CELL BIOCHEMISTRY AND FUNCTION
VOL.
10: 153-158 (1992)
Plant Peroxisomes: Recent Studies on Function and Biosynthesis HELMUT KINDL Depurtment of' Chemistry, University of Marburg, W-3550 Marburg, Germany
fungal pathway for 2-trans-enoyl-CoA, are presented in Figure 1. The plant peroxisome constitutes a metabolic comSaturated fatty acids characteristic of animal partment which has unique features with respect to triglycerides are present in plants in minor the structural organization of the constituent cata- amounts. Their degradation proceeds via 2-translysts. Glyoxysome, a peroxisomal form encompass- enoyl-CoA (shown in middle of the upper part of ing both fatty acid oxidation and the glyoxylate Figure 1). However, in plant lipids the polyunsaturcycle, is a model system of compartmentation and ated fatty acids dominate. 3-cis-Enoyl-CoA originlimited exchange of intermediates. Progress has ating during metabolism of 9-cis-enoyl-CoA, e.g. been achieved primarily in unravelling the primary from linoleic acid, reaches the main route via structures of peroxisomal enzymes via cDNA se- isomerization to 2-trans-enoyl-CoA. 4-cis-Enoylquences and in finding new metabolic routes. This CoA, in fungi converted to 3-trans-enoyl-CoA via review concerns recent work and stresses two fields: 2,4-dienoyl-CoA and a reductase reaction, is metathe multitude of mechanisms for the chain shorten- bolized in plants as indicated by the thick arrows: ing of poly-unsaturated fatty acids and the biosyn- chain shortening to 2-cis-enoyl-CoA and converthesis of the various forms of peroxisomes. sion to 2-trans-enoyl-CoA via D-3-hydroxyacylCoA. For the further metabolism of 2-trans-enoylCoA, this multifunctional protein takes over the STRUCTURAL ORGANIZATION OF task of hydration to L-3-hydroxyacyl-CoA and ENZYMES CATALYSING FATTY ACID subsequent oxidation to 3-ketoacyl-CoA (left part DEGRADATION of the figure). Thiolase which is an independent The glyoxysome is a metabolic compartment enzyme in plant glyoxysomes is responsible for the highly efficient in transforming lipid into building consecutive cleavage. During the past years, it blocks for gluconeogenesis.' The substrates for this became evident that epimerization actually proconversion are different forms of fatty acids, and ceeds by a two-step mechanism including two kinds thus the glyoxysome has to be competent by pro- of hydratases. This was shown for liver .~ viding enzymes to cope with the supply of the metabolism' and for plant g l y c o ~ o m e sIsomerase various fatty acids. Two areas of fatty acid metabo- activity in liver peroxisomes seems to be confined lism have been covered most thoroughly and thus to the multifunctional pr0tei1-1.~Plant peroxisomes provided some surprises. One point concerns the possess, besides the isomerase as part of the large way in which enzymatic activities handling cis- form of the multifunctional protein, a very active unsaturated fatty acids are arranged on proteins. monofunctional form. In contrast to this kind of metabolic organizaThe other real surprise was when it became evident that fungi utilize a pathway 2-trans-enoyl-CoA + tion in plants, fungi seem to rely on a pathway D-3-hydroxyacyl-CoA --+ 3-ketoacyl-CoA for de- which is outlined on the right part of the figure and grading the standard fatty acids. Both features, the symbolized with thin arrow^.^'^ A bifunctional route of 4-cis-enoyl-CoA originating during the protein converts 2-trans-enoyl-CoA via D-3-hyddegradation of fatty acids with a double bond roxyacyl-CoA into 3-ketoacyl-CoA. The primary extending from an even numbered C-atom and the structures of the respective proteins from NeuroINTRODUCTION
0263-64X4/92/030153-06$08.00 I 1992 by John Wiley & Sons, Ltd
154
H. KINDL
>
4-cis-Enoyl-CoA
3-trans-EnoyCCoA
Acyl-CoA 2-cis-Enoyl-CoA
'u
2-trans-EnoyCCoA
3 cis-EnoyCCoA
Figure 1. Summary of pathways used for degradation of 4-cis-enoyl-CoA and 3-enoyl-CoA. Both plant pathways via 2-cis-enoylCoA (thick arrows) and fungal pathway from 2-trans-enoyl-CoA (very thin arrows) are stressed in addition to the steps belonging to the standard equipment of peroxisomes. HY: hydratase domain; R: reductase domain.
sposa crassa, Saccharomyces cervisiue and Cundida tropicalis are very similar. Another interesting feature of fatty acid degradation in plant glyoxysomes is the multiple location of enzyme activities on several differently organized proteins. The activity of the r>-specifichydratase is located on both multifunctional proteins (MFP I and M F P 11) and on two forms of a monofunctional dimeric protein (symbolized in Figure 1, central art).^,^ The isomerase activity is present on a monofunctional dimeric protein and on the larger form of the multifunctional proteins, i.e. M F P 11. Recently, the purification of the peroxisoma1 A2,A3-enoyl-CoA isomerase acting on 3-cisenoyl-CoA and 3-trans-enoyl-CoA and being selective towards C,, and C,, was achieved.' I t is noteworthy that the trifunctional protein (MFP I) and the tetrafunctional protein ( M F P 11) differ markedly in their domain structure. New results revealed that peroxisomes are capable of degrading branched-chain carboxylic acids.' Dicarboxylic acids" and products of lipoxygenase action' ' may be degraded in peroxisomes.
STRUCTURES OF OTHER PEROXISOMAL ENZYMES Catalase is encoded by more than one gene. The three genes in maize are expressed in a development
and tissue specific mode. CAT1 is expressed during kernel maturation while scutellum CAT212 encodes a protein destined for glyoxysomes. CAT3 is activated in leaves and codes for an enzyme sornehow associated with mitochondria. This gene is expressed in a circadian rhythm.13 In cotton seed,l4." two different subunits are assembled into five tetrameric isoforms of catalase. Glyoxysomal sunflower catalase16-' is composed of several isoforms of different charge. A 56 kDa peptide, the primary transition product, is the subject of processing. Processing takes place also with the 59 kDa peptide which appears after light induction and is characteristic of leaf-type peroxisomes. It is pertinent to point out that catalases may be composed of different subunits, as well as subunits devoid of heme. Catalases of different sizes, depending on the growth in the dark or light, have been observed in Cucurbitaceae.' The ratio of catalase and peroxidase activities may vary from plant to plant.20 The primary structures of glycolate oxidase21.22 and hydroxypyruvate r e d ~ c t a s ehave ~ ~ been elucidated. The expression of plant glycolate oxidase in yeast led to an enzymatically a~tive-protein.'~ For glycolate oxidase, the knowledge of its tertiary structure provides advantage^.^^ The molecular organization of the genes coding for malate ~ y n t h a s e , ~ ~hydroxypyruvate -~' reductase3' and isocitrate l y a ~ has e ~ been ~ characterized. 8 7 1 9
PLANT PEROXISOMES
155
0,-dependent rudimentary redox chains which imported equally well into glyoxysomes and peroxare present in membranes of glyoxysomes have isomes. Many peroxisomal proteins are characterized by been assayed during stages of organelle d e ~ e l o p m e n t Urate . ~ ~ oxidase previously charac- a conserved tripeptide motif at the C-terminus. The terized as plant n ~ d u l i n has ~ ~ been further -S-K-L* motif was first noticed by work on plant .~~ investigations provided i n ~ e s t i g a t e dand ~ ~ compared with a non-induced urate o x i d a ~ eExtensive form.36 The content of urate oxidase in many evidence that the C-terminal motif is required for The application of antibodies ratissues depends on activating stimuli originating the from other living organism^.^^-^^ Activated oxygen ised against the motif peptide proved species such as superoxide anion may influence However, mistargeting has to be considered, as intracellular stress and the proliferation of exemplified by the transfer of firefly luciferase to yeast mit~chondria.'~Targeting signals for properoxis~mes.~~ For leaf peroxisomes, a remarkable suborganel- teins not having a C-terminal -S-K-L motif may lar compartmentation has been d e m ~ n s t r a t e dIt. ~ ~still reside at the carboxy terminus as found in the . ~other ~ cases, became clear that even isolated peroxisomes lack- case of rat liver acyl-CoA o x i d a ~ eIn ing an intact boundary membrane can channel more than one targeting signal sequence was detected within the m ~ l e c u l e . ~ ~ ~ ~ ~ their metabolites within the remaining structures. In contrast, cleavable presequences seem to exist Lipid transfer proteins4' as peroxisomal components, so far uncharacterized in their physiological as topogenic signals of two particular peroxisomal role, seem to be a target for future work. The same proteins. In their cytosolic precursor form both holds true for transport ATPases. The transfer of malate dehydrogenase and thiolase possess an certain proteins and other molecules may proceed N-terminal presequence with the following in a fashion different from the secretory machinery. property : 8-60 M -QRL-HL_ESS-.f The accumulation of glyoxysomal enzymes does Membrane proteins of the mdr family (mdr for multidrug resistance) which cleave ATP during a not take place in a concerted way. Differences in flip-over from one compartment to the other have the rates of translational and several post-translabeen characterized in the rat41 and detected in tional events mean that the appearance of mature glyoxylate cycle enzymes does not proceed plants.42 concomitantly.61.62An important but not yet fully understood factor in assembly of organelle enzymes is the folding of the imported protein. As a model BIOSYNTHESIS OF GLYOXYSOMES AND for the stepwise transition towards a functional LEAF PEROXISOMES enzyme, the function of chaperones as outlined in The biosynthesis of peroxisomes and glyoxysomes the case of citrate ~ y n t h a s emay ~ ~ be considered. proceeds by the translocation of peptides from the cytosol into pre-existing m i c r ~ b o d i e s . 'In * ~fat~~~~ storing seeds, the total space of glyoxysomes in- TRANSITION FORMS OF PEROXISOMES creases markedly during germination. Although the During their lifetime plant cells undergo several number of glyoxysomes may remain constant be- drastic conversions paralleled by transition OF intween the late state of seed maturation, dry seed tracellular structures and biochemical functions. and germinating seed, the amount of glyoxysomal Distinct forms of peroxisomes are observed during enzymes increases as the organelle increases in size. the formation and maturation of seeds, in dry seeds, The membrane lipids required for this enlargement and during mobilization of storage compounds in are acquired from lipid bodies being degraded at germinating seeds. In certain instances, the cells that stage.4s The transition from embryogeny to which house the nutrients during the etiolated stage g e r m i n a t i ~ nshows ~~ changes in transcriptional may become green in the light. Hence, leaf peroxactivities. isomes appear simultaneously with chloroplasts. Over the past 10 years, experiments related to Later, tissues undergo alterations towards senesmechanisms and components of protein import cence and that means reappearance of glyoxysomal have continued. However, compared to the early investigations47948 no marked progress was achieved. Leaf peroxisomal proteins, i.e. hydroxy- *Single letter code for Ser-Lys-Leu. pyruvate reductase4' and glycolate ~ x i d a s e , ~are ' ?Met-Clu Arg Leu-His Leu-Glu Ser Ser-.
156
properties. Intermediary forms characterized by a dual set of enzymes, the set of proteins of the preceding form and the constituents of the newly appearing form, are to be ~ o n s i d e r e d . ' ~ , ~ ~ During transition of glyoxysomes to leaf-type peroxisomes, a switch in the expression of catalase genes is observed. In addition, each primary translation product gives rise to several processed forms. Thus, the transition state is distinguished by the presence of a number of heteromeric hybrid i ~ o f o r m s . 'A ~ thorough kinetic investigation of a great number of other parameters changing during the transition to leaf pe roxi ~ome s'~ revealed that, for a substantial period of time, the set of leaf-type peroxisomal proteins is expressed and transferred to the organelle while the glyoxysome-type proteins are still synthesized and transferred to the transition-type peroxisome. Even if the transition towards the leaf peroxisomal form has proceeded for some time, it is still feasible to reverse the process by transferring the tissue into the dark.64 Senescence is not only characterized by a decrease in glycolate oxidase activity22 but also by the de nouo synthesis of glyoxysomal enzymes. This has been demonstrated in green t i s s ~ eand ~ ~for, ~ petals. Flowers represent a tissue with short lifetime and with many parameters of ~ e n e s c e n c e The .~~ enzyme activity of malate synthase and isocitrate lyase increases more than 150-fold during the last stage of flowering while the protein content decreases. The stage of senescing may be reached even under continuous light for several days.68 Experiments with senescing leave^^^.^' indicate that photorespiration decreases. Superoxide dismutase seems to be very active in the senescence-related form of microbodie~.~' Transition from one form of peroxisome into another implies that the protein constituents of the former form are removed. At present, it is not known how this process is governed. However, there are indications that proteases are induced in the lytic ~ o m p a r t m e n t ' ~or directed to the organelle.' In senescence, which essentially induces a degradative intracellular rearrangement, enzymes like lipoxygenase initiate the disruption of membranes and compartments. By this means, the movement of metabolites to storage tissues is facilitated, and an anaplerotic role of the glyoxylate cycle becomes predominant. It is pertinent to point out that peroxisomal transition forms also exist during the seed maturation. This was recentlv exemplified for cotton seed.73 The formation
VOL.
10: 153-158 (1992)
Plant Peroxisomes: Recent Studies on Function and Biosynthesis HELMUT KINDL Depurtment of' Chemistry, University of Marburg, W-3550 Marburg, Germany
fungal pathway for 2-trans-enoyl-CoA, are presented in Figure 1. The plant peroxisome constitutes a metabolic comSaturated fatty acids characteristic of animal partment which has unique features with respect to triglycerides are present in plants in minor the structural organization of the constituent cata- amounts. Their degradation proceeds via 2-translysts. Glyoxysome, a peroxisomal form encompass- enoyl-CoA (shown in middle of the upper part of ing both fatty acid oxidation and the glyoxylate Figure 1). However, in plant lipids the polyunsaturcycle, is a model system of compartmentation and ated fatty acids dominate. 3-cis-Enoyl-CoA originlimited exchange of intermediates. Progress has ating during metabolism of 9-cis-enoyl-CoA, e.g. been achieved primarily in unravelling the primary from linoleic acid, reaches the main route via structures of peroxisomal enzymes via cDNA se- isomerization to 2-trans-enoyl-CoA. 4-cis-Enoylquences and in finding new metabolic routes. This CoA, in fungi converted to 3-trans-enoyl-CoA via review concerns recent work and stresses two fields: 2,4-dienoyl-CoA and a reductase reaction, is metathe multitude of mechanisms for the chain shorten- bolized in plants as indicated by the thick arrows: ing of poly-unsaturated fatty acids and the biosyn- chain shortening to 2-cis-enoyl-CoA and converthesis of the various forms of peroxisomes. sion to 2-trans-enoyl-CoA via D-3-hydroxyacylCoA. For the further metabolism of 2-trans-enoylCoA, this multifunctional protein takes over the STRUCTURAL ORGANIZATION OF task of hydration to L-3-hydroxyacyl-CoA and ENZYMES CATALYSING FATTY ACID subsequent oxidation to 3-ketoacyl-CoA (left part DEGRADATION of the figure). Thiolase which is an independent The glyoxysome is a metabolic compartment enzyme in plant glyoxysomes is responsible for the highly efficient in transforming lipid into building consecutive cleavage. During the past years, it blocks for gluconeogenesis.' The substrates for this became evident that epimerization actually proconversion are different forms of fatty acids, and ceeds by a two-step mechanism including two kinds thus the glyoxysome has to be competent by pro- of hydratases. This was shown for liver .~ viding enzymes to cope with the supply of the metabolism' and for plant g l y c o ~ o m e sIsomerase various fatty acids. Two areas of fatty acid metabo- activity in liver peroxisomes seems to be confined lism have been covered most thoroughly and thus to the multifunctional pr0tei1-1.~Plant peroxisomes provided some surprises. One point concerns the possess, besides the isomerase as part of the large way in which enzymatic activities handling cis- form of the multifunctional protein, a very active unsaturated fatty acids are arranged on proteins. monofunctional form. In contrast to this kind of metabolic organizaThe other real surprise was when it became evident that fungi utilize a pathway 2-trans-enoyl-CoA + tion in plants, fungi seem to rely on a pathway D-3-hydroxyacyl-CoA --+ 3-ketoacyl-CoA for de- which is outlined on the right part of the figure and grading the standard fatty acids. Both features, the symbolized with thin arrow^.^'^ A bifunctional route of 4-cis-enoyl-CoA originating during the protein converts 2-trans-enoyl-CoA via D-3-hyddegradation of fatty acids with a double bond roxyacyl-CoA into 3-ketoacyl-CoA. The primary extending from an even numbered C-atom and the structures of the respective proteins from NeuroINTRODUCTION
0263-64X4/92/030153-06$08.00 I 1992 by John Wiley & Sons, Ltd
154
H. KINDL
>
4-cis-Enoyl-CoA
3-trans-EnoyCCoA
Acyl-CoA 2-cis-Enoyl-CoA
'u
2-trans-EnoyCCoA
3 cis-EnoyCCoA
Figure 1. Summary of pathways used for degradation of 4-cis-enoyl-CoA and 3-enoyl-CoA. Both plant pathways via 2-cis-enoylCoA (thick arrows) and fungal pathway from 2-trans-enoyl-CoA (very thin arrows) are stressed in addition to the steps belonging to the standard equipment of peroxisomes. HY: hydratase domain; R: reductase domain.
sposa crassa, Saccharomyces cervisiue and Cundida tropicalis are very similar. Another interesting feature of fatty acid degradation in plant glyoxysomes is the multiple location of enzyme activities on several differently organized proteins. The activity of the r>-specifichydratase is located on both multifunctional proteins (MFP I and M F P 11) and on two forms of a monofunctional dimeric protein (symbolized in Figure 1, central art).^,^ The isomerase activity is present on a monofunctional dimeric protein and on the larger form of the multifunctional proteins, i.e. M F P 11. Recently, the purification of the peroxisoma1 A2,A3-enoyl-CoA isomerase acting on 3-cisenoyl-CoA and 3-trans-enoyl-CoA and being selective towards C,, and C,, was achieved.' I t is noteworthy that the trifunctional protein (MFP I) and the tetrafunctional protein ( M F P 11) differ markedly in their domain structure. New results revealed that peroxisomes are capable of degrading branched-chain carboxylic acids.' Dicarboxylic acids" and products of lipoxygenase action' ' may be degraded in peroxisomes.
STRUCTURES OF OTHER PEROXISOMAL ENZYMES Catalase is encoded by more than one gene. The three genes in maize are expressed in a development
and tissue specific mode. CAT1 is expressed during kernel maturation while scutellum CAT212 encodes a protein destined for glyoxysomes. CAT3 is activated in leaves and codes for an enzyme sornehow associated with mitochondria. This gene is expressed in a circadian rhythm.13 In cotton seed,l4." two different subunits are assembled into five tetrameric isoforms of catalase. Glyoxysomal sunflower catalase16-' is composed of several isoforms of different charge. A 56 kDa peptide, the primary transition product, is the subject of processing. Processing takes place also with the 59 kDa peptide which appears after light induction and is characteristic of leaf-type peroxisomes. It is pertinent to point out that catalases may be composed of different subunits, as well as subunits devoid of heme. Catalases of different sizes, depending on the growth in the dark or light, have been observed in Cucurbitaceae.' The ratio of catalase and peroxidase activities may vary from plant to plant.20 The primary structures of glycolate oxidase21.22 and hydroxypyruvate r e d ~ c t a s ehave ~ ~ been elucidated. The expression of plant glycolate oxidase in yeast led to an enzymatically a~tive-protein.'~ For glycolate oxidase, the knowledge of its tertiary structure provides advantage^.^^ The molecular organization of the genes coding for malate ~ y n t h a s e , ~ ~hydroxypyruvate -~' reductase3' and isocitrate l y a ~ has e ~ been ~ characterized. 8 7 1 9
PLANT PEROXISOMES
155
0,-dependent rudimentary redox chains which imported equally well into glyoxysomes and peroxare present in membranes of glyoxysomes have isomes. Many peroxisomal proteins are characterized by been assayed during stages of organelle d e ~ e l o p m e n t Urate . ~ ~ oxidase previously charac- a conserved tripeptide motif at the C-terminus. The terized as plant n ~ d u l i n has ~ ~ been further -S-K-L* motif was first noticed by work on plant .~~ investigations provided i n ~ e s t i g a t e dand ~ ~ compared with a non-induced urate o x i d a ~ eExtensive form.36 The content of urate oxidase in many evidence that the C-terminal motif is required for The application of antibodies ratissues depends on activating stimuli originating the from other living organism^.^^-^^ Activated oxygen ised against the motif peptide proved species such as superoxide anion may influence However, mistargeting has to be considered, as intracellular stress and the proliferation of exemplified by the transfer of firefly luciferase to yeast mit~chondria.'~Targeting signals for properoxis~mes.~~ For leaf peroxisomes, a remarkable suborganel- teins not having a C-terminal -S-K-L motif may lar compartmentation has been d e m ~ n s t r a t e dIt. ~ ~still reside at the carboxy terminus as found in the . ~other ~ cases, became clear that even isolated peroxisomes lack- case of rat liver acyl-CoA o x i d a ~ eIn ing an intact boundary membrane can channel more than one targeting signal sequence was detected within the m ~ l e c u l e . ~ ~ ~ ~ ~ their metabolites within the remaining structures. In contrast, cleavable presequences seem to exist Lipid transfer proteins4' as peroxisomal components, so far uncharacterized in their physiological as topogenic signals of two particular peroxisomal role, seem to be a target for future work. The same proteins. In their cytosolic precursor form both holds true for transport ATPases. The transfer of malate dehydrogenase and thiolase possess an certain proteins and other molecules may proceed N-terminal presequence with the following in a fashion different from the secretory machinery. property : 8-60 M -QRL-HL_ESS-.f The accumulation of glyoxysomal enzymes does Membrane proteins of the mdr family (mdr for multidrug resistance) which cleave ATP during a not take place in a concerted way. Differences in flip-over from one compartment to the other have the rates of translational and several post-translabeen characterized in the rat41 and detected in tional events mean that the appearance of mature glyoxylate cycle enzymes does not proceed plants.42 concomitantly.61.62An important but not yet fully understood factor in assembly of organelle enzymes is the folding of the imported protein. As a model BIOSYNTHESIS OF GLYOXYSOMES AND for the stepwise transition towards a functional LEAF PEROXISOMES enzyme, the function of chaperones as outlined in The biosynthesis of peroxisomes and glyoxysomes the case of citrate ~ y n t h a s emay ~ ~ be considered. proceeds by the translocation of peptides from the cytosol into pre-existing m i c r ~ b o d i e s . 'In * ~fat~~~~ storing seeds, the total space of glyoxysomes in- TRANSITION FORMS OF PEROXISOMES creases markedly during germination. Although the During their lifetime plant cells undergo several number of glyoxysomes may remain constant be- drastic conversions paralleled by transition OF intween the late state of seed maturation, dry seed tracellular structures and biochemical functions. and germinating seed, the amount of glyoxysomal Distinct forms of peroxisomes are observed during enzymes increases as the organelle increases in size. the formation and maturation of seeds, in dry seeds, The membrane lipids required for this enlargement and during mobilization of storage compounds in are acquired from lipid bodies being degraded at germinating seeds. In certain instances, the cells that stage.4s The transition from embryogeny to which house the nutrients during the etiolated stage g e r m i n a t i ~ nshows ~~ changes in transcriptional may become green in the light. Hence, leaf peroxactivities. isomes appear simultaneously with chloroplasts. Over the past 10 years, experiments related to Later, tissues undergo alterations towards senesmechanisms and components of protein import cence and that means reappearance of glyoxysomal have continued. However, compared to the early investigations47948 no marked progress was achieved. Leaf peroxisomal proteins, i.e. hydroxy- *Single letter code for Ser-Lys-Leu. pyruvate reductase4' and glycolate ~ x i d a s e , ~are ' ?Met-Clu Arg Leu-His Leu-Glu Ser Ser-.
156
properties. Intermediary forms characterized by a dual set of enzymes, the set of proteins of the preceding form and the constituents of the newly appearing form, are to be ~ o n s i d e r e d . ' ~ , ~ ~ During transition of glyoxysomes to leaf-type peroxisomes, a switch in the expression of catalase genes is observed. In addition, each primary translation product gives rise to several processed forms. Thus, the transition state is distinguished by the presence of a number of heteromeric hybrid i ~ o f o r m s . 'A ~ thorough kinetic investigation of a great number of other parameters changing during the transition to leaf pe roxi ~ome s'~ revealed that, for a substantial period of time, the set of leaf-type peroxisomal proteins is expressed and transferred to the organelle while the glyoxysome-type proteins are still synthesized and transferred to the transition-type peroxisome. Even if the transition towards the leaf peroxisomal form has proceeded for some time, it is still feasible to reverse the process by transferring the tissue into the dark.64 Senescence is not only characterized by a decrease in glycolate oxidase activity22 but also by the de nouo synthesis of glyoxysomal enzymes. This has been demonstrated in green t i s s ~ eand ~ ~for, ~ petals. Flowers represent a tissue with short lifetime and with many parameters of ~ e n e s c e n c e The .~~ enzyme activity of malate synthase and isocitrate lyase increases more than 150-fold during the last stage of flowering while the protein content decreases. The stage of senescing may be reached even under continuous light for several days.68 Experiments with senescing leave^^^.^' indicate that photorespiration decreases. Superoxide dismutase seems to be very active in the senescence-related form of microbodie~.~' Transition from one form of peroxisome into another implies that the protein constituents of the former form are removed. At present, it is not known how this process is governed. However, there are indications that proteases are induced in the lytic ~ o m p a r t m e n t ' ~or directed to the organelle.' In senescence, which essentially induces a degradative intracellular rearrangement, enzymes like lipoxygenase initiate the disruption of membranes and compartments. By this means, the movement of metabolites to storage tissues is facilitated, and an anaplerotic role of the glyoxylate cycle becomes predominant. It is pertinent to point out that peroxisomal transition forms also exist during the seed maturation. This was recentlv exemplified for cotton seed.73 The formation
