation (n > 10). Asterisks indicate significant difference compared with Col-0 (one-way ANOVA followed by Dunnett's test, P < 0.05).

Col-0, wild type; a1-1 a2-1, cyp735a1-1 cyp735a2-1; a1-2 a2-2, cyp735a1-2 cyp735a2-2; DAG, days after germination; CI, primary cauline branch; RI, primary rosette branch;ABL, axillary bud leaves

bMeasurement was done on 21st day after germination (16 h-light/8 h-dark cycles)

Gene code Gene name Col-0 cyp735a1-2 cyp735a2-2 ipt3 ipt5 ipt7 Col-0 cyp735a1-2 cyp735a2-2 ipt3 ipt5 ipt7At3g16857 ARR1 1 ± 0.12 1.02 ± 0.09 1.37 ± 0.19* 1 ± 0.08 0.94 ± 0.18 1.09 ± 0.2At4g16110 ARR2 1 ± 0.11 0.73 ± 0.09* 1.25 ± 0.24* 1 ± 0.13 0.74 ± 0.12* 1.03 ± 0.22At1g59940 ARR3 N.D. N.D. N.D. 1 ± 0.32 1.05 ± 0.24 1.37 ± 0.2At1g10470 ARR4 1 ± 0.11 0.14 ± 0.02* 0.29 ± 0.05* 1 ± 0.09 0.75 ± 0.12* 0.33 ± 0.04*At3g48100 ARR5 1 ± 0.18 0.29 ± 0.06* 0.13 ± 0.03* 1 ± 0.07 1.28 ± 0.31 0.4 ± 0.06*At5g62920 ARR6 1 ± 0.18 0.04 ± 0.01* 0.22 ± 0.05* 1 ± 0.08 0.91 ± 0.2 0.38 ± 0.05*At1g19050 ARR7 1 ± 0.17 0.05 ± 0.01* 0.25 ± 0.04* 1 ± 0.08 0.84 ± 0.13* 0.08 ± 0.01*At2g41310 ARR8 1 ± 0.12 0.81 ± 0.12 0.46 ± 0.06* 1 ± 0.07 0.94 ± 0.19 0.66 ± 0.1*At3g57040 ARR9 1 ± 0.21 1.45 ± 0.26 0.72 ± 0.32* 1 ± 0.06 0.77 ± 0.14 0.79 ± 0.16*At1g74890 ARR15 1 ± 0.22 0.05 ± 0* 0.05 ± 0.02* 1 ± 0.12 1.65 ± 0.29 1 ± 0.09

Col-0, wild type, type-A ARR, ARR3-ARR9, ARR15; type-B ARR, ARR1, ARR2, N.D., not detected.

Table S3. Expression levels of cyokinin inducible type-A ARR genes and non-cytokinin-responsive type-B ARR genes in the shoot and root of Col-0,cyp735a1-2 cyp735a2-2, and ipt3 ipt5 ipt7, related to Figure 3

*, statistically significant difference compared with Col-0 (P < 0.01, one-way ANOVA followed by Dunnett's test)

Shoot RootSeedlings were grown on MGRL agar plates for 14 days before harvest. Data are means ± standard deviation (n = 4) relative to Col-0.

Col-0 CYP735A2-ox line 1 CYP735A2-ox line 9 Col-0 CYP735A2-ox line 1 CYP735A2-ox line 9tZ 6.36 ± 0.84 3.93 ± 1.61* 4.14 ± 0.66* 1.68 ± 0.1 2.36 ± 0.22* 2.28 ± 0.15*tZR 11.31 ± 2.23 9.22 ± 1.99 9.14 ± 1.47 2.98 ± 0.34 2.72 ± 0.18 2.54 ± 0.25tZRPs 178.2 ± 37.6 153.16 ± 23.1 182.16 ± 26.91 8.73 ± 0.79 11.53 ± 0.83* 10.84 ± 1.1*cZ 0.27 ± 0.02 0.13 ± 0.02* 0.12 ± 0.01* 0.64 ± 0.04 0.52 ± 0.04* 0.5 ± 0.07*cZR 0.53 ± 0.04 0.37 ± 0.07* 0.36 ± 0.05* 1.57 ± 0.44 1.58 ± 0.4 1.62 ± 0.3cZRPs 3.58 ± 0.47 1.3 ± 0.11* 1.57 ± 0.28* 5.97 ± 0.63 5.24 ± 1.75 4.62 ± 0.76DZ 0.09 ± 0.01 1.5 ± 0.35* 1.42 ± 0.15* 0.08 ± 0.01 0.25 ± 0.03* 0.28 ± 0.02*DZR 0.06 ± 0.01 0.93 ± 0.15* 0.9 ± 0.17* N.D. 0.1 ± 0.01 0.12 ± 0.02DZRPs 1.22 ± 0.32 24.9 ± 3.72* 26.33 ± 3.25* N.D. 0.61 ± 0.09 0.75 ± 0.12iP 0.19 ± 0.03 0.01 ± 0* 0.01 ± 0* 0.07 ± 0.01 0.03 ± 0* 0.02 ± 0*iPR 0.22 ± 0.09 0.01 ± 0* 0.01 ± 0* 0.24 ± 0.03 0.06 ± 0.01* 0.03 ± 0.01*iPRPs 60.5 ± 16.52 3.55 ± 0.84* 3.21 ± 0.76* 5.81 ± 0.54 1.83 ± 0.17* 1.23 ± 0.21*tZ7G 48.62 ± 6.07 93.11 ± 4.52* 98.02 ± 14.47* 24.21 ± 1.77 30.03 ± 1.08* 30.79 ± 1.54*tZ9G 56.4 ± 4.69 78.31 ± 15.47* 84.6 ± 13.25* 20.55 ± 2.17 26.16 ± 1.18* 27.54 ± 1.57*tZOG 12.01 ± 1.11 17.52 ± 1.49* 18.25 ± 1.93* 14.58 ± 1.44 20.68 ± 1.24 21.62 ± 0.72cZOG N.D. N.D. N.D. N.D. N.D. N.D.tZROG 1.57 ± 0.14 1.63 ± 0.08 1.82 ± 0.16* 0.33 ± 0.02 0.76 ± 0.06* 0.79 ± 0.04*cZROG 1.08 ± 0.11 0.79 ± 0.08* 0.78 ± 0.08* 0.26 ± 0.02 0.27 ± 0.03* 0.29 ± 0.02*tZRPsOG 0.23 ± 0.05 0.3 ± 0.06 0.38 ± 0.08* N.D. 0.1 ± 0.04 0.09 ± 0.04cZRPsOG N.D. N.D. N.D. N.D. N.D. N.D.DZ9G 0.33 ± 0.03 6.45 ± 0.24* 5.92 ± 0.4* 0.66 ± 0.05 2.67 ± 0.09* 3.05 ± 0.07*iP7G 10.32 ± 0.83 0.31 ± 0.01* 0.32 ± 0.02* 5.1 ± 0.29 1.43 ± 0.14* 0.91 ± 0.04*iP9G 1.93 ± 0.17 0.05 ± 0.02* 0.06 ± 0.01* 1.11 ± 0.09 0.29 ± 0.03* 0.17 ± 0.02*iP-type CK 73.16 ± 17.63 3.93 ± 0.87* 3.61 ± 0.79* 12.33 ± 0.92 3.63 ± 0.33* 2.37 ± 0.26*tZ-type CK 314.69 ± 52.73 357.18 ± 48.31 398.51 ± 58.93* 73.08 ± 6.03 94.35 ± 3.65* 96.49 ± 3.49*cZ-type CK 5.5 ± 0.65 2.59 ± 0.29* 2.91 ± 0.45* 8.45 ± 1.07 7.6 ± 2.16 7.04 ± 1.06Total CK 395.06 ± 71.39 397.49 ± 53.92 439.6 ± 64.14 93.86 ± 5.88 105.58 ± 4.64* 105.9 ± 4.75*

Col-0, wild type; gFW, gram fresh weight; tZ, trans-zeatin; tZR ,tZ riboside; tZRPs, tZ ribotides; cZ, cis-zeatin; cZR, cZ riboside; cZRPs, cZ ribotides; DZ, dihydrozeation; DZR, DZ riboside; DZRPs, DZribotide; iP, N6-(∆2-isopentenyl)adenine; iPR, iP riboside; iPRPs, iP ribotides; tZ7G, tZ-7-N-glucoside; tZ9G, tZ-9-N-glucoside; tZOG, tZ-O-glucoside; cZOG, cZ-O-glucoside; tZROG, tZR-O-glucoside;cZROG, cZR-O-glucoside; DZ9G, DZ-9-N-glucoside; iP7G, iP-7-N-glucoside; iP9G, iP-9-N-glucoside; N.D., not detected.

Shoots and roots were harvested from 3 week-old plants grown on soil and 2 week-old seedlings grown on 1/2MS vertical agar plates, respectively. Data are means ± standard deviation (n = 4).

Table S4. Cytokinin concentrations in CYP735A2 overexpressing transgenic plants, related to Figure 5

Root

*, significantly different from Col-0 as assessed by one-way ANOVA followed by Dunnett's test at P < 0.05.

Shootpmol/gFW

pmol/gFW (Scion) Col-0 cypDM cypDM Col-0(Stock) Col-0 cypDM Col-0 cypDM

tZ 0.77 ± 0.08a N.D. 0.6 ± 0.07ab 0.43 ± 0.13b

tZR 0.77 ± 0.07a 0.03 ± 0.02b 0.8 ± 0.09a 0.4 ± 0.16c

tZRPs 16.63 ± 5.03a 0.27 ± 0.06b 15.63 ± 1.56ac 7.28 ± 3.23bc

cZ 0.22 ± 0.22 0.45 ± 0.03 0.16 ± 0.05 0.43 ± 0.11cZR 0.47 ± 0.36 0.69 ± 0.11 0.48 ± 0.09 0.81 ± 0.16cZRPs 2.68 ± 1.33 2.16 ± 0.34 2.85 ± 0.55 3.93 ± 0.92DZ N.D. N.D. N.D. N.D.DZR 0.03 ± 0.02 0.01 ± 0.01 0.01 ± 0 0.02 ± 0.02DZRPs 0.08 ± 0.03 0.02 ± 0.01 0.06 ± 0.02 0.06 ± 0.03iP 0.47 ± 0.22a 0.53 ± 0.02a 0.12 ± 0.02b 0.38 ± 0.08a

iPR 0.47 ± 0.25 0.28 ± 0.05 0.71 ± 0.27 0.52 ± 0.14iPRPs 39.38 ± 1.61ab 30.73 ± 3.82bc 27.85 ± 2.32c 44.34 ± 5.84a

tZ7G 20.29 ± 5.06ab 0.89 ± 0.15c 23.63 ± 4.7a 12.4 ± 3.94b

tZ9G 9.14 ± 2.43a 0.72 ± 0.09b 9.79 ± 2.14a 5.42 ± 1.65ab

tZOG 4.7 ± 0.9a 0.5 ± 0.06b 5.3 ± 0.5a 4.32 ± 2.42a

tZROG 0.77 ± 0.11a 0.06 ± 0.01b 0.89 ± 0.1ac 0.65 ± 0.43abc

DZ9G 0.11 ± 0.05ab 0.01 ± 0.01a 0.13 ± 0.01b 0.1 ± 0.05ab

iP7G 17.17 ± 1.2a 59.28 ± 5.85b 17.54 ± 2.75a 26.09 ± 1.62a

IP9G 1.07 ± 0.06a 3.93 ± 0.49b 1.02 ± 0.14a 1.56 ± 0.18a

tZ-CK 53.06 ± 11.69a 2.47 ± 0.17b 56.64 ± 6.06a 30.91 ± 8.94c

iP-CK 58.57 ± 1.18a 94.76 ± 5.26b 47.24 ± 4.44a 72.89 ± 5.32c

cZ-CK 3.36 ± 1.91 3.3 ± 0.33 3.49 ± 0.66 5.16 ± 1.14Total 114.99 ± 14.77 100.53 ± 5.75 107.37 ± 11.16 108.97 ± 15.4

Table S5. Cytokinin concentrations in the scion of intergrafts between wild type and cyp735a1 cyp735a2, related to Figure 7Grafted plants between wild type (Col-0) and cyp735a1-2 cyp735a2-1 (cypDM) were grown for 45 days and rosette leaves wereharvested for cytokinin quantification. Data are means ± standard deviation (n = 4).

Different lower case letters indicate statistically significant differences (P < 0.01, Tukey's HSD test).

gFW, gram fresh weight; tZ, trans-zeatin; tZR ,tZ riboside; tZRPs, tZ ribotides; cZ, cis-zeatin; cZR, cZ riboside; cZRPs, cZ ribotides;

DZ, dihydrozeation; DZR, DZ riboside; DZRPs, DZ ribotide; iP, N6-(∆2-isopentenyl)adenine; iPR, iP riboside; iPRPs, iP ribotides; tZ7G,tZ-7-N-glucoside; tZ9G, tZ-9-N-glucoside; tZOG, tZ-O-glucoside; cZOG, cZ-O-glucoside; tZROG, tZR-O-glucoside; cZROG, cZR-O-glucoside; DZ9G, DZ-9-N-glucoside; iP7G, iP-7-N-glucoside; iP9G, iP-9-N-glucoside; N.D., not detected.

Supplemental Experimental Procedures

Plant materials

The cyp735a1-1 (SALK_063956), cyp735A1-2 (SALK_093028), cyp735a2-1

(SALK_077856), and cyp735a2-2 (SALK_028195) lines were obtained from the

Arabidopsis Biological Resource Center. The positions of T-DNA insertions were

determined to be in the fourth intron (position +2691/+2692) and the fifth exon

(position +3156/+3157) of the CYP735A1 coding sequence in cyp735a1-1 and

cyp735a1-2, respectively, and in the second intron (position +1170/+1171) and the

third intron (position +2622/+2623) of the CYP735A2 coding sequence in

cyp735a2-1 and cyp735a2-2, respectively. The nucleotide positions are reported

relative to the inferred initiation codon. Homozygous mutant plants were isolated by

PCR with gene- and T-DNA-specific primer sets. The AGI identifiers for the genes

described in this article can be found in The Arabidopsis Information Resource

database (see http://www.arabidopsis.org) under the following accession numbers:

ACT2 (AT3G18780), ACT8 (AT1G49240), ARR1 (At3g16857), ARR2 (At4g16110),

ARR3 (At1g59940), ARR4 (At1g10470), ARR5 (At3g48100), ARR6 (At5g62920),

ARR7 (At1g19050), ARR8 (At2g41310), ARR9 (At3g57040), ARR15 (At1g74890),

AHK2 (At5g35750), AHK3 (At1g27320), AHK4 (At2g01830), CYP735A1

(At5g38450), CYP735A2 (At1g67110), IPT3 (At3g63110), IPT5 (At5g19040), IPT7

(At3g23630).

The primer sequences for mutant isolation were:

cyp735a1-1

gene-specific

F (5' to 3') ATGTTGCTTACTATATTAAAATCACTCC

R (5' to 3') ATCCTCATGAAACCAATGGCTTC

cyp735a1-1

T-DNA-specific

F (5' to 3') GCGTGGACCGCTTGCTGCAACT


cyp735a1-2

gene-specific

F (5' to 3') TAACATAGTCATAGTCGGCACAGA


cyp735a1-2

T-DNA-specific

F (5' to 3') TAACATAGTCATAGTCGGCACAGA

R (5' to 3') GCGTGGACCGCTTGCTGCAACT

cyp735a2-1

gene-specific

F (5' to 3') ATGATGGTTACATTAGTACTAAAGTACG

R (5' to 3') CTTCATAGATCAAGTGGCTTC

cyp735a2-1

T-DNA-specific


R (5' to 3') CTTCATAGATCAAGTGGCTTC

cyp735a2-2

gene-specific

F (5' to 3') ATGAGTAAAGGTGATCTAGTTGAC

R (5' to 3') TATGTCTTCAAATGCCATTCTTGG

cyp735a2-2

T-DNA-specific


R (5' to 3') TATGTCTTCAAATGCCATTCTTGG

ahk2-2

gene-specific

F (5' to 3') GTCTATAACTTGTGAGCTCTTGAATC

R (5' to 3') GCTCGTGTCATAGACAGCAAAGGTC

ahk2-2

T-DNA-specific

F (5' to 3') GTCTATAACTTGTGAGCTCTTGAATC

R (5' to 3') ATAACGCTGCGGACATCTAC

ahk3-3

gene-specific

F (5' to 3') CTTGTGATTGCGTTACTTGTTGCAC

R (5' to 3') GCAGGCCTATGGTCCACAACCACAG

ahk3-3

T-DNA-specific

F (5' to 3') CTTGTGATTGCGTTACTTGTTGCAC

R (5' to 3') TGGTTCACGTAGTGGGCCATCG

cre1-12

gene-specific

F (5' to 3') GGAGAGCCTTCACCGGTTAGGG

R (5' to 3') AAGCTCTTGCATTTCATGGAAATC

cre1-12

T-DNA-specific

F (5' to 3') GGAGAGCCTTCACCGGTTAGGG

R (5' to 3') TGGTTCACGTAGTGGGCCATCG

Growth conditions

For studies on seedlings, wild type and mutants were grown on MGRL-based or

1/2MS vertical agar plates containing 1% sucrose (Inaba et al., 1994). Adult plants

were grown either on rockfiber blocks (Nittobo) or on soil (Metro-Mix 350, Sun Gro)

at 22°C under fluorescent light (100 µmol m-2 s-1, 16 h light/8 h dark). The

hydroponic culture was conducted as described (Taniguchi et al., 1998).

Arabidopsis transformation

Agrobacterium tumefaciens strain EHA105 was used as a host strain. Arabidopsis

plants were transformed using the floral dip method, as described elsewhere

(Clough and Bent, 1998). Transgenic seedlings were recovered on MS plates

containing 1% sucrose and 50 µg L–1 kanamycin.

Complementation analysis

Genomic fragments encompassing the putative promoter, coding region, and

terminator of the CYP735A genes (-2311 to +4315 for CYP735A1 and -2381 to

+4724 for CYP735A2 relative to the inferred initiation codon) were amplified with

PrimeSTAR GXL DNA polymerase (Takara), and the specific primer sets

gCYP735A1 and gCYP735A2, respectively. The fragments were cloned into pBI101

(Clontech) in place of the GUS gene, and introduced into the cyp735a1-1

cyp735a2-1 mutant.

To test the ability of externally applied cytokinins to complement the

cyp735a double mutant phenotype, wild-type and cyp735a1-2 cyp735a2-2 mutant

seeds were sown on soil. Cytokinins were applied daily by spraying solutions

containing trans-zeatin (tZ), N6-(∆2-isopentenyl)adenine (iP), or dihydrozeatin (DZ) at

various concentrations.

Transgenic plants overexpressing CYP735A2

The CYP735A2 cDNA was amplified with PrimeSTAR GXL DNA polymerase

(Takara) and the specific primer set CYP735A2-ox. The amplified fragment was

cloned into pBI121 (Clontech) linked to the cauliflower mosaic virus (CaMV) 35S

promoter, and introduced into wild-type Arabidopsis Col-0, generating CYP735A2-ox

transgenic lines.

Construction of promoter:GUS reporter lines

The CYP735A1 promoter (ProCYP735A1; -1358 to -1 bp relative to the inferred

initiation codon) and CYP735A2 promoter (ProCYP735A2; -3927 to -1 bp) were

amplified with PrimeSTAR GXL DNA polymerase (Takara) and the specific primer

sets ProCYP735A1 and ProCYP735A2, respectively. The fragments were

introduced into the pBI101 binary vector (Clontech) to generate the promoter:GUS

fusion genes, ProCYP735A1:GUS and ProCYP735A2:GUS. The fusion genes were

introduced into wild-type Arabidopsis Col-0, generating ProCYP735A1:GUS and

ProCYP735A2:GUS transgenic lines.

The primer sequences for vector construction

The primer sequences used for vector construction are:

Primer set Purpose Primer sequence

ProCYP735A1 GUS assay 5'-GTCGACAGAGCAAGGTTGTTTAACTCGG-3'

5'-GGATCCTTTTGGGTTTTTTCTTTTCTTCTTC-3'

ProCYP735A2 GUS assay 5'-CGTCTCGTCGACATGAATCACTTTTTTGGGT-3'

5'-CGTCTCGGATCCTTTTTAATTTCTTTGTTTTAT-3'

gCYP735A1 Complementation 5'-CTCGAGAAAACTAATTGTTTGTCTCTGG-3'

5'-CCATTTAATCCGACTAAGACC-3'

gCYP735A2 Complementation 5'-GCTTGTTTTCTCGAGATTATTATGATGTG-3'

5'-GTTTTAGCACACGATGAAAGTTCC-3'

CYP735A2-ox Overexpression 5'-TCTAGATGATGGTTACATTAGTACTAAAGTACG-3'

5'-GAGCTCCCTTCATAGATCAAGTGGCTTC-3'

Semi-quantitative and quantitative PCR

Semi-quantitative PCR was conducted using TaKaRa ExTaq (Takara) DNA

polymerase with the basic cycle conditions of 94°C for 30 sec, 56°C for 45 sec, and

72°C for 90 sec. To amplify double-stranded DNA in a semi-quantitative manner,

PCR reactions were performed with various cycle numbers. Quantitative PCR was

performed on a StepOnePlus Realtime PCR system (Applied Biosystems) with the

KAPA SYBR Fast qPCR kit (KAPA Biosystems).

The primer sequences for semi-quantitative and quantitative PCR are:

CYP735A1 Semi-quantitative 5'-ATGTTGCTTACTATATTAAAATCACTCC-3'

(At5g38450) 5'-ATCCTCATGAAACCAATGGCTTC-3'

CYP735A2

(At1g67110) Semi-quantitative

5'-ATGATGGTTACATTAGTACTAAAGTACG-3'

5'-CTTCATAGATCAAGTGGCTTC-3'

ACT2

(AT3G18780) Semi-quantitative

5'-TGTCCTCCTCACTTTCATCAGC-3'

5'-CATCAATTCGATCACTCAGAGC-3'

CYP735A2

(At1g67110) Quantitative

5'-ATGGTGTCCCTTCCGTTGAACA-3'

5'-GAGGGTAAAGTCTTAATGACTCGT-3'

ACT8

(AT1G49240) Quantitative

5'-AACATTGTGCTCAGTGGTGG-3'

5'-GTGGTGCCACGACCTTAATC-3'

ARR1


5'-GTGTTACCGACAAGTTACACTA-3'

5'-AGTTCTGGTTGGAAGTTGTTAT-3'

ARR2


5'-AACTACTCACTCGGTCTTCAAT-3'

5'-CTTGGTAAGAAACTGATACTGG-3'

ARR3


5'-GAATGTAATGACTCGTATCGAC-3'

5'-ACTTTAACGTCTCTCGTCAAGT-3'

ARR4


5'-GTTGACTGTTTCGACTGAATC-3'

5'-GTCATCATCTTCATCGTCTATC-3'

ARR5


5'-GCTGTTGATGATAGTATGGTTG-3'

5'-AGATATTGTAAAGCTCTTGTCG-3'

ARR6


5'-GAAGTTATGCTACCGAGGAAG-3'

5'-TACGATCAACGTGACTGTCGT-3'

ARR7


5'-AAGCCATTCTAACAAGAGAAAG-3'

5'-GAACATGAAGAGTCCTTGATAG-3'

ARR8


5'-GTGTCTAAACCGGAGATAGAAG-3'

5'-ACTACTCAACATTGGTTCAAGT-3'

ARR9


5'-TGCAACAAGATCTGCTATTAGT-3'

5'-TGCTCTATCAGTTGAAATCCC-3'

ARR15


5'-ATCTCCATCATCATCATCAAC-3'

5'-GACTCTAATTTGATCCTCTTGG-3'

Quantification of plant hormones

Extraction and determination of cytokinins, auxin (IAA), and abscisic acid from about

100 mg plant tissue were performed using an ultra-performance liquid

chromatograph coupled with a tandem quadrupole mass spectrometer equipped

with an electrospray interface as described previously (Kojima et al., 2009; Kojima

and Sakakibara, 2012). In the results reported, the category iP-type cytokinin

comprises iP, iP-riboside, iP-riboside 5’-phosphates, iP-7-N-glucoside, and

iP-9-N-glucoside; the category tZ-type cytokinin comprises tZ, tZ-riboside,

tZ-riboside 5’-phosphates, tZ-7-N-glucoside, tZ-9-N-glucoside, tZ-O-glucoside, and

tZR-O-glucoside; and the category cZ-type cytokinin comprises cZ, cZ-riboside,

cZ-riboside 5’-phosphates, cZ-O-glucoside, and cZR-O-glucoside.

Histological analysis

For Lugol's and propidium iodide staining, roots were treated as described previously

(Ueda et al., 2011). Fresh and GUS-stained samples were fixed and then embedded

in Technovit 7100 resin (Heraeus Kulzer) as instructed by the manufacturer.

Sections were produced using a Leica RM2165 microtome (Leica). Sections were

stained with Toluidine blue O when necessary, and observed under an Olympus

BX51 microscope (Olympus).

Chlorophyll assay

For the chlorophyll retention assay, Col-0 and cyp735a1-2 cyp735a2-2 seedlings

were grown on MS agar plates for 21 days. Fifth rosette leaves were floated on

water and kept in the dark at 22°C for the indicated periods. For the age-dependent

senescence assay, Col-0 and cyp735a1-2 cyp735a2-2 plants were grown on soil.

Chlorophyll quantification was performed as described previously (Porra et al.,

1989). Briefly, chlorophyll levels were determined by extraction with

N,N-dimethylformamide for 24 h in the dark followed by spectrophotometry.

Supplemental References

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16,

735-743.

Inaba, K., Fujiwara, T., Hayashi, H., Chino, M., Komeda, Y., and Naito, S. (1994).

Isolation of an Arabidopsis thaliana mutant, mto1, that overaccumulates soluble

methionine (temporal and spatial patterns of soluble methionine accumulation).

Plant Physiol 104, 881-887.

Kamada-Nobusada, T., and Sakakibara, H. (2009). Molecular basis for cytokinin

biosynthesis. Phytochemistry 70, 444-449.

Kojima, M., Kamada-Nobusada, T., Komatsu, H., Takei, K., Kuroha, T., Mizutani, M.,

Ashikari, M., Ueguchi-Tanaka, M., Matsuoka, M., Suzuki, K., et al. (2009). Highly

sensitive and high-throughput analysis of plant hormones using MS-probe

modification and liquid chromatography-tandem mass spectrometry: an application

for hormone profiling in Oryza sativa. Plant Cell Physiol 50, 1201-1214.

Kojima, M., and Sakakibara, H. (2012). Highly sensitive high-throughput profiling of

six phytohormones using MS-probe modification and liquid chromatography-tandem

mass spectrometry. Methods Mol Biol 918, 151-164.

Porra, R.J., W.A., T., and Kriedemann, P.E. (1989). Determination of accurate

extinction coefficients and simultaneous equations for assaying chlorophylls a and b

extracted with four different solvents: verification of the concentration of chlorophyll

standards by atomic absorption spectroscopy. Biochem Biophys Acta 975, 384-394.

Taniguchi, M., Kiba, T., Sakakibara, H., Ueguchi, C., Mizuno, T., and Sugiyama, T.

(1998). Expression of Arabidopsis response regulator homologs is induced by

cytokinins and nitrate. FEBS Lett 429, 259-262.

Ueda, M., Matsui, K., Ishiguro, S., Kato, T., Tabata, S., Kobayashi, M., Seki, M.,

Shinozaki, K., and Okada, K. (2011). Arabidopsis RPT2a Encoding the 26S

Proteasome Subunit is Required for Various Aspects of Root Meristem Maintenance,

and Regulates Gametogenesis Redundantly with its Homolog, RPT2b. Plant Cell

Physiol 52, 1628-1640.

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