Effects Of Iron-deficiency Stress On The Related Genes Expression Of The Nitrogen Metabolism And GA Signal Transduction Of Pear

Iron is one of the essential micronutrient for plants and plays an important role in many physiological processes of plant,such as the photosynthesis,respiration,protein and nucleic acid synthesis and so on.Plants have developed certain adaptive II mechanisms to increase Fe-uptake capacity under Fe-deficiency conditions.:Dicotyledonous and non-graminaceous species develop a reduction-based strategy I mechanism for Fe mobilization and acquisition;graminaceous species develop a chelation-based strategy II mechanism.Under the Fe deficiency conditions,the growth of the plants was restricted.The most evident effect of iron-deficiency is a marked decrease in the amounts of chlorophylls.And Fe defiency also has an effect on the other nutrient absorbing,such as nitrogen,phouphorus,potassium and so on.Under the Fe deficiency conditions,Pear root excretion of protons into the rhizosphere through the activation of specific plasma membrane-bound proton H⁺-ATPase from the epidermal cells of the roots,which lowers the pH of the soil solution and increases Fe solubilisation;an increased Ferric Chelate Reductase activity which reduces Fe3+ to Fe2+ at the root surface;Fe2+ transport across root cell membranes through a specific iron-regulated transporter?IRT?.After the H⁺ proton excretion,electrochemical potential changes inside and outside the cell,and it may has an effect on absorbing of the NO₃⁺ and K⁺.As we all know,pear absorb nutrition mainly based on the rootstock of the tree.So our research was based on the regulation of nitrogen metabolism of ‘Dangshansuli' pear?Pyrus bretschneideri Rehd.?under Fe deficiency,and the research about changment of GA concentration and its gene expression of signal transduction has not been reported.Tissue culture seedling of Birch-leaf Pear as materials,the genes of Fe absorbtion and nitrogen metabolism were cloned,and Ferric Chelate Reductase activity,nitrate reductase activity,nitrite reductase activity and so on,were all measured.The related gene expression level of the Iron absorbtion and nitrogen metabolism were analysised by the real time PCR.In addition,we measured the GA concentration and the gene expression of signal transduction of 'Dangshansuli' pear leaf which in different iron deficiency chlorosis degree.1.Determination of Birch-leaf Pear rapid propagation system: MS+6-BA 2.5mg/L+IAA 0.15 mg/L+GA3 0.05 mg/L was the most optimum initial culture medium.The most optimum proliferation culture medium is MS+6-BA 0.5 mg/L + IAA 0.6mg/L+GA3 0.6 mg/L;and the optimum rooting culture medium was 1/2 MS+IBA 0.5mg/L.2.Four cDNA sequence of Fe storage protein family?Fer1,Fer2,Fer3 and Fer4?,Low-affinity Transport Systerms of nitrate transport genes?NRT1,NRT1.2 and NRT1.3?,High-affinity Transport Systerms of nitrate transport genes?NRT2?,Ferric Chelate Reductase?FRO2?,nitrate reductase gene?NR?,nitrite reductase gene?NIR?,potassium channel gene?SKOR,TP3 and TP11?,Ferredoxin gene?Fd1,Fd2 and Fd3?,glutamate synthase?NADH-GOGAT and Fd-GOGAT?;H⁺-ATPase?HA2?;glutamine synthetase gene?GILE?,GA oxidase gene?GA2ox?were all cloned.Among them were 21 genes cDNA full length sequence and part sequence of one gene.3.The expression level of IRT and FRO2 were rapidly increased in the early iron-deficiency,and the Ferric Chelate Reductase?FCR?enzyme in Birch-leaf Pear root was also increased.The expression level of HA2 gene was subsequently up-regulated.The expression level of NRT1.3 and NRT1.2 gene were significantly upregulated in the early iron-deficiency stage.Otherwise,the expression level of NRT1 and NRT2 were not obviously response under iron-deficiency condition.The expression of SKOR was upregulated under iron-deficiency,but the expression level of TP11 and TP3 had no obviously response under iron-deficiency condition.4.Glutamine synthetase gene?GILE?expression upregulated,but its GS activity downregulated;the expression level of NADH-depedent electronic donor NADH-GOGAT was upregulated in the early iron-deficiency stage;the activities of Fd-GOGAT and GOGAT enzyme were all downregulated.And the Fd-GOGAT was a key glutamate synthase gene in iron-deficiency;but the expression Fd1,Fd2 and Fd3 were not down-regulated.5.The results showed that the GA content in leaves increased as the iron content decreased in medium.The levels of relative expression of GA2 ox gene at different degree of iron-deficiency didn't increased as GA contents raised.The levels of relative expression of the four alleles of GID1 all increased as degree of iron-deficiency become severe,there were positive corelation between GA content and levels of relative expression of the four alleles of GID1.Only the levels of relative expression of DELLA1 increased as the degree of iron-deficiency,that was most sensitive to the stress of iron-deficiency.And the iron-deficiency could accelerate the synthesis of GA and would not promote active GA into inactive ones.