Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation

Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products.     

Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.     

Plant-derived smoke stimulates germination of four herbaceous species common in temperate regions of Europe

Germination responses to aerosol smoke and its aqueous solution (smoke–water) were studied in laboratory for six species common throughout temperate Europe: four annual weeds from the Brassicaceae family (Camelina microcarpa, Capsella bursa-pastoris, Descurainia sophia and Sisymbrium orientale), and two perennial Plantago species (P. lanceolata and P. media) inhabiting natural grasslands. It was hypothesized that smoke enhances germination for these species despite they are not fire-adapted. Both smoke treatments increased the final germination percentage for C. bursa-pastoris and D. sophia by fourfold to fivefold, for C. microcarpa by 73–224 % and for P. lanceolata by 26–41 % compared to the control. S. orientale and P. media showed high (>80 %) germination in the control with no significant response to smoke–water. These results confirm the occurrence of smoke-stimulated germination among species of the European natural flora inhabiting non-fire-prone habitats, suggest more successful seedling recruitment for smoke-responsive species with climate change-driven increase in fire frequency and can be used in the cultivation of the species or in weed control.     

Variation of the seed endophytic bacteria among plant populations and their plant growth‐promoting activities in a wild mustard plant species,Capsella bursa‐pastoris

Recent studies have revealed that some bacteria can inhabit plant seeds, and they are likely founders of the bacterial community in the rhizosphere of or inside plants at the early developmental stage. Given that the seedling establishment is a critical fitness component of weedy plant species, the effects of seed endophytic bacteria (SEB) on the seedling performance are of particular interest in weed ecology. Here, we characterized the SEB in natural populations of Capsella bursa‐pastoris, a model species of weed ecology. The composition of endophytic bacterial community was evaluated using deep sequencing of a 16S rDNA gene fragment. Additionally, we isolated bacterial strains from seeds and examined their plant growth‐promoting traits. Actinobacteria, Firmicutes, Alpha‐, and Gammaproteobacteria were major bacterial phyla inside seeds. C. bursa‐pastoris natural populations exhibited variable seed microbiome such that the proportion of Actinobacteria and Alphaproteobacteria differed among populations, and 60 out of 82 OTUs occurred only in a single population. Thirteen cultivable bacterial species in six genera (Bacillus, Rhodococcus, Streptomyces, Staphylococcus, Paenibacillus, Pseudomonas) were isolated, and none of them except Staphylococcus haemolyticus were previously reported as seed endophytes. Eight isolates exhibited plant growth‐promoting traits like phosphate solubilization activity, indole‐3‐acetic acid, or siderophore production. Despite the differences in the bacterial communities among plant populations, at least one isolated strain from each population stimulated shoot growth of either C. bursa‐pastoris or its close relative A. thaliana when grown with plants in the same media. These results suggest that a weedy plant species, C. bursa‐pastoris, contains bacterial endophytes inside their seeds, stimulating seedling growth and thereby potentially affecting seedling establishment.     

Monitoring population and gene pool dynamics of the annual species <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis> (Brassicaceae): a review of relevant species traits and the initiation of a long-term genetic monitoring programme

Species that colonise habitats on an annual basis are important for studying and understanding evolutionary changes and adaptations in the course of environmental shifts, caused, for instance, by global change phenomena. These species are characterised by a weedy ecology enabling them to react fast to environmental changes. As a model species, we selected Capsella bursa-pastoris (Brassicaceae, Shepherd’s Purse), which is annual to biennial, predominantly selfing and closely related to the genetic model plant Arabidopsis thaliana. Differentiation and adaptation in C. bursa-pastoris are shortly reviewed. Based on this knowledge, two Botanical Gardens are currently arranging long-term sites characterised by annual ploughing for the genetic monitoring of C. bursa-pastoris. Demographic parameters will be monitored as well, i.e. different life-cycle phases such as flowering, fruiting, and the soil seed bank. Furthermore, seeds of at least 20 individuals will be sampled per year. Isozyme analyses, flow cytometry, AFLPs, SNPs as well as microsatellites will be utilised to characterise changes in genetic diversity patterns over time. In general, the objective of our genetic monitoring of C. bursa-pastoris is to address the following questions: In the light of future research, our objectives are to study whether the genetic structure and diversity of C. bursa-pastoris alter over time, whether a possible invasion of ecotypes/genotypes of Sheperd’s purse is traceable using genetic markers, and whether it is possible to relate variation in reproductive patterns to respective candidate genes, which may be useful for monitoring.     

The temperature response of CO2 assimilation, photochemical activities and Rubisco activation in Camelina sativa, a potential bioenergy crop with limited capacity for acclimation to heat stress

The temperature optimum of photosynthesis coincides with the average daytime temperature in a species’ native environment. Moderate heat stress occurs when temperatures exceed the optimum, inhibiting photosynthesis and decreasing productivity. In the present study, the temperature response of photosynthesis and the potential for heat acclimation was evaluated for Camelina sativa, a bioenergy crop. The temperature optimum of net CO₂ assimilation rate (A) under atmospheric conditions was 30–32?°C and was only slightly higher under non-photorespiratory conditions. The activation state of Rubisco was closely correlated with A at supra-optimal temperatures, exhibiting a parallel decrease with increasing leaf temperature. At both control and elevated temperatures, the modeled response of A to intercellular CO₂ concentration was consistent with Rubisco limiting A at ambient CO₂. Rubisco activation and photochemical activities were affected by moderate heat stress at lower temperatures in camelina than in the warm-adapted species cotton and tobacco. Growth under conditions that imposed a daily interval of moderate heat stress caused a 63?% reduction in camelina seed yield. Levels of cpn60 protein were elevated under the higher growth temperature, but acclimation of photosynthesis was minimal. Inactivation of Rubisco in camelina at temperatures above 35?°C was consistent with the temperature response of Rubisco activase activity and indicated that Rubisco activase was a prime target of inhibition by moderate heat stress in camelina. That photosynthesis exhibited no acclimation to moderate heat stress will likely impact the development of camelina and other cool season Brassicaceae as sources of bioenergy in a warmer world.     

The coexpression of two desaturases provides an optimized reduction of saturates in camelina oil

Reducing the saturate content of vegetable oils is key to increasing their utility and adoption as a feedstock for the production of biofuels. Expression of either the FAT5 16 : 0-CoA desaturase from Caenorhabditis elegans, or an engineered cyanobacterial 16 : 0/18 : 0-glycerolipid desaturase, DES9*, in seeds of Arabidopsis (Arabidopsis thaliana) substantially lowered oil saturates. However, because pathway fluxes and regulation of oil synthesis are known to differ across species, translating this transgene technology from the model plant to crop species requires additional investigation. In the work reported here, we found that high expression of FAT5 in seeds of camelina (Camelina sativa) provided only a moderate decrease in saturates, from 12.9% of total oil fatty acids in untransformed controls to 8.6%. Expression of DES9* reduced saturates to 4.6%, but compromised seed physiology and oil content. However, the coexpression of the two desaturases together cooperatively reduced saturates to only 4.0%, less than one-third of the level in the parental line, without compromising oil yield or seedling germination and establishment. Our successful lowering of oil saturates in camelina identifies strategies that can now be integrated with genetic engineering approaches that reduce polyunsaturates to provide optimized oil composition for biofuels in camelina and other oil seed crops.     

Diverse roles of glycine-rich RNA-binding protein 7 in the response of camelina (<Emphasis Type="Italic">Camelina sativa</Emphasis>) to abiotic stress

Camelina sativa L. is an oilseed crop used as a potential low-cost biofuel resource. Despite the economic and agricultural benefits of this crop, studies demonstrating the physiological and genetic response of camelina to changing environmental conditions are limited. In this study, three stress-responsive glycine-rich RNA-binding proteins (GRPs) in camelina—named CsGRP7a, CsGRP7b, and CsGRP7c—were isolated, and their functional roles in stress responses were characterized. The three CsGRP7 genes had similar nucleotide and deduced amino acid sequences, and contained an N-terminal RNA-recognition motif and a C-terminal glycine-rich region. The CsGRP7 genes were ubiquitously expressed in all plant tissues, and CsGRP7 proteins were localized to both the cytoplasm and the nucleus. The expression of CsGRP7 genes was markedly upregulated by cold stress, whereas their expression was only slightly affected by salt or dehydration stress. Analysis of CsGRP7a-expressing transgenic Arabidopsis thaliana and camelina plants revealed that CsGRP7a plays a positive role in cold stress tolerance, but a negative role in salt or drought stress tolerance. All three CsGRP7s harbored RNA chaperone activity. Collectively, these data indicate that the stress-responsive CsGRP7s harbor RNA chaperone activity and play different roles in the plant response to abiotic stresses.     

Overexpression of patatin‐related phospholipase AIIIδ altered plant growth and increased seed oil content in camelina

Camelina sativa is a Brassicaceae oilseed species being explored as a biofuel and industrial oil crop. A growing number of studies have indicated that the turnover of phosphatidylcholine plays an important role in the synthesis and modification of triacylglycerols. This study manipulated the expression of a patatin‐related phospholipase AIIIδ (pPLAIIIδ) in camelina to determine its effect on seed oil content and plant growth. Constitutive overexpression of pPLAIIIδ under the control of the constitutive cauliflower mosaic 35S promoter resulted in a significant increase in seed oil content and a decrease in cellulose content. In addition, the content of major membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, in 35S::pPLAIIIδ plants was increased. However, these changes in 35S::pPLAIIIδ camelina were associated with shorter cell length, leaves, stems, and seed pods and a decrease in overall seed production. When pPLAIIIδ was expressed under the control of the seed specific, β‐conglycinin promoter, the seed oil content was increased without compromising plant growth. The results suggest that pPLAIIIδ alters the carbon partitioning by decreasing cellulose content and increasing oil content in camelina.     

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa

Main conclusion

Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.     

Phylogenetic signatures of adaptation: The Arabis hirsuta species aggregate (Brassicaceae) revisited

The mustard family, Brassicaceae, is well-known for its homoplasy in almost any morphological character at practically all taxonomic levels. The genus Arabis, within the largest tribe of the Brassicaceae, is such an example comprising numerous para- and polyphyletic groups of taxa. Past research during the last 15 years has unraveled many phylogenetic relationships among the ∼550 (or more) species within the notoriously difficult tribe Arabideae. The European Arabis hirsuta species aggregate has remained unexplored, however. Herein we analyze phylogenetic relationships using nuclear ITS and plastid DNA sequences of Eurasian Arabis to characterize Hairy rock cress (A. hirsuta) and its relatives. Representative geographic sampling is used to study character and trait evolution, and bioclimatic data are used to differentiate between species. Our overview puts European Arabis into a reliable evolutionary framework, and we provide some striking insights into evolutionary trends and correlating morphological characters from seeds and flowers with environmental data such as climate variables and elevation. We demonstrate independent parallel evolution of sets of traits, and, therefore, we could further elaborate our previous findings that within tribe Arabideae high speciation rates are correlated with perennial growth form and occurrence at higher elevation. Finally some taxonomical remarks are provided to give added context.     

Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa

Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production.     

CbCBF from Capsella bursa-pastoris enhances cold tolerance and restrains growth in Nicotiana tabacum by antagonizing with gibberellin and affecting cell cycle signaling

Plant cells respond to cold stress via a regulatory mechanism leading to enhanced cold acclimation accompanied by growth retardation. The C-repeat binding factor (CBF) signaling pathway is essential for cold response of flowering plants. Our previously study documented a novel CBF-like gene from the cold-tolerant Capsella bursa-pastoris named CbCBF, which was responsive to chilling temperatures. Here, we show that CbCBF expression is obviously responsive to chilling, freezing, abscisic acid, gibberellic acid (GA), indoleacetic acid or methyl jasmonate treatments and that the CbCBF:GFP fusion protein was localized to the nucleus. In addition, CbCBF overexpression conferred to the cold-sensitive tobacco plants enhanced tolerance to chilling and freezing, as well as dwarfism and delayed flowering. The leaf cells of CbCBF overexpression tobacco lines attained smaller sizes and underwent delayed cell division with reduced expression of cyclin D genes. The dwarfism of CbCBF transformants can be partially restored by GA application. Consistently, CbCBF overexpression reduced the bioactive gibberellin contents and disturbed the expression of gibberellin metabolic genes in tobacco. Meanwhile, cold induced CbCBF expression and cold tolerance in C. bursa-pastoris are reduced by GA. We conclude that CbCBF confers cold resistance and growth inhibition to tobacco cells by interacting with gibberellin and cell cycle pathways, likely through activation of downstream target genes.     

Nickel and zinc effects,accumulation and distribution in ruderal plants Lepidium ruderale and Capsella bursa-pastoris

Ruderal plants can grow in polluted areas, but little is known about heavy metal accumulation and distribution in them. Here Ni and Zn accumulation, distribution and effects were investigated in Lepidium ruderale and Capsella bursa-pastoris grown at 5–30 µM Ni(NO₃)₂ or 10–80 µM Zn(NO₃)₂. Metal contents were measured by flame atomic absorption spectrophotometry and tissue distribution of metals was studied histochemically. Ni was more toxic than Zn for both plants. When metal-induced growth-inhibiting effects were compared at various metal concentrations in solution, L. ruderale was more tolerant to Ni, whereas C. bursa-pastoris to Zn. However, when compared at similar Zn or Ni contents in roots, root growth of C. bursa-pastoris was more tolerant than that of L. ruderale. On the contrary, at similar Zn or Ni contents in shoots, shoot growth of L. ruderale was more tolerant. Both plants are excluders maintaining low metal levels in shoots. In roots, Ni located in protoplasts while Zn was also detected in cell walls. Metal accumulation in root apices resulted in growth inhibition. Ni accumulation in root cortex constrained metal translocation into central cylinder and then to shoots, where it located only in conductive tissues and epidermis, particularly in leaf trichomes of C. bursa-pastoris. Zn was translocated to shoots more actively and distributed in all shoot tissues, being accumulated in leaf vascular bundles and epidermis. To conclude, these patterns of Ni and Zn distribution are aimed at metal sequestration in roots and leaf epidermis, thus keeping mesophyll from metal penetration and pigment degradation.     

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.