Luigi De Bellis

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are β-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete β-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform β-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid β-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid β-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete β-oxidation of acyl-CoA.

Localization of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons.

Crude particulate homogenates from leaves of barley (Hordeum vulgare L.), rice (Oryza sativa L.), leaf-beet (Beta vulgaris var. cicla L.) and pumpkin (Cucurbita pepo L.) cotyledons were separated on sucrose density gradients. The peroxisomal fractions appeared at a buoyant density of 1.25 g·cm−3 and contained most of the activities of catalase (EC 1.11.1.6), and hydroxypyruvate reductase (EC 1.1.1.81) on the gradients. In peroxisomal fractions from detached leaves and green cotyledons incubated in permanent darkness we detected the presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), key enzymes of the glyoxylate cycle, and β-oxidation activity (except in pumpkin). As proposed by H. Gut and P. Matile (1988, Planta 176, 548–550) the glyoxylate cycle may be functional during leaf senescence, and the presence of two key enzymes indicates a transition from leaf peroxisome to glyoxysome; for pumpkin cotyledons in particular a double transition occurs (glyoxysome to leaf peroxisome during greening, and leaf peroxisome to glyoxysome during senescence).

Peroxisomal enzyme activities in attached senescing leaves

Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice (Oryza sativa L.) and wheat (Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and β-oxidation activities. Leaf peroxisomes from dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence may represent a reverse transition of the peroxisomes into glyoxysomes.

Glycoxylate cycle enzyme activities are induced in senescent pumpkin fruits

The presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) was investigated in detached pumpkin fruits (Cucurbita pepo L., var. Alberello di Sarzana). After incubation of pumpkin fruit slices in permanent darkness, the enzyme activities appeared after only 1 day and then declined to zero at day 6. Catalase (EC 1.11.1.6) specific activity increased during the first 2 days of incubation and decreased thereafter. Hydroxypyruvate reductase (EC 1.1.1.81) and 3-hydroxyacyl CoA dehydrogenase (E.C. 1.1.1.35) specific activities changed very little, but NADP+-dependent isocitrate dehydrogenase (E.C. 1.1.1.42) and fumarase (E.C. 4.2.1.2) specific activities increased during the first day and declined thereafter. Sucrose density gradient fractionation of cell organelles showed that isocitrate lyase and malate synthase are localized in peroxisomal fractions. The presence of the two key enzymes of the glyoxylate cycle was confirmed by immunoblotting, both in crude extracts and in peroxisomal fractions. It is concluded that following detachment and dark incubation, a transition from peroxisomes to glyoxysomes occurs in pumpkin fruit.

Functional transformation of plant peroxisomes.

Peroxisomes in higher plant cells are known to differentiate into at least three different classes, namely, glyoxysomes, leaf peroxisomes, and unspecialalized peroxisomes, dependending on the cell types. In germinating fatty seedlings, glyoxysomes that first appear in the etiolated cotyledonary cells are functionally transformed into leaf peroxisomes during greening. Subsequently, the organelles are transformed back into glyoxysomes during senescence of the cotyledons. Flexibility of function is a distinct feature of plant peroxisomes. This article briefly describes recent studies of the regulatory mechanisms involved in the changes of the function of plant peroxisomes.

Molecular characterization of an Arabidopsis acyl-coenzyme a synthetase localized on glyoxysomal membranes

In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the β-oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designatedAtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). TheAtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that theAtLACS6 is localized on glyoxysomal membranes.AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and β-oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid β-oxidation activating the fatty acids.

EXPRESSION OF GLYOXYLATE CYCLE GENES IN CUCUMBER ROOTS RESPONDS TO SUGAR SUPPLY AND CAN BE ACTIVATED BY SHADING OR DEFOLIATION OF THE SHOOT

When cucumber roots are excised and incubated without a carbon source, isocitrate lyase (ICL) and malate synthase (MS) mRNAs increase significantly in amount. However, if sucrose is added to the excised roots, the mRNAs do not accumulate. Hairy roots obtained by transformation with Agrobacterium rhizogenes show the same response. Transgenic hairy roots containing the Icl and Ms gene promoters fused to the GUS reporter gene, have very low GUS activity which increases dramatically when roots are incubated in the absence of sugar, indicating regulation at the transcriptional level. Staining of sugar-deprived roots shows that GUS activity is concentrated mainly in root tips and lateral root primordia, where demand for carbohydrate is greatest. In order to determine if Icl and Ms genes are expressed in roots of whole plants under conditions which may occur in nature, cucumber plants were subjected to reduced light intensity or defoliation. In both cases increases were observed in ICL and MS mRNAs. These treatments also reduced root sugar content, consistent with the hypothesis that sugar supply could control expression of Icl and Ms genes in roots of whole plants.

Distinct cis-acting sequences are required for the germination and sugar responses of the cucumber isocitrate lyase gene

Recent data have shown that distinct DNA sequence elements direct the germination and sugar responses of the cucumber (Cucumis sativus, L.) malate synthase (Ms) gene (Sarah et al. (1996) Mol. Gen. Genet., 250, 153-161). Such information is, however, lacking for the isocitrate lyase (Icl) gene which is coordinately regulated with Ms. Deletions from the 5 end of the Icl promoter were therefore created specifically to address this question. Analysis of expression in seeds of transgenic Nicotiana plumbaginifolia plants showed that whereas a promoter sequence of 2.9 kilobases (kb) produced a normal germination response, deletion to -1568 base pairs (bp) dramatically reduced this response. Examination of the sugar response employed a transgenic cucumber root system. In this case, the 2900 bp and 1568 bp promoters both gave a strong sugar response, but further deletion to -1367 bp eliminated the response. Therefore, the germination and sugar responses of the Icl gene require distinct cis-acting elements, located respectively upstream and downstream of -1568 bp. This observation is consistent with distinct signal transduction systems regulating gene expression in each case.

Evidences of glyoxylate cycle in peroxisomes of senescent cotyledons

Abstract The metabolic pathway of the glyoxylate cycle has been investigated in peroxisomes isolated from senescent pumpkin ( Cucurbita sp.) cotyledons. β-oxidation activity, as well as activities of glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) were detected. In order to establish if there is a channelling of acetyl CoA into the glyoxylate cycle, peroxisomes have been incubated with various substrates. The incubations show acetyl CoA utilization by the glyoxylate cycle. When the incubation medium for citrate formation is used, all the label from [1- 14 C]acetyl CoA is recovered in citrate, whereas only 18% of the added radioactivity is recovered in malic acid (by isocitrate lyase and malate synthase) after a long incubation time (3 h). Only by feeding peroxisomes with [1,5- 14 C]citric acid and exogenous aconitase (EC 4.2.1.3) can a weak formation of other organic acids (glyoxylate and succinate) be note. The requirement for exogenous aconitase to carry out the peroxisomal glyoxylate cycle points towards the isocitric acid step as a crucial factor for the operation of the global cycle.

Molecular Characterization of Aconitase in Etiolated Pumpkin Cotyledons

Aconitase (EC 4.2.1.3) is an enzyme that catalyzes the reaction of reversible conversion between citrate and isocitrate. In cotyledons of oil-storage plants such as pumpkin, aconitase was thought to exist not only in mitochondria as a member of the tricarboxylic acid cycle but also in glyoxysomes as a member of glyoxylate cycle. The glyoxylate cycle is an important pathway for producing sucrose from reserved lipid at early stage of seedling growth under the cooperation of other metabolic pathways such as s-oxidation.