Studies On Molecular Mechanism Of Tension Wood Formation In Betula Luminifera Using RNA-Seq

Betula luminifera is not only a high-quality timber species with a priority to the promotion, but also an ideal materials for forest genetic study. Improvement of wood properties is one of the important contents of its breeding nowadays. However, it is a urgently solved problem for Betula luminifera and other forest trees how to accelerate the its genetic improvement. The solving of this problem require understanding the genetic basis of wood traits. Recently, RNA-seq was successfully applied to transcriptome profiling of non-model woody plants, which has been a powerful tool to discover the key canditidate genes controlling the economic traits. Therefore, in the present study, special phenotypes of tension wood (TW) were investigated for Betula luminifera, and redistributions of endogenous hormones were analyzed within the early stages of a tension time course. Meanwhile, in order to decipher the molecular mechanism of wood formation, gene expression during the same stage were characterized based on the de novo characterization of the Betula luminifera transcriptome using RNA-Seq technology. The main results are summarized as follows:1. After40days bending, TW fibres present a conspicuously thickened inner layer of the cell wall, whose double wall was averagely1.5times the thickness of opposite wood (OW) fibres. The results of double-stained with Safranin-Astra blue showed that a gelatinous G-layer deposited in the lumen side of secondary cell wall of TW. Fibre lengths and cellulose contents of TW were both obviously greater than those of OW, but lignin contents showed the converse tendency. Sucrose and fructose contents of TW were higher than those of O W within the early stages of a tension time course. It indicated that more C was already allocated to cellulose during early stage of TW formation. During the same stage, IAA contents of TW were all lower than those of OW, but difference between two regions reached significant level after7days bending. The distributions of GA and BR were same as IAA. In addition, although ZR contnets showed a rising tendency as bending progressed, their differences between TW and OW were insignificant for each harvest time. The results suggested that TW formation could be not initiated by the redistributions of these endogenous hormones.2. Transcriptomes from eight Betula luminifera tissues were analysed using Illumina HiSeqTM2000sequencing platform. Approximately2.1×107paired-end reads were obtained, generating1.9G bp sequencing data. These short reads were further assembled into54,777unique sequences (i.e. Unigenes) with an average length of443bp, whose length totally amounted to24.20Mb. Of the Unigenes,35,920(65.8%) had homologs in the NCBI non-redundant and Swiss-Prot protein database, which corresponded to24,482unique protein accessions. Of these annotated Unigenes,7.954and9,997were assigned to gene ontology classes and clusters of othologous groups, respectively. A total of15,427(28.2%) Unigenes were mapped into125pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. According to the biosynthetic pathways of cellulose and lignin currently, the majority of genes coding related enzymes were identified by targeted searches using these annotations. And many of these genes appeared to form multigene families. Novel candidate genes of two metabolic pathways were discovered. Fifteen genes related to cellulose and lignin biosynthesis were cloned and validated by RT-PCR. Overall28Unigenes corresponding to these genes were found by alignment, which covered different regions of subject genes respectively. Furthermore, their expression patterns in different organs were analysed by qRT-PCR to explore their putative functions.3. On the basis of transcriptome data, eight full-length CesA cDNAs from Betula luminifera were isolated using RACE. The predicted proteins encoded by these genes are composed of985to1,103amino acid residues, each of which contains all of motifs characteristic of plant CESA proteins. Eight predicted proteins share limited amino acid identity with each other, ranging from55to82%. Conversely, deduced BlCESAs exhibit81to92%identity with the corresponding PtriCESAs from Populus trichocarpa. The phylogenetic analysis showed that BlCESA1, BlCESA3and BlCESA4belonged to three clades linked to secondary cell wall synthesis, and BlCESA2, BlCESA5, BlCESA6, BlCESA7and BlCESA8belonged to three clades containing the most CESAs associated with primary cell wall synthesis. Gene expression analysis using qRT-PCR indicated that transcripts of BICesAl, BlCesA3and BlCesA4were most abundant in xylem of lignified stem, and presented the similar expression patterns uder the treatments of hormones and sucrose. It implied that proteins encoded by three genes were the parts of the secondary wall CesA complex. But expression patterns of BICesAl, BlCesA3and BlCesA4were slightly different in response to mechanical stress. Although their expression levels in the TW samples were obviously higher than values in the control, three genes exhibited different expression patterns in the OW samples. BlCesA2, BlCesA6and BICesA7predominantly expressed in leaf and phoem. BlCesA8strongly expressed not only in leaf and phoem but also in cambium, which had a higher sequence similarity with BlCesA6. And expression changes of two genes were different from each other under the treatments of hormones, sucrose and bending. Besides involving in biosynthesis of secondary cell wall, BlCesA8might have the other functions.4. Expression patterns of genes between tension and normal woods were analyzed using RNA-Seq technology during the early stage of TW formation (from6h to7d of a tension time course). Of44,680identified Unigenes,4,567were differentially espressed during this stage, which were mapped to1,151pathways through Interactive Pathways analysis. The results of GO comparisons showed that these differentially espressed Unigenes were assigned into40sub-categories, and suggested that metabolic processes were enhanced and catalytic activity increased under the treatment of bending. Furthermore, systematic expression analysis of21genes were conducted, which related to cellulose and lignin biosynthesis. Of163encoded Unigenes,109were differentially espressed during the early stage of TW formation,69.7%of which specifically or preferentially expressed in secondary xylem. Majority of Unigenes involved in cellulose biosynthesis were up-regulated during TW formation, concurrent with cellulose content change. A gene co-expression network for17up-regulated Unigenes was constructed, and BIMYB2, BlOFP1and BlOFP2with similar expression trends were discovered. Many Unigenes encoding the important enzymes of phenylpropanoid pathways alao presented the up-regulated expression, which preferentially expressed in secondary xylem. However, majority of Unigenes annotated as CCR, CAD and SAD were down-regulated during the same stage, which was in accord with the reduced lignin content of TW samples. In addition, expression of many Unigenes annotated as auxin response factors and induced proteins were already enhanced during early stage of TW formation, meanwhile more than half Unigenes encoding auxin efflux carriers were significantly down-regulated.The above results not only laid the foundations for further dissection of wood formation mechanism, but also provided the important candidate genes into development of molecular markers and MAS in Betula luminifera.