Small and round seed 5 gene encodes alpha-tubulin regulating seed cell elongation in rice
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 androundseed 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
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 (Small androundseed 5). The results clearly demonstrated that Srs5 encodes alpha-tubulin and regulates cell elongation in rice seed.
Characterization of the Srs5 mutant
SRS5 gene encodes alpha-tubulin
Srs5 regulates cell elongation independently of BR signal transduction
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 Twisteddwarf 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.
Our study demonstrated that short seed mutants can be classified into two types: those with reduced cell numbers, e.g., d1, and those with reduced cell length, e.g., d61 (Figure 2A, C, and 2D). This facilitates classification of novel seed mutants. The short seed phenotype of Srs5 was demonstrated to be caused by reduced cell length, as in d61; however, the additive phenotype of the double mutant indicated that SRS5 and D61 regulate seed length via different mechanisms. To evaluate the mechanisms regulating seed length, observation of microtubule arrangement and analysis of double mutants among srs1, srs3, Srs5, and various BR mutants need to be performed.
Plant materials and growth conditions
Kyudai No. 37 was first identified at Kyushu University and maintained in Togo Field, Nagoya University. Its genetic background is unknown. A japonica cultivar, Taichung 65, was used as the WT plant. d1-1 was identified as spontaneous mutant 'Daikoku' and substituted its genetic background into Taichung 65 by backcrossing with Taichung 65 as a recurrent parent at Kyushu University. d61-2 was obtained by MNU treatment of Taichung 65. F2 and two parental lines were sown at the beginning of April. The seedlings of all plants were transplanted at the beginning of May into a paddy field at the Research Center for Bioresources Development in Fukui, Japan. They were then grown under natural conditions. Transgenic plants were grown in a closed greenhouse under natural sunlight. Room temperature was maintained at 30°C from 09:00 to 18:00 and 25°C from 18:00 to 09:00.
Linkage analysis of SRS5
Primer sequences used in this study
Production of transgenic plants
The BAC clone containing the SRS5 gene was screened from the BAC library (constructed by the CUGI BAC/EST Resource Center) using four PCR primers, alpha-tub-5kb-up, alpha-tub-intron, alpha-tub-exon, and alpha-tub-5kb-down.
The BAC clone OSJNBa0014D2 was partially digested by Sau 3AI and cloned into the Bam HI site of binary vector pYLTAC7 (Liu et al. 1999) (provided by RIKEN BioResource Center). This clone contains the 7.26 kb upstream region from the transcriptional start site of the Srs5 gene and the 1.13 kb downstream region from the end of the 3'UTR region of the SRS5 gene. The binary vector was transformed into Agrobacterium tumefaciens strain EHA105 (Hood 1993) by electroporation, and Srs5 mutants were transformed as reported previously (Ashikari et al. 2005). Srs5 mutants containing empty vectors were used as controls.
RNA isolation and RT-PCR
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). cDNAs were synthesized from total RNA using the SuperScript III system (Invitrogen, Carlsbad, CA, USA).
For quantification of SRS5 mRNA, real-time RT-PCR was carried out using SYBR Premix Ex Taq ™ II (TAKARA Bio, Inc., Tokyo, Japan). Two primers, RT-alpha-tub, were used to quantify Srs5 expression; a further two, RT-OsUbiquitin, were used to quantify OsUbiquitin1 expression (accession No. Os06g0681400). The Thermal Cycler Dice Real Time System (TAKARA Bio, Inc.) was used for quantification for real-time RT-PCR.
This work was supported in part by the Funding Program for Next Generation World-Leading Researchers (NEXT Program) [GS-024] and the Ministry of Agriculture, Forestry and Fisheries of Japan [Genomics for Agricultural Innovation IPG-0002].
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