Study On The Developmental Mechanism Of Zanthoxylum Bungeanum Prickle Based On Morphology And Multi-Omics Analysis

Zanthoxylum bungeanum Maxim.is a member of the Rutaceae family.It’s an aromatic plant well known for both its medicinal and food properties.Due to its excellent adaptability to the ecological environment,it’s widely distributed in China and Southeast Asian.As an important economic forest,the existence of prickles leads to long time and high cost to harvest.Therefore,breeding no-prickle Zanthoxylum bungeanum has become an urgent problem.Three types were selected for experiments:wild prickly Zanthoxylum bungeanum（PZB）,wild smooth Zanthoxylum bungeanum（SZB）and grafted smooth Zanthoxylum bungeanum（GSZB）.Using the methods of Fourier transform infrared spectroscopy,optical microscopy,transmission electron microscopy,transcriptomics and metabolomics to explore the development mechanism of prickle from the perspective of morphological and multi-omics analysis,at the same time,providing a reliable basis for the breeding of Zanthoxylum bungeanum.The main conclusions are as follows:1.The FTIR absorption spectrum of prickle was different from that of bark and stalk.The absorption spectra of prickles had band at 1617 cm^-1 and 1110 cm^-1,while the bark and stalk had strong band at 3319 cm^-1 and 1999 cm^-1.According to the band correspondence,the associated-NH₂ or-NH bond could cause a band to appear at 3319 cm^-1,which means there were compounds contain such chemical bonds in bark and stalk.Besides the 1110cm^-1 band was caused by C-O-C stretching and symmetric vibration of the ester linkage,or-CH stretching in aromatic ring.In addition,the well-defined narrow band at 1617 cm^-1was attributed to C=C aromatic ring vibration,which indicated that the prickles contain a lot of aromatic compounds.2.The differences in microstructure of prickle and young stems were studied by light microscopy.The results showed that the growth direction of prickle cells and stem cells was at a vertical angle,and there was an obvious dividing line between them,called abscission zone.It was precisely because of the existence of abscission zone that the prickles were easily peeled off from young stems.In addition,the pseudo-color image analysis showed that prickle cells were similar in color to xylem cells and epidermal cells,while stem cells were similar in color to parenchyma cells,indicating that prickle cells were lignified earlier than young stem cells.3.The ultrastructural differences between prickles and young stems were investigated by transmission electron microscopy.There were lots of crystalline substances in the vacuoles of the epidermal cells of prickle,but there were not such substances in the vacuoles of the epidermal cells of stems.Combined with the results of optical microscopy,it is preliminarily judged that these dark crystals were the precursors of lignin synthesis.In addition,there were plasmodesmata between cells in the abscission zone,and the cells near the two sides of the abscission zone had no obvious difference in ultrastructure.4.Through transcriptome data analysis,GO terms and candidate genes that affect the development of prickles were screened out.Significantly enriched GO terms were:meristem development（GO:0048507）,response to gibberellin（GO:0009739）,transcription regulator activity（GO:0140110）,etc.Besides,the 9 candidate genes were:Zb YABBY2,Zb YABBY1,Zb YABBY5,Zb WRKY28,Zb AZG2,Zb LOG5,Zb IAA33,Zb Gh16,Zb Gh16X1.The candidate genes were tested by q RT-PCR,and the real-time fluorescence quantitative results were highly consistent with the RNA-Seq results,which verified the reliability of the RNA-Seq results.5.Through metabolome data analysis,the KEGG pathway and 30 differential metabolites related to prickle development were screened out.Significantly enriched KEGG pathways were:cutin,suberine and wax biosynthesis（map00073）,phenylalanine metabolism（map00360）,plant hormone signal transduction（map04075）,etc.Among the30 differential metabolites,Lyso PE,Lyso PC,and 10,16-dihydroxypalmitic acid,etc.were higher expressed in PZB,while stachyose,2’-Deoxyinosine-5’-monophosphate,and Protocatechuic acid-4-O-glucoside^*were higher expressed in the treatment of SZB and GSZB.