Effects Of Melatonin On The Lily Bulblet Development With Cloning And Functional Analysis Of LoBPM3 And LoRAV Gene

Efficient cultivating of lily bulbs into commercial bulbs is an effective way to help the lily industry upgrade.At present,the basic laws of bulb development have not been elaborated fully,and the technical measures to promote bulb development need to be improved.This study confirmed that melatonin is beneficial to the development of lily bulblets through cell histology,physiology and molecular biology experiments.Two melatonin-responsive genes,Lo BPM3 and Lo RAV,were further cloned,and the biological functions of Lo BPM3 and Lo RAV genes in melatonin-mediated development of lily bulblets were clarified.This study provides a new research direction for exploring the development of lily bulblets,and provides a theoretical basis for scientific regulation of bulb development.The main results are as follows:1.Melatonin promoted cell proliferation and enhances cytokinin signaling,which in turn promoted scale primordial initiation of lily bulblets;meanwhile the melatonin promoted root development also.The lily bulblets were treated with 20μM melatonin and cultivated.The height of bulb apical meristem was higher than that of control significantly.Scale primordia were generated at the basal flanks of the growth point,and the number of basal cells was increased significantly.The expression of the genes involved in melatonin synthesis,meristem development,cell proliferation,and cytokinin signaling were up-regulated significantly,while the expression of cell proliferation-suppressing genes were inhibited.In addition,melatonin increased bulb basal rooting regeneration rate and average root length significantly,and up-regulated the expression of the genes related auxin transport and root primordia regeneration.2.Melatonin promoted conversion fron protein-to-sugar and slowed the differentiation of immature scale cells,which in turn promoted carbohydrate accumulation in lily bulblets.The contents of total soluble sugar,trehalose and starch in the bulbs treated with melatonin were increased significantly,while the protein content was decreased significantly,and the content of total amino acid increased first and then decreased.The expression of genes involved in trehalose,starch synthesis and gluconeogenesis pathways were up-regulated significantly,while the expression of genes involved in starch degradation were inhibited.In addition,melatonin also reduced the cell size of immature scales,and the content of H₂O₂,and superdismutase oxidase（SOD）activity,and enhanced catalase（CAT）activity significantly.The expression of genes involved in cell differentiation and expansion was inhibited,and the expression of genes related to reactive oxygen species scavenging was up-regulated.3.Melatonin promoted growth of the lily bulblet and carbohydrate accumulation in tissue culture.When the lily bulblets were inoculated into the medium containing 20μM melatonin,the scales in the lily bulblets grew faster,the bulb diameter was larger,and the contents of starch and trehalose were increased significantly.At the same time,the expression of genes involved in cell proliferation,trehalose,starch synthesis and gluconeogenesis pathways were up-regulated significantly;while the expression of genes involved in cell proliferation inhibition and starch degradation were inhibited.4.Two melatonin-responsive genes,Lo RAV and Lo BPM3,were cloned in lily.Melatonin can promoted the expression of Lo BPM3 and inhibited the expression of Lo RAV.Lo RAV belongs to the AP2/B3 transcription factor gene family,with a full length of 1056 bp,encoding 351 amino acids,the deduced protein molecular weight is of 38.906 KD,and the theoretical isoelectric point（p I）is 9.4.Lo BPM3 belongs to the BPM gene family and contains two domains,MATH and BTB-POZ,with a full length of 1248 bp,encoding 415 amino acids.The deduced protein molecular weight is 46.045 KD,and the theoretical isoelectric point（p I）is 5.92.Both Lo RAV and Lo BPM3 are nuclear proteins and have no self-activation activity of transcription factors.5.Analyzed the function of Lo BPM3 and Lo RAV genes in melatonin-mediated bulb development.Transient overexpression of Lo RAV upregulate the expression of starch degradation gene Lo AMY and inhibition the expression of cell proliferation inhibitory gene Lo TCP4,and the starch accumulation gene Lo AGPase,and the cell proliferation genes Lo CYCB2-1 and Lo PCNA;Attenuates the regulation of Lo RAV and promoted the expression of Lo CYCB2-1,Lo PCNA recovered to the expression level of wild-type when the Lo RAV-OE treated with by 20μM melatonin,but had no effect on the expression of Lo AGPase and Lo AMY.Lo BPM3 interacted with Lo RAV in yeast cells and is located upstream of Lo RAV.Transient overexpression of Lo BPM3 inhibited the expression of Lo RAV,Lo AMY,and Lo TCP4 and upregulated the expression of Lo CYCB2-1.In conclusion,melatonin mainly promoted the development of lily bulblet through protein degradation and cell proliferation pathways,among which cell proliferation is the key,and the protein degradation is an important inducing factor.Lo RAV/Lo BPM3 is an important regulator of melatonin-mediated bulb cell proliferation pathway.These will provides a reference for research the development of lily bulbs.