RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content, Alters Transcripts of Phytic Acid Biosynthetic Genes, and Modulates Mineral Distribution in Rice Seeds
Received date: 2019-03-11
Accepted date: 2019-06-18
Online published: 2020-03-31
Phytic acid is the principal storage form of phosphorus in plant seeds and an essential signalling molecule in several regulatory processes of plant development. However, it is known as an anti-nutrient compound owing to its potent chelating property. Thus, reducing the phytic acid content in crops is desirable. Studies involving regulation of MIPS and IPK1 genes to generate low phytate rice have been reported earlier. However, the functional significance of OsITPK and the effect of its down-regulation on phytic acid content and the associated pleiotropic effects on rice have not yet been investigated. In this study, tissue specific RNA interference (RNAi)-mediated down-regulation of a major ITPK homolog (OsITP5/6K-1) resulted in 46.2% decrease in phytic acid content of T2 transgenic seeds with a subsequent 3-fold enhancement in the inorganic phosphorus content. Silencing of OsITP5/6K-1 altered the transcript levels of essential phytic acid pathway genes, without significantly affecting the transcript levels of other OsITPK homologs. Furthermore, the mapping of elements through X-ray microfluorescence analysis revealed significant changes in the spatial distribution pattern and translocation of elements in low phytate seeds. Additionally, low phytate polished seeds exhibited 1.3-fold and 1.6-fold enhancement in iron and zinc content in the grain endosperm, respectively. Silencing of OsITP5/6K-1 also altered the amino acid and myo-inositol content of the transgenic seeds. Our results successfully established that RNAi-mediated silencing of OsITP5/6K-1 gene significantly reduced the phytate levels in seeds without hampering the germination potential of seeds and plant growth. The present study provided an insight into the mechanism of phytic acid biosynthesis pathway.
Karmakar Aritra, Bhattacharya Sananda, Sengupta Shinjini, Ali Nusrat, Nath Sarkar Sailendra, Datta Karabi, K. Datta Swapan . RNAi-Mediated Silencing of ITPK Gene Reduces Phytic Acid Content, Alters Transcripts of Phytic Acid Biosynthetic Genes, and Modulates Mineral Distribution in Rice Seeds[J]. Rice Science, 2020 , 27(4) : 315 -328 . DOI: 10.1016/j.rsci.2020.05.007
| [1] | Ali N, Paul S, Gayen D, Sarkar S N, Datta K, Datta S K. 2013a. RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice. Rice, 6: 12. |
| [2] | Ali N, Paul S, Gayen D, Sarkar S N, Datta K, Datta S K. 2013b. Development of low phytate rice by RNAi mediated seed- specific silencing of inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene ( IPK1). PLoS One, 8: e68161. |
| [3] | Aggarwal S, Kumar A, Bhati K K, Kaur G, Shukla V, Tiwari S, Pandey A K. 2018. RNAi-mediated downregulation of inositol pentakisphosphate kinase (IPK1) in wheat grains decreases phytic acid levels and increases Fe and Zn accumulation. Front Plant Sci, 9: 259. |
| [4] | Bernfeld P.1955. Amylases α and β. In: Colowick S P, Kalpan N O. Methods in Enzymology. New York: Academic Press: 149-158. |
| [5] | Bohn L, Meyer A S, Rasmussen S K. 2008. Phytate: Impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ: Sci B, 9(3): 165-191. |
| [6] | Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72: 248-254. |
| [7] | Bregitzer P, Raboy V. 2006. Effects of four independent low- phytate mutations in barley ( Hordeum vulgare L.) on seed phosphorus characteristics and malting quality. Cereal Chem, 83(5): 460-464. |
| [8] | Chen P S, Toribara T Y, Warner H. 1956. Microdetermination of phosphorus. Anal Chem, 28: 1756-1758. |
| [9] | Coelho C M M, Tsai S M, Vitorello V A. 2005. Dynamics of inositol phosphate pools (tris-, tetrakis- and pentakisphosphate) in relation to the rate of phytate synthesis during seed development in common bean ( Phaseolus vulgaris). J Plant Physiol, 162(1): 1-9. |
| [10] | Datta K, Koukolikova-Nicola Z, Baisakh N, Oliva N, Datta S K. 2000. Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems. Theor Appl Genet, 100(6): 832-839. |
| [11] | Drakakaki G, Marcel S, Glahn R P, Lund E K, Pariagh S, Fischer R, Christou P, Stoger E. 2005. Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol Biol, 59(6): 869-880. |
| [12] | Duarte R F, Prom-u-thai C, Amaral D C, Faquin V, Guilherme L R G, Reis A R, Alves E. 2016. Determination of zinc in rice grains using DTZ staining and ImageJ software. J Cereal Sci, 68: 53-58. |
| [13] | Feng X G, Yoshida K T. 2004. Molecular approaches for producing low-phytic-acid grains in rice. Plant Biotechnol, 21(3): 183-189. |
| [14] | Furtado A, Henry R J. 2005. The wheat Em promoter drives reporter gene expression in embryo and aleurone tissue of transgenic barley and rice. Plant Biotechnol J, 3(4): 421-434. |
| [15] | Galani S, Aman A, Qader S A U. 2011. Germination potential index of Sindh rice cultivars on biochemical basis, using amylase as an indicator. Afr J Biotechnol, 10: 18334-18338. |
| [16] | Huang N, Angeles E R, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennet J, Khush G S. 1997. Pyramiding of bacterial blight resistance genes in rice: Marker-assisted selection using RFLP and PCR. Theor Appl Genet, 95(3): 313-320. |
| [17] | Karmakar S, Molla K A, Chanda P K, Sarkar S N, Datta S K, Datta K. 2016. Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight. Planta, 243(1): 115-130. |
| [18] | Kaur H, Verma P, Petla B P, Rao V, Saxena S C, Majee M. 2013. Ectopic expression of the ABA-inducible dehydration-responsive chickpea L-myo-inositol 1-phosphate synthase 2 (CaMIPS2) in Arabidopsis enhances tolerance to salinity and dehydration stress. Planta, 237(1): 321-335. |
| [19] | Kim S, Tai T. 2011. Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach. Mol Genet Genom, 286(2): 119-133. |
| [20] | Kuwano M, Ohyama A, Tanaka Y, Mimura T, Takaiwa F, Yoshida K T. 2006. Molecular breeding for transgenic rice with low- phytic-acid phenotype through manipulating myo-inositol 3-phosphate synthase gene. Mol Breeding, 18(3): 263-272. |
| [21] | Kuwano M, Mimura T, Takaiwa F, Yoshida K T. 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 Biotechnol J, 7(1): 96-105. |
| [22] | Larson S R, Rutger J N, Young K A, Raboy V. 2000. Isolation and genetic mapping of a non-lethal rice ( Oryza sativa L.) low phytic acid 1 mutation. Crop Sci, 40: 1397-1405. |
| [23] | Liu J C, Ockenden I, Truax M, Lott J N A. 2004. Phytic acid- phosphorus and other nutritionally important mineral nutrient elements in grains of wild-type and low phytic acid (lpa1-1) rice. Seed Sci Res, 14(2): 109-116. |
| [24] | Liu Q L, Xu X H, Ren X L, Fu H W, Wu D X, Shu Q Y. 2007. Generation and characterization of low phytic acid germplasm in rice ( Oryza sativa L.). Theor Appl Genet, 114(5): 803-814. |
| [25] | Livak K J, Schmitteng T D. 2001. Analysis of relative gene expression data using a real-time quantitative PCR and 2-ΔΔCT method. Methods, 25(4): 402-408. |
| [26] | Loewus F A, Murthy P P N. 2000. myo-Inositol metabolism in plants. Plant Sci, 150: 1-19. |
| [27] | Lott J N A, Greenwood J S, Batten G D. 1995. Mechanisms and regulation of mineral nutrient storage during seed development. In: Kigel J, Galili G. Seed Development and Germination. New York: Marcel Dekker: 215-235. |
| [28] | Lucca P, Hurrell R, Potrykus I. 2001. Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet, 102: 392-397. |
| [29] | Miller G L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 31: 426-428. |
| [30] | Molla K A, Karmakar S, Chanda P K, Ghosh S, Sarkar S N, Datta S K, Datta K. 2013. Rice oxalate oxidase gene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice. Mol Plant Pathol, 14(9): 910-922. |
| [31] | Pilu R, Panzeri D, Gavazzi G, Rasmussen S K, Consonni G, Nielsen E. 2003. Phenotypic, genetic and molecular characterization of a maize low phytic acid mutant ( lpa 241). Theor Appl Genet, 107(6): 980-987. |
| [32] | Raboy V, Gerbasi P F, Young K A, Stoneberg S D, Pickett S G, Bauman A T, Murthy P P N, Sheridan W F, Ertl D S. 2000. Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1. Plant Physiol, 124(1): 355-368. |
| [33] | Raboy V. 2003. myo-Inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry, 64(6): 1033-1043. |
| [34] | Raboy V. 2009. Approaches and challenges to engineering seed phytate and total phosphorus. Plant Sci, 177(4): 281-296. |
| [35] | Raj Bhandari M, Kawabata J. 2006. Cooking effects on oxalate, phytate, trypsin and alpha amylase inhibitors of wild type yam tubers of Nepal. J Food Compos Anal, 19: 524-530. |
| [36] | Sakai H, Iwai T, Matsubara C, Usui Y, Okamura M, Yatou O, Tarada Y, Aoki N, Nishida S, Yoshida K T. 2015. A decrease in phytic acid content substantially affects the distribution of mineral elements within rice seeds. Plant Sci, 238: 170-177. |
| [37] | Shi J R, Wang H Y, Hazebroek J, Ertl D S, 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(5): 708-719. |
| [38] | Shi J R, Wang H Y, Schellin K, Li B L, Faller M, Stoop J M, Meeley R B, Ertl D S, Ranch J P, Glassman K. 2007. Embryo- specific silencing of a transporter reduces phytic acid content of maize and soybean seeds. Nat Biotechnol, 25: 930-937. |
| [39] | Sivaprakash K R, Krishnan S, Datta S K, Parida A K. 2006. Tissue-specific histochemical localization of iron and ferritin gene expression in transgenic indica rice Pusa Basmati ( Oryza sativa L.). J Genet, 85(2): 157-160. |
| [40] | Sparvoli F, Cominelli E. 2015. Seed biofortification and phytic acid reduction: A conflict of interest for the plant? Plants, 4(4): 728-755. |
| [41] | Stein N, Herren G, Keller B. 2001. A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breeding, 120(4): 354-356. |
| [42] | Suzuki M, Tanaka K, Kuwano M, Yoshida K T. 2007. Expression pattern of inositol phosphate-related enzymes in rice ( Oryza sativa L.): Implications for the phytic acid biosynthetic pathway. Gene, 405: 55-64. |
| [43] | Woo L, Maher W. 1995. Determination of phosphorus in turbid waters using alkaline potassium peroxodisulphate digestion. Anal Chim Acta, 315(1): 123-135. |
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