Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14.

Mutations in peroxisome biogenesis proteins (peroxins) can lead to developmental deficiencies in various eukaryotes. PEX14 and PEX13 are peroxins involved in docking cargo-receptor complexes at the peroxisomal membrane, thus aiding in the transport of the cargo into the peroxisomal matrix. Genetic screens have revealed numerous Arabidopsis thaliana peroxins acting in peroxisomal matrix protein import; the viable alleles isolated through these screens are generally partial loss-of-function alleles, whereas null mutations that disrupt delivery of matrix proteins to peroxisomes can confer embryonic lethality. In this study, we used forward and reverse genetics in Arabidopsis to isolate four pex14 alleles. We found that all four alleles conferred reduced PEX14 mRNA levels and displayed physiological and molecular defects suggesting reduced but not abolished peroxisomal matrix protein import. The least severe pex14 allele, pex14-3, accumulated low levels of a C-terminally truncated PEX14 product that retained partial function. Surprisingly, even the severe pex14-2 allele, which lacked detectable PEX14 mRNA and PEX14 protein, was viable, fertile, and displayed residual peroxisome matrix protein import. As pex14 plants matured, import improved. Together, our data indicate that PEX14 facilitates, but is not essential for peroxisomal matrix protein import in plants.

Peroxisome-associated matrix protein degradation in Arabidopsis.

Peroxisomes are ubiquitous eukaryotic organelles housing diverse enzymatic reactions, including several that produce toxic reactive oxygen species. Although understanding of the mechanisms whereby enzymes enter peroxisomes with the help of peroxin (PEX) proteins is increasing, mechanisms by which damaged or obsolete peroxisomal proteins are degraded are not understood. We have exploited unique aspects of plant development to characterize peroxisome-associated protein degradation (PexAD) in Arabidopsis. Oilseed seedlings undergo a developmentally regulated remodeling of peroxisomal matrix protein composition in which the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS) are replaced by photorespiration enzymes. We found that mutations expected to increase or decrease peroxisomal H(2)O(2) levels accelerated or delayed ICL and MLS disappearance, respectively, suggesting that oxidative damage promotes peroxisomal protein degradation. ICL, MLS, and the beta-oxidation enzyme thiolase were stabilized in the pex4-1 pex22-1 double mutant, which is defective in a peroxisome-associated ubiquitin-conjugating enzyme and its membrane tether. Moreover, the stabilized ICL, thiolase, and an ICL-GFP reporter remained peroxisome associated in pex4-1 pex22-1. ICL also was stabilized and peroxisome associated in pex6-1, a mutant defective in a peroxisome-tethered ATPase. ICL and thiolase were mislocalized to the cytosol but only ICL was stabilized in pex5-10, a mutant defective in a matrix protein import receptor, suggesting that peroxisome entry is necessary for degradation of certain matrix proteins. Together, our data reveal new roles for PEX4, PEX5, PEX6, and PEX22 in PexAD of damaged or obsolete matrix proteins in addition to their canonical roles in peroxisome biogenesis.

Arabidopsis iba response5 suppressors separate responses to various hormones.

Auxin controls numerous plant growth processes by directing cell division and expansion. Auxin-response mutants, including iba response5 (ibr5), exhibit a long root and decreased lateral root production in response to exogenous auxins. ibr5 also displays resistance to the phytohormone abscisic acid (ABA). We found that the sar3 suppressor of auxin resistant1 (axr1) mutant does not suppress ibr5 auxin-response defects, suggesting that screening for ibr5 suppressors might reveal new components important for phytohormone responsiveness. We identified two classes of Arabidopsis thaliana mutants that suppressed ibr5 resistance to indole-3-butyric acid (IBA): those with restored responses to both the auxin precursor IBA and the active auxin indole-3-acetic acid (IAA) and those with restored response to IBA but not IAA. Restored IAA sensitivity was accompanied by restored ABA responsiveness, whereas suppressors that remained IAA resistant also remained ABA resistant. Some suppressors restored sensitivity to both natural and synthetic auxins; others restored responsiveness only to auxin precursors. We used positional information to determine that one ibr5 suppressor carried a mutation in PLEIOTROPIC DRUG RESISTANCE9 (PDR9/ABCG37/At3g53480), which encodes an ATP-binding cassette transporter previously implicated in cellular efflux of the synthetic auxin 2,4-dichlorophenoxyacetic acid.

The IBR5 phosphatase promotes Arabidopsis auxin responses through a novel mechanism distinct from TIR1-mediated repressor degradation.

BACKGROUND: In Arabidopsis, INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5), a putative dual-specificity protein phosphatase, is a positive regulator of auxin response. Mutations in IBR5 result in decreased plant height, defective vascular development, increased leaf serration, fewer lateral roots, and resistance to the phytohormones auxin and abscisic acid. However, the pathways through which IBR5 influences auxin responses are not fully understood. RESULTS: We analyzed double mutants of ibr5 with other mutants that dampen auxin responses and found that combining ibr5 with an auxin receptor mutant, tir1, enhanced auxin resistance relative to either parent. Like other auxin-response mutants, auxin-responsive reporter accumulation was reduced in ibr5. Unlike other auxin-resistant mutants, the Aux/IAA repressor reporter protein AXR3NT-GUS was not stabilized in ibr5. Similarly, the Aux/IAA repressor IAA28 was less abundant in ibr5 than in wild type. ibr5 defects were not fully rescued by overexpression of a mutant form of IBR5 lacking the catalytic cysteine residue. CONCLUSION: Our genetic and molecular evidence suggests that IBR5 is a phosphatase that promotes auxin responses, including auxin-inducible transcription, differently than the TIR1 auxin receptor and without destabilizing Aux/IAA repressor proteins. Our data are consistent with the possibility that auxin-responsive transcription can be modulated downstream of TIR1-mediated repressor degradation.

Identification and functional characterization of Arabidopsis PEROXIN4 and the interacting protein PEROXIN22.

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.

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.

Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824.

The cyclopropane fatty acid synthase gene (cfa) of Clostridium acetobutylicum ATCC 824 was cloned and overexpressed under the control of the clostridial ptb promoter. The function of the cfa gene was confirmed by complementation of an Escherichia coli cfa-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing cfa were introduced into C. acetobutylicum hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the cfa gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the cfa-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of cfa results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a marR homologous gene preceding the cfa gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.