Grain setting defect1, encoding a remorin protein, affects the grain setting in rice through regulating plasmodesmatal conductance

Abstract

Effective grain filling is one of the key determinants of grain setting in rice (Oryza sativa). Grain setting defect1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a transfer DNA insertion mutant (gsd1-Dominant) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests that the phenotype of the gsd1-Dominant mutant is caused by defects in the grain-filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with rice ACTIN1 in association with the PD callose binding protein1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice.

Figure 1.

Phenotypic comparison between the wild type and the homozygous gsd1-D mutant. A, Comparison of the wild type (left) and gsd1-D mutant panicles (right) at the grain-ripening stage. B to D, Comparison of the grain setting rate (B), 1,000-grain weight (C), and grain thickness (D) between the wild type and the gsd1-D mutant. Error bars show the means ± se (Student’s t test, n = 15 in B, n = 10 in C, and n = 25 in D). The single asterisk represents P < 0.05, and double asterisks represent P < 0.01. E and F, Iodine-potassium iodide (I₂-KI) staining of starch at the second internode of the wild type (E) and gsd1-D (F) in the grain matured plants. G and H, Scanning electron microscopy observation of the second internode sections of the wild type (G) and gsd1-D (H) in the grain matured plants. I and J, Mature seeds from the wild type (I) and the gsd1-D mutant (J). PC, Parenchyma cell; SG, starch granule; VB, vascular bundle; WT, wild type. Bars = 3 cm in A; 200 μm in E and F; 50 μm in G and H; and 2 mm in I and J.

Figure 2.

Sugar content comparison between the wild type and the gsd1-D mutant at the 5 DAF stage. A, Content of Suc, Glc, Fru, and starch in the flag leaf blades of the wild type and the gsd1-D mutant at the ED. B, Content of Suc, Glc, Fru, and starch in filling grains of the wild type and the gsd1-D mutant at the ED. C, Content of sugar in phloem exudate at different collection time after the ED. D, Decline of the ¹³C-labeled Suc in the flag leaf blades after photosynthesis of the flag leaf blade feed with ¹³CO₂. E, Increase of the ¹³C-labeled Suc in the flag leaf sheaths after photosynthesis of the flag leaf blade feed with ¹³CO₂. F, Increase of the ¹³C-labeled Suc in phloem exudate after photosynthesis of the flag leaf blade feed with ¹³CO₂. The results in A to C are means ± se from six individual plants, whereas the results in D to F are means ± se of three biological independent samples. Double asterisks indicate that the difference between the wild type and the gsd1-D mutant is statistically significant at P < 0.01 by the t test. WT, Wild type.

Figure 3.

Molecular cloning and expression of GSD1. A, Diagram of the GSD1 (Os04g52920) gene located on chromosome 4 and the T-DNA insertion position. Exons are shown as black boxes. In the gsd1-D mutant, T-DNA is inserted at the 462 bp upstream of ATG. Primers used in the genotype analysis are indicated by black arrows. F1 and R1 are gene-specific primers, and R2 is a T-DNA-specific primer. B, Diagram of the GSD1 protein domains. Numbers indicate positions of amino acids. C, T-DNA insertion confirmation and genotype analysis. D, Quantitative reverse transcription (RT)-PCR analysis of GSD1 expression in various tissues of the wild type and the gsd1-D mutant. The rice actin1 gene was used as a reference for normalization. Results are means ± se of three individual samples. E, Comparison of GSD1 protein abundance in the leaf blade, leaf sheath, P1, and P2 between the wild type and the gsd1-D mutant. Actin is used as a loading control. The leaf blade and leaf sheath were collected from 2-month-old rice. F, Comparison of GUS activity between pGSD1-L-GUS and pGSD1-S-GUS. The results are means ± se of three biological independent samples. CCD, Coiled-coil domain; Chr, chromosome; LB, leaf blade; LS, leaf sheath; P1, booting panicle (7 d before flowering); P2, grain-filling panicle (5 DAF); P3, grain-filling panicle (10 DAF); R, root; Remorin_C, C-terminal residue; Remorin_N, N-terminal residues; S, stem.

Figure 4.

Comparison of wild-type and GSD1 overexpressed transgenic plants. A, Wild-type, GSD1 overexpression (GSD1OX-18), and GSD1 suppression (GSD1AS-7) plants at the grain-filling stage. B, Mature panicles of wild-type, GSD1OX-18, and GSD1AS-7 plants. C to E, I₂-KI starch staining in the second internode of the wild type (C), GSD1OX-18 (D), and GSD1AS-7 (E) in the grain matured plants. F to H, Statistical analyses of the grain setting rate (F), 1,000-grain weight (G), and grain thickness (H) in the wild type, GSD1OX, and GSD1AS. Twenty panicles were analyzed for the grain setting rate. Ten independent 1,000-grain samples were analyzed for 1,000-grain weight. Sixty seeds were analyzed for seed size. Values are means ± se. Different lowercase letters indicate a significant difference at P < 0.01 by ANOVA. I, Quantitative RT-PCR analyses of the GSD1 expression in the stem of wild-type, GSD1OX, and GSD1AS transgenic plants at the tillering stage. The results are means ± se of three individual samples. J, Comparison of GSD1 expression in various tissues of the wild type, the gsd1-D mutant, and GSD1OX by quantitative RT-PCR analysis. The rice actin1 gene was used as a reference for normalization. The relative gene expression level in the wild-type leaf blade was set as 1. The results are means ± se of three individual samples. K, Comparison of GSD1 expression in protein levels in the leaf blade, root, and P1 of the wild type, the gsd1-D mutant, and GSD1OX. Actin is used as a loading control. The leaf blade, root, and P1 were collected from 2-month-old rice. LB, Leaf blade; LS, leaf sheath; P1, booting panicle (about 7 d before flowering); R, root; S, stem; WT, wild type. Bars = 20 cm in A; 3 cm in B; and 50 μm in C to E.

Figure 5.

Export of soluble sugars from the flag leaf at 5 DAF in transgenic rice plants. A to C, Content of Suc (A), Glc (B), and Fru (C) in wild-type, GSD1OX, and GSD1AS plants. Values are means ± se from six individual plants. D, Sugar content in phloem exudate of wild-type, GSD1OX, and GSD1AS plants. The results are means ± se of three individual samples. Different lowercase letters indicate a significant difference at P < 0.01 by ANOVA. WT, Wild type.

Figure 6.

GSD1 is expressed in companion cells demonstrated by promoter analysis and immunolocalization. A to H, Histochemical localization of GSD1 promoter-GUS activity in pGSD1-L-GUS transgenic rice. GUS expression in the root (A), cross sectioned stem (B), leaf blade (C), young spikelet (D), matured spikelet (E), and immature seed at 10 DAF with the inset for cross section (F). Cross sections of rice culm (G) and longitudinal sections of rice culm (H). I to K, Immunolocalization by GSD1-specific antibodies. GSD1 is localized in phloem companion cells in the wild type (I) and in the gsd1-D mutant (K). Detection in the wild type by preimmune IgG (J). L, GUS staining in culm of the pGSD1-S-GUS transgenic rice. CC, Companion cell; CV, commissural vein; DV, dorsal vascular bundle; LV, large vein; MV, midvein; Ph, phloem; SE, sieve element; SV, small vein; Xy, xylem. Bars = 500 μm in A to F; 100 μm in the F inset; and 50 μm in G to L.

Figure 7.

GSD1 is localized to PM and PD. A to D, GSD1 fused to GFP (GFP-GSD1) was expressed in tobacco leaf epidermal cells. GFP-GSD1 fluorescence detected on PM (A), callose staining with aniline blue indicating PD sites (B), and merged image showing that GSD1 localizes to PM and PD (C). Arrowhead indicates PD sites on the PM. D, GFP-GSD1 fluorescence detection after plasmolysis. E and F, Control GFP was expressed in onion epidermal cells. Control free GFP fluorescence signal (E). GFP control fluorescence merged with DIC image (F). G to I, GSD1 fused with GFP (GFP-GSD1) was expressed in onion epidermal cells. GFP-GSD1 fluorescence on the PM (G), GFP-GSD1 fluorescence merged with the DIC image (H), and image after plasmolysis treatment (I). J to L, Representative immunogold labeling of GSD1 and quantitative analysis of gold particles. GSD1 is localized to the channel of PD (J) and at the neck regions of PD (K) of the companion cells in 4-week-old rice shoot tissues. Quantification of gold particles is depicted in L. Arrowheads indicate immunogold labeling particles. CC, Companion cell; CW, cell wall; DIC, differential interference contrast; HS, Hechtian strand; N, nucleus; SE, sieve element. Bars = 50 μm in A to C and E to I and 500 nm in J and K.

Figure 8.

Overexpression of GSD1 hampers PD permeability. A to C, Confocal images showing CFDA loaded onto the adaxial surface of tobacco leaves. D to F, CF spread observed on the abaxial surface. Signals from 35S:mCherry (A and D), 35S:GSD1:mCherry (B and E), and 35S:PDLP5:mCherry (C and F) transformants. G, Quantification of the CF movement in 35S:mCherry, 35S:GSD1:mCherry, and 35S:PDLP5:mCherry transformants. More than 20 plants were used per assay, and more than three repeats were performed. The extent of dye diffusion was quantified by measuring the fluorescent diffusion area. The relative dye diffusion in 35S:mCherry transformants was set as 100%. Values are means ± se from 12 individual samples. H to J, Confocal images showing chemical staining of callose using aniline blue in 35S:mCherry (H), 35S:GSD1:mCherry (I), and 35S:PDLP5:mCherry (J) transformants. K, Quantification of the callose staining intensity in the 35S:mCherry, 35S:GSD1:mCherry, and 35S:PDLP5:mCherry transformants. Values are means ± se from 20 individual samples. Different lowercase letters in G and K indicate significant difference at P < 0.01 by ANOVA test. Bars = 500 μm in A to F and 50 μm in H to J.

Figure 9.

GSD1 interacts with OsACT1 at PD. A, mCherry-GSD1 and PDLP1-mCherry were cotransformed with GFP-OsACT1 into tobacco leaves, respectively. Confocal images show overlapping OsACT1 GFP fluorescence signals and PD marker protein PDLP1 mCherry signals. OsACT1 GFP signals and GSD1 mCherry signals also overlap. B, Interaction of GSD1 with OsACT1 was detected by BiFC. YN-OsACT1 (YN, N-terminal part of YFP), PDLP1-YN, and YN were cotransformed with YC-GSD1 (YC, C-terminal part of YFP) into tobacco leaves, respectively. YFP signals were detected in YN-OsACT1 and YC-GSD1 coexpressed tobacco leaves. No YFP fluorescence signal was detected in PDLP1-YN and YC-GSD1 or YN and YC-GSD1 coexpressed tobacco leaves. C, Interaction between GSD1-Myc and OsACT1-Flag was detected by Co-IP. GSD1-Myc and OsACT1-FLAG were expressed or coexpressed in tobacco leaves. Total protein extracts were separately immunoprecipitated with anti-Myc antibody-coupled agarose beads and anti-Flag antibody-coupled agarose beads. Proteins from the crude lysates and immunoprecipitated proteins were detected with anti-Myc antibodies and anti-Flag antibodies, respectively. D, YN-OsACT1, YC-GSD1, and PDCB1-mCherry were cotransformed into tobacco leaves. YFP and mCherry signals were detected using a confocal microscope. Overlaid YFP and mCherry fluorescence signals indicate that the GSD1/OsACT1 complex colocalized with PD callose binding protein PDCB1.

Figure 10.

A proposed model for GSD1 modulation of PD permeability. GSD1 encodes a PM protein, remorin, which is colocalized in PD with PD-specific proteins: GPI-anchored callose binding protein PDCB1, which is located at the neck region of PD (Simpson et al., 2009); and PD-located proteins PDLP1 and PDLP5 (Thomas et al., 2008; Lee et al., 2011). GSD1 interacts with OsACT1, which is associated with the desmotubule structure in PD. GSD1 may play a role in connecting the desmotubule and PM in the PD neck regions of companion cells, modulating the PD aperture through its expression levels and PM anchor. CW, Cell wall; DT, desmotubule; ER, endoplasmic reticulum.