Figure 1.
The dwt1 mutant plants display morphological defects.
A. Morphology of the wild type and dwt1 plants after heading. Bar = 10 cm. B. Mature main shoots with leaves removed from the culm. Arrowheads point to the nodes. Bar = 10 cm. C. Mature tillers with leaves removed from the culm. Arrowheads point to the nodes. Bar = 10 cm. D. Close-up view of the internodes after heading stage. From left to right: the 2nd node of the wild type, the 2nd internode, the 2nd and 3rd internodes, the 2nd, 3rd, and 4th internodes of dwt1 mutant. Bar = 0.5 cm. E. Frequency of normal and short internodes in wild-type and dwt1 main shoot (MS) and tiller shoot (TS). 2nd: only 2nd internode short; 2nd, 3rd: both 2nd and 3rd internodes short; 2nd, 3rd, 4th: all 2nd, 3rd, 4th internodes short. Elongation pattern of main shoots and tillers of both wild type and dwt1 mutants were evaluated at mature stage, and 25 main shoots and 100 tillers of wild type and 25 main shoots and 143 tillers of mutant plants were observed. F. Morphology of panicles from the main shoot (MS) and tillers (TS) of wild type (WT) and dwt1 after heading. Bar = 2 cm.
Figure 2.
The replanted main shoot and tiller of dwt1 reproduce the main-shoot-dominance phenotype.
A. Morphology of mature plants developed from replanted main shoot of the wild type (RPwM), replanted tillers of the wild type (RPwT), replanted main shoot of dwt1 (RPdM) and replanted tillers of dwt1 (RPdT). Bar = 5 cm. B. The culm length of both main shoots (MS) and tillers (TS) of replanted plants. Culm length of 15 main shoots and 60 tillers of wild-type plants, 15 main shoots and 55 tillers of mutant plants were measured at mature stage. Error bars indicate SD, and the very significant differences from the wild type are marked (**p<0.01, Student's t test).
Figure 3.
dwt1 has defects in cell elongation and cell proliferation.
A. The second internode of mature wild-type (left) and dwt1 (right) plants. Bar = 1 cm. B. The length of second internode at mature stage. n = 30. Error bars indicate SD, and the very significant differences from wild type are marked (**p<0.01, Student's t test). C and D. Longitudinal sections through the middle of the second internode of the wild type (C) and dwt1 (D) after the heading stage. Bar = 100 µm. E. Longitudinal and transverse length of the cells in the second internode. 12 second internodes from 3 individual mature plants were used, and 10 cells of each internode were measured. Error bars indicate SD, and the very significant differences from wild type are marked (**p<0.01, Student's t test). F. Cell number of the whole second internodes in mature plants. n = 5. Internode sample harvested from 5 individual plants were used for section. Error bars indicate SD, and the very significant differences from wild type are marked (**p<0.01, Student's t test).
Figure 4.
Molecular characterization of DWT1.
A. Map-based cloning of the DWT1 gene. The chromosomal region containing DWT1 is diagrammed as the top line with molecular markers shown above the line. The number below the corresponding markers indicates the numbers of recombinants between the markers and DWT1. The BAC clones are shown as overlapping lines. B. Structure of the DWT1 gene. The mutant sequence has one nucleotide C763 deletion in the second exon. Black boxes indicate exons, white boxes indicate UTRs and lines indicate introns. The grey box shows the homeobox domain. C. The plant stature of dwt1, the wild type, and complemented transgenic plants (DWT1-COM). Bar = 10 cm. D. The morphology of the second internodes of dwt1, wild type, and complemented transgenic plants (DWT1-COM). Bar = 2 cm. E–H. Nuclear localization of DWT1 protein. YFP fluorescence image (E), light view (F), PI stained image (G) and overlay of the three images (H) of Nicotiana benthamiana leaf epidermal cell transformed with the 35S:DWT1-YFP construct.
Figure 5.
Expression pattern of DWT1 transcripts and proteins.
A. qRT-PCR analysis of DWT1 gene expression levels in the wild-type tissues including coleoptiles (36 h after seed germination), root tips, mature leaf and sheath, the second internode during elongating (1 cm in length), segments of the upper (U), middle (M), and lower (L) parts of the second internode with 3 cm in length, dormant tiller bud, panicle (less than 1 cm in length), spikelet at stage Sp8 during development of pistil [63], young embryo (10 days after fertilization), and callus with 20–days regeneration. Rice ACTIN1 (OsACTIN1) was used as a control. Error bars indicate SD. n = 3. B. qRT-PCR analysis of DWT1 gene expression levels in young panicle of wild type. This experiment was biologically repeated three times, and 30 young panicles of the main shoot (MP, Length = 5 mm), and the tiller (TP, Length = 5 mm) respectively were used in each test. Error bars indicate SD, and the significant differences from panicle of main shoot are marked (**p<0.01, Student's t test). C–L. In situ hybridization of DWT1. Signals were detected in the primary branch meristem (E), secondary branch meristem (C, F), top portion of the panicle (G), shoot apical and radical apical of young embryo (I), and endodermis and exodermis of root tip (K). D, H, J and L were corresponding sections hybridized with the sense probe.I,II,III,IV, the first, second, third and the forth internode, respectively; pb, primary branch meristem; sb, secondary branch meristem; yl, young leave; fm, floral meristem; ifm, inflorescence meristem; exd, exodermis layer; end, endodermis layer. M–R. YFP fluorescence image of the branch meristem (M–O) and the internode (P–R) in transgenic plants expressing pDWT1:DWT1-YFP. M and P are fluorescence image, N and Q are light view, O and R are overlapping of fluorescence image and light view. S. Western-blot analysis of DWT1 protein levels. DWT1 protein was analyzed by immunoblotting using an anti-DWT1 antibody using mature flag leaf (L, 3 leaves from 3 different plants), elongating internodes (IN_A, length = 0.5 cm; IN_B, length = 3 cm, 10 internodes from different plants) and young panicle (P, length less than 2 mm, 50 panicles from different plants). β-tubulin was used as a control. These experiments were biologically repeated three times.
Figure 6.
DWT1 affects the expression of genes related to cell division and cell elongation.
A. Identification of GO biological process categories for the genes differentially expressed in dwt1 shorter internodes. The negative logarithm (base 10) of the adjusted P value was used as the bar length. B. qRT-PCR confirmed the differential expression of genes involved in cell division and cell elongation in the elongating second internode of the wild type and dwt1. . Rice ELONGATION FACTOR 1 ALPHA (EF1α) gene was used as a control. This experiment was biologically repeated three times, and 10 elongating second internodes of tillers (Length = 0.5 cm) were used for each biological repeat. Error bars indicate SD, and the significant differences from the wild type are marked (**p<0.01, Student's t test).
Figure 7.
OsGA20OX genes have increased expression in dwt1.
Transcript levels of OsGA20OX1, OsGA20OX2, OsGA20OX3 and OsGA20OX4 in the second internodes of the wild type and dwt1. The EF1α gene was used as a control. Three sequential developmental stages of elongating internode of wild type (Length = 0.5 cm, 1 cm and 3 cm) and dwt1 at the corresponding developmental stage of wild type were analyzed. This experiment was biologically repeated three times, and 10 internodes were used for each biological repeat. Error bars indicate SD. Significant differences from the wild type are marked (*P<0.05, ** P<0.01, Student's t test).
Figure 8.
The dwt1 tiller internodes are insensitive to GA treatment, and DWT1 may act downstream of SLR1 in the tiller internode elongation.
A. Morphological comparison of the wild type and dwt1 treated with mock solution or 100 µM GA3. 20 plants were uses for each treatment and representative images are shown. Bar = 10 cm. B. The second internodes of the wild type and dwt1 treated with mock solution or 100 µM GA3. Representative images of tiller internodes are shown. Bar = 1 cm. C. The internode length of the wild type and dwt1 treated with mock solution or 100 µM GA3. Length of 20 tiller internodes were measured 20 days after treatment. Error bars indicate SD, and the significant differences from no GA treated control are marked (** P<0.01, Student's t test). D. qRT–PCR analysis of OsGA20OX genes in the elongating internode of the wild type and dwt1 treated with mock solution or 100 µM GA3. EF1α gene was used as a control. This experiment was biologically repeated three times, and 5 internodes (Length = 5 mm) were used for each biological repeat. Error bars indicate SD. Significant differences from the wild type are marked (* P<0.05, ** P<0.01, Student's t test). E. Phenotype of basal internodes in the wild type, dwt1, slr1 and dwt1 slr1double mutant. Arrows point to the nodes on the culm. Bar = 1 cm.