Os03g0407400

The rice Os03g0407400 was reported as GS3 in 2006 by researchers from China.

Annotated Information

Gene Symbol

• Os03g0407400 <=> OsGS3,GS3,GRAIN SIZE 3

Function

• GS3, one of the genes regulating seed length, also regulates stigma length and participates in stigma exsertion in rice. GS3 mRNA is expressed in the basal part of the young stigma, and a nonsense mutation in the second exon of GS3 causes an increase in cell number, resulting in elongation of the stigma.[1] Figure 1 showed introgressed chromosome segment in AIS22 contributed to an increase in stigma length and, as a result, the frequency of stigma exsertion was increased by comparing AIS22 with Asominori and IR24.[1] A transgenic experiment (Figure 2) showed GS3 was the most likely candidate for the gene causing this difference between AIS22 and Asominori.[1]

Figure 1: Stigma traits in Asominori, AIS22 and IR24. (A) Morphology of stigmas of Asominori, AIS22 and IR24. Scale bar, 500µm. (B)Stigma exsertion in Asominori, AIS22 and IR24. Arrows indicate glumes retaining stigmas outside after enclosing the palea and lemma.

Figure 2: Morphology of stigmas of GS3-containing transgenic positive (+) and negative (−) plants. Scale bar, 500µm.

• The wild-type isoform is composed of four putative domains: a plant-specific organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family cysteine-rich domain, and a von Willebrand factor type C (VWFC) in the C terminus. These domains function differentially in grain size regulation. The OSR domain is both necessary and sufficient for functioning as a negative regulator. The wild-type allele corresponds to medium grain. Loss of function of OSR results in long grain. The C-terminal TNFR/NGFR and VWFC domains show an inhibitory effect on the OSR function; loss-of function mutations of these domains produced very short grain.[3]

Expression

•GS3 consists of five exons that encode a novel protein with several conserved domains, including a phosphatidylethanolamine-binding protein (PEBP)-like domain, a transmembrane region, a putative tumor necrosis factor receptor/ nerve growth factor receptor family domain, and a von Willebrand factor type C domain. A complementation test demonstrated that a C-to-A nonsense mutation in the second exon of GS3causes the long-grain phenotype (Takano-Kai et al.2009). GS3 is highly expressed in young panicles but is not expressed in leaves or panicles after flowering (Takano-Kai et al.2009).[5]

•A GS3 promoter::GUSfusion construct into Nipponbare and GUS staining at various stages of stigma development were used to estimate the mRNA expression of GS3in the pistil.[1]

•GUS expression was observed in the basal part of young stigmas, and the strongest GUS expression was found in the basal part of stigmas and the upper part of styles. GUSexpression was not detected in the pistil (Figure. 3).

Figure 3: GUS staining of pistils transformed with the GS3promoter::GUSfusion construct and development of the stigma. (A) Pistils from panicles approximately 7 cm, 10 cm, 13 cm and 15 cm long and after heading. Scale bar, 500µm. (B) Relationship between panicle length and SNBP (stigma non-brush shaped part) length. Error bars indicate SE (n=4).

•All the pistils from GS3 promoter::GUStransgenic plants showed the same GUS expression pattern. The period of greatest GS3 mRNA expression in the stigma coincided with the stage at which the stigma elongated dramatically(Figure. 3).[1] GS3 decreases the number of stigma cell(Figure 4).

Figure 4:Cell lengths associated with cell number on SNBP (stigma non-brush-shaped part) fromthe basal part of mature stigma. (A) An example of fluorescent image of the stigma tomeasure cell length and number of cells in the mature stigma. Scale bar, 500µm. (B) Cell length averaged from consecutive parts of five cells each along the longitudinal axis of the SNBP of transgenic plants with and without transgene, Asominori, and AIS22. Diagrams of each stigma are shown below the histogram. Error bars indicate SE (n=20).

•GS3 was highly expressed in young panicle, and the signal gradually decreased with panicle development(Figure 5 A, B and C). GS3 was also highly expressed in root tips (Figure 5 D). Weak signals were observed in other tested tissues, including embryo, shoot apical meristem, leaf, and stem(Figure 5 E, F, G A and H). GS3 mRNA preferentially accumulated in panicles less than 5 cm in length in both genotypes, whereas the transcript level was low in other tissues assayed(Figure 5 J). The expression level and pattern were not the cause of the loss of function of GS3in Minghui 63.[3]

Figure 5:Expression pattern ofGS3.(A–H) Expression ofGS3in various tissues revealed by in situ hybridization: (A) panicle at stage of pollen mother cell formation, (B) panicle at pollen mother cell meiosis, (C) panicle at pollen grain filling, (D) root at trefoil stage, (E) stem at heading stage, (F) leaf at tillering stage, (G) endosperm and embryo 14 d afterflowering, and (H) longitudinal section of 7-d-old seedling. (I) Negative control by hybridizing the young panicle with the sense probe. All tissues were sampled from Zhenshan 97 (medium grain). (Scale bars, 200 μm.) (J) Comparative expression pattern of GS3in Minghui 63 and NIL(c7) in various organs using real-time RT-PCR analysis: S, seedling at trefoil stage; R, root at trefoil stage; L, leaf at tillering stage; St, stem at heading stage; P1, young panicle<1 cm in length; P2, young panicle<5 cm in length; P3, young panicle 5–10 cm in length; P4, panicle before heading>10 cm in length; P5, panicle 5 d after heading; Pl, plumule

•In order to investigate effects on grain size of the various domains assessed using transgenics, seven constructs were made by domain deletions(Figure 6 A). VWFC domain seemed to have a larger effect of inhibiting OSR than did TNFR and that these two domains addedto each other in regulating OSR for grain size(Figure 6 A and B). Further, VWFC has a general role of inhibiting the effect of OSR in regulating plant growth and grain size(Figure 6 C, D, E and F).[3]

Figure 6 Effects on grain size of the various domains assessed using transgenics. (A) Schematic representation of the coding sequences of the constructs with one or more domains deleted; (B) grains of the T1generation transgenic plants compared with Minghui 63. OSR, organ size regulation domain; M, putative transmembrane domain; T, TNFR/NGRF domain; V, VWFC domain. Green box represents the polypeptide sequence resulting from frameshift. (CandD) Effect of VWFC in inhibiting OSR in regulating growth of plant and grain size. From left to right: Minghui 63, transgenic plants overexpressing thefl-cDNA (FL), or the truncated cDNA (OMT). (E) Northern blot analysis ofGS3transcripts in wild-type plants and in two positive segregants from respective T1generations. (F) Grain length of T1 plants overexpressingfl-cDNA (FL) or the truncated cDNA (OMT). + and−, positive and negative segregants. Data are given as mean±SEM. Student’sttest was used to generate thePvalues.

•RT–PCR and genetic transformation were used to analysis the expression of GS3. GS3 gene regulates grain size in rice during the early phases of panicle development while spikelets are elongating(Figure 7 A and B). GUS expression was observed in panicles up until 5 days before heading, but the signal was not detected in either flowering panicles or leaves(Figure 7 C and D).[4]

Figure 7: Expression analysis ofGS3using RT–PCR and genetic transformation. (A) RT–PCR ofGS3from Asominori, AIS22, and Nipponbare using young panicles (-3 cm) for RNA extraction. Numbers in parentheses indicate cycles of PCR. Actin cDNA was amplified as a control. (B) RT–PCR of GS3 from Asominori using panicles -3, 5, 7, 10, and 13 cm and after heading and a flag leaf for RNA extraction. (C and D)GUSstaining of transgenic plants transformed with the GS3 promotor:GUSfusion construct showing panicles before flowering (C) and a panicle at flowering with a flag leaf (D). Bars, 1 cm.

Evolution

Genetic transformation was used to demonstrate that the dominant allele for short grain complements the long-grain phenotype. A C to A mutation in the second exon of GS3(A allele) was associated with enhanced grain length inOryza sativa but was absent from other Oryza species. Linkage disequilibrium (LD) was elevated and there was a 95.7% reduction in nucleotide diversity (up) across the gene in accessions carrying the A allele, suggesting positive selection for long grain. Haplotype analysis traced the origin of the long-grain allele to aJaponicalike ancestor and demonstrated introgression into theIndicagene pool. A critical role for GS3 in defining the seed morphologies of modern subpopulations of O. sativaand enhances the potential for genetic manipulation of grain size in rice.[4]

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Labs working on this gene

(1)Plant Breeding Laboratory, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1, Hakozaki, Higashi, Fukuoka 812-8581, Japan

(2) National Key Laboratory of Crop Genetic Improvement and College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

(3) School of Plant Biology, and International Centre for Plant Breeding Education and Research, The University of Western Australia, Crawley, WA 6009, Australia

(4) National Center for Gene Research, Shangai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China

(5) Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, P. R. China

(6) Plant Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

(7) Department of Plant Sciences, University of Arizona, Tucson, Arizona, 85721

(8) Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

References

1. Noriko Takano-Kai; Kazuyuki Doi; Atsushi Yoshimura. GS3 participates in stigma exsertion as well as seed length in rice. Breeding Science, 2011, 61(3): 244-250

2. Chongrong Wang; Sheng Chen; Sibin Yu. Functional markers developed from multiple loci in GS3 for fine marker-assisted selection of grain length in rice. Theoretical and Applied Genetics, 2011, 122(5): 905-913

3. Hailiang Mao; Shengyuan Sun;Jialing Yao;Chongrong Wang;Sibin Yu;Caiguo Xu;Xianghua Li;Qifa Zhang. Linking differential domain functions of the GS3 protein to natural variation of grain size in rice. Proceedings of the National Academy of Sciences, 2010, 107(45): 19579-19584

4. Noriko Takano-Kai; Hui Jiang; Takahiko Kubo; Megan Sweeney; Takashi Matsumoto; Hiroyuki Kanamori; Badri Padhukasahasram; Carlos Bustamante; Atsushi Yoshimura;Kazuyuki Doi; Susan McCouch. Evolutionary History of GS3, a Gene Conferring Grain Length in Rice. Genetics, 2009, 182(4): 1323-1334

5. Chuchuan Fan; Yongzhong Xing; Hailiang Mao; Tingting Lu; Bin Han; Caiguo Xu; Xianghua Li; Qifa Zhang. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theoretical and Applied Genetics, 2006, 112(6): 1164-1171