Article Figures & Data
Figures
- Figure 1.
Positional Cloning of the IFL1 Gene.
(A) Diagram of the YAC (yUP7H9 and yUP17C2) and BAC (MIUP24) clones spanning the region in which the markers 7H9L and 17C2 are located. The ifl1 locus previously was mapped to a region between 7H9L and 17C2.
(B) Fine mapping of the ifl1 locus. The BAC clone MIUP24 was used to develop CAPS markers for fine mapping the ifl1 locus. The ifl1 locus was narrowed to a 0.3-centimorgan (cM) region covered by markers mup24RI-6A and mup24B2.
(C) DNA fragments used for complementation of the ifl1 mutation. Four overlapping DNA fragments covering the IFL1 locus were introduced into the ifl1 mutant. It was found that the DNA fragment B completely rescued the ifl1 mutation. (–) indicates no rescue; (+) indicates a complete rescue of the ifl1 mutation. The numbers in the parentheses indicate the number of plants with the indicated phenotype out of the total number of transgenic plants examined.
- Figure 2.
The 10-kb DNA Fragment B Rescues the Phenotypes of the ifl1 Mutant.
(A) A cross-section of an ifl1 stem showing the absence of normal interfascicular fibers. The arrow indicates the position at which interfascicular fibers are normally formed.
(B) An ifl1 mutant plant showing the pendent stem phenotype.
(C) A cross-section of a stem from an ifl1 mutant plant transformed with DNA fragment B showing the presence of normal interfascicular fibers.
(D) An ifl1 mutant plant transformed with the DNA fragment B showing a wild-type phenotype.
co, cortex; e, endodermis; if, interfascicular fiber; ph, phloem; pi, pith; s, sclerenchyma; x, xylem. Bars in (A) and (C) = 5 μm.
- Figure 3.
Structure of the IFL1 Gene and the Nature of the Mutations in the ifl1 Alleles.
(A) A schematic representation of the exon and intron organization of the IFL1 gene. The IFL1 gene is 4193 bp long, starting from the start codon (designated nucleotide 1) to the stop codon (designated nucleotide 4193). Single nucleotide mutations were found in both ifl1 (the G at nucleotide position 2086 is changed to an A) and ifl1-2 (the C at nucleotide position 635 is changed to a T). Black boxes denote exons. Lines between black boxes denote introns.
(B) Effect of the single nucleotide mutation in the ifl1 mutant on the translation of the predicted protein. Shown are residues around the mutation site. The G-to-A transition at the intron splicing acceptor site resulted in aberrant splicing. This aberrant splicing leads to a coding frameshift, thus generating a premature stop codon at the eighth codon after the mutation site. The mutated nucleotides are indicated by asterisks. Boxed sequences are introns that are spliced out in the mature mRNA. Dots denote nucleotides in the intron.
(C) Effect of the single nucleotide mutation in the ifl1-2 mutant on the translation of the predicted protein. Shown are residues around the mutation site. The C-to-T transition creates a nonsense mutation. The mutated nucleotides are indicated by asterisks.
(D) Creation of a new MseI site in the mutant ifl1 gene. A DNA fragment covering the ifl1 mutation site was PCR-amplified from the wild type and the ifl1 mutant genomic DNA. After digestion with MseI, DNA fragments were separated on an agarose gel containing ethidium bromide and visualized under UV light. The DNA fragment amplified from the ifl1 mutant was cut into two pieces due to the creation of a new MseI site (TTAG is changed to TTAA) by the ifl1 mutation (lane i), whereas the DNA fragment amplified from the wild type was not cut (lane w).
- Figure 4.
Amino Acid Sequence Deduced from the IFL1 cDNA.
The GenBank accession number for the IFL1 cDNA sequence data is AF188994.
- Figure 5.
Comparison of the Domains of the IFL1 Protein and the Other Group III HD-ZIP Proteins.
(A) Alignment of the amino acid sequences in the HD and ZIP motifs. Amino acid residues identical to the ones in IFL1 are marked with asterisks. Amino acid sequences involved in the formation of the helix-turn-helix structure in the HD of IFL1 are boxed. The invariant amino acid residues in the HD are marked with solid triangles. The ZIP motif is labeled, and the leucine residues that occur at every seventh position in the ZIP motif are marked with open triangles.
(B) Diagrams of the domains of IFL1 and the other group III HD-ZIP proteins. All of these proteins are similar in length, and they have the same domain organization. The names of individual domains are indicated above the IFL1 diagram. The numbers below each diagram indicate the amino acid positions of each domain.
(C) Diagrams of the domains of the truncated ifl mutant proteins. The deduced ifl1 mutant protein contains intact HD and ZIP motifs and a partial (244 amino acid residues) C-terminal region. The deduced ifl1-2 mutant protein contains an intact HD and most of the ZIP motif (one ZIP repeat is missing). The entire C-terminal region is missing in the ifl1-2 mutant protein. Gln110Stop denotes the glutamine at residue 110 in the wild-type protein that was changed to a stop codon in the ifl1-2 mutant protein.
- Figure 6.
Nuclear Localization of the IFL1-GUS Fusion Proteins in Onion Epidermal Cells.
Full-length IFL1 or different domains of IFL1 were fused in-frame to the N terminus of GUS in the expression vector pBI221. Plasmid DNA was delivered into onion cells by using a particle bombardment procedure. After 16 hr of incubation, cells were stained for GUS activity (shown as blue) and observed under a differential interference contrast microscope.
(A) The full-length IFL1-GUS fusion protein showing GUS staining only in the nucleus.
(B) The HDZ-GUS fusion protein showing GUS staining only in the nucleus.
(C) The C-terminal region-GUS fusion protein showing GUS staining in the cytoplasm.
(D) The control GUS protein showing staining in the cytoplasm.
- Figure 7.
RNA Gel Blot Analysis of IFL1 Gene Expression in Arabidopsis Organs.
Total RNA was isolated from different organs of Arabidopsis plants and used for RNA gel blot analysis. The gene-specific probe containing the 5′; untranslated region of the IFL1 gene was used to probe the blot. Equal loading of RNA in each lane was confirmed by hybridizing the blot with the 18S rDNA probe. The seedlings were 3 weeks old. Mature leaves, roots, and flowers were from 8-week-old plants. Stems I and II were from 4- and 8-week-old plants, respectively.
- Figure 8.
Expression Pattern of the IFL1 Gene Revealed by Using the IFL1 Promoter Activity Assay and in Situ mRNA Localization.
The 2.3-kb IFL1 gene promoter was inserted upstream of the GUS coding sequence in the expression vector pBI121. Agrobacteria containing the expression construct were used to transform Arabidopsis plants, and transgenic plants were selected and used for GUS activity assays. GUS activity is revealed by blue staining. Signals corresponding to mRNA localization appear as black patches that result from the silver enhancement.
(A) and (B) Sections from the upper (A) and lower (B) parts of a stem from a 5-week-old transgenic plant. GUS staining was mainly associated with interfascicular regions and vascular bundles.
(C) to (E) A young root (C), a young leaf (D), and a cotyledon (E) showing GUS staining in vascular strands.
(F) In situ localization of IFL1 mRNA showing signals in the interfasicular regions and vascular bundles in a stem section.
(G) A control section incubated with the sense IFL1 RNA probe showing the absence of signals.
co, cortex; ir, interfascicular region; v, vein; vb, vascular bundle. Bar in (A) = 11 μm for (A), (B), (F), and (G).
- Figure 9.
Effects of the ifl Mutations on the Vascular Differentiation in Inflorescence Stems.
Thin sections were prepared from stems of 7-week-old wild-type, ifl1, and ifl1-2 plants and stained with toluidine blue to reveal anatomy.
(A) to (C) Sections from the top segments of the stems from the wild-type (A), ifl1 (B), and ifl1-2 (C) plants. No xylary fibers were evident in the vascular bundles in all of these plants.
(D) to (F) Sections from the middle segments of the stems from the wild type (D), ifl1 (E), and ifl1-2 (F) plants. Xylary fibers were evident in both the wild-type and the ifl1 plants but not in the ifl1-2 plants.
(G) to (I) Sections from the basal segments of the stems from the wild type (G), ifl1 (H), and ifl1-2 (I) plants. In the bottom parts of the stems, although the vascular bundles in the stems of the wild-type plants developed many xylary fibers, the vascular bundles in the stems of both ifl1 and ifl1-2 mutant plants were devoid of xylary fibers.
if, interfascicular fiber; ph, phloem; v, vessel element; X, xylem; xf, xylary fiber. Bar in (A) = 5 μm for (A) to (I).
