Physiological And Molecular Mechanisms Of Differences In Mycorrhizal Growth Responses And Functional Analysis Of Related GmSWEET6 Genes In Soybean

Soybean is an important food and oil crop.Phosphorus(P)is one of the essential macronutrients for plant growth,and low P bioavailability in soils is a major limiting factor for soybean yield and quality.Symbiosis with arbuscular mycorrhizal fungi(AMF)can help plants to acquire P and other nutrients from the soils,and in return,the plants offer carbon to the fungi.Previous studies have shown that there are significant differences in the growth responses of plants inoculated with different AMF species,but the underlying physiological and molecular mechanisms are still unclear.In this study,firstly,an experiment with different P treatment,soybean genotypes and AMF strains was conducted to study the differences in soybean growth,P acquisition and photosynthesis under different P conditions.Secondly,combined with transcriptomic sequencing,the growth differences,carbon allocation and gene expression profiles were analyzed in soybean plants inoculated with different AMF strains or different clones of the same strain.Furthermore,the functional analysis of the identified key gene GmSWEET6 was performed in order to explore the possible physiological and molecular mechanisms of mycorrhizal growth differences of soybean plants inoculated with different AMF strains.The main results are as follows:1.In the experiment with different P treatment,soybean genotypes and AMF strains,high P significantly increased biomass and shoot P content in soybean.There were significant differences in symbiotic effects between different AMF strains and soybeans genotypes.Under low P(LP)conditions,the biomass and root P content were significantly increased in both YC03-3 and HN112 plants inoculated with Rhizophagus intraradices(Ri)and Glomus custos(Gc),but was unchanged in soybean plants inoculated with Glomus aggregatum(Ga)compared with non-inoculated treatment(NM).Moreover,high P increased the photosynthesis rate under NM conditions in soybean.Under LP conditions,the photosynthesis rate were significantly higher in the soybean plants inoculated with Ri and Gc compare with NM and Ga plants.However,there were no significant genotypic differences in P contents and photosynthetic rates between two soybean genotypes.It indicated that the difference in mycorrhizal growth response depends mainly on AMF species,but not on genotypes in soybean.2.In the experiments with different AMF strains and different clones of the same strain,Inoculation with highly cooperative Rhizophagus intraradices(Ri,with about 45% AM colonization and 10 spores per gram soil)and low-cooperative Glomus aggregatum with high colonization(Ga H,with about 70% AM colonization and 15 spores per gram soil)significantly increased the shoot biomass and P content of HN112 and HN89,but inoculation with low-cooperation Glomus aggregatum with low colonization(Ga L,with about 25% AM colonization and 6 spores per gram soil)had no significant influence on the shoot and root biomasses of the two soybean genotypes.AM colonization of the soybean plants inoculated with Ri and Ga L was obviously higher than the Ga L plants.Moreover,different inoculation treatments had different effects on the expression levels of phosphate transporter genes(PTs)in soybean.Compared with NM treatment,inoculation of Ri and Ga H significantly reduced the expression of LP-inducible and root-specific Gm PT4,but the expression of which in Ga L roots was not significantly changed.The three inoculation treatments significantly increased the expression of mycorrhiza-inducible Gm PT8 and Gm PT9,and the expression of mycorrhiza-inducible Gm PT10 was significantly up regulated by the inoculation of Ga H and Ga L.The growth of Ga L plants was not improved although the expression of mycorrhiza-inducible Gm PTs responsible for mycorrhizal P uptake pathway can be up-regulated under the inoculation of different AMF strains and different clones of the same strain,and the direct P uptake pathway and the mycorrhiza P uptake pathway were both open in Ga L inoculation plants.Therefore,the difference in expression patterns of Gm PTs under the three different inoculation treatments does not explain the difference in mycorrhizal growth response of soybean.3.RNA-seq sequencing of HN89 inoculated with different AMF strains and different clones of the same strain were performed to compare the differences in gene expression profiles among different inoculation treatments.GO enrichment analysis showed that the transcripts up-regulated in all inoculated roots were mainly related to proteolysis,defense response,response to biotic stimulus,membrane,ubiquitin ligase complex,peptidase activity,electron carrier activity and carboxypeptidase activity.In contrast,the transcripts upregulated only in Ri and Ga H roots were largely associated with response to oxidative stress,oxidation-reduction process,transmembrane transport,integral component of membrane,intrinsic component of membrane,membrane,membrane part,antioxidant activity,peroxidase activity and oxidoreductase activity.KEGG enrichment analysis showed that the up-regulated genes that were significantly enriched in Ri,Ga,and Ga L roots were mainly genes involved in fatty acid metabolism pathway,such as genes encoding FATTY ACYL-ACP THIOESTERASE M(Fat M),KETOACYL-ACP SYNTHASE II(KASII)and MALONYL Co A-ACP TRANSACYLASE(MCAT),conversely,the up-regulated genes that were only significantly enriched in Ri and Ga H roots were mainly genes involved in pentose phosphate pathway,carbon fixation in photosynthetic organisms,fructose and mannose metabolism,glycolysis/gluconeogenesis and glycan synthesis and metabolism,including invertase,GmSWEET6 and GmSWEET15.Moreover,the concentrations of glucose,fructose and sucrose in both shoots and roots of Ri and Ga H plants were significantly higher than those of Ga L plants.It indicated that the difference in mycorrhizal growth response could be attributed to the difference in metabolism and transport of carbohydrates at different inoculation treatments in soybean.4.Functional analysis of the identified key gene GmSWEET6 was further carried out.The GUS protein driven by the GmSWEET6 promoter was mainly expressed in the cortical cells containing arbuscules and the vascular bundle,suggesting that GmSWEET6 might be responsible for sugar unloading at soybeans roots and then transportation to symbiotic interfaces.The subcellular localization of tobacco epidermic cells indicated that GmSWEET6 was mainly localized on the plasma membrane,and the soybean transgenic hairy root obtained by hypocotyl transformation and inoculated with AMF further proved that GmSWEET6 protein might be localized on the pre-symbiotic membrane.The yeast functional complementary test confirmed that GmSWEET6 functioned in sucrose transport.These results suggested that GmSWEET6 might mediate sucrose transport from plant roots to the pre-symbiotic space of mycorrhiza.5.Compared with wild-type and control plants,overexpression of GmSWEET6 significantly reduced the biomass,P content,glucose and fructose concentrations of both shoots and roots,and the root sucrose concentration at NM treatments,but these physiological indicators were restored to normal levels after inoculation with AMFs except the glucose and fructose concentrations in the roots of OX1.Moreover,at NM treatments,the fructose concentration in the root exudates of the plants overexpressing GmSWEET6 was significantly higher than that of the control and RNAi lines,indicating that overexpression of GmSWEET6 might lead to sugar efflux from plant roots,and then inhibit plant growth.AMF inoculation might promote the partition of sugar to AMF,and thereby significantly improve soybean growth.6.The plant height of soybean plants overexpressing GmSWEET6 significantly decreased.Meanwhile,some of the GA2 oxidase genes involved in the decomposition of gibberellin(GA)were up-regulated in leaves and roots,while some of the GA20 oxidase genes involved in GA synthesis were down-regulated in leaves or roots.It indicates that overexpression of GmSWEET6 might also affect the synthesis,decomposition and transport of GA,and thereby result in a decrease in plant height.In summary,the difference in mycorrhizal growth response of inoculated soybean plants depended mainly on AMF species rather than soybean genotype,and was mainly due to the differential expression of the related gene involved in sugar metabolism and transport when inoculated with different AMF inoculums.After inoculated by AMF inoculum with high colonization capabilities,the expression of GmSWEET6 in plant roots was up-regulated,which promoted sugar transport to AMF,and then significantly improved P nutrition and stimulated photosynthesis in plants,and thereby led to increase in plant biomass and P content.This study identified a key sugar transporter GmSWEET6 on mycorrhizal pre-symbiotic membrane,which was responsible for carbohydrate transport from soybean roots to pre-symbiotic space,and its function was analyzed.Furthermore,this study revealed the physiological and molecular mechanisms of mycorrhizal growth differences of soybean plants inoculated with different AMF inoculums.Therefore,these results will provide a theoretical basis for regulating the resource allocation between plants and AMF,and maximizing the positive effect of AMF symbiosis on plant growth.