| Iron is 1 of the 16 essential nutrients for plant growth and development which plays an important role in plant photosynthesis,respiration protein and nucleic acid synthesis and many other physiological metabolism.Under neutral and alkaline conditions,the iron effectiveness is low leading to precipitation of Fe(OH)3.At high pH conditions,content of active-iron in solution reduced 1000 times,with the increase of each pH unit.The Yellow River Old valley region is the main pear production area in China,the soil pH is between 7.5 to 8.0 where iron deficiency is serious.The study shows the iron deficency also affect the absorption of other mineral nutrients.With the content of the iron in yellow leaves of 'Dangshansuli' pear decreased heavily,the nitrogen content also decreased.The obvious correlation exited between these two.Iron works as the activator to many enzymes during the nitrogen metabolisms as NR,NIR,GOGAT.Therefore,research of the interaction between the different mineral nutrition under the iron deficiency provides highly theoretical and practical value.Nitrogen metabolism is one of the fundamental physiology processes in plants.The main way of nitrogen assimilation in plants is through the reduction of nitrate to ammonium and then directly involved in the synthesis and transformation of amino acids.During this period,nitrate reductase(NR),nitrite reductase(NiR),glutamate synthase(GOGAT)and glutamine synthetase(GS)and other key enzymes are involved in the catalysis and regulation.Amino acid works as the main substrate for the synthesis of proteins in the cell and after modification classification,transportation and storage of the protein,it turns out to be an integral part of the plant organism.The study of the effect of the iron deficiency on the activity of the related genes and proteins is not rare to see.But the report of its effect on the content of the nitrogen,activity of the related genes,proteins existed in the nitrogen metabolism is still not found.In order to discuss the influences of iron deficiency on photosynthetic rate,chlorophyll fluorescence characteristics,related enzymes activity and genes expression in nitrogen metabolism,the chlorosis leaves under different degrees of iron stress in 'Dangshansuli' pear were taken as experiment materials,The content of Fe,N were determined and the correlation of iron with nitrogen in the leaves were analyzed,the photosynthetic rate,chlorophyll fluorescence characteristics and Fe,N metabolism-related enzymes activity were determined at each degrees of iron deficiency and analyze the effects of iron deficiency on these physiological indexes.Fe3+ reductase gene(FCR),nitrate reductase gene(NR),nitrite reductase gene(NiR),glutamate synthase gene(GOGAT)and glutamine synthetase gene(GS)were cloned,and to analyze the relative expression of the different degree of iron deficiency by Real-time PCR technology.The main results had been achieved were as follows.1.In leaves under defferent degrees of iron stress,there was a significant positive correlation between Fe content and the content of N,of that the coefficients were all at 0.934;It had been demonstrated that the leaf chlorosis on new shoot due to iron deficiency can affect the absorption of nitrogen.2.The development of 'Dangshansuli pear' leaves are seriously affected by iron deficiency,resulting in net photosynthetic rate decreased,then reducing the photosynthetic capacity of leaves;The intercellular CO2 concentration increased indicating that the decrease of photosynthetic rate was not caused by non-stomatal limitation;The effects of leaves chlorosis was firstly manifest that the structural of oxygen evolving complex(OEC)was damaged,then the donor side of PSII and the reaction center were subject to different degrees of damage,causing the electron transport chain is blocked and reducing the efficiency of light absorption and utilization.3.Under the iron stress conditions,the FCR activity was significantly lower than the control.with the degree of iron stress increased,the FCR activity showed a gradual upward trend,and reached a very significant difference;The NR activity and NiR activity were all significantly lower than the control,with the degree of iron stress increased,the NR activity and NiR activity both showed a gradual upward trend,and reached a very significant difference;The GOGAT activity of mild yellowing leaves was significantly higher than the control,but with the degree of iron stress increased,the GOGAT activity showed a downward trend,and The GOGAT activity of moderate yellowing and severe yellowing leaves has no significant difference compared to the control;mild yellowing,The GS activity of mild yellowing and moderate yellowing leaves has no significant difference compared to the control,but the GS activity of severe yellowing leaves was significantly lower than the control.4.Ferric chelate reductase gene(FCR),nitrate reductase gene(NR),nitrite reductase gene(NiR),glutamine synthetase gene(GAGAT)and Glutamine synthase(GS)are got from pear genome database,with the original sequences,primers were designed to amplify FRO,NR,Ni R,GS and GAGAT sequences.Bioinformatics analysis showed that,FCR gene sequence coding region(CDS)of 2166 bp,encoding721 amino acids,The protein encoded may be located in the plasma membrane,it is hydrophobic protein,and the secondary structure of the protein containing 45.91%alpha helix;NR gene sequence coding region(CDS)of 2709 bp,encoding 902 amino acids,The protein encoded may be located in the cytoplasmic,it is hydrophobic protein,and the secondary structure of the protein containing 29.05% alpha helix;Ni R gene sequence coding region(CDS)for 1758 bp,encoding 585 amino acids,The protein encoded may be located in the nucler,it is hydrophobic protein,and the secondary structure of the protein containing 37.44% alpha helix;GOGAT gene sequence coding region(CDS)of 4887 bp,encoding 1628 amino acids,The protein encoded may be located in the cytoplasmic,it is hydrophilic protein,and the secondary structure of the protein containing 39.31% alpha helix;GS gene sequence coding region(CDS)of 1071 bp,encoding 356 amino acids,The protein encoded may be located in the cytoplasmic,it is hydrophilic protein,and the secondary structure of the protein containing28.37% alpha helix.5.The trends of FCR,NR,NiR,GOGAT and GS activity are consistent with their genes expression,indicating that the expression of genes is beneficial to the increase of enzyme activity,the iron stress caused by yellowing affected the metabolism nitrogen. |
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