Functional Conservation And Diversification Of The Soybean E1 Gene And Its Homologs In Legumes

The maturity gene E1 plays a major role in regulation of flowering time in soybean. In this study, we analyzed the gene and protein constructures of the E1 gene and its homologs, revealed as well as the evolutionary relationship among them. We also studied the function of E1, PvE1 L and MtE1 L gene in flowering regulation using genetic transformation method to reveal the functional conservation and diversification of these genes. The major results are as following:1. Predicted the genes and proteins constructure of the E1 gene and its homologs and analyzed evolutionary relationships among these genesTotal 8 sequences highly homologous to the E1 gene were retrieved in legumes(we refer them including E1 as “E1 gene family”). All eight sequences are intron-free genes that code proteins containing a conserved putative bipartite nuclear location signal(NLS) and a domain distantly related to the plant-specific B3 domain(B3-like domain). Comparing with B3-like domains in other proteins, the MtE1 L and LjE1 L contains an extra helix, and the CcE1L2 contains two extra helices, which might be related to the genes function. Besides, the result of phylogenetic analysis shows that the nine genes mainly devided into two clades, and one clade includes Lj E1 L, MtE1 L and Ca E1 L,the other clade includes E1, E1 La, E1 Lb, PvE1 L, CcE1L1 �'� CcE1L2, which is associate with Hologalegina and Millettioid/Phaseoloid in taxology. Colinearity analysis and the divergent time estimation indicated that the PvE1 L gene in common bean shared the closest relationship with E1 gene except for E1 La and E1 Lb. The E1, E1 La, and E1 Lb genes are derived from two duplication events.2. Functional performance of The E1, PvE1 L and Mt E1 L in transgenic soybean or Medicago mutants.Upon successful cloning of E1, PvE1 L and Mt E1 L genes and construction of the plant expression vectors, we transformed these gene constructs into the soybean cultivar Dongnong 50(e1-as) using Agrobacterium-mediated cotyledon-node explant method. In transgenic E1 and PvE1 L soybean lines, target genes(E1 or PvE1L) were high expressed, both exhibited similar but later flowering time phenotype in long-day(LD) and short-day(SD) conditions. Also transgenic lines appeared taller with more nodes. However, the length of inter-node among transgenic E1 or PvE1 L soybean lines remained unchanged. In contrast, there was no noticeable changes in the flowering time between Mt E1 L transgenic soybean lines and wild-type plants in both LDs and SDs. Intriguingly, the MtE1 L transgenic lines exhibited a dwarf phenotype with less and shorter inter-nodes than wild-type plants in LDs and SDs. In medicago, the Mt E1 L was strongly expressed in the expanded trifoliate leaves in LDs. Meanwhile, the MtE1 L expression was expreesed biomdally in LDs, peaked at 4h after dawn(ZT4) and at dusk(ZT16). These results demonstrate that the Mt E1 L gene may have circadian rhythm. The medicago mte1 l mutants were late flowering, in which the MtE1 L gene was structurally disrupted by Tnt1 insertion. In conclusion, the function of E1 and PvE1 L in the control of flowering might be conserved, but that of MtE1 L might be diverged.3. The function of E1 gene in the control of flowering may depend on soybean genetic context.The sequences highly homologous to the E1 gene were retrieved only in legumes. We transformed the E1 gene into the Arabidopsis Columbia ecotype(Col-0) and rice cultivar Longjing 11 using floral dip method and Agrobacterium-mediated method, respectively. The results of genotype and expression analysis indicated that there was no significant differences in the flowering time between transgenic Arabidopsis lines(including E1 overexpression lines and vector-only transgenic lines) and wild-type plants, in addition, the expression level of CO and FT, key intergraters in control of flowering in Arabidopsis, was similar in LDs and SDs. The heading time among all lines were similar, and the expression of Hd1 and Hd3 a, which are two important regulator in the control of flowering in rice, is similar in SDs. Based on these results, although the maturity gene E1 has a key role in natural variation for flowering in soybean, ectopic expression of E1 gene in Arabidopsis(a long-day plant) and rice(a short-day monocotyledon) did not affect their phenotype in flowering time, revealing that the function of E1 gene in the control of flowering may depend on soybean genetic context.In conclusion, we analyzed the evolutionary relationship in E1 gene family. The function of E1 gene in the control of flowering may depend on soybean or soybean close-related genetic context. The PvE1 L gene is an ortholog of E1 gene, and the function of PvE1 L gene in the control of flowering regulation was similar to the E1 gene, as a flowering repressor. In the contrast, the MtE1 L gene may act as a promoter in the control of flowering in Medicago. The function of E1 gene family in the regulation of photoperiodic flowering might be assoiated with lineage specification.