Os11g0247300

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Annotated Information

Small and round seed 5 gene encodes alpha-tubulin regulating seed cell elongation in rice

Function

Seed size is an important trait in determinant of rice seed quality and yield. In this study, we report a novel semi-dominant mutant Small and round seed 5 (Srs5) that encodes alpha-tubulin protein. Lemma cell length was reduced in Srs5 compared with that of the wild-type. Mutants defective in the G-protein alpha subunit (d1-1) and brassinosteroid receptor, BRI1 (d61-2) also exhibited short seed phenotypes, the former due to impaired cell numbers and the latter due to impaired cell length. Seeds of the double mutant of Srs5 and d61-2 were smaller than those of Srs5 or d61-2. Furthermore, SRS5 and BRI1 genes were highly expressed in Srs5 and d61-2 mutants. These data indicate that SRS5 independently regulates cell elongation of the brassinosteroid signal transduction pathway.

Expression

Seed size and weight are important traits for rice yield (Song and Ashikari 2008, Takeda and Matsuoka 2008). Several quantitative trait loci (QTLs) affecting seed size have been identified, namely GW2 encoding a RING-type protein that functions as an E3 ubiquitin ligase (Song et al. 2007), qSW5 encoding a novel protein with no known domains (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 a wider seed phenotype, and loss of GS3 function leads to a longer seed phenotype, both resulting in increased yield.

Causal genes of the small (or short) seed mutants have also been identified, namely d1 (also named RGA1) encoding the heterotrimeric G protein alpha 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 another type of cytochrome P450 involved in BR synthesis (Hong et al. 2003, Hong et al. 2005), d61 (also named OsBRI1) encoding the BR receptor (Yamamuro et al. 2000), srs1 encoding a novel protein that has no known functional domains (Abe et al. 2010), and finally, srs3 encoding a kinesin 13 protein (Kitagawa et al. 2010). During seed formation in rice, it was demonstrated that D1 regulates cell number (Izawa et al. 2010), and SRS1 and SRS3 regulate cell length (Abe et al. 2010, Kitagawa et al. 2010). From these observations, SRS1 and SRS3 seem to affect seed size through signaling pathways other than G-protein signal transduction.

Although several genes regulating seed size have been identified, their molecular network underlying seed formation remains unclear. Here we report molecular cloning of a novel small and round seed mutant in Srs5 (

Evolution

In linkage analysis, we could not clearly distinguish seed size in F2 seed derived from distant cross between Srs5 and Kasalath. This was likely to be caused by background difference between indica and japonica. To overcome this, we used a chromosome segment substitution line (CSSL), which is a plant series that possesses relatively large chromosome segments of donor parent chromosomes in the recurrent parental chromosome background (Yano and Sasaki 1997, Yano 2001, Ebitani et al. 2005, Ashikari and Matsuoka 2006, Fukuoka et al. 2010). CSSLs can be used to achieve high accuracy in phenotyping in F2 populations. In fact, we could make classification of two seed size, wild and mutant type, in the F2 population derived from a cross between Srs5 and a CSSL.

Genetic analysis of the F2 population derived from a cross between Srs5 and SL233 demonstrated that the Srs5 gene act as semi-dominant manner. This semi-dominant effect was also confirmed in complementation test. Although The Srs5 mutants carrying WT SRS5 gene showed longer seeds than that of the plants containing empty vector, the degree of recovery was not completely same as WT (Figure 4B). The reason that the rescue by WT SRS5 gene was partial may be due to compete between WT and mutation gene products or incomplete conformation of the tubulin complex.

In this study, we demonstrated that the SRS5 gene encodes alpha-tubulin, which has been reported to be the causal gene of the rice mutation

T w i sted d warf 1(Tid1) (Sunohara 2009). The Tid1 mutation acts as a semi-dominant gene by affecting the interaction of alpha and beta tubulin. Since Srs5 was also a semi-dominant mutation, it was likely caused by incomplete conformation of the tubulin complex. Tid1 shows right helical growth, in addition to a semi-dominant dwarf phenotype. Additionally, Arabidopsis Lefty1 and Lefty2 mutations in genes orthologous to SRS5 also show semi-dominant and left helical growth (Thitamadee et al. 2002). These two mutants were gain-of-function alleles and exhibited similar twisted plant phenotypes. As the Srs5 mutant does not exhibit a twisted phenotype, different mutations in alpha-tubulin seem to lead to different phenotypes. In spikelets, higher accumulation of SRS5 mRNA was detected in the Srs5 mutant than in the WT (Figure 5A). This seems to compensate for the reduced function of alpha-tubulin protein. Higher expression of SRS5 was also detected in d61-2 but not in d1-1 (Figure 5B and 5C). Furthermore, BRI1 gene highly expresses in Srs5 and d61-2 but not in d1-1 (Figure 5B and 5C). These results suggest that the expression of SRS5 and BRI1 genes are compensated by sensing the cell elongation inhibition in the SRS5 and d61-2 mutants, although SRS5 and BRI1 genes regulate cell elongation independently (Figure 6D). Three other alpha-tubulin genes are present in the rice genome, and they share a high homology (Sunohara et al. 2009). In organs that exhibited no significant change in phenotype in the Srs5 mutant, these alpha-tubulins might work redundantly to maintain rice body planning.

Labs working on this gene

Shuhei Segami1, Izumi Kono23, Tsuyu Ando24, Masahiro Yano5, Hidemi Kitano6, Kotaro Miura1* and Yukimoto Iwasaki1*

1 Faculty of Biotechnology, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka, Eiheiji-Town, Fukui 910-1195, Japan

2 Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan

3 Laboratory of Synaptic Plasticity and Connectivity RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

4 National Institute of Agrobiological Science, 2-1-1 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

5 National Institute of Agrobiological Science, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634 Japan

6 Bioscience and Biotechnology Center, Nagoya University, Furo, Chikusa, Nagoya, Aichi 464-8601, Japan

References

Abe Y, Mieda K, Ando T, Kono I, Yano M, Kitano H, Iwasaki Y (2010) The SMALL AND ROUND SEED1 (SRS1/DEP2) gene is involved in the regulation of seed size in rice. Genes Genet Syst 85:327-39

Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M (2005) Cytokinin oxidase regulates rice grain production. Science 309:741-5

Small and round seed 5 gene encodes alpha-tubulin regulating seed cell elongation in rice Shuhei Segami1, Izumi Kono23, Tsuyu Ando24, Masahiro Yano5, Hidemi Kitano6, Kotaro Miura1* and Yukimoto Iwasaki1*

Ashikari M, Matsuoka M (2006) Identification, isolation and pyramiding of quantitative trait loci for rice breeding. Trends Plant Sci 11:344-50

Structured Information