Roles for ALDH10 enzymes in γ-butyrobetaine synthesis, seed development, germination, and salt tolerance in Arabidopsis.

Plant genomes generally contain two aldehyde dehydrogenase 10 (ALDH10) genes, which encode NAD+-dependent enzymes. These oxidize various aminoaldehydes that are produced by the catabolism of amino acids and polyamines. ALDH10s are closely related to the animal and fungal trimethylaminobutyraldehyde dehydrogenases (TMABADHs) that are involved in the synthesis of γ-butyrobetaine, the precursor of carnitine. Here, we explore the ability of the Arabidopsis thaliana proteins AtALDH10A8 and AtALDH10A9 to oxidize aminoaldehydes. We demonstrate that these enzymes display high TMABADH activities in vitro. Moreover, they can complement the Candida albicans tmabadhΔ/Δ null mutant. These findings illustrate the link between AtALDH10A8 and AtALDH10A9 and γ-butyrobetaine synthesis. An analysis of single and double knockout Arabidopsis mutant lines revealed that the double mutants had reduced γ-butyrobetaine levels. However, there were no changes in the carnitine contents of these mutants. The double mutants were more sensitive to salt stress. In addition, the siliques of the double mutants had a significant proportion of seeds that failed to mature. The mature seeds contained higher amounts of triacylglycerol, facilitating accelerated germination. Taken together, these results show that ALDH10 enzymes are involved in γ-butyrobetaine synthesis. Furthermore, γ-butyrobetaine fulfils a range of physiological roles in addition to those related to carnitine biosynthesis.

Cloning and molecular characterization of three lysophosphatidic acid acyltransferases expressed in flax seeds.

In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.

Physiology of L-carnitine in plants in light of the knowledge in animals and microorganisms.

L-carnitine is present in all living kingdoms where it acts in diverse physiological processes. It is involved in lipid metabolism in animals and yeasts, notably as an essential cofactor of fatty acid intracellular trafficking. Its physiological significance is poorly understood in plants, but L-carnitine may be linked to fatty acid metabolism among other roles. Indeed, carnitine transferases activities and acylcarnitines are measured in plant tissues. Current knowledge of fatty acid trafficking in plants rules out acylcarnitines as intermediates of the peroxisomal and mitochondrial fatty acid metabolism, unlike in animals and yeasts. Instead, acylcarnitines could be involved in plastidial exportation of de novo fatty acid, or importation of fatty acids into the ER, for synthesis of specific glycerolipids. L-carnitine also contributes to cellular maintenance though antioxidant and osmolyte properties in animals and microbes. Recent data indicate similar features in plants, together with modulation of signaling pathways. The biosynthesis of L-carnitine in the plant cell shares similar precursors as in the animal and yeast cells. The elucidation of the biosynthesis pathway of L-carnitine, and the identification of the enzymes involved, is today essential to progress further in the comprehension of its biological significance in plants.

Acylcarnitines participate in developmental processes associated to lipid metabolism in plants.

MAIN CONCLUSION: Plant acylcarnitines are present during anabolic processes of lipid metabolism. Their low contents relatively to the corresponding acyl-CoAs suggest that they are associated to specific pools of activated fatty acids. The non-proteinaceous amino acid carnitine exists in plants either as a free form or esterified to fatty acids. To clarify the biological significance of acylcarnitines in plant lipid metabolism, we have analyzed their content in plant extracts using an optimized tandem mass spectrometry coupled to liquid chromatography method. We have studied different developmental processes (post-germination, organogenesis, embryogenesis) targeted for their high requirement for lipid metabolism. The modulation of the acylcarnitine content was compared to that of the lipid composition and lipid biosynthetic gene expression level in the analyzed materials. Arabidopsis mutants were also studied based on their alteration in de novo fatty acid partitioning between the prokaryotic and eukaryotic pathways of lipid biosynthesis. We show that acylcarnitines cannot specifically be associated to triacylglycerol catabolism but that they are also associated to anabolic pathways of lipid metabolism. They are present during membrane and storage lipid biosynthesis processes. A great divergence in the relative contents of acylcarnitines as compared to the corresponding acyl-CoAs suggests that acylcarnitines are associated to very specific process(es) of lipid metabolism. The nature of their involvement as the transport form of activated fatty acids or in connection with the management of acyl-CoA pools is discussed. Also, the occurrence of medium-chain entities suggests that acylcarnitines are associated with additional lipid processes such as protein acylation for instance. This work strengthens the understanding of the role of acylcarnitines in plant lipid metabolism, probably in the management of specific acyl-CoA pools.

The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi.

Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and γ-butyrobetaine (γ-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled γ-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology.

The effect of carnitine on Arabidopsis development and recovery in salt stress conditions.

Carnitine exists in all living organisms where it plays diverse roles. In animals and yeast, it is implicated in lipid metabolism and is also associated with oxidative stress tolerance. In bacteria, it is a major player in osmotic stress tolerance. We investigate the carnitine function in plants and our present work shows that carnitine enhances the development and recovery of Arabidopsis thaliana seedlings subjected to salt stress. Biological data show that exogenous carnitine supplies improve the germination and survival rates of seedlings grown on salt-enriched medium, in a manner comparable to proline. Both compounds are shown to improve seedling survival under oxidative constraint meaning that they may act on salt stress through antioxidant properties. A transcriptome analysis of seedlings treated with exogenous carnitine reveals that it modulates the expression of genes involved in water stress and abscisic acid responses. Analyses of the abscisic acid mutants, aba1-1 and abi1-1, indicate that carnitine and proline may act through a modulation of the ABA pathway.

Prenatal diazepam exposure alters respiratory control system and GABAA and adenosine receptor gene expression in newborn rats.

In experimental animals, prenatal diazepam exposure has clearly been associated with behavioral disturbances. Its impact on newborn breathing has not been documented despite potential deleterious consequences for later brain development. We addressed this issue in neonatal rats (0-2 d) born from dams, which consumed 2 mg/kg/d diazepam via drinking fluid throughout gestation. In vivo, prenatal diazepam exposure significantly altered the normoxic-breathing pattern, lowering breathing frequency (105 vs. 125 breaths/min) and increasing tidal volume (16.2 vs. 12.7 mL/kg), and the ventilatory response to hypoxia, inducing an immediate and marked decrease in tidal volume (-30%) absent in controls. In vitro, prenatal diazepam exposure significantly increased the respiratory-like frequency produced by pontomedullary and medullary preparations (+38% and +19%, respectively) and altered the respiratory-like response to application of nonoxygenated superfusate. Both in vivo and in vitro, the recovery from oxygen deprivation challenges was delayed by prenatal diazepam exposure. Finally, real-time PCR showed that prenatal diazepam exposure affected mRNA levels of alpha1 and alpha2 GABAA receptor subunits and of A1 and A2A adenosine receptors in the brainstem. These mRNA changes, which are region-specific, suggest that prenatal diazepam exposure interferes with developmental events whose impact on the respiratory system maturation deserves further studies.

Consequences of prenatal exposure to diazepam on the respiratory parameters, respiratory network activity and gene expression of alpha1 and alpha2 subunits of GABA(A) receptor in newborn rat.

Diazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network.

Carnitine is associated with fatty acid metabolism in plants.

The finding of acylcarnitines alongside free carnitine in Arabidopsis thaliana and other plant species, using tandem mass spectrometry coupled to liquid chromatography shows a link between carnitine and plant fatty acid metabolism. Moreover the occurrence of both medium- and long-chain acylcarnitines suggests that carnitine is connected to diverse fatty acid metabolic pathways in plant tissues. The carnitine and acylcarnitine contents in plant tissues are respectively a hundred and a thousand times lower than in animal tissues, and acylcarnitines represent less than 2% of the total carnitine pool whereas this percentage reaches 30% in animal tissues. These results suggest that carnitine plays a lesser role in lipid metabolism in plants than it does in animals.

Disruption of AtOCT1, an organic cation transporter gene, affects root development and carnitine-related responses in Arabidopsis.

In animals, organic cation/carnitine transporters (OCTs) are involved in homeostasis and distribution of various small endogenous amines (e.g. carnitine, choline) and detoxification of xenobiotics such as nicotine. Here, we describe the characterization of AtOCT1, an Arabidopsis protein that shares most of the conserved features of mammalian plasma membrane OCTs. Transient expression of an AtOCT1::GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts supported localization in the plasmalemma. AtOCT1 functionally complemented the Deltacit2/Deltaagp2p yeast strain that is defective in plasma membrane carnitine transport. Disruption of AtOCT1 in an Arabidopsis oct1-1 knockout mutant affected both the expression of carnitine-related genes and the developmental defects induced by exogenous carnitine. RT-PCR and promoter-uidA fusion analysis showed that AtOCT1 was expressed in vascular tissues of various organs and at sites of lateral root formation. Correlating with this expression pattern, oct1-1 seedlings grown in vitro exhibited a higher degree of root branching than the wild-type, showing that the disruption of AtOCT1 affected root development under certain conditions.

A bipartite system for the constitutive and inducible expression of high levels of foreign proteins in plants.

We have developed combined transgene/virus vector systems for the expression of heterologous proteins in plants. The systems are based on the bipartite RNA plant virus, cowpea mosaic virus (CPMV), and involve the amplification of integrated copies of either full-length or deleted versions of RNA-2 carrying a foreign gene. In the case of plants transgenic for full-length versions of RNA-2 carrying the green fluorescent protein (GFP), amplification can be achieved by supplying RNA-1 either exogenously or by crossing. This allows either inducible or constitutive expression of the foreign gene and results in an infection that can be passaged to further plants. Replication of deleted versions of RNA-2 harbouring GFP requires the presence of both RNA-1 and a suppressor of gene silencing, a function which we show can be supplied by HcPro from potato virus Y. Replication of the deleted versions of RNA-2 can be achieved by supplying the suppressor and RNA-1 either exogenously or by crossing, showing that this system can also be used in an inducible and constitutive format. The use of deleted forms of RNA-2 has the advantage that no infectious virus is produced, providing an effective method of biocontainment. The CPMV-based systems have advantages over existing plant expression systems in terms of the expression levels obtainable and the simplicity and flexibility of use, and should be of great practical benefit in the development of plants as bioreactors.

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies.

To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.

An antibody derivative expressed from viral vectors passively immunizes pigs against transmissible gastroenteritis virus infection when supplied orally in crude plant extracts.

To investigate the potential of antibody derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed a small immune protein (SIP) in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The epsilonSIP molecule consisted of a single-chain antibody (scFv) specific for the porcine coronavirus transmissible gastroenteritis virus (TGEV) linked to the epsilon-CH4 domain from human immunoglobulin E (IgE). In some constructs, the sequence encoding the epsilonSIP molecule was flanked by the leader peptide from the original murine antibody at its N-terminus and an endoplasmic reticulum retention signal (HDEL) at its C-terminus to allow the expressed protein to be directed to, and retained within, the endoplasmic reticulum. Western blot analysis of samples from Nicotiana clevelandii or cowpea tissue infected with constructs revealed the presence of SIP molecules which retained their ability to dimerize. The analysis of crude plant extracts revealed that the plant-expressed epsilonSIP molecules could bind to and neutralize TGEV in tissue culture, the levels of binding and neutralization reflecting the level of expression. Oral administration of crude extracts from SIP-expressing plant tissue to 2-day-old piglets demonstrated that the extracts which showed the highest levels of in vitro neutralization could also provide in vivo protection against challenge with TGEV.

Cowpea mosaic virus-based systems for the production of antigens and antibodies in plants.

Cowpea mosaic virus (CPMV) is a bipartite RNA plant virus which has proved to be useful both for epitope presentation and as a polypeptide expression system. For epitope presentation, short antigenic sequences are expressed on the surface of the assembled virus. Chimaeric virus particles produced in this way can stimulate protective immunity in experimental animals. For polypeptide expression, we have created a vector in which foreign sequences can be inserted near the 3' end of RNA-2 and have successfully expressed a number of polypeptides in plant tissue. To extend the utility of the CPMV-based systems, we have recently developed a combined virus vector/transgenic expression system in which RNA-2 expressed from a transgene is replicated by RNA-1.

A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells.

To investigate the role of subunit assembly in the intracellular deposition of multimeric recombinant proteins, we expressed a partially humanized secretory immunoglobulin in rice endosperm cells and determined the subcellular locations of the assembled protein and its individual components. Transgenic rice plants expressing either individual subunits or all the subunits of the antibody were generated by particle bombardment, and protein localization was determined by immunoelectron microscopy. Assembly of the antibody was confirmed by immunoassay and coimmunoprecipitation. Immunolocalization experiments showed no evidence for secretion of the antibody or any of its components to the apoplast. Rather, the nonassembled light chain, heavy chain and secretory component accumulated predominantly within endoplasmic reticulum-derived protein bodies, while the assembled antibody, with antigen-binding function, accumulated specifically in protein storage vacuoles. These results show that the destination of a complex recombinant protein within the plant cell is influenced by its state of assembly.