Cloning And Functional Analysis Of Arabidopsis ANR1 Homologous Genes (OsMADS25, OsMADS27 And OsMADS57)in Rice(Oryza Sativa)

MADS-box transcription factors are vital regulators participating in plant growth and development process and the functions of most of them are still unknown. ANR1 is a key gene regulating lateral root development in response to an external nitrate signal in Arabidopsis. There are five ANR1-like genes in Oryza sativa, namely OsMADS23, OsMADS25, OsMADS27, OsMADS57 and OsMADS61, all of which belong to the AGL17 clade. We firstly investigated the responsiveness of these genes in response to fluctuations in nitrogen (N), phosphorus (P), sulfur (S) and hormones by using quantitative real-time PCR (qRT-PCR). These five MADS-box genes have been shown to have a different range of responses to the nutrient supply. According to the results of quantitative real-time PCR, we chose and cloned three nitrate inducible genes (OsMADS25, OsMADS27 and OsMADS57) and used molecular biology methods to analyze their roles in controlling root development. The main results are as follows:1. Firstly, we investigated root expression patterns of OsMADS25 in response to N, P, and S fluctuations using quantitative real-time PCR. The expression of OsMADS25 was induced by NO3- supply and sulfur (S) starvation, but down-regulated by NH4+, phosphorus (P) starvation and S resupply. The expression of OsMADS25 was also induced by auxin but did not respond to cytokinin. The OsMADS25 protein was found mainly in nucleus.OsMADS25-overexpressing lines showed significantly increases in primary root (PR) length and lateral root (LR) number as well as lateral root growth and LR density in presence of nitrate in Arabidopsis. In addition, overexpression of OsMADS25 in Arabidopsis significantly increased shoot and root fresh weight in the presence and absence of NO3-, but this positive effect was much stronger in the presence of NO3-. These observations suggest that OsMADS25 is a key transcriptional factor controls root development through NO3--signaling in Arabidopsis and that OsMADS25 is a positive regulator stimulates LR, PR and shoot growth by NO3-. Over-expression of OsMADS25 in transgenic rice plants significantly promoted lateral and primary root growth as well as LR number and LR density in presence of nitrate, while down-regulation of OsMADS25 produced the opposite effect. These findings suggest that OsMADS25 is a key transcriptional factor controls root development through NO3--signaling in rice. In addition, down-regulation of OsMADS25 in transgenic rice significantly decreased the primary root and shoot length as well as nitrate accumulation and suppressed the expression of nitrate transporter genes at high rates of nitrate supply (10 mM), while over-expression of OsMADS25 produced the opposite effect. The nitrogen and phosphorus contents of 4-week-old rice seedlings were not affected by OsMADS25. However, down-regulation of OsMADS25 in transgenic rice plants significantly decreased nitrate and sulfate contents in shoots as well as nitrate, phosphate and sulfate contents in roots. We found that OsMADS25 was also involved in salt-stress response in rice and Arabidopsis.2. We investigated the root expression patterns of OsMADS27 in response to N, P, S fluctuations and hormones. The expression of OsMADS27 was induced by NO3-supply and sulfur (S) starvation, but down-regulated by NH4+, phosphorus (P) starvation and S resupply. The expression of OsMADS27 was induced by auxin and did not respond to cytokinin. OsMADS25 protein localized mainly in nucleus. OsMADS27-overexpressing line in Arabidopsis showed significantly increases in PR length and LR number but no effect on LR length per unit PR length. OsMADS27 had no influences on nitrogen and phosphorus contents of 4-week-old rice seedlings, while OsMADS27-interfering line significantly suppressed nitrate, phosphate and sulfate contents in roots.3. The expression of OsMADS57 was induced by NO3- supply and S deprivation, but down-regulated by NH4+ and S resupply. The expression of OsMADS57 was also induced by auxin and did not respond to cytokinin. Seedlings of wild type Col-0 (wild type) and OsMADS57-overexpressing line were cultured vertically on agar plates containing a range of NO3- concentrations. In the presence of NO3-, overexpression of OsMADS57 showed significantly increase in LR length/unit PR length while there was no effect in the absence of NO3-. OsMADS57 significantly promoted PR growth and LR number when supplied with 0.2 mM NO3-.Taken together, our findings suggest that OsMADS25 is a positive regulator to control root and shoot growth in nitrate signaling in rice and Arabidopsis, and OsMADS27 and OsMADS57 also regulates root growth through the NO3--signaling pathway in Arabidopsis. Through this research, our results provide new evidences to study root architecture in rice and improve nitrogen use efficiency as well as provide new genetic resources for improving crop nitrogen use efficiency and plant molecular breeding.