Functional Characterization Of AOX And UCP In Floral Thermogenesis

Thermogenesis has been observed in some plant taxa,and the heat produced spontaneously by thermogenic plants can maintain flowers at higher temperatures than the ambient environment.Floral thermogenesis not only could promote the release of floral fragrance,but also can attract insects to visit flowers.It has been increasingly realized that floral thermogenesis ensures the development of reproductive,and has various biological functions in improving seed maturing.Previous studies reported that AOX and UCP proteins are likely involved in the regulation of floral thermogenesis,but its regulatory mechanism remains unclear.Therefore,the process of plant floral thermogenesis and its regulation mechanism required further study.In this study,two different types of floral thermogenesis plants,Magnolia denudata Desr.and Nelumbo nucifera Gaertn.were used as research materials.Firstly,by analyzing the tertiary structure,phylogenetic and conserved domain of AOX and UCP proteins in M.denudata and N.nucifera,it is found that AOX protein was produced obvious divergence during the evolution between animals and plants.UCP proteins are both conserved in the evolution of plants and animals and have highly similar protein domains.Secondly,the temperature in the flowering process was detected continually by using infrared imaging for the thermoregulatory plant of N.nucifera.These results demonstrated that the epidermis of the receptacle was the thermogenesis position,and the thermogenesis peak consisted of three phases:early pistil stage,pistil stage and stamen stage.By using scanning electron microscopy,it is found that the cell morphology and structure changed obviously after temporal floral closure and re-opening,and the stomatal density increased significantly.In addition,Western Blot results showed no difference in UCP protein expression from epidermis of the receptacle,while AOX protein expression was markedly increasing at the peak of the thermogenesis process.For the pseudo-thermoregulatory plant of M.denudata,infrared thermography technology was applied to monitor the flowering process.The results confirmed that two peaks of heat production periods appeared in the pistil and stamen stage.The protein expression level was experimented by Western Blot and quantification data showed that the expression of AOX and UCP protein was significantly changed in the process of heat production.AOX protein was significantly expressed at the peak of heat production from stigma tissue.Given the link between Western Blot results and detection of citric acid content in M.denudata by high performance liquid chromatography,it indicated that the citric acid content increased significantly during the peak heat generation period.Scanning electron microscopy was used to image the gynoecium of M.denudata.The results showed that there was a distinct cell gap between the epidermises of the gynoecium at the peak stage of heat generation.In summary,AOX proteins are conservatively evolved between N.nucifera and M.denudata,high divergence exists among non-thermogenic Arabidopsis thaliana plants,but UCP proteins are more conservative in evolution.During the process of floral thermogenesis in N.nucifera,temporal floral closure and re-opening phenomenon of the flower organs coincide with the heat generation mechanism.In addition,before and after temporal floral closure and re-opening of the petal,the N.nucifera produces obvious cell gap and forms high-density stomata accompanied by cell bulges.This movement ensures the plant to maintain sufficient oxygen and increases internal and external gas exchange therefore promoting insect visits and maintaining normal activities.Protein expression assay showed that AOX protein might play a leading role in the peak stage of heat production in N.nucifera.Floral thermogenesis of M.denudata is always accompanied with strong fragrance.By using HPLC analyses,it is found that increased AOX respiration substantially promoted production of citric acid,which provided fundamental metabolite for biosynthesis of VOCs.For citric acid is an essential substrate for the synthesis of floral components,which indicated the accumulation of citric acid in the peak heat period may cause increasing expression of AOX protein.Through the regulation of AOX protein,citric acid promotes the release of a large amount of floral scents during the heat production.Moreover,the changes of cell morphology of gynoecium further enhanced the gas exchange inside and outside the tissues during heat generation.Therefore,the thermogenic plant can attract insects to visit flowers and promoting reproductive development.This study conducts in-depth research on the floral thermogenesis from bioinformatics,plant physiology,cell biology and biochemistry and provides novel pespectives for further interpreting the mechanism of floral thermogenesis and accurately understanding the energy regulation in thermogenic plant.