| Some plants can actively generate heat and regulate the temperature during the flowering,so that the flower temperature is significantly higher than the ambient temperature.Floral thermogenesis plays important roles in promoting the successful reproduction of thermogenic plants.Mitochondrion,as a key factor for energy metabolism,plays an essential role in the regulation of heat generation.Mitoflash is a mitochondrial dynamic change event in which a large amount of superoxide bursts in the mitochondrial matrix and lasts for a certain period of time.Taking mitochondria as basic energy metabolism units,this study focused on the dynamic changes and functions of mitoflash in floral thermognenesis.In this study,thermogenic Nelumbo nucifera was majorly used for investigations,and the thermogenic plant Magnolia denudata was used for some verification.High-resolution infrared imaging technology was used to determine the key thermogenic timing and the major thermogenic tissues of N.nucifera flowers.Transmission electron microscopy was used to investigate the morphological changes,density and distribution of mitochondria in thermogenic and non-thermogenic cells.High-resolution laser confocal microscopy imaging was used to capture real-time dynamics of mitochondria,ROS production and mitochondrial membrane potential respectively.By analyzing mitochondrial fluorescence intensity changes and the trend of mitochondrial membrane potential oscillation frequency in thermogenic and non-thermogenic cells,the dynamics of mitoflash were characterized.The Neo Fox fluorescent probe technology and Extracellular Flux Analyzer were used to measure the respiratory activity of thermogenic and non-thermogenic tissues.Using inhibitors of different respiratory pathways,the main respiratory pathway of thermogenic tissues are clarified.In summary,the results in this study as follows:（1）Infrared imaging of N.nucifera showed that the non-thermogenesis period is bud stage with immatured receptacle;thermogenesis period is femal stage with receptive receptacle.The temperature of the flower part reaches 34°C during floral thermogenesis,and it can be about 10°C higher than the ambient temperature,and the heat generation peaks at 13:00-15:00 pm.The main themogenic part is the receptacle.（2）The thermogenic cells showed notable changes of mitochondria during thermogenesis.The number of mitochondria was significantly higher than that of non-thermogenic cells.as well as the ratio of mitochondrial length to width.The mitochondria in the thermogenic cells are mostly distributed around the vacuole and the cell edge close to the cell membrane.（3）This study reported the mitoflash classification in thermogenic cells.According to the number of mitoflah ROS bursts that occurred suddenly within 100 s and continued for a period of time with an increase rate ofΔF/F0﹥0.2,the types of mitoflash are divided into single mitoflash and multiple mitoflash.The frequency of multiple mitoflash in thermogenic cells is significantly higher than that of single mitoflash.The oscillating frequency of mitochondrial membrane potential in Magnolia thermogenic cells is significantly higher than that of non-thermogenic cells.According to itochondrial membrane depolarization that occurred suddenly within 100 s and resumed with an decrease rate ofΔF/F0﹥0.2,The types of potential oscillations are divided into single mitochondrial membrane potential oscillations and multiple mitochondrial membrane potential oscillations.The frequency of multiple mitochondrial membrane potential oscillations in the receptacle is significantly higher than that of single mitochondrial membrane potential oscillations.（4）It is found that ROS in thermogenic cell keep a higher level than non-thermogenic cell,and the co-localization results of the ROS in thermogenic cells are mainly derived from mitochondria.The mitochondrial ROS fluorescence intensity in thermogenic cells is 205.47±6.94,which is significantly higher than that in non-thermogenic cells,and the mitochondrial ROS activity in thermogenic tissues and cells is stronger.（5）Thermogenic tissues showed higher respiratory rates than non-thermogenic tissues did.In the thermogenic receptacle,the contribution of the AOX pathway to total respiration reached 61.38%,while,the contribution was 16.37%in non-thermogenic tissue of receptacle.In summary,the increased biogenesis of mitochondria was observed in company with notable morphological changes,suggesting increased activity of mitochondria during floral thermogenesis of N.nucifera.Especially,the increased activity of mitoflash generated large amounts of ROS,contributing to a significant accumulation of ROS in thermogenic cells.We further found increased AOX levels in thermogenic tissues with enhanced mitoflash activities.Considering that AOX is capable of removing excess ROS in cells,AOX provides a protection to maintain normal physiological status of the cell.However,the increased AOX also led to production of heat in these thermogenic cells.This study provided a new insight into regulation of floral thermognenesis. |
