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First published online 2 March 2005
doi: 10.1242/dev.01709

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Diversification of gene function: homologs of the floral regulator FLO/LFY control the first zygotic cell division in the moss Physcomitrella patens

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
² National Institute for Basic Biology, Okazaki 444-8585, Japan
³ Department of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan

View larger version (20K): [in a new window]   Fig. 1. Genomic structures of PpLFY1 and PpLFY2 in the wild type and the targeted lines. The gene structures of the wild-type PpLFY1 (A) and PpLFY2 (B). Restriction enzyme recognition sites are indicated on each gene: b, BglII; e, EcoRI. In A,B,D-G the boxes indicate exons and the lines between the boxes, introns; circles, putative start codons and squares, putative stop codons. The DNA fragments that were used as probes in the Southern analyses are indicated with thick lines below each gene in A and B. (C) Southern analyses of PpLFY1 and PpLFY2 in the wild type. The restriction enzymes, probes, and stringency used are indicated above or below each image. (D-G) Genomic structures of PpLFY1 (D,F) and PpLFY2 (E,G) in PpLFY1-GUS lines (D), PpLFY2-GUS lines (E), PpLFY1-dis and PpLFY1-PpLFY2-dis lines (F), PpLFY2-dis and PpLFY1-PpLFY2-dis lines (G). uidA, uidA coding region; nos-ter, nopaline synthase polyadenylation signal; NPTII, NPTII expression cassette; HPT, HPT expression cassette. The sense directions of uidA, nptII, and hpt are indicated with arrows. The scale bar applies to A,B,D-G.

View larger version (53K): [in a new window]   Fig. 2. Expression patterns of PpLFY1 and PpLFY2 mRNA, analyzed by semi-quantitative RT-PCR. The tissues analyzed and the number of PCR cycles used are indicated above each photograph. The glutaraldehyde dehydrogenase gene (GAPDH) was used as a control for the amount of template DNA added.

View larger version (110K): [in a new window]   Fig. 3. Histochemical detection of GUS activity in PpLFY1-GUS lines (A-L) and PpLFY-2-GUS lines (M-AB). (A,M) A young gametophore. (B,N) A gametophore with leaves. The apices of the main (arrow) and lateral shoots (arrowhead) are indicated. (C-F,O-R) A developing archegonium. GUS activity was detected in the egg cell (arrow) as well as in the archegonium tissue. (G,S) Antheridia. The tissue with GUS activity is the shoot apex. (H,T) A young sporophyte (bracket). (I,U-W) A young sporophyte isolated from an archegonium. (J) A young sporophyte with aberrant morphology. (K) An abnormal sporophyte with double sporangia. (L,X-AB) A developing sporophyte. The upper, middle, and lower parts differentiate into the sporangium (Sp), the seta (Se) and the foot (F), respectively (X). Scale bars: 50 µm in A,C-M, O-AB; 1 mm in B,N.

View larger version (50K): [in a new window]   Fig. 4. The phenotypes of the single and double disruptants of the PpLFY1 and PpLFY2 genes. (A) A sporophyte (arrow) is formed on the shoot apex of a gametophore. (B-E) Gametophores of wild-type P. patens (B), PpLFY1-dis-1 (C), PpLFY2-dis-1 (D), and PpLFY1-PpLFY2-dis-2 (E) lines, photographed from above. The sporangia are ocher to dark brown spheres, two of which are indicated in each panel by arrowheads. Scale bars: 1 mm.

View larger version (71K): [in a new window]   Fig. 5. Development of the egg cell, zygote and embryo. Ventral parts of open archegonia in the wild type (A-G) and the PpLFY double disruptants (H-K) were observed by light microscopy of a semi-thin section (A) or by CLSM (B-K). Nuclei (A,B,D-H,J,K, arrows) and sperm (C,I, arrowheads). White and brown boxes below images represent uncolored (A,B,H) and brown-colored (C-G,I-K) neck canals. (A-C,H,I) An egg cell before (A,B,H) or after (C,I) sperm invasion. (D,J) An unexpanded zygote. (E) An expanded zygote at the first cell division. (F) A two-cell embryo. (G,K) An unfertilized egg cell. Scale bars: 10 µm in A, 25 µm in B-K.

View larger version (111K): [in a new window]   Fig. 6. Development of sporophytes. Sporophytes of the wild type (A-D) and the PpLFY double disruptants (E-K) were observed by light microscopy of the 5 µm sections (A,B,D,H-K) or by stereoscopic microscopy (C,E-G). (A,B) A developing sporophyte (bracket) within an archegonium venter. (C,D) A mature sporophyte. A columella (co; tissue within the double-headed arrow) and a capsule wall (cw; the layer of cells between the two arrows) were differentiated. (E-G) An aberrant sporophyte. The arrow in E indicates an undifferentiated archegonium. (F) A sporophyte with double sporangia. (G) A withered and dehisced sporophyte. (H) A young sporophyte (bracket) that is arrested in development within an archegonium. (I-K) Sporophytes with an abnormal sporangium, seta and foot in abnormal morphology. Double-headed arrows indicate aberrant columella (co) and capsule walls (cw) differentiated (J,K). Single headed arrows indicate differentiated spores. Scale bars: 100 µm.