Screening Of Ammonium Uptake Releted Genes In Rice And Understanding The Regulatory Mechanism Behines Ammonium Uptake In Arabidopsis

Nitrogen is an important nutrient which accounted for 2% of the dry weight of plants,and plays an important role in crop production. Nitrate(NO3-) and ammonium(NH4+) are major nitrogen source for plants. Plants can uptake ammonium ions directly through ammonium transporters, or accumulate ammonium ions by the reduction of NO3-. In this study, we focus on isolation ammonium uptake related genes in ammonium-tolerant rice and understanding the regulatory mechanism behinds ammonium uptake in ammonium-sensitive Arabidopsis, and do the work as following:1. Screening ammonium uptake related genes in riceAccording to the previously research, higher concentration of ammonium will lead to the tip coiling of the seminal roots. In this study, more than 600 mutants were achieved using a tissue culture-mediated plant regeneration system from the seeds carrying Ac and Ds elements.7 ammonium-sensitive mutants were isolated from the Ds insertion-mutant library. In addition,the stability of root phenotypes were investigated, to test whether tip coiling phenotype can transmitted to F2 and the segregation ratio follow the genetic laws of Mendel. The results showed that 3 mutants were proved in Mendelian inheritance.For the m1 mutant, i PCR(inverse PCR) analysis and sequencing approach were used to obtain the following information: in m1 mutant, the Ds was inserted in the second intron of cytosol glutamine synthetase 1;2(GS1;2, Os03g0223400), and this gene was known to assimilate ammonium in the cell. GS1;2 is consist of 11 exons and 10 introns. This is the first time to link root development and ammonium assimilation by a key GS gene.2. The regulation of ammonium uptake by brassinosteroid(BR) signaling in ArabidopsisIn this study, we identified that activation of BR signaling negatively regulates AMT1 and GS/GOGAT gene expressions. The regulatory mechanism of NH4+-mediated AMT1 suppression reported that N-metabolites rather than NH4+ itself has effects on AMT1 transcription. BES1 is a key BR signaling transcription factor regulates global downstream gene expressions. NH4+-mediated GS/GOGAT gene expression patterns in En2 and bes1-D revealed that an ammonium-dependent GS/GOGAT induction was partially inhibited in a BES1 negative dominant mutant bes1-D. NH4+ re-supply suppresses AMT1 expressions, but NH4+-mediated AMT1 repression is inhibited in bes1-D. However, glutamine application suppressed AMT1 in both En2 and bes1-D, suggesting that BES1 inhibits NH4+-mediated induction of GS/GOGAT, which in turn inhibits NH4+-mediated suppression of AMT1 in bes1-D.3. Interaction between photoreceptors and BR signaling in ArabidopsisLight is a cue for plant growth and development, and is recognized by photoreceptors.According to the previous studies, the expression level of AMT1 in daytime is higher than in dark in Arabidopsis, BR hormones regulate diverse aspects of plant growth and development,including photomorphogenesis and the interaction between light and BR signaling. Thus, we explored the relationship between photoreceptors, AMT1 and BR signaling in this section.(1) The expression of AMT1 affected in the photoreceptor mutants: the expression of AMT1;1in phy B9(far-red light receptor mutant), phy A211/phy B9(red and far-red light receptor double mutant) and phot1-2/cry1-2(blue light receptor quadruple mutant) is higher compared with control plants. In addition, in phy A211(red light receptor mutant), phot1-2and cry1-2(blue light receptor double mutant), AMT1;1 expression is similar to control one.(2) Relationship between photoreceptors and BR signaling: the cop1-4 and HY5 ox mutants differed in their BR responses under dark conditions, suggesting that BR-promoted cop1-4hypocotyl elongation in the dark is not due to overexpression of HY5. It might be due to the interaction between COP1 and BZR1, recently identified. The responses of photoreceptor mutants to BR demonstrated that phy A211, phy A211/phy B9 and phot1-2/cry1-2 are significantly insensitive to exogenously supplied BR. The response of photoreceptor mutants,especially phy B9, phy A211/phy B9, cry1-2 and phot1-2/cry1-2 was similar to that of BR-treated plants growing under light.To further analyze BR effects on photoreceptor mutants we applied the BR biosynthesis inhibitor PCZ(propiconazole). The cry1-2 and phot1-2/cry1-2 mutants were clearly insensitive to PCZ, while phy B9 and phy A211/phy B9 were slightly insensitive to 100 n M PCZ. However, a high PCZ concentration(2 μM) completely inhibited cry1-2 and phot1-2/cry1-2 hypocotyl elongation growing under the light, indicating that hypocotyl growth in cry1-2 and phot1-2/cry1-2 mutants requires BR activity. Analysis of BR marker gene expression in the photoreceptor mutants showed that BR biosynthetic and signaling genes are regulated differently in the photoreceptor mutants, suggesting that photoreceptors somehow regulate the BR signaling pathway. Without PCZ treatment, BR marker gene expression in phot1-2/cry1-2 was similar to that in the bzr1-D mutant, but PCZ treatment fully suppressed the SAUR15 and ACS5 expression in phot1-2/cry1-2; in bzr1-D, on the other hand, PCZ did not completely inhibit SAUR15 and ACS5 expression. These data are correlated with the results on PCZ-mediated hypocotyl elongation in phot1-2/cry1-2 and bzr1-D. The results also suggest that photoreceptors negatively regulate BR signaling. In contrast, photoreceptor expression in bri1-5(BR-insensitive) and bzr1-D(BR-sensitive) mutants indicated that BR signaling negatively regulates photoreceptors at transcription level.4. Overexpression of a SNARE protein At BS14 b alters BR response in ArabidopsisAt BS14 b as a SNARE protein, its transcript level is significantly up-regulated after ammonium treatment. Overexpression of At BS14 b exhibited short petiole and hypocotyl lengths in Arabidopsis. According to the previous studies, the hypocotyl and petiole elongation in plants is regulated by phytohormone, such as BR and Gibberellic acid(GA). In this study, At BS14 b ox plants were insensitive to exogenously supplied BR, and BR-mediated suppression of biosynthetic genes(BR6OX2, CPD and DWF4) was inhibited. Split GFP and mating based split ubiquitin yeast hybrid assays showed that At BS14 b interacts with MSBP1 but not BRI1 and BAK1, suggesting a possible regulatory model by which At BS14 b regulates MSBP1 endocytosis and BR signaling.