| Lotus(Nelumbo nucifera Gaertn.)is a basal angiosperm,it is very conservative in the evolutionary process and still retains many original characters and phenomena.Therefore,it is of great significance to study the biological basis of lotus in plant phylogeny.In this paper,Honghu seed lotus variety "Man Tian Xing" was used as research material,and we used the methods of field morphological observation and recording,anatomical microscope,making temporary sections and paraffin sections,histochemical stain,pollen germination in vitro,infrared probe thermocouple temperature measurement,infrared imaging,bioinformatics methods and so on to preliminarily study the thermogenesis effect and pollination biology of N.nucifera.The main conclusions of this study are as follows:1.The flowering process of N.nucifera could be divided into five stages: bud stage,bud enlargement stage,initial-flowering stage,loosing-pollen stage and flower wilting stage.The anthesis of a single flower lasted about three to five days.Flower organs exhibited specific morphological characteristics at each stage during flower development procedure.2.N.nucifera had the characteristics of dichogamy and pistil matured earlier than stamen.The pistil matured at the initial-flowering stage,the stamens matured at loosing pollen stage,and initial-flowering stage was 24 h earlier than loosing pollen stage.Pistils and stamens remained mature status at the same time about 6 hours.Therefore,N.nucifera could be cross-pollinated or self-pollinated.The dichogamy mechanism had avoided the self-crossing phenomenon of lotus plants to some extent.The observation showed that the lotus flower was opened at 6 o'clock in the morning and closed at around 14 o'clock in the afternoon.It experienced a second opening and closing phenomenon until the petals withered.3.N.nucifera had significant thermogenesis phenomenon during anthesis process.Infrared thermometers and thermocouple probes were used to detect the heat generating parts of N.nucifera.The result showed that heating commenced at bud enlargement stage and terminated at wilting stage.When the environment temperature ranged from 24?to 40?,the temperature of the receptacle was always maintained at 30?~35? during thermogenic stage.However,the temperature of the flower organs in wilting stage no longer maintained a steady state and will fluctuated with the environment temperature.The constant temperature of receptacle is of great significance for pollen development,pollen maturity,successful fertilization and pollen tube growth.4.Before and after flowering themogenesis,the morphology and number of stomatal apparatus in the epidermis of receptacle and petal base changed significantly.The numbers of epidermal stomatal apparatus on the upper surface and side of receptacle and the outer surface of petal base increased and the pore diameter of stomata increased.Stomatas,located in the epidermis of plants,are gas exchange channel between plants and the external environment.The stomatas in receptacle and petal increased at thermogenesis stages,which could not only promote the exchange rate of gas and maintain the high heat output,but also promote the release of the special odor of lotus flower,thereby attracting more insects to pollinate them.5.Alternate Oxidase(AOX)is a mitochondrial terminal oxidase that is closely related to floral thermogenesis.A total of 7 AOX genes were identified by in silico method.Seven conservative motifs of AOX proteins in N.nucifera were predicted by MEME software.Cis-acting element analysis showed that they mainly contain various light response,hormone response,low temperature,drought response,defense and stress response cis-acting elements.The phylogenetic tree analysis indicated that they were divided into 5 branches.The most AOX proteins of N.nucifera were mainly clustered in the G1 branch,while NnAOX4 and NnAOX1 were respectively located in the G4 and G5 branches.The other four branches showed a phenomenon of aggregation of AOX protein in monocot or dicot.This study showed that AOX proteins play an extremely important role in the growth and development of N.nucifera.6.Pollen germination experiment in vitro was performed.The results showed that the pollen vigor of N.nucifera had great differences in different periods of flowering.The pollen of bud stage and bud enlargement stage were not mature enough to germinate in vitro.Pollen in the loosing pollen stage had the fastest germination rate and the highest pollen germination rate,the strongest pollen vigor in vitro culture and the germination time was only about 1.5h.Pollen in initial-flowing stage had similar germination rate and half germination speed with loosing pollen stage.Five temperature gradients were set and the pollens of loosing pollen stage were taken for germination experiment in vitro.The results showed that the germination rate was the lowest under the condition of 24 °C and the highest at the condition of 32 °C.The results showed that floral thermogenesis could maintain the temperature of the floral organs(especially the reproductive organs)in an appropriate temperature range,so as to ensure the high germination rate of pollen and rapid pollen tube elongation.7.N.nucifera is a typical beetle pollinating plant.This study tracked the behavior and frequency of visiting insects during flowering.The results showed that there were many insects visiting lotus flower,including forage grass insects,ladybug insects,beetle insects,fly insects and bee insects.During the female stage,the numbers of visiting insects were small,mainly beetle insects.During the bisexual stage,the number of fly insects and bee insects were the highest.During the flower withering stage,the main visiting insects were bees.Analyzing the behavior of these visiting insects,the results showed that beetles and bees as well as flies were potential pollinators of N.nucifera. |
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