Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.

Genetic evidence suggests that plant peroxisomes are the site of fatty acid ␤-oxidation and conversion of the endogenous auxin indole-3-butyric acid (IBA) to the active hormone indole-3-acetic acid. Arabidopsis mutants that are IBA resistant and sucrose dependent during early development are likely to have defects in ␤-oxidation of both IBA and fatty acids. Several of these mutants have lesions in peroxisomal protein genes. Here, we describe the Arabidopsis pex6 mutant, which is resistant to the inhibitory effects of IBA on root elongation and the stimulatory effects of IBA on lateral root formation. pex6 also is sucrose dependent during early seedling development and smaller and more pale green than WT throughout development. PEX6 encodes an apparent ATPase similar to yeast and human proteins required for peroxisomal biogenesis, and a human PEX6 cDNA can rescue the Arabidopsis pex6 mutant. The pex6 mutant has reduced levels of the peroxisomal matrix protein receptor PEX5, and pex6 defects can be partially rescued by PEX5 overexpression. These results suggest that PEX6 may facilitate PEX5 recycling and thereby promote peroxisomal matrix protein import.

Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid ␤-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid ␤-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels ␤-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adult pxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for ␤-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation.Peroxisomes are small, ubiquitous organelles encased in a single lipid bilayer that contain hydrogen peroxide-producing oxidases and catalases to inactivate reactive molecules (for review, see Gerhardt, 1992; Kindl, 1993; Olsen, 1998; Tabak et al., 1999). Arabidopsis and other oilseed plants ␤-oxidize longchain fatty acids (LCFAs) in peroxisomes to provide energy during germination. Plant peroxisomes also contain enzymes that act in photorespiration (Olsen, 1998) and the catabolism of branched-chain amino acids (Gerhardt, 1992; Zolman et al., 2001). In addition, seedlings and senescing tissues contain specialized peroxisomes called glyoxysomes that convert acetyl-coenzyme A (CoA) to succinate, which is transported to the mitochondria where it fuels the tricarboxylic acid cycle (Gerhardt, 1992; Olsen, 1998).Mammals metabolize fatty acids in both mitochondria and peroxisomes, and each organelle shortens a distinct subset of fatty acids (Lazarow, 1993; Tabak et al., 1999). In contrast, plants and yeast catabolize fatty acids exclusively in peroxisomes (Gerhardt, 1992; Kindl, 1993). Because peroxisomes lack DNA, proteins required for ␤-oxidation and other peroxisomal processes are translated in the cytoplasm and then imported (Olsen, 1998;Subramani, 1998; Tabak et al., 1999). Peroxisomal matrix proteins contain one of two peroxisomal targeting signals (PTSs). The PTS1 is made up of the amino acids "SKL" (or a conserved variant) at the extreme C termini of peroxisomal matrix-bound proteins (Gould et al., 1989). The PEX5 recept...

Auxin is an important plant hormone that plays significant roles in plant growth and development. Although numerous auxin-response mutants have been identified, auxin signal transduction pathways remain to be fully elucidated. We isolated ibr5 as an Arabidopsis indole-3-butyric acid-response mutant, but it also is less responsive to indole-3-acetic acid, synthetic auxins, auxin transport inhibitors, and the phytohormone abscisic acid. Like certain other auxin-response mutants, ibr5 has a long root and a short hypocotyl when grown in the light. In addition, ibr5 displays aberrant vascular patterning, increased leaf serration, and reduced accumulation of an auxin-inducible reporter. We used positional information to determine that the gene defective in ibr5 encodes an apparent dual-specificity phosphatase. Using immunoblot and promoterreporter gene analyses, we found that IBR5 is expressed throughout the plant. The identification of IBR5 relatives in other flowering plants suggests that IBR5 function is conserved throughout angiosperms. Our results suggest that IBR5 is a phosphatase that modulates phytohormone signal transduction and support a link between auxin and abscisic acid signaling pathways.

Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana  peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitin-conjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression of either gene individually failed to rescue the corresponding yeast mutant. Therefore, it is likely that the Arabidopsis proteins can function similarly to the yeast PEX4–PEX22 complex, with PEX4 ubiquitinating substrates and PEX22 tethering PEX4 to the peroxisome. However, the severe sucrose dependence of the pex4 pex22 mutant is not accompanied by correspondingly strong defects in peroxisomal matrix protein import, suggesting that this peroxin pair may have novel plant targets in addition to those important in fungi. Isocitrate lyase is stabilized in pex4 pex22, indicating that PEX4 and PEX22 may be important during the remodeling of peroxisome matrix contents as glyoxysomes transition to leaf peroxisomes.

Genetic evidence suggests that indole-3-butyric acid (IBA) is converted to the active auxin indole-3-acetic acid (IAA) by removal of two side-chain methylene units in a process similar to fatty acid boxidation. Previous studies implicate peroxisomes as the site of IBA metabolism, although the enzymes that act in this process are still being identified. Here, we describe two IBA-response mutants, ibr1 and ibr10. Like the previously described ibr3 mutant, which disrupts a putative peroxisomal acyl-CoA oxidase/ dehydrogenase, ibr1 and ibr10 display normal IAA responses and defective IBA responses. These defects include reduced root elongation inhibition, decreased lateral root initiation, and reduced IBA-responsive gene expression. However, peroxisomal energy-generating pathways necessary during early seedling development are unaffected in the mutants. Positional cloning of the genes responsible for the mutant defects reveals that IBR1 encodes a member of the short-chain dehydrogenase/reductase family and that IBR10 resembles enoyl-CoA hydratases/isomerases. Both enzymes contain C-terminal peroxisomaltargeting signals, consistent with IBA metabolism occurring in peroxisomes. We present a model in which IBR3, IBR10, and IBR1 may act sequentially in peroxisomal IBA b-oxidation to IAA.

). † These authors contributed equally to this work. SummaryIndole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal b-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA b-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.

The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal ␤-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes ␤-hydroxyisobutyryl-CoA. We demonstrated that a human ␤-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes ␤-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered ␤-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.