Fatty acid beta-oxidation in germinating Arabidopsis seeds is supported by peroxisomal hydroxypyruvate reductase when malate dehydrogenase is absent

Peroxisomal malate dehydrogenase (PMDH) oxidises NADH produced by fatty acid beta-oxidation during seed germination and seedling growth. Arabidopsis thaliana beta-oxidation mutants exhibit seed dormancy or impaired seed germination and failure of seedlings to degrade triacylglycerol (TAG), but the pmdh1 pmdh2 null mutant germinates readily and degrades TAG slowly during seedling growth. We reasoned that in the pmdh1 pmdh2 mutant an alternative means of oxidising NADH operates to allow a slow rate of beta-oxidation, such as NADH and NAD⁺ transport across the peroxisomal membrane or activity of another peroxisomal oxido-reductase. Here we show that peroxisomal hydroxypyruvate reductase (HPR) is present in germinating seeds and although knocking out HPR has little effect on germination and early seedling growth, when knocked out in combination with PMDH it exacerbates the pmdh1 pmdh2 phenotype. It greatly increases the proportion of dormant seeds and reduces the rate of seed germination. Seedlings have increased sucrose dependence and resistance to 2,4-dichlorophenoxybutyric acid (2,4-DB), and slower rate of TAG breakdown. When PMDH is absent, malate is lower in amount in germinating seeds and when HPR is also absent, serine (the immediate precursor of hydroxypyruvate) is much higher. These results indicate that HPR can oxidise NADH at sufficient rate in the absence of PMDH to support beta-oxidation and hence seed germination. We conclude that while HPR normally plays little role in seed germination our results support the growing body of evidence that peroxisomal NADH cannot be exported to the cytosol for oxidation but is oxidised by resident oxido-reductases.     

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

Peroxisomal ATP Import Is Essential for Seedling Development in Arabidopsis thaliana

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other β-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.The β-oxidation of fatty acids, a process that exclusively occurs within peroxisomes in plants and yeast, plays an important role in storage oil mobilization to support seedling establishment of oilseed plants, such as Arabidopsis thaliana (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Upon germination, fatty acids are released from storage oil triacylglycerol (TAG) by lipolysis, degraded via β-oxidation in specialized peroxisomes, termed glyoxysomes, and subsequently converted to sucrose, which drives growth and development until seedlings become photoautotrophic (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Before the fatty acids can enter β-oxidation, they are imported into peroxisomes by a peroxisomal ATP binding cassette (ABC) transporter, variously known as CTS (COMATOSE), At PXA1 (Arabidopsis peroxisomal ABC transporter), or PED3 (peroxisomal defective 3) and hereafter referred to as CTS (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002). Subsequently, the imported fatty acids are activated by esterification to CoA. This ATP-dependent reaction within peroxisomes is catalyzed by long-chain acyl-CoA synthetases 6 and 7 (LACS6 and LACS7, respectively), which are named according to their substrate specificity for long-chain fatty acids, which are significant components of seed storage oil in Arabidopsis (Fulda et al., 2002, 2004).In Saccharomyces cerevisiae, two mechanisms exist for import and activation of fatty acids, depending on chain length (Hettema et al., 1996). Long-chain fatty acids (C16 and C18) are converted to acyl-CoA esters in the cytosol prior to transport by the heterodimeric peroxisomal ABC transporter, Pxa1p/Pxa2 (Hettema et al., 1996). By contrast, short- and medium-chain fatty acids (≤C14) that enter the peroxisomes by passive diffusion or by an unknown transport protein are activated within peroxisomes (Hettema et al., 1996). The possibility cannot be excluded, though, that CTS imports the corresponding CoA derivatives, as is the case for the yeast Pxa1p/Pxa2p heterodimer (Hettema et al., 1996; Verleur et al., 1997), implicating a cytosolic activation of the fatty acids, catalyzed by a hitherto unknown enzyme. The actual substrates transported by CTS in Arabidopsis have not yet been experimentally determined (Theodoulou et al., 2006). However, the sucrose-dependent seedling growth phenotype of the lacs6 lacs7 double knockout mutant demonstrated that peroxisomal activation is essential for lipid mobilization to provide energy for early seedling growth (Fulda et al., 2004). The lacs6 lacs7 mutant is impaired in the degradation of fatty acids, leading to growth arrest shortly after germination (Fulda et al., 2004).Besides fatty acid mobilization, β-oxidation is also involved in generation of signaling molecules, such as the phytohormones auxin and fatty acid–derived jasmonic acid (JA) (Zolman et al., 2000; Schaller et al., 2004; Delker et al., 2007). By analogy to fatty acids released from storage oil, the precursors of these signaling molecules require CoA esterification before they can enter β-oxidation (Baker et al., 2006; Goepfert and Poirier, 2007). While the enzymes responsible for ATP-dependent activation of natural auxin (indole butyric acid [IBA]) and proherbicide 2,4-dichlorophenoxybutyric acid (2,4-DB) are currently unknown, several enzymes belonging to the acyl-activating enzyme (AAE) family have been implicated in jasmonate biosynthesis (Schneider et al., 2005; Koo et al., 2006; Kienow et al., 2008). Moreover, several as yet uncharacterized members of the large AAE family carry a putative peroxisome targeting signal (PTS) and thus might be good candidates to activate the additional β-oxidation substrates within peroxisomes (Shockey et al., 2002, 2003).In the case where activation of fatty acids or other substrates takes place within peroxisomes, the question arises as to how these ATP-dependent reactions are supplied with ATP. It is currently unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether they depend on external ATP to supply energy-dependent reactions within peroxisomes. So far, transport proteins that supply plant peroxisomes with energy for fatty acid oxidation have not been characterized. However, in bakers'' yeast, a peroxisomal adenine nucleotide transporter, ANT1, that is required for the ATP-dependent activation of medium-chain fatty acids inside peroxisomes has been characterized (Palmieri et al., 2001).ATP transport proteins play an important role in the distribution of the primary agent coupling endergonic and exergonic reactions in every cellular compartment (Winkler and Neuhaus, 1999). In Arabidopsis and other plants, various adenine nucleotide carriers have been identified at the molecular level. The mitochondrial ADP/ATP carrier mediates the export of ATP that is synthesized in the mitochondrion to provide energy for cellular metabolism (Heimpel et al., 2001; Haferkamp et al., 2002). The plastidial ATP/ADP transporter (nucleotide transporter) is involved in ATP uptake by both chloroplasts and heterotrophic plastids, to enable the nocturnal ATP supply required for chlorophyll biosynthesis (Reiser et al., 2004; Reinhold et al., 2007), as well as by heterotrophic plastids to drive starch biosynthesis (Batz et al., 1992; Tjaden et al., 1998). Yet another ATP/ADP antiporter located in the endoplasmic reticulum (ER) membrane provides energy by importing ATP into the ER for the accumulation of ER-related storage lipids and proteins (Leroch et al., 2008).In this study, we identified two novel peroxisomal adenine nucleotide carrier proteins (PNC1 and PNC2) from Arabidopsis. Colocalization studies demonstrated that these proteins are targeted to peroxisomes. Yeast complementation and in vitro ATP uptake assays showed that both PNC1 and PNC2 catalyze the counterexchange of ATP with AMP. Using an inducible RNA interference (RNAi) repression strategy, we further established several transgenic Arabidopsis lines with reduced expression levels of both PNC1 and PNC2. Our results showed that import of ATP into peroxisomes that is catalyzed by PNC1 and PNC2 is essential for activation of fatty acids during seedling germination and plays a role in other β-oxidation reactions in peroxisomes, such as auxin metabolism. Analysis of PNC1 and PNC2 repression lines further indicates that no other ATP generating systems exist inside plant peroxisomes and that ATP import is the only way to supply the peroxisomal matrix with ATP.     

Light quality influences germination, root growth and hypocotyl elongation in somatic embryos but not in seedlings of norway spruce

Summary We studied how light from different light sources influences germination and postgerminative growth of plants from somatic embryos and seeds of Norway spruce (Picea abies [L.] Karst). Somatic embryos of three spruce genotypes and seeds were subjected to light from commercially available light sources: Philips TLD Blue 18W/18 (BL), Osram Fluora (FL), Philips Cool White TL 50W/33 (CW), Osram Warm White 18W/30 (WW), Philips Yellow 36W/16 (YE) and Philips TLD Red 36W/15 (RE), 18 h a day, with a photon flux (PAR) at 30 μmol m⁻² s⁻¹. After 6 wk the germination frequencies of the somatic embryo-derived plantlets were 50% under BL and 98% under RE. The corresponding mean root lengths were 6.7 and 15.4 mm. In somatic embryo-derived plantlets cultured under BL, FL, CW and WW, both roots and hypocotyls turned brown, presumably due to production of phenolic substances. Browning was less severe in somatic embryo-derived plantlets cultured under RE and YE. Under RE, the epicotyl elongated in 37% of the plantlets after 6 wk, compared with 70% under the other light sources. Seed germination and postgerminative seedling growth was modestly influenced by light from these light sources. RE and WW initially delayed germination as compared with BL, FL and CW, but after 2 wk, more than 90% of the seeds had germinated under all light sources. In conclusion, germination and postgerminative growth of somatic embryos of spruce is sensitive to differences in light quality, whereas seed germination and seedling growth is not.     

Genetic Dissection of Peroxisome-Associated Matrix Protein Degradation in Arabidopsis thaliana

Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H₂O₂, which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP–ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.     

Spores of microorganisms

Bacillus cereus spores labelled with radioactive diaminopimelic acid (DAP) in different phases of spore development germinated and the fate of the individual DAP-containing fractions during germination and postgerminative development was studied. Envelope structures not depolymerized during germination were formed in the prespore stage. During maturation of the spores, structures degraded during germination and released into the medium were mainly formed. Some of the DAP-containing material remained in the cells in a labile form during germination and could probably be re-utilized in synthesis of the cell wall during postgerminative development.     

Abscisic Acid Represses Growth of the Arabidopsis Embryonic Axis after Germination by Enhancing Auxin Signaling

Under unfavorable environmental conditions, the stress phytohormone ABA inhibits the developmental transition from an embryo in a dry seed into a young seedling. We developed a genetic screen to isolate Arabidopsis thaliana mutants whose early seedling development is resistant to ABA. Here, we report the identification of a recessive mutation in AUXIN RESISTANT1 (AUX1), encoding a cellular auxin influx carrier. Although auxin is a major morphogenesis hormone in plants, little is known about ABA–auxin interactions during early seedling growth. We show that aux1 and pin2 mutants are insensitive to ABA-dependent repression of embryonic axis (hypocotyl and radicle) elongation. Genetic and physiological experiments show that this involves auxin transport to the embryonic axis elongation zone, where ABA enhances the activity of an auxin-responsive promoter. We propose that ABA represses embryonic axis elongation by potentiating auxin signaling in its elongation zone. This involves repression of the AUXIN INDUCIBLE (Aux/IAA) gene AXR2/IAA7, encoding a key component of ABA- and auxin-dependent responses during postgerminative growth.     

Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores'' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore''s genome is free of damage.     

Immunogold labelling indicates high catalase concentrations in amorphous and crystalline inclusions of sunflower (Helianthus annuus L.) peroxisomes

Summary Immunogold labelling and electron microscopy were used to investigate whether catalase was present in peroxisomal inclusions, the composition of which has not yet been determined in plant cells. In the mesophyll cells of sunflower (Helianthus annuus L.) cotyledons, the catalase gold label was confined to peroxisomes. At day 2 of postgerminative growth in darkness, peroxisomes were free of inclusions, and the matrix was homogeneously labelled with gold particles. Thereafter, amorphous inclusions appeared, but by day 5 of growth, conspicuous crystalline inclusions (cores) were the predominant type. This developmental change, first observed in cotyledons grown in continuous light between day 2.5 and 5, also took place in cotyledons kept in permanent darkness. Both amorphous and crystalline inclusions showed a much higher immunogold label than did the peroxisomal matrix, indicating that catalase was a component of both types of peroxisomal inclusions. In contrast to catalase, the immunogold label of glycolate oxidase was almost completely absent from cores and was confined to the peroxisomal matrix. Together with reports on the absence of other enzymes from peroxisomal inclusions in sunflower and other species (Vaughn, 1989) our results suggest that catalase is a major constituent of amorphous and crystalline peroxisomal inclusions in plants.     

Arabidopsis DAYU/ABERRANT PEROXISOME MORPHOLOGY9 Is a Key Regulator of Peroxisome Biogenesis and Plays Critical Roles during Pollen Maturation and Germination in Planta

Pollen undergo a maturation process to sustain pollen viability and prepare them for germination. Molecular mechanisms controlling these processes remain largely unknown. Here, we report an Arabidopsis thaliana mutant, dayu (dau), which impairs pollen maturation and in vivo germination. Molecular analysis indicated that DAU encodes the peroxisomal membrane protein ABERRANT PEROXISOME MORPHOLOGY9 (APEM9). DAU is transiently expressed from bicellular pollen to mature pollen during male gametogenesis. DAU interacts with peroxisomal membrane proteins PEROXIN13 (PEX13) and PEX16 in planta. Consistently, both peroxisome biogenesis and peroxisome protein import are impaired in dau pollen. In addition, the jasmonic acid (JA) level is significantly decreased in dau pollen, and the dau mutant phenotype is partially rescued by exogenous application of JA, indicating that the male sterility is mainly due to JA deficiency. In addition, the phenotypic survey of peroxin mutants indicates that the PEXs most likely play different roles in pollen germination. Taken together, these data indicate that DAU/APEM9 plays critical roles in peroxisome biogenesis and function, which is essential for JA production and pollen maturation and germination.     

The Origin, Development and Germination of Bulbils in Two Dioscorea Species

The origin, development and germination of aerial bulbils ofDioscorea alata and D. bulbifera are described and the significanceof the primary nodal complex (PNC) in bulbil ontogeny discussed.Further evidence is provided for the view that the PNC, or itsinitial, is the site of renewed growth in Dioscorea species. Dioscorea species, yams, bulbil germination, primary nodal complex     

Yarrowia lipolytica AAL genes are involved in peroxisomal fatty acid activation

In yeast, β-oxidation of fatty acids (FAs) essentially takes place in peroxisomes, and FA activation must precede FA oxidation. In Saccharomyces cerevisiae, a single fatty-acyl–CoA-synthetase, ScFaa2p, mediates peroxisomal FA activation. We have previously shown that this reaction also exists in the oleaginous yeast Yarrowia lipolytica; however, the protein involved in this process remains unknown. Here, we found that proteins, named Aal proteins (Acyl/Aryl-CoA-ligases), resembling the 4-coumarate–CoA-ligase-like enzymes found in plants are involved in peroxisomal FA activation in Y. lipolytica; Y. lipolytica has 10 AAL genes, eight of which are upregulated by oleate. All the Aal proteins contain a PTS1-type peroxisomal targeting sequence (A/SKL), suggesting a peroxisomal localization. The function of the Aal proteins was analyzed using the faa1Δant1Δ mutant strain, which demonstrates neither cytoplasmic FA activation (direct result of FAA1 deletion) nor peroxisomal FA activation (indirect result of ANT1 deletion, a gene coding an ATP transporter). This strain is thus highly sensitive to external FA levels and unable to store external FAs in lipid bodies (LBs). Whereas the overexpression of (cytoplasmic) AAL1ΔPTS1 was able to partially complement the growth defect observed in the faa1Δant1Δ mutant on short-, medium- and long-chain FA media, the presence of Aal2p to Aal10p only allowed growth on the short-chain FA medium. Additionally, partial LB formation was observed in the oleate medium for strains overexpressing Aal1ΔPTS1p, Aal4ΔPTS1p, Aal7ΔPTS1p, and Aal8ΔPTS1p. Finally, an analysis of the FA content of cells grown in the oleate medium suggested that Aal4p and Aal6p present substrate specificity for C16:1 and/or C18:0.     

Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.     

Involvement of glutathione peroxidase 1 in growth and peroxisome formation in Saccharomyces cerevisiae in oleic acid medium

Saccharomyces cerevisiae is able to use some fatty acids, such as oleic acid, as a sole source of carbon. β-oxidation, which occurs in a single membrane-enveloped organelle or peroxisome, is responsible for the assimilation of fatty acids. In S. cerevisiae, β-oxidation occurs only in peroxisomes, and H₂O₂ is generated during this fatty acid-metabolizing pathway. S. cerevisiae has three GPX genes (GPX1, GPX2, and GPX3) encoding atypical 2-Cys peroxiredoxins. Here we show that expression of GPX1 was induced in medium containing oleic acid as a carbon source in an Msn2/Msn4-dependent manner. We found that Gpx1 was located in the peroxisomal matrix. The peroxisomal Gpx1 showed peroxidase activity using thioredoxin or glutathione as a reducing power. Peroxisome biogenesis was induced when cells were cultured with oleic acid. Peroxisome biogenesis was impaired in gpx1? cells, and subsequently, the growth of gpx1? cells was lowered in oleic acid-containing medium. Gpx1 contains six cysteine residues. Of the cysteine-substituted mutants of Gpx1, Gpx1^C36S was not able to restore growth and peroxisome formation in oleic acid-containing medium, therefore, redox regulation of Gpx1 seems to be involved in the mechanism of peroxisome formation.