Figure 3 - uploaded by Makoto Hayashi
Content may be subject to copyright.
Immunoelectron microscope analysis of At LACS6. Immu- nostaining of etiolated cotyledons from Arabidopsis seedlings grown for 5 d in the dark. Antisera against At LACS6 (A) and catalase (B) were employed after IgG-purification. g, Glyoxysome; m, mitochon- drion; e, etioplast. The arrowhead indicates the staining on the glyoxysomal membrane (15-nm gold particles). Bar ϭ 1 m.
Source publication
In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-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-C...
Contexts in source publication
Context 1
... etiolated cotyledon from 5-d- old seedlings were fixed and thin sections were pro- cessed employing antisera against AtLACS6, pumpkin catalase, and nonimmune serum. The label- ing shown in Figure 3A was exclusively localized in glyoxysomes, particularly on glyoxysomal mem- brane (arrow), whereas the catalase is localized in the glyoxysomal matrix. No gold particles were observed in other organelles such as mitochondria and etio- plasts (Fig. 3A). ...
Context 2
... antisera against AtLACS6, pumpkin catalase, and nonimmune serum. The label- ing shown in Figure 3A was exclusively localized in glyoxysomes, particularly on glyoxysomal mem- brane (arrow), whereas the catalase is localized in the glyoxysomal matrix. No gold particles were observed in other organelles such as mitochondria and etio- plasts (Fig. 3A). No gold particles in any organelles were found in the case of nonimmune serum (data not shown). These data suggest that AtLACS6 is lo- calized on glyoxysomal ...
Context 3
... glyoxysomal localization was confirmed by the immuno-electron microscope analysis of Arabidopsis etiolated cotyledons, the signal being localized on glyoxysomal membranes (Fig. 3). This indication is consistent with the difficulties for AtLACS6 purifica- tion by traditional methods. The strict association of AtLACS6 with glyoxysomal membranes suggests that AtLACS6 also may have an additional regula- tory role in fatty acid transport in glyoxysomes by an unknown interaction with other factors. In fact, pre- vious ...
Similar publications
A cDNA clone for pumpkin acyl-CoA oxidase (EC1.3.3.6; ACOX) was isolated from a λgt11 cDNA library constructed from poly(A)+ RNA extracted from etiolated cotyledons. The inserted cDNA clone contains 2313 nucleotides and encodes a polypeptide of 690
amino acids. Analysis of the amino-terminal sequence of the protein indicates that the pumpkin acyl-C...
Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane. We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into the...
Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the g...
In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant...
Citations
... Fatty acid degradation begins with long-chain acyl-CoA synthetase (ACSL, EC:6.2.1.3), which activates the transformation of free fatty acids into acyl-CoA; this step is followed by a β-oxidation cycle that serially breaks down acetate units [30]. The expression of alcohol dehydrogenase (adh, EC:1.1.1.1), ...
Purpose: Epimedium brevicornu Maxim. is a perennial persistent C3 plant of the genus Epimedium Linn. in the family Berberaceae that exhibits severe physiological and morphological seed dormancy. We placed mature E.brevicornu seeds under nine stratification treatment conditions and explored the mechanisms of influence by combining seed embryo growth status assessment with related metabolic pathways and gene co-expression analysis.
Results: We identified 3.9 °C as the optimum cold-stratification temperature of E.brevicornu seeds via a chilling unit (CU) model. The best treatment was variable-temperature stratification (10/20 °C, 12/12 h) for 120 d followed by low-temperature stratification (4 °C) for 90 d. A total of 63801 differentially expressed genes were annotated to 2587 transcription factors (TFs) in 17 clusters in two comparisons. Genes specifically highly expressed in the dormancy release treatment group were significantly enriched in embryo development ending in seed dormancy and fatty acid degradation, indicating the importance of these two processes. Coexpression analysis implied that the TF GRF had the most reciprocal relationships with genes, and multiple interactions centered on zf-HD and YABBY as well as on MYB, GRF, and TCP were observed.
Conclusion: In this study, analyses of plant hormone signal pathways and fatty acid degradation pathways revealed changes in key genes during the dor- mancy release of E.brevicornu seeds, providing evidence for the filtering of E.brevicornu seed dormancy-related genes.
... Additionally, we observed the up-regulation of SCP-2 (sterol carrier protein 2) and HMG-CoA synthase (hydroxymethylglutaryl-CoA synthase). Specific, SCP-2 binds and transports lipid ligands such as long-chain fatty acids and their CoA thioesters 4 (Hayashi et al., 2002) and phospholipids (Shimazu et al., 2013). HMG-CoA synthase catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), an intermediate in cholesterol synthesis and ketogenesis (Shimazu et al., 2010). ...
Global warming is predicted to increase prolonged thermal challenges for aquatic ectotherms, i.e. it causes metabolic performance declines, impacts food intake, and finally causes impaired growth. In this research work, we investigated whether a tropical fish, Danio rerio (zebrafish), could tolerate prolonged thermal challenges and whether the temperature increase has a significant impact on growth and metabolism. To answer our questions, we evaluate the metabolomic performance, a question that has received little attention so far, using differential chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS). Three groups of fish were exposed to various temperatures of 27.6 ± 2°C, 30.7 ± 2°C or 32.2 ± 2°C during 270 days post fecundation (dpf) to evaluate the impact of the temperature increase on the growth and metabolomic performance. The results obtained demonstrated different metabolomic changes in response to acclimation to the different temperatures. After 270 days, the fish maintained at the highest tested temperature (32°C) showed reduced growth, reduced condition factor, and elevated levels of metabolites associated with amino acid catabolism and lipid metabolism pathways in the liver and intestine compared with fish kept at lower temperatures (27.6 ± 2°C). These findings demonstrate an explicit redistribution of energy stores and protein catabolism in fish at the highest temperature, thus showing a preference for maintaining length growth during limited energy availability. Moreover, here we also screened out both the marker metabolites and the altered metabolic pathways to provide essential insights to ascertain the effects of the water temperature increase on the growth and development of tropical fish.
... In Arabidopsis, the breakdown of TAGs is initiated with the release of their constituents, FA and glycerol, by the TAG lipase, SUGAR DEPENDENT1 (Eastmond, 2006). The released FAs are imported into the peroxisome by PEROXISOMAL ABC TRANSPORTER1 (Zolman et al., 2001;Hayashi et al., 2002) and enter the β-oxidation cycle (Graham, 2008). Subsequently, FAs are broken down into acetyl-CoA which can be used for carbohydrate synthesis via the glyoxylate cycle and gluconeogenesis in seed germination and seedling development (Graham, 2008). ...
Acyl-CoA binding proteins (ACBPs) constitute a well-conserved family of proteins in eukaryotes that are important in stress responses and development. Past studies have shown that ACBPs are involved in maintaining, transporting and protecting acyl-CoA esters during lipid biosynthesis in plants, mammals and yeast. ACBPs show differential expression and various binding affinities to acyl-CoA esters. Hence, ACBPs can play a crucial part in maintaining lipid homeostasis. This review summarises the functions of ACBPs during the stages of reproduction in plants and other organisms. A comprehensive understanding on the roles of ACBP during plant reproduction may lead to opportunities in crop improvement in agriculture.
... Based on our results LACS6 and 7 (Clade IV) were predicted to be localized in plastid and peroxisomes. While experimental data revealed that LACS6 and LACS7 are found to be involved in peroxisomal β-oxidation and seed development (Hayashi et al., 2002;Fulda et al., 2004). Our analysis predicted that LACS9 (Clade V) is located in the plastid. ...
In plants, Long-chain acyl-CoA synthetases (LACSs) play key roles in activating fatty acids to fatty acyl-CoA thioesters, which are then further involved in lipid synthesis and fatty acid catabolism. LACSs have been intensively studied in Arabidopsis, but its evolutionary relationship in green plants is unexplored. In this study, we performed a comprehensive genome-wide analysis of the LACS gene family across green plants followed by phylogenetic clustering analysis, gene structure determination, detection of conserved motifs, gene expression in tissues and subcellular localization. Our results identified LACS genes in 122 plant species including algae, low land plants (i.e., mosses and lycophytes), monocots, and eudicots. In total, 697 sequences were identified, and 629 sequences were selected because of alignment and some duplication errors. The retrieved amino acid sequences ranged from 271 to 1056 residues and diversified in intron/exon patterns in different LACSs. Phylogenetic clustering grouped LACS gene family into six major clades with distinct potential functions. This classification is well supported by examining gene structure and conserved motifs. Also, gene expression analysis and subcellular localization substantiate with clade division in the phylogeny, indicating that the evolutionary pattern is visible in their functionality. Additionally, experimental analysis of lacs2 mutant validated that LACS2 plays key roles in suberin synthesis. Thus, our study not only provides an evolutionary mechanism underlying functional diversification but also lays the foundation for further elucidation of the LACS gene family.
... Degradation of fatty acids begins with the activation of acyl-CoAs by the enzyme acyl-CoA synthetase (ACS; Hayashi et al. 2002) that follows with β-oxidation cycle to decompose acetate units one after the other (Hayashi et al. 2002). In our study, ACS was a common candidate gene between NDG and SL. ...
... Degradation of fatty acids begins with the activation of acyl-CoAs by the enzyme acyl-CoA synthetase (ACS; Hayashi et al. 2002) that follows with β-oxidation cycle to decompose acetate units one after the other (Hayashi et al. 2002). In our study, ACS was a common candidate gene between NDG and SL. ...
Background
Genome-wide association study (GWAS) has become an accepted and powerful method for understanding the associations between phenotypes and genotypes. In agricultural production, uniform and rapid germination is an important prerequisite in crop production. Here, a rice (Oryza sativa L.) GWAS with 33,934 SNPs (MAF > 0.05) for eight germination traits including germination percentage (GP), shoot (SL) and root length (RL), root (RFW) and shoot fresh weight (SFW), root (RDW) and shoot (SDW) dry weight, and number of days to germinate (NDG) was performed to define genomic regions influencing seed germination.
Results
Loci (43) with 70 significant germination-associated markers were detected across all rice chromosomes. Some of novel candidate associated genes were: LOC_Os01g26210 (OsWAK6) co-located with qGR-1 that is seed vigor QTL, LOC_Os07g23944 (GH31) with an α-glucosidases /starch lyase activity; id7000519 marker that corresponds to a gene cluster containing glutathione S-transferase and glucan endo-1,3-β-glucosidase co-located with qAG3 germination-related marker, LOC_Os06g47640 (calmodulin-related calcium sensor protein 29) involved in the inhibition of ABA during seed germination, and id4006430 marker that corresponds to a gene cluster containing three GH17 hydrolytic enzyme that are co-located with qHD4 and qGI1 markers.
Conclusion
The germination process is an initial and important step in the production of agricultural products, especially for rice, which is a crop plant that is grown in flooded lands. Here, the genetic diversity of rice genotypes was put under scrutiny for germination. Our GWAS results identified several likely candidate genes for germination traits that will greatly contribute to our understanding of the genetic complexity underlying the corresponding traits. The associated genes with the germination traits can be generally classified as hydrolytic enzymes and regulatory proteins that can directly or indirectly influence germination.
... Studies of glyoxysomes in the following years focused on aspects other than the aconitase enzyme, such as beta oxidation enzymes, targeting and mutant production in Arabidopsis [5,[36][37][38][39][40]. In addition, further studies of aconitase demonstrated that tobacco aconitase, like the animal counterpart, is inhibited by nitric oxide (NO), suggesting that NO is also able to modulate the enzyme in plants [41], that the repression of both mitochondrial and cytosolic tomato aconitase has drastic effects on photosynthesis and on fruit yield [42], and that plant aconitase acts as a mediator of oxidative stress and the regulation of cell death [43], although the role of the IRE binding protein has not been confirmed [35]. ...
After the discovery in 1967 of plant glyoxysomes, aconitase, one the five enzymes involved in the glyoxylate cycle, was thought to be present in the organelles, and although this was found not to be the case around 25 years ago, it is still suggested in some textbooks and recent scientific articles. Genetic research (including the study of mutants and transcriptomic analysis) is becoming increasingly important in plant biology, so metabolic pathways must be presented correctly to avoid misinterpretation and the dissemination of bad science. The focus of our study is therefore aconitase, from its first localization inside the glyoxysomes to its relocation. We also examine data concerning the role of the enzyme malate dehydrogenase in the glyoxylate cycle and data of the expression of aconitase genes in Arabidopsis and other selected higher plants. We then propose a new model concerning the interaction between glyoxysomes, mitochondria and cytosol in cotyledons or endosperm during the germination of oil-rich seeds.
... Prior to their import into peroxisomes, fatty acids released by SDP1 first have to be activated to their acyl-CoA esters by a long-chain acyl-CoA synthetase (LACS) [120]. The PEROXISOMAL ABC-TRANSPOR-TER1 (PXA1) then imports the acyl-CoAs [121][122][123][124] into the peroxisomal matrix and apparently cleaves the acyl-CoA thioester bond during this process [125]. Free fatty acids are once again activated to acyl-CoAs by the peroxisomal-matrix-localized LACS6 and LACS7 enzymes, and then degraded by β-oxidation [120]. ...
Plant oils represent an energy-rich and carbon-dense group of hydrophobic compounds. These oils are not only of economic interest, but also play important, fundamental roles in plant and algal growth and development. The subcellular storage compartments of plant lipids, referred to as lipid droplets (LDs), have long been considered relatively inert oil vessels. However, research in the last decade has revealed that LDs play far more dynamic roles in plant biology than previously appreciated, including transient neutral lipid storage, membrane remodeling, lipid signaling, and stress responses. Here we discuss recent developments in the understanding of LD formation, turnover and function in land plants and algae.
... It is a critical process in FA metabolism in prokaryotes and eukaryotes, and involves a two-step reaction. In the first step, free FAs react with ATP to generate a acyl-AMP, after which the acyl-CoA thioester bond is formed and AMP is released in the following step [9,10]. LACSs belong to the AMP binding protein (AMPBP) super family and mainly catalyze the synthesis of acyl-CoAs with acyl chain lengths of 12-20 carbons [11]. ...
... In higher plants, LACSs have several isoforms, which perform diverse biological functions in different cellular compartments [10,[18][19][20][21]. In Arabidopsis, there are nine LACS genes that participate in FA and glycerolipid metabolism. ...
... AtLACS1 and AtLACS4 showed a synergistic effect in the process of proper pollen coat formation [25]. AtLACS6 and AtLACS7 were involved in peroxisomal βoxidation and successful seedling establishment [10,26]. LACS9 is localized to the chloroplast envelope and is involved in the production of acyl-CoA [21], and its function partially overlaps that of LACS1 and LACS4 in TAG biosynthesis and lipid trafficking from the ER to the plastid, respectively, in Arabidopsis [18,27]. ...
Background:
Triacylglycerols (TAGs) are the main composition of plant seed oil. Long-chain acyl-coenzyme A synthetases (LACSs) catalyze the synthesis of long-chain acyl-coenzyme A, which is one of the primary substrates for TAG synthesis. In Arabidopsis, the LACS gene family contains nine members, among which LACS1 and LACS9 have overlapping functions in TAG biosynthesis. However, functional characterization of LACS proteins in rapeseed have been rarely reported.
Results:
An orthologue of the Arabidopsis LACS2 gene (BnLACS2) that is highly expressed in developing seeds was identified in rapeseed (Brassica napus). The BnLACS2-GFP fusion protein was mainly localized to the endoplasmic reticulum, where TAG biosynthesis occurs. Interestingly, overexpression of the BnLACS2 gene resulted in significantly higher oil contents in transgenic rapeseed plants compared to wild type, while BnLACS2-RNAi transgenic rapeseed plants had decreased oil contents. Furthermore, quantitative real-time PCR expression data revealed that the expression of several genes involved in glycolysis, as well as fatty acid (FA) and lipid biosynthesis, was also affected in transgenic plants.
Conclusions:
A long chain acyl-CoA synthetase, BnLACS2, located in the endoplasmic reticulum was identified in B. napus. Overexpression of BnLACS2 in yeast and rapeseed could increase oil content, while BnLACS2-RNAi transgenic rapeseed plants exhibited decreased oil content. Furthermore, BnLACS2 transcription increased the expression of genes involved in glycolysis, and FA and lipid synthesis in developing seeds. These results suggested that BnLACS2 is an important factor for seed oil production in B. napus.
... When the seed starts to germinate, TAG lipase on the oil body membrane degrades TAG into glycerol and fatty acids (Eastmond 2006). The fatty acids are then imported into the glyoxysome by PED3/CTS/PXA1, an ABC transporter found in the glyoxysomal membrane (Hayashi et al. 2002b), and activated to acyl-CoA by acyl-CoA synthetases (Hayashi et al. 2002a). Acyl-CoA is then catabolized into succinate by two metabolic pathways, namely fatty acid b-oxidation and the glyoxylate cycle (Mori and Nishimura 1989, Kato et al. 1995, Kato et al. 1996, Mano et al. 1996, Hayashi et al. 1998a, Kato et al. 1998, Hayashi et al. 1999, Hayashi et al. 2001. ...
Regulation of sucrose-starch interconversion in plants is important to maintain energy supplies necessary for viability and growth. Arabidopsis mutants were screened for aberrant responses to sucrose to identify candidates with a defect in the regulation of starch biosynthesis. One such mutant, fpgs1-4, accumulated substantial amounts of starch in non-photosynthetic cells. Dark-grown mutant seedlings exhibited shortened hypocotyls and accumulated starch in etioplasts when supplied with exogenous sucrose/glucose. Similar starch accumulation from exogenous sucrose was observed in mutant chloroplasts, when photosynthesis was prevented by organ culture in darkness. Molecular genetic analyses revealed that the mutant was defective in plastidial folylpolyglutamate synthetase, one of the enzymes engaged in folate biosynthesis. Active folate derivatives are important biomolecules that function as cofactors for a variety of enzymes. Exogenously-supplied 5-formyl-tetrahydrofolate abrogated the mutant phenotypes, indicating that the fpgs1-4 mutant produced insufficient folate derivative levels. In addition, the antifolate agents methotrexate and 5-fluorouracil induced starch accumulation from exogenously-supplied sucrose in dark-grown seedlings of wild-type Arabidopsis. These results indicate that plastidial folate suppresses starch biosynthesis triggered by sugar influx into non-photosynthetic cells, demonstrating a hitherto unsuspected link between plastidial folate and starch metabolism.
... These data strongly suggest that the BnLIP protein is one of the lipases responsible for hydrolysis of TAGs stored in rapeseed seeds, as it has been shown for lipases from other species (Eastmond, 2004(Eastmond, , 2006. The free fatty acids produced are transported to the glyoxysome, where they undergo β-oxidation to generate acetyl-CoA, which is finally converted into glucose through the glyoxylate cycle and gluconeogenesis (Hayashi et al., 2002;Eastmond, 2006; and references therein). The synthesized glucose will sustain early seedling growth until the photosynthetic machinery is functional. ...
Lipases catalyze the hydrolysis of ester bonds in triacylglycerides, generating glycerol and free fatty acids. These enzymes are encoded by extremely complex gene families, and appear to fulfil many different biological functions. Although they are present in all types of organisms, available information on plant lipases is still very limited, as compared to their bacterial and animal counterparts. A full-length clone, BnLIP, encoding a putative lipase, has been isolated by PCR amplification of Brassica napus genomic DNA, with oligonucleotide primers derived from the sequence of an Arabidopsis thaliana homologue. The clone included an open reading frame of 1581 bp encoding a polypeptide of 526 amino acids, with a calculated molecular mass of 59.5 kDa. Analysis of the deduced protein sequence, sequence alignment with homologous proteins from related plant species, and a phylogenetic analysis revealed that the BnLIP protein belongs to the ‘classical’ GxSxG-motif lipase family. RT-PCR assays indicated that the BnLIP gene is expressed specifically, but only transiently, during seed germination: the lipase mRNA was not present at detectable levels in ungerminated seeds, was detected only three days after seed imbibition, but its levels decreased rapidly afterwards. No expression was observed in roots, stems or leaves of adult plants. This expression pattern suggests that BnLIP is one of the lipases involved in the hydrolysis of triacylglycerides stored in rapeseed seeds, ultimately providing nutrients and energy to sustain seedling growth until photosynthesis is activated.
