Establishment And Application Of A Novel Experimental System For Studying The Photoperiodic Response Of Soybean Using Cotyledon-Only-Plant As Material

Soybean(Glycine max)is a short-day plant(SDP)that sensitives to photoperiodic response.It is commonly used as a model plant for SD photoperiodic response studies.The intact plants are main materials for photoperiodic response studies in soybean,which occupy a large space and have a long growth cycle.These features limit the expansion of the sample size and the improvement of research efficiency.In the current study,we obtained a kind of ‘cotyledon-only plant(COP)’ with only two cotyledons and hypocotyls in the above-ground by removing the newborn stem and leaves.We confirmed their photoperiodic sensitivity by investigating the flowering and maturity phenotype and the expression level of flowering-time related genes.Then,we conducted photo-thermal interaction assays and the grafting experiments using COPs as stocks to improve the survival rate of regenerated shoots which as scions.Also,we established the regulation network of photoperiodic response mechanisms in the early seedling stage using cotyledons as the material.The main results are as follow:1.The plant height of the COPs of photoperiod-sensitive soybean variety Zigongdongdou(ZGDD)is only 1/5 and 1/3 of that of the intact plants under LD and SD conditions,respectively.The maximum diameter is less than 1/2 of the intact plants under LD and SD conditions,which occupying a much smaller volume than the intact plants.Compared with intact plants,cotyledon longevity was significantly longer.Specially,cotyledon longevity of ZGDD COPs were significantly longer than those of intact plants(16.9 ± 1.7 d and 17.4 ± 0.8 d)under both LD and SD conditions.This longer longevity facilitates the continuous observation and sampling for a long time.2.The flowering time of ZGDD COPs was 39.37 ± 2.5 d under SD conditions,while maintaining continuous vegetative growth during the life cycle under LD conditions.Heihe27(HH27)COPs,a photoperiod-insensitive soybean variety,flowered under both LD and SD conditions(28.1±1.7 d and 30.5±5.9 d).The flowering time of ZGDD intact and COPs were62.5±9.3 d and 43.1±6.6 d,respectively,when transferred to LD conditions after 15 d of SD treatment.Neither ZGDD COPs nor intact plants flowered in the night break.Four soybean varieties(HH27,Heihe43,Zhonghuang30 and Zhonghuang39)with different photoperiodic sensitivity were selected for sowing experiments under natural conditions.The results showed that the flowering time of COPs was slightly later than that of intact plants,but the trend of flowering time in different photoperiods was the same.This proved that COPs had a similar photoperiodic response as intact plants.3.Gene expression analysis showed that the expression of E1,Gm FT1 a,Gm FT2 a and Gm FT5 a,key genes in the photoperiodic flowering pathway,was tightly regulated by photoperiod in the cotyledons of COPs.This expression is also resemble to that of intact plants.4.In this study,we also created a ‘single cotyledon plant’ with only one cotyledon and root.Then,we analyzed the expression level of Gm FT2 a in the cotyledon and found that Gm FT2 a was also regulated by photoperiod.Specially,the expression level was significantly higher under SD conditions than LD conditions,indicating the photoperiodic response of cotyledons.5.We examined the photo-thermal response using COPs generated in this study.These plants were grown under different photoperiods with different temperature combinations,including of SD+26 °C,SD+22 °C,LD+26 °C and LD+22 °C.The soybean varieties Zhonghuang39 and Jidou17 were selected and assayed.We found that the flowering time was significantly delayed under low temperature and LD conditions,flowed by SD+26 °C,SD+22 °C,LD+26 °C and LD+22 °C.QPCR analysis of flowering-time related genes showed that Gm FT2 a,Gm FT1 a,E1,Gm ELF3 a and E2 were regulated both by photoperiod and temperature,while Gm PIF4a/b/c/d/e were mainly affected by temperature.The flowering time phenotypes and expression patterns of flowering-time related genes were also observed in the intact plants,demonstrating the reliability of COPs as an experimental system for photoperiodic response studies.6.We conducted grafting experiments which COPs as stocks and transgenic regenerated shoots as scions.The survival rate of the regenerated plants was increased from 43.8±3 % to78.8±1.7 %,which significantly improving the efficiency of the genetic transformation of soybean.7.We performed RNA-seq using cotyledons as samples under different photoperiodic conditions.663 differentially expressed genes were identified,including photoperiodic pathway genes such as Gm FT2 a,Gm FT1 a and E1,whose functions have been verified,as well as some potential genes involved in the photoperiodic regulation of soybean.Enrichment analysis revealed that these genes are mainly involved in photoperiodism,phytohormone pathways and circadian rhythms.Expression of the main photoperiod-regulated genes was analyzed.E3,E4,Gm ELF3a(J),Gm LCL1/2/3/4,Gm PRR3 a,Gm TOC1,Gm COL1a/b and Gm COL2a/b were expressed in the germinating cotyledons that had not yet been exposed to light,whereas E1,E1 La,E1Lb,Gm FT1a/b and Gm FT2 a require exposure to light to be expressed.8.Weighted gene co-expression network analysis(WGCNA)identified two modules associated with photoperiod and screened key transcription factors b ZIP,NAC,ERF,WRKY,BHLH and others for photoperiod regulation.By combining transcription factor enrichment analysis,a network of soybean photoperiod regulation was constructed.