Sacha inchi (Plukenetia volubilis L.) is a shrub native to Amazon rainforests that’s of commercial interest as its seeds contain 35–60% edible oil (dry weight). This oil is one of the healthiest vegetable oils due to its high polyunsaturated fatty acid content and favourable ratio of omega-6 to omega-3 fatty acids. De novo transcriptome assembly and comparative analyses were performed on sacha inchi seeds from five stages of seed development in order to identifying genes associated with oil accumulation and fatty acid production. Of 30,189 unigenes that could be annotated in public databases, 20,446 were differentially expressed unigenes. A total of 14 KEGG pathways related to lipid metabolism were found, and 86 unigenes encoding enzymes involved in α-linolenic acid (ALA) biosynthesis were obtained including five unigenes encoding FATA (Unigene0008403), SAD (Unigene0012943), DHLAT (Unigene0014324), α-CT (Unigene0022151) and KAS II (Unigene0024371) that were significantly up-regulated in the final stage of seed development. A total of 66 unigenes encoding key enzymes involved in the synthesis of triacylglycerols (TAGs) were found, along with seven unigenes encoding PDCT (Unigene0000909), LPCAT (Unigene0007846), Oleosin3 (Unigene0010027), PDAT1 (Unigene0016056), GPDH (Unigene0022660), FAD2 (Unigene0037808) and FAD3 (Unigene0044238); these also proved to be up-regulated in the final stage of seed development.

The ω-3 fatty acid desaturase (FAD3) gene encodes a rate-limiting enzyme in the synthesis of α-linolenic acid. In this study, homologous cloning was used to obtain the full-length sequence of the PvFAD3 gene of Plukenetia volubilis. The full-length DNA sequence was 1871 bp long, with 8 exons and 7 introns. The structural analysis of the amino acid sequence revealed that the PvFAD3 protein contained three histidine-conserved regions and an endoplasmic reticulum retention signal. The real-time reverse transcription-polymerase chain reaction performed for determining the expression patterns of the PvFAD3 gene in different tissues of P. volubilis showed that PvFAD3 expression was highly expressed in the fast oil accumulation stage of seed. The analysis of subcellular localization assay in epidermal cells of tobacco (Nicotiana benthamiana) leaves showed that the PvFAD3 protein was mainly localized in the endoplasmic reticulum. Seed-specific overexpression vectors were constructed, and Agrobacterium-mediated genetic transformation was performed to obtain transgenic tobacco plants overexpressing PvFAD3. The results of fatty acid assays performed using harvested seeds showed a significant increase in α-linolenic acid content, a dramatic decrease in linoleic acid content, and an obvious increase in oil content in transgenic tobacco seeds. Collectively, the PvFAD3 gene of P. volubilis was confirmed as a key enzyme gene for α-linolenic acid synthesis; thus, indicating that the PvFAD3 gene can be used for fatty acid fraction improvement in oilseed plants.

Cellulose is the world’s most abundant renewable energy resource, and a variety of cellulose synthase genes are involved in the biosynthesis of cellulose. In the process of cellulose synthesis, all cellulose synthases are interrelated and act synergistically. In this study, we analyzed the contents of cellulose, hemicellulose, and lignin in the different parts and tissues of E. grandis. The results showed that the cellulose content had greater differences among three different heights. On this basis, we carried out the transcriptome-wide profiling of gene expression patterns using RNA sequencing. A total of 2066 differentially expressed genes were identified for three pairwise comparisons between three different heights, most of which were related to the programmed photosynthetic membrane and photosystem. A total of 100 transcripts of CSs (58 CesA and 42 Csl) were obtained from transcriptome libraries. The expression pattern of these genes indicated that different CS genes had a wide range of expression profiles. A phylogenetic analysis of 135 reference CS genes showed that the CSs of E. grandis were clustered into six major groups (CesA1-9, CslA, CslB/H, CslD, CslE, and CslG). Based on the weighted gene co-expression network analysis, a dual-directional regulation mechanism between Csl and CesA proteins in the cellulose biosynthesis was identified. The gene expression profile analysis, using qRT-PCR in different tissues of E. grandis, demonstrated that the CSs were highly expressed in xylem, and CesAs had a higher relative expression than Csls. The analysis of sequence similarity combined with the expression pattern indicated that the CesA1, 3, and 6 transcripts were associated with the biosynthesis of the secondary cell wall, and CesA4, 5, and 7 transcripts were more likely to associate with the biosynthesis of the primary cell wall. Finally, the qRT-PCR analysis confirmed the expression of 11 selected CSs in three different parts of E. grandis.

Cellulose, an essential structural component in the plant cell wall and a renewable biomass resource, plays a significant role in nature. Eucalyptus’s excellent timber tree species (including Eucalyptus grandis Hill) provide many raw materials for the paper and wood industries. The synthesis of cellulose is a very complex process involving multiple genes and regulated by various biological networks. However, research on regulating associated genes and non-coding RNAs during cellulose synthesis in E. grandis remains lacking. In this study, the wood anatomical characteristics and chemical indexes of E. grandis were analyzed by taking three different parts (diameter at breast height (DBH), middle and upper part of the trunk) from the main stem of E. grandis as raw materials. The role of non-coding RNAs (Long non-coding RNA, lncRNA; Micro RNA, miRNA; Circle RNA, circRNA) on regulating candidate genes was presented, and the network map of ceRNA (Competing endogenous RNA) regulation during wood cellulose biosynthesis of E. grandis was constructed. The transcriptome sequencing of nine samples obtained from the trunk of the immature xylem in E. grandis at DBH, middle and upper parts had a 95.81 G clean reading, 57,480 transcripts, 7365 lncRNAs, and 5180 circRNAs. Each sample had 172–306 known miRNAs and 1644–3508 new miRNAs. A total of 190 DE-lncRNAs (Differentially expressed long non-coding RNAs), 174 DE-miRNAs (Differentially expressed micro RNAs), and 270 DE-circRNAs (Differentially expressed circle RNAs) were obtained by comparing transcript expression levels. Four lncRNAs and nine miRNAs were screened out, and the ceRNA regulatory network was constructed. LncRNA1 and lncRNA4 regulated the genes responsible for cellulose synthesis in E. grandis, which were overexpressed in 84K (Populus Alba × Populus glandulosa) poplar. The cellulose and lignin content in lncRNA4-oe were significantly higher than wild type 84K poplar and lncRNA1-oe. The average plant height, middle and basal part of the stem diameter in lncRNA4-oe were significantly higher than the wild type. However, there was no significant difference between the growth of lncRNA1-oe and the wild type. Further studies are warranted to explore the molecular regulatory mechanism of cellulose biosynthesis in Eucalyptus species.

Plukenetia volubilis is a highly promising plant with high nutritional and economic values. In our previous studies, the expression levels of ricin encoded transcripts were the highest in the maturation stage of P. volubilis seeds. The present study investigated the transcriptome and proteome profiles of seeds at two developmental stages (Pv-1 and Pv-2) using RNA-Seq and iTRAQ technologies. A total of 53,224 unigenes and 6026 proteins were identified, with functional enrichment analyses, including GO, KEGG, and KOG annotations. At two development stages of P. volubilis seeds, 8815 unique differentially expressed genes (DEGs) and 4983 unique differentially abundant proteins (DAPs) were identified. Omics-based association analysis showed that ribosome-inactivating protein (RIP) transcripts had the highest expression and abundance levels in Pv-2, and those DEGs/DAPs of RIPs in the GO category were involved in hydrolase activity. Furthermore, 21 RIP genes and their corresponding amino acid sequences were obtained from libraries produced with transcriptome analysis. The analysis of physicochemical properties showed that 21 RIPs of P. volubilis contained ricin, the ricin_B_lectin domain, or RIP domains and could be divided into three subfamilies, with the largest number for type II RIPs. The expression patterns of 10 RIP genes indicated that they were mostly highly expressed in Pv-2 and 4 transcripts encoding ricin_B_like lectins had very low expression levels during the seed development of P. volubilis. This finding would represent valuable evidence for the safety of oil production from P. volubilis for human consumption. It is also notable that the expression level of the Unigene0030485 encoding type I RIP was the highest in roots, which would be related to the antiviral activity of RIPs. This study provides a comprehensive analysis of the physicochemical properties and expression patterns of RIPs in different organs of P. volubilis and lays a theoretical foundation for further research and utilization of RIPs in P. volubilis.