6.5—
Peroxisomes
Glycollic acid is produced in large amounts in the chloroplast, as a by-product of the reactions of carbon dioxide fixation. The formation of glycollate has been proposed to occur by the oxidation of ribulose-1,5-bisphosphate (RBP) by molecular oxygen, a reaction which would produce a two carbon fragment of phosphoglycollic acid and a three carbon fragment of phospholgyceric acid (PGA) instead of two molecules of PGA resulting from the normal carboxylation of RBP by CO2 . It has been found that RBP carboxylase acts as an oxygenase in the presence of molecular oxygen and that phosphoglycollate and PGA are produced in this reaction in vitro (Andrews et al., 1973). Thus the enzyme RBP carboxylase can act as an oxygenase or as a carboxylase and the formation of phosphoglycollic acid is favoured by high partial pressures of oxygen. Glycollate is produced from phosphoglycollate by the action of phosphoglycollate phosphatase, a chloroplast enzyme. Glycollate is also produced, in vitro, from fructose-6-phosphate by the action of the chloroplast enzyme, transketolase, which may be due to the oxidation, by hydrogen peroxide, of the glycolaldehyde-thiamine pyrophosphate, an intermediate complex in this enzyme reaction (Bradbeer & Racker, 1961). Glycollate is released from the chloroplast into the cytosol, where it is further metabolized in a specific metabolic pathway, the initial reactions of this pathway being located in the peroxisome.
6.5.1—
The Glycollate Pathway
The pathway of glycollate metabolism in leaves has been elucidated by infiltrating excised leaves or leaf discs with 14 C-labelled glycollate or other intermediates of the pathway. Glycollate is converted rapidly to glyoxyllate which may be oxidized non-enzymatically to carbon dioxide and formic acid, in the presence of hydrogen peroxide. In this reaction the carbon dioxide is derived from the carboxyl carbon of glycollate and the formate from the methyl carbon (Tolbert & Burris, 1950). However the presence of catalase, which breaks down peroxide, is thought to preclude such a total degradation of glyoxyllate in the peroxisome and the supply of 14 C-labelled glycollate or glycoxyllate to leaf tissue in the light has been found to give rise initially to labelled glycine and serine and subsequently to labelled glyceric acid hexoses and sucrose (Tolbert, 1963; see Fig. 6.7).
Infiltration of [2-14 C] glycollate into leaves in the light was found to give [2-14 C] glycine but serine was found to be labelled in the 2 and 3 carbons (Tolbert & Cohan, 1953; see Fig. 6.7). From this evidence it was concluded that two molecules of glycine give rise to one molecule of serine with a loss of one molecule of carbon dioxide; the 1 and 2 carbons of serine are derived from carbons 1 and 2 of glycine respectively, while the 3 carbon of serine is derived from the 2 carbon of glycine and carbon dioxide arises from carbon 1 of glycine. In wheat leaves in light [3-14 C] serine was converted to [3-14 C] glycerate
Figure 6.7
The glycollate pathway. The labelling pattern of intermediates of the
pathway ane hexose is indicated for when they are derived from
[1-'4 C]-glycollate (
and this was incorporated into hexose presumably by the reactions of the Embden-Meyerhof-Parras pathway (Rabson et al., 1962). This pathway of hexose formation from glycollate has been confirmed by the finding that [2-14 C] glycollate gives rise to glycose labelled in the 1, 2, 5 and 6 carbons and [3-14 C] serine to glucose labelled in the 1 and 6 carbons (Jiminez et al., 1.962).
The glucollate pathway is therefore gluconeogenic in light. The conversion of glyceric acid derived from glycollate, to glucose and sucrose is inhibited by DCMU an inhibitor of Photosystem II (Miflin et al., 1966). In the dark, supplied [14 C]-glycollate is converted into TCA cycle acids rather than sugars which may be due to an oxidation of pyruvate derived from glyceric acid, or may result from a direct conversion of glyoxyllate to malate.
Glycollate produced during photosynthesis in the presence of 14 CO2 is usually found to be uniformly labelled i.e. both carbons have the same specific activity. This distribution of radioactivity would be expected if the glycollate was derived from carbons 1 and 2 of ribulose-1,5-bisphosphate. Consequently all the compounds arising from glycollate are uniformly labelled. Serine, in particular, has been found to be uniformly labelled while phosphoglyceric acid was predominately carboxyl labelled, indicating that serine was produced from glycollate rather than directly from phosphoglyceric acid formed in CO2 -fixation (Rabson et al., 1962). The uniformly labelled glyceric acid formed in this pathway in turn produces uniformly labelled hexoses instead of the 3,4-14 C-hexoses resulting from incorporation of 14 C from the photosynthetic carbon cycle.
The operation of the glycollate pathway in leaves has been shown by tracer experiments but it has been confirmed by the detection of enzymes in leaves which are necessary for some reactions of the pathway. The initial reaction, the oxidation of glycollate to glyoxyllate is catalysed by glycollate oxidase, an enzyme first isolated by Zelitch and Ochoa (1953). It has FMN as the prosthetic group and utilizes molecular oxygen as the electron acceptor. The enzyme is competitively inhibited by bisulphite addition compounds of aldehydes, a -hydroxy-sulphonates, which have the general structure R-CHOH-SO3 H and
are therefore structural analogues of glycollate. The most commonly used a -hydroxysulphonate is a -hydroxypyridylmethane sulphonate (HPMS) and treatment of leaf discs or infiltration of excised leaves with this compound results in the accumulation of glycollic acid while having no effect upon the rate of CO2 -fixation. Experiments of this type have shown that 40 to 70: of the carbon fixed in photosynthesis will accumulate as glycollate in tissues treated with HPMS and these results have been interpreted as indicating that a large fraction of the carbon fixed in photosynthesis is metabolized by this pathway.
Transaminases are present in leaves which catalyse two steps of the pathway: a glutamate-glyoxyllate transaminase catalysing the formation of glycine is widespread in plant tissues, and a serine-pyruvate aminotransferase catalysing the formation of serine to hydroxypyruvate is found in leaves. The conversion of glycine to serine is catalysed by serine hydroxymethyltransferase which has been found in pea and wheat leaves (Cossins & Sinha, 1966). This reaction is inhibited by isonicotinyl hydrazide, and treatment of leaf tissue with this compound during photosynthesis in 14 CO2 causes an accumulation of [14 C] glycine and [14 C] glycollate and a decrease in the incorporation of 14 C into glucose (Miflin et al., 1966). Use of this inhibitor thus provides additional evidence for the operation of the pathway.
Two types of glyoxyllate reductase have been found to occur in leaves. One, an NADP-linked enzyme, is thought to be located in the chloroplast, while a second NAD-linked enzyme is present in the cytoplasm and is referred to as hydroxypyruvate reductase since the enzyme isolated from some sources has a higher activity to hydroxypyruvate than to glyoxyllate. The enzyme appears to catalyse the reduction of hydroxypyruvate to glycerate in the glycollate pathway.
6.5.2—
Metabolic Reactions of the Peroxisome
It was generally accepted that the enzymes of the glycollate pathway were soluble proteins of the cytosol and the failure of several attempts to localize these enzymes, particularly glycollate oxidase, in discrete organelles confirmed this idea. The first successful localization of enzymes of glycollate metabolism in a discrete organelle was achieved by Tolbert and coworkers at Michigan State University (Tolbert et al., 1968). They demonstrated that sucrose density gradient centrifugation of spinach homogenates separated three bands of particles: broken chloroplasts, mitochondria, and small bodies distinctly separated from, and denser than the other organelles. Electron microscopic examination of these bodies showed them to be organelles bounded by a single unit membrane and since they closely resembled peroxisomes from animal cells in size and morphology, they were referred to as leaf peroxisomes. The most important finding was that these peroxisomes contained the bulk of the activity of the glycollate oxidase, catalase and hydroxypyruvate reductase (NAD-glyoxyllate reductase) of the gradient, while cytochrome c oxidase was specifically located in the mitochondrial fraction. Peroxisomes were later
isolated from the leaves of nine other plant species including tobacco, maize, and sugarcane, and all contained the same enzyme complement (Tolbert et al., 1969). These studies therefore demonstrated that processes of glycollate oxidation and peroxide breakdown were localized together in an organelle discrete from the chloroplast. Electron microscope studies, particularly by Newcomb, have since revealed the presence of peroxisomes in leaves of many plant species (e.g. Frederick & Newcomb, 1969).
Further studies in Tolbert's laboratory have shown that other enzymes of the glycollate pathway are also localized in the peroxisome. Two aminotransferases, glutamate-glyoxyllate aminotransferase catalysing the conversion of glyoxyllate to glycine, and serine-pyruvate aminotransferase catalysing the conversion of serine to hydroxypyruvate, were found in peroxisomes isolated from leaves of various species. Serine hydroxymethyltransferase is the only enzyme of the glycollate pathway not found in the peroxisome, and is probably located in the mitochondrion. Supply of [14 C] glycollate and [14 C] glyoxyllate to isolated peroxisomes gave rise only to [14 C] glycine and while oxygen uptake occurred, no 14 CO2 release was detected (Kisaki & Tolbert, 1969).
The glycollate pathway appears to require enzymic steps located in three subcellular organelles (Fig. 6.8). Glycollate, formed in the chloroplast is
Figure 6.8
The distribution of the reactions of the glycollate
pathway among organelles of the leaf cell.
oxidized to glyoxyllate in the peroxisome. The glyoxyllate may then be exported to the chloroplast where it could be reduced to glycollate by the action of NADP-dependent glyoxyllate reductase which is located specifically in the chloroplast (Tolbert et al., 1970). Such a coupling of alternate oxidation and reduction reactions. a 'glycollate-glyoxyllate shuttle', has been proposed as a mechanism for controlling the levels of reduced NADP in the chloroplast, but no unequivocal evidence for such a reaction in vivo has been found. Glycine is formed from glyoxyllate in the peroxisome and then transferred to the
mitochondrion where it is converted to serine, with a concomitant loss of carbon dioxide. The conversion of serine to glycerate can then be accomplished by enzymes localized in the peroxisome. Further metabolism of glycerate to hexose appears to be confined to the chloroplast since these reactions would be initiated by the formation of phosphoglyceric acid, a reaction catalysed by phosphoglycerate phosphatase which is located in the chloroplast.
The metabolism of glycollate by leaf tissue results in the release of carbon dioxide, but the exact site of this CO2 release in the cell is still a controversial question. It has been proposed that CO2 is evolved by the mitochondria during the conversion of glycine to serine, since [14 C] glycine is as good a precursor as [14 C] glycollate for 14 CO2 evolution in leaves and the 14 CO2 evolved is derived from the carboxyl groups of these compounds. The presence of catalase in the peroxisomes is thought to minimize the non-enzymatic oxidation of glyoxyllate by H2 O2 to formate and carbon dioxide and no evolution of CO2 by isolated peroxisomes from glycollate or glyoxyllate could be detected by some workers (Kisaki & Tolbert, 1970). This loss of CO2 from glycine, which amounts to only 25% of the total carbon passing through the glycollate pathway, would not account for the large losses of CO2 which occur as a result of the photorespiration of glycollate in leaves. However the amount of catalase present in the peroxisome may not preclude the non-enzymatic oxidation of glyoxyllate and it has been shown that both [14 C] glycollate and [1-14 C] glyoxyllate can be decarboxylated by peroxisomal fractions at pH 8.0 (Halliwell & Butt, 1974). An enzyme is also present in chloroplasts which catalyses the decarboxylation of glyoxyllate to formic acid and CO2 (Zelitch, 1972). Present evidence indicates therefore that three subcellular organelles have the capacity to decarboxylate components of the glycollate pathway and each may contribute to the production of the carbon dioxide in photorespiration (Fig. 6.8).
6.5.3—
Photorespiration
The oxidation of glycollate in the peroxisome is accompanied by a consumption of oxygen and ultimately results in the release of carbon dioxide. The net result is a respiratory gas exchange where the substrate of respiration is glycollate rather than glucose. Since glycollate is only formed in light this respiration is light-dependent and is called photorespiration.
The direct measurement of oxygen uptake during a net photosynthetic release of oxygen, or CO2 release during net photosynthetic CO2 fixation is impossible. However, indirect methods have demonstrated that photorespiration occurs in plants and that the rate of CO2 loss in light is higher than that in the dark. Measurements of photorespiration rates vary considerably with the assay method used and can only be considered as approximations of the magnitude of the process. Nevertheless, it is becoming clear that the process of photorespiration has great importance in decreasing the rate of net photosynthesis in many plants.
Photorespiration can be detected by measuring the flux, that is the simultaneous uptake and release, of oxygen or carbon dioxide of photosynthesizing tissue by using isotopic tracer methods. The uptake of 18 O2 by leaves during photosynthesis has been detected but experiments using 18 O2 have generally given equivocal results which have been difficult to interpret.
The measurement of carbon dioxide flux during photosynthesis is much more convenient and depends upon the accurate measurement of carbon dioxide concentration in the atmosphere using an infra-red gas analyser and a simultaneous measurement of the total activity of supplied 14 CO2 with an ion counter or Geiger-Müller counter. When a plant is placed in a closed system in light, there is a rapid uptake of carbon dioxide and the CO2 concentration of the atmosphere around the plant decreases to a concentration at which the uptake of CO2 exactly balances the output of CO2 by the plant (Fig. 6.9). This concentration of carbon dioxide is called the CO2compensation point of the plant and is usually measured in parts per million (ppm) of CO2 in air. Plants such as tobacco, sunflower and wheat have compensation points, ranging from 35 to 100 ppm indicating a marked loss of CO2 (i.e. photorespiration) during photosynthesis, but others such as maize and sugarcane have compensation points of 3 to 10 ppm indicating a low loss of CO2 or a low photorespiration rate. If photosynthetic carbon fixation is similarly measured in a closed-system but in an atmosphere containing 14 CO2 , the CO2 arising from, the plant by photorespiration will, over short time periods, be 12 CO2 . Thus, while a decrease in the CO2concentration of the atmosphere around the plant will occur, the radioactivity of 14 CO2 in the atmosphere will appear to decrease at a faster rate because of the efflux of unlabelled CO2 from the plant, and continues to decrease even after the compensation point is reached (Fig. 6.9a). In a plant which
Figure 6.9
The concentration of CO2 and 14 CO2 and the specific activity of 14 CO2 around
a detached sunflower leaf (a) and a detached maize leaf (b) during illumination
and in subsequent darkness in an atmosphere of 21% oxygen and at 21°.
(Reproduced with permission from Hew et al., 1969.)
is photorespiring, therefore, a decrease in the specific radioactivity of 14 CO2 will occur, while in a plant with no photorespiration the uptake of 14 CO2 will not be accompanied by an efflux of 12 CO2 and the specific radioactivity of the 14 CO2 in the atmosphere will remain constant (Fig. 6.9b). In the dark the specific radioactivity of the 14 CO2 decreases with the efflux of 12 CO2 and the rate of decrease of this activity is a measure of dark respiration. The rate of photorespiration as measured by these methods has been shown to be 1.5 to 2.5 times that of dark respiration, while in plants with low photorespiration, such as maize, loss of CO2 in light is only a fraction of the rate of CO2 loss in the dark. This method clearly indicates that photorespiration occurs in some species while it is absent in others.
Another method which has been used to detect differences in the rate of photorespiration and dark respiration was devised by Zelitch (1968). In this method leaf discs are allowed to fix 14 CO2 for a period of 45 to 60 minutes. The remaining 14 CO2 is then quickly flushed out of the closed system, and the release of 14 CO2 from the tissue into CO2 -free air is measured over short periods in the light and the dark. The method is based on the assumptions that the rate of 14 CO2 loss is a measure of total CO2 loss i.e. that the specific radioactivity of the CO2 evolved remains constant over short time periods and that low CO2 tensions have no effect on the loss of CO2 . These assumptions may not be valid for all photosynthetic tissues but within these limitations the method is a very rapid and sensitive means for detecting photorespiration. Photorespiratory loss of CO2 in tobacco has been shown by this method to be 2 to 5 times higher than in the dark while CO2 loss from maize is not detectable (Fig. 6.10a). This method has been similarly used to detect photorespiration in other species.
Figure 6.10a
A comparison between the release of 14 CO2 by tobacco and maize discs
in the light and the dark after previously being supplied 14 CO2 in light.
(b). The effect of a -hydroxysulponate on the release of
14 CO2 from tobacco leaf discs in the light and dark.
(Reproduced with permission from Zelitch, 1968.)
Photorespiration is not simply a stimulation of dark respiration since the two process of photorespiration and dark respiration respond differently to changes in oxygen concentration. Dark respiration of leaves has an optimal rate at about 2% oxygen in air and any increase in the O2 concentration up to 100% does not increase the rate (Fig. 6.11). Net photosynthesis however is inhibited
Figure 6.11
The effect of oxygen concentration on the rate of photorespiration
(PR) and dark respiration (RD ) of detached soybean leaves.
(Reproduced with permission from Forrester et al., 1966.)
by oxygen, a phenomenon called the Warburg effect, and this is attributable to an increase in the rate of photorespiration rather than to an inhibition of photosynthesis per se . A reduction in the oxygen concentration in the atmosphere around a leaf from the ambient 21% O2 of air has been shown to lower the compensation point and increase the rate of net photosynthesis, while an increase in concentration raises the compensation point and lowers net photosynthesis (Forrester et al., 1966). Plants with photorespiration have been found to evolve CO2 at a high rate when they are transferred to darkness after a period of photosynthesis. This post-illumination CO2 burst lasts for a few minutes before a steady dark respiration is established and the magnitude of the burst has been found to depend on the light intensity and oxygen concentration during the previous period of photosynthesis: the CO2 -burst increases with an increase in light intensity and decreases with a decrease in O2 concentration (Tregunna et al., 1961). This burst has been explained as being due to the continued slow oxidation, in the dark, of a product produced in photosynthesis after the uptake of CO2 by photosynthesis has stopped. Photorespiration therefore appears to have different characteristics than dark respiration and to have a different substrate.
The substrate for photorespiration is thought to be glycollic acid. Glycollic
acid production in leaves and in chloroplasts is stimulated by an increase in oxygen concentration as is photorespiration, while the inhibition of glycollate oxidation by HPMS has been found to lower the rate of photorespiration in leaf discs to that of dark respiration (Fig. 6.10b). Photorespiratory loss of CO2 is also stimulated by the infiltration of leaves with glycollic acid while acetate has no effect on this rate. Thus the oxidation of glycollate, mediated by the leaf peroxisomes results in a photorespiratory loss of carbon dioxide by the leaf.
Plants with low compensation points are said to lack photorespiration. These plants are C4 plants, that is, the primary carboxylation step is catalysed by phosphoenolpyruvate carboxylase rather than by RBP carboxylase. This efficient fixation of CO2 has been postulated to preclude the formation of glycollate, but maize for example, has been shown to possess a glycollate pathway (Osmond, 1969) and peroxisomes occur in the mesophyll cells of the leaf. Presumably in such plants glycollate may be formed and oxidized, but the efficiency of refixation of the CO2 resulting from glycollate oxidation is such that no CO2 is released from the plant and hence no photorespiration is detected.
Estimates from various plant species suggest that from 15 to 40% of the carbon fixed in photosynthesis is lost by photorespiration. There is no unequivocal evidence to show that energy in the form of ATP is recovered during the oxidation of glycollate and the process appears to be a wasteful one in terms of energy conservation. An important question therefore arises: does photorespiration serve a useful function or is it simply an inevitable consequence of photosynthesis being carried on in an atmosphere of 21% oxygen? It has been suggested that photorespiration might act as a 'safety valve' for the plant, in that, under conditions of high light intensity and low carbon dioxide concentration, the oxidation of glycollate would consume both excess reduced NADP+ and excess oxygen, which would serve to protect the chloroplast from photo-oxidative damage. If the process of photorespiration imposes limitations on the growth of plants, it would be expected that there would have been some selective pressure to eliminate it by natural selection during the evolution of the higher plants. The occurrence of C4 plants, which have low photorespiration, may represent such an evolutionary step to correct for the presence of photorespiration by the development of an efficient CO2 -fixation mechanism. Since variations in the rate of photorespiration occur within a single species (Zelitch, 1971) it may be possible to select artificially for low photorespiration in crop plants and consequently increase net photosynthetic productivity and crop yield.