Molecular Mechanism Of Nitrogen Fertilizer In Increasing The Spikelet Number Per Panicle Of Rice

The number of spikelet per panicle is one of the most important traits in rice productivity determination. Nitrogen fertilizer application before panicle initiation is well known to increase spikelet number, but exactly how inflorescence development regulated by nitrogen fertilizer remains unclear. In the study, we conducted a series of experiments with different nitrogen levels. The role of nitrogen fertilizer application before panicle initiation in increasing the number of spikelet per panicle was studied. First, the genes of cytokinin (CTK) metabolism responsing to nitrogen fertilizer were identified by qRT-PCR. In addition, the proteome of root response to nitrogen/CTK were studied by two-dimensional electrophoresis, and the differentially expressed genes response to nitrogen fertilizer were found by the techniques of proteome and transcriptome. PAP2is one of the differentially expressed genes. The regulation of PAP2in panicle development is quite similar to nitrogen fertilizer. Finally, the pathway of PAP2was studied. The main results were shown as following:1. The expression of cytokinin synthetic genes (OsIPTs) was measured by qRT-PCR. We found the expression level of OsIPTs was increased by nitrogen fertilizer, but not obviously changed in leaves and roots. These results suggest that nitrogen fertilizer enhances local cytokinin synthesis to increase flower numbers in the panicles of rice. Localized cytokinin biosynthesis is an important response to nitrogen in rice growth and development.2. Twenty-eight proteins were identified by two-dimensional electrophoresis and mass spectrometry. The proteins expressed similarly by nitrogen and CTK. These proteins were categorized into classes related to energy and metabolism, etc. Energy gives the largest functional category, suggesting that the glycolysis (2enzymes detected) and tricarboxylic acid cycle (6enzymes detected) are accurately regulated by nitrogen and CK, thus promoting the synthesis of amino acid in root. 3. The proteome of young panicle response to nitrogen fertilizer application before panicle initiation was studied. Sixty-three differently expressed proteins were successfully identified by mass spectrometry. It is worthwhile to note that the largest functional category is proteolysis (18%).4. The regulation of nitrogen fertilizer was studied by transcriptome research. More than10,000genes was found their expression in leaves and panicle. We analyze the expression of genes which identified by map-based cloning. These genes are also involved in the regulation of plant height, tillering, heading date and seed setting rate. Plant height and spikelets were regulated by OsGAEl, OsPHl, and OsGASRl. Tillering and spikelets were regulated by MIP1, OsGS2, OsEATB, and OsAP2-39. Heading date and spikelets were regulated by PAP2, OsCO3, OsCOL4, OsDofl2, OsHDT1, and OsHOl. Seed setting rate and spikelets were regulated by OsPhtl;8and ASP1. This is consistent with the multiple effects of nitrogen fertilizer.5. The transcript level of PAP2was down-regulated by nitrogen fertilizer in leaves and young panicle. Our results indicate that nitrogen fertilizer can regulate panicle development through the pathway of PAP2. The effect of PAP2on panicle development and heading date was similar to that of nitrogen fertilizer. OsWRKY60and OsWOX9are downstream genes of PAP2. The two genes can regulate the differentiation of branch and spikelet. Flowering can be regulated by OsWRKY60. The gene is a direct upstream regulatory factor of Hd3a and RFTl. OsWRKY60and OsWOX9are indicated two key regulators in panicle development of rice from the results.