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Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

ABSTRACT: Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T₄) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T₄ seeds compared to NT seed controls. A significant increase in the contents of Fe²⁺ (15.4%, 21.7%), Zn²⁺ (7.45%, 11.15%) and Ca²⁺ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

SUBMITTER: Kumar A

PROVIDER: S-EPMC6533290 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

Kumar Awadhesh A Kumar Varun V Krishnan Veda V Hada Alkesh A Marathe Ashish A C Parameswaran P Jolly Monica M Sachdev Archana A

Scientific reports 20190523 1

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we t ...[more]

PMID: 31123331

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Project description:BackgroundSeed germination is important to soybean (Glycine max) growth and development, ultimately affecting soybean yield. A lower seed field emergence has been the main hindrance for breeding soybeans low in phytate. Although this reduction could be overcome by additional breeding and selection, the mechanisms of seed germination in different low phytate mutants remain unknown. In this study, we performed a comparative transcript analysis of two low phytate soybean mutants (TW-1 and TW-1-M), which have the same mutation, a 2 bp deletion in GmMIPS1, but show a significant difference in seed field emergence, TW-1-M was higher than that of TW-1 .ResultsNumerous genes analyzed by RNA-Seq showed markedly different expression levels between TW-1-M and TW-1 mutants. Approximately 30,000-35,000 read-mapped genes and ~21000-25000 expressed genes were identified for each library. There were ~3900-9200 differentially expressed genes (DEGs) in each contrast library, the number of up-regulated genes was similar with down-regulated genes in the mutant TW-1and TW-1-M. Gene ontology functional categories of DEGs indicated that the ethylene-mediated signaling pathway, the abscisic acid-mediated signaling pathway, response to hormone, ethylene biosynthetic process, ethylene metabolic process, regulation of hormone levels, and oxidation-reduction process, regulation of flavonoid biosynthetic process and regulation of abscisic acid-activated signaling pathway had high correlations with seed germination. In total, 2457 DEGs involved in the above functional categories were identified. Twenty-two genes with 20 biological functions were the most highly up/down- regulated (absolute value Log2FC >5) in the high field emergence mutant TW-1-M and were related to metabolic or signaling pathways. Fifty-seven genes with 36 biological functions had the greatest expression abundance (FRPM >100) in germination-related pathways.ConclusionsSeed germination in the soybean low phytate mutants is a very complex process, which involves a series of physiological, morphological and transcriptional changes. Compared with TW-1, TW-1-M had a very different gene expression profile, which included genes related to plant hormones, antioxidation, anti-stress and energy metabolism processes. Our research provides a molecular basis for understanding germination mechanisms, and is also an important resource for the genetic analysis of germination in low phytate crops. Plant hormone- and antioxidation-related genes might strongly contribute to the high germination rate in the TW-1-M mutant.

Dataset Information

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

Publications

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean.

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