Effects Of GmCYP707A3b And GmCYP707A1a On The Nodulation And Nitrogen Fixation In Soybean

With the worsening global environments and climate changes,the increasing occurrences of drought stress and soil salinization have significantly influenced soybean(Glycine max)production by affecting the nitrogen fixation and grain yield.Breeding of soybean varieties with enhanced tolerance against drought and salinity stresses by using various biotechnologies has become targets of many plant scientists.However,our understanding of soybean nitrogen fixation and tolerance against these stresses is still very limited.As an important stress hormone,ABA plays essential roles in various physiological processes during plant responses to drought and salinity stresses.We here characterized two P450 genes,GmCYP707A3 b and GmCYP707A1 a,involved in ABA metabolic pathways,to understand how they influence ABA catabolism and affect the nodulation and nitrogen fixation,as well as drought and salt stresses.We identified two GmCYP707 A family proteins through bioinformatic analysis,and then cloned GmCYP707A3 b and GmCYP707A1 a from soybean cultivar ―Tianlong N0.1‖ nodules.To examine how these genes aftect soybean nodulation,we made the overexpression and RNA interference(RNAi)knockdown constructs for these two genes.We then estabolished the chimerical soybean plants with wild-type shoots and transgenic hairy roots through transformation with Agrobacterium rhizogenes(K599)harboring GmCYP707A3 b or GmCYP707A1 a overexpression and RNAi knockdown vectors.We further examined how overexpression or knockdown of these two genes in transgenic hairy roots could influence the nodulation after infection with Sinorhizobium fredii strain USDA205 for 28 days.We also used these chimerical soybean plants to test how transgenic haory roots overexpressing or knockdown of these two genes affect their tolerances against drought and salt stresses.To gain further undertansidng of the functions of these two genes,we constructed the promoters of GmCYP707A3 b and GmCYP707A1 a driven expression of GUS reporter and transformed them in soybean hairy roots.Both GmCYP707A3 b and GmCYP707A1 a are expressed in root pericycle and in nodules of epidermal layer.GmCYP707A3 b and GmCYP707A1 a GFP fusions were localized to the cytoplasm and membrane,respectively.Our results also showed that the ABA and IAA contents increased,but JA content decreased in GmCYP707A3 b knockdown hairy roots,which also synthesized more isoflavones,had higher expression levels of GmCHS8 and GmCHI1 A,and gave more nodules and higher expression of noduation genes than the GUS control did.However,knockdown of GmCYP707A1 a in soybean hairy roots also increased the ABA and JA contents,but reduced IAA levels,decreased the nodule number and nitrogen fixation rate.The opposite phenotypes in knockdown of these two homolog genes may be due to the different functions and mechanisms between GmCYP707A3 b and GmCYP707A1 a,which worthy of further investigation.Even though,silencing GmCYP707A3 b and GmCYP707A1 a in chimerical soybean plants enhanced their tolerance to drought and salt stresses.Our studies,using transgenic technology to silence GmCYP707A3 b could enhance soybean resistance against drought and salinity stresses,improve nitrogen fixation rate of nodules meanwhile.These results may provide the molecular tools for breeding a soybean variety with improved adapation capability against drought and salt stresses.