A model for restriction of cell proliferation by VLCFA synthesis.

<p>VLCFA synthesis in the epidermis confines cytokinin (CK) synthesis to the vasculature via non-autonomous signals, and restricts cell proliferation. VLCFA is also used for the synthesis of cuticular wax; thus, cuticle formation and cell proliferation are coordinately controlled by VLCFA synthesis during shoot development. Green and blue lines represent the epidermis and the vasculature, respectively.</p

Quantification of phytohormones.

<p>Amounts of phytohormones were measured in 3- and 5-d-old wild-type seedlings grown in the absence (w/o) or presence of cafenstrole (30 nM or 3 µM). <i>pas2-1</i> was grown without cafenstrole. Data are presented as mean ± SD (<i>n</i>≥3).</p><p>DAG, days after germination; IAA, indoleacetic acid; n.d., not detected.</p

Overproliferation phenotypes of <i>pas2-1</i> mutants.

<p>(A) True leaf of a 2-wk-old <i>pas2-1</i> mutant seedling. (B) 5-d-old wild-type and <i>pas2-1</i> seedlings. (C) Cross sections of 5-d-old hypocotyls. (D) Transverse sections of shoot apices of 7-d-old seedlings. Bars, 1 mm (A), 500 µm (B), and 100 µm (C, D).</p

Reduced VLCFA synthesis increases the expression of <i>IPT3</i>.

<p>Expression patterns of <i>ProIPT3:GUS</i>. Aerial parts of 5-d-old seedlings grown in the absence or presence of cafenstrole (A), and transverse sections of shoot apices (B) and cotyledons (C). <i>pas2-1</i> grown in the absence of cafenstrole is shown for comparison. <i>ad</i>, adaxial side of cotyledons. Bars, 1 mm (A) and 100 µm (B, C).</p

<i>PAS2</i> expression in the epidermis is essential for plant development.

<p>(A) Quantification of <i>PAS2</i> expression levels in 5-d-old seedlings. The mRNA levels were normalized to <i>TUBULIN4</i>. The expression level in wild-type expressing <i>ProATML1:PAS2RNAi</i> is indicated as a relative value, with that in wild-type set to 1. Data are presented as mean ± SD (<i>n</i> = 3). (B) 5-d-old seedlings of wild-type and wild-type expressing <i>ProATML1:PAS2RNAi</i> with a severe phenotype. Transverse sections of shoot apices of 7-d-old seedlings are shown in (C). (D) 5-d-old seedlings of <i>pas2-1</i> and <i>pas2-1</i> expressing <i>ProATML1:PAS2–GUS</i>. (E) Transverse section of the shoot apex of a 5-d-old <i>pas2-1</i> seedling expressing <i>ProATML1:PAS2–GUS</i>. Bars, 1 mm (B, D), 200 µm (C), and 100 µm (E).</p

Cytokinin content and cell proliferation in LCFA- and VLCFA-related mutants.

<p>(A) Biosynthetic pathways for producing VLCFAs and cuticular wax. Enzymes and regulatory factors associated with each step are indicated. (B) Cytokinin content in various mutants. Amounts of tZ and iP were measured in 7-d-old whole seedlings, while 14-d-old seedlings were used for <i>fdh-13</i> due to phenotype-dependent identification of homozygous plants in the segregating generation. The tZ and iP levels are indicated as relative values, with those in wild-type (L<i>er</i> for <i>cer4-1</i> and <i>fdh-13</i>, and Col-0 for the others) set to 1. Data are presented as mean ± SD (<i>n</i> = 3). (C) Transverse sections of shoot apices of 7-d-old seedlings. <i>fdh-13</i> and its control (L<i>er</i>) were observed with 10-d-old seedlings. Bar, 100 µm.</p

Phenotypes of cafenstrole-treated seedlings.

<p>(A) 12-d-old wild-type seedlings grown in the absence (w/o) or presence of cafenstrole (30 nM or 3 µM). <i>pas2-1</i> grown in the absence of cafenstrole is shown for comparison. Lower images show the upper region of hypocotyls. (B) Magnified view of a true leaf of 2-wk-old wild-type seedlings grown in the presence of 3 µM cafenstrole. (C) Cross sections of 7-d-old hypocotyls grown in the absence (w/o) or presence of 30 nM cafenstrole. (D) Transmission electron microscopy analysis of the L1 layer of the SAM. 3-d-old wild-type seedlings grown in the absence (w/o) or presence of 30 nM or 3 µM cafenstrole were observed. Note that, in <i>pas2-1</i>, the L1 layer is not covered with cuticular wax. Arrows indicate the electron-dense cuticular layer. Bars, 2 mm (A, upper panel), 500 µm (A, lower panel), 1 mm (B), 100 µm (C), and 2 µm (D).</p

Epidermis-specific expression of <i>PAS2</i>.

<p>(A–C) GUS staining of transgenic plants carrying <i>ProPAS2:GUS</i>. Transverse section of the shoot apex of a 5-d-old seedling (A), and cross section of the shoot apex of a 10-d-old seedling (B) and inflorescence stem of a 3-wk-old seedling (C). (D) In situ hybridization of <i>PAS2</i>. A transverse section of the shoot apex of a 7-d-old wild-type seedling was hybridized with a <i>PAS2</i> antisense probe. (E) Expression pattern of <i>ProPAS2:PAS2–GUS</i> in the shoot apex of a 5-d-old seedling. Bars, 100 µm.</p

Cafenstrole-induced leaf expansion is suppressed in the <i>ipt3;5;7</i> mutant.

<p>(A) 11-d-old seedlings of wild-type and the <i>ipt3;5;7</i> triple mutant grown in the absence (−) or presence (+) of 30 nM cafenstrole. Bar, 5 mm. (B) First leaves of 11-d-old seedlings grown in the absence (white bars) or presence (green bars) of 30 nM cafenstrole were measured for leaf blade area, cell area, and cell number. Data are presented as mean ± SD (<i>n</i>≥13). Significant differences between non-treatment and 30 nM cafenstrole treatment were determined by Student's <i>t</i>-tests: ***, <i>p</i><0.001; the other differences are not significant (<i>p</i>>0.05).</p

Reduced VLCFA synthesis increases the expression of <i>CDKB2;1</i> and <i>ARR6</i>.

<p>(A) Transverse sections of shoot apices of 5-d-old seedlings expressing <i>ProCDKB2;1:NT–GUS</i>. <i>pas2-1</i> grown in the absence of cafenstrole is shown for comparison. (B, C) Expression patterns of <i>ProARR6:GUS</i>. Aerial parts of 5-d-old seedlings grown in the absence (w/o) or presence of cafenstrole (30 nM or 3 µM) (B) and transverse sections of shoot apices (C). Bars, 100 µm (A, C), and 1 mm (B).</p