The Expression Of Nitrogen Uptake And Metabolism Related Genes In Sophora Japonica L.and Its Relationship With The Aging Of Ancient Trees

Plant morphology,physiology and gene expression are affected by abiotic stresses.The root system is the main organ for plants to absorb nutrients.Plants can adapt to the changing environment by adjusting root nutrient uptake.Nitrogen（N）is one of the essential macronutrients for plant growth and development.N uptake and utilization are important biological activities that can affect the stress adaptability and senescence process of plants.Sophora japonica L.is a native leguminous tree species in China.Because of its large crown,dense foliage,high stress tolerance and long lifespan,S.japonica is widely used for urban and rural greening in northern China.S.japonica has a long lifespan of thousands of years,which accounts for a significant proportion of ancient tree resources in China.As one of the few leguminous tree species that cannot form nodules to fix N,S.japonica could survive under various extreme environmental stress,in which the regulation of N absorption and metabolism may play an important role.Due to the lack of available genetic data,studies on the response of N uptake and utilization of S.japonica to abiotic stresses are limited.Additionally,it has not been known how the N uptake and metabolism capacity of old S.japonica trees changes with the tree age.In this study,roots of S.japonica seedlings treated with short-term abiotic stresses were collected for transcriptome sequencing.The responses of gene pathways were analyzed.Meanwhile,genes related to N uptake and metabolism of S.japonica were excavated via Bioinformatics analyses.Then,physiological and biochemical techniques and real-time quantitative PCR were used to study the N uptake and metabolism regulation strategy of S.japonica seedlings under abiotic stresses in hydroponic systems.Finally,fine roots of healthy ancient S.japonica trees of different ages（200、1000、2000 years old）in the field were collected to study the changes in N uptake and utilization with the increase of the tree age.Correlation analysis was also used to explore the influencing factors of N absorption of ancient trees.This study revealed the molecular response mechanism of N uptake and metabolism in S.japonica to abiotic stresses and its relationship with aging.The main results and conclusions of this study are as follows:1.RNA-seq was conducted for S.japonica roots grown under short-term 20%polyethylene glycol（PEG）6000-induced drought stress under normal N and N starvation conditions（1 and 0 m M NH₄NO₃,respectively）.In each of the libraries,we generated more than 25 million clean reads,which were then de novo assembled to 46852 unigenes with an average length of 1310.49 bp.According to KEGG enrichment analyses,genes involved in“phenylpropanoid biosynthesis”and“biosynthesis of amino acids”pathways were upregulated,irrespective of the stress treatments.Upregulated N metabolism genes were enriched upon drought,and downregulated photosynthesis genes were enriched under N starvation.Compared with the normal N condition,N starvation may aggravate drought by downregulating transcripts in the“carbon metabolism”,“ribosome”,“arginine biosynthesis pathway”,“oxidative phosphorylation”and“aminoacyl-t RNA biosynthesis”pathways.Through the bioinformatics analysis,a total of 25 key genes related to N uptake and metabolism of S.japonica were identified.2.S.japonica seedlings were subjected to drought（5%PEG-6000）,salinity（75m M Na Cl）,or low N（0.01m M NH₄NO₃）stress for 3 weeks in a semi-hydroponic phenotyping platform.Salinity and low N negatively affected plant growth,while drought promoted root growth and inhibited aboveground growth.The NH₄⁺/NO₃^-ratio increased under all three treatments with the exception of a reduction in leaves under salinity.Drought enhanced NH₄⁺assimilation with increased Glutamate synthase（GOGAT）activity,although Glutamine synthetase（GS）activity remained unchanged,whereas salinity and low N inhibited NH₄⁺assimilation with decreased GS activity under salt stress and decreased GOGAT activity under low N treatment.In roots,AMT1.1 AMT2.1a and AMT2.1b（Ammonium transporter）showed higher transcription under drought;most NRTs were upregulated under salinity;expressions of NRT2s（Nitrate transporter）and AMTs were highly induced by low N stress.These results indicated that the growth of S.japonica seedlings was affected as a result of the changed N uptake and metabolism process under abiotic stresses,however,the regulation mechanism under drought was different with that under salinity.Drought stress enhanced NH₄⁺uptake by up-regulating the expression of AMTs,and accelerated NH₄⁺assimilation by increasing GOGAT activity,so the root growth of seedlings was promoted.Under salinity,although the increased NO₃^-absorption caused by up-regulation of most NRTs as well as enhuanced NO₃^-assimilation were observed,inhibited NH₄⁺assimilation caused by the decreased GS activity resulted in a limitation of the root growth.3.A hydroponic experiment was designed in which S.japonica seedlings were supplied with sole NH₄⁺or sole NO₃^-nutrition under salt stress.The growth and N metabolism performance were investigated.In the absence of Na Cl,plants fed by NH₄⁺showed better root growth than NO₃^-,but with no difference in aerial parts.Salinity inhibited the root growth of NH₄⁺-fed plants and the shoot growth of NO₃^--fed plants,while the total N accumulation was suppressed under either N form.Specifically,in NH₄⁺-fed plants,salinity significantly increased the net photosynthetic rate,root NH₄⁺content and root antioxidant enzyme activities,while higher NR activities but lower GS activities were observed in both leaves and roots.In NO₃^--fed plants,salinity suppressed the net photosynthetic rate,leaf antioxidant enzyme activities,and leaf GS activity,whereas root GOGAT activity was increased.Under salinity,AMT1.1 and AMT2.1a were upregulated in the leaves of NH₄⁺-fed plants,but AMT1.2 and AMT2.1a were downregulated in the roots.In NO₃^--fed plants,the transcripts of NPFs in the leaves and most NRTs in the roots were promoted by salinity.Overall,these results suggested that S.japonica plants grew better in NH₄⁺than NO₃^-medium in the absence of Na Cl.In response to salinity,as a result of the down-regulation of AMT1.2 and AMT2.1a as well as reduced GS activity,the NH₄⁺uptake and assimilation process were inhibited in the roots of NH₄⁺-fed plants,thus the root growth of seedlings was restricted.In the leaves of NO₃^--fed plants,although the expressions of NRT1s were increased,the growth of aboverground parts was inhibited by salinity due to reduced net photosynthetic rate and slowed NH₄⁺assimilation.4.Fine roots of around 200,1000,and 2000-year-old healthy S.japonica were used to study the changes in N uptake and utilization with the increase of the tree age.Key N metabolic enzyme activities in ancient trees over one thousand-year-old were significantly higher than that in 200-year-old trees.The expressions of N-related key genes increased with the tree age.These results indicated that the healthy 2000-year-old trees still have strong N absorption and metabolism capacity.Correlation analysis showed that the expressions of analyzed genes were positively correlated with NO₃^-content in the soil,suggesting that NO₃^-may be the driving factor for the expression of those N-related genes when S.japonica grows older.In summary,N uptake and metabolism related genes regulate the process of N uptake,N transport,N assimilation and N accumulation,through which S.japonica seedlings could adjust both the aerial and root growth to improve their resistance to the changes of environmental conditions.The expressions of N-related key genes increased with the tree age.2000-year-old healthy S.japonica trees may still have strong N absorption and metabolism capacity.These results clarified the responses of N uptake and metabolism of S.japonica to abiotic stresses and its relationship with the aging of ancient trees,which provided a theoretical basis for the studies on resistance mechanism and the protection of ancient trees.