Abstract To date, more than one-hundred taxa of green unicellular coccoid organisms have been wro... more Abstract To date, more than one-hundred taxa of green unicellular coccoid organisms have been wrongly included under the genus Chlorella. This is mainly due to the incomplete characterization resulting from studies that have not adopted and integrated the description of cell morphology, metabolic characteristics and genomic features. In this work, a new native Chlorella-like microalgal strain named “SEC_LI_ChL_1”, is described through a multimethod study. This strain was isolated from a pond where municipal landfill leachate flowed before wastewater treatment. The molecular characterization and the phylogenetic reconstructions based on two different DNA marker regions (i.e. the concatenated SSU and ITS rRNA gene and the complete 18S rRNA gene) were combined with light and transmission electron microscope observations, in addition to the analysis of metabolic traits under four different growing conditions (i.e. autotrophy, heterotrophy, mixotrophy and reduced autotrophy). The two separate phylogenetic analyses highlighted that the strain falls within the Chlorella-Micractinium clade, but with two different placements. According to the morphological-ultrastructural and metabolic analyses, “SEC_LI_ChL_1” shares some features with different Chlorella-like microalgae. Interestingly, two peculiar traits rarely or never previously described in Chlorella-like microalgal strains were observed: the association of the cells with endocellular bacteria, and the presence of doublets (i.e. two-celled microalgae aggregates not due to cell division).
doi: 10.3389/fpls.2015.00080 Promotion of arsenic phytoextraction efficiency in the fern Pteris v... more doi: 10.3389/fpls.2015.00080 Promotion of arsenic phytoextraction efficiency in the fern Pteris vittata by the inoculation of As-resistant bacteria: a soil bioremediation perspective
This study is aimed at evaluating the effects of bioaugmentation by means of soil addition with a... more This study is aimed at evaluating the effects of bioaugmentation by means of soil addition with arsenic resistant PGPB on the efficiency of phytoextraction capability of the fern Pteris vittata grown on an arsenopyrite contaminated soil, with respect to the metalloid of interest
In the present study, the effect of PGP (plant growth-promoting) bacterial inocula, resistant to ... more In the present study, the effect of PGP (plant growth-promoting) bacterial inocula, resistant to arsenic, on the efficiency of Pteris vittata - grown on an arsenopyrite contaminated soil - in phytoextracting this metalloid was evaluated. The research was carried out on soil samples collected from an industrial site located in Tuscany (Italy), highly polluted with arsenic and heavy metals (Pb, Fe, Zn and Cd). At first, the characterization of the bacterial community acclimated to As was carried out starting from enrichment cultures in presence of either arsenite or arsenate. Several strains were isolated in pure culture and phylogenetically identified through 16S rRNA analysis. Afterwards, each strain was tested for its capability of growing in presence of high As levels and MIC (Minimum Inhibitory Concentration) values for both arsenite and arsenate were determined. Then, bacterial isolates were checked for the occurrence of As-resistance genotypes, namely ars and aox genes. Three strains - namely Ochrobactrum cytisi E, Pseudomonas putida O and Achromobacter marplatensis P - were chosen for bioaugmentation tests. Phytoremediation trials using Pteris vittata plants grown in As contaminated soil were performed under glasshouse conditions for six months. The persistence of bacterial inocula in soil was assessed through PCR-DGGE analysis
Zuccarini P., Ciurli A., Alpi A., Hegedusova K.: Hydraulic and chemical mechanisms in the respons... more Zuccarini P., Ciurli A., Alpi A., Hegedusova K.: Hydraulic and chemical mechanisms in the response of Pinus pinaster A i t. to conditions of water stress. Ekologia (Bratislava), Vol. 30, No. 4, p. , 2011. The increase of temperatures at a global scale constitutes a threaten, among the others, for forest ecosystems, and imposes a deeper comprehension of plant behaviour towards drought stress, in order to better plan a sustainable management of water resources. Young plants of Pinus pinaster Ait. were exposed to prolonged drought stress, during which their xylem ABA concentration, leaf water potential and stomatal conductance were measured at regular intervals on the top branches; the same measurements were taken on a sample of control, non stressed plants. At the same time, plant transpiration rate was measured in response to different ABA concentrations and light regimes. Plant responses to variations of environmental parameters were measured, too: leaf T° vs. atmospheric T°; “E” vs...
According to the concept of circular economy, food industry wastewater should be considered a sec... more According to the concept of circular economy, food industry wastewater should be considered a secondary raw material that could be exploited, thus reducing the resources escaping from the production system. We aimed to assess the circular sustainability of dairy wastewater treatment by evaluating the possibility of transforming it from a cost to a valuable resource. We tested the non-photosynthetic WSZL mutant of Euglena gracilis as a bio-remediating organism for raw dairy wastewater to recover the resources herein present for biomass growth and simultaneously provide a source of paramylon, a high commercial value glucan. This mutant is an obligate osmotroph, which can synthesize paramylon up to 90% of the cell dry weight with the proper carbon source. We tested the mutant on the wastewater produced by a small dairy facility located near Pisa (Italy) using an indoor benchscale fermenter allowing the alga to grow inside a dialysis tubing immersed in the effluent. We monitored the effluent variation of biochemical oxygen demand (BOD 5 ), soluble chemical oxygen demand (COD), total suspended solids (TSS), ammonia nitrogen (NH 3 -N), nitrate nitrogen (NO 3 -N), orthophosphate (PO 4 3--P), electric conductivity and pH. The alga can reduce the pollutant concentration to values allowing indirect discharge of the wastewater into public sewage plants and can be therefore considered a promising organism for tertiary treatment of dairy wastewater. The cells produced about 2.6 g L -1 of paramylon from 3.2 g L -1 biomass, an amount greater compared with that produced by batch cultures, confirming the possibility of recovering wastewater resources and transforming them in a high-value product.
A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for... more A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for the in-situ, phytoextraction, of a dumping site with residues from the roasting process of arseno-pyrite is reported. The main characteristic of this site is the high content of both the As metalloid and heavy metals (e.g., Al, Fe, Cu, Co, Cr, Pb). Two poplar clones (Populus deltoides 'Dvina' and Populus x canadensis 'Orion') and Pteris vittata (Chinese brake fern) were planted in the contaminated soil both ex situ in pots and in situ. Plant survival, As accumulation in plant tissues, leaf content of pigments, soluble proteins, activity of catalase and SH-groups in both roots and leaves were evaluated during a 24-month study period. Both poplar and fern plants exhibited an increase in the activity of catalase and SH group contents when grown in the presence of pyrite ashes. The results showed that the co-planting system (arsenic-hyperaccumulator fern Pteris vittata and Populus clones) was suitable for phytoextraction of multi-contaminated dumping sites. Agronomic measures such as irrigation, soil tillage and amendments also seem to be necessary for the successful establishment of poplar trees and ferns in contaminated soils in order to enhance plant growth through the improvement of soil conditions.
A greenhouse pot experiment was carried out to evaluate the efficiency of arsenic phytoextraction... more A greenhouse pot experiment was carried out to evaluate the efficiency of arsenic phytoextraction by the fern Pteris vittata growing in arsenic-contaminated soil, with or without the addition of selected rhizobacteria isolated from the polluted site. The bacterial strains were selected for arsenic resistance, the ability to reduce arsenate to arsenite, and the ability to promote plant growth. P. vittata plants were cultivated for 4 months in a contaminated substrate consisting of arsenopyrite cinders and mature compost. Four different experimental conditions were tested: (i) non-inoculated plants; (ii) plants inoculated with the siderophore-producing and arsenate-reducing bacteria Pseudomonas sp. P1III2 and Delftia sp. P2III5 (A); (iii) plants inoculated with the siderophore and indoleacetic acid-producing bacteria Bacillus sp. MPV12, Variovorax sp. P4III4, and Pseudoxanthomonas sp. P4V6 (B), and (iv) plants inoculated with all five bacterial strains (AB). The presence of growth-promoting rhizobacteria increased plant biomass by up to 45% and increased As removal efficiency from 13% without bacteria to 35% in the presence of the mixed inoculum. Molecular analysis confirmed the persistence of the introduced bacterial strains in the soil and resulted in a significant impact on the structure of the bacterial community.
Iron-bearing sulphide minerals are largely responsible for the generation of mine drainage and he... more Iron-bearing sulphide minerals are largely responsible for the generation of mine drainage and hence the release of heavy metals and arsenic into the environment. Roasted arsenopyrite in dumped ash from the production of sulphuric acid has raised significant health and environmental concern. Current technologies applied for the remediation of arsenic-contaminated sites are expensive and environmentally disruptive. Nevertheless, phytoremediation through arsenic-hyperaccumulating ferns such as Pteris vittata L. and Populus hybrids has gained interest due to its cost-effectiveness and environmental soundness. In this respect, we have reported a preliminary study on plant-dependent restoration of a soil used as dumping site for roasted pyrite ashes making an 29 m high hill, in Scarlino industrial area in South Tuscany (IT). The ash hill has been removed and taken away as dangerous material. For the subsidence phenomenon several meters of ashes remain under the soil-level. The flat soil ...
Aquatic macrophytes play a central role in preserving the ecological equilibrium of shallow lakes... more Aquatic macrophytes play a central role in preserving the ecological equilibrium of shallow lakes and in the restoration of eutrophic lakes that have switched to phytoplankton-dominated turbid water. Massaciuccoli Lake, a shallow lake located along the Tuscan coast in Italy, has shown a constant and progressive simplification of the submerged plant community, for anthropogenic reasons, leading, in recent years, to turbid water. The growth and nutrient absorption capability of two macrophyte species, Myriophyllum verticillatum L. and Elodea canadensis Michaux, in the lake was investigated, with the prospect of a future lake restoration programme centred on their replacement. Mesocosm experiments were conducted to monitor the plant growth and nutrient (NO 2 -, NO 3 -, NH 4 + , Ntot, PO 4 3-, Ptot) content in the plant dry matter and water at the beginning and at the end of the trial. Bacterial activity was analysed in the water in order to verify the possible nutrient absorption contribution by organisms other than plants. Both M. verticillatum and E. canadensis showed satisfactory growth and nutrient reduction in the water body. Moreover, their different growth patterns suggested that optimal replacement can be performed with their introduction in two steps, starting with M. verticillatum, which shows the best capacity to colonise the aquatic environment, due to its tendency towards lengthening.
The presence of the two glyoxylate cycle marker enzymes, isocitrate lyase (ICL, EC 4.1.3.1) and m... more The presence of the two glyoxylate cycle marker enzymes, isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), was investigated in cotyledons during the post-germinative growth of tomato (Lycopersicon esculentum Mill., cv Chico III) seedlings. Both ICL and MS increased in the dark and declined after illumination. Cotyledons from seedlings grown in the dark for 7 days were employed for sucrose density gradient fractionation of cell organelles. It was shown that ICL and MS are localized in glyoxysomal fractions. The presence of the two key enzymes of the glyoxylate cycle in tomato cotyledons was confirmed by immunoblotting both in crude extracts and in glyoxysomal fractions. The occurrence of such enzymes was also investigated in different tissues of tomato plants (leaves, fruits, roots and flowers). Although it was difficult to detect ICL and MS activities by spectrophotometric assays, immunoblot analysis showed the presence of both enzymes in senescent leaves and, surprisingly, in young expanding leaves, but not in mature leaves. The role of glyoxylate cycle enzymes in leaves and the value of further studies with tomato are discussed.
-chain acyl-CoA oxidases are -oxidation enzymes that are active on short-chain acyl-CoAs and tha... more -chain acyl-CoA oxidases are -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 -oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform -oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C 8 ). 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 (C 4 ), hexanoyl-CoA (C 6 ), and octanoyl-CoA (C 8 ). 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 -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 -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 -oxidation of acyl-CoA. Oilseed plants convert reserve oil to sucrose after germination. This unique type of gluconeogenesis occurs in the storage tissues of oilseeds, such as endosperms or cotyledons (1). The metabolic pathway involves many enzymes in several subcellular compartments, including lipid bodies, glyoxysomes (a specialized peroxisome), mitochondria, and the cytosol. Within the entire gluconeogenic pathway, the conversion of a fatty acid to succinate takes place within the glyoxysomes, which contain enzymes for fatty acid -oxidation and the glyoxylate cycle.
Abstract To date, more than one-hundred taxa of green unicellular coccoid organisms have been wro... more Abstract To date, more than one-hundred taxa of green unicellular coccoid organisms have been wrongly included under the genus Chlorella. This is mainly due to the incomplete characterization resulting from studies that have not adopted and integrated the description of cell morphology, metabolic characteristics and genomic features. In this work, a new native Chlorella-like microalgal strain named “SEC_LI_ChL_1”, is described through a multimethod study. This strain was isolated from a pond where municipal landfill leachate flowed before wastewater treatment. The molecular characterization and the phylogenetic reconstructions based on two different DNA marker regions (i.e. the concatenated SSU and ITS rRNA gene and the complete 18S rRNA gene) were combined with light and transmission electron microscope observations, in addition to the analysis of metabolic traits under four different growing conditions (i.e. autotrophy, heterotrophy, mixotrophy and reduced autotrophy). The two separate phylogenetic analyses highlighted that the strain falls within the Chlorella-Micractinium clade, but with two different placements. According to the morphological-ultrastructural and metabolic analyses, “SEC_LI_ChL_1” shares some features with different Chlorella-like microalgae. Interestingly, two peculiar traits rarely or never previously described in Chlorella-like microalgal strains were observed: the association of the cells with endocellular bacteria, and the presence of doublets (i.e. two-celled microalgae aggregates not due to cell division).
doi: 10.3389/fpls.2015.00080 Promotion of arsenic phytoextraction efficiency in the fern Pteris v... more doi: 10.3389/fpls.2015.00080 Promotion of arsenic phytoextraction efficiency in the fern Pteris vittata by the inoculation of As-resistant bacteria: a soil bioremediation perspective
This study is aimed at evaluating the effects of bioaugmentation by means of soil addition with a... more This study is aimed at evaluating the effects of bioaugmentation by means of soil addition with arsenic resistant PGPB on the efficiency of phytoextraction capability of the fern Pteris vittata grown on an arsenopyrite contaminated soil, with respect to the metalloid of interest
In the present study, the effect of PGP (plant growth-promoting) bacterial inocula, resistant to ... more In the present study, the effect of PGP (plant growth-promoting) bacterial inocula, resistant to arsenic, on the efficiency of Pteris vittata - grown on an arsenopyrite contaminated soil - in phytoextracting this metalloid was evaluated. The research was carried out on soil samples collected from an industrial site located in Tuscany (Italy), highly polluted with arsenic and heavy metals (Pb, Fe, Zn and Cd). At first, the characterization of the bacterial community acclimated to As was carried out starting from enrichment cultures in presence of either arsenite or arsenate. Several strains were isolated in pure culture and phylogenetically identified through 16S rRNA analysis. Afterwards, each strain was tested for its capability of growing in presence of high As levels and MIC (Minimum Inhibitory Concentration) values for both arsenite and arsenate were determined. Then, bacterial isolates were checked for the occurrence of As-resistance genotypes, namely ars and aox genes. Three strains - namely Ochrobactrum cytisi E, Pseudomonas putida O and Achromobacter marplatensis P - were chosen for bioaugmentation tests. Phytoremediation trials using Pteris vittata plants grown in As contaminated soil were performed under glasshouse conditions for six months. The persistence of bacterial inocula in soil was assessed through PCR-DGGE analysis
Zuccarini P., Ciurli A., Alpi A., Hegedusova K.: Hydraulic and chemical mechanisms in the respons... more Zuccarini P., Ciurli A., Alpi A., Hegedusova K.: Hydraulic and chemical mechanisms in the response of Pinus pinaster A i t. to conditions of water stress. Ekologia (Bratislava), Vol. 30, No. 4, p. , 2011. The increase of temperatures at a global scale constitutes a threaten, among the others, for forest ecosystems, and imposes a deeper comprehension of plant behaviour towards drought stress, in order to better plan a sustainable management of water resources. Young plants of Pinus pinaster Ait. were exposed to prolonged drought stress, during which their xylem ABA concentration, leaf water potential and stomatal conductance were measured at regular intervals on the top branches; the same measurements were taken on a sample of control, non stressed plants. At the same time, plant transpiration rate was measured in response to different ABA concentrations and light regimes. Plant responses to variations of environmental parameters were measured, too: leaf T° vs. atmospheric T°; “E” vs...
According to the concept of circular economy, food industry wastewater should be considered a sec... more According to the concept of circular economy, food industry wastewater should be considered a secondary raw material that could be exploited, thus reducing the resources escaping from the production system. We aimed to assess the circular sustainability of dairy wastewater treatment by evaluating the possibility of transforming it from a cost to a valuable resource. We tested the non-photosynthetic WSZL mutant of Euglena gracilis as a bio-remediating organism for raw dairy wastewater to recover the resources herein present for biomass growth and simultaneously provide a source of paramylon, a high commercial value glucan. This mutant is an obligate osmotroph, which can synthesize paramylon up to 90% of the cell dry weight with the proper carbon source. We tested the mutant on the wastewater produced by a small dairy facility located near Pisa (Italy) using an indoor benchscale fermenter allowing the alga to grow inside a dialysis tubing immersed in the effluent. We monitored the effluent variation of biochemical oxygen demand (BOD 5 ), soluble chemical oxygen demand (COD), total suspended solids (TSS), ammonia nitrogen (NH 3 -N), nitrate nitrogen (NO 3 -N), orthophosphate (PO 4 3--P), electric conductivity and pH. The alga can reduce the pollutant concentration to values allowing indirect discharge of the wastewater into public sewage plants and can be therefore considered a promising organism for tertiary treatment of dairy wastewater. The cells produced about 2.6 g L -1 of paramylon from 3.2 g L -1 biomass, an amount greater compared with that produced by batch cultures, confirming the possibility of recovering wastewater resources and transforming them in a high-value product.
A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for... more A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for the in-situ, phytoextraction, of a dumping site with residues from the roasting process of arseno-pyrite is reported. The main characteristic of this site is the high content of both the As metalloid and heavy metals (e.g., Al, Fe, Cu, Co, Cr, Pb). Two poplar clones (Populus deltoides 'Dvina' and Populus x canadensis 'Orion') and Pteris vittata (Chinese brake fern) were planted in the contaminated soil both ex situ in pots and in situ. Plant survival, As accumulation in plant tissues, leaf content of pigments, soluble proteins, activity of catalase and SH-groups in both roots and leaves were evaluated during a 24-month study period. Both poplar and fern plants exhibited an increase in the activity of catalase and SH group contents when grown in the presence of pyrite ashes. The results showed that the co-planting system (arsenic-hyperaccumulator fern Pteris vittata and Populus clones) was suitable for phytoextraction of multi-contaminated dumping sites. Agronomic measures such as irrigation, soil tillage and amendments also seem to be necessary for the successful establishment of poplar trees and ferns in contaminated soils in order to enhance plant growth through the improvement of soil conditions.
A greenhouse pot experiment was carried out to evaluate the efficiency of arsenic phytoextraction... more A greenhouse pot experiment was carried out to evaluate the efficiency of arsenic phytoextraction by the fern Pteris vittata growing in arsenic-contaminated soil, with or without the addition of selected rhizobacteria isolated from the polluted site. The bacterial strains were selected for arsenic resistance, the ability to reduce arsenate to arsenite, and the ability to promote plant growth. P. vittata plants were cultivated for 4 months in a contaminated substrate consisting of arsenopyrite cinders and mature compost. Four different experimental conditions were tested: (i) non-inoculated plants; (ii) plants inoculated with the siderophore-producing and arsenate-reducing bacteria Pseudomonas sp. P1III2 and Delftia sp. P2III5 (A); (iii) plants inoculated with the siderophore and indoleacetic acid-producing bacteria Bacillus sp. MPV12, Variovorax sp. P4III4, and Pseudoxanthomonas sp. P4V6 (B), and (iv) plants inoculated with all five bacterial strains (AB). The presence of growth-promoting rhizobacteria increased plant biomass by up to 45% and increased As removal efficiency from 13% without bacteria to 35% in the presence of the mixed inoculum. Molecular analysis confirmed the persistence of the introduced bacterial strains in the soil and resulted in a significant impact on the structure of the bacterial community.
Iron-bearing sulphide minerals are largely responsible for the generation of mine drainage and he... more Iron-bearing sulphide minerals are largely responsible for the generation of mine drainage and hence the release of heavy metals and arsenic into the environment. Roasted arsenopyrite in dumped ash from the production of sulphuric acid has raised significant health and environmental concern. Current technologies applied for the remediation of arsenic-contaminated sites are expensive and environmentally disruptive. Nevertheless, phytoremediation through arsenic-hyperaccumulating ferns such as Pteris vittata L. and Populus hybrids has gained interest due to its cost-effectiveness and environmental soundness. In this respect, we have reported a preliminary study on plant-dependent restoration of a soil used as dumping site for roasted pyrite ashes making an 29 m high hill, in Scarlino industrial area in South Tuscany (IT). The ash hill has been removed and taken away as dangerous material. For the subsidence phenomenon several meters of ashes remain under the soil-level. The flat soil ...
Aquatic macrophytes play a central role in preserving the ecological equilibrium of shallow lakes... more Aquatic macrophytes play a central role in preserving the ecological equilibrium of shallow lakes and in the restoration of eutrophic lakes that have switched to phytoplankton-dominated turbid water. Massaciuccoli Lake, a shallow lake located along the Tuscan coast in Italy, has shown a constant and progressive simplification of the submerged plant community, for anthropogenic reasons, leading, in recent years, to turbid water. The growth and nutrient absorption capability of two macrophyte species, Myriophyllum verticillatum L. and Elodea canadensis Michaux, in the lake was investigated, with the prospect of a future lake restoration programme centred on their replacement. Mesocosm experiments were conducted to monitor the plant growth and nutrient (NO 2 -, NO 3 -, NH 4 + , Ntot, PO 4 3-, Ptot) content in the plant dry matter and water at the beginning and at the end of the trial. Bacterial activity was analysed in the water in order to verify the possible nutrient absorption contribution by organisms other than plants. Both M. verticillatum and E. canadensis showed satisfactory growth and nutrient reduction in the water body. Moreover, their different growth patterns suggested that optimal replacement can be performed with their introduction in two steps, starting with M. verticillatum, which shows the best capacity to colonise the aquatic environment, due to its tendency towards lengthening.
The presence of the two glyoxylate cycle marker enzymes, isocitrate lyase (ICL, EC 4.1.3.1) and m... more The presence of the two glyoxylate cycle marker enzymes, isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), was investigated in cotyledons during the post-germinative growth of tomato (Lycopersicon esculentum Mill., cv Chico III) seedlings. Both ICL and MS increased in the dark and declined after illumination. Cotyledons from seedlings grown in the dark for 7 days were employed for sucrose density gradient fractionation of cell organelles. It was shown that ICL and MS are localized in glyoxysomal fractions. The presence of the two key enzymes of the glyoxylate cycle in tomato cotyledons was confirmed by immunoblotting both in crude extracts and in glyoxysomal fractions. The occurrence of such enzymes was also investigated in different tissues of tomato plants (leaves, fruits, roots and flowers). Although it was difficult to detect ICL and MS activities by spectrophotometric assays, immunoblot analysis showed the presence of both enzymes in senescent leaves and, surprisingly, in young expanding leaves, but not in mature leaves. The role of glyoxylate cycle enzymes in leaves and the value of further studies with tomato are discussed.
-chain acyl-CoA oxidases are -oxidation enzymes that are active on short-chain acyl-CoAs and tha... more -chain acyl-CoA oxidases are -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 -oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform -oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C 8 ). 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 (C 4 ), hexanoyl-CoA (C 6 ), and octanoyl-CoA (C 8 ). 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 -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 -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 -oxidation of acyl-CoA. Oilseed plants convert reserve oil to sucrose after germination. This unique type of gluconeogenesis occurs in the storage tissues of oilseeds, such as endosperms or cotyledons (1). The metabolic pathway involves many enzymes in several subcellular compartments, including lipid bodies, glyoxysomes (a specialized peroxisome), mitochondria, and the cytosol. Within the entire gluconeogenic pathway, the conversion of a fatty acid to succinate takes place within the glyoxysomes, which contain enzymes for fatty acid -oxidation and the glyoxylate cycle.
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