Molecular Genetics and Genomics

, Volume 286, Issue 2, pp 119–133 | Cite as

Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach

Original Paper

Abstract

The majority of phosphorus (P) in seeds is found in phytic acid (InsP6) which accumulates as the mixed salt phytate. InsP6 is generally considered to be an anti-nutrient and the development of low phytic acid (lpa) seed crops is of significant interest. We have employed a reverse genetics approach to examine the impact of disrupting genes involved in inositol phosphate metabolism on Arabidopsis seed InsP6 levels. Our analysis revealed that knockout mutations in three genes (AtITPK1, AtITPK4, and AtMIK/At5g58730) reduced seed InsP6 in addition to knockouts of four previously reported genes (AtIPK1, AtIPK2β, AtMRP5, and At5g60760). Seeds of these lpa mutants also exhibited reduced germination under various stress conditions. The greatest reduction in InsP6 (>70%) was observed in atmrp5 seeds which were also among the least sensitive to the stresses examined. Expression analysis of the lpa genes revealed three distinct patterns in developing siliques consistent with their presumed roles. Disruption of each lpa gene resulted in changes in the expression in some of the other lpa genes indicating that transcription of lpa genes is modulated by other constituents of InsP6 metabolism. While all the lpa genes represent possible targets for genetic engineering of low phytate seed crops, mutations in AtMRP5, AtMIK, and At5g60760 may be most successful for conventional approaches such as mutation breeding.

Keywords

Phytic acid Arabidopsis Inositol phosphate metabolism Kinases Reverse genetics 

Abbreviations

BLASTp

Basic local alignment search tool protein–protein

DAF

Days after fertilization

GADPH

Glyceraldehyde 3-phosphate dehydrogenase

HPIC

High-performance ion chromatography

Ins

Inositol

InsP

Inositol (poly)phosphate

PtdIns

Phosphatidylinositol(s)

lpa

Low phytic acid

P

Phosphorus

Pi

Inorganic phosphate

RT-PCR

Reverse transcription-polymerase chain reaction

SAIL

Syngenta Arabidopsis insertion lines

SALK

Salk Institute

T-DNA

Transfer DNA

Notes

Acknowledgments

This was supported by USDA Agricultural Research Service CRIS Project 5306-21000-016/017-00D (T.H.T.) and National Research Initiative Competitive Grant 2005-35301-15708 from the USDA Cooperative State Research, Education, and Extension Service (T.H.T.). We are thankful UC DAVIS DANR analytic lab for assisting with HPIC analysis and to V. Raboy and C. Andaya for critical reading of the manuscript and helpful suggestions for improvement. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Supplementary material

438_2011_631_MOESM1_ESM.pdf (191 kb)
Supplementary material 1 (PDF 190 kb)

References

  1. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657PubMedCrossRefGoogle Scholar
  2. Bentsink L, Yuan K, Koornneef M, Vreugdenhil D (2003) The genetics of phytate and phosphate accumulation in seeds and leaves of Arabidopsis thaliana, using natural variation. Theor Appl Genet 106:1234–1243PubMedGoogle Scholar
  3. Bohn L, Meyer AS, Rasmussen SK (2008) Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ Sci B 9:165–191PubMedCrossRefGoogle Scholar
  4. Brearley CA, Hanke DE (1996a) Inositol phosphates in the duckweed Spirodela polyrhiza L. Biochem J 314:215–225PubMedGoogle Scholar
  5. Brearley CA, Hanke DE (1996b) Metabolic evidence for the order of addition of individual phosphate esters in the myo-inositol moiety of inositol hexakisphosphate in the duckweed Spirodela polyrhiza L. Biochem J 314:227–233PubMedGoogle Scholar
  6. Brinch-Pedersen H, Sørensen LD, Holm PB (2002) Engineering crop plants: getting a handle on phosphate. Trends Plant Sci 7:118–125PubMedCrossRefGoogle Scholar
  7. Chang SC, Miller AL, Feng Y, Wente SR, Majerus PW (2002) The human homolog of the rat inositol phosphate multikinase is an inositol 1, 3, 4, 6-tetrakisphosphate 5-kinase. J Biol Chem 277:43836–43843PubMedCrossRefGoogle Scholar
  8. Choi H, Hong J, Ha J, Kang J, Kim SY (2000) ABFs, a family of ABA-responsive element binding factors. J Biol Chem 275:1723–1730PubMedCrossRefGoogle Scholar
  9. Clerkx EJM, El-Lithy ME, Vierling E, Ruys GJ, Blankestijn-DeVries H, Groot SPC, Vreugdenhil D, Koornneef M (2004) Analysis of natural allelic variation of Arabidopsis seed quality traits between the accessions Landsberg erecta and Shakdara, using a new recombinant inbred line population. Plant Physiol 135:432–443PubMedCrossRefGoogle Scholar
  10. Cromwell GL, Coffey RD (1991) Phosphorus—a key essential nutrient yet a possible major pollutant—its central role in animal nutrition. In: Lyons TP (ed) Biotechnology in the feed industry. Alltech Publications, Nicholasville, pp 133–145Google Scholar
  11. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Reptr 1:19–21CrossRefGoogle Scholar
  12. Doria E, Galleschi L, Calucci L, Pinzino C, Pilu R, Cassani E, Nielsen E (2009) Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant. J Exp Bot 60:967–978PubMedCrossRefGoogle Scholar
  13. Dorsch JA, Cook A, Young KA, Anderson JM, Bauman AT, Volkmann CJ, Murthy PPN, Raboy V (2003) Seed phosphorus and inositol phosphate phenotype of barley low phytic acid genotypes. Phytochem 62:691–706CrossRefGoogle Scholar
  14. Giuliano G, Pichersky E, Malik VS, Timko MP, Scolnik PA, Cashmore AR (1988) An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene. Proc Natl Acad Sci USA 85:7089–7093PubMedCrossRefGoogle Scholar
  15. Hitz WD, Carlson TJ, Kerr PS, Sebastian SA (2002) Biochemical and molecular characterization of a mutation that confers a decreased raffinosaccharide and phytic acid phenotype on soybean seeds. Plant Physiol 128:650–660PubMedCrossRefGoogle Scholar
  16. Huang N, Sutliff TD, Litts JC, Rodriguez RL (1990) Classification and characterization of the rice alpha-amylase multigene family. Plant Mol Biol 14:655–668PubMedCrossRefGoogle Scholar
  17. Josefsen L, Bohn L, Sørensen MB, Rasmussen SK (2007) Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily. Gene 397:114–125PubMedCrossRefGoogle Scholar
  18. Kim S, Tai TH (2010) Genetic analysis of two OsLpa1-like genes in Arabidopsis reveals that only one is required for wild-type seed phytic acid levels. Planta 232:1241–1250PubMedCrossRefGoogle Scholar
  19. Kim SY, Chung HJ, Thomas TL (1997) Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system. Plant J 11:1237–1251PubMedCrossRefGoogle Scholar
  20. Kim S, Andaya C, Goyal S, Tai T (2008a) The rice OsLpa1 gene encodes a novel protein involved in phytic acid metabolism. Theor Appl Genet 117:769–779PubMedCrossRefGoogle Scholar
  21. Kim SI, Andaya C, Newman J, Goyal S, Tai T (2008b) Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK. Theor Appl Genet 117:1291–1301PubMedCrossRefGoogle Scholar
  22. Kuwano M, Mimura T, Takaiwa F, Yoshida KT (2009) Generation of stable ‘low phytic acid’ transgenic rice through antisense repression of the 1d-myo-inositol 3-phosphate synthase gene (RINO1) using the 18 kDa oleosin promoter. Plant Biotech J 7:96–105CrossRefGoogle Scholar
  23. Larson SR, Rutger JN, Young KA, Raboy V (2000) Isolation and genetic mapping of a non-lethal rice (Oryza sativa L.) low phytic acid 1 mutation. Crop Sci 40:1397–1405CrossRefGoogle Scholar
  24. Lemtiri-Chlieh F, MacRobbie EA, Webb AA, Manison NF, Brownlee C, Skepper JN, Chen J, Prestwich GD, Brearley CA (2003) Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proc Natl Acad Sci USA 100:10091–10095PubMedCrossRefGoogle Scholar
  25. Lessard PA, Allen RD, Bernier F, Crispino JD, Eujiwara T, Beachy RN (1991) Multiple nuclear factors interact with upstream sequences of differentially regulated β-conglycinin genes. Plant Mol Biol 16:397–413PubMedCrossRefGoogle Scholar
  26. Loewus MW, Loewus FA (1982) myo-inositol-1-phosphatase from the pollen of Lilium longiflorum Thunb. Plant Physiol 70:765–770PubMedCrossRefGoogle Scholar
  27. Loewus F, Murthy P (2000) myo-inositol metabolism in plants. Plant Sci 150:1–19CrossRefGoogle Scholar
  28. Majumder A, Biswas B (1973) Metabolism of inositol phosphates part V—biosynthesis of inositol phosphates during ripening of mung bean (Phaseolus aureus) seeds. Indian J Exp Biol 11:120–123Google Scholar
  29. Menkens AE, Cashmore AR (1994) Isolation and characterization of a fourth Arabidopsis thaliana G-box-binding factor, which has similarities to Fos oncoprotein. Proc Natl Acad Sci USA 91:2522–2526PubMedCrossRefGoogle Scholar
  30. Mitsuhashi N, Ohnishi M, Sekiguchi Y, Kwon YU, Chang YT, Chung SK, Inoue Y, Reid RJ, Yagisawa H, Mimura T (2005) Phytic acid synthesis and vacuolar accumulation in suspension-cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations. Plant Physiol 138:1607–1614PubMedCrossRefGoogle Scholar
  31. Murphy AM, Otto B, Brearley CA, Carr JP, Hanke DE (2008) A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens. Plant J 56:638–652PubMedCrossRefGoogle Scholar
  32. Nagy R, Grob H, Weder B, Green P, Klein M, Frelet-Barrand A, Schjoerring JK, Brearley C, Martinoia E (2009) The Arabidopsis ATP-binding cassette protein AtMRP5/AtABCC5 is a high affinity inositol hexakisphosphate transporter involved in guard cell signaling and phytate storage. J Biol Chem 284:33614–33622PubMedCrossRefGoogle Scholar
  33. Nunes AC, Vianna GR, Cuneo F, Amaya-Farfán J, de Capdeville G, Rech EL, Aragão FJ (2006) RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content. Planta 224:125–132PubMedCrossRefGoogle Scholar
  34. Odom AR, Stahlberg A, Wente SR, York JD (2000) A role for nuclear inositol 145-trisphosphate kinase in transcriptional control. Science 287:2026–2029PubMedCrossRefGoogle Scholar
  35. Raboy V (2002) Progress in breeding low phytate crops. J Nutr 132:503S–505SPubMedGoogle Scholar
  36. Raboy V (2003) myo-Inositol-123456-hexakisphosphate. Phytochem 64:1033–1043CrossRefGoogle Scholar
  37. Raboy V, Gerbasi PF, Young KA, Stoneberg SD, Pickett SG, Bauman AT, Murthy PP, Sheridan WF, Ertl DS (2000) Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. Plant Physiol 124:355–368Google Scholar
  38. Rutger J, Raboy V, Moldenhauer K, Bryant R, Lee F, Gibbons J (2004) Registration of KBNT lpa1–1 low phytic acid germplasm of rice. Crop Sci 44:363CrossRefGoogle Scholar
  39. Saiardi A, Caffrey JJ, Snyder SH, Shears SB (2000) Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae. FEBS Lett 468:28–32PubMedCrossRefGoogle Scholar
  40. Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C, Clarke JD, Cotton D, Bullis D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel T, Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14:2985–2994PubMedCrossRefGoogle Scholar
  41. Sharpley AN, Charpa SC, Wedepohl R, Sims JY, Daniel TC, Reddy KR (1994) Managing agricultural phosphorus for protection of surface waters: issues and options. J Environ Qual 23:437–451CrossRefGoogle Scholar
  42. Shi J, Wang H, Wu Y, Hazebroek J, Meeley RB, Ertl DS (2003) The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. Plant Physiol 131:507–515PubMedCrossRefGoogle Scholar
  43. Shi JR, Wang HY, Hazebroek J, Ertl DS, Harp T (2005) The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds. Plant J 42:708–719PubMedCrossRefGoogle Scholar
  44. Shi J, Wang H, Schellin K, Li B, Faller M, Stoop JM, Meeley RB, Ertl DS, Ranch JP, Glassman K (2007) Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds. Nat Biotech 25:930–937CrossRefGoogle Scholar
  45. Stephens LR, Irvine RF (1990) Stepwise phosphorylation of myo-inositol leading to myo-inositol hexakisphosphate in Dictyostelium. Nature 346:580–583PubMedCrossRefGoogle Scholar
  46. Stevenson-Paulik J, Odom AR, York JD (2002) Molecular and biochemical characterization of two plant inositol polyphosphate 6-/3-/5-kinases. J Biol Chem 277:42711–42718PubMedCrossRefGoogle Scholar
  47. Stevenson-Paulik J, Bastidas RJ, Chiou S-T, Frye RA, York JD (2005) Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc Natl Acad Sci USA 102:12612–12617PubMedCrossRefGoogle Scholar
  48. Sun Y, Thompson M, Lin G, Butler H, Gao Z, Thornburgh S, Yau K, Smith DA, Shukla VK (2007) Inositol 1, 3, 4, 5, 6-pentakisphosphate kinase from maize: molecular and biochemical characterization. Plant Physiol 144:1278–1291PubMedCrossRefGoogle Scholar
  49. Suzuki M, Tanaka K, Kuwano M, Yoshida KT (2007) Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): Implications for the phytic acid biosynthetic pathway. Gene 405:55–64PubMedCrossRefGoogle Scholar
  50. Sweetman D, Johnson S, Caddick SEK, Hanke DE, Brearley CA (2006) Characterization of an Arabidopsis inositol 13456-pentakisphosphate 2-kinase (AtIPK1). Biochem J 394:95–103PubMedCrossRefGoogle Scholar
  51. Sweetman D, Stavridou I, Johnson S, Green P, Caddick SE, Brearley CA (2007) Arabidopsis thaliana inositol 1, 3, 4- triphosphate 5/6—kinase 4 AtITPK4 is an outlier to a family of ATP-grasp fold protein from Arabidopsis. FEBS Lett 581:4165–4171PubMedCrossRefGoogle Scholar
  52. Tan X, Calderon-Villalobos LIA, Sharon M, Zheng C, Robinson CV, Estelle M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446:640–645PubMedCrossRefGoogle Scholar
  53. Tesnier K, Strookman-Donkers HM, van Pijlen JG, van der Geest AHM, Bino RJ, Groot SPC (2002) A controlled deterioration test for Arabidopsis thaliana reveals genetic variation in seed quality. Seed Sci Technol 30:149–165Google Scholar
  54. Ulmasov T, Hagen G, Guilfoyle TJ (1999) Dimerization and DNA binding of auxin response factors. Plant J 19:309–319PubMedCrossRefGoogle Scholar
  55. Verbsky JW, Wilson MP, Kisseleva MV, Majerus PW, Wente SR (2002) The synthesis of inositol hexakisphosphate: characterization of human inositol 1, 3, 4, 5, 6-pentakisphosphate 2-kinase. J Biol Chem 277:31857–31862PubMedCrossRefGoogle Scholar
  56. Verbsky JW, Chang SC, Wilson MP, Mochizuki Y, Majerus PW (2005) The pathway for the production of inositol hexakisphosphate in human cells. J Biol Chem 280:1911–1920PubMedCrossRefGoogle Scholar
  57. Wilson MP, Majerus PW (1997) Characterization of a cDNA encoding Arabidopsis thaliana inositol 134-trisphosphate 5/6-kinase. Biochem Biophys Res Comm 232:678–681PubMedCrossRefGoogle Scholar
  58. Xia HJ, Brearley C, Elge S, Kaplan B, Fromm H, Muller-Roeber B (2003) Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex. Plant Cell 15:449–463PubMedCrossRefGoogle Scholar
  59. Xu J, Brearley CA, Lin WH, Wang Y, Ye R, Mueller-Roeber B, Xu ZH, Xue HW (2005) A role of Arabidopsis inositol polyphosphate kinase, AtIPK2alpha, in pollen germination and root growth. Plant Physiol 137:94–103PubMedCrossRefGoogle Scholar
  60. York JD, Odom AR, Murphy R, Ives EB, Wente SR (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 285:96–100PubMedCrossRefGoogle Scholar
  61. Zhang ZB, Yang G, Arana F, Chen Z, Li Y, Xia HJ (2007) Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2beta) is involved in axillary shoot branching via auxin signaling. Plant Physiol 144:942–951PubMedCrossRefGoogle Scholar
  62. Zhao HJ, Liu QL, Fu HW, Xu XH, Wu DX, Shu QY (2008) Effect of non-lethal low phytic acid mutations on grain yield and seed viability in rice. Field Crop Res 108:206–211CrossRefGoogle Scholar

Copyright information

© Springer-Verlag (outside the USA) 2011

Authors and Affiliations

  1. 1.Crops Pathology and Genetics Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Department of Plant Sciences – MS 1University of CaliforniaDavisUSA

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