?-Aminobutyric Acid (GABA) Alleviates Ammonium Toxicity And Their Mechanisms In Rice Seedlings

The ammonium toxicity in plant is an old phenomenon around the world.It has also become an issue of global ecological and economic importance for human intervention in the nitrogen cycle.Rice is known as an ammonium(NH4+)-tolerant species.Nevertheless,rice can suffer NH4+ toxicity,and excessive use of nitrogen(N)fertilizer has raised NH4+ in many paddy soils to levels that reduce vegetative biomass and yield.Faced with such high ammonium environment,it is imminent to find a way to improve the resistance of rice plant to high ammonium.Rice seedlings were more sensitive to ammonium,so in this study,we used the rice seedlings as materials,established the suitable sterile research training mode,found that GABA can alleviate the ammonium toxicity of rice seedlings.GABA plays a wide role in plants,which has an important physiological function in many plants under stress,but to improve the plant resistance to excessive ammonium is first reported.Based on the obvious phenotype,we used growth data to examine NH4+ stress and its alleviation by GABA and investigated the role that GABA played in alleviating the ammonium toxicity of rice seedlings,using microscope,non-invasive micro-test technique,and other environmental molecular biological approaches.Firstly,we demonstrated that elevated levels of exogenous NH4+ mainly inhibited the development of total root length and root area,and as a result,root biomass decreased significantly,leaf blade gradually appeared chlorosis and necrosis,shoot biomass gradually reduced as well.Moreover,the content of K,Ca,Mg,Fe,Si,Zn significantly reduced,the culture medium was seriously acidulated and root tips membrane integrity were damaged.The alleviation of GABA on ammonium toxicity in rice seedlings was also reflected in these aspects:GABA improved the inhibitory effect of high ammonium on rice root,increased biomass of rice seedlings both root and shoot,and raised the content of Ca,Mg,Fe,Si and Zn,relieved root zone acidification,kept the root tip cell membrane complete.Research on the key enzymes of ammonium assimilation showed:with the increase of the concentration of NH4+,GS activity in root of rice seedlings gradually increased,GOGAT and GDH activity was highest in 5.76 mM then began to decrease.The situation of the shoot is:GS activity reached the highest at 2.88 mM,and then began to decrease,the activity of GOG AT reached a maximum at 5.76 mM,and then decreased,the activity of GDH enhanced gradually with the increase of the concentration of ammonium.It was suggested that in order to assimilation NH4+ quickly,preventing excessive NH4+ produce harmful effects on plants,rice seedlings maintain high level activities of GS\GOGAT and GDH,but not enough as ammonium concentration increased.We used GS inhibitor MSO to treatment seedlings,found that MSO can restore the inhibition of root growth of rice seedlings under high ammonium.Moreover,roots of rice seedlings is markedly inhibited by exogenous Gln,which can be restored by 2-OG.The results showed that:the important reason for the roots growth inhibition by high ammonium in rice seedlings was not NH4+itself,it was Gln,the downstream metabolites of NH4+ It is indicated that there is a direct relationship between root growth inhibition of rice seedling under high ammonium and insufficient carbon skeleton induced by a large number of NH4+ assimilation.NH4+ and H+ fluxes were measured using the Scanning Ion-selected Electrode Technique(SIET),revealed that GABA promoted the NH4+ efflux and reduced efflux of H+under excessive ammonium(2.88 mM NH4+).Supplemented by pH indicator media staining and determination of culture medium of pH,proved that GABA alleviated the rice root zone acidification caused by high ammonium.At the same time,the expression NH4+transporter OSAMT1;2 were measured using RT-PCR,the result displayed that GABA inhibited OSAMT1;2 expression,indicating that GABA also inhibited the absorption of NH4+ in rice seedlings under excessive ammonium.GABA reduced the activities of GS/GOGAT,GDH in root of rice seedlings under excessive ammonium(2.88 mM NH4+),indicating that the addition of GABA reduced the level of NH4+ assimilation.We further proved that GABA reduced the root expression of GS1.1,GS1.2 and NADH-GOGAT1.1 under excessive ammonium(2.88 mM NH4+),suggesting that the regulation of GABA on the activities of GS/GOGAT partly contributed to the transcriptional level.GABA improved the shoot GS activity of rice seedlings,decreased the activity of GOGAT,and had little effect on GDH activity under excessive ammonium(2.88 mM NH4+).Detection of gene expression showed that GABA promoted the expression of GS1.1,GS1.2,but inhibited the expression of NADH-GOGAT1.1,NADH-GOGAT1.2.In short,the mechanism of GABA-dependent alleviation of ammonium toxicity in rice seedlings is:GABA reduced the NH4+ net absorption of rice seedlings,free NH4+ content decreased,reducing free NH4+ toxicity to rice,and the assimilation of NH4+ is reduced,thereby reducing the loss on C skeleton in rice seedlings under high ammonium.Furthermore,GABA may supply energy to the rice seedlings that consumed by invalid cycle of NH4+ through the TCA cycle.