PEX11 mediates intralumenal vesicle formation in peroxisomes

Tharp, Nathan E.; An, Chelsea; Hwang, James; Shad, Nayeli S.; Wright, Zachary J.; Bartel, Bonnie

doi:10.1038/s41467-026-71873-3

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Published: 21 April 2026

PEX11 mediates intralumenal vesicle formation in peroxisomes

Nature Communications volume 17, Article number: 3538 (2026) Cite this article

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Abstract

Peroxisomes are eukaryotic organelles that compartmentalize crucial metabolic reactions. Peroxisome size, shape, and number are governed by the peroxisomal membrane protein PEX11. PEX11 is encoded in multiple isoforms across diverse eukaryotes, including five in Arabidopsis, but the functional distinctions among these isoforms are largely uncharacterized. Here we report null pex11 mutants in plants expressing reporters that mark peroxisome membranes and lumen to illuminate distinct functions for PEX11 isoforms. We find that PEX11C/D/E promotes the formation of peroxisomal intralumenal vesicles, limits peroxisome size throughout development, and is required for efficient fatty acid β-oxidation in germinating seedlings. Unlike the pervasive roles of PEX11C/D/E, we find that PEX11A/B promotes the formation of peroxisomal intralumenal vesicles and limits peroxisome enlargement specifically during seedling lipid mobilization. Complete loss of the PEX11 family confers seedling lethality, even though peroxisomes remain abundant. Our findings reveal that Arabidopsis PEX11 isoforms shape internal peroxisome membranes and have distinct functions in cellular physiology that are essential for plant development. These results extend the roles of PEX11 beyond its canonical function in peroxisome division.

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Introduction

Peroxisomes are metabolically plastic organelles that compartmentalize specialized metabolism. In addition to reactive oxygen species catabolism and fatty acid β-oxidation¹, peroxisomes harbor various reactions, depending on the organism or tissue, including plasmalogen synthesis in mammals² and phytohormone maturation in plants¹. Most multicellular organisms cannot survive without peroxisomes. For example, human peroxisome biogenesis disorders can be lethal in infancy^3,4, and plant peroxisomes are essential for embryogenesis and for catabolizing the fats stored in lipid droplets that fuel early seedling development¹.

New peroxisomes arise from the endoplasmic reticulum^5,6 or are generated from pre-existing peroxisomes via division^7,8,9. The proteins required for peroxisome biogenesis, division, and import of membrane and lumenal proteins are known as peroxins (PEX proteins), and a core set of peroxins is conserved among metazoans, fungi, and plants¹⁰. In lumenal protein import, cytosolic receptor peroxins bind to peroxisome targeting signals (PTSs) on proteins destined for the peroxisome lumen and accompany their cargo into the organelle, with the assistance of membrane-embedded peroxins, before exiting to facilitate further import rounds^1,11,12.

A prominent, conserved peroxin implicated in peroxisome division is PEX11. PEX11 is a transmembrane protein thought to coordinate peroxisomal division by tubulating the membrane^13,14 and recruiting the fission machinery that executes scission^{9,11,15,16,17,18}. The N-terminal domain of Penicillium chrysogenum PEX11 can induce liposome tubulation¹⁴, and Saccharomyces cerevisiae (baker’s yeast) and Physcomitrella patens (moss) pex11 mutants contain fewer, larger peroxisomes, consistent with fission defects^19,20,21. Similarly, downregulating Arabidopsis thaliana PEX11 transcripts using RNA interference diminishes peroxisome number and increases peroxisome size^16,22,23. Conversely, overexpressing human^13,24,25,26, plant^13,16,23,27, or yeast^13,28 PEX11 isoforms can induce peroxisome tubulation or increase peroxisome abundance.

Unlike other peroxins, all examined eukaryotes with peroxisomes encode multiple PEX11 isoforms^10,29, including five in Arabidopsis (PEX11A-E), three in S. cerevisiae (Pex11, Pex25, and Pex27), and three in humans (PEX11α, β, and γ). Yeast mutants lacking individual PEX11 isoforms display enlarged peroxisomes, and the removal of all three homologs impairs import of peroxisomal enzymes^19,20. PEX11β, the most prominent mammalian isoform, alters peroxisome abundance²⁵ and is critical for neuronal function³⁰ and epidermal cell differentiation³¹, whereas PEX11α is dispensable for peroxisome function³², and PEX11γ overexpression does not alter peroxisome abundance³². However, how various PEX11 isoforms function individually and in combination remains incompletely understood.

There may be roles for PEX11 beyond peroxisome division. Peroxisomes are born from complex ER “nest” domains⁶, a process that presumably requires the severing of nascent organelles from the ER, and PEX11 has been implicated in de novo peroxisome formation in fungi²⁹. Moreover, vesicles within peroxisomes are observed in plants^{6,33,34,35,36}, yeast^37,38, and animals^39,40. We recently used fluorescent reporters separately labeling peroxisomal membranes and lumen to visualize intralumenal vesicle (ILV) formation³⁶ and peroxisome biogenesis from ER nests⁶ in Arabidopsis seedlings. Peroxisomal ILVs form via endosomal sorting complex required for transport (ESCRT)-assisted invagination of the outer membrane and are hypothesized to aid in mobilizing fats stored in lipid droplets³⁶. However, the full mechanism of ILV formation, including the peroxisome-specific components, remains unknown.

In this study, we discovered unanticipated roles for PEX11 in peroxisome biology by examining various combinations of null pex11 mutants in Arabidopsis, an ideal model for elucidating peroxisome structure and function. Arabidopsis seedlings are well-suited to live-cell microscopy and contain remarkably large peroxisomes compared to yeast or mammals^36,41, allowing visualization of peroxisome substructures using light microscopy³⁶. Moreover, quantitative physiological assays can report peroxisome function in intact seedlings⁴². We found that disrupting various combinations of PEX11 isoforms conferred enlarged peroxisomes lacking ILVs, failure to mobilize stored fats, and severe physiological defects, including seedling death. Our data reveal that PEX11 isoforms have distinct and overlapping membrane-shaping roles beyond peroxisome fission in Arabidopsis.

Results

PEX11 promotes peroxisomal ILV formation and limits peroxisome size

Disrupting PEX11 increases peroxisome size in various systems, including yeast^19,28,43 and plants^16,21,22,23. Given our discovery of pervasive ILVs in Arabidopsis peroxisomes³⁶, the involvement of PEX11 in membrane shaping, and the recent connection of PEX11 to an ESCRT component⁴⁴, we sought to determine whether PEX11 might function in ILV formation in addition to its canonical role in peroxisome division.

As in other eukaryotes, PEX11 is encoded in a multigene family in Arabidopsis¹⁰. PEX11A-B and PEX11C-E form distinct subfamilies (Supplementary Fig. 1a, b)^22,23 with similar predicted structures (Supplementary Fig. 1e, f). The primary sequences of PEX11C, PEX11D, and PEX11E are over 80% identical to each other, whereas PEX11A and PEX11B are ~29% identical to each other and less similar to members of the PEX11C-E clade (Supplementary Fig. 1). To explore the roles of these subfamilies, we separately generated PEX11A-B or PEX11C-E null mutations using a plant-optimized CRISPR-Cas9 system⁴⁵ to simultaneously express two or three guide RNAs (gRNAs) (Supplementary Fig. 1d). To enable visualization of peroxisome lumen and membrane, we generated mutations in plants carrying a transgene constitutively expressing mRuby3 fused to a C-terminal PTS1 (mRuby3-PTS1) and mNeonGreen fused to the C-terminal membrane anchor of PEX26 (mNeonGreen-mPTS^PEX26)³⁶ (Fig. 1a). From the progeny of the Cas9/gRNA-expressing plants, we obtained various single and higher-order pex11 mutants carrying early frameshift mutations (Supplementary Table 1, 2) and lacking the Cas9 transgene. Immunoblot analysis using an antibody raised to a conserved region of PEX11E⁴⁶ revealed the expected ~27-kDa protein in pex11cd, pex11 de, and pex11abce mutants but not in the pex11cde mutant (Fig. 2a, Supplementary Fig. 2a, b). These data indicate that the antibody recognizes all isoforms in the PEX11C/D/E clade and confirm that our pex11c, pex11d, and pex11e frameshift mutations (Supplementary Table 1, 2) prevent accumulation of the corresponding full-length proteins.

Fig. 1: Arabidopsis PEX11 genes control peroxisome size and ILV content. — **Fig. 1: Arabidopsis *PEX11* genes control peroxisome size and ILV content.**

**Fig. 2: PEX11 isoforms differentially contribute to plant development and physiology.**

We compared peroxisomes in the parental wild-type reporter line with those in the pex11a pex11b double mutant, henceforth referred to as pex11ab, and a mutant lacking the other three PEX11 proteins (pex11c pex11d pex11e; pex11cde). We imaged the abundant peroxisomes in cotyledons, embryonic leaves packed with lipid droplets that fuel seedling growth before photosynthesis begins, and found dramatically enlarged peroxisomes when either subset of PEX11 genes was disrupted (Fig. 1b, c).

As wild-type seedlings develop, cotyledon peroxisomes enlarge as lipid stores are mobilized and then shrink as lipid stores are depleted (Fig. 1d-f)^36,41. In our analysis, wild-type peroxisomes expanded from an average diameter of about 3 µm at day 2 to 5 µm at day 3 before returning to 3 µm by day 7 (Fig. 1d-f). Similarly, pex11ab peroxisomes expanded as seedlings developed and were smaller again by day 7 (Fig. 1d-f). However, pex11ab peroxisomes attained markedly larger maximum sizes than wild-type peroxisomes, with almost half of pex11ab peroxisomes exceeding 10 µm in diameter at day 5 (Fig. 1d-f). To determine whether the restoration of smaller peroxisomes in older pex11ab seedlings reflected shrinkage of individual peroxisomes, we followed peroxisomes in pex11ab mutants using time-lapse microscopy. In 3-day-old hypocotyls, we observed individual pex11ab peroxisomes shrinking over time (Fig. 1i, Supplementary Movie 1). In contrast to wild-type and pex11ab peroxisomes, the dramatic enlargement of pex11cde peroxisomes continued throughout the time course. Remarkably, peroxisomes in older pex11cde cotyledons sometimes exceeded 20 µm in diameter (Fig. 1d-f). Although these enlarged peroxisomes remained mostly spherical (Supplementary Fig. 2f), some were misshapen due to limitations imposed by the surrounding cell walls (Fig. 1d, asterisks).

Strikingly, ILVs were less apparent or absent in the large peroxisomes of higher-order pex11 mutants. As seedlings matured, the enlarged peroxisomes in pex11ab and pex11cde contained notably sparse ILVs, and the few internal membranes that remained often appeared to be tiny ILVs or membrane remnants (Fig. 1c, d). However, the lack of PEX11A/B or PEX11C/D/E did not completely block ILV formation, as the small peroxisomes found in very young (2-day) pex11 mutant seedlings appeared to contain internal membranes (Fig. 1d), and pex11ab peroxisomes appeared to acquire internal membranes as they shrank (Fig. 1i, Supplementary Movie 1).

Because the pex11ab and pex11cde mutants displayed such dramatic peroxisome phenotypes (Fig. 1), we also examined other allele combinations. We found similarly increased peroxisome size and reduced internal membrane content in multiple different pex11a pex11b allele combinations (Supplementary Fig. 2c). Moreover, cotyledons of pex11a and pex11b single mutants sometimes displayed slightly enlarged peroxisomes that appeared to have fewer ILVs than wild-type peroxisomes (Supplementary Fig. 2d, e). However, this expansion was not as extensive as in the pex11ab double mutants (Supplementary Fig. 2c-e), indicating that PEX11A and PEX11B are partially redundant. In contrast, peroxisomes in pex11cd and pex11de mutants resembled wild type rather than pex11cde in both size and ILV content (Supplementary Fig. 2d), indicating that PEX11C, D, and E function redundantly.

Large peroxisomes could result from reduced ILV formation (because ILVs form from the outer peroxisomal membrane^6,36) or from decreased peroxisome division, which would presumably decrease peroxisome numbers. Although we did observe fewer peroxisomes in cotyledons and hypocotyls of 2- or 3-day-old pex11cde mutants than in wild type or pex11ab (Fig. 1g, Supplementary Fig. 4c), we did not find significantly altered peroxisome numbers in older pex11 cotyledons (Fig. 1g), suggesting that PEX11-mediated fission cannot explain all pex11 phenotypes. Moreover, the total volume occupied by peroxisomes in cotyledons of 3-day-old pex11ab mutants and 5- and 7-day-old pex11cde mutants was dramatically elevated (Fig. 1h), which is inconsistent with defects in fission alone. We concluded that both PEX11A/B and PEX11C/D/E limit peroxisome size during seedling development by supporting peroxisomal ILV formation.

PEX11C/D/E facilitates lipid mobilization, plant growth, and peroxisomal protein import

To assess the protein import competence of the aberrant pex11 peroxisomes, we compared our mutants to wild type and pex14-1, a mutant with a frameshift mutation near the middle of the PEX14 gene that prevents accumulation of full-length PEX14 and efficient lumenal protein import (Supplementary Fig. 2a, 4a)⁴⁷. As a quantitative proxy for import, we monitored the processing of a PTS2-containing protein, peroxisomal malate dehydrogenase (PMDH). Because the N-terminal PTS2 region is removed in peroxisomes, mutants with inefficient import accumulate the cytosolic precursor protein, which can be visualized by immunoblotting^48,49,50. We found that, like pex14⁴⁷, pex11cde displayed incomplete PMDH processing (Fig. 2a, b), suggesting that PEX11C/D/E directly or indirectly supports lumenal protein import.

To evaluate the metabolic competence of pex11 peroxisomes, we used peroxisome-related physiological assays. Peroxisomes are the sole site of β-oxidation in plants⁵¹, and dysfunctional peroxisomes inefficiently access fixed carbon stored in lipid droplets, conferring reduced seedling growth that can be partially restored by the provision of sucrose^52,53. The wild-type reporter line displayed slightly reduced growth compared to untransformed Arabidopsis (Fig. 2c-g), whereas pex11cde mutants displayed compromised growth in both the light and the dark that was partially rescued by sucrose supplementation (Fig. 2c, d, Supplementary Fig. 3a, b). The pex11cde growth defects resembled those of pex14-1, suggesting reduced peroxisome function. In contrast, the pex11a, pex11b, pex11ab, pex11cd, and pex11de seedlings had at most mild growth defects compared to the parental line (Fig. 2c, d, Supplementary Fig. 3a). The pex11cde growth defects persisted into adulthood and were generally more severe than those displayed by pex14. For example, pex11cde plants had small rosettes, short stature, and decreased survival after transplantation to soil (Fig. 2e-h). In contrast, pex11ab resembled the parental line in these assays (Fig. 2e-h). We concluded that the PEX11C/D/E clade, unlike PEX11A/B, is required throughout plant development.

In a second readout of peroxisome metabolism, we monitored responses to a phytohormone precursor, indole-3-butyric acid (IBA). Because peroxisomes β-oxidize IBA into active auxin, which inhibits root⁵³ and hypocotyl elongation⁵⁴, and promotes lateral root formation⁵³, peroxisomal dysfunction limits IBA responsiveness. Like pex14, the IBA responsiveness of the pex11cde mutant was clearly impaired; pex11cde hypocotyls and primary roots elongated similarly with or without IBA supplementation (Fig. 2c, Supplementary Fig. 3a, b). In contrast, various single and double pex11 mutants had negligible defects in these assays (Supplementary Fig. 3a), and pex11ab hypocotyls were only slightly less IBA-responsive than the wild-type reporter line (Fig. 2c, Supplementary Fig. 3a, b). When we monitored lateral root promotion by IBA, pex11ab resembled the parental line, whereas pex11cde displayed intermediate IBA responsiveness compared to pex14, which did not generate lateral roots in response to IBA (Supplementary Fig. 3c). The severe physiological defects of pex11cde and the minor defects of pex11ab (Fig. 2) suggest that the two PEX11 clades have distinct roles despite both mutants displaying enlarged cotyledon peroxisomes (Fig. 1).

Arabidopsis peroxisomes expand during seedling development^36,41. This enlargement requires the transfer of fats from lipid droplets, as peroxisomes in mutants lacking the PXA1 (ABCD1) fatty acid transporter remain small during seedling development³⁶. Similarly, peroxisomes in a mutant lacking SDP1, a prominent triacylglycerol (TAG) lipase⁵⁵, were smaller than wild-type peroxisomes during seedling development (Supplementary Fig. 4a). In contrast, mutants lacking certain fatty acid β-oxidation enzymes accumulate excessively expanded peroxisomes during seedling development, with some of these mutants exhibiting no marked physiological defects^{36,41,52,56,57}. Because pex11ab and pex11cde mutants both displayed large peroxisomes (Fig. 1), but only pex11cde showed physiological defects associated with inefficient lipid mobilization (Fig. 2), we monitored lipid droplet utilization in pex11 mutants. We found abundant lipid droplets in pex11 mutants and wild-type cotyledons at 3 days, but by 7 days, when lipid droplets were sparse in wild-type and pex11ab cotyledons, numerous lipid droplets remained in pex11cde mutants (Fig. 3a). Moreover, OLEOSIN1 (OLE1), a lipid droplet coat protein that is degraded as lipids are mobilized^58,59, remained abundant in 7-day-old pex11cde and pex14 seedlings after it had been degraded in wild type (Fig. 3b, c). We quantified lipid retention by monitoring seedling TAG levels using thin-layer chromatography. Unlike in pex11ab, we observed elevated TAG levels in 5- and 7-day-old pex11cde seedlings compared to wild type (Fig. 3d). To assess the global impacts of PEX11 on seedling lipids, we used lipidomics (Supplementary Fig. 5). Lipid content was not notably altered in 7- or 12-day-old pex11ab seedlings (Fig. 3e, Supplementary Fig. 5a, 6). In contrast, the chloroplast membrane lipids digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG) were reduced in older pex11cde seedlings (Fig. 3e, Supplementary Fig. 5, 6), commensurate with the delayed development of this mutant (Fig. 2). Most notably, 7- and 12-day-old pex11cde seedlings, like sdp1 and pex14, had markedly elevated TAG and diacylglycerol (DAG) levels compared to the wild-type reporter line (Fig. 3e, Supplementary Fig. 5, 6). Intriguingly, 12-day-old pex11cde seedlings also had moderately increased levels of the membrane lipid phosphatidylcholine (PC), an alteration that was not observed in sdp1 or pex14 mutants (Fig. 3e, Supplementary Fig. 5, 6). Together with the sucrose dependence of pex11cde (Fig. 2c, d), these findings confirm that PEX11C/D/E is needed for efficient lipid mobilization during seedling development, whereas PEX11A/B makes at most minor contributions to this process.

**Fig. 3: PEX11 isoforms differentially contribute to lipid metabolism.**

PEX11A/B promotes ILV formation and restrains peroxisome size during lipid mobilization

We directly tested whether lipid mobilization was necessary for the extreme peroxisome expansion observed in pex11 mutants by inhibiting lipid mobilization with diphenyl methylphosphonate (DMP)⁶⁰. We found that wild-type and pex11ab seedlings grown on DMP contained more abundant lipid droplets (Supplementary Fig. 4b) than mock-treated seedlings, confirming that DMP impaired lipid mobilization (Fig. 4a). These lipid droplet differences were not apparent in pex11cde mutants (Supplementary Fig. 4b), which already displayed impaired TAG mobilization (Fig. 3). Peroxisome numbers were unaffected when seedlings were grown on DMP (Supplementary Fig. 3c), suggesting that the lipid mobilization inhibitor does not impact peroxisome proliferation. Strikingly, DMP fully suppressed peroxisome enlargement in both pex11ab and wild-type seedlings (Fig. 4b, c). In marked contrast, pex11cde peroxisome size was unaffected by DMP (Fig. 4b, c).

**Fig. 4: PEX11 isoforms play different roles in shaping peroxisome membranes during lipid mobilization.**

To confirm that lipid mobilization drives pex11ab peroxisome enlargement, we genetically disabled lipid mobilization in our reporter line and the pex11ab mutant. We used CRISPR-Cas9 to introduce frameshift mutations in the genes encoding three lipid mobilization enzymes (Fig. 4a): SDP1, a TAG lipase⁵⁵; PXA1, the peroxisomal fatty-acyl-CoA transporter^61,62; or PED1, a thiolase acting in β-oxidation⁵². We found that 3-day-old sdp1, pxa1, and ped1 seedlings contained smaller peroxisomes than wild type or pex11ab (Fig. 4d). Moreover, when pex11ab mutants lacked PXA1 or PED1, peroxisome enlargement was markedly diminished (Fig. 4d). Similarly, when pex11ab mutants lacked SDP1, most peroxisomes appeared similar in size to sdp1 mutants (Fig. 4d). However, a subpopulation of peroxisomes in sdp1 pex11ab mutants were large and lacked ILVs (Fig. 4d), perhaps because another TAG lipase, SDP1-like⁶³, remained present. The observation that pharmacological (Fig. 4b, c) or genetic (Fig. 4d) inhibition of lipid mobilization suppressed the large-peroxisome phenotype of pex11ab mutants indicates that TAG catabolism is necessary for the excessive pex11ab peroxisome enlargement and confirms that PEX11A/B restrains peroxisome expansion and promotes ILV formation during lipid mobilization. In contrast, the robust peroxisome expansion observed in pex11cde, even when lipid mobilization is impaired (Fig. 4b, c), reveals that PEX11C/D/E uses a different mechanism to limit peroxisome enlargement.

ILVs in wild-type seedlings appear to move freely within the peroxisome lumen, whereas subpopulations of enlarged peroxisomes in β-oxidation mutants sometimes contain ILVs that appear affixed to the outer membrane adjacent to lipid droplets³⁶. Similarly, we observed a subpopulation of enlarged peroxisomes in older ped1 mutant seedlings with ILVs sequestered to the peroxisome membrane adjacent to lipid droplets (Fig. 4e), consistent with the hypothesis that peroxisomal ILVs function in lipid mobilization^6,36. Moreover, some peroxisomes in 7-day-old ped1 pex11ab mutants were enlarged and contained ILVs apposed to lipid droplets (Fig. 4e). The presence of ILVs in these expanded ped1 pex11ab peroxisomes supports our time-lapse data showing that PEX11A/B is not absolutely required for ILV formation (Fig. 1i, Supplementary Movie 1) and suggests that β-oxidation of ILV lipids might contribute to the apparent ILV deficit in pex11ab mutants.

Because peroxisome expansion in pex11ab, but not pex11cde, required active lipid mobilization (Fig. 4), we examined peroxisomes in tissues with different dependencies on β-oxidation. For example, “true” leaves form post-embryonically from the shoot apical meristem (Fig. 5a) and rely on photosynthesis rather than stored lipids for energy⁶⁴. We found that pex11ab leaf peroxisomes resembled wild-type peroxisomes, whereas pex11cde leaf peroxisomes were enormous and lacked ILVs (Fig. 5b), supporting the conclusion that PEX11C/D/E restrains peroxisome enlargement independently of lipid mobilization.

Fig. 5: PEX11 genes differentially control peroxisome size and ILV formation in true leaves and roots. — **Fig. 5: *PEX11* genes differentially control peroxisome size and ILV formation in true leaves and roots.**

The pattern of enlarged peroxisomes in pex11 roots was also linked to lipid mobilization. Germinating seedling roots utilize lipid droplets deposited during embryogenesis, whereas roots in older plants rely on photosynthate from the shoot. In the root meristem, daughter cells arising from dividing stem cells are displaced as the root grows downward (Fig. 5a). Thus, the primary root provides a chronological record of root cell birth and differentiation⁶⁵, with the oldest root cells adjacent to the hypocotyl. pex11ab seedlings only displayed enlarged peroxisomes in mature root cells (Fig. 5c), which descended from embryonic root cells that were packed with lipid droplets. As in older cotyledon cells (Fig. 1d), these enlarged pex11ab mutant peroxisomes mostly lacked ILVs (Fig. 5c). In contrast, pex11cde mutants already contained larger peroxisomes than pex11ab or wild-type seedlings in the young cells of the root meristem (Fig. 5c). Most of these enlarged peroxisomes in pex11cde root meristem contained abundant ILVs, demonstrating that PEX11C/D/E is not essential for ILV formation in these cells. Similarly, in the young differentiation zone (Fig. 5a), where cells exit the elongation zone and begin to form root hairs⁶⁵, pex11cde peroxisomes were enlarged with some ILVs (Fig. 5c). In the mature differentiation zone, however, the large peroxisomes of pex11cde mutants contained intralumenal membranes that often appeared as stringy or clumped internal membranes rather than the usual spherical vesicular architecture in cells from younger root zones (Fig. 5c), suggesting that PEX11C/D/E is needed to correctly shape or maintain newly forming ILVs. The distinct peroxisome expansion defects observed in the corresponding pex11 mutants suggest that PEX11A/B and PEX11C/D/E have different functions.

PEX11A/B and PEX11C/D/E support ILV formation and restrain peroxisome size via distinct mechanisms

Because varying expression could contribute to different pex11 mutant phenotypes, we examined PEX11 transcript levels in an RNA-seq atlas⁶⁶. All five PEX11 isoforms were expressed during seed germination, with PEX11D and E transcripts more abundant than PEX11A, B, or C transcripts (Supplementary Fig. 7). Additionally, PEX11A, C, and E transcripts were present at similar levels in 1-day-old seedling roots, whereas PEX11D transcripts were low in roots but more abundant in cotyledons and hypocotyls than the other transcripts (Supplementary Fig. 7).

The observation of dramatically enlarged peroxisomes in pex11ab mutant cotyledons (Fig. 1) despite abundant PEX11D transcripts in cotyledons (Supplementary Fig. 7d) suggested that the differences observed in pex11ab and pex11cde mutants originated from functional differences among the corresponding proteins. To test this possibility, we expressed mTagBFP2-tagged versions of PEX11B or PEX11E (Fig. 6a) from an estradiol-inducible promoter⁶⁷ in pex11ab and pex11cde mutants. Immunoblotting following growth on estradiol revealed that mTagBFP2-HA-PEX11E accumulated to higher levels than mTagBFP2-HA-PEX11B in the selected transgenic lines (Fig. 6b).

**Fig. 6: PEX11 isoforms have distinct functions.**

As expected from prior studies showing tagged Arabidopsis PEX11 isoforms at peroxisomal membranes^23,27, both mTagBFP2-HA-PEX11B and mTagBFP2-HA-PEX11E co-localized with the mNeonGreen-mPTS^PEX26 peroxisomal membrane marker (Fig. 6c, d, Supplementary Fig. 8). We found that the subset of pex11ab cells that accumulated mTagBFP2-PEX11B following estradiol induction showed restored peroxisome size and ILV formation, whereas adjacent cells without mTagBFP2-HA-PEX11B still contained large peroxisomes lacking ILVs (Fig. 6c). In contrast, expression of mTagBFP2-HA-PEX11E in pex11ab seedlings did not restore ILV content. Instead, peroxisomes in seedlings that appeared to have lower mTagBFP2-HA-PEX11E levels were ringed with mTagBFP2-HA-PEX11E, but largely lacked ILVs, like the original pex11ab mutant (Fig. 6c, fourth column). Even seedlings with higher mTagBFP2-HA-PEX11E accumulation, which contained smaller, clustered, and occasionally elongated (tubulated) peroxisomes (or peroxules), often lacked peroxisomal ILVs (Fig. 6c, sixth column, Supplementary Movie 2). The ability of the PEX11E reporter to decrease peroxisome size in pex11ab without restoring peroxisomal ILV content indicates that PEX11A/B has a role in ILV formation that cannot be performed by PEX11E. Intriguingly, expressing yeast Pex11 (mTagBFP2-HA-ScPex11) complemented pex11ab peroxisome size and ILV defects (Fig. 6e), suggesting conserved PEX11 function in other eukaryotes.

In reciprocal experiments, expression of mTagBFP2-HA-PEX11E clearly restored pex11cde peroxisome size to resemble wild type, whereas pex11cde cells accumulating mTagBFP2-HA-PEX11B did not display appreciably altered peroxisomes compared to neighboring cells that did not accumulate the reporter (Fig. 6d). Together, these complementation experiments demonstrate that the peroxisome morphology defects observed in the pex11 mutants stemmed from deficiencies in the corresponding PEX11 genes, that the N-terminal tag did not notably interfere with PEX11 function in regulating peroxisome size, and that PEX11A/B and PEX11C/D/E play distinct roles in sculpting peroxisomal membranes.

PEX11 is essential for seedling survival in Arabidopsis

To determine if PEX11C/D/E also perform redundant roles with PEX11A/B in peroxisome biology, we sought higher-order pex11 mutants. We used our three-gRNA CRISPR system to target PEX11C-E in a pex11ab mutant and identified several viable quadruple mutants (Supplementary Table 1, 2). Growth of pex11abcd quadruple mutant seedlings was similar to pex11ab mutants, with a slight defect when grown without sucrose, whereas pex11abce mutants had a moderate growth defect and some sucrose dependence but were less impaired than the pex11cde mutant (Supplementary Fig. 3a). Genotyping the adult progeny of a homozygous pex11abcd mutant that was also heterozygous for a pex11e mutation did not reveal any homozygous pex11abcde quintuple mutants, indicating that the absence of all PEX11 isoforms conferred embryonic or seedling lethality. We did not observe shriveled or missing seeds in seedpods from pex11 hemiquintuple (pex11a/a b/b c/c d/d E/e) mutants, suggesting that, unlike many peroxin null alleles^{68,69,70,71,72,73}, the quintuple pex11 mutant did not display embryonic or gametophytic lethality. Indeed, when we genotyped seedlings from a pex11 hemiquintuple parent, we found pex11abcde seedlings at the expected Mendelian ratio ( ~ 25%; Fig. 7b). However, the tiny pex11abcde quintuple mutant seedlings were even smaller than pex11cde mutants on sucrose-supplemented medium (Fig. 7a) and did not survive after transfer to soil, indicating that PEX11C/D/E and PEX11A/B functions are together essential for plant survival.

**Fig. 7: PEX11 is essential for seedling development and peroxisome function.**

Like pex11abcd and pex11cde mutants, pex11abcde mutants contained large peroxisomes with sparse ILVs compared to wild type (Fig. 7c, d). Occasionally, the massively enlarged peroxisomes in pex11abcde mutants appeared to compromise the final cell division needed for stomate formation. Guard cells and pavement cells arise from post-embryonic cell divisions from the same meristemoid precursors⁷⁴, and the two guard cells that form a stomate are small compared to the pavement cells that comprise most of the epidermis. The extreme peroxisome enlargement in the pex11abcde mutant sometimes resulted in normally spherical peroxisomes becoming elongated due to the limits imposed by the guard cell walls (Fig. 7c). In some instances, these elongated pex11abcde peroxisomes were positioned perpendicularly to the stomatal opening and appeared to have occluded the final symmetrical cell division that produces two guard cells from a guard mother cell (Fig. 7c, last column). The presence of constrained peroxisomes in deformed stomata suggests that the PEX11 family prevents peroxisomes from occupying an excessive volume, thereby allowing cell division.

Surprisingly, peroxisomes remained even more abundant in pex11abcde than in wild-type seedlings (Fig. 7d, e), conclusively demonstrating that PEX11 is not essential for peroxisome proliferation in Arabidopsis cotyledons. The enhanced defects of pex11abcde mutants imply that both PEX11 clades cooperatively regulate peroxisome size and morphology.

Discussion

By generating pex11 null mutants in Arabidopsis, we confirmed a role for PEX11 in limiting peroxisome size and discovered that peroxisomal ILV abundance is dependent on PEX11. Among the five Arabidopsis PEX11 isoforms, PEX11A/B limits peroxisome size during the lipid mobilization that drives early seedling growth, whereas PEX11C/D/E is essential for normal lipid metabolism and plant growth, and limits peroxisome size in diverse tissues even in the absence of lipid mobilization (Fig. 7f). Together, these distinct PEX11 functions in maintaining peroxisome size and morphology are essential for plant life.

PEX11 is typically thought to initiate peroxisome division. However, the dramatically increased peroxisome size in pex11ab mutants was not accompanied by similarly decreased peroxisome abundance (Fig. 1), which would follow division defects. Furthermore, inhibiting lipid mobilization prevented pex11ab (and wild-type) peroxisome enlargement (Fig. 4), indicating that this enlargement required fatty acids from lipid droplets. Similarly, yeast pex11 mutant peroxisomes are small unless grown on oleate-containing media¹⁹, and yeast Pex11 is implicated in fatty acid import⁷⁵. Our observation that yeast Pex11 can restore ILV content and peroxisome size in the Arabidopsis pex11ab mutant (Fig. 6e) hints that the connections between PEX11, peroxisome size, and lipid mobilization observed in other systems^16,19,21,22 may, at least in part, reflect peroxisomal ILV formation during lipid mobilization (Fig. 7f).

Catabolism of the fatty acids liberated from TAG at lipid droplet-peroxisome contact sites requires membrane-localized transporters to import fatty acids into peroxisomes^76,77. Because long-chain fatty acids are virtually insoluble in water⁷⁸, it is unlikely that these fatty acids would be imported like water-soluble metabolites. Instead, long-chain fatty acids might insert into the peroxisomal membrane and become accessible to the lumen through peroxisomal fatty-acid transporters acting to flip fatty acids to the inner leaflet of the lipid bilayer⁷⁶. ILVs could then assist lipid import by bringing membranes laden with free fatty acids or fatty acyl-CoAs into the peroxisome lumen (Fig. 7f). Indeed, peroxisome membranes in young cotton seedlings contain abundant free fatty acids derived from lipid droplet TAG⁷⁹. Moreover, ESCRT disruptions impair not only peroxisomal ILV formation but also lipid mobilization, and β-oxidation enzyme mutants display ILV defects (Fig. 4e)³⁶. Although the composition of ILV membranes is unknown, our data support the idea that peroxisomal ILV membranes might harbor lipids from lipid droplets.

The propensity of long-chain fatty acids or fatty acyl-CoAs to permeabilize and destabilize membranes⁸⁰ would presumably limit the concentration of free fatty acids in ILV membranes. Perhaps some TAG-derived lipids are converted into phospholipids and delivered to the peroxisome membrane to fuel ILV formation, rather than being immediately hydrolyzed to free fatty acids. This idea is supported by the observation that cotton seedling peroxisomes contain phospholipids derived from lipid droplet TAG⁷⁹. Phospholipids incorporated into the peroxisome bilayer could enter peroxisomes via ILV invagination, be degraded by intraperoxisomal phospholipases, and finally catabolized through β-oxidation. Drosophila pex2 and pex16 mutants accumulate phospholipid precursors and are deficient in phospholipid degradation products⁸¹. Similarly, 12-day-old pex11cde mutant seedlings had increased PC levels (Fig. 3e, Supplementary Fig. 6), hinting that phospholipid degradation may be a normal peroxisomal process. This model would explain why a yeast peroxisomal phospholipase mutant displays peroxisomal ILVs⁸² and defects in β-oxidation⁸³, and why an Arabidopsis peroxisomal β-oxidation enzyme mutant appears to accumulate peroxisomal ILVs (Fig. 4e)⁵².

Given these connections between lipid mobilization, peroxisome enlargement, and ILV formation, it was curious that the notable pex11ab peroxisomal ILV defects (Fig. 1) were not accompanied by the severe physiological defects that are hallmarks of impaired β-oxidation (Fig. 2). However, unlike pex11cde mutants, and like wild-type peroxisomes, pex11ab peroxisomes shrank and acquired internal membranes as seedlings developed (Fig. 1), indicating that ILV formation is only partially disrupted in the absence of PEX11A/B. Moreover, genetically impairing β-oxidation by introducing a ped1 mutation into the pex11ab mutant revealed ILVs adjacent to lipid droplets in enlarged peroxisomes (Fig. 4e), again demonstrating that ILVs can form without PEX11A/B and suggesting that these ILVs are broken down by the PED1 thiolase. Perhaps, without PEX11A/B, inefficient ILV formation during lipid mobilization results in excessive peroxisome enlargement, while the continued degradation of the ILVs that do form allows fat utilization and seedling growth.

Unlike pex11ab, very young pex11cde tissues contained fewer peroxisomes than wild type (Fig. 1g, Supplementary Fig. 4c), consistent with a role for PEX11C/D/E in peroxisome proliferation. Moreover, expressing mTagBFP2-HA-PEX11E in the pex11ab mutant resulted in numerous small peroxisomes (Fig. 6c), indicating that PEX11E can increase peroxisome abundance. We also detected tubulated membranes in pex11ab cells with higher mTagBFP2-HA-PEX11E accumulation, consistent with prior studies demonstrating that PEX11 overexpression can induce peroxisome tubulation, which has been hypothesized to precede division^{13,23,24,26,27}. However, the striking peroxisome expansion in older pex11cde mutant cotyledons was not accompanied by decreased peroxisome numbers, and reduced peroxisome fission cannot completely account for the dramatic increase in total peroxisome volume in this tissue (Fig. 1, Supplementary Fig. 2f). We conclude that PEX11C/D/E, like PEX11A/B, has roles beyond peroxisome division, which might include regulation of pexophagy^84,85 and/or biogenesis from the ER^6,29,86,87 (Fig. 7f).

Like in pex11ab mutants, the enlarged peroxisomes in pex11cde mutants often appeared to lack ILVs (Fig. 1c). The enlarged pex11cde peroxisomes, even in tissues where β-oxidation is not particularly active (e.g., true leaves (Fig. 5b) and young root cells (Fig. 5c)), suggest a role for peroxisomal ILVs beyond lipid mobilization. Indeed, blocking lipid droplet mobilization did not prevent the excessive enlargement of pex11cde peroxisomes (Fig. 4b, c). Perhaps peroxisome enlargement in pex11cde mutants derives from increased phospholipid transfer from a non-lipid droplet source. For example, non-vesicular lipid transfer from the ER supports the expansion of multiple compartments^88,89, including peroxisomes^6,90,91,92, and might supply pex11cde peroxisomes with excess lipids. Alternatively, a failure of newly synthesized peroxisomes to efficiently detach from the ER might contribute to excessive peroxisome expansion in pex11cde (Fig. 7f). Either hypothesis could explain the increased PC levels in pex11cde (Fig. 3e, Supplementary Fig. 6).

pex11cde seedlings, unlike pex11ab, inefficiently mobilized lipid droplets, leading to TAG retention and sucrose-dependent seedling growth (Figs. 2, 3, Supplementary Fig. 6). Lipid droplet TAG is hydrolyzed by the peroxisomal membrane lipase SDP1⁵⁵ to form DAG and a free fatty acid, which is transported into the peroxisome via PXA1⁶¹ for β-oxidation (Fig. 4a). PEX11E interacts with FREE1⁴⁴, an early-acting plant-specific ESCRT protein⁹³, which might initiate the ESCRT cascade to form peroxisomal ILVs. Intriguingly, FREE1 also interacts with SDP1⁴⁴. Perhaps PEX11C/D/E binding to FREE1 promotes FREE1-SDP1 interactions to assist the lipid droplet–peroxisome contacts that facilitate lipid mobilization in addition to initiating the ESCRT cascade that forms peroxisomal ILVs.

In addition to bringing effector proteins to the peroxisome, PEX11 may have intrinsic properties that contribute to pex11 mutant phenotypes. For example, predicted Arabidopsis PEX11 structures reveal a small channel through the six predicted alpha-helices (Supplemental Fig. 1c), and yeast Pex11 acts as a non-selective channel that transfers small solutes in vitro⁷⁵. Intriguingly, a nonselective cation channel on the lysosome membrane, TMEM63A, allows lysosomal membrane deformation by releasing cationic osmoticants when lysosome membranes experience mechanical tension caused by increased turgor pressure⁹⁴. If an osmoticant normally released by PEX11 builds up within the organelle, perhaps pex11 mutant peroxisomes experience heightened turgor pressure, which impedes the membrane deformation that would allow ILV formation or peroxisome division. It will be interesting to learn whether Arabidopsis PEX11 acts as an ion channel, which solute(s) it transports, and whether PEX11 isoforms have different specificities.

Interestingly, pex11cde mutants displayed PTS2-processing defects (Fig. 2a, b), suggesting a direct or indirect role for PEX11C/D/E, and perhaps ILVs, in lumenal protein import. Indeed, a potential import role for ILVs was hypothesized when peroxisomes were originally found to import oligomeric proteins³⁷. Perhaps ILV invagination assists canonical peroxisomal import¹² by sequestering cargo that is too large to import efficiently, potentially explaining the presence of PTS-containing proteins in a subset of ILVs³⁶. The IBA resistance and plant growth defects observed in pex11cde mutants (Fig. 2, Supplementary Fig. 3) could stem from defects in lumenal protein import and/or the inefficiencies that could result from dramatically expanding the volume of the peroxisome lumen (Fig. 1h, Supplementary Fig. 2f). For example, the activity of DEG15, the enzyme responsible for cleaving PTS2-containing proteins^48,49,50, could be reduced in the diluted lumen of expanded peroxisomes.

In addition to the distinct functions of Arabidopsis PEX11A/B and C/D/E, the seedling lethality of the pex11 quintuple mutant (Fig. 7a, b) could reflect overlapping functions or an essential role of cross-clade oligomers. All Arabidopsis PEX11 isoforms interact with each other¹⁶, and PEX11 hetero-oligomers could mediate additional protein interactions and functions. pex11 quintuple mutant peroxisomes in 7-day-old cotyledons were enlarged and devoid of ILVs (Fig. 7c, d), reinforcing the critical role for PEX11 in ILV formation. Remarkably, pex11 quintuple mutants contained even more peroxisomes than wild type (Fig. 7d, e), indicating that Arabidopsis PEX11 is not essential for building peroxisomes. Moreover, the cellular crowding that ensues upon excessive peroxisome expansion could disrupt the functions of other organelles and cellular processes, including cell division (Fig. 7c). Beyond roles in development, plant PEX11 genes are differentially induced upon biotic and abiotic stresses^46,95,96, and it will be interesting to learn the roles of PEX11 isoforms and peroxisomal ILVs under various stress conditions.

Other examined eukaryotes also have multiple PEX11 isoforms¹⁰, and the distinctions between Arabidopsis PEX11 clade functions prompt the question of whether PEX11 isoforms function similarly in other species. The small size of peroxisomes in many organisms makes it nearly impossible to resolve sub-peroxisomal structures using light microscopy, and employing pex11 mutants to allow imaging of larger peroxisomes⁸³ would fail to reveal ILVs if the ILV-promoting function of PEX11 is conserved. Prior studies have shown partial complementation of yeast pex11 growth defects by some Arabidopsis PEX11 isoforms^23,97. Yeast Pex11 is more closely related to Arabidopsis PEX11A/B than PEX11C/D/E¹⁰, and we found that mTagBFP2-HA-ScPex11 complemented Arabidopsis pex11ab peroxisome size and ILV defects (Fig. 6e) hinting that the enlarged peroxisomes observed in yeast pex11 mutants¹⁹ could stem from impaired ILV formation. It will be interesting to learn if PEX11-dependent peroxisomal ILV formation is conserved in other organisms that display large peroxisomes upon PEX11 dysfunction.

In summary, we employed live-cell microscopy to demonstrate that Arabidopsis PEX11 isoforms differentially sculpt peroxisomal membranes to regulate organellar function. The PEX11 roles in ILV formation illuminated here may provide insight into PEX11 functions in other systems, including the pathophysiology of human PEX11 deficiencies^{98,99,100,101,102}, and inform future studies uncovering additional functions of internalized peroxisomal membranes.

Methods

Plant growth conditions and physiological assays

All plant lines were in the Columbia-0 accession of Arabidopsis thaliana. pex14-1⁴⁷ and the dual fluorescent reporter line marking peroxisome lumen (mRuby3-PTS1) and membrane (mNeonGreen-mPTS^PEX26)³⁶ were described previously. sdp1-8 (SALK_201353C) was generated by the Salk Institute Genomic Analysis Laboratory¹⁰³ and obtained from the Arabidopsis Biological Resource Center at The Ohio State University.

Plants were grown under continuous white light at 22 °C unless otherwise indicated. Seeds were surface sterilized and stratified for 1-3 days in 0.1% agar in the dark at 4 °C. Seeds were sown on plant nutrient (PN) media¹⁰⁴ supplemented with 0.5% sucrose (PNS). Media was solidified using 0.6% or 1% agar when growing seedlings horizontally or vertically, respectively. After 7-12 days, seedlings were transplanted to soil for seed production.

For light-grown sucrose dependence and IBA assays, seeds were sown on PNS supplemented with 10 μM IBA (from a 100 mM stock dissolved in ethanol), or PN or PNS supplemented with the same amount of ethanol, and grown vertically under continuous yellow-filtered light¹⁰⁵ for 7 days. For dark-grown sucrose dependence and IBA assays, seeds were germinated under continuous white light for 1 day. Seeds were then plated on PNS supplemented with 30 μM IBA, or PN or PNS supplemented with the same amount of ethanol, and incubated for 1 day under yellow-filtered light before plates were wrapped in foil and placed vertically for 4 days. Plates were scanned using a flatbed scanner, and seedling hypocotyls (dark-grown assays) or roots (light-grown assays) were measured using ImageJ/FIJI (version 1.54p). Seedlings that did not germinate were not measured, except in Supplementary Fig. 3a, where seedlings that did not germinate were quantified as 0.

For lateral root assays, seedlings were grown vertically on PNS under continuous white light for 4 days before being transferred to PNS supplemented with 8 μM IBA, or PNS supplemented with the same amount of ethanol, and grown under yellow-filtered light for an additional 4 days, after which lateral roots that had emerged from the primary root were counted using a dissecting microscope, and the primary root length was measured to the nearest 1 mm using a ruler.

Seedlings and adult plants were photographed using an Apple iPhone camera. Image brightness was corrected using Adobe Photoshop (version 26.11). Representative 7-day-old seedlings grown on PN or PNS were transferred to a fresh plate and photographed. For pex11 quintuple mutant seedlings, 7-day-old PNS-grown seedlings from a pex11a/a b/b c/c d/d E/e parent were photographed, and homozygous quintuple mutants were identified via genotyping. For adult plant analyses, 8-day-old seedlings were transplanted from PNS to soil, and plant survival was monitored twice a week over 35 days. Rosettes were photographed at 25 days. To measure rosette diameter, a circle encompassing the rosette was drawn using FIJI, and Microsoft Excel (version 2508) was used to calculate the diameter from the circumference.

Computational methods

Arabidopsis PEX11 protein sequence alignment, phylogenetic cladogram, and percent identity matrix were generated using MegAlign (DNAStar version 10.0.1). The sequence alignment was performed using the ClustalW method (BLOSUM series protein weight matrix). The AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/)^106,107 was used to generate structural predictions. The TmAlphaFold Transmembrane Protein Structure database (https://tmalphafold.ttk.hu/)¹⁰⁸ was used to predict transmembrane domains and the position of PEX11 in the membrane. ENDscript 2.0 (https://endscript.ibcp.fr/ESPript/ENDscript/)¹⁰⁹ was used to delineate secondary structure elements. ChimeraX (version 1.10.1)^110,111 was used to render PEX11 structures and determine root mean square deviation (RMSD) scores with the Matchmaker function using the Needleman-Wunsch alignment algorithm and the BLOSUM-62 similarity matrix.

DNA methods

The CRISPR-Cas9-gRNA constructs were generated in the pAGM55261 vector⁴⁵. The CHOPCHOP application (https://chopchop.cbu.uib.no/)¹¹² was used to identify sgRNA targets in the coding region of Arabidopsis PEX11A-E, SDP1, PXA1, and PED1 genes (Supplementary Table 3). To target single genes (SDP1, PXA1, and PED1), complementary gRNA oligos with BsaI handles were annealed. To target multiple PEX11 genes, GenScript synthesized constructs containing tandem PEX11A/B and PEX11C/D/E gRNAs flanked by BsaI cut sites. To generate CRISPR-Cas9 constructs, annealed sgRNA oligos or synthesized plasmids were cloned into BsaI sites of the pAGM55261 vector (Addgene 153210)⁴⁵ using Golden Gate assembly (New England Biolabs, E1601) to yield Cas9-expressing vectors with gRNA sequences flanked by a U6 promoter and gRNA scaffold.

Constructs containing mTagBFP2-HA-AtPEX11B or mTagBFP2-HA-AtPEX11E were generated from a p35S-mTagBFP2-HA, pACT2-tdTomato-PTS1, pUBQ10-mNeonGreen-mPTS^PEX26 trifluorescent construct in pUC57⁶. The full-length AtPEX11B (At3g47430) and AtPEX11E (At3g61070) genes (exons and introns) were PCR amplified using Q5 high-fidelity DNA polymerase (New England Biolabs, M0491) and Gibson assembly primers (Supplementary Table 4) from A. thaliana genomic DNA and cloned into the NcoI site (downstream of the HA tag) of the trifluorescent construct via Gibson assembly (New England Biolabs, E2611). The resultant plasmids were digested with XhoI and religated with Quick Ligase (New England Biolabs, M2200) to remove 35S promoter and then digested with AvrII and NheI and religated to remove pACT2-tdTomato-PTS1 and pUBQ10-mNeonGreen-mPTS^PEX26. To generate mTagBFP2-HA-ScPEX11, ScPEX11 (YOL147C) was PCR amplified from W303 S. cerevisiae genomic DNA using Gibson assembly primers (Supplementary Table 4) and cloned into mTagBFP2-HA-AtPEX11E cut with NcoI (to remove AtPEX11E) using Gibson assembly. The mTagBFP2-HA-PEX11 entry vectors were recombined into the pFZ19⁶⁷ plasmid using LR Clonase II (Invitrogen, 11791-020) to generate plant transformation vectors designed to inducibly express PEX11 variants using β-estradiol.

Plasmids were verified by sequencing via assembled Oxford Nanopore reads by Plasmidsaurus (Eugene, OR).

Arabidopsis transformation

Constructs were transformed into Agrobacterium tumefaciens GV3101 (containing the pMP90 plasmid)¹¹³ by electroporation. Plants were transformed using a modified version of the floral dip method¹¹⁴. To generate pex11 clade mutants, Agrobacterium strains containing CRISPR-Cas9 constructs targeting PEX11A/B or PEX11C/D/E were used to transform plants expressing mRuby3-PTS1 and mNeonGreen-mPTS^PEX26 reporters³⁶. To generate sdp1, pxa1, and ped1 mutations, Agrobacterium strains containing CRISPR-Cas9 constructs targeting SDP1, PXA1, or PED1 were used to transform wild-type and pex11ab mutants expressing the mRuby3-PTS1 and mNeonGreen-mPTS^PEX26 reporters. To generate pex11 quintuple mutants, pex11ab mutants were transformed with an Agrobacterium strain containing a CRISPR-Cas9 construct targeting PEX11C/D/E.

For complementation experiments, the pex11ab mutant was transformed with Agrobacterium strains containing constructs for expressing mTagBFP2-HA-AtPEX11B, mTagBFP2-HA-AtPEX11E, or mTagBFP2-HA-ScPex11. Transformants carrying mTagBFP2-HA-PEX11 constructs were selected by either hygromycin resistance or estradiol-inducible mTagBFP2 expression. The pex11cde mutant was transformed directly with the Agrobacterium strain carrying the mTagBFP2-HA-AtPEX11E construct. To obtain pex11cde expressing mTagBFP2-HA-AtPEX11B, pex11c/c d/d E/e plants were transformed, and homozygous pex11cde mutants expressing mTagBFP2-HA-AtPEX11B were obtained from the progeny of the original transformants. Transgenic plants containing mTagBFP2-HA-PEX11 variants were confirmed via PCR-based genotyping (Supplementary Table 5).

CRISPR-Cas9 mutagenesis and genotyping

Transformants containing a CRISPR-Cas9-gRNA construct were selected as red fluorescent (from the pOLE1-tRFP on the Cas9 transgene) T₁ seeds and genotyped by PCR-amplifying the targeted region from cauline leaves. Amplicons were Sanger-sequenced (Azenta/Genewiz) to identify plants with mutations of interest. In the next generation, non-red-fluorescent T₂ seeds (lacking the Cas9 transgene) were selected, and T₂ plants were genotyped by Sanger sequencing of PCR amplicons (Supplementary Table 6).

DNA was extracted from seedlings or leaves for genotyping by placing a cauline leaf or multiple seedlings into 8-well PCR strip tubes. 25 μL of 50 mM NaOH was added to each tube and incubated for 15 min at 100 °C. 25 μL of neutralization buffer (200 mM Tris, 1 mM EDTA, pH 8.0) was added to each tube, tissue was dispersed into the solution using pipette tips on a mutli-channel pipette, and 2.5 uL was used as PCR template.

To confirm genotypes of seed stocks, DNA was extracted from seeds using a modified protocol^115,116. Approximately 100 seeds were frozen on dry ice and ground before adding 200 μL Edwards buffer (200 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, and 0.5% SDS). Samples were centrifuged for 5 minutes at 15,800 x g, and the supernatant was transferred to a new tube before adding 200 μL of isopropanol. Samples were mixed by inversion and then centrifuged at 15,800 x g for 5 minutes. Pellets were dissolved in 100 μL H₂O, and 1-2 μL was used as PCR template.

Microscopy

To screen for mTagBFP2 expression in seedlings or to screen for the Cas9 transgene via pOLE1-tRFP in seeds, fluorescence was detected using a Leica MZ10F fluorescence stereomicroscope with band-pass filters for ET Blue, GFP3, and ET DsRed.

Live-cell confocal scanning microscopy imaging experiments used seedlings grown on PNS unless otherwise noted. For lipid mobilization inhibition experiments, seedlings were grown vertically on PNS supplemented with 25 μM DMP (from a 50 mM stock in DMSO) or PNS supplemented with the same volume of DMSO (mock) for 3 days before imaging. To image pxa1 mutants, seed coats were gently nicked (after stratification but before plating) using tweezers to promote germination. To induce tagged-PEX11 expression in pex11 mutants, seedlings with and without the PEX11 transgene were grown vertically on PNS supplemented with 25 μM β-estradiol (from a 100 mM stock in DMSO) for 3 days before imaging. To image peroxisomes in pex11 quintuple mutants, seedlings from a pex11a/a b/b c/c d/d E/e parent were imaged and then genotyped to identify quintuple mutant seedlings.

To stain lipid droplets, seedlings were incubated in microcentrifuge tubes containing 100 μM monodansylpentane (Abcepta, SM1000b) in 50 mM Tris-HCl (pH 7.6) for at least 20 minutes with rocking.

For confocal imaging, seedlings were mounted in water or lipid droplet dye solution on glass slides (VWR, 48311-951) with 0.16-mm coverslips (VWR, 48393-241). For time-lapse imaging, seedling hypocotyls were excised, placed onto Lab-Tek II 2-well chamber slides (ThermoFisher, 155379), and overlaid with a thin layer of PN with 0.6% agar for imaging.

Fluorescence was captured using an Olympus FV3000RS inverted laser scanning confocal microscope equipped with FV31S-SW Fluoview Acquisition software (version 2.6.1.243) and multialkali spectral GaAsP detectors. An UPLXAPO 60x/1.42 oil-immersion objective was used for imaging experiments except Fig. 1b, which used an UPLXAPO 10X/0.40 objective.

When imaging two reporters, mNeonGreen was excited with a 488-nm laser and emission was captured between 500-540 nm, and mRuby3 was excited with a 561-nm laser and emission was captured between 570-620 nm. Each channel was imaged on separate imaging tracks with the brightfield channel imaged simultaneously with mRuby3.

When imaging three reporters or two reporters and lipid droplet dye, mTagBFP2 or monodansylpentane was excited with a 405-nm laser and emission was captured between 412-475 nm, mNeonGreen was excited with a 488-nm laser and emission was captured between 500-540 nm, and mRuby3 was excited with a 561-nm laser and emission was captured between 571-612 nm. Each channel was imaged on separate imaging tracks with the brightfield channel imaged simultaneously with mNeonGreen.

All images were acquired as z-stacks. Images were displayed as a single slice from a z-stack or as maximum-intensity projections. All imaging was done with sequential line scanning with no line averaging. Images were acquired with 16-bit depth at 1024 ×1024 resolution.

At least three seedlings of each genotype from each time point or condition were imaged for each experiment. Selected confocal images represent events that were observed in multiple cells from at least three different seedlings in the T₂ or T₃ generation. For imaging experiments with pex11ab expressing mTagBFP2-HA-PEX11E, 8 seedlings were observed that had varying effects on peroxisome morphology, which appears to be influenced by the amount of mTagBFP2-HA-PEX11E. Three images were selected to represent the effects of varied levels of mTagBFP2-HA-PEX11E.

Image analysis and peroxisome quantification

Orthogonal representations of cotyledon peroxisomes were generated in FIJI using files trimmed to contain 16 slices acquired using 3-µm steps.

To quantify peroxisome size and number, images from three seedlings were analyzed for each condition. The peroxisome membrane (green) channel was used to enable inclusion of both pre-peroxisomes (lacking the lumenal marker) and mature peroxisomes (enclosing the lumenal marker). For quantifying peroxisomes in mock- or DMP-treated seedling hypocotyls (Fig. 4b, c, Supplementary Fig. 4c), 160 ×80 μm regions of the peroxisome membrane channel were used to generate maximum-intensity projections from 10 slices with 1-µm steps. For quantifying peroxisomes in cotyledons (Figs. 1d–h and 7d, e), 120 ×120 μm regions of the peroxisome membrane channel were quantified using maximum-intensity projections from 10 slices with 1-µm steps [Fig. 1 (2-, 3-, and 5-day time points); Fig. 7] or 10 slices with 3-µm steps [Fig. 1 (7-day time point)]. FIJI was used to generate maximum-intensity projections, and the oval tool was used to manually outline peroxisomes, count peroxisomes, and calculate areas. Peroxisomes on the border of an image were included if the peroxisome appeared to be spherical and >50% of the peroxisome area was included. Microsoft Excel was used to calculate diameter from area. Peroxisomes that appeared to be moving excessively (based on examination of the z-stack files) and peroxisome clusters containing ambiguous individual peroxisomes outlines were counted but were excluded from diameter calculations. For peroxisome size stacked bar charts, peroxisome diameters for each time point were binned by size, converted to percentages, and the mean of three replicate percentages was plotted. To account for the 3-fold larger step size for the 7-day time point in Fig. 1, peroxisome numbers were normalized by dividing by 3. To calculate total peroxisome volume, the mean peroxisome diameter from each replicate was converted into spherical volume, multiplied by the number of peroxisomes for that replicate, and normalized to the mean wild-type peroxisome volume at 2 days.

Immunoblotting

Seedlings grown on PNS medium (physiological assays) or PNS supplemented with 25 μM β-estradiol (complementation experiments) were frozen on dry ice and stored at −80 °C until processing. Protein was extracted from ground, frozen plant tissue by adding 3 volumes of sample buffer [435 mM Tris pH 8.5, 3.5% (w/v) lithium dodecyl sulfate, 870 μM EDTA, 17.4% (w/v) glycerol, 48 μM Coomassie blue G250, and 65 mM dithiothreitol] and then incubating the samples at 100 °C for 5 min. The samples were centrifuged at 18,500 x g for 5 min at 4 °C, and then equal volumes of supernatants were loaded on Bolt 10% (w/v) polyacrylamide Bis-Tris gels (Invitrogen) alongside diluted pre-stained markers (New England Biolabs, P7719). The gels were electrophoresed in MOPS running buffer [50 mM MOPS, 50 mM Tris pH 7.7, 1 mM EDTA, 0.1% (w/v) SDS] containing a 1:400 dilution of NuPAGE Antioxidant (Invitrogen). The proteins were transferred from the gel to Hybond-ECL nitrocellulose membranes (Amersham, Protran Premium 0.45 μm NC; 10600003) using a GenScript eBlot L1 transfer system (GenScript, L00686). Membranes were air-dried overnight before blocking for at least 1 hour at room temperature with 8% (w/v) nonfat dry milk in TBST [20 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20]. After blocking, membranes were incubated at 4 °C with primary antibodies diluted in 8% milk in TBST for at least 1 day with rocking. Primary antibodies were mouse anti-HSC70 (1:50,000; Stressgen SPA-817), rat anti-HA (1:100; Sigma 11867431001 Roche 3F10), rabbit anti-PEX11E⁴⁶ (1:1000), rabbit anti-PEX14 (1:10,000; Agrisera AS08 372), rabbit anti-PMDH2⁸⁴ (1:10,000), or rabbit anti-OLE1 (1:2000; Cedarlane CLAS20-4412). After incubation with primary antibodies, membranes were washed three times for 5 minutes in TBST and then incubated with secondary antibodies diluted in 8% milk in TBST for at least 1 hour at room temperature. Secondary antibodies were horseradish-peroxidase (HRP)-linked goat anti-mouse (1:2500 or 1:5000; Invitrogen 31430), goat anti-rat (1:1000; Invitrogen A10549), or goat anti-rabbit (1:2500 or 1:5000; LI-COR 926-80011). Membranes were imaged with WesternSure Premium Chemiluminescent substrate (LI-COR, 926-95000) using an Odyssey Fc imaging system (LI-COR, 2801-02). Membranes were sequentially probed with primary antibodies recognizing proteins of different sizes without stripping. Quantification was performed using Image Studio software (LI-COR, version 5.2).

Lipid analysis

TAG levels were monitored by thin-layer chromatography (TLC). 5 mg of seeds were plated on PNS in triplicate for each genotype and time point and harvested after 3, 5, or 7 days, frozen on dry ice, and stored at −80 °C. Lipids were extracted using a modified Folch extraction method^6,117. Tissue was ground in 1.7 mL microcentrifuge tubes after being frozen in liquid nitrogen. 500 μL of methanol/chloroform/formic acid (2:1:0.1, v/v/v) was added to ground sample and vortexed for 10 minutes at room temperature. 250 μL of phase separation solution (1 M KCl, 0.2 M H₃PO₄) was added. Samples were vortexed, then centrifuged at 18,000 x g for 5 minutes at room temperature. Equal volumes of the lower phase were spotted onto TLC plates (silica gel 60, Sigma, 1.05721 plates) and placed into a glass chamber containing a mobile phase consisting of hexane/diethyl ether/acetic acid (70:30:1, v/v/v). TAG was visualized by dipping plates into 5.0% H₂SO₄, drying until water evaporated, and heating at approximately 100 °C for 5-10 minutes until color developed. For TAG quantification, TLC plates were scanned using a flatbed scanner and converted into 32-bit grayscale TIF images, which were quantified using Image Studio software (LI-COR).

For lipidomic analysis, approximately 40-50 mg of seeds for each genotype were plated on PNS plates for each time point. Five replicates of 5-40 mg of seedling tissue were harvested at 3, 7, and 12 days, frozen in liquid nitrogen, and stored at −80 °C. Lipidomics analysis was performed by the Kansas Lipidomics Research Center (Manhattan, KS)¹¹⁸ to monitor 359 lipids. Harvested seedlings were transferred to a vial containing 0.8 ml isopropanol with 0.01% BHT preheated to 75 °C and incubated for 15 min. Lipids were extracted by shaking at room temperature for 24 h after adding 2.4 mL of chloroform/methanol/water (40:55:5, v/v/v). The seedling tissue was removed to a new vial, dried, and weighed. The volume of extracted lipid corresponding to 0.085 mg dry tissue was evaporated and dissolved in 300 µL chloroform/methanol/300 mM ammonium acetate in water (30:66.5:3.5, v/v/v) along with internal standards for direct-infusion electrospray ionization tandem mass spectrometry analysis using a Sciex 6500+ mass spectrometer. Quality control samples, which were identical pooled samples containing internal standards, and internal-standard-only samples were placed at regular intervals among the experimental samples¹¹⁸. In positive mode, the ion spray voltage was 5500 V, the curtain gas was 35 psi, the source temperature was 100 °C, the ion source gas (GAS1) was 45 psi, the ion source gas (GAS2) was 45 psi, the declustering potential was 100 V, and the entrance potential was 10 V. In negative mode, parameters were the same except the ion spray voltage was −4500 V, the declustering potential was −100 V, and the entrance potential was −10 V. The collision gas was nitrogen¹¹⁸. Multiple reaction monitoring (MRM) data were processed and exported to Excel using MultiQuant software (version 3.0.2). Lipids were targeted based on the masses of intact ions (m/z) and individual fragment ions. Data were quantified using LipidomeDB Data Calculation Environment (http://lipidome.bcf.ku.edu:8080/Lipidomics/). No response factors were used in the quantification; 1 nmol = the amount of lipid that produced the same mass spectral intensity as 1 nmol of the best-matched internal standard. The 203 lipids with a coefficient of variation less than 0.3 (calculated from repeated infusion of the quality control samples throughout the analysis) and that were present above the detection limit of 0.0005 nmol for the mean of pooled replicate samples were included in the quantitative analyses (Supplementary Data 1). Data were processed and visualized using the “ggplot2” and “tidyverse” packages in RStudio (version 2025.05.1 + 513). For principal component analysis, the data were normalized using the Z-score method in RStudio. For bar and stacked bar charts, the percentages of total lipid content for each lipid type were summed, and the means of the five biological replicates were plotted.

Statistical analysis

Statistical tests were performed using JMP Student Edition (version 18.2.1). For the adult plant survival plot, the Mantel-Cox (log-rank) test was performed to compare the difference in survival curves using pairwise comparison between each genotype and the wild-type reporter utilizing Bonferroni correction to adjust p-values. Two-tailed t tests were used to compare peroxisome diameter of DMP-treated seedlings. Chi-square analysis with Pearson’s test was used to show that the pex11a/a b/b c/c d/d e/e mutations did not confer embryonic lethality. One-way ANOVA was performed to compare peroxisome diameter, number, and volume; sucrose dependence; IBA responsiveness; rosette diameter; PMDH processing and OLE1 levels; TAG levels in pex11 mutant time course TLC assays; and percentages of total lipid content for each lipid type. When p-values were <0.05, Tukey’s post hoc test was used to compare means and assign homogenous subsets. For lipidomics volcano plots, one-factor statistical analysis and probabilistic quotient normalization to the wild-type reporter line were performed in MetaboAnalyst 6.0, and the resultant fold-changes and p-values were exported for visualization in RStudio. Data graphed using GraphPad Prism or RStudio were imported into Adobe Illustrator (version 29.8.1) for figure assembly.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data supporting the findings of the study are available within the main text or supplementary information. Source data are provided with the paper. Source data are provided with this paper.

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Acknowledgements

We thank Alison Baker for the PEX11E antibody and Gabrielle Buck, Makaela Jackson, Sayuri Jayawardena, Isabella Kreko, Durreshahwar Muhammad, Ana Swearingen, and Cody Upton for useful feedback on the manuscript. We are grateful to Ruth Welti and Mary Roth of the Kansas Lipidomics Research Center Analytical Laboratory for lipidomics analysis and advice. We thank Spencer Freeman for insights on organelle turgor modulation. This work was supported by the National Institutes of Health (R35GM130338 to BB and F31GM150269 to NET), the Robert A. Welch Foundation (C-1309 to BB), a National Science Foundation REU grant 2244041 (to CA), and the American Society of Plant Biologists SURF program (to CA). Lipidomics instrument acquisition and method development were supported by the National Science Foundation (including support from the Major Research Instrumentation program; most recent award DBI-2511193), the USDA National Institute of Food and Agriculture (Hatch/Multi-State project 7001195), and Kansas State University.

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James Hwang
Present address: School of Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
Nayeli S. Shad
Present address: Department of Public Health Sciences, Biological Sciences Division, The University of Chicago, Chicago, IL, USA
Zachary J. Wright
Present address: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

Authors and Affiliations

Biosciences Department, Rice University, Houston, TX, USA
Nathan E. Tharp, Chelsea An, James Hwang, Nayeli S. Shad, Zachary J. Wright & Bonnie Bartel

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Nathan E. Tharp
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Chelsea An
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James Hwang
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Nayeli S. Shad
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Zachary J. Wright
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Bonnie Bartel
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N.E.T., Z.J.W., and B.B. conceived and designed the experiments; N.E.T., C.A., J.H., and N.S.S. performed the experiments; N.E.T. and C.A. analyzed the data; and N.E.T, Z.J.W., and B.B. wrote the manuscript with input from all authors.

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Correspondence to Bonnie Bartel.

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Tharp, N.E., An, C., Hwang, J. et al. PEX11 mediates intralumenal vesicle formation in peroxisomes. Nat Commun 17, 3538 (2026). https://doi.org/10.1038/s41467-026-71873-3

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Received: 18 December 2025
Accepted: 01 April 2026
Published: 21 April 2026
Version of record: 21 April 2026
DOI: https://doi.org/10.1038/s41467-026-71873-3