Mechanism Analysis Of G Protein Signaling Pathway Regulating Salinity And Nitrogen Stress Responses In Plants

Plants have evolved multiple machnisms to adapt to biotic and abiotic stress in the erratic environment.The traditional heterotrimeric G protein consisted of one G??GPA1?,one G??AGB1?,three G??AGG1-3?and one regulator of G protein signaling?RGS1?.The three-member family of Arabidopsis extra-large G proteins?XLG1-3?defines the prototype of an atypical G?subunit in the heterotrimeric G protein complex.The interactome of the traditional G protein indicated important roles in stress responses.Recent evidence indicates that XLG subunits operate along with its G??dimer in stress responsiveness,however downstream targets of activated XLG proteins in the stress pathways are unknown.G protein signaling pathway involved in root morphology,cell prolifteration,ER stress,salinity stress and nitrogen use efficiency.While the mechanisms of stress responses are still unknown.To investigate the main function of XLGs in plants and the mechanisms of stress responses,our work focused on three aspects,including the interactome screen of XLGs,the mechanisms of G protein regulating salinity stress and nitrogen stress.The main results were listed as below.1.To assemble a set of candidate XLG-targeted proteins,a yeast two-hybrid complementation-based screen was performed using XLG protein baits to query interactions between XLG and partner protein found in glucose-treated seedlings,roots,and Arabidopsis cells in culture.Seventy-two interactors were identified and greater than 60%of a test set displayed in vivo interaction with XLG proteins.Gene co-expression analysis shows that greater than 70%of the interactors are positively correlated with the corresponding XLG partners.Gene Ontology enrichment for all the candidates indicates stress responses and posits a molecular mechanism involving a specific set of transcription factor partners AtSZF1/2 to XLG.The subcellular localization of the XLG proteins in the nucleus,endosome,and plasma membrane is dependent on the specific interacting partner.2.AtSZF1/2 encoding two transcription factors and the expression level was stimulated by salt stress.The double mutants of AtSZF1/2 was hypersensitive to salt stress.The expression level of AtSZF1/2 were inhibited in xlg1/2/3 and agb1-2 mutants,and enhanced in rgs1-2 mutants compared to the Col.The G protein mutants agb1-2 and xlg1/2/3 were hypersensitive to salt stress while rgs1-2 was hyposentive to salt stress.The results above indicated that G protein signaling pathway regulated the salt stress by modulating the expression of AtSZF1/2.3.We used rice and Arabidopsis G protein mutants to study the external C/N responses,root architecture,nitrogen uptake and translocation and primary nitrate responses in nitrogen stress.?1?In different C/N treatment,the plants showed different responses to the C/N ratio in the environment.The plants showed no responses to the extracellular nitrogen supply without sucrose.The G protein mutants showed different responses to the C/N ratio.The results showed that the agb1-2,xlg1/2/3 and xlg/gpa1 mutants was hypersensitive to high C/N ratio and the rgs1-2 mutants showed the opposite results.The G protein signaling pathway could regulate the plant growth in response to the extracellular C/N supply.?2?The G?subunit RGA1 null mutants DK22 was used for the 3-Dimentional root architecture studies.We found that nitrogen inhibits the root growth through several aspects,such as the root horizontal projection area?network area?,perimeter,total length,but the root diameter?verage root width?did not change.Nitrogen influenced the root spatial distribution by inhibiting horizontal growth and promoting vertical expansion.The dynamic changes of the rice G protein mutant DK22 from day 7 to day 9 was different from the wild type in the bushiness and spatial distribution.The agb1-2 mutant in Arabidopsis lacked the inhibitory effect of nitrate on root growth.The heterotrimeric G protein complex regulates the inhibitory effect on root growth caused by high nitrogen supply and root spatial distribution in response to different nitrogen concentrations.?3?The plates results showed that agb1-2 mutants had robust root growth and weak shoot growth under limited nitrogen supply.The ¹⁵N tracing results showed agb1-2 mutants exhibited defected nitrogen translocation through root to shoot under sufficient nitrogen supply;rgs1-2 mutants showed increased source to sink translocation under low nitrogen supply.The transcriptional analysis of the agb1-2 and rgs1-2 showed that the primary nitrate resposes of the G protein mutants did not altered.G protein regulate nitrogen responses in a stress triggered manner,not nitrate responses.The RT-PCR results showed that G?subunit and XLG1/2/3 are essential component during the nitrogen treatment,just as the salinity stress responses.?4?The transcriptome analysis of the Arabidopsis roots treated with 10mM nitrate for 3h after 3 days nitrogen starvation showed that in the G protein mutants the primary nitrate responses did not vanish,only a few nitrogen metabolism related genes changed.The mainly differentially expressed genes were related to stress and photosynthesis.In conclusion,XLGs were mainly involved in the stress responses in the plants.G protein signaling pathway regulates the salinity stress responses through the genes expression level of SZF1/2.G proteins sculpt the root architecture via the root to shoot N translocation.G proteins also responded to nitrogen stress through transcriptional level of the photosynthesis and stress related genes.