De novo characterization of the Chinese fir (Cunninghamia lanceolata) transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.

BACKGROUND: Chinese fir (Cunninghamia lanceolata) is an important timber species that accounts for 20-30% of the total commercial timber production in China. However, the available genomic information of Chinese fir is limited, and this severely encumbers functional genomic analysis and molecular breeding in Chinese fir. Recently, major advances in transcriptome sequencing have provided fast and cost-effective approaches to generate large expression datasets that have proven to be powerful tools to profile the transcriptomes of non-model organisms with undetermined genomes. RESULTS: In this study, the transcriptomes of nine tissues from Chinese fir were analyzed using the Illumina HiSeq™ 2000 sequencing platform. Approximately 40 million paired-end reads were obtained, generating 3.62 gigabase pairs of sequencing data. These reads were assembled into 83,248 unique sequences (i.e. Unigenes) with an average length of 449 bp, amounting to 37.40 Mb. A total of 73,779 Unigenes were supported by more than 5 reads, 42,663 (57.83%) had homologs in the NCBI non-redundant and Swiss-Prot protein databases, corresponding to 27,224 unique protein entries. Of these Unigenes, 16,750 were assigned to Gene Ontology classes, and 14,877 were clustered into orthologous groups. A total of 21,689 (29.40%) were mapped to 119 pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The majority of the genes encoding the enzymes in the biosynthetic pathways of cellulose and lignin were identified in the Unigene dataset by targeted searches of their annotations. And a number of candidate Chinese fir genes in the two metabolic pathways were discovered firstly. Eighteen genes related to cellulose and lignin biosynthesis were cloned for experimental validating of transcriptome data. Overall 49 Unigenes, covering different regions of these selected genes, were found by alignment. Their expression patterns in different tissues were analyzed by qRT-PCR to explore their putative functions. CONCLUSIONS: A substantial fraction of transcript sequences was obtained from the deep sequencing of Chinese fir. The assembled Unigene dataset was used to discover candidate genes of cellulose and lignin biosynthesis. This transcriptome dataset will provide a comprehensive sequence resource for molecular genetics research of C. lanceolata.

Genome-wide identification and characterization of OVATE family proteins in Betula luminifera reveals involvement of BlOFP3 and BlOFP5 genes in leaf development.

Ovate family proteins (OFP) are plant-specific transcription factors involved in regulating morphologies of the lateral organs, plant growth and development. However, the functional roles of OFP genes in Betula luminifera, an important timber tree species, are not well studied. In this study, we identified 20 BlOFP genes and analyzed their phylogenetic relationship, gene structure, conserved motifs, and cis-elements. Further, expression analysis indicates that BlOFP genes were up-regulated in leaves on the one-year-old branch compared to leaves on the current-year branch and bract, except BlOFP7, BlOFP11, BlOFP14 and BlOFP12. The overexpression of BlOFP3 and BlOFP5 in Arabidopsis thaliana not only resulted in a slower growth rate but also produced sawtooth shape, flatter and darker green rosette leaves. Further investigation showed that the leaf thickness of the transgenic plants was more than double that of the wild type, which was caused by the increasement in the number and size of palisade tissue cells. Furthermore, the expression analysis also indicated that the expressions of several genes related to leaf development were significantly changed in the transgene plants. These results suggested the significant roles of BlOFP3 and BlOFP5 in leaf development. Moreover, protein-protein interaction studies showed that BlOFP3 interacts with BlKNAT5, and BlOFP5 interacts with BlKNAT5, BlBLH6 and BlBLH7. In conclusion, our study demonstrates that BlOFP3 and BlOFP5 were involved in leaf shape and thickness regulation by forming a complex with BlKNAT5, BlBLH6 and BlBLH7. In addition, our study serves as a guide for future functional genomic studies of OFP genes of the B. luminifera.

Identification and characterization of two bamboo (Phyllostachys praecox) AP1/SQUA-like MADS-box genes during floral transition.

Bamboo (Bambusoideae) is by far the largest member of the grass family Poaceae, which is vital to the economy of many countries in the tropics and subtropics. However, the mechanism of flowering of bamboo (Phyllostachys praecox) is still unknown. In this study, we isolated two novel genes from P. praecox and evaluated their functional characteristics. The sequence and phylogenetic analysis indicated that these two genes, named PpMADS1 and PpMADS2, belong to FUL3 and FUL1 clade of Poaceae AP1/SQUA-like genes, respectively. The PpMADS2 possesses a truncated C terminus lacking the highly conserved paleoAP1 motif. It was further confirmed that the truncated C-terminal region was produced by natural sequence deletion in exons, but not by alternative splicing. Ectopic expression of PpMADS1 and PpMADS2 significantly promoted early flowering through upregulation of AP1 in Arabidopsis. Yeast two-hybrid experiments demonstrated that AP1 protein can interact with PpMADS1 but not PpMADS2, suggesting that these two genes may act differently in signaling early flowering of bamboo plants. RT-qPCR and in situ hybridization analysis revealed distinct expression patterns of these two genes in vegetative and reproductive tissues of bamboo. Taken together, our results suggest that both PpMADS1 and PpMADS2 are involved in floral transition, and PpMADS2 might play more important roles than PpMADS1 in floral development of Phyllostachys praecox.

Molecular Characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Family in Betula luminifera.

As a major family of plant-specific transcription factors, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes play vital regulatory roles in plant growth, development and stress responses. In this study, 18 SPL genes were identified and cloned from Betula luminifera. Two zinc finger-like structures and a nuclear location signal (NLS) segments were existed in the SBP domains of all BlSPLs. Phylogenetic analysis showed that these genes were clustered into nine groups (group I-IX). The intron/exon structure and motif composition were highly conserved within the same group. 12 of the 18 BlSPLs were experimentally verified as the targets of miR156, and two cleavage sites were detected in these miR156-targeted BlSPL genes. Many putative cis-elements, associated with light, stresses and phytohormones response, were identified in the promoter regions of BlSPLs, suggesting that BlSPL genes are probably involved in important physiological processes and developmental events. Tissue-specific expression analysis showed that miR156-targeted BlSPLs exhibited a more differential expression pattern, while most miR156-nontargeted BlSPLs tended to be constitutively expressed, suggesting the distinct roles of miR156-targeted and nontargeted BlSPLs in development and growth of B. luminifera. Further expression analysis revealed that miR156-targeted BlSPLs were dramatically up-regulated with age, whereas mature BlmiR156 level was apparently declined with age, indicating that miR156/SPL module plays important roles in vegetative phase change of B. luminifera. Moreover, yeast two-hybrid assay indicated that several miR156-targeted and nontargeted BlSPLs could interact with two DELLA proteins (BlRGA and BlRGL), which suggests that certain BlSPLs take part in the GA regulated processes through protein interaction with DELLA proteins. All these results provide an important basis for further exploring the biological functions of BlSPLs in B. luminifera.

Molecular cloning, expression analyses and primary evolution studies of REV- and TB1-like genes in bamboo.

Most cultured bamboos are perennial woody evergreens that reproduce from rhizomes. It is unclear why some rhizome buds develop into aerial bamboo shoots instead of new rhizomes. REVOLUTA (REV)-like Class III homeodomain leucine-zipper (HD-Zip) proteins and TEOSINTE BRANCHED1 (TB1)-like transcription factors have been shown to play regulatory roles in meristem initiation and outgrowth. We cloned and analyzed the bamboo (Phyllostachys praecox C.D. Chu & C.S. Chao.) REV- (PpHB1) and TB1-like (PpTB1) gene. Gene expression was mainly detected by in situ hybridization. PpHB1 expression was detected in the tips of lateral buds, on the adaxial portion of the leaf and within the developing procambium, indicating its close correlation to rhizome bud formation and procambial development. PpTB1 expression was mainly detected on the top of buds at later developmental stages, suggesting it was more likely involved in bud outgrowth. Meristem genes might therefore serve as specific molecular markers of rhizome bud development and could be useful in studies designed to elucidate the mechanisms underlying bamboo shoot development. In addition, meristem genes such as TB1-like sequences may be useful in phylogenetic analyses of bamboo species.