Integration of omics approaches to understand oil/protein content during seed development in oilseed crops.

Oilseed crops, especially soybean (Glycine max) and canola/rapeseed (Brassica napus), produce seeds that are rich in both proteins and oils and that are major sources of energy and nutrition worldwide. Most of the nutritional content in the seed is accumulated in the embryo during the seed filling stages of seed development. Understanding the metabolic pathways that are active during seed filling and how they are regulated are essential prerequisites to crop improvement. In this review, we summarize various omics studies of soybean and canola/rapeseed during seed filling, with emphasis on oil and protein traits, to gain a systems-level understanding of seed development. Currently, most (80-85%) of the soybean and rapeseed reference genomes have been sequenced (950 and 850 megabases, respectively). Parallel to these efforts, extensive omics datasets from different seed filling stages have become available. Transcriptome and proteome studies have detected preponderance of starch metabolism and glycolysis enzymes to be the possible cause of higher oil in B. napus compared to other crops. Small RNAome studies performed during the seed filling stages have revealed miRNA-mediated regulation of transcription factors, with the suggestion that this interaction could be responsible for transitioning the seeds from embryogenesis to maturation. In addition, progress made in dissecting the regulation of de novo fatty acid synthesis and protein storage pathways is described. Advances in high-throughput omics and comprehensive tissue-specific analyses make this an exciting time to attempt knowledge-driven investigation of complex regulatory pathways.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato.

Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.

Correlation between transcript abundance of the RB gene and the level of the RB-mediated late blight resistance in potato.

Numerous disease-resistance genes have been cloned and characterized in various plant species. Only a few of these reported genes were transcriptionally induced or had enhanced transcription upon pathogen infection. Here, we report that transcription of the RB gene, which was cloned from the wild potato species Solanum bulbocastanum and confers resistance to potato late blight, was significantly increased after inoculation with the late blight pathogen Phytophthora infestans. Different RB transgenic lines showed different levels of resistance, which were correlated with the amounts of RB transcript in the transgenic plants. Different transgenic lines also showed different patterns of RB transcription 1, 3, and 5 days after P. infestans inoculation. Interestingly, the RB gene showed a higher basal level of transcription and a more dramatic transcriptional increase upon inoculation in S. bulbocastanum than in all potato transgenic lines. Our results revealed a predictive correlation between transcript abundance of the RB gene and the level of the RB-mediated late blight resistance. High level of resistance was associated with a combination of rapid RB transcript induction immediately after pathogen infection followed by the steady production of RB transcript. Thus, the transcription level of the RB gene provides a valuable marker for selecting and deploying RB-containing potato lines for late blight control.

Sgt1, but not Rar1, is essential for the RB-mediated broad-spectrum resistance to potato late blight.

BACKGROUND: Late blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes. RESULTS: We previously cloned a late blight resistance gene, RB, from a diploid wild potato species Solanum bulbocastanum. Transgenic potato lines containing a single RB gene showed a rate-limiting resistance against all known races of Phytophthora infestans, the late blight pathogen. To better understand the RB-mediated resistance we silenced the potato Rar1 and Sgt1 genes that have been implicated in mediating disease resistance responses against various plant pathogens and pests. The Rar1 and Sgt1 genes of a RB-containing potato clone were silenced using a RNA interference (RNAi)-based approach. All of the silenced potato plants displayed phenotypically normal growth. The late blight resistance of the Rar1 and Sgt1 silenced lines were evaluated by a traditional greenhouse inoculation method and quantified using a GFP-tagged P. infestans strain. The resistance of the Rar1-silenced plants was not affected. However, silencing of the Sgt1 gene abolished the RB-mediated resistance. CONCLUSION: Our study shows that silencing of the Sgt1 gene in potato does not result in lethality. However, the Sgt1 gene is essential for the RB-mediated late blight resistance. In contrast, the Rar1 gene is not required for RB-mediated resistance. These results provide additional evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.

Phenotypic and transcriptomic changes associated with potato autopolyploidization.

Polyploidy is remarkably common in the plant kingdom and polyploidization is a major driving force for plant genome evolution. Polyploids may contain genomes from different parental species (allopolyploidy) or include multiple sets of the same genome (autopolyploidy). Genetic and epigenetic changes associated with allopolyploidization have been a major research subject in recent years. However, we know little about the genetic impact imposed by autopolyploidization. We developed a synthetic autopolyploid series in potato (Solanum phureja) that includes one monoploid (1x) clone, two diploid (2x) clones, and one tetraploid (4x) clone. Cell size and organ thickness were positively correlated with the ploidy level. However, the 2x plants were generally the most vigorous and the 1x plants exhibited less vigor compared to the 2x and 4x individuals. We analyzed the transcriptomic variation associated with this autopolyploid series using a potato cDNA microarray containing approximately 9000 genes. Statistically significant expression changes were observed among the ploidies for approximately 10% of the genes in both leaflet and root tip tissues. However, most changes were associated with the monoploid and were within the twofold level. Thus, alteration of ploidy caused subtle expression changes of a substantial percentage of genes in the potato genome. We demonstrated that there are few genes, if any, whose expression is linearly correlated with the ploidy and can be dramatically changed because of ploidy alteration.