The Wx gene encodes AD glucose starch glycosyl transferase and controls amylase synthesis in the endosperm of the grass family. Amylose accumulated in the rice endosperm is mainly regulated by the amount of Wx gene products. Amylose content is an important factor affecting the eating quality of rice, which varies markedly with countries and cultures. In rice, two Wx alleles, Wxa and Wxb, which regulate the amylose content widely. The endosperms homozygous for Wxa produce 10-fold more Wx gene products than those for Wxb, resulting in higher amylose content in the former. Wxb predominates in the Japonica type while Wxa in the India type and its relatives. The importance of the amylose content in grain quality requires that the allelic states in parents be examined before hybridization and that unfavorable alleles in early generations after hybridization be eliminated. However, the level of Wx gene expression and amylose synthesis is also controlled by other genes controlling the amylose content. Especially, du genes are known to reduce the level of the Wx products as well as amylose in eh endosperm.
Eight dull mutants that lower the amylose content of rice endosperm as well as waxy mutant and a cultivar with common grains were crossed in a dialyze manner. The amylose content of F 1 and F 2 seeds was determined on the basis of single grain analysis. It was concluded that the low amylose content of dull mutants is under monogenic recessive control. Alleles for low amylose content are located at five loci designated as du-1, du-2, du-3, du-4 and du-5. These loci are independent of wx locus located on chromosome 6. The five du loci have an additive effect in lowering the amylose content. Two loci, du-1 and du-4, were found to be located on chromosomes 7 and 4, respectively.
The Wx gene expression plays a major role in determining the amylose content in the rice endosperm, although Wx alleles and their trans-acting genes like du genes are also known to genetically control the amylose content.
- 1.In endosperm, du-1 and du-2 reduce the amount of spliced Wxb transcripts. They affect either the splicing of intron 1 or other post-transcriptional steps in processing of the the Wxb transcripts.
- 2.Both du-1 and du-2 mutations cause inefficient splicing of the mutant Wxb transcripts that have three new weak 5′ splice sites due to a mutation at the 5′ splice site of intron 1, but not of the non-mutatedWxa transcripts.
- 3.Both du-1 and du-2 have differential effects on Wxb splicing in endosperm and pollen. Results suggest that neither du-1 nor du-2 encodes general factors required for pre-mRNA splicing.
- 1. Plant material
A japonica rice cultivar, Kinmaze, its mutant lines, du-1 (EM12) and du-2 (EM2) ( Yano et al. 1988 ), and other rice plants were all grown in a greenhouse under a light–dark cycle consisting of 14 h of light and 10 h of dark. Transgenic rice plants carrying the Wxa gus transgene were produced by electro oration of protoplasts isolated from embryogenic suspension cultures according to the method described previously. Transgenic plants of the R2 generation were crossed with dull mutants. F1 plants were selfed, and the F2 seeds and plants were screened for opaque dull endosperm and the presence of the Wxa-gus transgene by Southern blot analysis. F2 plants homozygous for both dull and Wxa-gus were selfed, and immature F3 seeds were analyzed by RNA gel blot analysis.
- 2. RNA gel blot analysis
Total RNA was prepared from immature seeds 15 days after pollination. RNA (10 μg) was separated by electrophoresis in 1% agarose gels containing formaldehyde, blotted and hybridized with a waxy cDNA probe that had been labeled with [α-32P] dCTP, using the Multiprime DNA labelling system (Amersham). The probes used were a 1.3 kb BglII fragment of Wx cDNA (exon 5 to exon 14), a 0.9 kb EcoRI fragment of RBE1 cDNA, a full length AGPP cDNA, a full length gus and the exon 2 of the rice actin 1. After the membrane was washed, an autoradiography was obtained by exposing the membrane to an X-ray film.
- 3. Competitive RT–PCR analysis of Wx transcripts
A competitor DNA was constructed by inserting a 61 bp DNA fragment into the BalI site in exon 3 of Wxa cDNA cloned into a pGEM-T vector. The competitor DNA was transcripted into RNA by the T7 promoter using Riboprobe Combination Systems. The competitor RNA was quantitated by absorbance at 260 nm. Total RNA was extracted from immature seeds and mature anther ( Chomczynski & Sacchi, 1987). Serial dilutions of the competitor RNA were co-amplified with 500 ng of total RNA using RNA PCR Kit (Takara) and LA Taq with GC Buffer (Takara). PCR cycle conditions followed by 25 or 30 cycles of 94°C for 30 sec, 68°C for 30 sec, 72°C for 1 min. To detect amplified products another two rounds of amplification were performed. The first amplification was 30 cycles with primers, 5′-ACCATTCCTTCAGTTCTTTG-3′, and 5′-TCCGTAGATCTTCTCACC-GG-3′, and the second cycle was 25 or 30 cycles with primers, 5′-CAGTTCTTTGTCTATCTCAAGACAC-3′ and 5′-CGTACCGAGGAG-AGATCACC-3′. The first primer pairs were used for amplification of endosperm RNA. After PCR reactions, amplified DNA was electrophoresed in 2% agarose gels and visualized by ethidium bromide staining. Quantitation of the amount of Wxb mRNA was performed as described previously.
- 4. RT–PCR analysis of other transcripts
RNA was amplified with 500 ng of total RNA using RNA PCR Kit (Takara). PCR amplifications were performed using RBE1-specific primer RB214 in exon 3, 5′-CATGGTGACTGTTGTGGAGG − 3′, and primer RB1500 in exon 6, 5′-CTATCAGGAATGGCCATTGC-3′, and AGPP-specific primer AG335 in exon 2, 5′-AACCGTCACCTGTCAA-GAGC-3′, and primer AG1203 in exon 7, 5′-TGAGTCCTCTATTATT-GCGCC-3′. PCR cycle conditions followed by 25 cycles of 94°C for 30 sec, 65°C for 30 sec, 72°C for 1 min. Amplified DNA was electrophoresed in 2% agarose gels and visualized by ethidium bromide staining.
- 5. Protein gel blot analysis
Proteins were extracted from mature seeds and anthers using a previously published protocol. For protein gel blot analysis, proteins were separated by 10% SDS-PAGE and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore). After blocking with 1% BSA, the blots were probed by an antibody against rice WAXY protein ( Hirano & Sano, 1991). The immunoreactive protein was detected using an ECL Western blotting detection kit). An autoradiography was obtained by exposing the membrane to Hyperfilm-ECL. The detected signal was quantified by Densitograph (ATTO).
Uniqueness of du-1 and du-2 mutations: our studies indicate that rice du-1 and du-2 mutations reduce the splicing efficiencies of intron 1 at two 5′ cryptic splice sites present in Wxb transcripts having + 1T rather than G. Furthermore, they seem to have no effect on the Wxa transcripts that have the normal 5′ splice site of intron 1. Moreover, these mutations do not appear to influence the splicing of transcripts derived from the three other genes examined. These findings suggest that du-1 and du-2 mutations may be uniquely uncovered due to the 5′ splice site mutation present in the Wxb allele of rice. However, our results do not exclude the possibility that gene products of du-1 and du-2 play roles in the splicing of other transcripts because they may quantitatively affect the splicing efficiencies of other transcripts or they may be redundant in particular tissues and/or developmental stages. In addition, dull mutants with low or intermediate amylose contents were not found in indica rice cultivars, most of which carry Wxa allele. These observations support the hypothesis that dull mutations only arise from plants having waxy genes with mutant 5′ splice sites. A mutant Drosophila lacking a splicing factor, SRp55, is lethal. In contrast, because the waxy gene is a non-essential gene, our studies may have identified mutations of factors affecting the splicing of waxy transcripts. Mutant gene was allelic to du2 but the gene was designated as du2-2 as its phenotype was distinct. Nearisogenic lines (NILs) with different combinations of alleles at the Wx and du2-2 loci were then established and evaluated for the Wx gene expression. Waxy or chalky endosperms due to du2-2 were detected only in response to Wxb but not to Wxa, which may account for the fact that no du variants were detected in the Indica type.
Two lines of evidence suggest that both du-1 and du-2 mutations affect the splicing of Wxb transcripts rather than post-transcriptional steps after splicing. First, they do not affect the processing of Wxa transcripts that are almost identical to Wxb transcripts including the 3′ UTR. Second, du-1 and du-2 show tissue-specific effects with respect to splice site selection and the abundance of spliced Wxb mRNA. These results are consistent with the current model on the regulation of pre-mRNA splicing based on studies in mammals and Drosophila as discussed below. 1. Possible roles of Du-1 and Du-2 proteins in splicing A number of protein factors have been identified in vertebrates and yeast that are involved in the regulation of pre-mRNA splicing. For instance, SR proteins (reviewed in Manley & Tacke, 1996) are involved in constitutive and regulated splicing, are tissue-specifically regulated, and have been shown to bind purine-rich splicing enhancers present in exons. SR proteins have been identified in plants and have been shown to function as splicing factors in in vitro splicing assays with mammalian cell extracts. More recently, atSRp30, an Arabidopsis SR protein with a strong similarity to SF2/ASF, was shown to have an in vivo role in the regulation of pre-mRNA splicing in plants. The selection of the 5′ splice site in early transcripts of SV40 has been shown to be regulated by SR proteins, and different SR proteins promote splicing at the two different 5′ splice sites. ASF/SF2, one of the best-characterized human SR proteins, has been shown to bind purine-rich exonic enhancers ( Tacke & Manley, 1995). Interestingly, examination of the base sequences of exon 1 of Wxb revealed the presence of an octamer, GGAAGAAC, which has a complete match with the consensus of the purine-rich exonic splicing enhancer ( Watakabe et al. 1993 ), RGAAGAAC, where R is any purine ( Tacke & Manley, 1995), at 17 bases upstream of the authentic splice site ( Isshiki et al. 1998 ). Based on these, it is an intriguing possibility that du-1 and du-2 code for splicing factor(s) such as SR proteins, binding to this putative splicing enhancer and stabilizing the splicing complex at two weak splice sites generated by mutation in Wxb pre-mRNA. Binding of these splicing factors may not be required for a strong wild-type 5′ splice site. Since multiple forms of SR proteins are also known in plants, our failure to identify the effects of du-1 and du-2 on the splicing of other transcripts in either endosperm or pollen may have resulted from their redundant functions. Alternatively, du-1 and du-2 may encode more specific splicing regulators such as TRA or TRA 2, which are involved in sex-specific splicing of dsx transcripts inDrosophila ( Heinrichs & Baker, 1995; Tian & Maniatis, 1993), or hnRNP A1, which acts antagonistically to ASF/SF2 in the selection of the 5′ splice site. The effects of du-1 and du-2 are tissue specific. Although both mutations equally affect splicing efficiencies at the two 5′ splice sites in endosperm, in pollen the du-1effect is greatly enhanced, whereas du-2 has little effect on splicing efficiencies. A tissue-specific difference in splicing efficiency was previously reported for a retrotransposon-induced mutant waxy gene of maize. The transcripts of wxG, in which a retrotransposon is inserted in intron 8, are 30-fold more efficiently spliced in pollen than in endosperm. This result suggests the presence of splicing factors that act differently in endosperm and pollen, and that the gene products of du-1 and du-2 may belong to these factors. 2. Use of the Wxbgene and dull mutations in studies of regulated splicing in plants The study of splicing, particularly the identification of splicing regulators, is severely hampered by the lack of an in vitro system in plants. The Wxb system of rice described in this investigation may be a useful model system to study tissue-specific and regulated splicing in plants. Two weak 5′ splice sites were generated by natural mutations, and the selection of the splice site was tissue specifically regulated. Extragenic mutations influencing 5′ splice site selection in a tissue-specific fashion were available. A putative splicing enhancer which may interact with splicing regulators was present between the two weak 5′ splice sites of exon 1 ( Isshiki et al. 1998 ). The effects of variously modified cis-elements on splicing efficiency and splice site selection can be tested by transfection into rice protoplasts ( Isshiki et al. 1998 ). Having isolated three SR protein genes of rice, we are currently testing whether they may modulate splicing efficiency and selection of the two 5′ splice sites of the Wxb mRNA in rice protoplasts. Such studies should help us to understand the regulation of alternative splicing in the Wxb gene of rice.
Labs working on this gene
- 1. Ministry of Education, Science, Sports and Culture, Japan.
- 2. Laboratory of Plant Breeding, Faculty of Agriculture, Hokkaido University, Japan.
- 3. Biological Resources R&D Center, Fukui Prefectural University, Japan.
- 4. Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Japan.
- 5. Yokohama Research Center, Mitsubishi Chemical Co, Japan.
- 6. Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Japan.
- 7. Hokuriku National Agriculture Experiment Station, Japan.
- 8. Institute of Biological Chemistry, Washington State University, Washington.
- 9. Department of Biochemistry, Michigan State University, Michigan.
- 10. Department of Cell and Molecular Genetics, Scottish Crop Research Institute, UK.
- 1. M. Yano, K. Okuno, H. Satoh and T. Omura. Chromosomal location of genes conditioning low amylose content of endosperm starches in rice, Oryza sativa L. Theoretical and Applied Genetics, 1988, 76(2): 183-189.
- 2. Hikaru Satoh, Takeshi Omura. New Endosperm Mutations Induced by Chemical Mutagens in Rice Oryza sativa L. Japanese Journal of Breeding, 1981, 31(3): 316-326.
- 3. Le-Viet Dung, Ichiho Mikami, Etsuo Amano, etal. Study on the Response of dull endosperm 2-2, du2-2, to Two Wx Alleles in Rice. Breeding Science, 2000, 50(3): 215-219.
- 4. Masayuki Isshiki, Midori Nakajima, Hikaru Satoh, etal. Dull: rice mutants with tissue-specific effects on the splicing of the waxy pre-mRNA. The Plant Journal, 2000, 23(4): 451-460.
- 5. Anderson J, Hnilo J, Larson R, etal. The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme. J Biol Chem. 1989, 264(21):12238-12242.
- 6. Nakata P, Greene T, Anderson J, etal. Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits. Plant Mol Biol. 1991, 17(5):1089-1093.
- 7. Burton R, Johnson P, Beckles D, etal. Characterization of the genes encoding the cytosolic and plastidial forms of ADP-glucose pyrophosphorylase in wheat endosperm. Plant Physiol. 2002, 130(3):1464-75.
low amylose content mutant dul
Similar to CEL5=CELLULASE 5 (Fragment)
NM_001111411.1 GI: 162463770 GeneID: 541657
Zea mays dull endosperm1 (du1), mRNA.
ORGANISM Zea mays
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; PACMAD clade; Panicoideae; Andropogoneae; Zea.
|Sequence Coding Region||
<gbrowseImage1> name=NC_008401:762215..764081 source= Zea mays Chromosome07 preset=GeneLocation </gbrowseImage1>
<gbrowseImage2> name=NC_008401:762215..764081 source= Zea mays Chromosome07 preset=GeneLocation </gbrowseImage2>
<aaseq> MEMVLRSQSPLCLRSGPVLIFRPTVAGGGGGTQSLLRTTRFARR RVIRCVVASPGCPNRKSRTASPNVKVAAYSNYAPRLLVESSSKKSEHHDSSRHREETI DTYNGLSGSDAAELTSNRDVEIEVDLQHISEEELPGKVSINASLGEMETVDEAEVEED KFEVDTSGIVLRNVAVREVDPKDEHNAKDVFVVDSSGTAPDNAAVEEVVDEAEVEEDM VDVDILGLDLNNATIEEIDLMEEALLENFDVDSPGNASSGRTYGGVDELGELPSTSVD CIAINGKRRSLKPKPLPIVRFQEQEQIVLSIVDEEGLIASSCEEGQPVVDYDKQEENS TAFDEQKQLTDDFPEEGISIVHFPEPNNDIVGSSKFLEQKQELDGSYKQDRSTTGLHE QDQSVVSSHGQDKSIVGVPQQIQYNDQSIAGSHRQDQSIAGAPEQIQSVAGYIKPNQS IVGSCKQHELIIPEPKKIESIISYNEIDQSIVGSHKQDKSVVSVPEQIQSIVSHSKPN QSTVDSYRQAESIIGVPEKVQSITSYDKLDQSIVGSLKQDEPIISVPEKIQSIVHYTK PNQSIVGLPKQQQSIVHIVEPKQSIDGFPKQDLSIVGISNEFQTKQLATVGTHDGLLM KGVEAKETSQKTEGDTLQATFNVDNLSQKQEGLTKEADEITIIEKINDEDLVMIEEQK SIAMNEEQTIVTEEDIPMAKVEIGIDKAKFLHLLSEEESSWDENEVGIIEADEQYEVD ETSMSTEQDIQESPNDDLDPQALWSMLQELAEKNYSLGNKLFTYPDVLKADSTIDLYF NRDLSAVANEPDVLIKGAFNGWKWRFFTEKLHKSELAGDWWCCKLYIPKQAYRMDFVF FNGHTVYENNNNNDFVIQIESTMDENLFEDFLAEEKQRELENLANEEAERRRQTDEQR RMEEERAADKADRVQAKVEVETKKNKLCNVLGLARAPVDNLWYIEPITTGQEATVRLY
WVFADGPPGSARNYDNNGGHDFHATLPNNMTEEEYWMEEEQRIYTRLQQERREREEAI KRKAERNAKMKAEMKEKTMRMFLVSQKHIVYTEPLEIHAGTTIDVLYNPSNTVLTGKP EVWFRCSFNRWMYPGGVLPPQKMVQAENGSHLKATVYVPRDAYMMDFVFSESEEGGIY DNRNGLDYHIPVFGSIAKEPPMHIVHIAVEMAPIAKVGGLGDVVTSLSRAVQDLGHNV EVILPKYGCLNLSNVKNLQIHQSFSWGGSEINVWRGLVEGLCVYFLEPQNGMFGVGYV YGRDDDRRFGFFCRSALEFLLQSGSSPNIIHCHDWSSAPVAWLHKENYAKSSLANARV VFTIHNLEFGAHHIGKAMRYCDKATTVSNTYSKEVSGHGAIVPHLGKFYGILNGIDPD IWDPYNDNFIPVHYTCENVVEGKRAAKRALQQKFGLQQIDVPVVGIVTRLTAQKGIHL IKHAIHRTLERNGQVVLLGSAPDSRIQADFVNLANTLHGVNHGQVRLSLTYDEPLSHL IYAGSDFILVPSIFEPCGLTQLVAMRYGTIPIVRKTGGLFDTVFDVDNDKERARDRGL EPNGFSFDGADSNGVDYALNRAISAWFDARSWFHSLCKRVMEQDWSWNRPALDYIELY RSASKL</aaseq>
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