Mechanism Of Corolla Closure Of Ipomaea Purpurea

Flower is an important reproductive organ in angiosperms.The morphological structure and movement of the flower(the process of flower opening and senescence)are closely related to the successful reproduction of the plant.Flower movement generally includes pistil movement,stamen movement and corolla movement,which are closely related to the entire life process of the flower.The movement of the corolla is inseparable from its morphological structure and gene expression in the flower.Ipomoea purpurea L.belongs to Pharbitis of the Convolvulaceae.It is an annual herbaceous twining plant and is a sympetal flower.The flowering period was roughly from June to October.In the flowering period,single flowers generally opened at around 4:00 a.m.,and closed at around 9:00 a.m.,until they were completely closed at around 12:00 a.m.When the corolla was open,it was trumpet-shaped,but when it is closed,it became an inverted bell shape,forming a closed chamber,and then withered,unlike some flowers which wilted directly from the open state.This experiment was to explore the changes in morphological structure and differentially expressed genes in the process of corolla closure.We used Ipomoea purpurea as experimental material to observe the corolla closure process of its single flower;used freehand slice method,scanning electron microscopy(with freezing table and fixed drying method),paraffin section and stereo microscope to observe morphological structure of the corolla during the closure process.We also used extraction and quality detection of total RNA of the corolla,the construction of m RNA-Seq library and high-throughput sequencing,the screening and verification of differentially expressed genes at different stages of the corolla and other methods to study the changes in gene expression.The purpose was to explore the morphological and anatomical significance of the formation of an inverted bell-shaped chamber of Ipomoea purpurea in the process of closure and to explore the mechanism of corolla closure at the molecular level,and to provide a theoretical basis for the study of corolla senescence closure in other species.The results of the study were as follows:(1)The movement rhythm of the corolla of Ipomoea purpurea caused a single flower to complete the process of opening to closure within 24 hours.Through observation,when the corolla of Ipomoea purpurea was just fully opened,it was the S0 stage.And the closure process was divided into seven stages,S1,S2,S3,S4,S5,S6,and S7.The flowers were trumpet-shaped when they were in full bloom,and the pink part of the upper half of the corolla gradually curled inward during the closure process until the entire corolla was closed.When the pistil and stamen of the flower,the top 1/3 part of the corolla,the top 2/3 part of the corolla,and the entire corolla were removed artificially,it was found that as long as the pink part of the corolla existed,the corolla could still curl inward normally despite the horizontal cut off.The study of the changes in the epidermal cells of the corolla found that the adaxial surface of the corolla were pink papillary motive cells with larger volume;the abaxial surface of the corolla were transparent irregular flat cells with smaller volume.And in the process of corolla closure,the degree of water loss and shrinkage of papillary epidermal cells was faster than that of flat cells on the abaxial surface.Therefore,from a cellular level analysis,during the process of corolla closure,the epidermal cells on the adaxial surface of the corolla lost water and shrank faster than the epidermal cells on the abaxial surface,resulting in unequal cell osmotic pressure on both sides,and the corolla curled and shrank inward.(2)The Ipomoea purpurea pertained to sympetalous flower with 5 corolla veins(vascular bundles in the petals),which formed by the convergence of two vascular tissues.In the natural state,it was observed that when insects gnawed off the pink part of the corolla and only the corolla veins were left,the corolla veins could still curl inward;when only 1/3 of the corolla veins were artificially cut and the pink part was left,the pink part that lost the corolla veins will not curl inward,and the remaining 2/3 of the corolla veins could still drive the corolla to curl inward;when four of the corolla veins were artificially cut(cut in the middle of the two vascular vessels between each corolla vein),the remaining corolla vein could drive the surroundings curled inward,and the cut corolla veins could not normally close inward;when five corolla veins were cut artificially,the entire corolla could not be closed inward.It was naturally falling and wilting.Through stereo microscope and paraffin section observation,the presence of vascular tissue in the corolla veins was confirmed.In summary,the cells were not the only reason for the closure of the corolla of Ipomoea purpurea.The vascular tissue of the corolla veins mainly provided the force of the corolla to curl inward,and then drove the entire corolla to close inward.(3)We took the pink part of the upper half of the corolla in S0,S1,S2,and S3 stages as experimental materials,extracted total RNA,and used RNA-Seq technology for transcriptome sequencing analysis.According to the expression of genes in different stages,it was found that there were 11081(3759 upregulated,7322 downregulated)DEGs from the S0 stage to the S1 stage.There were 15730(7596 upregulated,8134downregulated)DEGs from the S1 stage to the S2 stage.There were 11274(7499 upregulation,3775 downregulation)DEGs from the S2 stage to the S3 stage.According to the expression level of genes in different stages,the differential genes could be divided into 3 categories,a total of 6 clusters.The GO and KEGG enrichment analysis of differential genes found that the DEGs of Cluster 1,Cluster 2,Cluster 3,Cluster 4,Cluster 5 and Cluster 6 were enriched in 396,662,1039,1293,1174 and 260 GO items respectively(qvalue<0.05),the most significant of these enrichment items are cellular process,cell,membrane and catalytic activity,etc.These 6 clusters enriched 87,102,103,118,113,65 KEGG pathways.The most significant pathway enriched by DEGs was related to amino acid metabolism,sugar metabolism,chlorophyll metabolism,autophagy and circadian rhythm,such as fructose and mannose metabolism,carotenoid biosynthesis,pentose phosphate pathway,cutin,suberine and wax biosynthesis,autophagy,plant circadian rhythm,etc.These metabolic pathways are related to corolla senescence,indicating that senescence controlled the closure of the corolla.To sum up,the reason why Ipomoea purpurea formed an inverted bell-shaped chamber when closed was morphologically due to the movement of the corolla veins and the changes in the turgor pressure of the inner and outer epidermal cells of the corolla,and the molecular mechanism was due to the aging gene controlling its closure process.