Zbigniew Kaniuga

Isolation and characterization of cytosolic and chloroplast isoenzymes of Cu,Zn-superoxide dismutase from tomato leaves and their relationships to other Cu,Zn-superoxide dismutases

Abstract Two isoenzymes (I and II) of Cu,Zn-superoxide dismutase (EC 1.15.1.1) from tomato leaves have been purified to apparent homogeneity. The isoelectric points, amino acid compositions, metal contents and ultraviolet spectra, although mutually different, were reminiscent of those determined for other Cu,Zn-superoxide dismutases. The relative molecular masses are about 33 000 and 30 000 for isoenzymes I and II, respectively, as judged from SDS-polyacrylamide gel electrophoresis (two equal subunits), gel exclusion chromatography and sedimentation equilibrium centrifugation. Specific activities were 7200 and 6300 standard units for isoenzyme I and II, respectively. Isoenzyme I exhibited an optimum at pH about 7, giving the highest rate constant reported so far ( k I = (6–7) · 10 9 M −1 · s −1 ), while isoenzyme II activity was essentially pH-independent in the range between 5.8 to 7.8 ( K II = (3–3.5) · 10 9 M −1 · s −1 ). Azide caused 50% inhibition of isoenzymes I and II at concentrations of 10 and 6 mM, respectively, in contrast to 32 mM claimed to be characteristic for Cu,Zn-superoxide dismutase (Misra, H.P. and Fridovich, I. (1978) Arch. Biochem. Biophys. 189, 317–322). Isoenzyme I was more susceptible to heat, urea and hydrogen peroxide denaturation than isoenzyme II. Proteolytic digestions and in vitro subunit hybridization of the isoenzymes reveal differences in their structures. Compositional analysis of tomato and other Cu,Zn-superoxide dismutases suggest that: (i) chloroplastic enzymes are more closely related to each other than to their cytoplasmic counterparts from particular plants and (ii) the evolutionary rate of chloroplastic enzymes is lower than that of cytoplasmic ones.

Photosynthetic apparatus in chilling-sensitive plants. VII. Comparison of the effect of galactolipase treatment of chloroplasts and cold-dark storage of leaves on photosynthetic electron flow

1. Both galactolipase treatment of tomato chloroplasts and the cold and dark storage of leaves induce a large degradation of chloroplast monogalactosyl diacylglycerol and digalactosyl diacylglycerol as well as an accumulatwon of free fatty acids accompanied by the inhibition of Hill reaction activity with water as electron donor. All these changes are reversed upon illumination of the leaves. 2. Inhibition of diphenylcarbazide (DPC) leads to dichlorophenolindophenol (DCIP) activity by free fatty acids released following galactolipase treatment of chloroplasts isolated from either fresh or cold and dark-stored and illuminated leaves is almost completely reversed by either bovine serum albumin or Mn2+, while that in chloroplasts from the cold and dark-stored leaves is reversed by bovine serum albumin and Mn2+ only up to about 60 and 25%, respectively. 3. Fatty acids released during the treatment of chloroplasts with galactolipase affect the electron transport mainly in the same site as exogenous unsaturated fatty acids do, while those released due to endogenous galactolipase activity appear to affect also in the region damaged by either Tris washing of chloroplasts or the cold and dark treatment of leaves. 4. The loss of manganese from chloroplasts (Kaniuga, Z., Zabek, J. and Sochanowicz, B. (1978) Planta 144, 49-56) seems to be the main reason of cold and dark-induced inactivation of Hill reaction activity in chloroplasts of chilling-sensitive plants, while both the degradation of galactolipids and the accumulation of fatty acids are of secondary importance.

Galactolipase activity and free fatty acid levels in chloroplasts: Novel approach to characteristics of chilling sensitivity of plants

Both galactolipase activity and free fatty acid content are much higher in chloroplasts of chilling‐sensitive plants (bean, broad bean, tomato and maize) than in those of chilling‐resistant ones (spinach, pea and lettuce). However, monogalactosyldiacylglycerol and digalactosyldiacylglycerol content in chloroplasts of these two groups of plants relates neither to free fatty acid levels nor galactolipase activity. It seems that chloroplast galactolipase activity and free fatty acid level may be useful biochemical parameters discriminating chilling‐sensitive from chilling‐resistant plants.

Photosynthetic apparatus of chilling-sensitive plants. X. Relationship between superoxide dismutase activity and photoperoxidation of chloroplast lipids☆

Abstract (1) The rate of photoperoxidation of chloroplast lipids, as measured by malondialdehyde formation following the illumination of either leaves or chloroplast preparations, is found to be approx. 2-fold higher in chloroplasts from both cold- and dark-stored as well as stored and illuminated tomato leaves than in those from fresh leaves. (2) Enhanced lipid photoperoxidation can also be observed in chloroplasts from fresh leaves treated with cyanide as well as in superoxide dismutase-depleted chloroplasts following washing with Tris or Hepes. (3) Cyanide-sensitive superoxide dismutase activity is not detected in chloroplasts isolated from cold- and dark-stored leaves. Their illumination does not reactivate the enzyme activity. (4) On the basis of these observations, it is concluded that inactivation of chloroplast cyanide-sensitive superoxide dismutase due to cold and dark treatment of leaves, rather than diminished electron transport, is responsible for accelerated chloroplast lipid photoperoxidation.

Photosynthetic apparatus of chilling-sensitive plants. XI. Reversibility by light of cold- and dark-induced inactivation of cyanide-sensitive superoxide dismutase activity in tomato leaf chloroplasts☆

(1) The inactivation of cyanide-sensitive, copper- and zinc-containing superoxide dismutase activity in chloroplasts following cold and dark storage of both detached leaves and growing tomato plants is accompanied by a decrease in copper and zinc content in both chloroplast preparations and butanol extracts of the enzyme. In contrast, this treatment of chloroplast preparations affects neither superoxide dismutase activity nor copper and zinc content. (2) Copper- and zinc-containing superoxide dismutase is not reactivated following the 2–3 h illumination of cold- and dark-stored detached leaves. However, prolonged illumination of growing seedlings results in the restoration of both the enzyme activity and copper and zinc content in chloroplasts. (3) The data suggest that the dissociation of copper, and probably of zinc, from the enzyme during cold and dark treatment of either detached leaves or growing plants and reincorporation of the metals following the illumination of intact plants are responsible for the reversible inactivation of chloroplast cyanide-sensitive superoxide dismutase of chilling-sensitive plants.

Fatty-acid-induced release of manganes from chloroplasts☆

Abstract The effect of both endogenous and exogenous unsaturated free fatty acids on manganese release from chloroplasts of chill-resistant (spinach) and chill-sensitive (tomato, bean) plants was studied. The level of endogenous free fatty acids increased 2–3-fold during cold and dark storage of leaves of chill-sensitive plants and was accompanied by depletion of about 60% of total chloroplast manganese content. Similar effects were observed when accumulation of free fatty acids in chloroplasts was achieved by storage of growing tomato plants for a few days in the dark at room temperature. In contrast, the cold and dark treatment of leaves of chill-resistant plant (spinach) affected neither free fatty acid, manganese levels nor Hill-reaction activity in chloroplasts. Incubation of chloroplasts of both chill-sensitive and chill-resistant plants with bean leaf galactolipase resulted in an accumulation of free fatty acids and a release of approx. 60% of total manganese content. The same amount of total manganese content was released following 3 h incubation of chloroplasts with linolenic acid at fatty acid/chlorophyll ratio ( w w , 2:1–10:1). The efficiency of C18 unsaturated fatty acids/linolenic, linoleic, oleic on manganese release from chloroplasts was established in decreasing order C18:3 >C18:2 >C18:1. The results indicate that the inhibitory effect of both endogenous and exogenous fatty acids on Hill reaction depends on the release from chloroplasts of functionally active, loosely bound manganese. Thus, similarly to both Tris and hydroxylamine treatments of chloroplasts, the incubation of chloroplast preparations with unsaturated fatty acids may be a useful tool for manganese depletion of chloroplasts.

On the nature of endogenous substrate in rat-liver mitochondria

Abstract 1. Oxidation and phosphorylation coupled with endogenous substrate oxidation was studied in comparison with fatty-acid oxidation. Similar sensitivities to arsenite and malonate were observed for the oxidation of both endogenous substrate and fatty acid. The P:O ratio for the oxidation of endogenous substrate and fatty acids was found to be approx. 2.5. Endogenous substrate oxidation as well as fatty-acid oxidation was stimulated by NAD + . 2. The endogenous substrate was activated by both systems described by Van den Bergh for the activation of fatty acids. The carnitine-stimulated oxidation of endogenous substrate was observed only in the dinitrophenol-sensitive system. 3. The results presented indicate that fatty acids constitute the endogenous substrate in rat-liver mitochondria.

The Fatty Acid Composition of Phosphatidylglycerol and Sulfoquinovosyldiacylglycerol of Zea mays Genotypes Differing in Chilling Susceptibility

Summary The content and fatty acid composition of four major chloroplast lipids, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglycerol and sulfoquinovosyldiacylglycerol, have been measured in the intact leaves of maize inbred lines differing in chilling susceptibility. Visual assessment of chilling injury symptoms during post-chilling recovery of seedlings indicated that both lines CM 7 and Co 151 were more chilling susceptible than lines S 215 and EP l, exhibiting greater chilling tolerance. Fatty acid composition of chloroplast polar lipids was very similar and some small deviation in the content of individual fatty acids did not affect uniformity of the general pattern. In all four lines of maize the content of height-temperature melting fraction of phosphatidylglycerol (htm-PG) and double bond index of polar lipids were equal, while fatty acid composition of polar lipids was very similar despite of small deviations in the content of individual fatty acid. Although the level of the high-temperature melting fraction of sulfoquinovosyldiacylglycerol (htm-SQDG) was lower in CS and CT lines Co 151 and S 215, respectively, than in CS and CT lines CM 7 and EP 1, respectively, this difference could not be related to their chilling sensitivity. All of these data do not support the concepts that the level of htm-PG alone or in combination with low-temperature-melting lipid would differentiate the chilling sensitivity of maize inbred lines. It is likely that some other factors are responsible for differential injury symptoms observed in chilled maize seedlings upon recovery. Discrepancies between the level of htm-PG and chilling sensitivity in several other species as well as various effects of acclimation on the level of htm-PG and temperature of phase transition are discussed.

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin I. Reversal of the inhibition by diethyl ether extraction

Abstract 1. Reversal of antimycin inhibition of succinate oxidation was studied in diethyl ether-extracted heart-muscle preparation and rat-liver mitochondria, respectively. 2. The activity of succinate oxidase in ether-extracted preparations which were not treated with antimycin was completely restored after addition of cytochrome c . Under the same conditions the activity of succinate cytochrome c reductase was increased 2-3-fold. 3. Antimycin-inhibited activity of succinate oxidase and that of succinate cytochrome c reductase was completely restored by extraction of the preparations with diethyl ether providing that soluble cytochrome c was added. 4. Sensitivity of the heart-muscle preparations to antimycin was not affected by extraction with diethyl ether. 5. Antimycin which had been isolated from antimycin-treated preparations was not in any way modified and its capacity to inhibit succinate oxidation was preserved. 6. It was found that a complete reactivation of antimycin-inhibited succinate oxidase could be achieved by ether extraction even when some residual antimycin remained in the preparations. This residual antimycin could be removed by extraction with diethyl ether-ethanol solvent mixture but not by repeated extractions with diethyl ether alone. 7. The results suggest that the reversal of antimycin inhibition by diethyl ether extraction of inhibited preparation was due to the removal of the antibiotic from the site which was responsible for the inhibition of the electron transport chain. However, some bound inhibitor remained at another site which did not directly affect the inhibition.

DEGRADATION OF LEAF POLAR LIPIDS DURING CHILLING AND POST-CHILLING REWARMING OF ZEA MAYS GENOTYPES REFLECTS DIFFERENCES IN THEIR RESPONSE TO CHILLING STRESS. THE ROLE OF GALACTOLIPASE

Degradation of leaf polar lipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)] and chlorophyll (Chl) were studied in four Zea mays genotypes differing in chilling susceptibility following dark chilling and post-chilling rewarming at original growth conditions. Assessment of visual chilling injury symptoms during post-chilling rewarming differentiated maize inbred lines into chiling-sensitive (CS) CM7 and Co151 lines and chillingtolerant (CT) S215 and EP1 lines. Severity of chilling injury in CS and CT inbreeds were correlated with the extent of Chl and polar lipids degradation.Chilling for either 4 or 6 days followed by 4 days of rewarming caused more extensive degradation of total polar lipids content in CS than in CT lines. MGDG decreased mostly during chilling whereas DGDG dropped during rewarming only. Chl content was not affected during chilling but its large decrease, greater in CS than in CT lines, was observed upon rewarming. Extent of polar lipids breakdown in CS and CT inbreeds during chilling and post-chilling rewarming is correlated with galactolipase activity in chloroplasts (Kaniuga et al., 1998) and visual assessment of chilling injury.In view of the data it is likely that contribution of galactolipase activity induced during low-temperature stress of CS plants is an important factor responsible for thylakoid lipid degradation and development of chilling injury as postulated previously (Kaniuga 1997). It is suggested that genetically engineered reduction of galactolipase activity or elimination of the factors(s) involved in induction/stimulation of its activity during chilling might increase tolerance of CS species to chilling stress.