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.

Figure 1

Linear and circular map of pBIN-MIPS ihp construct. Restriction digestion of pBIN-MIPS ihp; Lane1: λ EcoRI + HindIII marker, Lane2: ~12 kb binary vector fragment and ~2 kb MIPS1 ihp fragment, Lane3: 1 kb ladder.

Figure 2

Linear and circular map of pBIN-MIPS AS. MIPS1 ORF (~1.5 kb) region introduced in antisense orientation under vicilin promoter at BamHI/XbaI site. Restriction digestion of pBIN-MIPSAS; Lane1: λ-EcoRI/HindIII marker, Lane2: BamHI/XbaI restriction showing ~12.5 kb binary vector fragment and ~1.5 kb MIPS1 antisense fragment, Lane3: PCR amplification of MIPS1 from pBIN-MIPS AS, Lane4: 1 kb ladder.

Figure 3

Colony PCR for screening positive colonies harboring constructs. (A) Colony PCR of Agrobacterium with Bar and MIPSAS specific primers showing ~500 bp bar gene and ~1.5 kb MIPS gene amplification. (B) Colony PCR of Agrobacterium with bar and MIPS ihp specific primers showing ~500 bp bar gene and ~750 bp MIPS fragments amplification.

Figure 4

A modified cotyledonary-node method using glufosinate as the selection agent. Figure (a–j): Seedling grown in germination, shoot induction, shoot elongation medium with glufosinate selection and finally transferred into rooting medium. Figure (k–o): Plantlets were hardened in sterilized pot mix in the tissue culture facility shifted to greenhouse where plants acclimatized and reached maturity level.

Figure 5

Screening of transgenic plants (A) using bar gene specific primers (B) based on phytate content. Phytate content in non-transgenic control (NT) and in T₁ transgenic seeds were analyzed. The symbol *Indicates significant differences at p = 0.05 (n = 3).

Figure 6

Leaf painting assay with glufosinate. Transgenic plants (a: AS; b: RNAi) showed resistance to Basta^® (100 mg/L) on 5^th days compared with NT control (c).

Figure 7

Expression analysis of transgenic rice plants. qRT-PCR analysis of T₄ progenies of lines MS14-28-12-29-3-5, MI51-32-22-1-13-6. (NT = non-transgenic controls).

Figure 8

Southern blot analysis of T₄ progenies (MS14-28-12-29-3-5 and MI51-32-22-1-13-6). Each line consist of 10 μg genomic DNA, digested with XhoI. The position and sizes of markers are indicated (NTC = Non-transgenic control).

Figure 9

Analysis of seed phytate content in non transgenic control (NTC) and transgenics (T₄). Phytate content of transgenic seeds (MS14-28-12-29-3-5 and MI51-32-22-1-13-6) compared to NTC seeds showed significant differences (P < 0.05).

Figure 10

Electron microscopic analysis of NT and transgenic (T₄) seeds. (a) Cotyledon sections of NTC (b) MS14-28-12-29-3-5 (c) MI51-32-22-1-13-6 lines. Phytin globoid (PG) cavities are the small white circular areas. (PSV-Protein storage vacoules; CW- Cell wall; LB-lipid bodies).

Figure 11

Bioavailability of metal ions in NT and transgenics (T₄). Bioavailability of divalent minerals (Fe²⁺, Zn²⁺ and Ca²⁺) using in vivo simulation model showed significant (P < 0.05) increase in Transgenics (MS14-28-12-29-3-5 and MI51-32-22-1-13-6) compared to NT control.

Figure 12

Various parameters considered for agronomic evaluation of T₄ transgenic plants grown in green house. Values are mean ± SE, n = 10 (P ≤ 0.002).