Os05g0186300

Gene Os05g0186300, namely OsNADP-ME3, is one of the cytoplasmic NADP-MEs of Rice NADP-MEs, and could improve plant resistance to abiotic stresses to a certain extent.

email me

Annotated Information

Function

OsNADP-ME3 is a malic enzyme, and malic enzymes widely exist in plant.Malic enzyme is a key enzyme in the KERBS CYCLE. This is very important to the metabolism of organisms.In plants, it takes part in photosynthesis,respiration,production of lipids and other important metabolic pathways,which is very important to keep the osmotic potential of plant cells, stabilize the PH of cytoplasm and keep the ions absorption of plant roots balanced.It is one fo the most important enzymes in life activities of plants.

The overexpression of OsNADP-ME3 in Arabidopsis thaliana obviously increases its stress resistance.OsNADP-ME3 was carried over into Arabidopsis plants. Through Northern detection,we find that No.5,No.9 and No.10 of strain are higher(Figure 1).

Figure 1 Expression of the NADP-ME3 in wild-type (WT) and transgenic lines Note: WT: Wide-type Arabidopsis; 1,2,4,5,6,7,8,9,10: Transgenic lines of pBI121-NADP-ME3

Choose these three strains to be materials, contrasted with WT.Through the changes of root length under stress situation of different concentrations of NaCl,Manntiol and H2O2 with these three strains, it shows that, there is no difference between WT and GM(Genetically Modified)(Figure 2A;Figure 2E;Figure 2I),but after 20d with NaCl, the growing states of GM was better than WT, and in medium of 150mmol/L NaCl, lateral roots of GM was more than that of WT, and the leaf was bigger than WT(Figure 4C).The measuring result of root length also showed that the growing states of GM was better than WT, and value exhibited that GM is 1cm longer than WT(Figure 4D).All indicated that OsNADP-ME3 can increase halotolerancy of plant to some extent.

Figure 2 Figure 4 Analysis of the resistance of the overexpressed transgenic plants with NADP-ME3 under salt stress Note: A,E,I: Controls; B~C: NaCl stress condition; D: The root lengths of transgenic plants under NaCl stress condition; F~G: Manntiol stress condition; H: The root lengths of transgenic plants under manntiol stress condition; J~K: H2O2 stress condition; L: The root lengths of transgenic plants under H2O2 stress condition

Under the condition of manntiol, the growing states of GM strains is better than that of WT, but it is not obvious under 250 mmol/L Manntiol(Figure 4F).When 275 mmol/L, it was a little better than WT(Figure 4G). The measuring result of root length also showed that the growing states between GM and WT was not obvious, which showed that OsNADP-ME3 may be not relative with drought tolerance.

Under the condition of H2O2,although the root length of GM strains and WT was not obvious when it was 1 mmol/L, leaf of former was bigger than the latter,and growing states the same. As H2O2 concentration was added up to 1.5 mmol/L, all were obvious. Not only the root of GM strain was longer than WT,but also the leaf was much bigger. This indicated that OsNADP-ME3 can increase the antioxidant capacity.

In summary, the GM strain grows better than WT under the condition of salt-stress and oxidative intimidation,which indicates that OsNADP-ME3 improve plant resistance to abiotic stresses to a certain extent. Correlative study shows that salt-stress can induce a great deal of active radical,and causes ROS oxidative intimidation.This moment, strain could regulate the balance of oxidation by antioxidant and antioxidase,while NADP-ME can produce NADPH in malic acid metabolism. NADP can offer reducing power to the metabolism of ROS, which could resist the harm to plant caused by salt-stress.

Expression

The expressions of NADP-ME3 mRNA under a variety of abiotic stresses were detected with Northern blot by extracting RNA from leaves and roots of rice with different adversity handling(Figure 3).

The results indicated that,under the condition of salt stress(80 mmol/L NaCl), the expression level of NADP-ME3 in the initial period of leaf stress is relatively high. With time increasing, it decreased gradually, and the decline trend was evident.Meantime, under the same condition, although the expression level is low in root at the initial stage, it gradually increased with the time prolonging, which demonstrated that there exists response relationship between NADP-ME3 and salt stress; under the condition of alkaline stress(30 mmol/L NaHCO3),in leaf, the expression level of NADP-ME3 in the initial 6 hours is relatively high, and then showed the trend of decreasing,while in root, it was increasing (6h),and then gradually decreased.After 24h,it increased again with irregular change,but at any time, it was higher than the contrast,which illustrated that OsNADP-ME3 expressed induced by NaHCO3; under the condition of drought stress(10% PEG),in leaf, the expression level of NADP-ME3 in the initial stage is highest.With stress time prolonging,the transciriptional level of mRNA decreased gradually,while in root, it is relatively high in the initial stage, and up to highest at 12h,then declined mildly.

In conclusion, at the exposure to NaCl, NaHCO3 and PEG, the expression level of NADP-ME3 changed with the different lengths of exposure duration. Therefore, the NADP-ME3 may be involved in response to abiotic stresses.

Figure 3 Changes of the expression level of NADP-ME3 gene from rice under the abiotic stresses

Evolution

Rice NADP-MEs are a multigene family of three cytoplasmic NADP-MEs and one plastidic NADP-ME. NADP-ME3 is of one of the cytoplasmic members . The cDNA sequence of the cloned NADP-ME3 is 2 240 bp, and the 5' untranslated region (UTR) is 151 bp while the 3' UTR is 376 bp. The open reading frame is 1 712 bp, encoding 570 amino acids.Homology analysis of the amino acid sequence in rice NADP-ME gene family(Figure 4).

Labs working on this gene

◑Key Laboratory of Ministry of Education for Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University.

◑Asian Natural Environmental Science Center (ANESC), University of Tokyo.

◑State Key Laboratory of Rice Biology,China National Rice Research Institute and Zhejiang University.

Figure 4 Homology analysis of the amino acid sequence in rice NADP-ME gene family