Analysis About The Temperature And Photoperiod Regulation Of Flowering Induction Mechanism In Lilium × Formolongi

Lilium×formolongi has a precocious flowering ability that can flower in 1 year from seeds without vernalization.It is an excellent material to study the flowering regulation mechanism and obtain early-flowering cultivars of lily.L.×formolongi was determined to be a facultative long-day(LD)plant.Under LD conditions,the seedlings can bolt 6 months after sowing with 9-10 rosette leaves and the flowering induction is mainly controlled by CO/FT mode.Previous researches have shown that temperature also plays a decisive role in the flowering time regulation of L.×formolongi.Therefore,in this study,the seedlings and bulbs of L.×formolongi were grown in the conditions with different temperature and photoperiod.The growth and flowering characteristics and expression patterns of flowering related genes were measured and analyzed.The transcriptome and metabonomics analysises were performed to explore the molecular and metabolic regulation mechanisms towards the effect of temperature on the photoperiodic flowering regulation of L.×formolongi.Then,the photoperiodic key genes LfCOLs family were cloned and their functions were examined via yeast two-hybrid and overexpression in Arabidopsis.The main results were as follows:(1)The temperature could reverse the flowering and growth characteristics regulated by photoperiod of L.×formolongi seedlings.High temperature(HTLD),especially of the root zone,inhibited bolting and flowering of the seedlings in LDs with more rosette leaves.Only 14.6% of the seedlings began to bolt one year after sowing.While during the low temperature and SD conditions of greenhouse in Beijing(NE),the seedlings could bolt with only 6-7 rosette leaves.The seedlings in HTLD at rosette leaves stage were transferred to NLD and NE.After transfer,the seedlings restored normal bolting and in NE the seedlings bolted more faster and intensively than in NLD.(2)The outer scales of seedlings with 1-2 internodes under NLD(light:dark 25℃:18℃),15-20 rosette leaves under HTLD for 10 months(light:dark root zone 32℃:20℃)and 1-2 internodes after transfer from HTLD to NE(light:dark 25℃:4℃)were collected for transcriptome sequencing.82 genes homologous to these involved in the flowering induction were found.The circadian clock and photoperiodic pathways and flowering integrators Lf FT1 and Lf SOC1 were inhibited in high temperature conditions and activated after cold exposure.Besides,in high temperature LDs,LfCCA1,LfCRY,LfCOP1,Lf FKF1,LfCOLs and Lf PIFs were disturbed at transcriptional level.After cold exposure,the expression of Lf FKF1,Lf GI,LfCOLs and Lf FT1 started earlier,though the expression levels were lower than in normal LDs,which indicated that low temperature increased the sensitivity to photoperiodic signals.(3)The middles leaves and outer scales in the three conditions above were collected for untargeted metabolome and plant hormone-targeted metabolome analyses.In HTLD,ABA,D-allose,pentose and glucuronate interconversions and glutathione metabolism in the leaves and ACC in the bulbs were significantly activated and acted as flowering inhibitors.While after cold exposure,TCA cycle was suppressed and SA,JA and some sugars(D-mannose,D-fructose,D-arabinose and maltotriose)levels were upregulated in the leaves,simultaneously with the vigorous fatty acid metabolism and increased linolenic acid and stearic acid in the bulbs.These metabolic regulations might provide energy to bolting in NE.(4)The large bulbs(perimeter 10-14 cm)and small bulbs(perimeter 5-8 cm)of L.×formolongi were submitted to cold exposure(4℃)for 0,3,5,7 or 9 weeks.Each group were planted respectively in LDs and SDs.The photoperiodic pathway was still the main pathway to regulate flowering in the bulbs.For small bulbs,LD conditions were necessary for flowering.For large bulbs,vernalization could partially replaced LDs.The expression levels of LfCOL13 were inhibited in the large bulbs after planting.The expression levels of LfCOL15 were inhibited in both large and small bulbs.(5)The plant expression vectors of LfCOL13,LfCOL14,LfCOL15 and LfCOL16 were constructed.The transgenic lines of these four genes all showed late flowering phenotype.The flowering time of transgenic plants of LfCOL15 were the latest.The transgenic plants grew slowly and obviously delayed flowering in both LDs and SDs.The expression level of At FT was suppressed.The next was LfCOL13 transgenic plants and the expression levels of At FT in both LDs and SDs were inhibited.The flowering time of transgenic plants with LfCOL16 was later than wild type in LDs and SDs.And the flowering time of transgenic plants with LfCOL14 was later than wild type only in SDs.(6)The functional analysis of LfCOLs family were detected via yeast two-hybrid.Among the flowering promotors,LfCOL5 physically interacted with most LfCOLs and Lf FKF1,Lf GI and Lf SPA1 proteins.It was the main flowering promotor in the photoperiodic pathway of L.×formolongi.Among the flowering inhibitors,LfCOL15 physically interacted with Lf FKF1,Lf GI and other LfCOLs proteins.The expression levels of At FKF1 and At GI were also reduced in the LfCOL15 transgenic plants.Thus LfCOL15 was the main flowering inhibitor in the photoperiodic pathway of L.×formolongi.Besides,the protein interaction characteristics of LfCOL13 and LfCOL16 were similar to LfCOL15,so the late-flowering genes might have functional redundancy.