Study On The Organogenesis Rule And Molecular Mechanism Of Underground-stem Bulblets In LA Hybrid Lily

Lily(Lilium spp.)is an important bulbous flower with various flower types,rich colors and excellent ornamental effect.In China,lilies are mainly cultivated as cut flowers at present,rarely used in public gardens.Bulbs of lilies with high ornamental value are usually degraded seriously,leading to the compelling replacement of bulbs every year and planting cost increase.Therefore,it is of great significance to cultivate new cultivars with strong self-propagation capacity to expand their application in the garden.Bulb,as an important commercial and asexual-reproductive organ of lily,attracts much attention and its formation mechanism has been a hot topic.The majority of researches forcus on the formation mechanism in vitro through tissue-culture and scale-cutting technology,whereas few of them concentrate on the natural proliferation of lily bulbs,espcially their specific molecular formation mechanism.LA hybrid lily‘Aladdin'has been identified to have very strong self-propagation ability under natural conditions,which can spontaneously form a large number of bulblets with good quality on underground stem.As a result,it is an optimal experimental material to study the formation mechanism of bulb in the nature.Therefore,in this study,the organogenesis rule and the molecular regulation mechanism of the unique underground-stem bulblets in L.‘Aladdin'was explored,especially the hormone regulation at the transcriptional level,laying the solid foundation for the future directional breeding of new lily cultivars with strong reproduction capacity.The main results obtained in this study are as follows:(1)The formation capacity of underground-stem bulblets in L.‘Aladdin'under natural conditions was preliminarily evaluated.Results showed that the average number,average weight and average diameter of bulblets in L.‘Aladdin'are significantly better than any other cultivar(p<0.05).As a result,L.‘Aladdin'is an ideal experimental material for investigating the regulation mechanism underlying the natural formation of new bulbs.Moreover,the organogenesis rule of underground-stem bulblets in L.‘Aladdin'was observed and showed that such bulblets can be first seen on the leaf axils of underground stem at stage IV(about 60 days after cultivation(DAC)).(2)Changes of endogenous hormones(ZR,IAA,ABA and GA₃)levels in axils of underground stem during bulblet formation were determined by enzyme-linked immunosorbent assay(ELISA).According to the changing trend of phytohormones,the whole forming period can be divided into two parts:one is from stage I to stage III(early period),at which IAA and ZR contents showed a significant upward trend and reached the peak at stage III;the other is from stage III to stage IV(later period),at which the levels of four endogenous hormones decreased obviously and fell to the lowest point at stage IV but ratioes of ZR/GA₃ and ZR/IAA were the highest.Based on the dramatic change of IAA in early and later period,it is suggested that IAA may have dual effects on bulb formation.In the whole process of underground-stem bulblet formation,ZR was significantly correlated with other three hormones,implying cytokinin might be the key node in hormone regulatory network during bulblet formation in L.‘Aladdin'.(3)Application of exogenous 6-BA can significantly promote underground-stem bulblet formation in L.‘Aladdin',mainly reflected in shortening the formation time and increasing the new bulblets number.In addition,spraying exogenous 6-BA can induce the de novo formation of stem bulbils on the middle and upper axils of aboveground stem,suggesting the 6-BA induction pathway is involved in the formation mechanism of new stem bulbs in L.‘Aladdin'.Furthermore,the HPLC-MS assay was used to determine the levels of phytohormones in leaf axils of aboveground stem at 0,10 and20 days after spraying 6-BA(treatment)or water(control).Results illustrated that the contents of IAA and GA₄ decreased significantly in 10-20 days(later period),opposite to that in the control,indicating that the decrease of IAA and GA₄ in later period is likely to be crucial to induce bulbil formation.Besides,t Z/GA₄,i P/GA₄,i PA/GA₄ and t Z/IAA,i P/IAA,i PA/IAA under 6-BA treatment were significantly higher than that in the control when bulbils formed at stage VI,further proving that higher relative content of cytokinin was beneficial to the formation of new stem bulbs.Therefore,it is not a single hormone playing a decisive role,but a suitable homeostasis among multiple hormones that directly affects the formation of lily bulbs,providing a new approach to study the molecular regulatory mechanism underlying bulblets formation in L.‘Aladdin'.(4)The potential effects of KNOTTED1-like homeobox(KNOX)transcription factors,which can simultaneously regulate several hormone metabolism and signal transduction pathways,on bulblet formation in L.‘Aladdin'were analyzed.Two La KNOX genes(La KNOX1 and La KNOX2)and one BEL1-like homeobox(BELL)family gene(La BEL1)were isolated from L.‘Aladdin'.Bioinformatics analysis,subcellular localization and transactivation assays of such three genes were performed.Moreover,results of quantitative real-time PCR(q RT-PCR)demonstrated that La KNOX1,La KNOX2and La BEL1 genes are hormone-responsive genes,which were significantly induced at leaf axils on underground stem when new bulblets form(stage IV).The correlation analysis revealed that the expression patterns of them were significantly correlated with the ratio of ZR/GA₃ and ZR/IAA(p<0.05)respectively.Furthermore,physical interactions among La KNOX1,La KNOX2 and La BEL1transcription factors and their conserved domains involved in the interaction were verified in vitro and in vivo by yeast two hybrid(Y2H)and bimolecular fluorescence complementary(Bi FC)assays.(5)La KNOX1,La KNOX2 and La BEL1 genes were transferred into Arabidopsis respectively for functional verification.In addition,due to the interactions between La KNOX1 and La KNOX2,La KNOX1 and La BEL1,they were also co-transformed into Arabidopsis respectively for functional verification.The contents of phytohormones in transgenic Arabidopsis were detected by HPLC-MS assay.Results illustrated that La KNOX1,La KNOX2,La BEL1,and their heterodimers can simultaneously regulate the levels of endogenous cytokinin(t Z,i P and i PA),gibberellin(GA₃,GA₄ and GA₇),auxin(IAA)and abscisic acid(ABA).Specifically,La KNOX1 transcription factor(TF)interacts with La BEL1 TF to enhance the inhibition to IAA level,while interacts with La KNOX2 to weaken such inhibition.In addition,the heterodimerization of La KNOX1 with La KNOX2 or La BEL1 TF resulted in an obvious decrease in GA₄ level and the significant increase in isopentenyl-type cytokinin(i P and i PA)content and t Z/IAA,i P/IAA,i PA/IAA,i P/GA₄ and i PA/GA₄,which is consistent with the alterations of phytohormones contents during the stem bulbs formation in L.‘Aladdin'.To sum up,the pupative molecular mechanism underlying the underground-stem bulblet formation in L.‘Aladdin'was proposed.In the process of undergroun-stem bulblets formation,the‘increased-decreased'changes of m RNA accumulation of La KNOX1 gene in the early period(stage I-stage III)could lead to the increase of t Z and IAA levels,while the decrease of GA₄ content.In the later period(stage III-stage IV),La KNOX1,La KNOX2 and La BEL1 genes were all induced substantially,resulting in the heterodimerization between La KNOX1 and La KNOX2,La KNOX1 and La BEL1.Additionally,as the expression of La KNOX2 is about three times of that of La KNOX1 and La BEL1 at stage IV,a large number of La KNOX2 proteins are likely to be left to function alone.In this case,based on the results of phytohormones levels changes in transgenic Arabidopsis,it is speculated that the contents of endogenous GA₃,GA₄ and IAA decline,and ratioes of t Z/IAA,i P/IAA,i PA/IAA,i P/GA₄and i PA/GA₄ increase in the later period.Finally,such alterations in several hormones concentrations induce underground-stem bulblets formation in LA hybrid lily‘Aladdin'.