Identification of Alternatively Spliced mRNA Variant during the Tension Wood Formation in Populus trichocarpa

• Main Authors: Hsu-Cheng Hsueh, 薛旭程
• Other Authors: Ying-Hsuan Sun
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/11428872588125134383

碩士 === 國立中興大學 === 森林學系所 === 104 === When woody angiosperm exposed to gravitational stress, tension wood would be formed at the up-side of the wood to counter act the exerted mechanical or gravitational stresses. Compare to the normal wood, the secondary cell wall of the tension wood would form a thick gelatinous cell wall layer (G-layer) which composed of more than 90% of cellulose crystalline. Such characteristics make the tension wood a suitable material for studying regulatory mechanism of cellulos biosynthesis. Alternative splicing (AS) has been demonstrated to regulate the biosynthesis of secondary cell wall. This research focued on identify alternative spliced genes in tension wood, and study the roles of AS in tension wood formation. Transcriptome analysis studied were first carried out to identify differentially expressed genes between tension wood and normal wood in Populus trichocarpa, the results showed that cellulose synthase (CesA) genes that catalyzed cellulose biosynthesis, were not up-regulated in tension wood, suggested that the increase of cellulose content were regulated by other mechanism. The up-regulation of FLA gene and down-regulation of cellulose hydrolase 5 (GH5) family in tension wood may have resulted in the highly crystallized cellulose in the G-layer. The down-regulation of PAL, HCT, 4CL and CAld5H contributed to the low lignin content in tension wood. Gene ontology (GO) enrichment analysis of the up-regulation genes in tension wood reveal that the tension wood formation is related to extension and motility of microtubule. Microtubule distribution are known to affect the position of cellulose biosynthesis. The results suggested that restructuring of cytoskeleton played an important role in tension wood. Transcription factors, on the other hand, are enriched in the down-regulated genes in tension wood. Among these down-regulated transcription factors are the PtrVNDs, homologous to a class of NAC transcription factors that regulate vessel cells differentiation in Arabidopsis. The decrease of vessels in the tension wood maybe due to the down-regulation of PtrVNDs. The analysis of differential expression of transcriptional isoform were carried out to identify alternative spliced gene. The results showed SP1L1 that related to directional extension of microtubule and ADF4 that related to actin filament depolymerization are among the differentially alternatively spliced genes, suggesting that cellulose biosynthesis in tension wood maybe regulated through AS of cytoskeleton related genes. The AS of C3H also were identified, suggesting that AS may also involved in regulating lignin biosynthesis in tension wood. The intron retention events were also studied during tension wood formation, and the results showed SP1L1, MAP and ARP, genes related to cytoskeleton, had retained intron in tension wood. The expression patterns of these alternative spliced genes and isoforms were analyzed in a tension wood induction time-course. The results showed the expression of isoform SP1L1_8.3 decrease along with tension wood formation, whereas its alternative spliced isoform SP1L1_8.7 conversely increase. The expression of isoform ADF4.1 was down-regulated in the early stages of tension wood induction. In contrast, ADF4.2 is up-regulate in later stages. These expression patterns of the AS genes suggested that AS of cytoskeleton related genes maybe a key regulatory mechanism of tension wood formation.