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First published online 13 August 2003
doi: 10.1242/dev.00644

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Involvement of auxin and a homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens

Keiko Sakakibara1,2, Tomoaki Nishiyama1, Naomi Sumikawa1, Rumiko Kofuji1,*, Takashi Murata1 and Mitsuyasu Hasebe1,2,{dagger}

1 National Institute for Basic Biology, Okazaki 444-8585, Japan
2 Department of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan



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  		Fig. 4. Genomic structures of Pphb7 in wild type and transformants and northern analysis of Pphb7. Genomic structure of Pphb7 in wild type (A), Pphb7-GUS (B), GFP-Pphb7 (C) and Pphb7 disruptant (D). The boxes and the lines between the boxes indicate the exons and introns. The circle and square indicate putative start and stop codons. The gray and black boxes indicate a homeodomain and a leucine-zipper motif. The uidA, sGFP, nos-ter, and nptII designate the uidA-coding region, GFP-coding region, nopaline synthase polyadenylation signal, and NPTII expression cassette, respectively. Each bracket above the genome structure indicates the region contained in the plasmid used for gene targeting. P, PmaCI;, S, SalI; X, XbaI; Ba, BamHI; Bg, BglII; E, EcoT14I. (E) Northern analysis of Pphb7. Poly(A)+ RNA (1.0 µg) from protonemata (P) and gametophores with rhizoids (G) of wild type was hybridized with cPphb7 probe in (A). As an internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a probe. (F-H) Southern analyses to confirm gene targeting. Genomic DNA of wild type (wt) and transformants was digested with BglII (F,H) or EcoT14I (G), and the gPphb7 probe was used. (F) Pphb7-GUS-1 and -2 lines (lanes 1,2); (G) GFP-Pphb7-1 to -10 lines (lanes 1-10); (H) Pphb7dis-1 to -5 lines (lanes 1-5).

 


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  		Fig. 1. Rhizoid development in the wild type. (A) A juvenile and (B) an adult leaf. (C) A juvenile gametophore with the first rhizoid (arrow). (D) An adult gametophore with 17 leaves. Lines G, K, L and M indicate transverse sections in G, K, L and M. The white and black arrows indicate the uppermost mid-stem rhizoid and the uppermost juvenile leaf, respectively. (E) A mid-stem rhizoid formed below an adult leaf. (F) The midrib cells (pink) and a mid-stem rhizoid (green). (G-M) Transverse sections of a gametophore with 18 leaves. The asterisk in G indicates the abaxial epidermal cell of a midrib in the same longitudinal cell file as the uppermost mid-stem rhizoid. (H) A line drawing of G showing hydroids in the center of the stem (red), leaf traces (green) and a midrib (blue). (I) A higher magnification of the midrib. (J) A line drawing of I showing epidermal cells (light blue), stereids (yellow), hydroids (pink) and deuters (orange). The asterisk in K indicates the uppermost mid-stem rhizoid. The single and double asterisks in L indicate the first and the second mid-stem rhizoid, respectively. The arrows in K and L indicate leaf trace cells. Scale bars: 100 µm for A,F,G,K-M; 250 µm for B,E; 50 µm for C; 1 mm for D; 20 µm for I.

 


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  		Fig. 2. The effects of exogenous auxin in wild type. (A) A gametophore cultured in 1 µM NAA for a week with adventitious gametophore (white arrow). (B) The uppermost mid-stem rhizoid of the gametophore (white arrow) in A. The black arrow indicates a midrib. (C-E) Gametophores grown with 0.1 (C), 1.0 (D), and 10 (E) µM NAA for 6 weeks. The positions of the transverse sections in F-I is indicated by the lines. The asterisks and arrows in F and G indicate rhizoids and cells of the leaf traces, respectively. Scale bars: in A, 1 mm for A; in C, 1 mm for C-E; in B, 100 µm for B; in F, 100 µm for F-I.

 


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  		Fig. 3. Rhizoid cells of wild type and Pphb7 disruptant. (A) A wild-type rhizoid composed of 4 cells, viewed with Nomarski optics. The single asterisk indicates a rhizoid apical cell and the double asterisk indicates a rhizoid subapical cell. (B-G) A rhizoid composed of 8 cells in wild type (B-D) and Pphb7dis-3 (E-G). The apical cell (B,E), third cell (C,F), and fifth cell (D,G) viewed with Nomarski optics (right), and imaged under UV excitation (left). The arrowheads indicate septa between the cells. Scale bar: in A, 100 µm for A; in B, 50 µm for B-G.

 


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  		Fig. 5. Histochemical detection of GUS activity in Pphb7-GUS-1 line. A juvenile gametophore without (A) and with (B) a basal rhizoid protrusion (arrow). (C) A gametophore cultured for 6 weeks on G medium. (D) Epidermal cells of an adult gametophore stem. (E) A mid-stem rhizoid initial cell with protrusion. (F) The apical and subapical cells of a rhizoid with 8 cells. (G,H) Apical parts of a chloronema (G) and a caulonema (H). (I) Gametophores cultured in 10 µM NAA solution for 6 hours. Scale bars: in A, 50 µm for A,B; in C, 1 mm for C,I; in D, 50 µm for D; in E, 25 µm for E; in F, 100 µm for F; in G, 50 µm for G,H.

 


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  		Fig. 6. Subcellular localization of the GFP-Pphb7 fusion protein. (A-D) A rhizoid from GFP-Pphb7-1. The apical (A), second (B), third (C) and fourth (D) cells were examined using Nomarski optics (left), by GFP fluorescence (middle), and by chlorophyll fluorescence (right). (E-H) A rhizoid that expressed an intact GFP under the control of the GH3 promoter (E-G) and a wild-type rhizoid (H-J). The apical (E,H), third (F,I), and fourth (G,J) cells were examined using Nomarski optics (left) and GFP fluorescence (right). Scale bar: 50 µm for all panels.

 


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  		Fig. 7. Effects of phytohormones, dehydration, and osmotic stress on Pphb7 expression. Pphb7 expression in gametophores treated with (A) exogenous NAA, (B) other phytohormones (BA, GA and ABA) and (C) dehydration and osmotic stress (6% mannitol). GAPDH was used as an internal control. Relative expression levels are indicated below each panel.

 


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  		Fig. 8. Rhizoids of the wild-type and Pphb7 disruptant lines. (A) Gametophores with 12 leaves in the wild type (left), Pphb7dis-2 (middle), and Pphb7dis-3 (right). (B-D) A Pphb7dis-3 gametophore with 18 leaves was serially sectioned at the position of the leaf above the uppermost mid-stem rhizoid (B), where the leaf merges to a stem (C), and where the uppermost mid-stem rhizoid (asterisk) emerges (D). The abaxial leaf epidermal cells (asterisk in B) and the stem epidermal cells (asterisk in C) in the same longitudinal cell file as the uppermost mid-stem rhizoid. Arrows in C indicate leaf traces. Gametophores of the Pphb7dis-3 grown for 6 weeks in the presence of 0.1 (E), 1.0 (F) and 10 (G) µM NAA. (H,I) Merged images of chlorophyll autofluorescence by UV excitation and SYBR Green I staining of chloroplasts in the fifth cell from the rhizoid apical cell of wild type (H) and Pphb7dis-3 (I). The yellow spots are chloroplast nucleoids. (J,K) Transmission electron micrographs of rhizoid chloroplasts from wild type (J) and Pphb7dis-3 (K). Scale bars: in A, 1 mm for A; in E, 1 mm for E-G; in B, 100 µm for B-D; in H, 2.5 µm for H,I; in J, 0.5 µm for J,K.

 


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  		Fig. 9. Comparison of the rhizoid characters of wild type and Pphb7 disruptant with or without NAA. (A) Frequency distributions of the light transmittance of rhizoids in the wild-type (white bars) and Pphb7dis-3 (gray bars) gametophores. The red, blue, and green channels from RGB images are shown. The means and standard deviations are indicated in brackets (n=100). (B) The number of chloroplasts per cell at each cell position from the rhizoid apical cell in the wild type (white circles) and Pphb7dis-3 (gray circles) (n=10). Bars indicate the standard deviations. (C) Frequency distribution of chloroplast size in rhizoid cells from the wild type (white bars) and Pphb7dis-3 (gray bars). The means and standard deviations are indicated in brackets (n=100).

 


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  		Fig. 10. Expression of the genes involved in photosynthesis. RT-PCR analyses of transcripts of photosynthesis genes in the wild type and Pphb7 disruptants. The number of PCR cycles is indicated to the right of each panel. Gametophores of the wild type (lane 1), rhizoids of the wild type treated without (lane 2) or with (lane 5) NAA, and rhizoids of Pphb7dis-2 and -3 treated without (lanes 3 and 4) or with (lanes 6 and 7) NAA.

 


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  		Fig. 11. A model of rhizoid development. (A) Arrows and barred lines indicate the positive and negative interactions, respectively. (B) An explanation for the different developmental patterns observed between mid-stem and basal rhizoids. The arrows indicate the hypothetical flow of an unknown factor.

 

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© The Company of Biologists Ltd 2003