Enzymological Characteristics Of Glutamate Synthase And Nitrogen Regulation On The Enzyme Activity In Sugar Beet(Beta Vulgaris L.)

Glutamate synthase (GOGAT), a key enzyme in nitrogen assimilation process in sugar beet (Beta vulgaris L.). Two varieties shuangfengl6and tiandanl were selected in this study. The isozyme of GOGAT (Fd-GOGAT and NADH-GOGAT) were purificated and separated in order to further know about nitrogen assimilation mechanism in sugar beet, by using three-steps method, ammonium sulfate precipitation, gel filtration and ion chromatograpHy, and the enzymological characteristics of GOGAT were clearly indicated. The regulation and control of nitrogen treatment to GOGAT was definited by investigating the accumulated dynamic of GOGAT in Function blades and roots on different sampling dates under five nitrogen levels (0,60,120,180and240kgN/hm2) and five rate of nitrogen form (NO3-:NH4+is:3:1,2:1,1:1,1:2,1:3) with N120, based on that below, a reasonable theoretical basis to improving the effective utilization of nitrogen and the quality of root would be perfected. The results were as followed:Purification of Fd-GOGAT and NADH-GOGAT respectively resulted in14.1and4.5-fold, Fd-GOGAT molecular weight was214kDa, it included two subunits, the molecular weight were respectively160kDa and54kDa. NADH-GOGAT had only a single subunit of170kDa, its molecular weight was183KDa. Fd-GOGAT was more stable than NADH-GOGAT, the suitable temperature was40℃, pH was7.7. Km values for L-glutamine, a-ketoglutarate and Fd were respectively2307μmol/L,940μmol/L, and2.062μmol/L. NADH-GOGAT was labile, its suitable temperature was35℃, pH was7.3. The Km values of NADH-GOGAT for glutamine, a-ketoglutarate, and NADH were3376μmol/L,2278μmol/L and4.79μmol/L, respectively.Fd-GOGAT take up78.04%of GOGAT activity in function blade, but the content in root is only16.39%. NADH-GOGAT is dominant in root, and the content is84.61%.The dynamics changes of Fd-GOGAT in leaf were single peak curves under the different nitrogen levels, the peak values appeared in late June. Changes of the NADH-GOGAT in the leaf and the root expressed double peak curves, the two peaks appeared in late June16and August20respectively, and the first peak of enzyme activity was higher than the later in the leaf, but it was opposite in the root. Nitrogen treatment obviously effected GOGAT activity, enzyme activities enhanced when nitrogen levels increased under a range of nitrogen levels, but lowered after exceeding the range. The highest activity of Fd-GOGAT and NADH-GOGAT in function blade was at the treatment of N180, and the NADH-GOGAT of root achieved the highest activity at N120treatment.Under the different nitrogen forms, the Fd-GOGAT activity in the leaf enhanced with the increase of nitrate nitrogen content, which achieved the maximum value with3:1(NO3-:NH4+), and the NADH-GOGAT activity in the leaf showed the same trend as well as Fd-GOGAT, but its maximal activity was with2:1(NO3-:NH4+). The NADH-GOGAT activity in roots was sensitive to ammonia nitrogen, the activity enhanced with the increase of NH4+content, the maximal value was1:2(NO3-:NH4+)The sugar content lowered while the root yield increased along with the increase of nitrogen levels in certain scope. Under the different nitrogen forms, although the sugar content enhanced as soon as the NH4+content increased, the increase of NO3-content could increase the root yield, the sugar yield achieved the maximal value at2:1(NO3-:NH4+.There were close relationship of GOGAT activity with beet yield and sucrose content, the correlation between Fd-GOGAT activity and beet yield was significantly positive (R=0.989**), and the correlation between Fd-GOGAT activity and sucrose content was negative (R=-0.645).