Multi-omics Analysis Of A Glossy Mutant Of Newhall Navel Orange "Gannan No.1" And Functional Verification Of Transcription Factor Csese3 In Regulation Of Wax And Jasmonic Acid Biosynthesis

Although rich citrus germplasms are found in nature,few of them possess many desirable traits at the same time under natural growth conditions.As the most widely cultivated species in the southern part of China,’Newhall’ navel orange(Citrus sinensis Osbeck ‘Newhall’)is enjoyed by consummers owing to the particular fruit-like fragrance and good performance in storage.However,imperfections still exist that the fruit surface is rough and dull,and the flavor often evaporates in a short time.Thus,waxing and packaging to polish the surface and prolong the shelf life are indispensable processes to guarantee industrial value,which would not only increase the costs but also exacerbate the “white pollution”.Identification and selection of citrus species that possess as many traits of interests based on the rich natural variations is an effective way to solve this problem.Our group previously reported a glossy mutant of Newhall navel orange(MT)in a garden of Jiangxi province in China.Compared with the wild-type(WT)growing on the same conditions,MT has better performance in glossiness,fragrance,colorfulness,and disease resistance in field growth and post-harvest stage.No significant differences were found after performing SSR analysis with 216 pairs of primers,and DNA-sequencing results showed 99.78% similarity between these two materials,and no obvious SNPs or indels were found.These results suggested a similar genetic background of MT and WT and that MT might be a bud mutant of wild Newhall orange.Previous metabolic analysis of WT and MT at the mature stage showed that wax content was significantly decreased while jasmonic acid,proline,and several terpene species were significantly increased in MT.Therefore,we infer that MT exhibited pleiotropic changes in lipid,primary and secondary-related branches at the same time.These altered metabolic pathways generated different levels of metabolites,which finally resulted in those desirable phenotypes.To better uncover the pleiotropic changes of MT,we performed lipidomics,metabolomics,and transcriptomic analysis on WT and MT in 90DAA(90 days after flowering),150 DAA,180DAA,and 210 DAA.We dissected the mechanisms underlying the formation of these desirable phenotypes,identified a key gene Cs ESE3 putatively involving in the phenotype alteration and verified its function.The main results obtained are as follows:1.The reduction of wax content is accompanied by the increase of the total phospholipids in MT,and the increased chloroplast lipids further activate the jasmonic acid biosynthetic pathway in MT.We observed almost no wax components,especially aldehyde and alcohols were accumulated in MT.We then performed lipidomic analysis on WT and MT and found that chloroplast lipids were in significantly higher levels in MT.36:6MGDG,36:6DGDG,and 36:2PG were the most abundant chloroplast lipids and the levels of them were 1.72,1.74,and 1.67 fold higher compared to WT in mature stage,respectively.The increase in phospholipid content has also been confirmed in transcription level.The eukaryotic lipid synthesis initiating gene Cs FAD2 was significantly up-regulated in the four developmental stages of MT.Two other important FADs,Cs FAD5 and Cs FAD7 were also significantly up-regulated in MT.The increase in chloroplast lipids resulted in the up-regulation of three DAD1 s,inclding Cs DAD1,Cs DAll2,and Cs DAll4,which would hydrolyze the excessive chloroplast lipids and release fatty acid precursors for jasmonic acid(JA)formation.Thus,we obsereved higher expression levels of JA-biosynthetic genes,including Cs LOX3,Cs AOS and Cs OPR and elevated levels of JA in MT compared to WT.2.The glycolysis and citrate degradation processes are significantly activated in MT,which supplied the excessive carbon sources required for lipids.Sesquiterpenoids were highly accumulated in MT and showed co-accumulative patterns with chloroplast lipids while most phenylpropanoids and flavonoids were reduced in MT and showed opposite accumulative patterns.Citric acid and citrate lyase(ACL)gene were increased by over two fold in MT,which would supply more cytosolic acetyl-Co A.We also found key genes in glycolysis pathways such as fructokinase 2(FLN2),fructose bisphosphate aldolase 3(TPI3),and 3 Pyruvate kinases(PK)were all significantly upregulated in MT.Secondary metabolic profiling found sesquiterpene valencene,caryophyllene,and two precursors of flavonoids,naringenin,and kaempferol were simultaneously highly accumulated in MT and positively correlated with 36-carbon chloroplast lipids.Most of the modified flavonoids,such as PMFs(polymethoxyflavonoids),Quercetin.O.Gala,Kaemperfol.O.Rha,as well as compounds in the phenylpropanoid pathway,including coumarin,p-coumaric acid were decreased in MT and negatively correlated with 36-carbon chloroplast lipids.3.A SHINE-like transcription factor,Cs ESE3 could activate wax and JA fromation in both citrus and ‘Micro Tom’ tomato.Cs ESE3 co-expressed with the key wax genes CER1-3,CER3 and almost all necessary genes in JA synthesis pathway,including PLIP1-2,LOX3,AOS,OPR in both transcriptomes.The sequence of Cs ESE3 was similar to the wax-regulating gene SHNs,but lack of complete ‘mm’ domain.Cs ESE3,not SHNs showed continuously down-regulated profiles in MT,and showed good consistency with variation of wax content.Overexpressing Cs ESE3 in citrus callus could active expression of key genes in wax and JA metabolic pathways including Cs CER3,Cs PLIP1 and Cs LOX3,etc.Levels of amino acids were also highly accumulated in overexpression lines,with Asparagine increasing by more than 17 fold.Tomato lines overexpressing Cs ESE3(OE)exhibited higher drought resistant ability and the fruits had more rough surface and longer glandular trichomes.Wax,jasmonic acid content were highly accumulated in two OE lines and key genes involved in these pathways such as Sl LACS1,Sl CER1,Sl OPR3,and Sl Tom Lo XD were significantly induced.Flavonoids were also largely increassed in OE lines,with the two precursors,naringenin and naringeninchalcone increasing by over7 fold.We transiently overexpressed Cs ESE3 in kumquat and found the gene could also significantly increase JA content.4.Cs ESE3 could bind to the GCC-Box element in the promoter of the JA-initiating phospholipase PLIP1,and activate its expression while could not promote the expression of three other DAD1 family genes that were significantly upregulated in MT.Luc experiments confirmed that Cs ESE3 could increase the activity of Cs PLIP1 promoter by more than 7 fold,which is significantly higher than the other lipid genes.Yeast one-hybrid and EMSA experiments further confirmed that Cs ESE3 can bind to the GCC-Box site in the PLIP1 promoter.The Luc experiment also found that Cs ESE3 could not activated the expression of the three DAD1 genes that were significantly up-regulated in MT.5.Expression of Cs ESE3 responds to ethylene.Ethylene content released from MT only took up 19.76% and 18.63% of that from WT in the year 2017 and 2018,which is consistent with the significant down-regulation of Cs ESE3 in MT.Moreover,we characterized two ACS genes involving in the direct formation of ethylene co-regulated with Cs ESE3.We also identified an ethylene response element(ATTTTAAA)presented in the 1.9K position from the Cs ESE3 gene,and found Cs ESE3 was induced by 2-fold when fruits were treated by exogenous ethylene.Thus,this gene might be an essentail intermediate point between JA and ethylene.