Modifying fatty acid profiles through a new cytokinin‐based plastid transformation system

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin‐based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3‐ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over‐expression of isopentenyl transferase. Over‐expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic‐based selection systems, and may be used in combination with a negative selectable marker as part of a two‐step selection system to obtain homoplasmic plant lines.     

cis-3-Hexenal production in tobacco is stimulated by 16-carbon monounsaturated fatty acids

Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Delta9 desaturase and an insect acyl-CoA Delta11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from alpha-linolenic acid (18:3). As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1(Delta9)) and palmitvaccenic acid (16:1(Delta11)), respectively. In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics. Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3. In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from alpha-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants. Although 16:1(Delta9) and 16:1(Delta11) had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression.     

Total fatty acid content of the plasma membrane of Saccharomyces cerevisiae is more responsible for ethanol tolerance than the degree of unsaturation

The effect of change in unsaturated fatty acid composition on ethanol tolerance in Saccharomyces cerevisiae overexpressing ScOLE1 (?9 fatty acid desaturase gene of S. cerevisiae), CaFAD2 (?12 fatty acid desaturase gene of Candida albicans), or CaFAD3 (ω3 fatty acid desaturase gene of C. albicans) was examined. ScOLE1 over-expression increased the total unsaturated fatty acid content and enhanced ethanol tolerance, compared with a control strain. In contrast, overexpression of CaFAD2 and CaFAD3, which led to production of linoleic acid (18:2) and α-linolenic acid (18:3), respectively, neither changed total unsaturated fatty acids nor enhanced ethanol tolerance. The total unsaturated fatty acid content rather than the degree of unsaturation is thus an important factor for ethanol tolerance.     

Plant plastid engineering

Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation.     

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Removal of antibiotic resistance genes from transgenic tobacco plastids

Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.     

Identification and characterization of an animal delta(12) fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae

We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae. The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C. elegans omega-3 fatty acid desaturase. In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions. This is the first report of a cloned animal Delta(12) desaturase gene. Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids. The increase of membrane fluidity brought about by this change in unsaturation was measured. The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress.     

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum)

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.     

Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.     

Precise excision of plastid DNA by the large serine recombinase Bxb1

Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP‐ and attB‐flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH‐PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear‐transformed transgenic tobacco plants expressing a plastid‐targeted Bxb1 recombinase were crossed with transplastomic pTCH‐PB plants, and the T₁ hybrids exhibited efficient excision of the target sequence. The Bxb1–att system should prove to be a useful tool for site‐specifically manipulating the plastid genome and generating marker‐free transplastomic plants.     

Cloning of a higher-plant plastid omega-6 fatty acid desaturase cDNA and its expression in a cyanobacterium.

Oligomers based on amino acids conserved between known plant omega-3 and cyanobacterium omega-6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine max and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant omega-6 and omega-3 desaturases but more than 48% similarity to cyanobacterial omega-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid omega-6 desaturase. The identity was supported by expression of the B. napus cDNA in cyanobacterium. Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 73 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 16:1(9c) fatty acid was converted primarily to 16:2(9c, 12) in vivo. Thus, the plant omega-6 desaturase, which utilizes 16:1(7c) in plants, can utilize 16:1(9c) in the cyanobacterium. The plastid and cytosolic homologs of plant omega-6 desaturases are much more distantly related than those of omega-3 desaturases.     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.     

A second functional delta5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum.

A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www. csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Delta5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino-acid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Delta5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing Delta5 fatty acid desaturase. As we have already reported one other Delta5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Delta5 fatty acid desaturase genes. The substrate specificities of the two functional Delta5 desaturases of D. discoideum were also examined.     

Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition

Background  

A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition.