Spatial and temporal expression analysis of D-myo-inositol 3-phosphate synthase (MIPS) gene family in Glycine max
Phytic acid, the principal storage form of phosphorus in plant seeds accounts for up to 60 to 80% of the total seed phosphorus content in soybean. Its accumulation increases linearly throughout seed development and it strongly chelates essential mineral cations and charged proteins decreasing their bioavailability. D-Myo-inositol 3-phosphate synthase (MIPS; EC 18.104.22.168), the evolutionarily conserved enzyme in plants, catalyzes the first and the rate limiting step in phytic acid biosynthetic pathway. Aiming at controlling the level of phytate, we monitored the differential expression profile of four, previously reported, members of the MIPS gene family in developing seeds and vegetative tissues of soybean by quantitative real-time PCR (qRT-PCR). Transcript levels were measured relative to the endogenous reference gene eEF-1á (eukaryotic elongation factor 1-alpha) using SYBER-Green. The qRT-PCR data analysis indicated that the expression of the four highly conserved MIPS genes is both temporally and spatially regulated, information much needed for reverse genetic applications. MIPS1 exhibited high transcript levels in the early developing cotyledons with the levels peaking at around 4 to 6 mm seed size stage. Despite of high level of nucleotide sequence conservation amongst the MIPS gene family members, MIPS2, MIPS3 and MIPS4 were poorly expressed in developing seed tissues, although their transcript levels were relatively high in the other organ tissues. MIPS1 was however moderately expressed in seedlings where MIPS2 showed relatively higher expression levels. Among the four isoforms, MIPS4 had the highest transcript levels in the leaf tissue. The data was clearly indicative of the fact that the four isoforms had diverged regulatory elements resulting in their differential expression. Of the four members of the MIPS gene family, MIPS1 is thus the major isoform that had high expression in the developing seed tissues and can be targeted using the dsRNA induced sequence specific RNA degradation mechanism for reduction of phytate levels without affecting the critical aspects of inositol metabolism in other tissues of the plant.
Key words: Soybean, MIPS isoforms, differential expression, endogenous reference gene, qRT-PCR.