| The problem of wood supply shortage is more obvious with the total prohibition of natural forest in China,so the development and utilization of woody bamboo has become one of the research hotspots.As the most widely distributed bamboo species,moso bamboo（Phyllostachys edulis）has a wide range of uses,including furniture,construction,papermaking and other aspects,especially,it has become a qualified substitute for wood by its virtue of good wood properties.In addition,bamboo pulp papermaking has the advantages of easy raw materials and smooth paper,which can replace part of wood pulp papermaking to a certain extent.However,the degradation of lignin in the process of papermaking has always been one of the difficulties to achieve green environmental protection in bamboo pulp and papermaking industry.Therefore,it is of great significance to regulate the wood quality of bamboo by means of bioengineering through studying the regulatory mechanism of bamboo lignification,elucidating the transcriptional regulation network of secondary wall biosynthesis,and exploring the key genes for lignin biosynthesis and regulation.In this study,combined with the physiological and biochemical changes during the lignification of bamboo shoots,an integrated approach of transcriptome,small RNA and degradome sequencing have been applied to investigate the expression patterns and mechanisms of mRNA and miRNA involved in lignification of moso bamboo shoots,and a genetic regulatory network has been constructed.A genome-wide identification and bioinformatics analysis of the MYBs in this network has been performed,and the key MYB involved in lignification（PeMYB123）were identified,which regulatory function involved in lignin biosynthesis has been validated using molecular biology method.The main research results are as follows:1.Physiological and biochemical changes during lignification of bamboo shootsAccording to the growth characteristics of bamboo shoots,5 shoot heights（1.0 m,2.0 m,4.0 m,6.0 m and 8.0 m）representing different growth stages during the rapid growth were chosen,and the most representative internode(13th)of each height shoot was used as materials.The histochemical staining analysis showed that the number of staining cells in the vascular bundle increased and the staining deepened with the increase of shoot height,indicating that the lignification degree in shoots gradually increased,which presented an S-shaped variable-speed deepening curve of"slow-rapid-slow".In addition,lignin content continued to increase with the increase of shoot height,while the distribution of lignin content in the upper,middle and lower portions of single internode was uneven,in which lignification degree and lignin content changed from’upper>middle>lower’to’lower>middle>up’at 8.0 m shoots.Phenylalanine ammonia-lyase（PAL,EC 4.3.1.5）and Laccase（LAC,EC 1.10.3.2）were key enzymes involved in lignin biosynthesis,which activities were upregulated with trend of increase first and then decrease along with the shoot height.2.Gene expression and regulatory network during bamboo shoot lignificationThere were 11504 differentially expressed genes（DEGs）found in the 13th internode of different height shoots using transcriptome sequencing,where most DEGs associated with cell wall and lignin biosynthesis were up-regulated,whereas some DEGs related to cell growth were down-regulated.A total of 1502 miRNAs including 1223 known and 279 novel ones were identified,of which 687 ones were differentially expressed using miRNA sequencin.In summary,in silico and degradome sequencing analyses indicated that 5756 genes were targeted by 691miRNAs.Through weighted gene co-expression network analysis（WGCNA）,five gene modules related to lignin biosynthesis were obtained.A regulatory network of lignin biosynthesis in moso bamboo was constructed with 11 miRNAs,22 transcription factors and 36 enzyme genes.MYB transcription factors are the core regulators of this network,which are regulated by NAC and directly regulates the downstream enzyme genes,including the 4-coumarate:Co A ligase（4CL）gene that determines the specific biosynthetic pathway into which the phenylpropane metabolic pathway enters.3.Genome-wide identification and expression analysis of MYB transcription factorA total of 222 PeMYBs,which included 214 typical R2R3-MYB proteins（2R-MYB）,six R1R2R3-MYB proteins（3R-MYB）and two 4R-like MYB protein（4R-MYB）,were identified in moso bamboo by using a genome-wide search strategy.The corresponding genes were named as PeMYB1～PeMYB214,PeMYB3R-1～PeMYB3R-6 and PeMYB4R-1～PeMYB4R-2 in turn.Gene structure analysis showed that PeMYBs were obviously different with exon numbers varied from 1 to 12.Phylogenetic analysis indicated that PeMYBs clustered into 31 subfamilies,of which 22 subfamilies had been divided into four functional classes.The members of seven subfamilies were predicted to have the regulatory function of the biosynthesis and deposition of lignin,cellulose and hemicellulose,which were responsible for the formation of secondary cell walls（SCWs）.In addition,almost all the PeMYBs showed significant tissue specificity based on RNA-seq data.Furthermore,quantitative real-time PCR（qPCR）analysis showed that the transcript abundance levels of 12 PeMYBs related to the biosynthesis and deposition of SCWs have changed significantly with the increasing degree of shoot lignification,indicating that these PeMYBs might play fundamental roles in SCW thickening and shoot lignification of bamboo.4.Functional validation of PeMYB123PeMYB123 is one of PeMYBs in the lignification regulatory network in moso bamboo.PeMYB123 was isolated from moso bamboo by RT-PCR,and its ORF was 969 bp encoding a protein of 322 amino acids with a typical R2R3 domain.The protein was homologous to the lignin regulator At MYB103 in Arabidopsis.PeMYB123 constitutively expressed in moso bamboo,and the highest level was in shoots with a trend of first rise and then decrease with the shoot height increasing,followed by in nodes and internodes,and it was low in culm sheaths,shoot buds and roots.The PeMYB123 transgenic Arabidopsis plants have been obtained through Agrobacterium mediated transformation.Compared with the wild type,the inflorescence stem diameter of PeMYB123 transgenic plants was thicker,which resulted in the increase of biomass,with the cellulose and hemicellulose content increased（the most up to 40.58%and 45.26%）,while the lignin content decreased（the most up to 18.88%）.Expression profiles assay showed a substantial induction in the expression of genes involved in cellulose and hemicellulose biosynthesis,while most of those involved in lignin biosynthesis were significantly suppressed.These results suggested that PeMYB123 may control lignification through regulating the expression of key enzyme genes.5.Identification of 4CL genes and its regulation by PeMYB123There were 15 homologous genes of 4CL in moso bamboo（Pe4CL1～Pe4CL15）.Phylogenetic analysis showed that all the Pe4CLs were clustered into two subfamilies（A and B）,and those in A were divided into three Types（Type Ⅰ～Ⅲ）.The binding sites（AC element and SMRE）of MYB were found in all the promoters of 15 Pe4CLs.Tissue specific expression analysis indicated that Pe4CLs had significantly different expression patterns in different tissues,as well as bamboo shoots at different growth stages.The results of qPCR demonstrated that 12Pe4CLs were upregulated with the increasing height of shoots except Pe4CL6 with a down-regulated expression pattern.Transcriptional activity results showed that the complete PeMYB123 and C-terminal had transcriptional activities,while N-terminal or R2R3 structural sequences had no transcriptional activities.The results of yeast one-hybrid experiment showed that PeMYB123 could bind to the promoters of Pe4CLs.These results indicated that PeMYB123may participate in lignin biosynthesis by regulating the transcription of Pe4CLs during lignification of moso bamboo,thereby affecting the deposition of SCW.Our findings have important scientific value for analyzing the molecular mechanism of bamboo lignin biosynthesis.In addition,it provides a reference for studying the lignification-related mechanisms in other monocotyledons,and provides important genetic resources for genetic engineering breeding of moso bamboo. |
