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A Role for Peroxisomes in Photomorphogenesis and Development of Arabidopsis

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Science  19 Jul 2002:
Vol. 297, Issue 5580, pp. 405-409
DOI: 10.1126/science.1073633

Abstract

The nuclear protein DET1 is a central repressor of photomorphogenesis in plants. We have identified the molecular lesion in ted3, a mutation that dominantly suppresses the phenotypes of det1-1. TED3 encodes a peroxisomal protein (AtPex2p) essential for Arabidopsis growth. Developmental defects and the abnormal expression of many genes indet1 are rescued by ted3. ted3 also partially suppresses another pleiotropic de-etiolated mutantcop1. Thus, peroxisomes, whose functions are still largely unexplored, play a key role in a photomorphogenetic pathway negatively regulated by the DET1 and COP proteins.

Plants use sophisticated signal transduction systems to sense and respond to environmental variation. The light signaling network, for example, consists of a complex web of interactions between multiple photoreceptors, early signaling factors, and central integrators to control the expression of hundreds of responsive genes (1). The Arabidopsis DET1, COP, and FUS proteins are proposed to be global repressors of photomorphogenesis, because their loss-of-function mutants develop as light-grown plants in the absence of light (2). Dark-growndet1 mutants have short hypocotyls, opened cotyledons, and developed chloroplasts and ectopically express many light-regulated genes (3). They also exhibit stress symptoms, such as anthocyanin accumulation and aberrant expression of stress-related genes. Light-grown det1 plants exhibit general growth defects, such as small, pale-green leaves and seedling lethality in strong alleles (3). DET1 encodes a 62-kD nuclear protein that regulates gene expression (4), yet its precise mechanism of action is still unknown.

To better understand how DET1 exerts its functions on photomorphogenesis and development, we identified several extragenic suppressors of det1-1, an intermediate-strength allele that is impaired in the splicing of intron 1 but still produces ∼2% of wild-type mRNA, and named them ted mutants. ted3is a dominant suppressor of det1 phenotypes that does not correct the splicing defect of det1-1 (5).

We fine-mapped the TED3 gene to a ∼130-kb region at the bottom of chromosome 1 (Fig. 1A). Sequencing of selected open reading frames (ORFs) in this interval inted3 revealed a mutation in an ORF with 333 amino acids (GenBank accession number AAG52254). Sequence comparison between the genomic fragment and the corresponding expressed sequence tag (EST) clone (N96573) suggested that this gene has eight exons (Fig. 1A) and encodes a 38-kD protein. The deduced TED3 protein has a C3HC4-type RING finger motif and two putative membrane-spanning domains (Fig. 1, A and C). A transition from G to A in ted3 caused a missense mutation of a valine to methionine substitution at residue 275, one amino acid upstream of the first cysteine of the putative RING finger domain (Fig. 1, A and C). To confirm that the nucleotide change in ted3 is responsible for the suppression of det1-1, we transformeddet1-1 with a transfer DNA (T-DNA) containing the mutant ted3 gene with its own flanking sequence (6). As shown in Fig. 1B, plants carrying the mutantted3 transgene [det1-1 (ted3)] were able to recapitulate the suppression phenotypes.

Figure 1

Cloning and sequence analysis ofTED3. (A) Fine mapping of the TED3gene. TED3 was mapped by analyzing 4168 chromosomes from F2 plants of ted3 det1-1 (Col-0) X Ler with molecular markers (22). In theTED3 gene, solid lines represent introns, black boxes represent exons, and triangles indicate positions of the T-DNA insertions described in the text. In the TED3 protein, the two putative transmembrane (TM) domains and the RING finger motif are shown as black boxes. The mutation in ted3 and its position relative to the start codon are indicated. (B) Suppression of det1-1 by the ted3 transgene. (Top) 6-day-old dark-grown seedlings. (Bottom) 8-day-old light-grown seedlings. (C) Alignment of TED3 with HsPex2p (P28328) and PpPex2p (X96945). Black boxes are identical residues; dotted lines indicate gaps. The two putative transmembrane domains in TED3 are underlined. Asterisks denote the conserved cysteine and histidine residues in the RING finger. The arrow indicates the position of theted3 mutation. (D) Phylogenetic analysis of the relationship between TED3 and other PEX2 proteins. GenBank accession numbers are as follows: PaPex2p, Podospora anserina, X87329; YlPex2p, Yarrowia lipolytica, U43081; ScPex2p,Saccharomyces cerevisiae, M86538; MmPex2p, Mus musculus, L27842; AtPex10p, AF119572. Sequence alignment and tree construction were performed with the use of the MegAlign program. The bottom scale indicates relative distance between sequences.

Database searches identified TED3 as a single-copy gene in Arabidopsis that is homologous (∼20 to 24% identity) to the yeast and mammalian PEX2 genes involved in peroxisome assembly and matrix protein import (7). An alignment of TED3 with its homologs from human and Pichia pastoris showed conserved residues along the length of these proteins (Fig. 1C). Phylogenetic analysis demonstrated that TED3 is more closely related to PEX2 proteins than it is to AtPex10p, theArabidopsis protein most similar (13% identity) to TED3 (Fig. 1D). AtPex10p also contains a RING finger domain and belongs to the PEX10 family of peroxisomal membrane proteins (7). AtPex10p and PEX2 proteins are ∼10 to 13% identical, suggesting that PEX2 and PEX10 are two distantly related gene families.

Present in almost all eukaryotes, peroxisomes are single membrane–bound organelles that perform a wide array of metabolic reactions such as β-oxidation of long-chain fatty acids (7). Functions of plant peroxisomes include lipid mobilization via β-oxidation and the glyoxylate cycle for the germination of oilseed (such as Arabidopsis) seedlings, photorespiration, and nitrogen metabolism in root nodules (8). To determine the localization of TED3/AtPex2p,Arabidopsis seedlings containing a TED3::GFP (green fluorescent protein) fusion protein driven by the 35S cauliflower mosaic virus promoter (9) were analyzed by fluorescence microscopy. TED3::GFP showed a punctate pattern of green fluorescence, which colocalized with the peroxisomal enzyme catalase (Fig. 2A), indicating that, like its yeast and mammalian counterparts, TED3/AtPex2p is peroxisomal.

Figure 2

TED3/AtPex2p is an essential peroxisomal protein. (A) Subcellular localization of TED3::GFP. Hypocotyl cells from dark-grown TED3::GFP seedlings were probed with cottonseed catalase antiserum and subsequently with Texas Red–conjugated secondary antibody (23). (B) Siliques from wild-type and plants heterozygous (HET) for the T-DNA insertion in the TED3 gene. (C) Comparison of 21-day-old plants. (Left) TED3 antisense line; (right) wild-type. (D) Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of TED3 (top) and Ubiquitin10(bottom) RNA from leaf, flower, silique, stem, root, 5-day light-, and dark-grown seedlings, lanes 1 to 7, respectively. (E toG) TED3::GUS expression in cotyledons, flower and seeds, respectively.

Yeast PEX2 knockouts, in which the PEX2 gene is disabled, do not grow on alternative carbon sources, such as oleic acid and methanol, whereas analogous knockouts in mammals exhibit severe neurological dysfunctions and infant death (7). To study the role of TED3/AtPex2p in plant growth and development, we isolated two independent lines containing a T-DNA insertion in the seventh intron or the eighth exon (the RING finger region) ofTED3 (Fig. 1A). In each case, of the ∼200 T2s and T3 progeny from the heterozygous T2s, 2/3 were heterozygous for the insertion and 1/3 had no insertion, whereas no homozygotes were found, suggesting embryonic lethality for the null mutants. This conclusion was reinforced when we observed smaller siliques with fewer seeds in the heterozygotes compared with their siblings without the insertion (Fig. 2B). Also, transgenic plants with the TED3 antisense construct (10) and much reduced TED3 transcript were dwarf-sized, pale, and sterile (Fig. 2C). TED3messenger RNA (mRNA) was detected in all tissues and in light- and dark-grown seedlings (Fig. 2D), indicating that TED3 is ubiquitously expressed throughout development. Transgenic lines carrying T-DNAs with a fusion of the TED3 promoter and the β-glucuronidase (GUS) reporter gene (11) revealed high-level expression of TED3::GUS in cotyledons, pollen, ovules, and seeds (Fig. 2, E to G), suggesting that TED3/AtPex2p is essential for Arabidopsis reproduction and development.

Given that ted3::GFP was still peroxisome localized (12), we asked whether peroxisomal activities were impaired in det1 and, if so, whether ted3 could correct these deficiencies. Because Arabidopsis seeds catabolize fatty acids into sugar in glyoxysomes (specialized peroxisomes), the ability to germinate and develop on sugar-free medium primarily reflects peroxisomal activity (13). Dark-grown wild-type seedlings on sugar-free medium had longer hypocotyls than on sugar-containing plates due to the inhibition of sucrose on hypocotyl elongation (Fig. 3A), whereasdet1-1 mutants were developmentally arrested without supplemental sugar. Almost all of these det1-1 seedlings failed to green or grow true leaves even after being transferred to light for 5 days (Fig. 3B). These glyoxysomal deficiencies indet1-1, however, could be rescued by ted3 (Fig. 3, A and B). We also performed a root elongation assay, in which the growth of det1-1 and ted3 on IBA (indole-3-butyric acid) was tested. Given that IBA can be converted to IAA (indole-3-acetic acid) in a process similar to β-oxidation in glyoxysomes, β-oxidation mutants would also be defective in the conversion of IBA to IAA and would show resistance to the inhibition of root elongation by IBA (14). det1-1 did show some degree of resistance to IBA, whereas ted3 det1-1 and wild-type plants responded to IBA in a similar way (Fig. 3C). Electron micrographs (15) showed some morphological differences indet1-1 peroxisomes. Peroxisomes in light-growndet1-1 plants usually had a less dense matrix compared with wild-type and ted3 plants (Fig. 3, D to G), indicating thatdet1 might be deficient in peroxisomal matrix proteins. Taken together, we conclude that det1 seedlings have defective peroxisomes, a trait that can be rescued by theted3 gain-of-function mutation. We also crosseddet1 to a loss-of-function peroxisomal mutantpxa1, which is defective in transporting fatty acids but has a fairly weak phenotype; i.e., it is slow-growing and smaller in size than wild-type plants (14). F1progeny exhibited conditional synthetic growth defects (in strong light, Fig. 3H), and double mutants showed embryonic lethality, further substantiating the close functional relations between DET1 and peroxisomes.

Figure 3

Peroxisomal defects in det1-1 are rescued by ted3. (A) Hypocotyl measurements of 6-day dark-grown seedlings on sugar-free medium. The y axis represents mean ratios of hypocotyl length between seedlings grown in the presence of 1.5% sucrose and without it. (B) Frequency of seedling establishment (percentage of seedlings that develop true leaves) on sugar-free plates. In (A) and (B), 1 to 4 represent wild-type Col-0, det1-1, ted3 det1-1, andted3 DET1 plants, respectively. (C) IBA dose-response curves of 7-day light-grown seedlings. The mean ratios of hypocotyl lengths between seedlings grown with and without IBA were plotted against increasing IBA concentrations. Diamond, Col-0; square, det1-1; triangle, ted3 det1-1; cross, ted3 DET1. (D to G) Electron micrographs of cotyledon cells from 5-day light-grown seedlings of wild-type, det1-1, ted3 det1-1, and ted3 DET1 plants. P, peroxisome. Bars, 0.5 μm. (H) Phenotypic comparison of pxa1, det1-1, and their F1 progeny grown under ∼300 μmol/m2s of white light. (I) Fluence rate response curves in white light. Mean ratios of hypocotyl lengths between 7-day-old seedlings grown in white light or complete darkness were plotted against increasing light fluence rates. Inset, 7-day light-grown seedlings. Error bars in (A) to (C) and (I) indicate standard deviations. (J) Western blot analysis of glyoxysomal proteins detected by Arabidopsis antibody to ICL and antibody to MS and cucumber antibody to pAPX, respectively. Wild-type,det1-1, ted3 det1-1, ted3 DET1, andTED3ox proteins are in lanes 1 to 5, respectively.

Supporting the view that ted3 may encode a hypermorphic mutation contributing to the restoration of peroxisomal activities, some phenotypic similarities were observed between ted3 and plants overexpressing TED3 (TED3ox) (16). In each case, light-grown seedlings had hypocotyls longer than the wild-type and were less responsive, albeit to different degrees, to the inhibition of hypocotyl elongation by light (Fig. 3I). To better understand the nature of the ted3mutation, we compared the level of some glyoxysomal enzymes, namely, isocitrate lyase (ICL), malate synthase (MS), and peroxisomal ascorbate peroxidase (pAPX), in wild-type, det1-1, andted3 plants. In dark-grown seedlings, the amount of these enzymes in ted3 det1 is markedly higher than indet1-1 (Fig. 3J, left). Although the RNAs were expressed at similar levels in light-grown wild-type and ted3 seedlings (12), their protein levels were much higher inted3 and TED3ox than in wild-type (Fig. 3J, right). These results suggest that the ted3 mutation may be hypermorphic and that it contributes to the stabilization of some peroxisomal proteins.

The profound suppression of det1 by ted3 prompted us to compare global gene expression profiles in wild-type,det1-1 and ted3 plants with the use of theArabidopsis oligoarray that contains ∼8300 genes (17). In dark-grown seedlings, over 900 genes were misregulated by more than threefold in det1-1 compared with wild-type, whereas the expression of ∼90% of them was restored or partially restored by ted3 (Fig. 4A). In the light, of the over 300 genes with expression altered threefold or greater in det1-1, ∼60% were restored to various degrees in ted3 det1-1(Fig. 4B). A significant portion of the genes suppressed byted3 is associated with various stress responses, including light stress (Fig. 4C, table S1), which suggests that det1mimics a light-stressed seedling. In addition, a number of peroxisomal genes and genes with functions related to peroxisomes were underexpressed in det1-1, including genes encoding alanine:glyoxylate aminotransferase, isocitrate lyase, glyoxylate II, and oleosin. Genes highly overexpressed included those encoding peroxisomal-3-keto-acyl-CoA thiolase, malate dehydrogenase, and the putative peroxisome proliferation factor PEX11 (table S1).

Figure 4

ted3 rescues the abnormal expression of many genes in det1-1 and partially suppressescop1. (A and B) Cluster analysis of genes induced or repressed at least threefold in det1-1 in the dark (A) or light (B). Color bar (bottom) indicates relative expression level. 1, wild-type; 2, det1-1; 3, ted3 det1-1. (C) Classification of genes whose aberrant expression in det1-1 was restored or partially restored byted3. (D) cop1 is partially suppressed by ted3. Shown are 6-day dark-grown (left) and 20-day light-grown (right) plants.

We have provided evidence that increased peroxisomal function can suppress the numerous morphological and gene-expression defects caused by mutations in DET1. Although the precise mechanism of its function is unknown, DET1 is a nuclear protein associated with chromatin, and it probably acts to regulate the expression of hundreds of genes by limiting promoter access to transcription factors (18). TED3/AtPex2p might act directly in a photomorphogenetic pathway negatively regulated by DET1, which includes the regulation of genes required for peroxisomal functions. This loss of regulation of peroxisomal gene expression in det1 mutants would result in defective peroxisomes, which may cause seedlings to de-etiolate. In the hypermorphic ted3 mutant, increased peroxisomal functions may lead to restoration of etiolation. Given the putative functions of PEX2, the mutant ted3 protein may increase trafficking and stabilization of peroxisomal proteins. Most misregulated genes are restored to their normal expression levels byted3, which suggests that there are signals generated from peroxisomes that regulate nuclear gene expression through negative feedback, because peroxisomes are known to release signal molecules to affect nuclear gene expression (19). The fact thatted3 partially suppressed another pleiotropic de-etiolated mutant, cop1 (Fig. 4D), but not det2, a de-etiolated mutant defective in brassinosteroid biosynthesis (5), indicates that this model may apply more generally to a photomorphogenetic pathway controlled by the DET1, COP, and FUS proteins. COP proteins control photomorphogenesis by mediating proteolysis of key transcriptional regulators, thereby limiting transcription factor availability to light-regulated promoters (2). Given the similar phenotypes of det1,cop, and fus mutants, and the fact thatcop1 mutants also show some peroxisomal defects in sugar and IBA response assays (12), DET1 and the COP proteins may act to regulate similar sets of genes with roles in light signaling, photosynthesis, and peroxisomal function.

Given the hypermorphic nature of the ted3 mutation, it is also possible that ted3 suppresses det1indirectly. Because det1 mutants exhibit many characteristics of light-stressed plants (3) (table S1), it is conceivable that increasing peroxisomal function might allowdet1 ted3 plants to manage light stress more effectively (20). This possibility seems less likely, because it is known that a number of stress conditions induce the proliferation of peroxisomes (21) rather than reduce their function as in thedet1 mutant. Thus, the suppression of det1 byted3 appears specific, not general, to the mutant phenotypes. Identification of ted3 as a mutant with increased peroxisomal function will be invaluable in elucidating the functions of PEX proteins in peroxisomal biogenesis and for addressing the roles of peroxisomes in the light signaling and stress response pathways of plants.

  • * To whom correspondence should be addressed. E-mail: chory{at}salk.edu

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