Bethany K. Zolman

Plant Peroxisomes: Biogenesis and Function

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.

Inputs to the Active Indole-3-Acetic Acid Pool: De Novo Synthesis, Conjugate Hydrolysis, and Indole-3-Butyric Acid b-Oxidation

The phytohormone auxin is important in virtually all aspects of plant growth and development, yet our understanding of auxin homeostasis is far from complete. Plants use several mechanisms to control levels of the active auxin, indole-3-acetic acid (IAA). Plants can synthesize IAA both from tryptophan (Trp-dependent pathways) and from a Trp precursor but bypassing Trp (Trp-independent pathways). Despite progress in identifying enzymes in Trp-dependent IAA biosynthesis, no single IAA biosynthetic pathway is yet defined to the level that all of the relevant genes, enzymes, and intermediates are identified. In addition to de novo synthesis, vascular plants can obtain IAA from the hydrolysis of IAA conjugates. IAA can be conjugated to amino acids, sugars, and peptides; endogenous conjugates that are active in bioassays and hydrolyzed in plants are likely to be important free IAA sources. Conjugation is also used to permanently inactivate excess IAA, and these conjugates may be distinct from the hydrolyzable conjugates. The peroxisomal (3-oxidation of endogenous indole-3-butyric acid (IBA) also can supply plants with IAA, which may account for part of the auxin activity of exogenous IBA. Compartmentalization of enzymes and precursors may contribute to the regulation of auxin metabolism. IAA obtained through de novo synthesis, conjugate hydrolysis, or IBA β-oxidation may have different functions in plant development, and possible roles for the IAA derived from the various pathways are discussed.

IBR5, a Dual-Specificity Phosphatase-Like Protein Modulating Auxin and Abscisic Acid Responsiveness in Arabidopsis

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 promoter-reporter 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.

Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes

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 β-oxidation. 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 peroxisomal-targeting signals, consistent with IBA metabolism occurring in peroxisomes. We present a model in which IBR3, IBR10, and IBR1 may act sequentially in peroxisomal IBA β-oxidation to IAA.

IBR3, a novel peroxisomal acyl-CoA dehydrogenase-like protein required for indole-3-butyric acid response

Indole-3-butyric acid (IBA) is an endogenous auxin that acts in Arabidopsis primarily via its conversion to the principal auxin indole-3-acetic acid (IAA). Genetic and biochemical evidence indicates that this conversion is similar to peroxisomal fatty acid β-oxidation, but the specific enzymes catalyzing IBA β-oxidation have not been identified. We identified an IBA-response mutant (ibr3) with decreased responses to the inhibitory effects of IBA on root elongation or the stimulatory effects of IBA on lateral root formation. However, ibr3 mutants respond normally to other forms of auxin, including IAA. The mutant seedlings germinate and develop normally, even in the absence of sucrose, suggesting that fatty acid β-oxidation is unaffected. Additionally, double mutants between ibr3 and acx3, which is defective in an acyl-CoA oxidase acting in fatty acid β-oxidation, have enhanced IBA resistance, consistent with a distinct role for IBR3. Positional cloning revealed that IBR3 encodes a putative acyl-CoA dehydrogenase with a consensus peroxisomal targeting signal. Based on the singular defect of this mutant in responding to IBA, we propose that IBR3 may act directly in the oxidation of IBA to IAA.

Disruption of Arabidopsis CHY1 Reveals an Important Role of Metabolic Status in Plant Cold Stress Signaling

To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter (CBF3-LUC). One mutant, chy1-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chy1 mutant. chy1-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chy1 mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chy1 plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wild-type but not in chy1. Constitutive CBF3 expression partially rescues the sensitivity of chy1-10 plants to dark treatment in the cold. The chy1 mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal beta-hydroxyisobutyryl (HIBYL)-CoA hydrolase needed for valine catabolism and fatty acid beta-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.

pex5 Mutants That Differentially Disrupt PTS1 and PTS2 Peroxisomal Matrix Protein Import in Arabidopsis

PEX5 and PEX7 are receptors required for the import of peroxisome-bound proteins containing one of two peroxisomal targeting signals (PTS1 or PTS2). To better understand the role of PEX5 in plant peroxisomal import, we characterized the Arabidopsis (Arabidopsis thaliana) pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has germination defects and is completely dependent on exogenous Suc for early seedling establishment, based on poor utilization of seed-storage fatty acids. This mutant also has delayed development and reduced fertility, although adult pex5-10 plants appear normal. Peroxisomal metabolism of indole-3-butyric acid, propionate, and isobutyrate also is disrupted. The pex5-10 mutant has reduced import of both PTS1 and PTS2 proteins, and enzymatic processes that occur in peroxisomes are disrupted. To specifically study the import and importance of PTS1 proteins, we made a truncated PEX5 construct lacking the PTS1-binding region (PEX5454). Transformation of this construct into pex5-10 resulted in the rescue of PTS2 import, thereby creating a line with PTS1-specific import defects. The pex5-10 (PEX5454) plants still had developmental defects, although restoring PTS2 import resulted in a less severe mutant phenotype. Comparison of pex5-10 and pex5-10 (PEX5454) phenotypes can separate the import mechanisms for enzymes acting in different peroxisomal processes, including indole-3-butyric acid/2,4-dichlorophenoxybutyric acid oxidation, isobutyrate and propionate metabolism, and photorespiration.

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions.

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4Col and acx1acx3acx4Col mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.

Auxin Input Pathway Disruptions Are Mitigated by Changes in Auxin Biosynthetic Gene Expression in Arabidopsis

Disruption of multiple auxin-input pathways reveals developmental roles for these auxin forms and a feedback loop necessary for maintaining optimal free hormone levels. Auxin is a phytohormone involved in cell elongation and division. Levels of indole-3-acetic acid (IAA), the primary auxin, are tightly regulated through biosynthesis, degradation, sequestration, and transport. IAA is sequestered in reversible processes by adding amino acids, polyol or simple alcohols, or sugars, forming IAA conjugates, or through a two-carbon elongation forming indole-3-butyric acid. These sequestered forms of IAA alter hormone activity. To gain a better understanding of how auxin homeostasis is maintained, we have generated Arabidopsis (Arabidopsis thaliana) mutants that combine disruptions in the pathways, converting IAA conjugates and indole-3-butyric acid to free IAA. These mutants show phenotypes indicative of low auxin levels, including delayed germination, abnormal vein patterning, and decreased apical dominance. Root phenotypes include changes in root length, root branching, and root hair growth. IAA levels are reduced in the cotyledon tissue but not meristems or hypocotyls. In the combination mutants, auxin biosynthetic gene expression is increased, particularly in the YUCCA/Tryptophan Aminotransferase of Arabidopsis1 pathway, providing a feedback mechanism that allows the plant to compensate for changes in IAA input pathways and maintain cellular homeostasis.

Peroxisomes as a Source of Auxin Signaling Molecules

Peroxisomes house many metabolic processes that allow organisms to safely sequester reactions with potentially damaging byproducts. Peroxisomes also produce signaling molecules; in plants, these include the hormones indole-3-acetic acid (IAA) and jasmonic acid (JA). Indole-3-butyric acid (IBA) is a chain-elongated form of the active auxin IAA and is a key tool for horticulturists and plant breeders for inducing rooting in plant cultures and callus. IBA is both made from and converted to IAA, providing a mechanism to maintain optimal IAA levels. Based on genetic analysis and studies of IBA metabolism, IBA conversion to IAA occurs in peroxisomes, and the timing and activity of peroxisomal import and metabolism thereby contribute to the IAA pool in a plant. Four enzymes have been hypothesized to act specifically in peroxisomal IBA conversion to IAA. Loss of these enzymes results in decreased IAA levels, a reduction in auxin-induced gene expression, and strong disruptions in cell elongation resulting in developmental abnormalities. Additional activity by known fatty acid β-oxidation enzymes also may contribute to IBA β-oxidation via direct activity or indirect effects. This review will discuss the peroxisomal enzymes that have been implicated in auxin homeostasis and the importance of IBA-derived IAA in plant growth and development.