Figure 1.
Phenotypic characterization of the sd37 mutant.
(A) Gross morphology of the sd37 mutant and 3037 (wild type) plants at 7 DAG. Bar = 1 cm. (B) Heading stage of the sd37 mutant and 3037 plants. Bar = 10 cm. (C) Internode lengths of the sd37 mutant and 3037 plants at the mature stage. P, panicle; I, first internode below panicle; II, second internode below panicle; III, third internode below panicle; and IV, fourth internode below panicle. Bar = 1 cm. (D) Panicle morphology of the sd37 mutant and 3037. Bar = 2 cm. (E) Grain morphology. The sd37 mutant plants have shorter and broader grains than 3037 plants. Bar = 5 mm (seeds). (F) Graph showing the root lengths of sd37 and 3037 plants during the first 14 days of development. Data are averages of 20 plants (± SD).
Table 1.
Morphological measurements of the wild-type (3037) and mutant (sd37) plants.
Figure 2.
Histological analysis of the aboveground parts of sd37 and 3037.
(A) The seedling phenotypes of sd37 and 3037 plants at 7 DAG. Arrows indicate the second leaf sheath. Bar = 1 cm. (B) Parenchyma cells in the second leaf sheaths of sd37 and 3037 plants. Bars = 0.05 mm. (C) The number of parenchyma cells in the second leaf sheaths of sd37 and 3037 plants. Error bars indicate ± SD (N = 20). A significant difference (**, P<0.01) was found between the sd37 and 3037 plants. (D) The length of parenchyma cells in the second leaf sheaths of sd37 and 3037 plants. Error bars indicate ± SD (N = 20). A significant difference (*, P<0.05) was found between the sd37 and 3037 plants. (E) Longitudinal sections through each stem internode of sd37 and 3037 plants. Arrows indicate the second internodes below the panicle. Bar = 1 cm. (F) Longitudinal sections of the middle of the second stem internode of sd37 and 3037 plants at the heading stage. Bars = 0.05 mm. (G) The length of the second stem internodes in sd37 and 3037 plants. Error bars indicate ± SD (N = 20). A significant difference (**, P<0.01) was found between the sd37 and 3037 plants. (H) The length of the parenchyma cells in the second internodes in sd37 and 3037 plants. Error bars indicate ± SD (N = 20). No significant difference (P>0.05) was found between the sd37 and 3037 plants. (I)–(J) Longitudinal sections through the SAMs of 3037 and sd37 plants. Arrows indicate the second internode. Bars = 0.05 mm. (K) Longitudinal cell number in the second internode below the SAM. Error bars indicate ± SD (N = 10). A significant difference (**, P<0.01) was found between the sd37 and 3037 plants.
Figure 3.
(A) Physical mapping of SD37. The numbers in parentheses indicate the number of recombinants. SD37 was localized to BAC AC13769. The presumed ORFs were predicted using Gramene. White boxes indicate UTRs, and the black box represents the solitary exon. (B) Different sizes of the CAPS markers for 3037 and sd37 are shown using genomic DNA. PCR products of the SD37 CDS were amplified using the OE-F and OE-R primers (Table S3) and digested using AlwI. (C) SD37 expression in leaves from 3037 and the sd37 mutant were assessed using RT-PCR. Rice UBQ1 was used as an internal control. (D) Protein structure of SD37. The arrowhead indicates the point mutation in the SRS2 region. (E) Rescue of the sd37 phenotype with the pCAMBIA1300_SD37pro:SD37 construct. One representative complementation line (pCAMBIA1300::SD37) is shown. Bar = 10 cm. (F) CAPS marker detection in 3037 (lane 1), sd37 (lane 2), and a complementation line (lane 3). Samples were analyzed by agarose gel electrophoresis.
Figure 4.
Subcellular localization of pJIT163_hGFP::SD37 using ER-mCherry and Golgi-mCherry in rice protoplast cells.
(A) pJIT163_hGFP::SD37. (B) ER-mCherry. (C) Visible light. (D) Merged image. (E) pJIT163_hGFP::SD37. (F) Golgi-mCherry. (G) Visible light. (H) Merged image. Bar = 5 µm.
Figure 5.
The expression pattern of SD37.
(A)–(B) SD37 expression levels were measured by RT-PCR and real-time PCR in various organs, including the root, culm, SAM, young leaf, booting panicle, and panicle. Expression values are the average of 10 samples ± SD. (C)–(G) GUS expression (blue staining) patterns in the p1391Z_SD37pro::GUS transgenic line in different organs. (C) Root cross-section; (D) seeds with coleoptile and radicle; (E) culm; (F) young leaf cross-section; and (G) booting panicle. (H)–(J) SD37 expression around the shoot apical meristem as revealed by RNA in situ hybridization. (H) Shoot apical meristem; (I) young leaf; and (J) young leaf (negative control) preparation examined with a sense SD37 probe.
Table 2.
Selected functionally classified and differentially expressed genes in the sd37 mutant compared with the 3037 (wild type) as revealed by microarray analysis.
Figure 6.
Gross morphology of SD37 transgenic plants and the relative amount of SD37 mRNA, as determined by real-time PCR.
(A) Gross morphology of the vector-control/Nipponbare; sd37 over-expressing transgenic line: PUBQ:sd37 in the Nipponbare genetic background; SD37 over-expressing transgenic line 1:PSD37:SD37 in the Nipponbare background; over-expressing transgenic lines 2–4: PUBQ:SD37 in the Nipponbare background. Bar = 10 cm. (B) Gross morphology of the SD37 RNA interference transgenic plants in 3037 and Nipponbare backgrounds at 30 days. Bar = 10 cm. (C) Relative amount of SD37 mRNA levels in the transgenic plants in (A) and (B), as determined by real-time PCR. (D) Quantitative measurement of the total second leaf sheath parenchyma cell number in the axial parenchyma cells in the second leaf sheath parenchyma (per leaf) of the RNAi-SD37 transgenic line and vector control. Error bars indicate ± SD (N = 10). A significant difference (**, P<0.01) was found between the RNAi-SD37 transgenic line and vector control.
Table 3.
Pollen viability in the wild-type (3037) and mutant (sd37) plants.