Cloning And Functional Analysis Of The Phytochrome Chromophore Synthesis Gene GmHY2 In Soybean

Plant height and flowering time are important agronomic traits of soybean[Glycine max（Linn）Merr.],which directly affect its ecological adaptability and yield.These traits are not only controlled by genetic factors but also influenced by external environmental signals.As the primary environmental signal,light can regulate plant morphology and development.Plants perceive changes in light quality and intensity through photoreceptors,and regulate downstream gene expression accordingly.Phytochromes are the most widely studied type of photoreceptor.In this study,a mutant named Glycine max long internode 1（Gmlin1）was screened using a forward genetic approach,which showed earlier flowering time and elongated internodes.The candidate gene related to phytochromes biosynthesis,GmHY2a（named according to the homologous gene HY2 in Arabidopsis）,was identified,and its molecular function was illustrated.The results are as follows:1.The Gmlin1 mutant exhibits elongated internodes and early floweringIn this study,we screened two mutants,Gmlin1-1 and Gmlin1-2,from the EMS-mutagenized Williams 82.Both mutants exhibited earlier flowering and increased plant height,which was caused by significantly elongated internodes.The internodes of the main stem showed elongated cells.Gmlin1-1 and Gmlin1-2 both flowered about 10days earlier,and their plant height was about 10 cm higher than that of the wild-type Williams 82.Genetic analysis of the mutants showed that the segregation ratio of wild type and mutants in the M2 generation of Gmlin1-1 was 3:1.The segregation ratio of wild type and mutants in the BC₁F₂ population,obtained by backcrossing Gmlin1-2with Williams 82,was also 3:1.These results suggested that both mutants were controlled by a single recessive gene.2.The phenotype of the Gmlin1 mutant is caused by the mutation of the GmHY2a geneTo identify candidate genes,bulked segregate analysis（BSA）was performed using the M₂ segregation population of Gmlin1-1 and the BC₁F₂ population of Gmlin1-2.In both BSA-seq results,mutations were located in the Glyma.02g304700 gene.In Gmlin1-1,the A in the 1550th base of the gene was substituted with G,resulting in alternate splicing of the transcript.In Gmlin1-2,the G in the 5862nd base was substituted with A,causing the replacement of the 255th Glycine with Glutamic acid.Two other alleles,Gmlin1-3 and Gmlin1-4,were identified in the mutant library.In Gmlin1-3,the 6107th base was deleted,causing a frameshift.In Gmlin1-4,the 5861st G to A mutation resulted in the substitution of the 255th Glycine with Arginine.When the mutants were crossed with each other,the F₁ plant phenotype was consistent with that of the parent,suggesting that the four mutants were caused by allelic mutations.Gene complementation in Gmlin1-1 restored the phenotype to wild type,suggesting that mutations in the Glyma.02g304700（GmHY2a）gene was responsible for the increased plant height and early flowering.3.GmHY2a is the major regulatory gene in soybean phytochrome synthesisThe GmHY2 gene encodes PΦB synthase,a key enzyme in phytochrome synthesis,with a conserved Fe＿bilin＿red domain that is widely present in photosynthetic organisms.Subcellular localization studies have shown that it is located in chloroplasts.The Williams 82 genome contains three homologous GmHY2 genes.GmHY2c（Glyma.14g136300）is a truncated gene with only 432 bp of the coding sequence,which is speculated to be a pseudogene.The genomic regions surrounding GmHY2a and GmHY2b（Glyma.14g009100）are highly syntenic,indicating that they evolved from the same ancestor through genome duplication.However,GmHY2b lacks a conserved sequence in the functional domain.Complementary experiments with luciferase showed that the binding ability of GmHY2b to the electron donor ferredoxin protein（GmFd2）is significantly weaker than that of GmHY2a.Therefore,it is speculated that GmHY2a is the major regulatory gene in phytochrome synthesis for soybeans.4.Mutation in GmHY2a was affect the function of phytochromeThe wild-type Williams 82 and Gmlin1 mutant both exhibit a skotomorphogenetic phenotype under dark condition without significant difference.Under continuous white light,the de-etiolation responses of the Gmlin1 mutant were weakened compared to WT,showed long hypocotyl and incompletely unfolded apical hook.Under single light conditions,the Gmlin1 mutant shows a weakened response to red light and far-red light compared to WT,indicating that the mutation of GmHY2a has affected the light response mediated by phytochrome.Soybean protoplast transient transformation system further proof that the mutation of GmHY2a reduces the amount of phytochrome GmPHYA and GmPHYB in nuclear.The above results indicate that the mutation of GmHY2a in the Gmlin1 mutant reduces the Pfr-type phytochrome in the nucleus,thus affecting the plant’s response to red and far-red light.5.Constitutive shade-avoidance response in the Gmlin1 mutant leads to internode elongationWhile the Williams 82 variety displays a shade avoidance response phenotype such as elongated hypocotyl under simulated shade（EOD-FR）treatment,this treatment did not cause significant growth changes in the Gmlin1-1 mutant compared to the control.Under normal light conditions,the expression levels of shade avoidance marker genes（GmIAA29,GmPIL1,and GmHB2）were increased in the Gmlin1-1 mutant compared to wild type.There was no significant difference in the expression of these genes between the EOD-FR treatment and the control in Gmlin1-1.These results suggest that the Gmlin1 mutant exhibits a constitutive shade avoidance response.Furthermore,we demonstrate that the upregulated transcription of auxin-and gibberellin-related genes in the Gmlin1 mutant promotes the elongation of the hypocotyl and internodes.6.Photoperiod sensitivity was reduced and flowering time was accelerated in the Gmlin1 mutantUnder long-day conditions,the flowering time of the Gmlin1 mutant was significantly earlier than that of the wild type.The photoperiod transfer experiment demonstrated that the Gmlin1 mutant had reduced sensitivity to photoperiod compared to the wild type.The GmHY2a mutation down-regulated the soybean E1 gene,which is central in the photoperiod pathway,and thus released the inhibition on downstream genes GmFT2a and GmFT5a by E1,promoting the advancement of flowering time.The mature seeds’morphology,size,seed weight per plant,and seeds per plant were measured,and the results showed no significant changes compared to Williams 82.In summary,this study cloned the soybean phytochrome biosynthesis gene GmHY2a using a forward genetic approach.This gene affects the light response ability of phytochrome and regulates the expression of auxin-and gibberellin-related genes,promoting internode elongation.Moreover,the reduced sensitivity to photoperiod and early flowering time of the Gmlin1 mutant make it a promising candidate for breeding new soybeans in high-latitude regions.