Molecular Mechanism Of Nitrogen Regulation Of Nitric Oxide Synthesis And Plant Growth And Development

Nitrogen is one of the essential nutrients for plant growth and participates in a variety of physiological and biochemical processes of plants.Nitric oxide（NO）is an important nitrogen metabolite that plays an important regulatory role in physiological processes such as plant growth and development,stress resistance and so on.Nitrate reductase（NR）is known to be the main source of NO enzymes in plants.Nitric oxide synthase（NOS）in animals catalyzes the direct synthesis of arginine（L-Arg）into NO,and the existence of similar NOS in plants is controversial.Arabidopsis cue1-6（The Arabidopsis chlorophyll a/b binding protein gene underexpressed 1）mutants have high accumulation of endogenous NO,while a large amount of phosphoenolpyruvate（PEP）accumulates in the cytoplasm.The content of PEP was reduced in noa1（The Arabidopsis NO-associated protein 1 is absent）mutants with less endogenous NO accumulation.It is speculated that PEP or phosphoenolpyruvate carboxylase（PEPC）may be involved in the synthesis of NO.NR uses nitrate nitrogen to synthesize NO,and stress will also induce plant synthesis of NO,but too much NO will inhibit plant growth,but there are few reports on the interaction between nitrogen and stress on NO synthesis.In this study,wild type Arabidopsis thaliana,PEPC deletion double mutant（ppc1/ppc2）and NR deletion double mutant（nia1/nia2）were used as materials to investigate the growth and development,NO synthesis,PEPC and NR enzyme activities and related gene expressions of Arabidopsis thaliana under different nitrogen forms and nitrogen levels combined with stress.To study the molecular mechanism of NO synthesis involving PEPC.The main findings of this paper are as follows:（1）PEPC has NOS activity,catalyzes the formation of NO by L-Arg,and PEP,Na HCO₃and NADPH（reduced nicotinamide adenine dinucleotide phosphate）are required in the reaction system.The enzyme kinetic parameters of this reaction are:maximum speed of enzyme reaction V_max=0.2 m M/min/mg protein,the Michael’s constant K_mis about 180μM,and the optimal Arg concentration is 800μM.The PEPC enzyme activity was determined on the noa1 mutant and cue1-6 mutant,and it was found that the PEPC enzyme activity was positively correlated with the NO content in its body.Animal NOS inhibitors L-NAME（N-nitro-L-arginine methyl ester）,AMG（aminoguanidine）and PEPC inhibitors L-malic acid and L-aspartic acid can inhibit the NOS activity of PEPC and are concentration-dependent.（2）Low nitrogen promoted root growth of Arabidopsis thaliana,high nitrogen inhibited root growth,and high NH₄⁺concentration caused ammonium toxicity.Low nitrogen is subjected to stress treatment at the same time,and excess NO will accumulate in Arabidopsis thaliana and inhibit plant growth.Through six nitrogen treatments,the results show that compared with CK,1/2N and 1/20N promoted root growth of Arabidopsis thaliana,while 2N inhibited root growth.Low concentration of NH₄⁺promoted the growth of Arabidopsis thaliana,which showed that main root elongation,lateral root number and hair number increased.High concentration of NH₄⁺resulted in ammonium toxicity,which showed that main root elongation was inhibited,fresh weight,dry weight and water content decreased,and malondialdehyde（MDA）content and relative conductivity increased.Compared with CK,NO content in Arabidopsis thaliana was significantly increased under other nitrogen treatments,and the highest content was found under 1/2 NH₄⁺treatment.The content of NO in Arabidopsis thaliana was significantly increased under the four stress treatments compared with the control,and the highest was found under the highlight treatment.When nitrogen drops to 1/20N,while undergoing stress treatment,excess NO is produced in Arabidopsis.（3）Under the four stress treatments,the enzyme activity of NR and PEPC was enhanced;The expression of PPC1,PPC2,NIA1 and NIA2 were all significantly upregulated.Compared with CK,the expression levels of NR enzyme activity and NIA1 and NIA2 genes decreased under 1/2 NH₄⁺treatment.Although PEPC enzyme activity was enhanced,the expression of PPC1 and PPC2 genes was inhibited,and the NO content in Arabidopsis thaliana was significantly higher than that in the control treatment,indicating that there were NR-dependent NO synthesis pathways,PEPC-dependent NO synthesis pathways,and NO synthesis pathways that did not depend on both.（4）Under the condition of 1/20N and 1/20 NH₄⁺,due to the reduction of nitrogen or the loss of nitrate nitrogen,the proportion of NO synthesized through the NR pathway decreases,which are 28.88%and 20.42%,respectively;the proportion of NO synthesized by PEPC pathway increased,42.47%and 51.55%,respectively;other pathways were 28.65%and 28.02%,respectively.Compared with CK,NO synthesized by PEPC pathway and NR pathway increased under stress.The proportion of NO synthesized by NR under cold and osmotic stress was higher,under high light and salt stress,the proportion of PEPC synthesized NO was higher.In conclusion,stress treatment with low nitrogen can produce excessive NO in plants and inhibit growth,while normal nitrogen supply can reduce excessive accumulation of NO under stress.In agricultural production,genetic methods can be used to screen and cultivate materials with low accumulation of NO,so as to improve crop yield and stress resistance.