Polyploid genome of <Emphasis Type="Italic">Camelina sativa</Emphasis>revealed by isolation of fatty acid synthesis genes

Background  

Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome.     

Genome size shifts: karyotype evolution in Crepis section Neglectoides (Asteraceae)

Plant genome size evolution is a very dynamic process: the ancestral genome of angiosperms was initially most likely small, which led to a tendency towards genome increase during evolution. However, findings in several angiosperm lineages demonstrate mechanisms that also led to genome size contraction. Recent molecular investigations on the Asteraceae genus Crepis suggest that several genomic reduction events have occurred during the evolution of the genus. This study focuses on the Mediterranean Crepis sect. Neglectoides, which includes three species with some of the smallest genomes within the whole genus. Crepis neglecta has the largest genome in sect. Neglectoides, approximately twice the size of the two species Crepis cretica and Crepis hellenica. Whereas C. cretica and C. hellencia are more closely related to each other than to C. neglecta the karyotypes of the latter species and C. cretica are similar, while that of C. hellenica differs considerably. Here, the karyotypic organisation of the three species is investigated with fluorescence in‐situ hybridisation and studied in a molecular phylogenetic framework based on the nuclear markers Actin, CHR12, CPN60B, GPCR1 and XTH23. Our findings further corroborate the occurrence of genome size contraction in Crepis, and suggest that the difference in genome size between C. neglecta and C. cretica is mostly due to elimination of dispersed repetitive elements, whereas chromosomal reorganisation was involved in the karyotype formation of C. hellenica.     

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss‐of‐function mutants revealed the importance of delta‐12‐desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.     

A microspore embryogenesis protocol for <Emphasis Type="Italic">Camelina sativa,</Emphasis> a multi-use crop

Camelina [Camelina sativa (L.) Crantz], a member of the Brassicaceae family, has a unique oil profile that has potential both for biofuels and as a food crop. It is essential to have a doubled haploidy protocol in order to enhance breeding of this crop for prairie conditions as well as improve the yield and quality characteristics. Microspore-derived embryos have been produced from Camelina sativa. Buds 1–3 mm in length were selected for culture. The microspores were isolated and purified in full-strength B5 extraction medium and cultured in NLN medium with 12.5% sucrose and 12.5% polyethylene glycol 4000 (PEG) without glutamine, at a density of 10,000 microspores per mL. Glutamine was added to the cultures 72 h after extraction to give a final concentration of 0.8 g/L. The microspore cultures were maintained at 24°C in the dark. After 28 days embryos were observed and these were regenerated to plants and selfed seed was produced. The highest embryogenic frequency achieved was 38 microspore-derived embryos from 100,000 microspores.     

Camalexin induction in intertribal somatic hybrids between Camelina sativa and rapid-cycling Brassica oleracea

  Camelina sativa, a wild relative of Brassica crops, is virtually immune to blackspot disease caused by Alternaria brassicicola. Intertribal somatic hybrids were produced between C. sativa and rapid-cycling Brassica oleracea as a step toward the transfer of resistance to this disease into Brassica vegetable crops. The plants recovered were confirmed as somatic hybrids by flow cytometry and RAPD analysis. All hybrids showed a morphology intermediate between the two parents. Rooted plants grew in soil up to 4–5 weeks, and some produced sterile flowers. Two of three hybrids tested showed a high level of resistance to  A. brassicicola. Resistance was correlated with the induction of high levels of the phytoalexin camalexin 48 h after inoculation, as in the resistant Camelina fusion partner. In contrast, susceptible somatic hybrids produced much lower levels of camalexin. Received: 2 April 1998 / Accepted: 14 July 1998     

h-TBP: an approach based on intron-length polymorphism for the rapid isolation and characterization of the multiple members of the β-tubulin gene family in <Emphasis Type="Italic">Camelina sativa</Emphasis> (L.) Crantz

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.     

Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds

Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6–13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

     

Constitutive or seed‐specific overexpression of Arabidopsis G‐protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa

Heterotrimeric G‐proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant‐specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%–40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops.     

Establishment of in vitro culture, plant regeneration, and genetic transformation of Camelina sativa

The results of establishing an in vitro culture, plantlet regeneration, and rooting of Camelina sativa cultivar Peremozhets and cultivar Mirazh are presented. The effective concentrations of sterilizing agents and the duration of plant material treatment were estimated. The phytohormone ratio, the sucrose concentration in the nutrient medium that induced the effective formation of C. sativa shoots, and the NAA concentration for plantlet rooting have been established. A method of Agrobacterium-mediated transformation of Camelina by using binary vector pGH217 carrying the reporter β-glucoronidase (gus) gene driven under the 35S CaMV promoter and nos-terminator, as well as the selective marker hpt gene conferring hygromycin-resistance in transgenic plant, was elaborated.     

Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles

Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed‐specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. Here we show by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of the oilseed crop Camelina sativa, indicating tissue‐specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over‐expression of an acyl–acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in Camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.     

Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas,Convolvulaceae) complex

• Genome size evolution and its relationship with pollen grain size has been investigated in sweet potato (Ipomoea batatas), an economically important crop which is closely related to diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, ten sweet potato varieties and two outgroup taxa.
• Nuclear DNA amounts were determined using flow cytometry. Pollen grains were studied using scanning and transmission electron microscopy.
• 2C DNA content of hexaploid I. batatas ranged between 3.12–3.29 pg; the mean monoploid genome size being 0.539 pg (527 Mbp), similar to the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was, respectively, 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida and I. triloba, 2C DNA content was 1.01–1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species, with 2C of 1.47–1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% AT bases.
• DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, the genome size and ploidy levels were correlated with pollen size. Our results allow us to propose alternative or complementary hypotheses to that currently proposed for the formation of hexaploid Ipomoea batatas.

     

Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa,using CRISPR/Cas9 gene editing

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T₂ and T₃ generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T₃ and T₄ generation Camelina seeds was associated with a combination of germ‐line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.     

Transformation of the oilseed crop Camelina sativa by Agrobacterium-mediated floral dip and simple large-scale screening of transformants

Camelina sativa is a promising under-exploited oilseed crop with potential to become a biofuel feedstock. The ability to transform C. sativa would allow for the rapid introduction of novel traits into this emerging crop. We report the development of an Agrobacterium-based floral dip transformation method, requiring no vacuum-infiltration step, with transformation efficiencies up to 0.8%. C. sativa cultivars Ames 26665, ??Calena?? A3U7761, Ames 1043, and ??Celine?? were tested using Agrobacterium tumefaciens strains GV3101, EHA105, and At503. Use of all strains and cultivars resulted in transformed plants; however, GV3101 was the only Agrobacterium strain and Ames 1043 the only C. sativa cultivar to yield transformed plants under all conditions tested. Progeny analysis revealed that in approximately 78% of the transformed plants, the transgene segregated as a single locus. Furthermore, a high-throughput, filter paper-based PCR method was developed to screen marker-free transformed plants. Together, these methods will allow for easier introduction of new genes into this promising oilseed crop.