水稻转录因子OsMADS57参与硝酸盐调控根系伸长的机制

Ideotype root architecture is important for efficient N acquisition in plant. The plant root system is extremely sensitive to variations in nitrogen form and availability in the soil. Rice plants have aerenchymatous tissue in their roots that allow partial oxygen to release into the soil. Thus nitrification occurs immediately in the aerobic niche of the rhizosphere or on the surface of the roots. Therefore, even in a flooded paddy soil, rice roots are actually exposed to the mixture of NH4+ and low NO3- concentration. Numerous experiments showed molecular mechanism for nitrate regulating root growth especial for lateral root proliferation in Arabiopsis. However, mechanism for nitrate regulating rice root growth is poorly understood, mainly due to ignoring nitrate nutrition in rice plant. The Arabidopsis ANR1 gene, which encodes a NO3- -inducible and root-specific member of the MADS-box family of transcription factors, was identified as a key component of the signal transduction pathway by which NO3- stimulates lateral root proliferation. The OsMADS57 MADS-box gene is one of the analogue genes for AtANR1. Our preliminary study showed that under 0.2 mM NO3- supply, Osmads57-TDNA mutants exhibited repressed the elongation of seminal and adventitious root, compared with wild type (WT). However, no difference was observed between mutants and WT under 5 mM NO3- and 0.2 mM NH4+ supplies. What role might OsMADS57 play in converting low NO3- stimulus at the root tip into an increased rate of elongation? First, Expression pattern in response to nitrogen availability and forms, and tissue localization analysis of OsMADS57 are used to verify its molecular function. To gain insight into how root elongation responds to low nitrate supply, root elongation was followed morphologically using the CYCB1.1::GUS marker for G2/M phase cells. It is reasonable to predict that root phenotype in mutant is due to the absence of the positive factors response to low nitrate concentration. One possibility is suggested by high affinity NO3--uptake systems including NRT2 homologs and their partner protein NAR2 homologs, whose expression will be analyzed in mutants and WT under N treatments to verify their role in modulating the rate of primary root elongation through OsMADS57. Strigolactones have been verified to play an important role in root elongation regulated by low nitrate condition. And auxin is key hormonal signals that control the cellular architecture of the primary root. Strigolactones' secretion and D genes and auxin distribution and related auxin transporters are analyzed to test strigolactones and auxin participating root elongation regulated by NO3-.

理想的根系性状是水稻高效吸收养分的基础，根系发育和土壤氮供应关系密切。强烈的根际硝化作用使水稻根系处于铵、低浓度硝酸盐营养中。目前硝酸盐调控根系生长的研究集中在旱地作物，而硝酸盐调控水稻根系生长的分子机制近乎空白。我们发现水稻MIKC型MADS-box转录因子MADS57的表达受硝酸盐水平调控，低浓度硝酸盐增强其表达。低硝水平下mads57突变体根系伸长被抑制，其机制并不清楚。以MADS57基因突变体和超表达材料为试材，研究不同氮处理下MADS57表达模式及根系响应，从细胞水平探讨其根系伸长变化原因；研究硝酸盐高亲和转运体基因表达，探明它们是否参与依赖于MADS57的低浓度硝酸盐调控水稻根系伸长；采用pDR5::GFP等转基因材料，研究独角金内酯、生长素及相关基因变化，揭示独角金内酯和生长素与MADS57调控根系伸长的关系，从生理和分子角度阐明MADS57参与硝酸盐调控水稻根系伸长的机制。

理想的根系性状是水稻高效吸收养分的基础，根系发育和土壤氮供应关系密切。强烈的根际硝化作用使水稻根系处于铵、低浓度硝酸盐营养中。目前硝酸盐调控根系生长的研究集中在旱地作物，而硝酸盐调控水稻根系生长的分子机制近乎空白。结果表明，水稻根系OsMADS57的表达部位在中柱，且表达受NO3-和NAA的诱导增强。与野生型水稻相比，0.2 mM NO3-处理下osmads57突变体种子根和不定根的伸长受到抑制；在5mM NO3-处理下野生型和突变体的根系差异不明显。与野生型水稻相比，0.2 mM NO3-条件下osmads57突变体根系PINs家族基因均上调， [3H]IAA向根尖运输增强，最终导致突变体根尖的生长素浓度显著大于野生型水稻。外源处理生长素后野生型水稻种子根的长度回补到突变体种子根的长度，同时野生型水稻种子根根尖的DR5::GUS表达增强到和突变体相同的水平，这些结果表明突变体种子根长度的变化依赖于生长素的变化。.有意思的是，研究结果表明OsMADS57参与调控硝酸盐从根系往地上部的运输。与野生型相比，osmads57突变体伤流液往地上部转运的硝酸盐量降低31%，而超表达材料要高出野生型1倍。15N-NO3-的吸收试验结果表明，与野生型相比，突变体材料15N在地上部/根系的分配比例下降40%，而超表达增加约40%。qPCR结果表明当0.2mM NO3-供应5分钟时，与野生型水稻相比，突变体材料根系中柱特异表达的硝酸盐高亲和运输蛋白基因OsNRT2.3a的表达显著降低，降幅为66%；0.2mM NO3-供应60分钟时，突变体材料根系其他三个NRT2家族基因的表达才显著降低。酵母单杂研究结果表明，OsMADS57能结合到OsNRT2.3a启动子区域。综上所述，OsMADS57在硝酸盐调控水稻根系伸长和硝酸盐往地上部的运输中发挥重要作用。