MINI SEED 2 (MIS2) Encodes a Receptor-like Kinase that Controls Grain Size and Shape in Rice

Abstract

Background: Grain size is a key agronomic trait that is directly associated with grain yield in rice. Although several genes related to grain size in rice have been identified, our understanding of the mechanism of grain development is still limited.

Results: In this study, we reported the characterization of a novel seed size mutant mini seed 2 (mis2), in which the grain showed reduced length, width and thickness along with wrinkled surface. Microscopic analysis revealed that the spikelet epidermal cell size was reduced but the cell number was increased in the mis2 mutant, suggesting that MIS2 controls grain size by coordinately regulating epidermal cell size and cell number. Map-based cloning revealed that MIS2 encodes a receptor-like kinase CRINKLY4 (CR4) which showed the highest expression in developing panicles. The MIS2 protein is localized primarily on the plasma membrane along with the endosome. However, the Arg258Gln mutation located in extracellular domain in the mis2 mutant disturbed its subcellular localization. Additionally, three major haplotypes of MIS2 were identified in the japonica, indica and aus rice cultivars. The 18-bp InDel (insertion and deletion) in the 5'-UTR (untranslated region) caused different expression level of MIS2 in haplotypes.

Conclusions: We reported a key role of OsCR4 in controlling grain size and shape by coordinately regulating epidermal cell size and cell number. The Arg258 in the extracellular seven-repeat domain is essential for the correct subcellular behavior and function of the OsCR4 protein.

Keywords: CRINKLY4; Epidermal cells; Grain size and shape; MIS2; OsCR4; Receptor-like kinase; Rice; Spikelets.

Fig. 1

Morphological characterization of the mis2 grains. (a) Mature grains from WT and mis2. Upper: unhulled grains; Lower: hulled garins. Scale bar = 2 mm. (b-e) Quantification data of grain length (b), grain width (c), grain thickness (d) and 1000-grain weight (e) of WT and mis2. * P < 0.05 and ** P < 0.01 by Student’s t test. Data are given as mean ± SD (n = 15)

Fig. 2

Comparison of the spikelet epidermal cells between WT and mis2. (a-d) Scanning electron micrographs (SEM) for the outer surfaces of lemma (a, b) and palea (c, d) of WT (a, c) and mis2 (b, d). CL and CW indicate epidermal cell length and cell width orientation, respectively. Bars = 100 μm. (e, f) The average length and width of the epidermal cells. (n = 10). (g) Comparisons of the calculated epidermal cell number of the lemma and palea in the grain-length (GL) and grain-width (GW) direction, respectively (n = 10). (h-k) SEM for the inner surfaces of lemma (h, i) and palea (j, k) of WT and mis2. Bars = 100 μm. (l, m) Cross sections of WT and mis2 spikelet hull. Bar = 50 μm. WLC, wart-like cells; SC, silicified cells; OPC, outer parenchyma cells; IPC, inner parenchyma cells. * P < 0.05 and ** P < 0.01 by Student’s t test. Data are given as mean ± SD (n = 20)

Fig. 3

Morphological comparison of seed surface between WT and mis2. (a, b) SEM for the surface of WT and mis2 seed. Bar = 0.5 mm. (c, d) Magnified views of the boxed region in (a, b), respectively. Bar = 50 μm

Fig. 4

Comparison of the interlocking cells, cell wall and cuticle layer between WT and mis2. (a, b) Transmission electron micrographs (TEM) for the interlocking cells of the palea and lemma in the WT and mis2. Bar =10 μm. (c, d) Magnified views of the boxed region in (a, b), respectively. Bar =5 μm. (e, f) Cuticle and cell wall of interlocking cells in the WT and mis2. Bar =1 μm. Cu, cuticle; CW, cell wall

Fig. 5

Map-Based cloning of MIS2 and genetic complementation. (a) The MIS2 locus was mapped to a region between markers M2 and M3 on chromosome 3. (b, c) The candidate gene was further delimited to a 213-kb genomic region between markers M7 and M8. Twenty-one candidate genes are predicted in this region. The numbers beneath the marker positions indicate the number of recombinants. (d) The MIS2 gene structure. White and green box represent untranslated region and coding sequence, respectively. Black lines represent introns. The start codon (ATG) and the stop codon (TGA) are indicated. A single nucleotide mutation from G to A in MIS2 resulted in an arginine-to-glutamine change. (e) Complementation of mature grain morphology. Three genomic DNA complementation transgenic lines are shown. Bar = 2.5 mm

Fig. 6

Expression pattern of MIS2. (a) Quantitative RT-PCR analysis showing the relative expression level of MIS2 in root, stem, leaf blade, leaf sheath, young panicle at various length stage, young spikelets and mature spikelets. The rice ubiquitin gene was used as an internal control. Values are given as mean ± SD (n = 3). (b-f) Expression pattern of GUS gene driven by the MIS2 promoter in root (b), stem (c), leaf blade (d), leaf sheath (e) and spikelets at different stages (f). Bar = 2 mm

Fig. 7

Subcellular behavior of MIS and MIS2^mu protein. (a) Three-dimensional model of the MIS2 extracellular repeat domain using PyMol. Seven repeats are labeled and Arg 258, which is mutated to Gln in mis2, is shown as a stick and colored purple. (b-e) Co-expression of MIS2 and ARA6 in tobacco leaf. The florescence of MIS2::GFP (b) and ARA6::mCherry (c) were detected and merged on the plasma membrane (d). The merged image (e) is a magnification view of the boxed region in (d). Line arrows indicate GFP-only vesicles, and dot arrows indicate co-localizations. Bar = 20 μm (b-d), Bar = 10 μm (e). (f-i) Co-expression of MIS2^mu and ARA6 in tobacco leaf epidermal cells. ARA6::mCherry is localized on plasma membrane (g). MIS2^mu::GFP localization is considerably changed (f, h). Magnification view shows GFP-labeled and mCherry-labeled compartments in cytoplasm and no colocalization is observed (I). Line arrows indicate GFP-only vesicles, respectively. Bar = 20 μm (f-h), Bar = 10 μm (i)

Fig. 8

Quantitative RT-PCR analysis of epidermal cell development, grain size and BRs related genes. (a) Relative expression fold change of ROC5 and related genes in WT and mis2. (b) Relative expression fold change of genes related to grain size and BRs in WT and mis2. Young inflorescence of ~ 1 cm was used for analysis. Rice ubiquitin gene was used as an internal control. **P < 0.01 and *P < 0.05 by Student’s t test. Values are given as mean ± SD (n = 3)

Fig. 9

Haplotype and origin analysis of MIS2. (a) MIS2 gene structure and natural variations among 524 rice accessions. (b) Relative expression level of Hap1 and Hap 2 in young panicle. (c-g) Comparison of grain length (c), grain width (d), length-width ratio (e), grain weight per 1000 (f) and grain surface area (g) among accessions containing Hap1, Hap2 and Hap3. The letter on histogram (a, b and c) indicate significant differences (P < 0.05) by ANOVA. (H) Geographic origin of accessions containing Hap1, Hap2 and Hap3. Hap1, Hpa2 and Hap3 are represented by red, blue and yellow circles, respectively