High production of recombinant protein using geminivirus-based deconstructed vectors in Nicotiana benthamiana.

We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plant-based production of recombinant proteins. Confirmation of the optimal combination by co-delivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a gene-silencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants.

Functional Characterization of the Effects of CsDGAT1 and CsDGAT2 on Fatty Acid Composition in Camelina sativa.

Triacylglycerols (TAGs) are the storage oils of plant seeds, and these lipids provide energy for seed germination and valuable oils for human consumption. Three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) and phospholipid:diacylglycerol acyltransferases participate in the biosynthesis of TAGs. DGAT1 and DGAT2 participate in the biosynthesis of TAGs through the endoplasmic reticulum (ER) pathway. In this study, we functionally characterized CsDGAT1 and CsDGAT2 from camelina (Camelina sativa). Green fluorescent protein-fused CsDGAT1 and CsDGAT2 localized to the ER when transiently expressed in Nicotiana benthamiana leaves. To generate Csdgat1 and Csdgat2 mutants using the CRISPR/Cas9 system, camelina was transformed with a binary vector carrying Cas9 and the respective guide RNAs targeting CsDGAT1s and CsDGAT2s via the Agrobacterium-mediated floral dip method. The EDD1 lines had missense and nonsense mutations in the CsDGAT1 homoeologs, suggesting that they retained some CsDGAT1 function, and their seeds showed decreased eicosaenoic acid (C20:1) contents and increased C18:3 contents compared to the wild type (WT). The EDD2 lines had a complete knockout of all CsDGAT2 homoeologs and a slightly decreased C18:3 content compared to the WT. In conclusion, CsDGAT1 and CsDGAT2 have a small influence on the seed oil content and have an acyl preference for C20:1 and C18:3, respectively. This finding can be applied to develop oilseed plants containing high omega-3 fatty acids or high oleic acid.

Ectopic Expression of Perilla frutescens WRI1 Enhanced Storage Oil Accumulation in Nicotiana benthamiana Leaves.

Vegetable oils are indispensable in human and animal diets and have been widely used for the production of detergents, lubricants, cosmetics, and biofuels. The seeds of an allotetraploid Perilla frutescens contain approximately 35 to 40% oils with high levels of polyunsaturated fatty acids (PUFAs). WRINKELD1 (WRI1) encoding an AP2/ERF-type transcription factor is known to upregulate the expression of genes involved in glycolysis and fatty acid biosynthesis and TAG assembly. In this study, two WRI1 isoforms, PfWRI1A, and PfWRI1B were isolated from Perilla and predominantly expressed in developing Perilla seeds. The fluorescent signals from PfWRI1A:eYFP and PfWRI1B:eYFP driven by the CaMV 35S promoter were detected in the nucleus of the Nicotiana benthamiana leaf epidermis. Ectopic expression of each of PfWRI1A and PfWRI1B increased the levels of TAG by approximately 2.9- and 2.7-fold in N. benthamiana leaves and particularly, the enhanced levels (mol%) of C18:2, and C18:3 in the TAGs were prominent with the concomitant reduction in the amounts of saturated fatty acids. The expression levels of NbPl-PKß1, NbKAS1, and NbFATA, which were known to be target genes of WRI1, significantly increased in tobacco leaves overexpressing PfWRI1A or PfWRI1B. Therefore, newly characterized PfWRI1A and PfWRI1B can be potentially useful for the enhanced accumulation of storage oils with increased PUFAs in oilseed crops.

Plant-made pharmaceuticals: exploring studies for the production of recombinant protein in plants and assessing challenges ahead.

The production of pharmaceutical compounds in plants is attracting increasing attention, as plant-based systems can be less expensive, safer, and more scalable than mammalian, yeast, bacterial, and insect cell expression systems. Here, we review the history and current status of plant-made pharmaceuticals. Producing pharmaceuticals in plants requires pairing the appropriate plant species with suitable transformation technology. Pharmaceuticals have been produced in tobacco, cereals, legumes, fruits, and vegetables via nuclear transformation, chloroplast transformation, transient expression, and transformation of suspension cell cultures. Despite this wide range of species and methods used, most such efforts have involved the nuclear transformation of tobacco. Tobacco readily generates large amounts of biomass, easily accepts foreign genes, and is amenable to stable gene expression via nuclear transformation. Although vaccines, antibodies, and therapeutic proteins have been produced in plants, such pharmaceuticals are not readily utilized by humans due to differences in glycosylation, and few such compounds have been approved due to a lack of clinical data. In addition, achieving an adequate immune response using plant-made pharmaceuticals can be difficult due to low rates of production compared to other expression systems. Various technologies have recently been developed to help overcome these limitations; however, plant systems are expected to increasingly become widely used expression systems for recombinant protein production.

The seed-specific transcription factor DPBF2 modulates the fatty acid composition in seeds.

Triacylglycerol (TAG), an ester derived from glycerol and three fatty acids (FAs), is synthesized during seed development and controlled by transcriptional regulation. We examined the mechanism regulating the FA composition of developing Arabidopsis thaliana seeds. The seed-specific DC3 PROMOTER-BINDING FACTOR2 (DPBF2) transcription factor was upregulated by LEAFY COTYLEDON2 (LEC2). DPBF2 showed transcriptional activity in yeast and localized to the nucleus in Arabidopsis protoplast cells. The Arabidopsis dpbf2-1 homozygous T-DNA mutant and transgenic lines overexpressing of DPBF2 using a seed-specific phaseolin promoter in wild-type (WT) Arabidopsis and in dpbf2-1 showed similar FA composition profiles in their seeds. Their 18:2 and 20:1 FA contents were higher, but 18:1 and 18:3 contents were lower than that of WT. Transcript levels of FATTY ACID DESATURASE2 (FAD2), FAD3, LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE1 (LPCAT1), LPCAT2, PHOSPHATIDYLCHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE (PDCT), and FATTY ACID ELONGASE 1 (FAE1) are increased in DPBF2-overexpressing seeds. Besides, PDCT and FAE1 were upregulated by DPBF2, LEC1-LIKE (L1L), and NUCLEAR FACTOR-YC2 (NF-YC2) transcriptional complex based on tobacco protoplast transcriptional activation assay. These results suggest that DPBF2 effectively modulates the expression of genes encoding FA desaturases, elongase, and acyl-editing enzymes for modifying the unsaturated FA composition in seeds.

Increasing Monounsaturated Fatty Acid Contents in Hexaploid Camelina sativa Seed Oil by FAD2 Gene Knockout Using CRISPR-Cas9.

Seed oils are used as edible oils and increasingly also for industrial applications. Although high-oleic seed oil is preferred for industrial use, most seed oil is high in polyunsaturated fatty acids (PUFAs) and low in monounsaturated fatty acids (MUFAs) such as oleic acid. Oil from Camelina, an emerging oilseed crop with a high seed oil content and resistance to environmental stress, contains 60% PUFAs and 30% MUFAs. Hexaploid Camelina carries three homoeologs of FAD2, encoding fatty acid desaturase 2 (FAD2), which is responsible for the synthesis of linoleic acid from oleic acid. In this study, to increase the MUFA contents of Camelina seed oil, we generated CsFAD2 knockout plants via CRISPR-Cas9-mediated gene editing using the pRedU6fad2EcCas9 vector containing DsRed as a selection marker, the U6 promoter to drive a single guide RNA (sgRNA) covering the common region of the three CsFAD2 homoeologs, and an egg-cell-specific promoter to drive Cas9 expression. We analyzed CsFAD2 homoeolog-specific sequences by PCR using genomic DNA from transformed Camelina leaves. Knockout of all three pairs of FAD2 homoeologs led to a stunted bushy phenotype, but greatly enhanced MUFA levels (by 80%) in seeds. However, transformants with two pairs of CsFAD2 homoeologs knocked out but the other pair wild-type heterozygous showed normal growth and a seed MUFAs production increased up to 60%. These results provide a basis for the metabolic engineering of genes that affect growth in polyploid crops through genome editing.

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

Current progress towards the metabolic engineering of plant seed oil for hydroxy fatty acids production.

KEY MESSAGE: Hydroxy fatty acids produced in plant seed oil are important industrial material. This review focuses on the use of metabolic engineering approaches for the production of hydroxy fatty acids in transgenic plants. Vegetable oil is not only edible but can also be used for industrial purposes. The industrial demand for vegetable oil will increase with the continued depletion of fossil fuels and ensuing environmental issues such as climate change, caused by increased carbon dioxide in the air. Some plants accumulate high levels of unusual fatty acids in their seeds, and these fatty acids (FAs) have properties that make them suitable for industrial applications. Hydroxy fatty acids (HFAs) are some of the most important of these industrial FAs. Castor oil is the conventional source of HFA. However, due to the presence of toxin ricin in its seeds, castor is not cultivated on a large scale. Lesquerella is another HFA accumulator and is currently being developed as a new crop for a safe source of HFAs. The mechanisms of HFA synthesis and accumulation have been extensively studied using castor genes and the model plant Arabidopsis. HFAs accumulated to 17% in the seed oil of Arabidopsis expressing a FA hydroxylase gene from castor (RcFAH12), but its seed oil content and plant growth decreased. When RcFAH12 gene was coexpressed with additional castor gene(s) in Arabidopsis, ~30% HFAs were accumulated and the seed oil content and plant growth was almost restored to the wild-type level. Further advancement of our understanding of pathways, genes and regulatory mechanisms underlying synthesis and accumulation of HFAs is essential to developing and implementing effective genetic approaches for enhancing HFA production in oilseeds.

Genistein production in rice seed via transformation with soybean IFS genes.

To produce genistein in rice, the isoflavone synthase (IFS) genes, SpdIFS1 and SpdIFS2 were cloned from the Korean soybean cultivar, Sinpaldalkong II as it has a higher genistein content than other soybean varieties. SpdIFS1 and SpdIFS2 show a 99.6% and 98.2% identity at the nucleotide level and 99.4% and 97.9% identity at the amino acid level, respectively, with IFS1 and IFS2 from soybean (GenBank accession Nos. AF195798 and AF195819). Plant expression vectors were constructed harboring SpdIFS1 or SpdIFS2 under the control of a rice globulin promoter that directs seed specific expression, and used to transform two rice varieties, Heugnam, a black rice, and Nakdong, a normal rice cultivar without anthocyanin pigment. Because naringenin, the substrate of SpdIFS1 and SpdIFS2, is on the anthocyanin biosynthesis pathway, the relative production rate of genistein was compared between SpdIFS-expressing transgenic Heugnam and Nakdong. Southern blot analysis of eight of the resulting transgenic rice plants revealed that the T0 plants had one to three copies of the SpdIFS1 or SpdIFS2 gene. The highest level of genistein content found in rice seeds was 103 µg/g. These levels were about 30-fold higher in our transgenic rice lines than the genistein aglycon content of a non-leguminous IFS-expressing transgenic tobacco petal, equaling about 12% of total genistein content of Sinpaldalkong II. There were no significant differences found between the genistein content in Heugnam and Nakdong transgenic rice plants.

Ectopic overexpression of castor bean LEAFY COTYLEDON2 (LEC2) in Arabidopsis triggers the expression of genes that encode regulators of seed maturation and oil body proteins in vegetative tissues.

The LEAFY COTYLEDON2 (LEC2) gene plays critically important regulatory roles during both early and late embryonic development. Here, we report the identification of the LEC2 gene from the castor bean plant (Ricinus communis), and characterize the effects of its overexpression on gene regulation and lipid metabolism in transgenic Arabidopsis plants. LEC2 exists as a single-copy gene in castor bean, is expressed predominantly in embryos, and encodes a protein with a conserved B3 domain, but different N- and C-terminal domains to those found in LEC2 from Arabidopsis. Ectopic overexpression of LEC2 from castor bean under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants induces the accumulation of transcripts that encodes five major transcription factors (the LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON1-LIKE (L1L), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) transcripts for seed maturation, and WRINKELED1 (WRI1) transcripts for fatty acid biosynthesis), as well as OLEOSIN transcripts for the formation of oil bodies in vegetative tissues. Transgenic Arabidopsis plants that express the LEC2 gene from castor bean show a range of dose-dependent morphological phenotypes and effects on the expression of LEC2-regulated genes during seedling establishment and vegetative growth. Expression of castor bean LEC2 in Arabidopsis increased the expression of fatty acid elongase 1 (FAE1) and induced the accumulation of triacylglycerols, especially those containing the seed-specific fatty acid, eicosenoic acid (20:1(Δ11)), in vegetative tissues.