Studies On The Pollination Biology And The Molecular Basis Of Fertility Of The Orchid Oberioides Microtatantha

Pollination biology is one of the hotspots in evolutionary biology and is important to understand the interaction and co-evolution between plants and biological or abiotic pollinators,and also between plants and environment.Orchidaceae is one of the most species diversity families with extremely diverse genetic and morphological variations.Up to date,many pollination systems and various pollination mechanisms have been found in Orchidaceae.These biological information on pollination provide the effective clues and the evidence for exploring and understanding the origin and evolution,the reproductive ecology and the population expansion of orchids.Oberonioides microtatantha is a small lithophytic species in Orchidaceae.It is mainly distributed in the low mountain area from East China to South China.Because of the adaptation to the habitat with low temperature and rainy in the period of flowering,the orchid has evolved the mechanism of water flow pollination and can adjust its female suitability by inducing functional female sterility to achieve its survival and reproduction in the extreme environment.In the study,the wild populations of O.microtatantha in Shixing,Guangdong Province and Ningbo,Zhejiang Province were investigated for five years and theri pollination mechanism and the molecular basis of fertility were discussed.The main results were as follows:(1)Under the wild conditions,O.microtatantha need a long-term aestivation to avoid the heat and drought stress.During this period,they only survive in the form of pseudobulbs,and rely on the moss and the lichens to shield themselves.The inflorescences usually finish their tissue differentiation in winter and are in full bloom when the cold spell comes in early spring.At this time,the habitat is mostly low temperature and rainy,which seriously restricts the animal activity,so that the orchid is lacking of the biologicalpollinators,while the flower feature and flowering pattern of the orchid has been adapted to the extreme environment and takes the water flow as its abiotic pollinator.With the field observation and the simulation experiments of water-flow polliation,the new pollination mechanism is confirmed in success.In the orchid,mixed mating is carried out by water flow to realize self-and cross-polination,and to achieve the double benefits from reproductive security and heterosis.The water flow pollination mechanism is different from the others hydrophily pollination and is the first report in Orchidaceae.(2)According to the results of manual pollination,it was showed that the fruit-set ratio of cross-pollinated flowers(11.38% ± 2.10%)was higher than that of self-pollinated flowers(4.87% ± 0.85%).In consideration of the complete inactivated pollinia is only 2.64% in total,it means that there is a significant phenomenon of female sterility in O.microtatantha.This study considered the female organ structurally-complete and its sterility was functional.Therefore,the sexual system of O.microtatantha is a functional andro-andromonoecy at the level of population,which is an adaptation to the unique habitats and the water-flow pollination.(3)There are differences in photosynthetic characteristics,soluble sugar content,starch accumulation and life history types among the three different groups.The apparent quantum efficiency(AQY)and the maximum net photosynthetic rate(Pmax)of the high-light fertile group(HF)were higher than those of the low-light sterile group(LNF)and the high-light sterile group(HNF),but the light saturation estimate(LK)and the light compensation point(LCP)were higher than those of the latter two groups.The soluble sugar content and the starch accumulation in the high-light fertile group were the highest.The results of quantitative classification based on PCA of life cycle forms in the three groups were V0.2427C0.3577S0.399,V0.2202C0.3562S0.4236 and V0.2760C0.3431S0.3809 respectively.This study showed that the response to light intensity was polymorphic in O.microtatantha and it would led to a fine-tuning for the life cycle form.A sufficient supply of photosynthetic assimilation products was help for improving the female fertility of the orchid.The light also would induce the morphological gradient resulting in the change of the quantitative traits of organs,such as petioles,ovaries and stigmas,which were closely related to fertility,and thus affect pollination.(4)Basing on RNAseq analysis,total 1184 differentially expressed unigenes were screened and 22 GO terms and 10 KEGG metabolic pathways were enriched.The results showed that the fertility-related genes involved in several physiological processes including photosynthesis,carbohydrate metabolism,signal transduction,histone modification,etc.Some potential key genes,including Lhcb,Sn RK1,FBP,ADPG,SAI,HMT and HDAC coding genes and products,were preliminarily screened.They played an important role in regulating the fertility of O.microtatantha.(5)The down-regulated expression of auxin response factor ARF3 and the up-regulated expression of auxin-repressed proteins in the sterile flowers suggested that auxin was closely related to the fertility.This study proposed a hypothesis that these auxin-related genes may regulate the transport of auxin from pollinia to ovary,interfere with auxin-mediated pollination signal to stimulate or inhibit the enlargement of ovary and the development of ovule.