Article Figures & Data
Figures
- Figure 1.
Structure of the T-DNA Insertional Mutants.
(A) Structure of the myb33 allele.
(B) Structure of the myb65 allele.
Arrows indicate the location of the neomycin-phosphotransferase II gene (K) and the uidA gene driven by the −60 35S Cauliflower mosaic virus promoter (G). LB, left border; RB, right border; R1, EcoRI; H3, HindIII. As determined by sequencing the T-DNA/plant DNA junctions, the 10 nucleotides of Arabidopsis DNA flanking the T-DNAs are shown.
- Figure 2.
Characteristics of the Male Sterile Phenotype of myb33 myb65.
(A) Flowers of the Columbia-0 ecotype photographed under bright-field microscopy.
(B) Flowers of the myb33 myb65 double mutant, photographed under bright-field microscopy.
(C) Three different bolts of myb33 myb65 that exhibit the sporadic setting of siliques, where the majority of siliques fail to set any seeds, but occasionally fully or partially filled siliques are set. Bolts are from plants grown at 22°C in a 12-h-day/12-h-night cycle and ∼90 μmol·m−2s−1 light intensity.
- Figure 3.
Comparison of the Development of Locules from Wild-Type and myb33 myb65 Plants Using Toluidine Blue Staining.
(A) to (E) Development in wild-type anthers.
(A) Early microsporocyte stage, with the pollen mother cells (pmc) surrounded by the four distinct cell layers, the epidermis (ep), endothecium (en), middle layer (m), and tapetum (t).
(B) PMCs begin to separate from one another and the locular space beginning to form.
(C) Tetrad stage, with the individual microspores separated by callose.
(D) Microspore stage. The tapetum shows signs of degradation and has a granular appearance.
(E) Mature pollen stage (tricellular pollen) before release from the anther. The tapetum has degenerated.
(F) to (J) Development in myb33 myb65 anthers.
(F) Development at the early microsporocyte stage appears indistinguishable from the wild type.
(G) At PMC stage, the tapetum (t) begins to enlarge.
(H) The locule becomes crowded, and the PMCs have an irregular shape.
(I) The tapetum continues to expand, and the PMCs begin to degrade.
(J) The tapetum occupies the majority of the locule, and the PMCs have degenerated.
Anthers are from plants grown at 22°C in a 12-h-day/12-h-night cycle and ∼90 μmol·m−2s−1 light intensity. All bars = 10 μm.
- Figure 4.
Further Microscopic Analysis of myb33 myb65 Anthers.
(A) Toluidine blue staining of a section showing the alternative developmental pathways within locules from the same myb33 myb65 anther.
(B) Alaline blue staining for callose in myb33 myb65 at the PMC stage under dark-field microscopy. Fluorescence indicates the presence of callose.
(C) Staining for callose in myb33 myb65 at the microspore stage. Wild-type development has proceeded in the locule on the left (no callose), whereas development has aborted in the locule on the right (callose present).
Anthers are from plants grown at 22°C in a 12-h-day/12-h-night cycle and ∼90 μmol·m−2s−1 light intensity. All bars = 10 μm.
- Figure 5.
Structure of the Constructs Used for Molecular Complementation, for MYB33 Expression Analysis, and for Overexpresion of MYB33 and mMYB33 Genes.
(A) Structure of the Landsberg erecta MYB33 genomic clone used for molecular complementation. This clone contains 1390 bp upstream of the putative transcription start site (arrow), which includes all the 5′ untranscribed regions of MYB33 and into the coding region of the upstream gene (At5g06090; the stop codon being −1833 relative to MYB33 start codon), 660 bp of 5′ untranslated region, including the first two introns (denoted I and II), 1833 bp of open reading frame, including two introns (denoted III and IV), and 611 bp of sequences 3′ of the MYB33 stop codon, which extends downstream 12 bp before the stop codon of the next gene (At5g060110; stop codon is +624 bp relative to MYB33 stop codon). Untranscribed regions and introns are shown as thin lines, untranslated regions as open boxes, and coding regions as closed boxes. Numbers shown are relative to the MYB33 start codon. The location of the miR159 target is indicated by a solid line. This construct was also used to overexpress MYB33 in wild-type plants.
(B) The Pro33:GUS transgene in the vector pBI101.1.
(C) The Pro33:GUS:3′end transgene in the vector backbone pBI101.1
(D) The 5′-MYB33:GUS transgene in the vector backbone pBI101.1.
(E) The MYB33:GUS transgene made in the vector pPZP200-Hygro.
(F) The mMYB33:GUS transgene made in the vector pPZP200-Hygro. The alteration to the nucleotide sequence in the miRNA target motif is shown, and nucleotides underlined are those differing from the wild type. The unaltered amino acid sequence is shown. The mMYB33 gene is identical but lacks the GUS reporter gene. H3, HindIII.
- Figure 6.
Expression of MYB33 and MYB65; GUS Staining of Transgenic Pro33:GUS, myb65 Enhancer Trap, and MYB33:GUS Arabidopsis Plants.
(A) GUS activity as determined by histochemical analysis in a Pro33:GUS Arabidopsis flower at floral stage 12.
(B) Pro33:GUS flower at floral stage 13.
(C) Pro33:GUS flower at floral stage 15.
(D) Pro33:GUS root of a 5-d-old plant.
(E) Wounded leaf of a Pro33:GUS plant.
(F) Seven-day-old Pro33:GUS seedling
(G) Transverse section of young anthers at the PMC stage of Pro33:GUS shown under dark-field microscopy. Pink crystals indicate GUS expression.
(H) A GUS-stained flower (at floral stage 15) from the myb65 enhancer trap line.
(I) A GUS-stained inflorescence of a MYB33:GUS plant.
(J) GUS-stained imbibed seeds from a MYB33:GUS plant.
(K) Transverse section of MYB33:GUS anthers at the archesporial cell stage.
(L) Early PMC stage.
(M) Meiosis.
(N) Young microspore stage.
(O) Stage of tapetal degeneration.
(P) Dehiscence.
a, anther; ab, abscission zone (receptacle); c, connective; fl, anther filament; o, ovule; p, pistil; rt, root tip; sp, sepal.
- Figure 7.
The miR159 Target Motif Is Highly Conserved and Is Required to Restrict MYB33 Expression for Normal Plant Development.
(A) Alignment of the miR159 target sequence (black line) and surrounding sequences of the GAMYB-like genes of Arabidopsis (AtMYB33, AtMYB65, AtMYB101, AtMYB97, and AtMYB120), as well as two other closely related genes (AtMYB81 and AtMYB104), and the barley (HvGAMYB) and rice (OsGAMYB) genes. Amino acid sequence above corresponds to the sequence encoded by MYB33 and MYB65. The asterisk indicates the third position in each codon.
(B) GUS staining of 3-d-old MYB33:GUS and mMYB33:GUS seedlings.
(C) GUS staining of an inflorescence from a mMYB33:GUS transgenic line.
(D) GUS staining of a 7-d-old MYB33:GUS seedling (48 h of staining).
(E) GUS staining of a 7-d-old mMYB33:GUS seedling (16 h of staining).
(F) GUS staining of a 7-d-old 5′-MYB33:GUS seedling (6 h of staining).
(G) Comparison of 5-d-old transgenic plants transformed with the wild-type MYB33 gene (left) and mMYB33 (right).
(H) Ten-day-old mMYB33 transformant that is failing to form proper leaves.
(I) Rosette of a 4-week-old plant transformed with the MYB33 gene.
(J) Rosette of a 4-week-old plant transformed with the mMYB33 gene.
(K) Side view of a 3-week-old mMYB33 plant exhibiting extreme leaf curling.
(L) Side view of a 4-week-old mMYB33 plant exhibiting a short primary bolt with a terminal flower.
(M) Comparison of a 4-week-old MYB33 transformant with mMYB33 transformants with phenotypic defects of increasing degrees.
ab, abscission zone; a, anther; fl, anther filament; p, pistal. Bars = 1 cm.
- Figure 8.
Model for the Control of MYB33 Expression.
The 5′ upstream region strongly promotes expression in flowers (receptacle, pistal, sepals, and anther filaments/connective), meristems, and root tips. Addition of region A or region C alone does not alter this expression pattern. Addition of these two regions together, along with region B, enables expression in anthers. The miR159 target sequence (miR) represses expression in meristems, root tips, and flowers but has no apparent effect on anthers. Region A, contains sequences from the ATG (+1) to +1024; region B, +1043 to the stop codon (+1832); region C, from the stop codon to the open reading frame of the gene downstream of MYB33.
Tables
Ecotype/Mutant Light Intensity (μmol·m−2s−1)a Total Number of Siliques/Plant Total Number of Filled Siliques/Plant Percentage of Filled Siliques Ws 95 55.0 ± 4.7 52.6 ± 4.7 96.2 ± 1.1 140 51.7 ± 12.2 49.4 ± 11.7 96.2 ± 1.3 210 99.3 ± 11.1 95.5 ± 10.5 96.2 ± 0.6 330 152.7 ± 15.3 152.6 ± 15.3 97.6 ± 0.5 Col 95 39.8 ± 6.7 35.0 ± 5.3 89.3 ± 1.5 140 61.8 ± 10.0 57.8 ± 9.4 93.3 ± 1.9 210 103.7 ± 11.2 97.5 ± 11.0 93.3 ± 1.7 330 83.0 ± 10.9 75.1 ± 10.9 89.9 ± 3.4 myb33 myb65 95 241.6 ± 37.7 7.1 ± 1.2 3.6 ± 0.9 140 129.6 ± 16.8 46.3 ± 7.13 35.5 ± 3.7 210 203.0 ± 39.2 107.3 ± 29.2 50.3 ± 3.5 330 176.1 ± 23.0 136.0 ± 21.1 75.1 ± 2.6 ↵a Light intensity at which the plants were grown.
Growth Temperaturea Ecotype/Mutant Total Number of Siliques/Plant Total Number of Filled Siliques/Plant Percentage of Filled Siliques 22°C Col 51.6 ± 5.5 45.9 ± 3.9 91.2 ± 2.0 Ws 49.7 ± 5.0 45.8 ± 4.3 92.8 ± 1.4 myb33 myb65 217.1 ± 14.8 5.0 ± 1.9 2.5 ± 0.7 16°C Col 101.3 ± 29.0 87.8 ± 24.2 89.6 ± 2.5 Ws 154.4 ± 13.1 152.4 ± 13.2 98.4 ± 0.8 myb33 myb65 112.3 ± 10.0 36.4 ± 5.4 32.5 ± 3.4 ↵a Temperature at which the plants were grown.
