| Strawberry(Fragaria Xananassa Duch.),an evergreen small-fruited berry crop that belongs to genus Fragaria in family Rosaceae,is an herbaceous perennial.It is widely cultivated all over the world and highly favored by consumers for fruit texture,flavor,nutritional qualities.Anthocyanins responsible for strawberry color are also related to fruit development,flavor formation and intrinsic quality.Moreover,anthocyanins are natural colorants and antioxidants,which have health benefit and economic value.Therefore,developing anthocyanin-rich strawberries has been attracting much attention.Light,especially light quality,and low temperature are two pivotal environmental factors for anthocyanin accumulation in plants.The main pattern of strawberry cultivation in China is protected cultivation,which often causes poor fruit coloration due to insufficient light.Red and blue lights are two main contributors to photosynthesis,so they are important to use in facility cultivation.Additionally,strawberry is easily susceptible to low temperature in winter and early spring,although there are protective measures.Therefore,this study used potted strawberries to explore anthocyanin synthesis and the expression mechanism of related genes in strawberry fruit exposed to different light quality and low temperature by high performance liquid chromatography(HPLC),quantitative real-time RT-PCR(qRT-PCR)and transcriptome sequencing.The main findings are as follows1.Two cultivated strawberry,’Toyonoka’(8x)and ’Tokun’(10x)used in this experiment have the bright red and pale yellowish-orange fruit color,respectively.After white light(control),red light,blue light and mixed light(red:blue=1:1)treatment,the anthocyanin content and expression level of related genes were detected.The results showed red light,blue light and mixed light significantly increased the content of total anthocyanins at beginning phase of coloration in two strawberry genotypes,suggesting that they can promote fruit coloring earlier in comparison to white light.At the late stage of coloration,the content of anthocyanin in ’Toyonoka’ and ’Tokun’ under mixed light treatment was noticeably higher than other light sources(mixed light>blue light>white light>red light)Furthermore,the red light and blue light had a remarkable influence on anthocyanin accumulation in ’Toyonoka’,but there were no significant differences among blue light,red light and white light in ’Tokun’.Therefore,differences in genotypes and varieties should be taken into consideration to make the most of light quality when light quality is applied to strawberry cultivation.2.In these two strawberry genotypes,the expression profile of MYB10 was regulated by light quality,accompanied by the similar change of CHS,DFR,ANS,UFGT gene expression levels and the anthocyanin contents,which suggested light quality may regulate anthocyanin biosynthesis via activation of FaMYB10 expression.Additionally,the expression levels of LAR and ANR responsible for proanthocyanidin biosynthesis were significantly induced by red light and mixed light in ’Toyonoka’,while mixed light played a significant role in ’Tokun’.These evidences provide a hint that reddish light probably contribute to proanthocyanidin accumulation.3.Compositions and contents of anthocyanins in ’Toyonoka’ under red light and blue light were detected at the early coloration stage using high performance liquid chromatography(HPLC).All fruit samples contained Pg3G(Pelargonidin 3-glucoside)and another anthocyanin Pg3MG(Pelargonidin-3-malonylglucoside)and had no detectable Cy3G(Cyanidin 3-glucoside).In contrast to the white light,blue light and red light obviously enhanced anthocyanin accumulation.The highest contents of total anthocyanins(136 μg·g-1)and Pg3G(122.18 μg·g-1)were detected in fruit treated with blue light,while the highest content of Pg3MG(19.20 μg·g-1)was detected in red light.Under all light conditions,Pg3G was the major anthocyanin and accounted for more than 80%of TA,whereas Pg3MG made up a smaller percentage.Red light and blue light can increase the Pg3MG content,but the proportion of Pg3MG in total anthocyanins decreased in both treatments.Therefore,the increase of total anthocyanins in strawberry fruit under light quality treatment mainly depends on the content of Pg3G.4.High-throughput sequencing was performed to further analyze overall transcriptional change and expression pattern of genes involving anthocyanin biosynthesis in ’Toyonoka’strawberry exposed to red light and blue light.Through pairwise comparisons,a total of 1402 DEGs(Differentially expressed genes)were identified between red light and white light,including 708(50.50%)up-regulated genes and 694(49.50%)down-regulated genes Among 5034 DEGs of blue light and white light,there were 2484(49.34%)up-regulated genes and 2550(50.66%)down-regulated genes.Between blue-light and red-light conditions,3764 DEGs were detected;of these,1931(51.30%)were up-regulated,and 1833(48.70%)were down-regulated.Although these DEGs were distributed in various primary and secondary metabolic pathways in plants,the light quality influenced the anthocyanin accumulation by regulating the expression level of photoreceptors(PHY,CRY,PHOT,etc.),light signal transduction elements(COP1,HY5,SPA,etc.),regulatory genes(MYB,bHLH,WD40,etc.)and structural genes(CHS,ANS,DFR,etc.)related to the anthocyanin biosynthetic pathway5.The objective of this experiment was to investigate the influences of low temperature(4℃)on anthocyanin accumulation during strawberry coloration.We determined the anthocyanin contents,and transcriptional abundances of MYBs and structural genes involving anthocyanin biosynthesis.Meanwhile,proanthocyanidin content,SOD activity and O2-production rate were also measured in this process.The results showed that O2-production rate increased during the significant accumulation of anthocyanins at low temperature(12-48 h)and then decreased,while the SOD activity remained low level at 12-72 h.Those suggested that anthocyanin possibly exerted an important influence on O2-scavenging in fruit under low-temperature stress.Simultaneously,ANS,UFGT and MYB10 showed higher expression levels and MYB1 expression was suppressed in fruits exposed to low temperature,suggesting that the increase of anthocyanin content might be caused by the regulatory genes(MYB10 and MYB1)modulating the ANS and UFGT structural genes expression6.Three different Microsoft Excel-based statistics algorithms,geNorm,NormFinder,and BestKeeper were used to assess the expression stability of seven candidate housekeeping genes in three sample groups including different tissues and fruit developmental stages,and fruit subjected to light quality or low temperature treatment.Our analysis suggested that the normalization results of target gene in qRT-PCR were more consistent when reference genes with stable expression were applied as internal controls,whereas the significant differences of normalization were caused by the most variable reference genes.The HISTH4,GAPDH,ACTIN1 and DBP expressed much more stably.However,the 18S,PIRUV and ACTIN2 were frequently presented as unstable genes.Especially,18S had very high abundance and variability.Hence,PIRUV,ACTIN2 and 18S were not recommended for target gene normalization in the given experimental conditions. |
