Study On Regulation Mechanism Of Nitrogen Application To Nitrogen Assimilation And Carbon Metabolism In Sugar Beet (Beta Vulgaris L.)

In order to systemically and deeply study the regulation mechanism of nitrogen （N）application levels and different NO-3/NH+4ratio with same N application levels on nitrogenassimilation and carbon metabolism in sugar beet,‘Shuangfeng16’（higher yield diploid varieties）and ‘Tianyan7’（higher sugar content diploid varieties）, two varieties were used in pot cultureexperiment with five N application levels and five different NO₃^-/NH₄⁺ratio treatments （1:1,1:2,1:3,2:1,3:1）. The changes of dry matter accumulation of sugar beet plants, the activities of keyenzymes of nitrogen assimilation （nitrate reductase NR, nitrite reductase NiR, glutaminesynthetase GS and glutamate synthase GOGAT） and carbon metabolism （RuBP carboxylase,sucrose phosphorus synthase SPS, sucrose synthase SS and invertase Inv） were analyzed, thedynamic changes of products accompanied with the enzyme activity of carbon and nitrogenmetabolism in sugar beet such as nutrition nitrogen AN, function nitrogen FN and structurenitrogen SN, glucose, fructose and sucrose in growing period. At the same time, through using theqRT-PCR, the expression of key enzymes gene for nitrogen metabolism of sugar beet regulated bynitrogen during the whole growth period was detected, and the regulation mechanism of nitrogenin nitroten metabolize on the transciriptional level was clear. Through the correlation analysisbetween the different nitrogen levels and different NO₃^-/NH₄⁺ratio with enzyme activity andmetabolites content, we preliminary study the competitive characteristics between nitrogenassimilation and sugar metabolism for photosynthetic products and energy in every growth stages,the most essential segment of produce competition, and the effect of competitive characteristics onthe sugar beet yield and quality. The results of this study contributed to progressive understandingof nitrogen assimilation mechanism on enzymatic and molecular level, providing the theoreticfoundation for regulating carbon and nitrogen assimilation by reasonably nitrogen application. Themain conclusions of this study were as follows:1. Activity of NR and NiR in sugar beet showed bimodal curve. NR activity in seedling stagewas the highest, second peak appeared at the foliage formation to root growth stage. The leaf NiRactivity peak were behind a sampling period of the NR, while root NiR activity peaks appeared atthe root growth and sugar accumulation initial stage. Activity of NR and NiR in leaf and root were highest under N180levels. NR and NiR activity reaching the highest at NO₃^-/NH₄⁺3:1show thathigh nitrogen and NO-3-N ratio has positive effect on NR and NiR, coupling relationship existsobviously and the NR and NiR.2. Activity of GS, NADH-GOGAT in sugar beet leaf and NADH-GOGAT in sugar beet roottuber all performance trend of bimodal curve, but activity of Fd-GOGAT in sugar beet leaf and GSin sugar beet root tuber all performance trend of unimodal curve, Fd-GOGAT of sugar beet roottuber were lowest. At the peak time, there is a certain synergy and complementarity relationsbetween GS and GOGAT，Fd-GOGAT and NADH-GOGAT. GS, Fd-GOGAT and NADH-GOGATin leaf were increased with the nitrogen level increasing, and decreased with the increase ofnitrogen level more than180kg hm^-2.Activity of GS, NADH-GOGAT in roots tuber also increasedwith the nitrogen level increasing, but reached the highest enzyme activity decreased rapidly after120kg hm^-2. The ratio of nitrogen with more high content of NO3--N leaves increased the activityof GS, GOGAT in leaf in the different forms of nitrogen treatment. The most suitable contents ofNO-3/NH+4to GS、Fd-GOGAT and NADH-GOGAT in leaves and GS and NADH-GOGAT in rootwere1:1,3:12:1and1:2, respectively. GS and GOGAT in root were more sensitive to NH4+, andthe enzyme activity increased with the NO3-/NH+4ratio decreasing. Activity of GS andNADH-GOGAT in root was best suited radio of NO₃^-/NH₄⁺is equal to1:2.3. Gene expression of NR, NiR, GS and GOGAT in sugar beet was different in time and space.Content of MRNA was relatively high corresponding with nitrogen treatment which having highenzyme activity. Nitrogen treatment can regulate activity of nitrogen metabolism enzymes in sugarbeet at the level of transcription.4. With reproductive process moves forward，contents of AN in sugar beet organs showedunimodal curve, AN of leaves and petiole reached peaking at foliage formation stage, AN of roottubes reached peaking in period of root growth.. With different nitrogen levels, N180was thehighest, in treatment of different nitrogen forms, the N3:1was the highest. The various organs ANcontent expressed as leaf> petiole> root in beet throughout the growing period. Expression of FNcontent in sugar beet leaves increased first and then decreased, and then increased and decreased,the peaks appeared at the formation stage of the leaves and the early stage of sugar accumulationof the root. The FN content in petioles and roots showed a single peak curve trend, the peaksappeared in the late of root growth and sugar accumulation period respectively. The levels ofnitrogen treatment performance for the N180> N120>N240>N60>N0, and the ammonium nitrateproportion is N2:1>N3:1>N1:1>N1:2>N1:3, FN content in each organ according to the reductionorder of leaf, root, petiole. Variation tendency of SN content in the whole growth period of sugarbeet’s different organs was roughly same, showed a single-peak curve. The SN content in thedifferent organs of two varieties of sugar beet showed low nitrogen （N0and N60） lower than highnitrogen （N120, N180and N240）, and high ammonium nitrate ratio （3:1,2:1and1:1） was higherthan that of low NH+4ratio （1:2and1:3）. Two varieties of FN content was leaf> petiole> root tuber. Nitrogen in the process of the assimilation process, the dynamic changes and transformationof three nitrogen forms was little difference between the two varieties. The highest proportion ofthe total nitrogen of various organs are two varieties SN content; leaves and petioles followed byAN, while FN was lowest, content of FN in root tuber was close to AN, this indicates that thenitrogen assimilation direction is structural nitrogen. In the comparison of three organs at eachgrowth period, the content of AN in SF16and TY7petiole was highest, the content of FN in roottuber was highest, and the content of SN in the leaves was highest. The content of three nitrogenforms were different could relationship with the differences of organs function.5. The leaf area improved in different degrees under different nitrogen levels and forms ofproportional treatments,180kg hm^-2and NO-3/NH+43:1mixed nitrogen had a significant effect（P<0.05）. At the same time the maximal photochemical quantum yield photosystem II （PSII）,ΦPSII, photochemical quenching coefficient （qP）, net photosynthetic rate and stomataconductance also showed a consistent variation. RuBP carboxylic activity was higher in120kghm^-2and nitrogen treatment NO₃^-/NH₄⁺under2:1mixed.6. Under different nitrogen treatments, the accumulation of sugar beet leaves and activity ofkey enzyme in glucose metabolism had promoted. Under the content of fertilization in180kg·hm^-2and NO₃^-/NH₄⁺3:1, accumulation of sugar components, SPS and SS decomposition and Invenzyme activity were high in sugar beet leaves, as well as the direction of.120kg·hm^-2fertilization,and the enzyme activity of the synthetic direction NO-3/NH+42:1beet leaves SS. SS plays animportant role in the formation of the beet earth source,120kg·hm^-2fertilizer NO-3/NH+4the2:1treatment can be effective in the early growth stage to adjust the allocation of sucrose direction,promoting the formation of the source and substance of the production, which is conducive to theincrease in the rate of fertility late tuber sugar，at the same time ensure SS synthetic directionvitality to maintain a higher level, to promote the expansion of the sugar beet photosyntheticsource and substance of production in the tuber sugar accumulation period. The SPS and SS of beetpetiole synthesis direction of enzyme activity in the180kg·hm^-2and the mixed nitrogenNO₃^-/NH₄⁺ratio of3:1fertilizer was greater, but enhanced synthesis enzyme unlimited direction ofSPS and SS. The decomposition direction of SS and Inv enzyme activity was reduced. A largenumber of the petiole sucrose may be accumulation, the detriment of the blade sucrose to enter thepetiole. The beet petiole root in SPS vitality synthetic direction was significantly higher than of theSS enzyme activity in the whole growth, and the accumulation in sugar petiole root was positivelycorrelated, and the effect is obvious, Beet root sugar accumulation by SS synthesis anddecomposition direction Inv enzyme synergy to regulate, and the reasonable nitrogen levels andNO₃^-/NH₄⁺ratio beet root sugar metabolism enzyme activity reached a high level, and beet sugarmetabolism enzyme to favor sucrose synthesis direction of development, the regulation of nitrogenfertilizer, sugar beet yield and quality up to the optimal level in production.7. In whole growth period, sugar beet nitrogen assimilation and carbon metabolism showed a correlation of different degree, effect of nitrogen assimilation and key indicators in carbonmetabolism on the yield changing with the growth period moving forward. Effect of exNR inleaves and SPS in leaves on root tuber yield was strong, effect of NiR in root and Inv in leaves onsugar yield was the most significant. The promoting effect of NiR in root and SS+on the contentof tuber sugar was most. In the foliage form of leaves, effect of NiR, RuBP carboxylase enzyme inroots and NiR, RuBP, SS-in leaves on sugar beet root yield was the most obvious. Effect ofFd-GOGAT in leaves, NADH-GOGAT in leaves, SPS in leaves, SS-in root and Inv in root onincreasing the sugar content was bigger contribution. Effect of NIR in leaves and Fd-GOGAT inleaves SS-in leaves SS-in root on sugar content was biggest. In the growth period, the promotingeffect of Fd-GOGAT in leaves on root yield of sugar beet positively. SS-in leaves, Inv in leavesand NiR in root had more contribution to root yield. The contribution of NADH-GOGAT in leaves,SS+in leaves, Inv in petioles and SS+in petioles and SS-in root to sugar content was big. Effectof NADH-GOGAT in leaves and Inv in root on sugar content and yield was great.8. Dry matter accumulation in sugar beet increased with the nitrogen level and nitratenitrogen in the mixed state increasing; especially in the late growth stage was more obvious.Nitrogen application amount of180kg hm^-2and mixed nitrogen with higher ratios NO-3-N（NO₃^-/NH₄⁺3:1） were in favor of conducive to build a strong nutrition and make morephotosynthetic product providing the material basis for the root in the enlargement growth period.SF16and TY7under180kg hm^-2treatment got the highest yield,120kg hm^-2treatment reachedsugar yield optimal with different nitrogen levels; SF16and TY7under N3:1treatment reached thehighest yield, but N2:1treatment reached the highest sugar yield in different ratios of nitrogenforms processing.