Study On Cellular Localization,Expression And Function Of Glutamine Synthetase Isozymes In Wheat

Wheat is one of the important food crops in China,and wheat production plays a decisive role in guaranteeing China's food security.Nitrogen fertilizer plays an important role in improving the yield and quality of wheat.However,the problem of low nitrogen use efficiency is widespread in China,which not only causes huge economic losses,but also causes environmental pollution.Improving nitrogen use efficiency is an effective way to achieve the goal of "reducing nitrogen and increasing yield" in wheat production.In order to improve nitrogen use efficiency,glutamine synthetase?GS?,a key enzyme for nitrogen assimilation,has become a research hotspot.GS is strictly regulated at multiple levels,but the most relevant reports focus on the m RNA level.So,a study on the expression pattern and localization of GS isozymes at the protein level will be more conducive to confirming and supplementing our knowledge of their functions.To study the localization and expression of individual TaGS isozymes?TaGS1;1,TaGS1;2,TaGS1;3 and TaGS2?at the protein level,we first used three-generation sequencing technology to analyze the transcription of TaGS isozymes and determine these highly expressed transcripts of TaGS isozymes.Then,the kinetic properties of individual TaGS isozymes expressed by the prokaryotic were analyzed,and the localization and expression of TaGS under different nitrogen nutrition were analyzed by using the specific antibodies of individual TaGS isoforms.In addition,the accumulation and transport characteristics of carbon and nitrogen assimilates in source-translocation-sink during grain filling period of wheat were analyzed.Finally,the localization,expression and function of individual TaGS isozymes in wheat grains were studied in detail.The main results are as follows:1.There are three alternative splicing transcripts of TaGS gene,among which the transcript of TaGS1;1-6B-4 is a newly discovered alternative splicing form of TaGS1;1.At the same time,by comparing the CPM values of individual TaGS transcripts,it was found that the main transcript of TaGS1;1,TaGS1;2,TaGS1;3 and TaGS2 are Traes CS6B02G327500.1,Traes CS4D02G240700.1,Traes CS4D02G047400.1 and Traes CS2B02G528300.2,respectively.2.In addition to TaGS2,TaGS1;1 with high affinity to substrate and TaGS1;3 with high catalytic activity were also localized in mesophyll,and may participate in cytoplasmic assimilation of NH₄⁺ released from photorespiration or absorbed by roots;TaGS1;2 was localized in xylem of leaves.In roots,although there were hundreds of times more TaGS1;1 than TaGS1;2 transcripts,the amount of TaGS1;1 subunit was not higher than that of TaGS1;2;NH₄⁺ inhibited TaGS1;1 expression but stimulated TaGS1;3 expression.In root tips,NO₃^- stimulated TaGS1;1,TaGS1;3,and TaGS2 expression in meristem,while NH₄⁺ promoted tissue differentiation and TaGS1;2 expression in endodermis and vascular tissue.Only TaGS1;2 was located in vascular tissue of leaves and roots,and was activated by Gln,suggesting a role in nitrogen transport.TaGS1;3 was induced by NH₄⁺ in root endodermis and mesophyll,suggesting a function in relieving NH₄⁺ toxicity.Thus,TaGS isoforms play distinct roles in nitrogen assimilation for their different kinetic properties,cellular localization,and response to nitrogen regimes.3.Before 16 days after flowering?DAF?,a large amount of soluble sugars and free amino acids were accumulated in the peduncle.Subsequently,a large amount of amino acids and soluble sugars imported into the grains through phloem were used for the synthesis of starch and protein in the grains.In addition to organic nitrogen,inorganic nitrogen also existed in phloem exudate.The output rate of NO₃^-from flag leaf into peduncle was similar to that from peduncle into ear,reached its maximum at 24 DAF and then rapidly decreasing.However,the output rate of NH₄⁺ from peduncle into ear was 8-100 times as high as that from flag leaf into peduncle.In addition,only TaGS1;2 is distributed in vascular bundles of flag leaves and peduncle.TaGS1;2 is mainly distributed in xylem of flag leaves,but it is mainly distributed in phloem of peduncle.These results indicated that NH₄⁺ was transported to the grain through the phloem of peduncle,TaGS1;2,which was localized in phloem of peduncle,participated in the process of assimilating NH₄⁺ into Gln.With the NH₄⁺ entering the grain,the content of NH₄⁺ in the grains was much higher than that of flag leaf and peduncle,indicating that the grains have higher tolerance to NH₄⁺.4.Immunofluorescence revealed that TaGS1;1,TaGS1;3,and TaGS2 were expressed in different regions of the embryo.In grain transporting tissues,TaGS1;2 was localized in vascular bundle,TaGS1;2 and TaGS1;1 were in chalaza and placentochalaza,TaGS1;1 and TaGS1;3 were in endosperm transfer cells,and TaGS1;3 and TaGS2 were in aleurone layer.GS exhibited maximum activity and expression at eight days after flowering?DAF?with peak glutamine content in grains;from then,NH₄⁺ increased largely from NO₃^-reduction,glutamate dehydrogenase?GDH?aminating activity increased continuously and the activities of GS and glutamate synthase?GOGAT?decreased,while only TaGS1;3 kept a stable expression in different TaGS isozymes.Hence,low-concentration grain NH₄⁺ is mainly assimilated by GS-GOGAT cycle at eight DAF,while high-concentration grain NH₄⁺ is assimilated by the GDH-GS pathway after eight DAF.TaGS1;3,located in aleurone layer and endosperm transfer cells,plays a key role in Gln into endosperm for gluten synthesis.Study on the localization and expression pattern of TaGS isozymes at the protein level will be more conducive to confirming and supplementing our knowledge of their functions.At the same time,the research on the localization and function of TaGS isozymes in grains is helpful to comprehensive understand the nitrogen assimilation process of grains,and provides new strategies for improving the nitrogen use efficiency.