The Molecular Mechanism Of The UV-B Dependent E3 Ligase GmILPA1 Regulating Plant Height Of Soybean

Soybean is an important food and economic crop,and an important source of human food,animal feed,and industrial raw materials.At present,the demand for soybeans in China is rapidly increasing,and the supply-demand contradiction is becoming increasingly prominent.Improving soybean production has become an urgent need to ensure national food security.Dense planting can improve soybean yield and effectively utilize planting area,with the core of reducing soybean plant height.Soybean plant height is closely related to its yield.Semi dwarf varieties can increase planting density and lodging resistance to increase soybean yield.Therefore,it is of great significance to isolate important genes regulating soybean plant height and analyze its regulation mechanism for improving soybean plant height and cultivating new varieties with high density tolerance.The plant height of crops is regulated by many factors,including plant hormones and environmental factors.Plant hormone gibberellin(GA)can promote stem elongation and increase plant height.Ultraviolet b(UV-B)can inhibit plant growth and cause plant dwarfing.Therefore,plant height is regulated by both GA and UV-B.At present,studies on the regulation of UV-B on GA synthesis and catabolism genes at the transcriptional level have been reported.However,studies on UV-B-mediated posttranslational modification of GA metabolism-related genes to regulate plant height have not been reported.In this paper,a recessive single gene-regulated dwarf mutant Gmilpal with large leaf angle was screened by using the EMS-induced mutant of soybean constructed in our laboratory.It only showed dwarf phenotype in UV-B.The target gene-APC/C E3 ubiquitin ligase complex subunit APC8 protein gene GmILPA1 was cloned,and its molecular mechanism of mediating gibberellin metabolism to regulate soybean plant height was analyzed.The main results are as follows:1).GmILPA1 positively regulates soybean plant height.In this study,a dwarf mutant Gmuid1 with shortened internode length was screened based on the highgeneration mutant produced by EMS mutagenesis of Hedou 12(H12).The mutant site of Gmuidl was located at 1,873,495 on chromosome 11 by map-based cloning and BSA-seq using the F2 hybrid population,which was located in the gene Gmlya.11 G026400.The base of this site was mutated from G to A,resulting in frameshift and premature termination of the amino acid sequence encoded by the target gene.GmILPA1(Glycine max Increased Leaf Petiole Angle1),which encodes E3 ubiquitin ligase APC/C subunit APC8,has been reported and named GmILPA1(Glycine max Increased Leaf Petiole Angle1).It was further confirmed GmILPA1 is the target gene by allelic hybridization and restorer line,and its mutation can cause Gmuid1 dwarf phenotype,indicates that GmILPA1 positively regulates soybean plant height.2).GmILPA1 promotes soybean plant height in a UV-B dependent manner.Compared with the wild-type H12,Gmilpa1-2(Gmuidl)exhibits an extreme dwarfing phenotype in the field.When H12 and Gmilpa1-2 are planted in a greenhouse,there is no significant difference in plant height between the H12 and Gmilpa1-2.It is speculated that the greenhouse blocks UV B,which may be the reason for the disappearance of Gmilpa1-2 dwarfing phenotype.Therefore,the H12 and Gmilpa1 mutants were planted in the laboratory under white light and UV B conditions,and it was found that under UV B conditions,Gmilpa1 can reproduce the dwarfing phenotype,but there is no difference in plant height between it and H12 under white light.Therefore,the results indicate that UV-B is an essential factor for dwarfism of Gmilpa1.3).GA can restore the dwarf phenotype of Gmilpa1 mutant under UV-B.Plant hormone GA can promote stem elongation and increase plant height.The analysis of endogenous GA of H12 and Gmilpa1-2 at V2 stage under white light and UV-B conditions showed that there was no significant difference in endogenous GA content under white light.While under UV-B conditions,the contents of active GA1 and GA4 and their precursors GA20 and GA9 in Gmilpa1-2 were significantly lower than those in H12,while the contents of inactive GA8 and GA34 were higher than those in H12.Exogenous application of 100 μM GA3 could restore the plant height of Gmilpa1-2 to H12 under UV-B,indicating that Gmilpa1 mutant was a GA-sensitive dwarf mutant.4).UV-B can promote the interaction between GmILPA1 and GmGA2ox-like.GmILPA1-interacting proteins GmGA2ox-like and GmUBL1 were obtained by yeast library screening.Next,we generated 3 lines overexpressing GmGA2ox-like,which displayed a dwarf phenotype compared to non-transgenic WT plants,we performed the in vitro enzymatic activity assay using GA1 and GA4 as the substrates.The results showed that GmGA2ox-like converted GA1 and GA4 to their corresponding 2βhydroxylated products GA8 and GA34,respectively.Yeast two-hybrid,BiFC and Co-IP experiments proved that GmILPA 1,GmGA2ox-like and GmUBL1 interacted with each other.Since the Gmilpa1 mutant showed a dwarf phenotype only under UV-B conditions,we used BiFC and Co-IP experiments to analyze whether the interaction intensity was affected by UV-B.The results showed that UV-B enhanced the interaction between GmILPA1 and GmGA2ox-like,but had no effect on the interaction between GmILPA1 and GmUBL1,GmUBL1 and GmGA2ox-like.5).GmILPA1 and GmUBL1 can mediate GmGA2ox-like ubiquitination.The ubiquitination modification of GmGA2ox-like was confirmed by N.benthamiana transient expression,in vitro ubiquitination experiment and soybean transgenic ubiquitin enrichment experiment,and the lysine 394 was determined to be the key site of ubiquitination modification.Further experiments showed that both GmILPA1 and GmUBL1 could mediate the ubiquitination modification of GmGA2ox-like.6).GmUBL1 can enhance the degradation of GmGA2ox-like by GmILPA1.In vivo degradation and Cell-free experiments of H12 and Gmilpa1-2 showed that under white light conditions,the content of GmGA2ox-like protein in H12 and Gmilpa1-2 remained unchanged and there was no difference.Under UV-B conditions,the content of GmGA2ox-like protein in H12 showed a downward trend,and the protease inhibitor MG 132 inhibited the degradation of GmGA2ox-like,while the content of GmGA2oxlike protein in Gmilpa1-2 remained unchanged.The results showed that GmILPA1 can target degradation of GmGA2ox-like by 26S proteasome,and the degradation process depends on UV-B.At the same time,the transient expression system of N.benthamiana confirmed that GmUBL1 could not mediate the degradation of GmGA2ox-like,but could enhance the degradation of GmGA2ox-like by GmILPA1.7).GmILPA1 underwent artificial selection during domestication.Fst,π and Tajima ’s D indicated that GmILPA1 was located in a strong domestication selection interval.GmILPA1 haplotype analysis of 444 soybean materials from wild,landrace and cultivated species showed that GmILPA1 could be divided into 6 haplotypes.Hap3 and Hap4 were unique haplotypes of wild species,indicating that the haplotype Hapl/2/5/6 had undergone artificial selection.Hap5 was the only haplotype with Indel in the promoter region.The plant height of Hap5 was shorter than that of Hap1/2/6,but the 100-grain weight and grain weight per plant were higher than that of Hap Hap1/2/6.By investigating the geographical distribution of different haplotypes,it was found that Hap1/6 was mainly distributed in the southern region with low UV-B intensity,and Hap5 was mainly distributed in the northeastern region with high UV-B intensity.The promoter sequence analysis of different haplotypes of GmILPA1 showed that the Hap1/2/6 promoter region contained a normal light-responsive cis-regulatory element,and transcriptional activity experiments showed that it could respond to UV-B-induced expression of GmILPA1,while the Hap5 promoter region lacked 13 bp,which did not respond to UV-B-induced expression of GmILPA1.It is speculated that the missing Indel of the response element is the main factor that Hap5 is insensitive to UV-B.Hap5 is suitable for the growth of Northeast China with high UV-B intensity.In summary,this study cloned the functional gene GmILPA1 regulating soybean plant height,revealed a new mechanism of UV-B-dependent E3 ligase GmILPA1 mediating gibberellin metabolism to regulate soybean plant height,and provided new clues for further understanding the adaptive growth mechanism of soybean to UV-B radiation.