Figure 1.
Structural organization of FaKNOX1 gene.
The schematic diagram represents the exon/intron boundaries of FaKNOX1 cDNA and its corresponding genomic sequence. The FaKNOX1 cDNA encodes a protein containing four domains: MEINOX domain, the GSE domain, the ELK domain and the homeodomain.
Figure 2.
Analyses of Class I KNOX genes.
(A) Alignment of FaKNOX1 amino acid sequence along with five representatives of Class I KNOX transcription factors. Black boxes indicate identical residues and the grey boxes indicate similar residues. The lines above the sequence mark the KNOX1, KNOX2, GSE and ELK domains and the homeodomain. As characteristic of TALE superclass transcription factors the homeodomain is represented by a three-amino-acid loop extension present between the first and second α-helices of the homeodomain. The letter ‘L’ and ‘T’ represent loop and turn, respectively. (B) The phylogenetic analysis of KNOX genes. Class II KNOX proteins were used as outgroup. Bootstrap values based on 1000 replicates are shown next to the branching points.
Figure 3.
Steady-state FaKNOX1 transcript accumulation patterns in various organs.
(A) Results obtained by RNA-gel-blot analysis and hybridization. (B) A corresponding comparative quantitative real-time PCR analysis. The flower sample was used as reference tissue and EF1a was used as reference gene. Error bars represent standard error of the mean derived from three replicates.
Figure 4.
Leaf development pattern of strawberry seedlings.
(A) The first four to five leaves of strawberry seedlings are simple, and then follow the familiar trifoliate leaves. (B) Development of strawberry (F. vesca) seedling showing the progression of leaf morphogeneisis from simple leaf to compound trifoliate leaf form. (C and D) Represent an enlarged view of developing stages of emerging trifoliate leaves (dotted regions on panel B). The arrow indicates the specific portion of leaf used for semi-quantitative RT-PCR analysis. (E) A comparative semi-quantitative RT-PCR showing that FaKNOX1 transcript was readily detectable in leaf samples harvested from 14 d to 47–50-d-old strawberry plants. Lanes 1–3 represent expression of FaKNOX1 in simple leaves of 14-d to 40-d-old seedlings and lanes 4–5 represent expression of FaKNOX1 in emerging trifoliate leaves of 45- and 47-d-old seedlings. FaUBQ was used as an internal control gene. Bars = 5 mm.
Figure 5.
In situ hybridization of FaKNOX1 in the vegetative meristem of strawberry seedlings.
The presence of FaKNOX1 is indicated by blue/black staining. Longitudinal section through the SAM of 2-leaf-stage seedling hybridized to a sense FaKNOX1 RNA probe (A) or antisense FaKNOX1 RNA probe (B). Longitudinal section through the SAM of 45-d leaf-stage seedling with emerging trifoliate leaf hybridized with a sense FaKNOX1 RNA probe (C) or an antisense FaKNOX1 probe (D and E). Bars = 1000 µm.
Figure 6.
(A) Phenotypic comparison of wild-type F. vesca plants with two representatives of the severe (M2) and conspicuous (M1) categories of FaKNOX1 RNAi transgenic lines. Representative of (B) M2 FaKNOX1 RNAi plants and (C) M1 FaKNOX1 RNAi plants. Bars = 1 cm.
Figure 7.
Leaf and flower morphology of a FaKNOX1 RNAi line.
(A) A wild-type leaf compared to different leaf forms of FaKNOX1 RNAi plants. The arrow indicates the leaf portion used for cross sections in Figure 7B. (B) Wild-type strawberry flower. (C) FaKNOX1 RNAi flower. Bars = 1 mm.
Figure 8.
Cellular organization of leaf of wild-type and FaKNOX1 RNAi plant.
Cross sections through the (A) wild-type expanded leaf, (B) M1 FaKNOX1 RNAi expanded leaf and (C) M2 FaKNOX1 RNAi expanded leaf to show the variation in the arrangement of different cell layers. AD, adaxial epidermis: PL, palisade layer; SL, spongy layer and AB, abaxial epidermis. Bars = 50 µm.
Figure 9.
Structural organization of the shoot apical meristem of wild-type and FaKNOX1 RNAi plants.
(A) Runner tip and emerging plantlet from the stolon of wild-type strawberry plants, (C) runner bud and (E) young developing plantlet from FaKNOX1 RNAi plants showing pale colored, narrow and elongated leaves. Transverse section through the shoot apical meristem of wild-type (B) and different stages from developing runner tip of FaKNOX1 RNAi plants (D and F). Asterisk indicates SAM. Bars = 50 µm.
Figure 10.
Arabidopsis plants overexpressing FaKNOX1.
Phenotype of wild-type Arabidopsis plants (A) and plants overexpressing FaKNOX1 (B and C). Overexpression lines present deeply lobed leaves, asymmetrical adaxial/abaxial growth, and a severe dwarf phenotype. (d) Flowers of FaKNOX1 overexpression lines show early abscission of petals and stamens that fail to develop (inset = wild-type flowers). Bars = 5 mm.
Figure 11.
Strawberry plants overexpressing FaKNOX1.
(A) Comparison of phenotypes of wild-type and FaKNOX1 overexpression transgenic plants. (B) Phenotype of wild-type leaf with leaf forms obtained from FaKNOX1 overexpression plant. (C) Comparison of phenotype of wild-type fruits with FaKNOX1 overexpressing fruit. Note that the petals of wild-type fruit have abscised prior to fruit expansion whereas the FaKNOX1 overexpression plants produce fruits where petals fail to abscise. Bars = 1 cm.