| Fruit sweetness is among the most important quality for fruits such as citrus and apple.It is determined by the levels of sugars and acids and their ratio.The content of organic acids,mainly citrate and malate,in fruits is a major determinant for the trait of sweetness or sugar/acid ratio.As increasing sugar contents leads to the shortening of the shelf life of the fruits,and thus manipulating the fruit acidity has become one of the major goals in horticultural studies and breeding fields.The content of organic acids in fruits is impacted by fruit types,genotypes,fruit development,environmental factors and management practices.In general,organic acids in fruits accumulate during fruit growth and development,and be utilized during fruit ripening.Therefore,the content of organic acids depends on the balance between their biosynthesis and degradation.Currently,the studies on the mechanism of organic acid accumulation in fruits have been focused on the synthesis,transport,storage and degradation of citrate and malate.Prior studies in our laboratory have taken advantage of four sweet orange(Citrus sinensis L.Osbeck)varieties with differing fruit acidity and available in the germplasm resources maintained in Citrus Research Institute of Chinese Academy of Agricultural Sciences,including Newhall and Xinhui(normal acid),Bingtang(low acid),and Succari(acidless).Using RNA sequencing,the transcriptomes of fruits at two developmental stages,I(cell division)and II(cell expansion),were obtained for integrated systems biology analysis.A gene co-expression network related to fruit acidity control was constructed.Analysis of this network has led to the identification of 15 acid-related candidate genes,which are predicted to be involved in regulatory functions such as transport,transcription,signaling,and stress response.In this thesis,similar systems biology studies were first performed for sugar content and sugar/acid ratio,respectively,with an aim of identifying key candidate genes related to sweetness control in fruits by constructing gene co-expression networks associated with sugar accumulation and sugar/acid ratio in expanding sweet orange fruits.In the meantime,three hub genes in the fruit acidity control network,CsAIL6、CsAHA10、CsTT8,were selected for further characterization,including expression vector construction and genetic transformation of a normal acidic sweet orange,Jincheng.Furthermore,by using transgenic strawberry fruit system,we confirmed the physiological function for one of the three genes(FvAIL6)in fruit acidity control.The main results are summarized below:1.Systems biology analysis of sugar/acid ratio-associated genesThe rationale for this part of study is that the sugar/acid ratio is a key determinant to fruit sweetness,although it remains debatable whether the sugar/acid ratio can be treated as a horticultural trait.By comparing the genes that are respectively correlated with acidity and sugar/acid ratios,we aimed to identify those hub genes specifically related to sugar/acid ratios,which will lay the foundation for future studies of sugar or acid accumulation.Comparative analysis of sugar content,acid content and sugar/acid ratio at two developmental stages in four orange varieties showed a small difference between these varieties.However,due to apparent increase in sugar contents in all four varieties at 142 days post anthesis(DPA),the sugar/acid ratio exhibited slightly different changes from 45 to 142 DPA in different orange varieties.Specifically,the ratio remains almost identical in Xinhui,a slight increase in Newhall,and dramatic increase in both Bingtang and Succari.To identify candidate genes related to the sugar/acid ratios,a total of 7,430genes showing differential expression between 45 and 142 DPA were subjected to correlation analysis between expression level and the sugar/acid ratios.This resulted in the identification of 72 genes strongly correlated with the ratios.These genes were then used for gene co-expression analysis,with the construction of various subnetworks based on distinct co-expression modules.Surprisingly,Venn diagram and co-expression module analyses very few of the sugar/acid ratio-associated genes belong to the acidity-related or sugar-correlated genes,strongly indicating that at least in these four sweet orange varieties a unique subset of genes might be involved in the control of fruit sugar/acid ratio independent of sugar and acid accumulation alone.2.Co-expression network analysis of genes in expanding citrus fruits.Based on our prior results of the fruit development and sugar or acid accumulation relationship in sweet oranges,we found that the sugar contents in fruits of four varieties all exhibit a dramatic increase from 45 to 142 DPA despite their differential acidity changes.Therefore,we re-analyzed the transcriptome data which are common across these four sweet orange varieties during this fruit developmental stage,with an aim of identifying a common subset of fruit sugar accumulation-related gens conserved in sweet orange varieties.We identified a total of 3,145 differentially expressed genes that are common across all four varieties.Using the gene co-expression network approach,we constructed GA and ethylene response subnetworks,as well as subnetworks of transcription factors and chromatin modification,and made prediction of possible physiological functions for those hub genes.GA response subnetwork shows that,cell division and cytoskeleton organization are among the most overrepresented GO terms,consistent with the role of GA in regulating cell division,cell expansion and cytoskeleton.Ethylene response subnetwork analysis indicates that ethylene response factors(such as ERF13)and other ethylene response genes(such as ALP and MYB14)play a potential role in sugar accumulation during early fruit development of sweet oranges.We then analyzed 257 transcription factor genes conserved in orange fruit development.Venn diagram analysis shows that transcription factor genes are involved in regulation of primary metabolic process,developmental process,and hormone response.Finally,we examined 137 genes classified as the GO term of chromatin modification.Among these genes,we found that four are involved in the processes of developmental regulation,metabolic regulation and hormone response.3.Expression and bioinformatics analysis and genetic transformation of CsAIL6,CsAHA10 and CsTT8 genes in sweet orange.CsAIL6 encodes a 632 amino acids-containing transcription factor,which belongs to the AP2 subfamily of AP2/ERF family,which structural domain is conserved.Analysis of the phylogenetic tree constructed using the deduced amino acid sequences shows that CsAIL6 is highly homologous to Arabidopsis proteins AtAIL6 and AtAIL7.In addition,the result of expression analysis indicates that CsAIL6 may negatively regulate the acid level in citrus fruits.CsAHA10(Cs1g16150),also called CitPH5 or CsPH8,encodes a952 amino acids-containing protein,belonging to a member of ATPase family.Phylogenetic analysis shows that another citrus gene,Cs1g16160,is highly homologous to CsAHA10.They both can be considered orthologs of AtAHA10 in Arabidopsis and PhPH5 in petunia.Prior results suggest that CsAHA10 is localized to the vacuolar membrane and that its transcriptional expression difference in high-and low-acid lemon varieties is controlled by CsTT8 transcription factor or other upstream regulatory proteins.CsTT8 or CitAN1 is predicted to encode transcription factor with 692 amino acids.Phylogenetic analysis of CsTT8 and its homologous proteins from other plants that encode bHLH transcription factors involved in anthocyanin biosynthesis showed that CsTT8 is orthologous to AtTT8 in Arabidopsis,PhAN1 in petunia and Zm R in maize,all belonging to the IIIf subfamily.Besides the aforementioned function of regulating the expression of CsAHA10 and CsECA4(which encodes a H~+-ATPase),bHLH proteins can form an MBW complex with MYB transcription factor and WD40 protein in the control of anthocyanin metabolism,which in turn impacts vacuolar pH and petal pigmentation.We have carried out the studies of genetic transformation of the above three genes into sweet orange Jincheng.Plant CRISPR/Cas9 vectors,over expression vectors and RNA interference(RNAi)vectors of CsAIL6,CsAHA10 and CsTT8 were constructed and introduced into Jincheng by Agrobacterium tumefaciens,resulting in 35 lines(OE)and 75lines(RNAi).However,these three genes are expressed by the organ-specific pattern.On the other hand,despite our effort of reducing the juvenile phase by grafting transgenic seedlings to adult orange plants,to date none of transgenic orange plants have flowered yet,which made impossible to evaluate the success of gene silencing and assess the fruit acidity.4.Functional characterization of FvAIL6 gene in strawberryDiploid strawberry has advantage over sweet orange given its shorter juvenile phase and rapid fruit development.In addition,strawberry and citrus have the same fruit developmental stages,with citrate being predominant in both types of fruits and both belonging to non-climacteric fruits.Thus,although the edible part of strawberry is different from citrus,they have a similar acid accumulation pattern in our previous study.Taken together,we choose to accelerate the process of functional characterization of FvAIL6 gee using a diploid strawberry(Fragaria vesca L.Yellow Wonder 5AF7).FvAIL6 exhibits organ-specific expression pattern,indicating its potential role in flower,fruit and root development.Plant RNAi vector of FvAIL6 was constructed and introduced into YW 5AF7 by Agrobacterium tumefaciens-mediated transformation,resulting in 23 lines.The result of qRT-PCR revealed that a total of three independent transgenic lines(CZ46-3,CZ46-31,and CZ46-68)had 30-55%lower transcript level of FvAIL6.The acid content of developing fruits(17 DPA)in CZ46-3 was 38%higher than that in the non-transformed wild-type control.Our further results showed that a similar38%higher acid content in CZ46-3,suggesting that the acidity change caused by FvAIL6RNAi is reproducible.Interestingly,in contrast to a dramatic increase of acid content from7 to 17 DPA in wild-type fruits,CZ46-3 fruits maintained a relatively high acid level.At7 DPA,CZ46-3 fruits had a 2.6-fold increase compared to the wild-type,although this difference reduced to only 13%at 27 DPA.This result suggests that FvAIL6,an ortholog of CsAIL6,likely acts as a negative regulator in fruit acidity control.This finding also indicates that the diploid strawberry can be used as a fruit model system to study physiological functions for those genes in fruits from citrus and other fruit trees with a relatively long juvenile phase.In summary,in this thesis project,exploratory or hypothesis-driven investigation was performed towards the mechanisms underlying fruit sweetnee control in the following three aspects:construction of gene coexpresson networks,respectively,for sugar/acid ratio and sugar accumulation during fruit expansion,and functional validation of the select hub genes in the fruit acidity control gene network previously constructed in our laboratory.Although the long juvenile phase in transgenic sweet orange lines prevented functional testing for those three genes,it was found that diploid strawberry plants transformed with FvAIL6 RNAi vector had fruits showing increased fruit acidity.This novel finding demonstrates that FvAIL6 acts as a negative regulator in fruit acidity control.Importantly,this result also shows that the previously constructed fruit acidity control gene network is reliable.Taken together,these results suggest the effectiveness of the research strategy which uses systems biology to identify key hub genes followed by their functional validation using reverse genetic approaches.Thus,the findings presented in this thesis research opens a new avenue for the future mechanistic study of fruit sweetness control via regulating the contents of sugar and acids and their ratios. |
