Metabolic And Molecular Basis Of Blueberry Leaf Color Turning In Response To Low Temperature And The Regulatory Roles Of Key Transcription Factor VcMYB9a

Blueberry(Vaccinium spp.)is a perennial shrub of the genus Vaccinium L.of Ericaceae.Its fruit is high in nutritional and health values and its external features display seasonal variations.Especially in Fall with the decrease of temperature,anthocyanins accumulate in blueberry leaves leading to changes in leaf colors from green to red,which are aesthetically pleasing.Therefore,recently blueberry shrubs are more and more used in landscaping and they are excellent materials for beautifying the environment.Anthocyanins are widely involved in many aspects of plant growth and development,such as improving plant resistance to environmental stresses such as low temperature and drought,attracting animals,promoting the spread of pollen and seeds and so on.Anthocyanin is mainly distributed in the vacuoles of leaves,flowers,fruits and other organs and tissues of higher plants.It has a high content in some aging leaves such as blueberry and maple,which is an important factor to determine the color of leaves.Anthocyanin is synthesized through phenylalanine pathway,which is jointly affected by transcription factor regulation,structural gene expression level and induction of external environmental factors.MYB is the most important transcription factor regulating anthocyanin synthesis and metabolism.MYB regulates plant anthocyanin biosynthesis by gonerning the expression level of structural genes(F3H,ANS,DFR,UFGT etc.)in metabolic pathway while temperature is one of the important environmental cues that can induce the accumulation of anthocyanins.However,the metabolic and molecular basis of blueberry leaves in response to low temperature induced leaf color transformation is not clear,and how Vc MYB transcription factor regulates anthocyanin biosynthesis in blueberry leaves and affects plant growth and development in response to low temperature induction is not clear.In addition,the functional research of important blueberry genes lacks an efficient and stable genetic transformation system,which also slows down the process of related investigations.In this study,I analyzed the metabolic and molecular basis of blueberry leaf color transformation in response to low temperature induction,and further explored the molecular mechanism of Vc MYB9a regulating anthocyanin biosynthesis in blueberry leaves in response to low temperature induction and affecting plant growth and development.In addition,an efficient and stable genetic transformation system of blueberry was constructed with diploid blueberry'Red Button',which was used to verify the function of blueberry Vc MYB9a gene.The main results are as follows:1.After low temperature treatment,in blueberry leaves,the contents of chlorophylls decreased and anthocyanins greatly accumulated,resulting in the change of leaf color from green to red.And the change of leaf color became more intense with the continuous or increased intensity of low temperature.Finally,leaf color changed from full green to bright red with high ornamental values,and anthocyanin was the main pigment that determined the color change of blueberry leaves.2.After low temperature treatment,the responses of blueberry to low temperature induction were significantly different among varieties.‘Duke'and other highbush blueberry varieties were low temperature sensitive varieties.Photosynthesis stopped rapidly in low temperature environment,chlorophyll content decreased sharply,anthocyanin accumulated,leaf color changed dramatically,and eventually leaves entered the aging period.Meanwhile,the contents of multiple amino acids in leaves increased and these amino acids were known to be involved in anthocyanin biosynthesis,inhibiting plant growth,promoting organ senescence and enhancing stress resistance.Investigations with ¹⁵N indicated that nutritional elements such as nitrogen,phosphorus and potassium returned to the branches of plants for storage.The branches with higher degrees of lignification had stronger resistance to low temperature.In contrast,‘Brightwell'and other varieties of rabbiteyes were not sensitive to low temperature stress.In these varieties,plants continued to grow,anthocyanin biosynthesis was less,and color conversion period was longer.Moreover,these varieties were coupled with slow nutrient reflux,low degree of lignification of new autumn shoots and vulnerability to frost damage,combined of which seriously affected the yield of berries in following years.This indicated that low temperature inhibited the growth and development of blueberry,and anthocyanin improved the ability of plants to resist low temperature stress.3.Targeted metabonomics analysis showed that green leaves of blueberry contained more Proanthocyanidin B1,Cyanidin-3-O-galactoside,Quercetin-3-O-glucoside and Cyanidin-3-O-arabinoside,whose contents continued to increase in the process of leaf color changing from green to red,while the red leaves contained mainly Cyanidin-3-O-galactoside and Cyanidin-3-O-arabinoside.This indicated that low temperature increased anthocyanin contents mainly by promoting the biosynthesis of Cyanidin-3-O-galactoside and Cyanidin-3-O-arabinoside in blueberry leaves.4.The heterologous expression of Vc MYB transcription factors in blueberry leaf color transformation induced by low temperature was transformed into apple callus.It was found that Vc MYB9a,Vc MYB9b and Vc MYB5d could significantly promote anthocyanin biosynthesis.Among them,the accumulation of anthocyanin in Vc MYB9a overexpression callus was the highest.Sequence alignment showed that the CDS sequences of three Vc MYB genes were highly consistent,with Vc MYB9b and Vc MYB5d might be Vc MYB9a alleles.Overexpression of Vc MYB9a in Arabidopsis resulted in the accumulation of anthocyanin,resulting in reddish rosettes.This indicates that the process of anthocyanin accumulation in blueberry slices induced by low temperature might be jointly regulated by multiple Vc MYB transcription factors,and Vc MYB9a might be the main gene regulating anthocyanin biosynthesis in blueberry leaves in response to low temperature induction.5.Through the optimization of adventitious bud regeneration system induced by blueberry leaf disc in vitro,it was found that WPM+2.0 mg·L^-1ZT+1.0 mg·L^-1IBA was suitable for tissue culture regeneration of various blueberry materials.The ploidy level of blueberry was negatively correlated with callus induction and differentiation efficiency.The diploid blueberry'Red Button'had advantages of short induction time,good differentiation quality,strong regeneration ability and large number of adventitious buds.Using the sterile leaf disc of'Red Button',the infection conditions were optimized by Agrobacterium-mediated,and the appropriate antibiotic concentration was screened.Finally,the transgenic materials were obtained,and the efficient and stable genetic transformation system of blueberry was established.It has been proved that'Red Button'is an ideal genetic transformation material and can be used as a model material for studying the function of blueberry genes.6.Quantitative detection of Vc MYB9a transformation materials showed that the expression levels of key structural genes(Vc PAL,Vc C4H,Vc4CL,Vc CHS,Vc CHI,Vc F3H,Vc DFR,Vc ANS and Vc UFGT)in blueberry anthocyanin metabolic pathway were regulated by Vc MYB9a.7.After Vigs-induced silencing and overexpression of Vc MYB9a,it was found that the anthocyanin content in the leaves of blueberry Vc MYB9a silencing lines decreased,the chlorophyll content increased,the leaves changed from dark red to bright green,and the biomass of plants increased.On the contrary,the anthocyanin content in the leaves of blueberry Vc MYB9a overexpression lines was significantly increased and the chlorophyll content was less,which changed leaf colors from bright green to dark red.The plant growth was inhibited,the biomass increased slowly,and the leaf senescence was accelerated.After low temperature treatment,it was found that the ability of blueberry Vc MYB9a overexpression lines to resist low temperature stress was significantly improved.The results of this study showed that Vc MYB9a could regulate the expression of anthocyanin metabolic pathway genes and promote the biosynthesis of anthocyanins after the induction of low temperature.The accumulation of anthocyanins created red colors in blueberry leaves,inhibited the growth and development of plants,increased the low temperature resistance of blueberries,and provided blueberry with higher ornamental values.