Functional Study Of FveNCED And FveHK Genes In The Anther And Fruit Development In Fragaria Vesca

Strawberries are perennial herbs,and their fruits,which are known as 'Queen of fruits',are bright-colored,nutrient-rich,soft and juicy.At present,the cultivated area of strawberry in China ranks first in the world.Strawberries are mainly grown in greenhouse and other facilities,but the deformity of strawberry fruits is a serious problem.One of the main reasons for deformity is that the pistils are not successfully fertilized.Pollen activity is the key factor for fertilization.Besides,the strawberry fruit development and ripening decide the fruit size,color,nutrient composition and other qualities.Therefore,the study of mechanisms of strawberry anther and fruit development and ripening is significant for improving strawberry quality.Woodland strawberry(Fragaria vesca)is thought as an ideal model plant for studying cultivated strawberry(F.× ananassa,octoploidy).F.vesca is a diploid strawberry.It has a simple genome and a relatively well-developed transformation system,and its characteristics in flower and fruit development are nearly the same as those of F.ananassa.In this study,we first presented a genome-wide analysis of the genes key for abscisic acid(ABA)synthesis(FveNCEDs),which are very important for F.vesca fruit ripening.Then we characterized their expression patterns in different tissues,different fruit ripening stages,as well as in response to different abiotic stresses.Besides,during these analyses,we found some of the cytokinin receptor genes(HKs)exhibited a rapidly increased expression during F.vesca fruit ripening.HKs play very important roles in the transduction of cytokinin(CK)signals.Thus,the features of FveHKs and their expression profiles in F.vesca were also investigated.Morever,RNA interference(RNAi)technique was used to silence individual FveHK genes for exploring their possible roles.Our main results are as follows:1.Three FveNCEDs were identified from F.vesca and they had different expression profiles.For example,FveNCED1 was the main gene expressed in F.vesca seedlings,different meristems,early-stage flowers,developing carpels,and pollinated styles.FveNCED2 showed a high expression during the early-stage fruit development,while expression of FveNCEDl and FveNCED2 was similar in anthers.FveNCED6 was only expressed in early-stage flowers and partial floral tissues.Besides,all of the 3 FveNCEDs were expressed in microspores,but they were almost not expressed in mature pollens.2.FveNCED2 was the unique gene that exhibited a highly and continuously up-regulated expression during F.vesca fruit ripening,which co-occurred with the increase of ABA content in F.vesca.The response of FveNCED1 was the most significant to different abiotic stresses.FveNCED6 was almost not expressed in these assays.All of these results suggest that FveNCEDl and FveNCED2 are the key genes for ABA biosynthesis in abiotic stress responses and fruit ripening,respectively.3.Four FveHKs were identified from F.vesca:FveHK2a,FveHK2b,FveHK3 and FveHK4.Protein sequence alignments between FveHK2a and FveHK2b showed 91.83%homology.However,their genes had different expression patterns in F.vesca.The expression of FveHK2a in reproductive organs was generally higher than FveHK2b,while it was opposite in most of the vegetative tissues.These results indicate that the functions of FveHK2a and FveHK2b may have diverged.FveHK3 had high expression in most tissues.FveHK4 exhibited high expression in most vegetative organs,while in reproductive organs,it was mainly expressed in anthers,unpollinated styles and embryos.Additionally,the expression of FveHK4 rapidly increased during F.vesca ripening,and was higher than the other FveHK genes in mature fruits.Besides,exogenous CKs could rapidly enhance the FveHK4 expression,while had only slightly effects on the other FveHK genes.4.Three specific RNAi vectors for FveHKs were constructed:FveHK2-RNAi,FveHK3RNAi and FveHK4-RNAi.The FveHK2-RNAi vector silenced both FveHK2a and FveHK2b due to their high similarity in the sequence.The results show that the vegetative growth of all the transgenic lines of the three FveHKs-RNAi vectors was similar with wild type(WT)plants in normal conditions.However,the senescence of FveHK2-RNAi detached leaves was advanced compared with WT in dark,suggesting that FveHK2 was the key CK receptor to negatively regulaete leaf senescence in F.vesca.Besides,the senescence and chlorophyll degradation in FveHK4-RNAi detached leaves were clearly delayed in dark.All of these results indicate the distinct roles of different FveHKs in the regulation of leaf senscence in F.vesca.5.The reproductive growth of FveHK2-RNAi and FveHK3-RNAi transgenic plants was normal,while the fruits of FveHK4-RNAi plants,including receptacles and achenes,were abortive.Pollen grains from WT could restore the fruit set of FveHK4-RNAi plants.All results of the microscope observation,including pollen staining and germination,show that more than 90%of pollen grains from FveHK4-RNAi mature anthers were shrunk,abnormal and sterile.Further study by scanning electron microscope(SEM)and transmission electron microscope(TEM)demonstrate that these abnormal pollen grains from FveHK4-RNAi were shrunk inward from germination holes and lost cytoplasm.Moreover,the exines of FveHK4RNAi pollens were thicker than those of WT pollens.All of these results indicate that FveHK4 plays an important role in F.vesca pollen development,and its knockdown would lead to male sterile.6.The observations of pollen grains at different stages indicate that the visible abnormity of FveHK4-RNAi pollens might start from S10c(S10 late)stage.The analyses of anther paraffin section and TEM demonstrate that the main characteristic of FveHK4-RNAi anthers was the delayed degradation of tapetum cells at S10 stage.RNA-seq and RT-qPCR analyses in S9 and S10 anthers show that the expression levels of a CK signalling gene(FveRR5)and several carbohydrate transport and metabolism genes(FveSTP1,FveBGAL7a,FveBGAL7b and FveBGLU17)significantly decreased in FveHK4-RNAi anthers.In contrast,the expression of two exine related genes(FveMS2 and FveCYP704B1)and one gibberellin GA-2 oxidase gene FveGA2ox1 significantly increased in S10 anthers of FveHK4-RNAi plants.These results suggest that these genes play a role in the male sterility of FveHK4RNAi plants.In conclusion,this study presents genome-wide characterizations and expression profilings of FveNCEDs and FveHKs in F.vesca.Furthermore,the functions of different FveHKs have been investigated by RNAi technology,and the roles of FveHK4 in anther development and possible mechanisms of the male sterility caused by FveHK4 RNAi have been analyzed.This study may provide a basis for further analysis of the roles of CKs and ABA on F.vesca growth and development,and give a reference for the improvement of strawberry yield and quality.