Abstract
In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.
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Selected References
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- Bagga S., Adams H. P., Rodriguez F. D., Kemp J. D., Sengupta-Gopalan C. Coexpression of the maize delta-zein and beta-zein genes results in stable accumulation of delta-zein in endoplasmic reticulum-derived protein bodies formed by beta-zein. Plant Cell. 1997 Sep;9(9):1683–1696. doi: 10.1105/tpc.9.9.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bosher J. M., Labouesse M. RNA interference: genetic wand and genetic watchdog. Nat Cell Biol. 2000 Feb;2(2):E31–E36. doi: 10.1038/35000102. [DOI] [PubMed] [Google Scholar]
- Chiba Y., Ishikawa M., Kijima F., Tyson R. H., Kim J., Yamamoto A., Nambara E., Leustek T., Wallsgrove R. M., Naito S. Evidence for autoregulation of cystathionine gamma-synthase mRNA stability in Arabidopsis. Science. 1999 Nov 12;286(5443):1371–1374. doi: 10.1126/science.286.5443.1371. [DOI] [PubMed] [Google Scholar]
- Christensen A. H., Quail P. H. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 1996 May;5(3):213–218. doi: 10.1007/BF01969712. [DOI] [PubMed] [Google Scholar]
- Chuang C. F., Meyerowitz E. M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4985–4990. doi: 10.1073/pnas.060034297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ciceri P., Castelli S., Lauria M., Lazzari B., Genga A., Bernard L., Sturaro M., Viotti A. Specific combinations of zein genes and genetic backgrounds influence the transcription of the heavy-chain zein genes in maize opaque-2 endosperms. Plant Physiol. 2000 Sep;124(1):451–460. doi: 10.1104/pp.124.1.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coleman C. E., Clore A. M., Ranch J. P., Higgins R., Lopes M. A., Larkins B. A. Expression of a mutant alpha-zein creates the floury2 phenotype in transgenic maize. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):7094–7097. doi: 10.1073/pnas.94.13.7094. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Damerval C., Le Guilloux M. Characterization of novel proteins affected by the o2 mutation and expressed during maize endosperm development. Mol Gen Genet. 1998 Feb;257(3):354–361. doi: 10.1007/s004380050657. [DOI] [PubMed] [Google Scholar]
- Galili Gad, Höfgen Rainer. Metabolic engineering of amino acids and storage proteins in plants. Metab Eng. 2002 Jan;4(1):3–11. doi: 10.1006/mben.2001.0203. [DOI] [PubMed] [Google Scholar]
- Geetha K. B., Lending C. R., Lopes M. A., Wallace J. C., Larkins B. A. opaque-2 modifiers increase gamma-zein synthesis and alter its spatial distribution in maize endosperm. Plant Cell. 1991 Nov;3(11):1207–1219. doi: 10.1105/tpc.3.11.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Habben J. E., Moro G. L., Hunter B. G., Hamaker B. R., Larkins B. A. Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8640–8644. doi: 10.1073/pnas.92.19.8640. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamilton Andrew, Voinnet Olivier, Chappell Louise, Baulcombe David. Two classes of short interfering RNA in RNA silencing. EMBO J. 2002 Sep 2;21(17):4671–4679. doi: 10.1093/emboj/cdf464. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hsieh J., Fire A. Recognition and silencing of repeated DNA. Annu Rev Genet. 2000;34:187–204. doi: 10.1146/annurev.genet.34.1.187. [DOI] [PubMed] [Google Scholar]
- Hunter Brenda G., Beatty Mary K., Singletary George W., Hamaker Bruce R., Dilkes Brian P., Larkins Brian A., Jung Rudolf. Maize opaque endosperm mutations create extensive changes in patterns of gene expression. Plant Cell. 2002 Oct;14(10):2591–2612. doi: 10.1105/tpc.003905. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hutvágner György, Zamore Phillip D. RNAi: nature abhors a double-strand. Curr Opin Genet Dev. 2002 Apr;12(2):225–232. doi: 10.1016/s0959-437x(02)00290-3. [DOI] [PubMed] [Google Scholar]
- Karchi H., Shaul O., Galili G. Lysine synthesis and catabolism are coordinately regulated during tobacco seed development. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2577–2581. doi: 10.1073/pnas.91.7.2577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kemper E. L., Neto G. C., Papes F., Moraes K. C., Leite A., Arruda P. The role of opaque2 in the control of lysine-degrading activities in developing maize endosperm. Plant Cell. 1999 Oct;11(10):1981–1994. doi: 10.1105/tpc.11.10.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim Cheol Soo, Woo Ym Young-min, Clore Amy M., Burnett Ronald J., Carneiro Newton P., Larkins Brian A. Zein protein interactions, rather than the asymmetric distribution of zein mRNAs on endoplasmic reticulum membranes, influence protein body formation in maize endosperm. Plant Cell. 2002 Mar;14(3):655–672. doi: 10.1105/tpc.010431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kodrzycki R., Boston R. S., Larkins B. A. The opaque-2 mutation of maize differentially reduces zein gene transcription. Plant Cell. 1989 Jan;1(1):105–114. doi: 10.1105/tpc.1.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lai Jinsheng, Messing Joachim. Increasing maize seed methionine by mRNA stability. Plant J. 2002 May;30(4):395–402. doi: 10.1046/j.1365-313x.2001.01285.x. [DOI] [PubMed] [Google Scholar]
- Levin J. Z., de Framond A. J., Tuttle A., Bauer M. W., Heifetz P. B. Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis. Plant Mol Biol. 2000 Dec;44(6):759–775. doi: 10.1023/a:1026584607941. [DOI] [PubMed] [Google Scholar]
- Llave Cesar, Xie Zhixin, Kasschau Kristin D., Carrington James C. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science. 2002 Sep 20;297(5589):2053–2056. doi: 10.1126/science.1076311. [DOI] [PubMed] [Google Scholar]
- Lohmer S., Maddaloni M., Motto M., Di Fonzo N., Hartings H., Salamini F., Thompson R. D. The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J. 1991 Mar;10(3):617–624. doi: 10.1002/j.1460-2075.1991.tb07989.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maddaloni M., Donini G., Balconi C., Rizzi E., Gallusci P., Forlani F., Lohmer S., Thompson R., Salamini F., Motto M. The transcriptional activator Opaque-2 controls the expression of a cytosolic form of pyruvate orthophosphate dikinase-1 in maize endosperms. Mol Gen Genet. 1996 Mar 20;250(5):647–654. doi: 10.1007/BF02174452. [DOI] [PubMed] [Google Scholar]
- Matzke M. A., Matzke A. J., Pruss G. J., Vance V. B. RNA-based silencing strategies in plants. Curr Opin Genet Dev. 2001 Apr;11(2):221–227. doi: 10.1016/s0959-437x(00)00183-0. [DOI] [PubMed] [Google Scholar]
- Mazur B., Krebbers E., Tingey S. Gene discovery and product development for grain quality traits. Science. 1999 Jul 16;285(5426):372–375. doi: 10.1126/science.285.5426.372. [DOI] [PubMed] [Google Scholar]
- Moore I., Gälweiler L., Grosskopf D., Schell J., Palme K. A transcription activation system for regulated gene expression in transgenic plants. Proc Natl Acad Sci U S A. 1998 Jan 6;95(1):376–381. doi: 10.1073/pnas.95.1.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parrish S., Fleenor J., Xu S., Mello C., Fire A. Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. Mol Cell. 2000 Nov;6(5):1077–1087. doi: 10.1016/s1097-2765(00)00106-4. [DOI] [PubMed] [Google Scholar]
- Schmidt R. J., Burr F. A., Burr B. Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. Science. 1987 Nov 13;238(4829):960–963. doi: 10.1126/science.2823388. [DOI] [PubMed] [Google Scholar]
- Schmidt R. J., Ketudat M., Aukerman M. J., Hoschek G. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. Plant Cell. 1992 Jun;4(6):689–700. doi: 10.1105/tpc.4.6.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shaver J. M., Bittel D. C., Sellner J. M., Frisch D. A., Somers D. A., Gengenbach B. G. Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1962–1966. doi: 10.1073/pnas.93.5.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimizu-Sato Sae, Huq Enamul, Tepperman James M., Quail Peter H. A light-switchable gene promoter system. Nat Biotechnol. 2002 Sep 3;20(10):1041–1044. doi: 10.1038/nbt734. [DOI] [PubMed] [Google Scholar]
- Song R., Llaca V., Linton E., Messing J. Sequence, regulation, and evolution of the maize 22-kD alpha zein gene family. Genome Res. 2001 Nov;11(11):1817–1825. doi: 10.1101/gr.197301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Song Rentao, Llaca Victor, Messing Joachim. Mosaic organization of orthologous sequences in grass genomes. Genome Res. 2002 Oct;12(10):1549–1555. doi: 10.1101/gr.268302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Song Rentao, Messing Joachim. Contiguous genomic DNA sequence comprising the 19-kD zein gene family from maize. Plant Physiol. 2002 Dec;130(4):1626–1635. doi: 10.1104/pp.012179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tavernarakis N., Wang S. L., Dorovkov M., Ryazanov A., Driscoll M. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat Genet. 2000 Feb;24(2):180–183. doi: 10.1038/72850. [DOI] [PubMed] [Google Scholar]
- Ueda T., Waverczak W., Ward K., Sher N., Ketudat M., Schmidt R. J., Messing J. Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein. Plant Cell. 1992 Jun;4(6):701–709. doi: 10.1105/tpc.4.6.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waterhouse P. M., Graham M. W., Wang M. B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13959–13964. doi: 10.1073/pnas.95.23.13959. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wesley S. V., Helliwell C. A., Smith N. A., Wang M. B., Rouse D. T., Liu Q., Gooding P. S., Singh S. P., Abbott D., Stoutjesdijk P. A. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 2001 Sep;27(6):581–590. doi: 10.1046/j.1365-313x.2001.01105.x. [DOI] [PubMed] [Google Scholar]
- Woo Y. M., Hu D. W., Larkins B. A., Jung R. Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression. Plant Cell. 2001 Oct;13(10):2297–2317. doi: 10.1105/tpc.010240. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zamore Phillip D. Ancient pathways programmed by small RNAs. Science. 2002 May 17;296(5571):1265–1269. doi: 10.1126/science.1072457. [DOI] [PubMed] [Google Scholar]