Functional Characterization Of More Axillary Branches (Max) Genes Involved In Strigolactone Biosynthesis And Signaling From Soybean (Glycine Max L.)

Strigolactones(SLs)are well-known plant secondary metabolites controlling different biological and physiological aspects of plants,including root growth,root hairs,shoot branching,and plant symbiotic interaction.Studies of branching mutants in Arabidopsis,pea,rice and petunia have revealed several key genes involved in strigolactone biosynthesis and signaling pathway.Despite the biological significance,the components of SLs biosynthesis and signaling have not been unequivocally explored in soybean.Considering the importance of the Strigolactones and their biosynthetic origin(from carotenoids),in the present study,we identified the putative components of SLs biosynthetic enzymes and signaling proteins in soybean genome.Soybean genome contains conserved MORE AXILLARY BRANCHING(MAX)homolog's,GmMAX1s,Gm MAX2 s,GmMAX3s,and GmMAX4s.These genes were selected on the basis of their expression level in different tissues of Glycine max.The tissue expression patterns are coincident with SLs synthesis in roots and signaling in other tissues under normal conditions.The functions of GmMAX1a,GmMAX2a,GmMAX3b and GmMAX4a were studied in mutant,complemented and over expression Arabidopsis lines.Phenotypic analysis revealed restoration of the particular phenotypes,such as shoot branching and shoot height,leaf shape,primary root length,and root hair growth,trichome growth and trichome length.Not only the phenotypes were restored,hormone levels were also significantly changed,such as reduced JA and ABA contents in all mutant leaves,but increased auxin levels in atmax1,atmax3 and atmax4 mutants.Over-expression of these Gm MAXs also altered the hormone contents in wild-type Arabidopsis.Abiotic stresses adversely affect the yield of many crops.Therefore,we also tested the possible function of SLs biosynthesis and signaling genes in Arabidopsis plants and soybean transgenic hairy roots.SLs biosynthesis and signaling genes in mutants' atmax1,atmax2,atmax3,atmax4 plants has a less potential of survival compared to wild type plants,more surprisingly,GmMAXs genes of SLs biosynthesis and signaling could significantly enhance the drought tolerance in transgenic hairy roots compared to GUS control under drought stress condition.The result exhibited hypersensitivity to a biotic drought stress compared to control.Suggesting that SLs biosynthesis and signaling components has a significant role in drought toleranceThe over expressed GmMAXs transgenic soybean hairy roots were developed and phenotype was analyzed under the microscope.Chimerical soybean hairy roots significantly increased roots hairs length compared to control.The transcriptomic analysis of GmMAX1a,GmMAX2a,GmMAX3b and GmMAX4a also showed altered expression of genes related to hormonal pathways.Hormones analysis indicated that GmMAX1a,GmMAX3b and GmMAX4a over expression hairy roots lines have reduced ABA level,but increased JA levels,and increased the auxin content in GmMAX1a and GmMAX4a.In GmMAX3a hairy roots,a non-significant change in auxin content was observed.The GmMAX2a signaling perception protein increased the ABA and JA but slightly decreased IAA contents than GUS control,where as the contrasting changes were observed in GmMAX3b-KD lines.The role of GmMAXs was further characterized in soybean nodulation with over expression and knockdown transgenic hairy roots.GmMAX1a,GmMAX2a,GmMAX3b and GmMAX4a over expression(GmMAX1a-OE,GmMAX2a-OE GmMAX3b-OE and GmMAX4a-OE)lines exhibited increased nodule number while as GmMAX3 b knockdown(GmMAX3b-KD)decreased the nodule number in transgenic hairy roots.The expression levels of several key nodulation genes were also altered in Gm MAXs transgenic hairy roots.The SLs Gm MAXs positively regulate soybean nodulation.Moreover,early nodulation genes such as GmNSP1a,GmNSP1? were highly expressed in GmMAXs chimerical soybean plants hairy roots after 48 hrs of infection with Bradyrhizobium japonicum strain USDA110 relative to GUS control.Further,subcellular localization study revealed that the SLs biosynthesis proteins GmMAX1a-GFP,GmMAX3b-GFP,GmMAX4a-GFP were localized in the chloroplast,while the GmMAX2a SL signaling factor localized in the nucleus.This study not only revealed the conservation of SLs biosynthesis and signaling in soybean,but also showed possible interactions between SLs and other hormone synthesis and signaling in coordinating plant development and soybean nodulation.GmMAX1a,GmMAX2a,GmMAX3b and GmMAX4a-mediated SLs biosynthesis and signaling may be involved in soybean nodulation by affecting both root hair formation and the interaction with rhizobia.