Cellulose is the most significant structural component of plant cell wall. Cellulose, polysaccharide containing repeated unbranchedβ(1-4) D-glucose units, is synthesized at the plasma membrane by the cellulose synthase complex (CSC) from bacteria to plants. The CSC is involved in biosynthesis of cellulose microfibrils containing 18 cellulose synthase (CesA) proteins. Macrofibrils can be formed with side by side arrangement of microfibrils. In addition, beside CesA, various proteins like the KORRIGAN, sucrose synthase, cytoskeletal components, and COBRA-like proteins have been involved in cellulose biosynthesis. Understanding the mechanisms of cellulose biosynthesis is of great importance not only for improving wood production in economically important forest trees to mankind but also for plant development. This review article covers the current knowledge about the cellulose biosynthesis-related gene family.

In the methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylose-5-phosphate synthase (DXS) is considered the key enzyme for the biosynthesis of terpenoids. In this study, PmDXS (MK970590) was isolated from Pinus massoniana. Bioinformatics analysis revealed homology of MK970590 with DXS proteins from other species. Relative expression analysis suggested that PmDXS expression was higher in roots than in other plant parts, and the treatment of P. massoniana seedlings with mechanical injury via 15% polyethylene glycol 6000, 10 mM H₂O₂, 50 μM ethephon (ETH), 10 mM methyl jasmonate (MeJA), and 1 mM salicylic acid (SA) resulted in an increased expression of PmDXS. pET28a-PmDXS was expressed in Escherichia coli TransB (DE3) cells, and stress analysis showed that the recombinant protein was involved in resistance to NaCl and drought stresses. The subcellular localization of PmDXS was in the chloroplast. We also cloned a full-length 1024 bp PmDXS promoter. GUS expression was observed in Nicotiana benthamiana roots, stems, and leaves. PmDXS overexpression significantly increased carotenoid, chlorophyll a, and chlorophyll b contents and DXS enzyme activity, suggesting that DXS is important in isoprenoid biosynthesis. This study provides a theoretical basis for molecular breeding for terpene synthesis regulation and resistance.

Transcription factors (TFs) play crucial regulatory roles in controlling the expression of the target genes in plants. APETALA2/Ethylene-responsive factors (AP2/ERF) are part of a large superfamily of plant-specific TFs whose members are involved in the control of plant metabolism, development and responses to various biotic and abiotic stresses. However, the AP2/ERF superfamily has not been identified systematically in Masson pine (Pinus massoniana), which is one of the most important conifer in southern China. Therefore, we performed systematic identification of the AP2/ERF superfamily using transcriptome sequencing data from Masson pine. In the current study, we obtained 88 members of the AP2/ERF superfamily. All PmAP2/ERF members could be classified into 3 main families, AP2 (7 members), RAV (7 members), ERF (73 members) families, and a soloist protein. Subcellular localization assays suggested that two members of PmAP2/ERF were nuclear proteins. Based on pine wood nematode (PWN) inoculated transcriptome and qPCR analysis, we found that many members of PmAP2/ERF could respond to PWN inoculation and PWN related treatment conditions in vitro. In general, members of the AP2/ERF superfamily play an important role in the response of Masson pine responds to PWN. Furthermore, the roles of the AP2/ERF superfamily in other physiological activities of Masson pine remain to be further studied.

The normalization of data by choosing suitable reference genes is fundamental for obtaining accurate and reliable results in quantitative real-time polymerase chain reaction (qPCR) analyses. In this study, the expression stability of 12 candidate reference genes of Pinus massoniana under different abiotic stresses was evaluated using four statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The results indicate that the following genes could be used as reference genes under different treatments: Actin 2 (ACT2) and F-box family gene (F-box) for salinity treatment, cyclophilin (CYP) and alpha-tubulin (TUA) for ABA treatment, actin 7 (ACT7) and CYP for drought treatment, actin 1 (ACT1) and ACT7 for cold treatment, ACT1 and CYP for heat treatment, and TUA and ACT2 for the “Total” group. To validate the suitability of the selected reference genes in this study, the Short-Root protein (SHR), Alpha-pinene synthase (APS), and Pyrabactin resistance-like protein (PYL) gene expression patterns were analyzed. The expression patterns had significant biases when the most unstable reference genes were used for normalization, compared with when the optimum reference gene or gene combinations were used for normalization. These results will be beneficial for further studies on gene transcription in early-stage, unlignified seedlings of P. massoniana.

Cellulose, the most abundant constituent material of the plant cell walls, is a major structural component of plant biomass. Manipulating cellulose synthesis (CesA) genes by genetic engineering technology, to increase cellulose production may thus offer novel opportunities for plant growth and development. To investigate this, here we produced transgenic “Populus 895 plants” overexpressing the cellulose synthase (CesA2) gene derived from Pinus massoniana under the control of constitutive 35S promoter, via Agrobacterium-mediated transformation. Relative expression levels of PmCesA2 were functionally characterized in poplar hybrid clone “Nanlin895” (Populus deltoides × Populus euramericana). The results demonstrated the transgenic lines showed enhanced growth performance with increased biomass production than did the untransformed controls. It is noteworthy that the overexpression of PmCesA2 in poplar led to an altered cell wall polysaccharide composition, which resulted in the thickening of the secondary cell wall and xylem width under scanning electron microscopy. Consequently, the cellulose and lignin content were increased. Hence, this study suggests that overexpression of PmCesA2 could be used as a potential candidate gene to enhance cellulose synthesis and biomass accumulation in genetically engineered trees.

Pinus massoniana Lamb, an economically important conifer tree, is widely distributed in China. WRKY transcription factors (TFs) play important roles in plant growth and development, biological and abiotic stress. Nevertheless, there is little information about the WRKY genes in P. massoniana. By searching for conserved WRKY motifs in transcriptomic RNA sequencing data for P. massoniana, 31 sequences were identified as WRKY TFs. Then, phylogenetic and conserved motif analyses of the WRKY family in P. massoniana, Pinus taeda and Arabidopsis thaliana were used to classify WRKY genes. The expression patterns of six PmWRKY genes from different groups were determined using real-time quantitative PCR for 2-year-old P. massoniana seedings grown in their natural environment and challenged by phytohormones (salicylic acid, methyl jasmonate, or ethephon), abiotic stress (H₂O₂) and mechanical damage stress. As a result, the 31 PmWRKY genes identified were divided into three major groups and several subgroups based on structural and phylogenetic features. PmWRKY genes are regulated in response to abiotic stress and phytohormone treatment and may participate in signaling to improve plant stress resistance. Some PmWRKY genes behaved as predicted based on their homology with A. thaliana WRKY genes, but others showed divergent behavior. This systematic analysis lays the foundation for further identification of WRKY gene functions to aid further exploration of the functions and regulatory mechanisms of PmWRKY genes in biological and abiotic stress in P. massoniana.