Studies On The Evolution,Expression Patterns And Functions Of Cytokinin Biosynthesis And Degradation Genes In Woodland Strawberry

Strawberry is known as the 'Queen of fruits' with bright color,unique flavor and high nutritional fruit.The short growth period and early bearing fruits of strawberry result in its significant economic benefits.In nowadays,China becomes a big country of strawberry production and consumption.And the cultivated area and annual output are at the forefront of the world.It is difficult to study the normal cultivated strawberry(Fragaria ×ananassa),because it is octoploid.Diploid woodland srawberry(Fragaria vesca)have the benefits of short growth period,high seed yield,high efficient transformation system,small sequenced genome and other advantages.And in addition to different fruit size,the flower structure characteristics,the flower organ arrangement and early fruit development of woodland srawberry are similar with cultivated strawberry,so it is an ideal model plant to study strawberry.Cytokinin is one of the most important hormones to regulate the growth,development and responding to stress of strawberry.The change of cytokinin concentration in plants was mainly controlled by two aspects:the cytokinin biosynthesis and degradation.Isopentenyl transferase(IPT)and cytokinin oxidase(CKX)are the key enzymes in the synthesis and degradation of cytokinin,respectively.Moreover,they are the two most important factors of determining the concentration of cytokinin.Therefore,studing the evolution and function of IPT and CKX genes in forest strawberry are significant for elucidating these two kinds of genes functioning strawberry fruit development and responding stress.In addtion,it is also important to analyze the function of cytokinin for cultivating new varieties of strawberry with excellent characters.1.IPT can be divided into ATP/ADP-IPTs and tRNA-IPTs according to the difference of catalytic substrates.Based on the domains search and validation,7 IPT genes were identified in the woodland strawberry,including 5 ATP/ADP-IPTs and 2 tRNA-IPTs.Sequence comparison and phylogenetic analysis with 171 IPT gene in 19 representative land plants reveals that only angiosperms have ATP/ADP-IPT gene,and ATP/ADP-IPT gene homologous do not exist in low plants:moss and selaginella.Moreover,the number of ATP/ADP-IPT genes in angiosperms varied greatly,ranging from 2 to 21.The number of ATP/ADP-IPT genes in strawberry was less than the mean number of all the investigated species(8.13).The tRNA-IPT gene is widely distributed in all the investigated land plants,and the number(2?3)is relatively constant in angiosperms.Therefore,ATP/ADP-and tRNA-IPT genes may have different evolutionary patterns in strawberry and other angiosperms.The exon/intron structure analysis of strawberry IPT genes showed that only one ATP/ADP-IPT gene contains 2 introns,and other ATP/ADP-IPT genes contain none of intron.In contrast,the two tRNA-IPT genes contain more than 15 introns.The same situation occurred in other angiosperms.The protein Motifs analysis showed that the two different kinds of IPT proteins had different Motif composition,while the Motif composition of the same kind of IPT protein was basically the same.In addition,we selected representing 58 species among six major kongdoms(including bacteria,archaea,protists,fungi,animal and plants)to analyze the origin of two types of IPT gene in strawberry.The identification and phylogenetic analysis showed that the two kinds of IPT genes had different origins:the tRNA-IPT gene is very ancient,widely distributed in all organisms;while ATP/ADP-IPT gene only existed in angiosperms,and probably appeared by the duplication and differentiation of type ? tRNA-IPT in common ancestor of angiosperms.In conclusion,the different function of ATP/ADP and tRNA-IPT genes in strawberry may result from the various gene structure,motif composition,origin and evolution mode.2.We further analyzed the expression patterns of two type IPT genes in strawberry.Strawberry flower and early fruits transcriptome data and RT-qPCR analysis of roots,leaves,the mid and late stage of fruits showed that tRNA-IPT genes exressed stably in different tissues/fruit development stages of strawberry flowers and fruits(i.e.,constitutively expressed);while ATP/ADP-IPT genes expressed only in parts of tissues/fruit development stages at very low or undetectable levels(i.e.,tissue-specific expressed).On the other hand,the two kinds of IPT genes did not show different expression patterns under the high temperature,low temperature,high salinity and water deficit stress conditions.Therefore,it is speculated that the two types of strawberry IPT genes may have different functions in different tissues or developmental stages of reproduction organs.The expression patterns analysis of other three representative(Amborella,maize and Arabidopsis)angiosperms,also support the differences between ATP/ADP-and tRNA-IPT gene in different tissues and organs.In Amborella(the earliest differentiated angiosperm),ATP/ADP-IPT genes almost did not expressed in the female flower bud,while in the core of angiosperm(strawberry,maize and Arabidopsis),more than half of ATP/ADP-IPT genes expressed in flowers.These results showed that the expansion and differentiation of ATP/ADP-IPT gene may be associated with floral organs complication.3.Cytokinin oxidase(CKX)is the only enzyme known as degrading cytokinin in plants.Eight CKX genes were identified in woodland strawberry.The phylogenetic analysis with the 101 CKX genes in other 8 terrestrial plants,it is found that there is only one homologous copy of the CKX gene in the common ancestor of the land plants.Therefore,in strawberry and other terrestrial plants,CKX genes expanded at same levels.The main CKX gene expansions in strawberry were whole genome replication and discrete replication.The number of introns in the CKX gene of strawberry changed little,ranging from 3 to 5;and the Motif composition of different CKX proteins were also consistent.The CKX gene/protein in other land plants also has similar characteristics.These results indicate that the CKX genes in land plants were relatively conservative in gene sequences,exon/intron structures and Motif composition.Transcriptome data analysis of strawberry flower and early fruits showed that the expression of CKX gene was varied among different tissues,as well as in different stages of the same tissue.Therefore,the functional differentiation of strawberry CKX gene may be reflected in the differentiation of expression patterns.4.The distribution of the CKX gene in six major kingdoms were further investigated.In addition to land plants,the CKX genes were found only in the genome of a few bacteria and an Excavate.There were no homologous sequences of CKX genes in archaea,protists(including protozoa and algae),fungi and animals.Phylogenetic analysis showed that all CKX genes in terrestrial plants formed a single gene cluster,which were closest related with the bacterial CKX genes.The motif composition of the CKXs of the land plants was consistent with the motif composition of the bacterial CKXs,which are different from the excavata CKX.These results suggest that the CKX gene in land plants probably originated from bacteria.There is no CKX gene in algae which is the sister lineage of land plants.Compared with other types of bacteria,the relationships of most CKX genes in cyanobacteria were far from the terrestrial plants,and the Motif compositions were also different.Furthermore,the differentiated time estimation showed that the differentiation time between the bacteria and terrestrial plants CKX genes differentiated was about 418 million years,which was consistent with the time of plants landing.Therefore,the CKX gene in land plants could not be derived from chloroplast genome.In summary,the CKX genes in land plant probably originated from bacteria;the common ancestor of terrestrial plants obtained CKX genes from bacteria horizontal gene transference through chloroplast pathway before the landing event.5.Over-expression of CKX gene has a great influence on plant growth and development.So the CKX gene was transformed into strawberry and tobacco by using the root-specific promoter to detect the expression of the CKX gene under drought tolerance and basically decrease the effect on the plant development.The drought treatment of wild type and transgenic tobacco,the groove on stem of transgenic tobacco is less than wild type.More importantly,4 days after re-water,the transgenic tobacco recovered from the drought stress,wilting leaves unfolded,and the groove of the stem disappeared,while the wild type plants did not.Overexpression of CKX gene enhances tobacco drought tolerance.Overexpression of CKX gene enhanced drought tolerance in tobacco.Root-specific over-expression of CKX gene in strawberry showed that strawberry root was longer than control,while the upper part was relatively shorter.The RT-qRCR analysis revealed that the expression of most cytokinin signaling pathway genes in transgenic strawberry were lower than that in the wild type.In addition,the GA biosynthesis genes(GA3 and GA20ox1)and the signal repressor protein(Della)expressed the root specific expression of CKX gene,reducing endogenous cytokinin concentration at the same time,it may also lead to a decrease of endogenous gibberellin concentration.Under drought treatment,the drought response gene(RD29)expressed significantly lower in transgenic strawberry roots and leaves than that in the wild type,indicating that under same extent drought condition,the effect of drought stress of transgenic strawberry were significantly smaller than that of wild type,the expression of CKX gene in strawberry increased tolerance to drought stress.