Effect Of Altering Phosphate Transporter Gene OsPT8 Expression On Content Of Phytic Acid And Middle/Micro- Nutrients In Rice Seeds

Phosphorus(P),one of the plant essential macromitrients,plays an important role in the growth and development of plants. Since P content is more than the required for cell metabolism during seed fbrmation,higher plants synthesize these excess P to the fbrm of phytic acid. Once synthesized,most phytic acid P is deposited as a mixed phytate or phytin salt of metallic element, such as calcium(Ca), magnesium(Mg), iron(Fe) and zinc (Zn).Consequently, P content has a close relationship with the Content of metallic nutrients in plant seeds. The previous researches on phytic acid of rice seeds were mainly fbcused on the improvement of rice quality and bio-availability of metallic nutrientsby changing the expression of key genes involving in phytic acid biosynthesis to reduce seeds phytic acid contents.In our previous study, we have shown that a phosphate transnporter gene OsPT8 is volved in phosphate(Pi) uptake and transport in rice throughout the entire growth period,especially in redistribution of P from vegetative organs to reproductive organs. However little is known about the effects of expression on rice seed phytic acid and nutrients content. In the present study, we investigated the relationship of OsPT8 expression, the contents of phytic acid P, middle- and micro- nutrients by RNA-interference knockdown of OsPT8 using endosperm specific promoter(Glub-1) and shoot specific promoter(RISSZI) for selective knockdown the expression of OsPT8 respectively in endosperm and aerial parts,and constitutive overexpression of OsPT8rS by using 35S promoterr. The main results were as follows:1. In comparison to the rice seeds of wild type(WT)(cv. Nipponbare),shoot specifically RNAi-knockdown of OsPT8(SSRi) or endosperm-specifically RNAi-knockdown of OsPT8(EnSRj) significantly decreased total P content in single seed.However, the change of content of phytate-P(PA-P) in the seed was not Consistent with thatof total P during seed development. In the seed filling stage, in comparison to WT, overexpression of OsPT8 (Oe) accumulated more P in seeds, however, the PA-P concentration decreased about 6-13%, indicating the suppression of phytic acids synthesis. In contrast, the shoot-specific knockdown of OsPT8 decreased the transport of P from vegetative organs to the seeds, promoted the synthesis of phytic acid, and resulted in 8.7% more phytic-P content in comparison to WT. The endosperm-specific knockdown of OsPT8 did not significantly affect total P transport from vergetative to reproductive organs and phytic synthesis in the seeds.2. To better understand the impact of changes in OsPT8 expression on phytic acid synthesis in rice seeds, we detected the expression of the putative key genes involved in phytic acid biosynthesis by qRT-PCR. In the late stage of seed development (28 days after flowering), the degree of OsPT8 expression knockdown in the seeds of SSRi lines reached by 71%-96% compared with WT. In these SSRi seeds, the transcriptional level of the genes coding Ins(3)P1 synthase (RINO1), Ins(1,3,4)P35-/6-kinase (OsITP5/6K-4), Ins PolyP 2-Kinase (OsIPK1) were respectively down-regulated by about 22%,40%, and 40%-80% in comparison to their expression levels in the WT seeds. In relative early development stage of the seeds (14 days after flowering), the degree of OsPT8 expression knockdown in the seeds of EnSRi lines reached by 55%-80% compared with WT. Interestingly, the transcriptional levels of the genes involving in phytic acid biosynthesis in the EnSRi seeds were largely up-regulated. RINO1 and OsITP5/6K-4 were up-regulated by 3-4 folds, and the OsIPK1 was 2-3 folds higher than their expression in WT seeds. In the late development stage of the seeds, the konckdown of OsPT8 was not significant in EnSRi, and little change of the phytic biosynthetic genes expression was observed.3. We analysed the content of some micro-essential nutrients in the seeds of both WT and OsPT8-RNAi lines. The positive relationship between Fe and Zn in the seeds was found, and such relationship was not significantly affected by OsPT8 knockdown either in the shoot or in the endosperm. The seeds of SSRi lines which accumulated more PA-P contained about 41% more Fe per unit seed weight higher than the WT. Overexpression OsPT8 reduced PA-P in the seeds, however, the iron in the seed was not significantly reduced. There was a similar trend of the change of Zn with Fe in the seeds of both the transgenic and WT lines. The seeds of both SSRi and EnSRi lines accumulated more Mg, about 39% and 20% more, respectively than WT seeds. However, Mg in the seeds was not significantly changed by overexpression OsPT8. Interestingly, no matter how the total P and PA-P in the seeds was changed by altering OsPT8 expression, the content of both Cu on the unit weight basis was not significantly affected. There was positive relationship between total P and Ca in the seeds of different transgenic and WT lines.