Molecular tailoring of farnesylation for plant drought tolerance and yield protection

Protecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the beta-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the alpha- or beta-subunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the non-transgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficit-induced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.     

The biochemistry of headgroup exchange during triacylglycerol synthesis in canola

Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFAs) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed oil synthesis. Loss of expression of BnROD1.A3 and BnROD1.C3 in a double mutant, or by RNA interference, reduced the PUFA content of the oil to 26.6% compared with 32.5% in the wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFAs that accumulate in the seed oil compared with 40% in Arabidopsis. Our results demonstrate the care needed when translating results from a model species to crop plants.     

Two seed coat-specific promoters are functionally conserved between <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

Canola is one of the most important cash crops in Canada, and a national project named “Designing Oilseeds for Tomorrow’s Market” was undertaken to improve seed meal quality of this strategically important crop. As a part of this project, our group is focusing on identifying seed coat-specific promoters for canola (Brassica napus). These promoters will be used to genetically modify canola seed coat to reduce or eliminate anti-nutritional components from the meal. The Arabidopsis thaliana BAN promoter (AtBANpro) and δVPE promoter (AtδVPEpro) were isolated and fused to GUS reporter gene to generate transgenic canola plants. These plants were analyzed by GUS staining and microtome sectioning which showed that both promoters are seed coat-specific in canola: AtBANpro in inner seed coat layer and AtδVPEpro in outer seed coat layer. Therefore, the two Arabidopsis promoters can be used to modify genes in seed coat of canola for further improving its seed qualities.     

Maternal control of seed weight in rapeseed (Brassica napus L.): the causal link between the size of pod (mother,source) and seed (offspring,sink)

Seed size/weight is one of the key traits related to plant domestication and crop improvement. In rapeseed (Brassica napus L.) germplasm, seed weight shows extensive variation, but its regulatory mechanism is poorly understood. To identify the key mechanism of seed weight regulation, a systematic comparative study was performed. Genetic, morphological and cytological evidence showed that seed weight was controlled by maternal genotype, through the regulation of seed size mainly via cell number. The physiological evidence indicated that differences in the pod length might result in differences in pod wall photosynthetic area, carbohydrates and the final seed weight. We also identified two pleiotropic major quantitative trait loci that acted indirectly on seed weight via their effects on pod length. RNA‐seq results showed that genes related to pod development and hormones were significantly differentially expressed in the pod wall; genes related to development, cell division, nutrient reservoir and ribosomal proteins were all up‐regulated in the seeds of the large‐seed pool. Finally, we proposed a potential seed weight regulatory mechanism that is specific to rapeseed and novel in plants. The results demonstrate a causal link between the size of the pod (mother, source) and the seed (offspring, sink) in rapeseed, which provides novel insight into the maternal control of seed weight and will open a new research field in plants.     

Effects of fall and spring seeding date and other agronomic factors on infestations of root maggots, Delia spp. (Diptera: Anthomyiidae), in canola

Several agronomic benefits can result from fall seeding of canola (Brassica spp.), but extensive research data are lacking on the potential impact of this practice on infestations of root maggots (Delia spp.) (Diptera: Anthomyiidae), which are major pests of the crop in western Canada. Field experiments making up 13 location by year combinations were conducted in central Alberta, Canada, from 1998 to 2001 to determine the effect of fall versus spring seeding of canola on root maggot damage. Depending on the experiment, interactions with seeding rate, seed treatment, timing of weed removal, and canola species (cultivar) also were investigated. Root maggot damage declined with an increase in seeding rate for plots seeded in May but not in fall or April. Susceptibility to infestation was greater for plants of Brassica rapa L. than Brassica napus L., but seed treatment had no effect on damage by these pests. Combined analysis using data from all experiment by location by year combinations indicated that seeding date had no significant effect on root maggot damage. The extended emergence of Delia spp. adults, which spans the appearance of crop stages vulnerable to oviposition regardless of seeding date, prevented reduced root maggot attack. Covariance analysis demonstrated the importance of increasing seeding rate for reducing root maggot infestations, a practice that can be especially beneficial for May-seeded canola when growing conditions limit the ability of plants to compensate for root maggot damage. Results determined with the small plot studies described here should be validated in larger plots or on a commercial field scale, but both the combined and covariance analyses indicate that seeding canola in fall does not predispose plants to greater damage by larval root maggots than seeding in spring.     

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.     

Drawing lines and borders: how the dehiscent fruit of Arabidopsis is patterned

The advent of fruits marked a key innovation in the evolution of flowering plants and helped generate a diverse array of mechanisms for seed dispersal. In the model plant, Arabidopsis thaliana, seed dispersal occurs through a process known as "pod-shatter" in which the fruit structure falls to pieces upon light mechanical pressures. This dispersal mechanism is dependent on the careful patterning of tissues in the fruit, which perform diverse functions that enable the fruit to open at maturation. Using the genetic power of Arabidopsis, many of the molecular components that help specify these tissues have been identified. Studies of the interactions among these genes have revealed a regulatory network that limits processes such as cell-cell separation and lignification to discreet regions of the fruit. Knowledge of these processes in a model fruit creates a foundation on which to build an understanding of the evolution of fruit form in other species and provides tools to engineer shatter-resistant seed pods to prevent crop loss in plants of agronomic importance such as canola.     

Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea

Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.     

A seed coat outer integument-specific promoter for Brassica napus

In search for seed coat-specific promoters for canola (Brassica napus), transgenic plants carrying a 2,121 bp fragment of Arabidopsis thaliana At4g12960 promoter (AtGILTpro) fused to the uidA reporter gene (GUS) were generated. Out of 7 independent events in transgenic canola plants raised, 2 exhibited GUS activity exclusively in the outer integument of the seed coat. GUS activity in other tissues was also observed in the remaining five transformants. Therefore, the AtGILT promoter can be used as a canola seed coat outer integument-specific promoter after the generation and selection of desired transformants from several transgenic lines.     

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.     

Identification,characterization and field testing of Brassica napus mutants producing high‐oleic oils

Producing healthy, high‐oleic oils and eliminating trans‐fatty acids from foods are two goals that can be addressed by reducing activity of the oleate desaturase, FAD2, in oilseeds. However, it is essential to understand the consequences of reducing FAD2 activity on the metabolism, cell biology and physiology of oilseed crop plants. Here, we translate knowledge from studies of fad2 mutants in Arabidopsis (Arabidopsis thaliana) to investigate the limits of non‐GMO approaches to maximize oleic acid in the seed oil of canola (Brassica napus), a species that expresses three active FAD2 isozymes. A series of hypomorphic and null mutations in the FAD2.A5 isoform were characterized in yeast (Saccharomyes cerevisiae). Then, four of these were combined with null mutations in the other two isozymes, FAD2.C5 and FAD2.C1. The resulting mutant lines contained 71–87% oleic acid in their seed oil, compared with 62% in wild‐type controls. All the mutant lines grew well in a greenhouse, but in field experiments we observed a clear demarcation in plant performance. Mutant lines containing less than 80% oleate in the seed oil were indistinguishable from wild‐type controls in growth parameters and seed oil content. By contrast, lines with more than 80% oleate in the seed oil had significantly lower seedling establishment and vigor, delayed flowering and reduced plant height at maturity. These lines also had 7–11% reductions in seed oil content. Our results extend understanding of the B. napusFAD2 isozymes and define the practical limit to increasing oil oleate content in this crop species.     

In search of the best pollinators for canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) production in Pakistan

To discover the pollinator community of canola (Brassica napus L.) and the best pollinators for canola production, an experiment was performed at the research farm of Bahauddin Zakariya University, Multan, Pakistan. The insect pollinator community was composed of 35 species in 3 orders and 14 families. Most of the bees (Hymenoptera) and a butterfly species (Lepidoptera) foraged for nectar, whereas all the flies (Diptera) foraged either for pollen or both nectar and pollen. Eight major pollinators were tested for their pollination efficiency. The nectar-robbing behavior of many species made it difficult to judge the efficiency of an insect on the basis of visitation rate and stay time; therefore, the amounts of pollen deposited and pollen harvested per visit were also measured. The single visit efficiency in terms of the number of seeds per pod revealed that Apis dorsata, A. florea, and Halictus sp. were superior for canola pollination, having Spears’ values of 1.62, 1.55 and 1.73, respectively. With the increase in the number of seeds per pod, seed weight per pod also increased, confirming the importance of these three pollinator species in canola production.     

Crop sequence effects on root maggot (Diptera: Anthomyiidae: Delia spp.) infestations in canola

Strong market demand for canola, Brassica napus L., has prompted some western Canadian producers to increase the frequency of this crop in rotations with other crop species, but the impact of this practice on canola insect pests has not been determined. Here, we investigate 12 cropping sequences involving canola over a 3-yr period (2008-2010 inclusive) at five locations across western Canada. Cropping sequences varied from continuous production of two herbicide-tolerant canola varieties, to production in two of 3 yr, to canola production in one of the 3 yr. Treatments analyzed were the frequency and timing of canola within the rotational sequence. Damage by larvae of root maggots (Diptera: Anthomyiidae: Delia spp.) to canola taproots increased as the study progressed, particularly in 2010 after canola had been grown continuously for 3 yr. Yield declined with continuous canola production, and differences were greatest in 2010. At mean canola crop prices for 2010, the yield reduction from continuous production amounted to economic losses of approximately Can$282-$377/ha. Crop quality, in terms of oil and protein concentrations of harvested seed, was affected more by crop variety than cropping sequence. Crop sequence effects for root maggot damage, yield, and seed quality were relatively stable in the presence of environmental (location) variation. Results of our study suggest that continuous canola production could be unsustainable over the long-term even though market forces currently provide incentive for this practice.     

Reduced abundance and earlier collection of bumble bee workers under intensive cultivation of a mass‐flowering prairie crop

One of the most commonly seeded crops in Canada is canola, a cultivar of oilseed rape (Brassica napus). As a mass‐flowering crop grown intensively throughout the Canadian Prairies, canola has the potential to influence pollinator success across tens of thousands of square kilometers of cropland. Bumble bees (Bombus sp.) are efficient pollinators of many types of native and crop plants. We measured the influence of this mass‐flowering crop on the abundance and phenology of bumble bees, and on another species of social bee (a sweat bee; Halictus rubicundus), by continuously deploying traps at different levels of canola cultivation intensity, spanning the start and end of canola bloom. Queen bumble bees were more abundant in areas with more canola cover, indicating that this crop is attractive to queens. However, bumble bee workers were significantly fewer in these locations later in the season, suggesting reduced colony success. The median collection dates of workers of three bumble bee species were earlier near canola fields, suggesting a dynamic response of colonies to the increased floral resources. Different species experienced this shift to different extents. The sweat bee was not affected by canola cultivation intensity. Our findings suggest that mass‐flowering crops such as canola are attractive to bumble bee queens and therefore may lead to higher rates of colony establishment, but also that colonies established near this crop may be less successful. We propose that the effect on bumble bees can be mitigated by spacing the crop more evenly with respect to alternate floral resources.     

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.     

Fine mapping of a major quantitative trait locus that regulates pod shattering in soybean

Legumes represent the second most important family of crop plants, accounting for ~27 % of the world’s crop production. While some legumes are grown as forages or vegetables, most crop legumes are grown for harvesting their nutritious seeds. The legume seeds are contained in the pod, which is composed of a single seed-bearing carpel that, when matures, splits open along two seams, a process called pod dehiscence or pod shattering. Pod shattering before or during harvest causes yield losses of grain legumes. Moreover, the dominant shattering trait of the wild progenitors is a limiting factor for efficient introgression of value-added traits into elite breeding lines. Knowledge of the genetic mechanisms underlying pod shattering will facilitate breeding of shattering-resistant varieties, expedite introgression of agronomically favorable traits from wild species to elite breeding lines, and enrich our understanding of the evolution of seed dispersal and crop domestication in diverse crop species. Here we report fine mapping of a major quantitative trait locus (designated as qPDH1) that regulates pod shattering in soybean (Glycine max). A combination of linkage and association mapping allowed us to delimit the qPDH1 locus within a 47-kb region on chromosome 16. The data reported here will facilitate positional cloning of the underlying gene and the development of breeder-friendly genetic markers for marker-assisted selection in soybean.     

Antisense suppression of deoxyhypusine synthase by vacuum-infiltration of Agrobacterium enhances growth and seed yield of canola

Deoxyhypusine synthase (DHS; EC 2.5.1.46) mediates the first of two enzymatic reactions that convert inactive eukaryotic translation initiation factor-5A (eIF-5A) to an activated form, thought to facilitate translation. A full-length cDNA clone encoding canola ( Brassica napus cv. Westar) DHS was isolated from a cDNA-expression library prepared from senescing leaves. Transgenic canola lines with suppressed DHS expression were obtained by introducing a transgene expressing antisense 3'-UTR canola DHS cDNA under the regulation of the constitutive cauliflower mosaic virus 35S (CaMV-35S) promoter. Transformed seed was obtained by vacuum infiltration of canola inflorescences using the protocol developed for Arabidopsis with modifications. The resultant transgenic plants had reduced levels of leaf DHS protein and exhibited delayed natural leaf senescence. Suppression of DHS also increased leaf size by 1.5- to two-fold and resulted in increases in seed yield of up to 65%. Moreover, the enhanced performance of transgenic plants reflected increased tolerance to chronic sublethal stress. When wild-type and transgenic plants were grown in 6-inch pots, the increase in seed yield accruing from suppression of DHS was approximately 4.5-fold greater than when the plants were grown in 12-inch pots. Thus, suppression of DHS appears to ameliorate the effects of sublethal stress engendered by growth in small containers.