Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities

The effect of increased Mn-superoxide dismutase (SOD) on antioxidant enzymes and metabolites was studied using transformed maize, TG1+ and TG2+. The progeny of the backcross of each of the primary transformants with the parental line generated two populations denoted M6884 and M6885. These were grown at optimal (25 degrees C) and sub-optimal (18, 14 and 10 degrees C) temperatures to assess the impact of elevated SOD activity on cold tolerance and the antioxidant defences in maize. The plants of the M6885 population had similar foliar SOD activities to the untransformed maize plants. Within the segregating M6884 population 50% of the plants had elevated SOD activity (up to four times the activity of the untransformed controls) and 50% of the plants contained the product of the transgene. In untransformed plants grown at 25 degrees C and 18 degrees C, SOD activity was not detectable in mesophyll extracts. Similarly, increased foliar SOD activity in the M6884 transformed maize did not lead to detectable mesophyll SOD activity. Increased foliar KCN-insensitive SOD activities were accompanied by enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities; enzymes which are localized exclusively in the leaf mesophyll tissues. Increased foliar SOD activity had no effect on the hydrogen peroxide, glutathione or ascorbate contents of the leaves. This suggests that increased recycling of reduced ascorbate was required to compensate for enhanced hydrogen peroxide production in transformed plants.     

Localization of nitrite and sulfite reductase in bundle sheath and mesophyll cells of maize leaves

The distribution of nitrite reductase (EC 1.7.7.1) and sulfite reductase (EC 1.8.7.1) between mesophyll ceils and bundle sheath cells of maize ( Zea mays L. cv. Seneca 60) leaves was examined. This examination was complicated by the fact that both of these enzymes can reduce both NO-₂ and SO^2-₃ In crude extracts from whole leaves, nitrite reductase activity was 6 to 10 times higher than sulfite reductase activity. Heat treatment (10 min at 55°C) caused a 55% decrease in salfite reductase activity in extracts from bundle sheath cells and mesophyll cells, whereas the loss in nitrite reductase activity was 58 and 82% in bundle sheath cells and mesophyll cell extracts, respectively. This result was explained, together with results from the literature, by the hypothesis that sulfite reductase is present in both bundle sheath cells and mesophyll cells, and that nitrite reductase is restricted to the mesophyll cells. This hypothesis was tested i) by comparing the distribution of nitrite reductase activity and sulfite reductase activity between bundle sheath and mesophyll cells with the presence of the marker enzymes ribulose-l, 5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoe-nolpyruvate carboxylase (EC 4.1.1.32), ii) by examining the effect of cultivation of maize plants in the dark without a nitrogen source on nitrite reductase activity and sulfite reductase activity in the two types of cells, and iii) by studying the action of S^2-on the two enzyme activities in extracts from bundle sheath and mesophyll cells. The results from these experiments are consistent with the above hypothesis.     

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

Localization of antioxidant enzymes in the cellular compartments of sorghum leaves

The localization of antioxidant enzymes between the mesophyll and bundle sheath cells were determined in sorghum (Sorghum vulgare L.) leaves. The activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) were assayed in whole leaf, mesophyll and bundle sheath fractions of sorghum leaves subjected to water-limited conditions. Drought was imposed by withholding water and the plants were maintained at different water potentials ranging from 0.5–2.0 MPa. The purity of the isolates was tested using the marker enzymes like RuBPcase and PEPcase. GR was mostly localized in mesophyll fraction, while SOD, APX and peroxidase were located in bundle sheath cells. Catalase was found to be equally distributed between the two cell types. Under water stress conditions, most of the SOD activity was found in the bundle sheath tissues. Little or no activity of the enzymes CAT, APX or POD was found in the mesophyll extracts when exposed to water stress. GR activity increased when exposed to low water regimes. From this study, it is clear that antioxidants are differentially distributed between the mesophyll and bundle sheath cells in sorghum leaves. Under water stress conditions, the mesophyll cells showed less damage from oxidative stress when compared to the bundle sheath cells. This is critical for determining the sensitivity of sorghum to extreme climatic conditions.     

Effects of moderate drought on ascorbate peroxidase and glutathione reductase activities in mesophyll and bundle sheath cells of maize

Mesophyll and bundle sheath cells of maize leaves ( Zea mays L.) both contain the enzymes ascorbate peroxidase (AP; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) which are involved in hydrogen peroxide detoxification. Since bundle sheath cells of maize are deficient in photosystem II and have high CO₂ levels, oxidative stress may be less severe in these cells than in mesophyll cells. The present study was conducted to determine if AP and GR activity levels preferentially increase in mesophyll cells relative to bundle sheath cells when plants are subjected to moderate drought. Although drought inhibited the growth of greenhouse-grown plants, it did not affect the levels of protein, chlorophyll or AP. GR was unaffected by drought in whole leaf tissue and mesophyll cells, but did increase slightly in bundle sheath cells. This slight increase is of questionable biological importance. AP and GR activity levels were similar in mesophyll cells, bundle sheath cells and in whole leaf tissue. The data suggest that moderate drought has little effect on enzymes of the hydrogen peroxide scavenging system and that mesophyll and bundle sheath cells may be exposed to similar levels of hydrogen peroxide.     

Differential Localization of Antioxidants in Maize Leaves

The aim of this work was to determine the compartmentation of antioxidants between the bundle-sheath and mesophyll cells of maize (Zea mays L.) leaves. Rapid fractionation of the mesophyll compartment was used to minimize modifications in the antioxidant status and composition due to extraction procedures. The purity of the mesophyll isolates was assessed via the distribution of enzyme and metabolite markers. Ribulose-1,5 bisphosphate and ribulose-1,5-bisphosphate carboxylase/oxygenase were used as bundle-sheath markers and phosphoenolpyruvate carboxylase was used as the mesophyll marker enzyme. Glutathione reductase and dehydroascorbate reductase were almost exclusively localized in the mesophyll tissue, whereas ascorbate, ascorbate peroxidase, and superoxide dismutase were largely absent from the mesophyll fraction. Catalase, reduced glutathione, and monodehydroascorbate reductase were found to be approximately equally distributed between the two cell types. It is interesting that, whereas H2O2 levels were relatively high in maize leaves, this oxidant was largely restricted to the mesophyll compartment. We conclude that the antioxidants in maize leaves are partitioned between the two cell types according to the availability of reducing power and NADPH and that oxidized glutathione and dehydroascorbate produced in the bundle-sheat tissues have to be transported to the mesophyll for re-reduction to their reduced forms.     

Comparative analysis of biochemical properties of mesophyll and bundle sheath chloroplasts from various subtypes of C4 plants grown at moderate irradiance

The photochemical characteristics of mesophyll and bundle sheath chloroplasts isolated from the leaves of C4 species were investigated in Zea mays (NADP-ME type), Panicum miliaceum (NAD-ME type) and Panicum maximum (PEP-CK type) plants. The aim of this work was to gain information about selected photochemical properties of mesophyll and bundle sheath chloroplasts isolated from C4 plants grown in the same moderate light conditions. Enzymatic as well as mechanical methods were applied for the isolation of bundle sheath chloroplasts. In the case of Z. mays and P. maximum the enzymatic isolation resulted in the loss of some thylakoid polypeptides. It was found that the PSI and PSII activities of mesophyll and bundle sheath chloroplasts of all species studied differed significantly and the differences correlated with the composition of pigment-protein complexes, photophosphorylation efficiency and fluorescence emission characteristic of these chloroplasts. This is the first report showing differences in the photochemical activities between mesophyll chloroplasts of C4 subtypes. Our results also demonstrate that mesophyll and bundle sheath chloroplasts of C4 plants grown in identical light conditions differ significantly with respect to the activity of main thylakoid complexes, suggesting a role of factor(s) other than light in the development of photochemical activity in C4 subtypes.     

Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures

In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co-ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 degrees C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.     

Low temperature-induced changes in antioxidative metabolism in rubber-producing shrub,guayule (<Emphasis Type="Italic">Parthenium argentatum</Emphasis> Gray)

Two-year-old rubber-producing shrub, guayule (Parthenium argentatum, cv. 11591), was treated with low temperature (15 ^°C). The leaves were harvested at regular intervals (0, 2, 4 and 6 days) and the contents of protective antioxidants (ascorbic acid, monodehydroascorbate and caroteniods) and antioxidative enzymes (superoxide dismutase, catalase, peroxidases, glutathione reductase and monodehydroascorbate reductase) were determined. Low temperature-induced significant increase in the contents of ascorbic acid, monodehydroascorbate and caroteniods as well as the activities of all antioxidative enzymes. The results show an increase in several components of the antioxidant system in cold-treated guayule plants, which may suggest a role in mitigating an increase in oxidative stress.     

Sulfate assimilation and glutathione synthesis in C<Subscript>4</Subscript> plants

Sulfate assimilation and glutathione synthesis were traditionally believed to be differentially compartmentalised in C₄ plants with the synthesis of cysteine and glutathione restricted to bundle sheath and mesophyll cells, respectively. Recent studies, however, showed that although ATP sulfurylase and adenosine 5′ phosphosulfate reductase, the key enzymes of sulfate assimilation, are localised exclusively in bundle sheath in maize and other C₄ monocot species, this is not true for the dicot C₄ species of Flaveria. On the other hand, enzymes of glutathione biosynthesis were demonstrated to be active in both types of maize cells. Therefore, in this review the recent findings on compartmentation of sulfate assimilation and glutathione metabolism in C₄ plants will be summarised and the consequences for our understanding of sulfate metabolism and C₄ photosynthesis will be discussed.     

Acclimation of mesophyll and bundle sheath chloroplasts of maize to different irradiances during growth

The regulation by light of the photosynthetic apparatus, and composition of light-harvesting complexes in mesophyll and bundle sheath chloroplasts was investigated in maize. Leaf chlorophyll content, level of plastoquinone, PSI and PSII activities and Lhc polypeptide compositions were determined in plants grown under high, moderate and low irradiances. Photochemical efficiency of PSII, photochemical fluorescence quenching and non-photochemical fluorescence quenching over a range of actinic irradiances were also determined, using chlorophyll a fluorescence analysis. Acclimation of plants to different light conditions caused marked changes in light-harvesting complexes, LHCI and LHCII, and antenna complexes were also reorganized in these types of chloroplasts. The level of LHCII increased in plants grown in low light, even in agranal bundle sheath chloroplasts where the amount of PSII was strongly reduced. Irradiance also affected LHCI complex and the number of structural polypeptides, in this complex, generally decreased in chloroplasts from plants grown under lower light. Surprisingly moderate and low irradiances during growth do not affect the light reaction and fluorescence parameters of plants but generated differences in composition of light-harvesting complexes in chloroplasts. On the other hand, the changes in photosynthetic apparatus in plants acclimated to high light, resulted in a higher efficiency of photosynthesis. Based on these observations we propose that light acclimation to high light in maize is tightly coordinated adjustment of light reaction components/activity in both mesophyll and bundle sheath chloroplasts. Acclimation is concerned with balancing light utilization and level of the content of LHC complexes differently in both types of chloroplasts.     

Lincomycin-induced alteration in the contents of chlorophyll-protein complexes of dimorphic maize chloroplasts and its effect on the temperature-induced spectral changes

The difference spectroscopy technique has been utilized to investigate the temperature-induced spectral changes in mesophyll and bundle sheath chloroplasts of maize ( Zea mays L. cv. Ganga-5) in order to assess the role of different pigment-protein complexes in the manifestation of temperature effect on the chloroplast membranes. Cooling and heating of both mesophyll and bundle sheath chloroplasts resulted in absorbance difference (AA) bands at similar wavelengths but the degree of absorb-ance changes were significantly higher in bundle sheath chloroplasts. For example, upon cooling to 7-8°C, positive AA bands were observed at 440, 490 and 680 nm in mesophyll chloroplasts and at 440, 495–500 and 680 nm in bundle sheath chloroplasts but the absorbance change at 680 nm was ca 2% in mesophyll chloroplasts, whereas it was ca 5% in bundle sheath chloroplasts, which have a lower content of light-harvesting pigment-protein complex. The role of chlorophyll-protein complexes was further investigated by monitoring the temperature-induced spectral changes of mesophyll and bundle sheath chloroplasts isolated from lincomycin-treated maize plants where lincomycin selectively inhibits the biosynthesis of specific chlorophyll-protein complexes. Results indicated that depletion of certain pigment-protein complexes in mesophyll chloroplasts made them more susceptible (a ca 4% vs ca 2% absorbance change upon cooling and a ca 6% vs ca 4% absorbance change upon heating) and less tolerant to temperature variation (a 76% vs 39% reversibility during ambient→Cooling→ambient temperature cycle). The data indicate that pigment-protein complexes play a significant role in protecting the chloroplast membranes against temperature variation.     

Chloroplast thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology

Tissue-specific effects of low growth temperature on maize chloroplast thylakoid protein accumulation were analysed using immunocytology. Sections of leaves from plants grown at 25 and 14°C were probed with antibodies to specific chloroplast thylakoid proteins from the four major protein multisubunit complexes of the thylakoid membrane followed by fluorescein-conjugated goat anti-rabbit antibodies. At a normal growth temperature of 25°C, the 32 kDa D1 protein of the photosystem II reaction centre and the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II are both accumulated to a greater degree in the mesophyll than in the bundle sheath chloroplasts. In contrast, subunit II of photosystem I, cytochrome f and the α- and β-subunits of ATP synthetase are predominant in the bundle sheath thylakoids at 25°C. A striking difference between the 25°C-grown and the 14°C-grown leaf tissue was the presence in the latter of (20–30%) cells whose chloroplasts apparently completely lack several of the thylakoid proteins. In plants grown at 14°C, the accumulation of the 33 kDa protein of the extrinsic oxygen-evolving complex of photosystem II was apparently unchanged, but other thylakoid proteins showed a significant reduction. The uneven distribution of proteins between the bundle sheath and mesophyll chloroplasts observed at 25°C was also maintained at 14°C. Reduction in the fluorescence at 14°C was manifested either as an overall reduction in the diffuse fluorescence across the chloroplast profiles or less frequently as a reduction to small discrete bodies of intense fluorescence. The significance of these results to low-temperature-induced reduction in the photosynthetic productivity of maize is discussed.     

Acclimation of mesophyll and bundle sheath chloroplasts of maize to different irradiances during growth

The regulation by light of the photosynthetic apparatus, and composition of light-harvesting complexes in mesophyll and bundle sheath chloroplasts was investigated in maize. Leaf chlorophyll content, level of plastoquinone, PSI and PSII activities and Lhc polypeptide compositions were determined in plants grown under high, moderate and low irradiances. Photochemical efficiency of PSII, photochemical fluorescence quenching and non-photochemical fluorescence quenching over a range of actinic irradiances were also determined, using chlorophyll a fluorescence analysis. Acclimation of plants to different light conditions caused marked changes in light-harvesting complexes, LHCI and LHCII, and antenna complexes were also reorganized in these types of chloroplasts. The level of LHCII increased in plants grown in low light, even in agranal bundle sheath chloroplasts where the amount of PSII was strongly reduced. Irradiance also affected LHCI complex and the number of structural polypeptides, in this complex, generally decreased in chloroplasts from plants grown under lower light. Surprisingly moderate and low irradiances during growth do not affect the light reaction and fluorescence parameters of plants but generated differences in composition of light-harvesting complexes in chloroplasts. On the other hand, the changes in photosynthetic apparatus in plants acclimated to high light, resulted in a higher efficiency of photosynthesis. Based on these observations we propose that light acclimation to high light in maize is tightly coordinated adjustment of light reaction components/activity in both mesophyll and bundle sheath chloroplasts. Acclimation is concerned with balancing light utilization and level of the content of LHC complexes differently in both types of chloroplasts.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening

The activities of nitrate reductase (EC1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC6.3.1.2), glutamate synthase (EC1.4.7.1) and NAD(P)H-dependent glutamate dehydrogenase (EC 1.4.1.3) were investigated in mesophyll and bundle sheath cells of maize leaves (Zea mays L.). Whereas nitrate and nitrite reductase appear to be restricted to the mesophyll and GDH to the bundle sheath, glutamine synthetase and glutamate synthase are active in both tissues.During the greening process, the activities of nitrate and nitrite reductase increased markedly, but glutamine synthetase, glutamate synthase and glutamate dehydrogenase changed little.Abbreviations BDH British Drug Houses - EDTA Ethylene diamine tetra-acetic acid - GDH Glutamate dehydrogenase - NADH Nicotinamide-adenine dinucleotide reduced form - NADPH Nicotnamide-adenine dinucleotide phosphate reduced form - PMSF Phenylmethyl sulphonyl fluoride