Os01g0848400

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  • The rice Os01g0848400 was first reported as qSH1 in 2006 by the researchers from Japan [1]. qSH1 encodes a BEL1-type homeobox protein.

Annotated Information

Function

  • The gene qSH1 is a major quantitative trait locus of seed shattering in rice. A single-nucleotide polymorphism (SNP) in the 5' regulatory region of the qSH1 gene caused loss of seed shattering owing to the absence of abscission layer formation.

Mutation

A mutation in the qSH1 gene alone resulted in complete loss of the abscission layer in the Nipponbare genetic background and that the Kasalath allele of qSH1 could rescue it


Structured Information

The causal gene of a novel small and round seed mutant phenotype (srs3) in rice was identified by map-based cloning and named the SRS3 gene. The SRS3 gene was grouped as a member of the kinesin 13 subfamily. The SRS3 gene codes for a protein of 819 amino acids that contains a kinesin motor domain and a coiled-coil structure. Using scanning electron microscopy, we determined that the cell length of seeds in the longitudinal direction in srs3 is shorter than that in the wild type. The number of cells of seeds in the longitudinal direction in srs3 was not very different from that in the wild type. The result suggests that the small and round seed phenotype of srs3 is due to a reduction in cell length of seeds in the longitudinal direction. The SRS3 protein, which is found in the crude microsomal fraction, is highly expressed in developing organs. Rice yield potential is determined by several factors including seed size (or weight), number of panicles per plant and number of seeds per panicle (Song and Ashikari 2008, Takeda and Matsuoka 2008). Significant progress has been made in our understanding of regulation of seed formation through molecular and genetic studies, and this knowledge can potentially be used for the improvement of rice yield. Causal genes of a large seed phenotype have been identified by quantitative trait locus analysis, namely GW2 encoding a RING-type protein which functions as an E3 ubiquitin ligase (Song et al. 2007), qSW5 encoding a novel protein with no known domain (Shoumura et al. 2008) and GS3 encoding a membrane protein with various conserved domains (Fan et al. 2006, Takano-Kai et al. 2009). Loss of GW2 and qSW5 function leads to enhanced seed width, and loss of GS3 function leads to enhanced seed length, both resulting in increased yield. Therefore, genes regulating seed size can potentially be used to increase overall yield. It will be important to reveal the biochemical pathways in which these genes function. Causal genes of the small (or short) seed phenotype have also been identified as a group of seed size-regulating genes by map-based cloning, namely RGA1 (also named D1) encoding the heterotrimeric G protein α subunit (Ashikari et al. 1999, Fujisawa et al. 1999), D11 encoding a cytochrome P450 involved in brassinosteroid (BR) biosynthesis (Tanabe et al. 2005), D2 and BRD2 encoding an another type of cytochrome P450 involved in BR synthesis (Hong et al. 2003, Hong et al. 2005) and D61 (also named OsBRI1) encoding the BR receptor (Yamamuro et al. 2000). These results suggested that the G protein and BR signaling pathways were important in the regulation of seed size. After these studies, it was tested whether these genes were useful to improve the seed size in rice. When a genetically modified chimeric gene for a constitutively active form of the heterotrimeric G protein α subunit (QL) was introduced into the rice mutant d1, which is defective for the α subunit gene, the seed length and weight were substantially increased in the transformants (Oki et al. 2005). The result suggested that the enhancement of G protein signaling increased the seed size in rice. It was shown in another study that when a chimeric gene for a sterol C-22 hydroxylase in maize BR biosynthesis (Zm-CYP) was expressed in rice plants the seeds were heavier in the transformants (Wu et al. 2008). The result suggested that a gene controlling the BR level may be useful for increasing grain yield in crop plants. Thus, the utilization of seed size-regulating genes by genetic engineering approaches has opened up the way to produce improved rice plants with heavier seeds. Although the heterotrimeric G protein and BR signaling pathways regulate seed size in rice, it is not clear whether other signaling pathway genes have similar effects. A search for new genes that regulate seed size would contribute to resolving this issue. It is likely that any newly discovered genes functioning in this manner would operate in the heterotrimeric G protein and BR signaling pathways. If this were the case, the new discoveries would help in analyzing the precise mechanism of these signaling pathways. If seed sizes could be increased by manipulating newly discovered genes, utilization of the new genes would also open up future possibilities for breeding improved rice plants with larger seeds. The presence of many unidentified genes regulating seed size has been shown in rice (Nagato and Yoshimura 1998). Previously we have reported the rough map position of a new causal gene for the small and round seed phenotype, one of the genes regulating seed size, namely SRS3 on chromosome 5 (Tanabe et al. 2007). Here we report that the SRS3 gene encodes a novel kinesin 13 protein.


This subsection of the ‘Protein attributes’ section indicates the type of evidence that supports the existence of the protein. Note that this subsection does not give information on the accuracy or correctness of the sequence(s) displayed. While it gives information on the existence of a protein, it may happen that the sequence slightly differs from genomic sequences, especially for sequences derived from gene model predictions. In UniProtKB there are 5 types of evidence for the existence of a protein: 1. Evidence at protein level 2. Evidence at transcript level 3. Inferred from homology 4. Predicted 5. Uncertain The value ‘Evidence at protein level’ indicates that there is clear experimental evidence for the existence of the protein. The criteria include partial or complete Edman sequencing, clear identification by mass spectrometry, X-ray or NMR structure, good quality protein-protein interaction or detection of the protein by antibodies. The value ‘Evidence at transcript level’ indicates that the existence of a protein has not been strictly proven but that expression data (such as existence of cDNA(s), RT-PCR or Northern blots) indicate the existence of a transcript. The value ‘Inferred by homology’ indicates that the existence of a protein is probable because clear orthologs exist in closely related species. The value ‘Predicted’ is used for entries without evidence at protein, transcript, or homology levels. The value ‘Uncertain’ indicates that the existence of the protein is unsure. Only the highest or most reliable level of supporting evidence for the existence of a protein is displayed for each entry. For example, if the existence of a protein is supported by both the presence of ESTs and direct protein sequencing, the protein is assigned the value ‘Evidence at protein level’. The ‘protein existence’ value is assigned automatically, based on the annotation elements present in the entry. The criteria used by this automatic procedure are listed in the document ‘Criteria used to assign the PE level of entries’.

Evolution The causal gene of a novel small and round seed mutant phenotype (srs3) in rice was identified by map-based cloning and named the SRS3 gene. The SRS3 gene was grouped as a member of the kinesin 13 subfamily. The SRS3 gene codes for a protein of 819 amino acids that contains a kinesin motor domain and a coiled-coil structure. Using scanning electron microscopy, we determined that the cell length of seeds in the longitudinal direction in srs3 is shorter than that in the wild type. The number of cells of seeds in the longitudinal direction in srs3 was not very different from that in the wild type. The result suggests that the small and round seed phenotype of srs3 is due to a reduction in cell length of seeds in the longitudinal direction. The SRS3 protein, which is found in the crude microsomal fraction, is highly expressed in developing organs. Chracterization of srs3, TCM 2092 and TCM 768 Previously, we reported mapping of the srs3 mutant, TCM 1173, derived from mutagenesis of Taichung 65, which was designated as the wild type (WT) herein. The plant type of the srs3 mutant was compared with that of the WT (Fig. 1 and Table 1). In addition to a small and round phenotype, the srs3 mutant exhibited shortened panicles and internodes. This suggests that the SRS3 gene regulates the development of many organs in rice body planning. From characteristic phenotypes such as small seeds and shortened panicles in srs3, it was considered that the SRS3 gene may have function in regulating cell length or cell numbe The two mutants TCM 2092 and TCM 768 were also compared with the WT (Fig. 1 and Table 1). TCM 2092 and TCM 768 had mutations in the SRS3 gene as described in Fig. 3, although a test to determine whether TCM 2092 and TCM 768 are allelic to srs3 remains to be carried out. The extent of severity of the abnormalities is in the order TCM 768, TCM 2092 and srs3. Thus, TCM 768 showed the most severe abnormalities, such as shortened seed, panicle and internode, reduced seed weight and leaf erection at the mature stages. In addition to these characteristics, reduced fertility was clear in TCM 768 (51.96%), although it is not clear in the srs3 mutant (88.13%) (Table 1). The SRS3 gene may have an important function in inflorescence development. One vague characteristic is the seed number per panicle. This was increased in srs3 (127%), but it was not clearly increased in TCM 768 (112%) (Table 1). It seems to be unclear whether the SRS3 gene regulates the seed number per panicle in rice. Comparison of cell length and cell number in the WT and srs3 mutant Because the srs3 mutant shows the small and round seed phenotype, we postulated two hypothesizes: (i) the cell length in the srs3 mutant is shorter than that of the WT; or (ii) the cell number in the srs3 mutant is less than that of the WT. To confirm this, the length and width of cells in the inner epidermal tissues of the lemma were analyzed by scanning electron microscopy (SEM). The length and width of 100 cells enclosed within a square were measured (Fig. 2A, B). The average cell length in the WT and srs3 was 115.6 ± 27. 6 and 97.6 ± 24.6 μm, respectively (Fig. 2C and Table 2). The cell length in srs3 was reduced by 15.6% compared with the WT. The difference in cell length between the srs3 mutant and the WT was significant with a P-value <0.001. On the other hand, cell width in srs3 was reduced by only 3% compared with the WT (Fig. 2D and Table 2). The difference in cell width between the srs3 mutant and the WT was not significant, with a P-value >0.01. The number of cells in the longitudinal and lateral plane of the seeds was estimated by dividing the tissue length and width by the cell length and width, respectively (Table 2). The lengthwise estimated cell number in seed of the WT and srs3 was 66.7 and 68.9, respectively. Thus the lengthwise cell number was not different between the WT and srs3, indicating that the shortened seed length in srs3 was due to a reduction of cell length, but not cell number. The widthwise estimated cell number in seeds of the WT and srs3 was 78.5 and 86, respectively, showing an increase of 10% in srs3. The results suggested that loss of SRS3 function may enhance cell division in the lateral direction of the lemma. Positional cloning of SRS3 Linkage mapping places the SRS3 locus on the short arm of chromosome 5 (Tanabe et al. 2007). Identification of the SRS3 gene was performed by map-based cloning using F2 plants from a cross between srs3, a japonica mutant, and Kasalath, an indica strain. The F2 plants segregated into two groups in a 3 : 1 ratio; one group showing the normal seed size and the other showing the small and round seeds of srs3. The 1,200 F2 plants bearing small and round seeds were used for high-resolution mapping of the SRS3 locus. The candidate genomic region of srs3 was delimited to a 21 kb interval between markers 3,130 and 3,151 with three recombinant plants (Fig. 3A). In the candidate region, three genes are annotated in the Rice Annotation Project Database (RAP-DB, http://rapdb.dna .affrc.go.jp/): Os05g0154600 (a zinc finger, RING-type protein), Os05g0154700 (a kinesin motor domain protein) and Os05g0154800 (U1snRNP-specific protein). As the expression levels of these genes in srs3 were not very different from those in the WT, the possibility that the expression levels of these three genes cause the small and round seed phenotype seems to be low. To identify the mutation in srs3, we have used >58 sets of PCR primers to generate and sequence cDNA fragments covering these open reading frames. The sequence polymorphism we found was in Os05g0154700, suggesting that this gene is the causal gene for the small seed phenotype of the srs3 mutant. We named the gene, SRS3 (Fig. 3B). The SRS3 cDNA contains an open reading frame of 819 codons. The SRS3 gene consists of 12 exons and 11 introns. The mutation in the srs3 mutant is a base substitution (C to T) in the ninth exon, resulting in a Leu492 to Phe492 substitution (Fig. 3C). TCM 2092 and TCM 768 have mutations in SRS3 The causal gene of the small and round seed mutant phenotype in TCM 2092 and TCM 768 was localized on the short arm of chromosome 5 (data not shown). Because the candidate causal gene in the srs3 mutant was localized on the short arm of chromosome 5, the SRS3 cDNA sequences in TCM 2092 and TCM 768 were analyzed (Fig. 3C). TCM 2092 has one base substitution (G to A) in the 11th intron of the SRS3 gene, resulting in abnormal splicing. The sequence of the abnormal transcript in TCM 2092 is indicated below the genome DNA sequence (Fig. 3C). The splicing site between exon 11 and exon 12 of the SRS3 gene, GT–AG, is indicated in blue (Fig. 3C). The GTAAG sequence observed between exon 11 and intron 11 of the SRS3 gene, which is a conserved intron splicing site (Irimia and Roy 2008), is indicated with a short dashed line. The GTAAG sequence in the WT is changed to GTAAA in TCM 2092. The mutated A in TCM 2092 is indicated in Fig. 3C in red. The sequences of the transcripts deduced from the WT and TCM 2092 are shown under the genomic sequence. From the comparison of the genomic DNA and the transcript in TCM 2092, a newly generated splicing site is shown as a filled triangle in the genome sequence of TCM 2092. The newly generated stop codon found in the transcript of TCM 2092 is underlined. The newly generated splicing sequence GTACT in TCM 2092, shown with a long dashed line, was not similar to the conserved sequence GTAAG and it was not clear why the sequence GTACT was preferred for splicing in TCM 2092. As a consequence of abnormal splicing, the mutated SRS3 protein in TCM 2092 is predicted to be 762 amino acids long. TCM 768 has a nonsense mutation (T to A) in exon 5, resulting in a truncated 243 amino acid long protein. Suppression of SRS3 gene expression and complementation test To confirm that SRS3 is the causal gene for the small seed phenotype of the srs3 mutant, we generated SRS3 knockdown mutants using RNA interference (RNAi)-mediated gene silencing (Fig. 4A). Seeds (T1 generation) set in three independent primary transformants (T0 generation) exhibited the characteristic small and round phenotype (Fig. 4B). The amounts of SRS3 proteins were greatly reduced in SRS3 knockdown transgenic plants (Fig. 4D). The result demonstrates that the causal gene for srs3 is the SRS3 gene. The seed sizes in silenced transgenic plants are summarized in Table 3. The seed length in silenced transgenic plants is reduced (from 5.14 to 5.25 mm) compared with that in the WT (7.16 mm). Seed width in silenced transgenic plants is slightly increased (from 3.72 to 3.76 mm) compared with that in the WT (3.23 mm). Next we performed a complementation test. The entire sequence of the SRS3 cDNA under the control of the ubiquitin promoter was introduced into TCM 2092 using Agrobacterium tumefaciens-mediated transformation (Fig. 4E). Transgenic plants expressing the induced SRS3 gene were confirmed by reverse transcription–PCR (RT–PCR). Seven independent transgenic plants out of 15 which were regenerated showed a rescued phenotype. A seed of a transgenic plant expressing the induced SRS3 gene (line 1) is shown in Fig. 4F. The seed sizes in two rescued transgenic plants are summarized in Table 3. Seed length in the transgenic plants expressing the SRS3 gene was increased (from 6.53 to 6.86 mm) compared with that in TCM 2092 (5.16 mm). Seed width in the transgenic plants expressing the SRS3 gene was reduced (from 3.14 to 3.32 mm) compared with that in TCM 2092 (3.69 mm). In this study, the transgenic plants expressing the induced SRS3 gene in TCM 2092 were not completely rescued up to the WT. The reasons are that the mutated SRS3 proteins in TCM 2092 may compete with the normal SRS3 proteins introduced or the ubiquitin promoter may not fully function in seed development. Transformation with a control vector that contained no insert had no apparent effect on TCM 2092 phenotype. These results suggested that the SRS3 gene rescued the small and round phenotype of TCM 2092. SRS3 is a putative kinesin 13 protein BLAST searches revealed that the deduced amino acid sequence of the SRS3 protein showed considerable similarity to that of previously reported kinesins from Arabidopsis and rice: 62% identity and 73% similarity with the AtKinesin13A protein from Arabidopsis thaliana (Lu et al. 2005), 69% identity and 81% similarity with AtKinesin13B from A. thaliana (Guo et al. 2009), and 62% identity and 73% similarity with Os01g0625200, a putative kinesin 13 protein from rice (Guo et al. 2009). Phylogenic analysis revealed that the SRS3 protein belongs to the kinesin 13 subfamily (Fig. 5). Expression of SRS3 Accumulation of SRS3 transcripts and SRS3 protein during the rice life cycle was investigated by real-time RT–PCR and Western blot. First, we found expression of SRS3 protein in all organs examined, namely aerial parts, roots, internodes and panicles before and after heading (Fig. 8A, left panel). We then examined the expression profile of SRS3 protein at various developmental stages. At the L4 stage, where the first (L1), second (L2) and third (L3) leaves have finished growing, the fourth leaf is in a developing stage (Fig. 8A, middle panel). The SRS3 protein was detected mostly in the fourth leave (L4) at this stage. This shows that the SRS3 protein is highly expressed in developing organs. The amount of SRS3 protein during development of the panicle was also studied (Fig. 8A, right panel). The results indicate that the amount of SRS3 protein is higher in developing panicles compared with more developed panicles (Fig. 8B). The results of real-time RT–PCR analyses of SRS3 mRNA are shown in Fig. 8C. The amount of SRS3 mRNA was normalized to the amount of OsUbiquitin1 mRNA. SRS3 mRNA expression did not seem to correlate with the expression profiles of the SRS3 protein in roots, leaves and young panicles.

Knowledge Extension

  • The loss of seed-shattering habit is thought to be one of the most important events in rice domestication, because the Beasy-to-shatter trait in wild relatives results in severe reduction in yield.
  • Over the course of human history, distinct grain-threshing systems have been developed in several different eras in local areas of the world, in accordance with the degree of seed shattering.
  • In current rice-breeding programs, this seed-shattering habit is still a target, especially in the construction of new indica (another subspecies of O. sativa) cultivars. Thus, seed-shattering habit is one of the most important agronomic traits in rice cultivation and breeding.

Labs working on this gene

  • Institute of the Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, 446-1 Ippaizuka, Kamiyokoba Tsukuba, Ibaraki 305-0854, Japan.
  • National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
  • Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan.

References

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Structured Information