Storage reserve mobilization in germinating oilseeds: Arabidopsis as a model system.

Germinating oilseeds break down fatty acids through peroxisomal beta-oxidation and convert the carbon into soluble carbohydrates through the glyoxylate cycle and gluconeogenesis. This interconversion is unique among higher eukaryotes. Using a combination of forward and reverse genetic screens, we have isolated mutants that compromise fatty acid breakdown at each step. These mutants exhibit characteristic, yet nonidentical, seedling establishment phenotypes that can be rescued by the provision of an alternative carbon source. In addition, we have recently shown that Arabidopsis seed's lipid breakdown occurs in two distinct tissues, the embryo and endosperm. The utilization of endospermic lipid reserves requires gluconeogenesis and transport of the resulting sugars to the germinating embryo. We discuss the potential of the Arabidopsis endosperm tissue as a simplified model system for the study of germination and lipid breakdown in germinating oilseeds.

The role of trehalose-6-phosphate synthase in Arabidopsis embryo development.

We previously showed that trehalose-6-phosphate synthase 1 (TPS1), which catalyses the first step in trehalose synthesis, is essential for embryo maturation in Arabidopsis. The tps1 mutant embryos develop more slowly than wild type. Patterning in the tps1 embryos appears normal but they do not progress past the torpedo stage to cotyledon stage, which is when storage reserves start to accumulate in the expanding cotyledons. Our initial data led to the hypothesis that trehalose metabolism plays a key role in regulating storage reserve accumulation by allowing the embryo to respond to the dramatic increase in sucrose levels that occurs at the torpedo stage of embryo development. More recent data demonstrate that while the tps1 mutant is blocked in the developmental progression of embryos from torpedo to cotyledon stage the expression of genes involved in the accumulation of storage reserves proceeds in a similar fashion to wild type. Thus it appears that induction of metabolic processes required for accumulation of storage reserves in tps1 occurs independently of the developmental stage and instead follows a temporal programme similar to wild-type seeds in the same silique.

Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings.

A phenotypic screen was employed to isolate Arabidopsis plants that are deficient in their ability to utilize or sense acetate. The screening strategy, based on resistance to the toxic acetate analogue monofluoroacetic acid, was adapted from one that has been used successfully to identify important metabolic and regulatory genes involved in acetate metabolism in fungi. Following conventions established from the fungal work, the mutants were called acn mutants for acetate non-utilization. Three highly resistant plant lines were the focus of genetic and physiological studies. Mutant acn1 appears to be a true acetate non-utilizing mutant, as it displays increased sensitivity to exogenous acetate. The progeny of the original acn2 mutant did not germinate, even in the presence of sucrose as an exogenous carbon source. The germination of seeds from the F3 generation depended on the sucrose concentration in the medium. Only a small proportion of seeds germinated in the absence of exogenous sucrose and in the presence of 100 mM sucrose, but up to 70% of seeds germinated on 20 mM sucrose. Mutant acn3 exhibited sensitivity to exogenous sucrose, showing significant chlorosis on medium containing 20 mM sucrose, but no chlorosis when grown in the absence of exogenous sucrose. This phenotype was alleviated if acetate was provided. The acn mutants demonstrate that disrupting organic acid utilization can have profound affects on carbohydrate metabolism.

Acyl-CoA measurements in plants suggest a role in regulating various cellular processes.

Acyl-CoA esters have been shown to be involved in regulating metabolism and cell signalling in bacteria, yeast and mammalian cells, but little is known about their role in plants. Using a new method for the sensitive detection and quantification of acyl-CoA esters, we have recently shown that acyl-CoA pools can be dramatically altered in transgenic oilseed rape embryos, engineered to produce medium-chain fatty acids, and in mutant Arabidopsis seedlings that are unable to mobilize storage lipid. The consequences of these alterations are discussed in the context of oil yield and organelle biogenesis and the possible role of acyl-CoAs in regulating these processes.

Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid beta-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings.

3-ketoacyl-CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid beta-oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identified a T-DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modified. Long-chain (C16 to C20) fatty acyl-CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long-chain thiolase activity. In addition, extracts from kat2 seedlings have significantly decreased activity with aceto-acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germination and seedling growth, indicating that KAT2 has broad substrate specificity. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal beta-oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies.

Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana.

Molecular genetic approaches in the model plant Arabidopsis thaliana (Col0) are shedding new light on the role and control of the pathways associated with the mobilization of lipid reserves during oilseed germination and post-germinative growth. Numerous independent studies have reported on the expression of individual genes encoding enzymes from the three major pathways: beta-oxidation, the glyoxylate cycle and gluconeogenesis. However, a single comprehensive study of representative genes and enzymes from the different pathways in a single plant species has not been done. Here we present results from Arabidopsis that demonstrate the co-ordinate regulation of gene expression and enzyme activities for the acyl-CoA oxidase- and 3-ketoacyl-CoA thiolase-mediated steps of beta-oxidation, the isocitrate lyase and malate synthase steps of the glyoxylate cycle and the phosphoenolpyruvate carboxykinase step of gluconeogenesis. The mRNA abundance and enzyme activities increase to a peak at stage 2, 48 h after the onset of seed germination, and decline thereafter either to undetectable levels (for malate synthase and isocitrate lyase) or low basal levels (for the genes of beta-oxidation and gluconeogenesis). The co-ordinate induction of all these genes at the onset of germination raises the possibility that a global regulatory mechanism operates to induce the expression of genes associated with the mobilization of storage reserves during the heterotrophic growth period.

Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues.

We report a novel, highly sensitive and selective method for the extraction and quantification of acyl CoA esters from plant tissues. The method detects acyl CoA esters with acyl chain lengths from C4 to C20 down to concentrations as low as 6 fmol in extracts. Acyl CoA esters from standard solutions or plant extracts were derived to their fluorescent acyl etheno CoA esters in the presence of chloroacetaldehyde, separated by ion-paired reversed-phase high-performance liquid chromatography, and detected fluorometrically. This derivitization procedure circumvents the selectivity problems associated with previously published enzymatic methods, and methods that rely on acyl chain or thiol group modification for acyl CoA ester detection. The formation of acyl etheno CoA esters was verified by mass spectrometry, which was also used to identify unknown peaks from chromatograms of plant extracts. Using this method, we report the composition and concentration of the acyl CoA pool during lipid synthesis in maturing Brassica napus seeds and during storage lipid breakdown in 2-day-old Arabidopsis thaliana seedlings. The concentrations measured were in the 3--6 microM range for both tissue types. We also demonstrate the utility of acyl CoA profiling in a transgenic B. napus line that has high levels of lauric acid. To our knowledge, this is the first time that reliable estimates of acyl CoA ester concentrations have been made for higher plants, and the ability to profile these metabolites provides a valuable new tool for the investigation of gene function.

Re-examining the role of the glyoxylate cycle in oilseeds.

Oil is the primary seed storage reserve in many higher plants. After germination, this reserve is mobilized in order to support growth during early seedling development. The glyoxylate cycle is instrumental in this metabolic process. It allows acetyl-CoA derived from the breakdown of storage lipids to be used for the synthesis of carbohydrate. Recently, Arabidopsis mutants have been isolated that lack key glyoxylate cycle enzymes. An isocitrate lyase mutant has provided the first opportunity to test the biochemical and physiological functions of the glyoxylate cycle in vivo in an oilseed species.

Plastid redox state and sugars: interactive regulators of nuclear-encoded photosynthetic gene expression.

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3', 4'-dichlorophenyl)-1,1'-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Stress induces peroxisome biogenesis genes.

Peroxisomes are the cellular location of many antioxidants and are themselves significant producers of reactive oxygen species. In this report we demonstrate the induction of peroxisome biogenesis genes in both plant and animal cells by the universal stress signal molecule hydrogen peroxide. Using PEX1-LUC transgenic plants, rapid local and systemic induction of PEX1-luciferase could be demonstrated in vivo in response to physiological levels of hydrogen peroxide. PEX1-luciferase was also induced in response to wounding and to infection with an avirulent pathogen. We propose a model in which various stress situations that lead to the production of hydrogen peroxide can be ameliorated by elaboration of the peroxisome compartment to assist in restoration of the cellular redox balance.

Promoter trapping of a novel medium-chain acyl-CoA oxidase, which is induced transcriptionally during Arabidopsis seed germination.

The first step of peroxisomal fatty acid beta-oxidation is catalyzed by a family of acyl-CoA oxidase isozymes with distinct fatty acyl-CoA chain-length specificities. Here we identify a new acyl-CoA oxidase gene from Arabidopsis (AtACX3) following the isolation of a promoter-trapped mutant in which beta-glucuronidase expression was initially detected in the root meristem. In acx3 mutant seedlings medium-chain acyl-CoA oxidase activity was reduced by 95%, whereas long- and short-chain activities were unchanged. Despite this reduction in activity lipid catabolism and seedling development were not perturbed. AtACX3 was cloned and expressed in Escherichia coli. The recombinant enzyme displayed medium-chain acyl-CoA substrate specificity. Analysis of beta-glucuronidase activity in acx3 revealed that, in addition to constitutive expression in the root axis, AtACX3 is also up-regulated strongly in the hypocotyl and cotyledons of germinating seedlings. This suggests that beta-oxidation is regulated predominantly at the level of transcription in germinating oilseeds. After the discovery of AtACX3, the Arabidopsis acyl-CoA oxidase gene family now comprises four isozymes with substrate specificities that encompass the full range of acyl-CoA chain lengths that exist in vivo.

The multifunctional protein AtMFP2 is co-ordinately expressed with other genes of fatty acid beta-oxidation during seed germination in Arabidopsis thaliana (L.) Heynh.

In germinating oilseeds peroxisomal fatty acid beta-oxidation is responsible for the mobilization of storage lipids. This pathway also occurs in other tissues where it has a variety of additional physiological functions. The central enzymatic steps of peroxisomal beta-oxidation are performed by acyl-CoA oxidase (ACOX), the multifunctional protein (MFP) and 3-ketoacyl-CoA thiolase (thiolase). In order to investigate the function and regulation of beta-oxidation in plants it is first necessary to identify and characterize genes encoding the relevant enzymes in a single model species. Recently we and others have reported on the cloning and characterization of genes encoding four ACOXs and a thiolase from the oilseed Arabidopsis thaliana. Here we identify a gene encoding an Arabidopsis MFP (AtMFP2) that is induced transiently during germination. The pattern of AtMFP2 expression closely reflects changes in the activities of 2-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase. Similar patterns of expression have previously been reported for ACOX and thiolase genes. We conclude that genes encoding the three main proteins responsible for beta-oxidation are co-ordinately expressed during oilseed germination and may share a common mechanism of regulation.

Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle.

The glyoxylate cycle is regarded as essential for postgerminative growth and seedling establishment in oilseed plants. We have identified two allelic Arabidopsis mutants, icl-1 and icl-2, which lack the glyoxylate cycle because of the absence of the key enzyme isocitrate lyase. These mutants demonstrate that the glyoxylate cycle is not essential for germination. Furthermore, photosynthesis can compensate for the absence of the glyoxylate cycle during postgerminative growth, and only when light intensity or day length is decreased does seedling establishment become compromised. The provision of exogenous sugars can overcome this growth deficiency. The icl mutants also demonstrate that the glyoxylate cycle is important for seedling survival and recovery after prolonged dark conditions that approximate growth in nature. Surprisingly, despite their inability to catalyze the net conversion of acetate to carbohydrate, mutant seedlings are able to break down storage lipids. Results suggest that lipids can be used as a source of carbon for respiration in germinating oilseeds and that products of fatty acid catabolism can pass from the peroxisome to the mitochondrion independently of the glyoxylate cycle. However, an additional anaplerotic source of carbon is required for lipid breakdown and seedling establishment. This source can be provided by the glyoxylate cycle or, in its absence, by exogenous sucrose or photosynthesis.

Application of a new method for the sensitive detection and quantification of acyl-CoA esters in Arabidopsis thaliana seedlings and mature leaves.

We report a novel, highly sensitive and selective method for the detection and quantification of acyl-CoA esters from Arabidopsis thaliana. Extracted acyl-CoA esters were derived to their fluorescent acyl-etheno-CoA esters, separated by ion-paired reversed-phase HPLC, and detected fluorometrically. We report the composition and concentration of the acyl-CoA pool in mature A. thaliana leaves, and during storage-lipid breakdown in 2-day-old seedlings. The concentrations measured were in the 1-4 microM range for both tissue types. To our knowledge, this is the first time that reliable estimates of acyl-CoA ester concentrations have been made for plants.

The Arabidopsis acyl-CoA oxidase gene family.

A family of acyl-CoA oxidase isozymes catalyse the first step in the peroxisomal fatty acid beta-oxidation spiral. Our group and others have recently characterized four genes from this family in the model oilseed Arabidopsis. These genes encode isozymes with different acyl-CoA substrate specificities, which together encompass the full range of fatty acid chain lengths that exist in vivo. Here we review the biochemical properties and physiological roles of the acyl-CoA oxidase isozymes.

Long-chain acyl-CoA oxidases of Arabidopsis.

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.

No induction of beta-oxidation in leaves of Arabidopsis that over-produce lauric acid.

Leaves from transgenic Brassica napus L. plants engineered to produce lauric acid show increased levels of enzyme activities of the pathways associated with fatty acid catabolism (V.A. Eccleston and J.B. Ohlrogge, 1998, Plant Cell 10: 613-621). In order to determine if the increases in enzyme activity are mirrored by increases in the expression of genes encoding enzymes of beta-oxidation, which is the major pathway of fatty acid catabolism in plants, the medium-chain acyl-acyl carrier protein (ACP) thioesterase MCTE from California bay (Umbellularia california) was over-expressed under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis thaliana (L.) Heynh. Arabidopsis was the most suitable choice for these studies since gene expression could be analyzed in a large number of independent MCTE-expressing lines using already well-characterized beta-oxidation genes. Levels of MCTE transcripts in leaves varied widely over the population of plants analyzed. Furthermore, active MCTE was produced as determined by enzymatic analysis of leaf extracts of MCTE-expressing plants. These plants incorporated laurate into triacylglycerol of seeds, but not into lipids of leaves as shown by gaschromatographic analysis of total fatty acid extracts. The expression levels of the beta-oxidation and other genes that are highly expressed during developmental stages involving rapid fatty acid degradation were measured. No significant difference in gene expression was observed among MCTE-expressing plants and transgenic and non-transgenic controls. To eliminate the possibility that post-translational mechanisms are responsible for the observed increases in enzyme activity acyl-CoA oxidase activity was also measured in leaves of MCTE-expressing plants using medium and long chain acyl-CoA substrates. No significant increases in either medium- or long-chain acyl-CoA oxidase activities were detected. We conclude that endogenous beta-oxidation is sufficient to account for the complete degradation of laurate produced in rosette leaves of Arabidopsis expressing MCTE.

Distinct cis-acting elements direct the germination and sugar responses of the cucumber malate synthase gene.

The malate synthase gene (ms) promoter in cucumber (Cucumis sativus L.) was investigated with the aim of distinguishing DNA sequences mediating regulation of gene expression by sugar, and expression following seed germination. Promoter deletions were constructed and their ability to direct expression of the beta-glucuronidase (gus) reporter gene was investigated in transgenic Nicotiana plumbaginifolia. Gene expression was assayed in germinating seeds and developing seedlings (the germination response) and in seedlings transferred from light into darkness with and without sucrose (the sugar response). As progressively more of the promoter was deleted from the 5' end, first the sugar response and then the germination response was lost. Thus, distinct regions of the promoter are required for carbohydrate control and for regulation of gene expression in response to germination. Sequence comparisons of the ms promoter with that of the isocitrate lyase gene (icl) of cucumber have previously identified four IMH(ICL-MS-Homology) sequences. One such sequence, IMH2, is shown here to be implicated in the sugar response of the ms gene. The 17 bp sequences which when deleted from the ms gene results in loss of the germination response, contains a 14 bp sequence which is similar to a sequence in the icl promoter, which we refer to as IMH5. Furthermore, this sequence has similarity with amdI9-like sequences in filamentous fungi, which confer facB-mediated acetate inducibility on several genes, including those encoding ICL and MS.