Peroxisome replication and proliferation in plants

Peroxisomes are organelles enclosed by a single membrane, having roles in β-oxidation of fatty acids, photorespiration, biosynthesis of phytohoromones and abiotic stress responses. These organelles harbour 50 enzymes species including catalase. Peroxisomes also contain reactive oxygen species (ROS) including hydrogen peroxide (H₂0₂). Peroxisomes are highly conserved within the eukaryotic domain and are highly dynamic relying on the actin cytoskeleton for their translocation in plants and yeast. They constantly proliferate to adjust their collective metabolism and morphology to meet cellular demands.

High levels of ROS can induce peroxisomal proliferation. Radicals originate from β-oxidisation, photosynthetic development and environmental stress. β-oxidation releases energy for post-germination growth. In photosynthetic development glyoxysomes convert to leaf peroxisomes to engage in photorespiration and H₂O₂ breakdown. Glyoxysomes metabolise glycolipids for germination which are not required when photosynthetic tissue develops. Plants develop a signalling response to stress using ROS. Peroxisomes can develop membrane extensions or peroxules in response to radicals. Peroxules interact with chloroplasts and mitochondria absorbing radicals.

Peroxisome replication is controversial with 2 models aiming to elucidate their replication. The Growth and Division Model suggests that peroxisome proteins are synthesised on free polyribosomes in the cytosol and are organised posttranslationally. The Semi-autonomous Model stipulates peroxisomes arise by 2 distinct networks; de novo biogenesis and by growth and fission of pre-existing peroxisomes. Despite ongoing research into this field there is still no conclusive evidence to suggest that one model descriptive than the other so the debate continues. 3 proteins implicated in peroxisomal proliferation, defined as the rapid increase in numbers in response to a stimulus and replication, defined as the gradual continuation of peroxisomes. PEROXIN11s (PEX) initiate peroxisome membrane elongation, DYANMIN-RELATED PROTEINs (DRP) undertake peroxisome constriction and FISSIONs (FIS) promote membrane fission.

Role of the Endoplasmic Reticulum in Peroxisomal Replication

Growth and Division and Semi-autonomous perspectives

In the Growth and Division Model, peroxisomes are considered fully autonomous increasing in mass and volume by posttranslational import of peroxisome proteins. New peroxisomes form from pre-existing peroxisomes by fission. Peroxisomes enlarge as a result of the endoplasmic reticulum (ER) serving as a source of lipids. The transportation of these lipids occurs through transport proteins or contact sites between the ER and peroxisome. This paradigm was challenged by in vivo trafficking studies of peroxisome membrane proteins (PMP). Some PMPs were found to sort indirectly to peroxisomes through the ER. Yeast mutants lacking fission initiator proteins PEX34p and PEX11p, lack peroxisomal structures. When PEX34p and PEX11p were reintroduced by recombination to mutants they formed peroxisomal assemblies.

The current paradigm for peroxisome replication combines facets of the original model, ER Vesciulation stipulating that peroxisomes solely originate from specialised regions of the ER, however this model lost credibility and subsequently became less popular and the Growth and Division Model. Latest data suggests peroxisomes are semi-autonomous. Two key pathways are considered necessary for the synthesis of peroxisomes; de novo synthesis from the ER and growth and fission of preceding peroxisomes. The Semi-autonomous Model elaborates further to elucidate the participation of preperoxisomes produced from vesicles and membrane fragments from the ER. Preperoxisomes transport cargo including phospholipids and PMPs which fuse to pre-existing peroxisomes which enlarge and divide. A unified model of peroxisome biogenesis may not be straightforward to attain as the processes governing de novo synthesis, growth and division may not be governed completely autonomously and these processes vary depending on species, physiological condition or cell type. While there is direct evidence for peroxisome de novo synthesis in yeast and mammals there is no evidence in plants. The ER appears to serve as a platform as trafficked membrane components are translocated by transporter proteins from the ER to pre-existing peroxisomes.

A remaining question is how de novo synthesis and fission are co-ordinately regulated to govern peroxisome profusion and division. The source of new peroxisomes is likely to be influenced by cellular states in past and present environmental circumstances. This can be extracted from the roles of peroxisomes in carbon source breakdown in the generation of ROS which may impair lipids and proteins, perhaps explaining discrepancies about peroxisome replication. In contradiction, yeast peroxisomes are delivered from de novo and pre-existing peroxisomes in wild type despite using fermentation.

Morphological Peroxisome Membrane Alterations: PEROXIN11a-e

In Arabidopsis thaliana 5 PEX11 genes have been identified; PEX11a-e. These are essential peroxisome proliferation regulators. PEX11 are subdivided into 3 main groups based on their sequence homology PEX11a, PEX11b and PEX11c-e. Overexpression of PEX11 homologs leads to higher peroxisome numbers, suggesting a role for PEX11 in promoting fission. Saccharomyces cerevisiae mutants lacking PEX11p exhibit reduced levels of peroxisomes and increased peroxisomal elongation. Arabidopsis PEX11a-e gene knockdowns, peroxisome numbers were found to rapidly decrease. Knocking down PEX11c-e, results in an increase in peroxisome volume. Knocking down PEX11a-b can lead to minor increases in peroxisome size.

It has been determined that PEX11 is not directly responsible for the fission of peroxisomes. PEX11a-e are directly responsible for the modification of membranes in preparation of fission. Overexpression of PEX11e leads to peroxisomal elongation superseding fission. It is stipulated that peroxisomal elongation occurs with interactions between PEX11 and phospholipids present on the outer membranes. PEX11p present in S. cerevisiae was found to harbour a comparable binding domain to peroxisome proliferator-activated receptor (PPAR) binding fatty acids. This suggests PEX11 directly binds to phospholipids present on the cytosolic face of peroxisomal membranes.

Cells with reduced levels of PEX11c-e increase in volume but do not elongate, indicating PEX11c-e are required for membrane elongation but not for peroxisome growth. A study revealed that peroxisomal elongation did not proceed with an increase in peroxisome frequency, yet PEX11 expression remained high. This reinforced PEX11’s role in elongation and not in fission. Another investigation uncovered that PEX11c-e had overlapping functions related to peroxisomal replication. It was revealed PEX11a-b did not play an obvious role during peroxisomal replication. Mutants overexpressing PEX11a-b were found to enlarge peroxisomes. This study verified that PEX11b played an essential role in the negative regulation during fission. PEX11a-b overexpression promotes peroxisome aggregation comparable to other outer PMPs. PEX11b in Arabidopsis has a primary role of inducing peroxisome numbers in response to light and in peroxisome maintenance. This confirmed that light had a possible regulatory role in peroxisome numbers.

Roles of FISSION1 and DYANMIN-RELATED PROTEINS 3 and 5

After PEX11a-e induces tubulation in peroxisomal membranes, division proceeds with membrane constriction and fission, mediated by FIS1 a membrane-anchored protein. FIS1 then recruits DRPs which are large GTPase. DRPs form oligomeric structures surrounding the tubulated membranes forcing fission or fusion through GTP hydrolysis.

There are 2 isoforms present in the FIS1 family (FIS1a-b) that have C-terminal tailed anchored proteins with a single transmembrane domain. FIS1 has a tetratricopeptide (TPR) repeating domain at its N-terminus. This TPR is a characterised domain which has an essential role in protein-protein interactions on the cytosolic surface of membranes. FIS1a-b have rate limiting functions at the fission phase of division. Overexpression of FIS1a-b results in a significant increase of peroxisomes and mitochondria. Silencing of FIS1a-b by siRNA in mammalian mutants results in reduced numbers of peroxisomes and mitochondria. This has yet to be confirmed in plants.

From mammalian homologs, FIS1 tethers to DRPs targeted to membranes but no direct evidence has been uncovered in plants. DRPs lack phospholipid binding domains. They are also believed to be cytoplasm proteins recruited to target membranes by anchoring proteins, supporting FIS1’s role. FIS1 is dual-targeted to peroxisome and mitochondria. Interestingly, FIS1 mammalian homologs were verified to uniformly distribute over peroxisomal membranes, putting some doubt on FIS1’s role as a receptor for DRP. Gene knockdowns of FIS1 in mammalian cells were reported to have little effect on peroxisome morphology. This remains to be confirmed in plants.

Homologs of FIS1 in mammals have been shown to recruit DRP1 on peroxisomal and mitochondrial membranes. This would result in the characterisation of a positive regulator of peroxisome fission. FIS1’s ability to directly and indirectly recruit DRP in Arabidopsis has yet to be verified. FIS1 is shared by fission machineries of both mitochondrial and peroxisomal division. This has led to much speculation about whether both organelles coordinate their division. These organelles share comparable metabolic pathways, notably lipid breakdown during germination and glycolate recycling. Orthologs of FIS1 Arabidopsis were identified as having a role in peroxisomal and mitochondrial division factors. Interactions between FIS1 and PEX11 have been observed in mammals and plants, suggesting a possible functional association between peroxisome fission machineries.

DRPs have been involved in peroxisome and mitochondrial division dependent of FIS1. Out of 16 Arabidopsis DRPs, 3 had role in peroxisome fission; DRP3a, DRP3b and DRP5. DRP3a-b were demonstrated to colocalise, playing major roles in fission. DRP5 along with DRP3 targets chloroplasts and peroxisomes and facilitates their division. DRP5 mutants contain distended chloroplasts and aggregated peroxisomes that have impaired division and function. DRP5 evolved more recently in plants and algae, whereas DRP3a-b originally formed from algae.

Peroxisome Proliferation in Response to Environmental and Intracellular Conditions

Mobilisation of Lipid Reserves during Germination

Without a stimulus for proliferation, peroxisomes remain homeostatic and replicate with a comparable rate to the cell cycle. In the presence of a stimuli including germination, peroxisome proliferation occurs by expression of PEX11b.

Peroxisome proliferation is required to metabolise stored lipids to enable germination. Activation of triacylglycerol (TAG) using coenzyme A is the initial step in peroxisome β-oxidation. TAG is actively transported by ABC transporter COMATOSE (CTS). Acyl-CoA oxidases (ACX1-6) were determined to govern the breakdown of long-chain lipids. After breaking down long-chain lipids, ACX1-6 products are oxidised by multi-functional proteins (MFP1-2) yielding trans-alkenes. Trans-alkene is then hydrated using enoyl-CoA hydratase. The alcohol of hydroxyacyl-CoA is then oxidised by nicotinamide adenine dinucleotide (NAD⁺). Acetyl-CoA is then cleaved by using 3-ketoacyl-CoA thiolase-2 (KAT2), yielding acyl-CoA. Acyl-CoA enters the citric acid cycle in mitochondria releasing energy for germination. To sustain this increase in β-oxidisation, peroxisome proliferation is induced by PEX11b which interacts with ACX1-6 through CTS.

ACX1-4 substrate specificities are as follows, ACX2 encodes for long, ACX1 medium-long, ACX3 medium and ACX4 encoding for short chains. ACX5-6 have yet to be characterised. ACX1-2 knockouts cause reductions in long-chain acetyl-CoA. Double mutants of ACX1-2 result in a sucrose-dependent seedling establishment and breakdown of CTS interactions. This affected PEX11b’s ability to induce proliferation in response to higher levels of β-oxidisation. Mutants of ACX3-4 uncovered a reduction of large, medium and short chain lipid breakdown.

Peroxisome defective 1 (PED) a thiolase mutant is activated during germination and early post-germinative growth. PED3, ABC transporter was found to promote germination by importing very long-chain fatty acids associated with the breakdown of stored lipids and precursors of jasmonic acid and Indole-3-acetic acid to peroxisomes. KAT2 mutates required an exogenous carbon source for post-germinative development, also demonstrated in long-chain acyl-CoA synthetase double mutants. CTS mutants have compromised lipid catabolism and raised levels of ACX during post-germinative growth, increased dormancy. CTS has been classified as forever dominant, implicating CTS involvement in the governance of dormancy and germination. CTS mutants can be rescued by using sucrose, however, germination cannot. Saccharomyces peroxisomes retain normal size in CTS mutants unlike CAT2 mutants.

Multi-functional protein 2 (MFP2) and PED3 mutants have enlarged peroxisomes whereas CTS mutants do not. This has led to speculation that intraperoxisomal accumulation of ACX1-6 may have roles in regulating peroxisome size, this remains to be verified in plants. In Yarrowia lipolytica ACX was found to interact with PEX11p and PEX16p to trigger peroxisome division when binding to peroxisomal membranes. Studies indicate PEX11p, PEX25p, PCD1 and CAT2 act together with ALDP to support the role of ACX in regulating peroxisomal proliferation in Saccharomyces homologs. PEX11αβγ mammalian homologs have been shown to interact with MFP, ACX and ALDP. In plants, speculation of these interactions only exists with little evidence. Little is known about plant factors regulating expression of genes involved in peroxisomal proliferation. No gene encoding for PPAR (mammals) or osteoclastic-activation factors homologs (fungi) have been found.

Light Induced Peroxisomal Proliferation

Light induces peroxisomal proliferation through phytochrome-A mediated pathway, regulated by far-red light. Transcription factor HYH activates PEX11b, a possible peroxisome proliferation factor gene. iRNA studies of PEX11b showed reduced numbers of peroxisomes during dark-to-light transitions. Microarray studies revealed light triggers the expression of PEX2, PEX11b and RING-type E3 ubiquitin ligase (REUL) involved in photomorphogenesis. Peroxisomes act like a hub for signaling molecules such as nitric oxide, promoting photomorphogenesis. Gain-of-function mutants of membrane-bound REUL uncovered that their suppression reduced their photomorphogenic responses. Conversely, light represses the expression of the glyoxylate cycle and β–oxidation genes, essential for initiating germination.

Organelles involved in photosynthesis need to develop and their genes need to be activated. During this transformation glyoxysomes which break down glycolipids covert to leaf peroxisomes, required for H₂O₂ reduction. This conversion occurs when glyoxysome-specific enzymes, malate synthase and isocitrate lyase are replaced with leaf peroxisome-specific enzymes, glycolate oxidase. After 30 minutes exposure to light, PEX5 and PEX7 expression rapidly increased peroxisome elongation, constriction and fission occurs within 2 hours in Arabidopsis seedlings. Therefore, to meet the demands of high photosynthesis and photorespiration, peroxisomal abundance increases with the immediate import of leaf peroxisome enzymes.

Light activated phytochrome-A is transported to the nucleus from the cytoplasm by FAR-RED ELONGATED HYOCOTYL1 (FHY1). Sustained exposure to far-red light induces the production of transcription factor HYH. Silencing PEX11b, results in the deficiency of light induced proliferation. Tissue specific functions of PEX11 have also been observed. Cryptochrome and phytochrome-B were verified to have minor roles in the activation of PEX11b. However, their roles are yet to be characterised.

Other microarray studies using Arabidopsis demonstrated that phytochrome-A may have a role in the transformation of glyoxysomes to leaf peroxisomes. It has been suggested phytochrome-A and B initiate hypocotyl growth. The level of phytochrome-A and B started to decline after 3 hours of white light. This correlates with the expression of PEX11b, supporting the concept that phytochrome-A has a primary role in the up-regulation of PEX11b in the initial hours of light exposure. Despite detecting the significance of far-red light and phytochrome-A in regulating peroxisomal proliferation, there appeared no obvious reduction in PEX11b expression in phytochrome-A mutants. It is possible other constituents of the phytochrome-A pathway have not been determined. It is yet to be identified if specific cis-elements in the promoter of PEX11b are required for activation in light.

Peroxisome Proliferation during Environmental Stress

Leaf and root peroxisomes of Solanum lycopersicum, antioxidant systems were up-regulated in response to environmental stresses. In Arabidopsis salt stress induced expression of 3 peroxisomal genes including KAT2, PEX1 and PEX10. However, PEX1, PEX10, KAT2, PEX11b roles in environmental stress responses are still to be fully explained.

As well as metabolised H₂O₂, peroxisomes also generate superoxides O^2- and NO^- radicals caused by their β-oxidation activity. The generation of ROS is often the first response to abiotic stresses, controlling programmed death cell, pathogen defense and stomatal behaviour. For cell survival, cells attempt to down-regulate ROS production, while simultaneously scavenging ROS. Photorespiration is thought to be integral to stress responses in chlorophyll for controlling ROS accumulation. Subtle control of ROS levels by peroxisomes enables ROS to act as a signal expressing damage response molecules. The exact signalling mechanisms in peroxisomes need to be determined.

Under normal conditions ROS production can be controlled by peroxisomal antioxidant enzymes in conjunction with other antioxidant enzymes elsewhere in the cell. The risk of cellular damage can arise when under stress. Peroxisomal ROS generation is enhanced and antioxidant systems depressed during environmental stress. H₂O₂ build-up can diffuse across a cell’s plasma membranes into the extracellular matrix, presenting a serious risk to neighbouring cells.

After exposure to radicals PEX1 and PEX10 expressions increase and induce peroxisome membrane extensions or peroxules. Peroxules frequently interact with chloroplasts and mitochondria which are the source of most radicals. Peroxules absorb radicals by their connections to chloroplasts and mitochondria. Consequently, these radicals are then transported to the peroxisome matrix where they are processed by catalase. As radicals increase within the cell, peroxules can elongate by expressing PEX11a-e, constrict by expressing DRP3a-b and 5 and divide by expressing FIS1, thus rapidly reproducing more peroxisomes. This process supports the semi-autonomous Model where the ER delivers cargo proteins to developing preperoxisomes.

In Summary

Though much advancement in peroxisome replication and proliferation has occurred, their origins in plants remain unclear. The Growth and Division and Semi- autonomous Models were developed to elucidate peroxisome replication. Evidence remains to be observed verifying whether neither or both models occur. PEX11a-e are known to initiate replication by elongating peroxisome membranes. DRP3-5 starts constricting membranes followed by FIS1a-b stimulating fission, thus completing replication. Peroxisome proliferation can be induced by several stimuli including β-oxidisation during germination, light and environmental stresses. Peroxisome numbers rapidly increase during germination, metabolising TAG to release energy. Far-red light can induce proliferation by the phytochrome-A pathway, governed by PEX11b. PEX11b has comparable activities to PPAR present in mammals. KAT2, PEX1 and PEX10 are implicated in inducing peroxules which frequently interact with chloroplasts and mitochondria to reduce ROS. Peroxules can then fragment and continue to grow and divide driving peroxisome proliferation.

A major challenge which remains is to determine if peroxisomes are synthesised de novo in plants. Specifically, is de novo synthesis and fission regulated co-ordinately influencing peroxisome profusion and division. FS1 is said to tether to DRPs in peroxisomal membranes. It would be interesting to find if FIS1 uniformly distribute over peroxisome membranes on plants which may put doubt on FIS1’s role as a receptor for DRPs. Plant regulators of peroxisomal replication genes remain unknown. PPAR homologs have not been uncovered in plants, perhaps PEX11b could be the focus of future study. PEX11a-e interactions with peroxisomal β-oxidisation genes remain uncharacterised. Cryptochrome and phytochrome-B roles in light induced proliferation continue to be uncharacterised.