Chilling response of plants: importance of galactolipase, free fatty acids and free radicals.

The chilling response of plants is complex and based on the interplay of two important metabolic processes--lipolytic degradation of membrane lipids and a set of oxidative reactions leading to lipid peroxidation and membrane damage evoked in chilling-sensitive (CS) plants subjected to low temperature and light. The effects of chilling of detached leaves and intact plants differ and are often neglected during experiments. In closely-related species, the activity of several constitutive enzymes (i.e. superoxide dismutase, ascorbate peroxidase and glutathione reductase) appears to be higher in chilling-tolerant (CT) than in CS species; while in several native, closely-related CS species, lipid acyl hydrolase (galactolipase) activity is higher than in CT species. Moreover, in chilling-insensitive (CI) plants, galactolipase activity is very low and is neither activated by detachment of leaves nor under stress conditions in growing plants. Dark and low-temperature treatments of detached leaves of CS species and post-chilling recovery of growing plants in the light activate galactolipase, which is responsible for the release of free fatty acids (FFA), the main substrates of peroxidation by lipoxygenase and free radicals. In several CS species, increased galactolipase activity is an important factor contributing to chilling susceptibility. Thus, it seems likely that enhancement of chilling tolerance may be achieved by genetically suppressing galactolipase in order to reduce both the degradation of chloroplast lipids and the level of released FFA, and thereby avoiding the deleterious action of their peroxidation products on plant tissues.

Galactolipase activity but not the level of high-melting-point phosphatidylglycerol is related to chilling tolerance in differentially sensitive Zea mays inbred lines.

Galactolipase activity, the level of high-melting-point phosphatidylglycerol (HMP-PG) as well as degradation of lipids during chilling and rewarming were studied in seedlings of maize inbred lines with different chilling responses. In aged chloroplasts of chilling-sensitive (CS) lines, galactolipase activity was considerably higher than that determined in aged chloroplasts isolated from chilling-tolerant (CT) ones. Chilling of seedlings at 5 °C for 6 days induced neither loss of chlorophyll content nor visible changes in the leaves, while a slight decline in total acyl lipid content by about 15.5% and 12.5% in CS and CT lines, respectively, was observed. Among total acyl lipids, only monogalactosyldiacylglycerol (MGDG) levels were decreased significantly upon chilling. Following return to the original growth conditions for 4 days, visible chilling injury in seedlings as well as essential differences in the decrease in total acyl lipids by about 53% and 20% in CS and CT lines, respectively, were found. These changes were accompanied by more extensive degradation of MGDG, digalactosyldiacylglycerol and phosphatidylglycerol in CS than in CT lines. As the levels of HMP-PG in fresh leaves were the same in all four lines of maize, it seems that galactolipase activity and not the level of HMP-PG is related to chilling response in maize.

Galactolipase and chilling sensitivity of plants.

Galactolipase is a lipid acyl hydrolase (EC 3.1.1.26) acting predominantly on galactolipids which constitute up to 80% of total acyl lipids in chloroplast membrane. Evidence is presented on the involvement of this enzyme in plant response to chilling via degradation of membrane lipids and the increase of free fatty acids, associated with reduced oxygen evolution in the Hill reaction. The occurrence of two pools of fatty acids has been hypothesized. Analysis of numerous plant species showed higher galactolipase activity in the chilling-sensitive than in the chilling-resistant plants. Differences in the pH-dependence curve and in the response to detergents of galactolipases from these two groups of plants suggest heterogeneity of the enzyme. Referring to the hypothesis concerning the role of high melting-point fatty acids of phosphatidylglicerol molecular species in chilling sensitivity the data are presented against generalization of this hypothesis.

Discrimination between chilling-sensitive and chilling-resistant plants based on measurements of free fatty acid accumulation and inactivation of oxygen evolution in aged chloroplasts.

The effect of aging of isolated chloroplasts of two chilling-sensitive (CS) and three chilling-resistant (CR) plants on the inactivation of oxygen evolution and accumulation of free fatty acids (FFA) was studied at 30 degrees C, pH 5.5 or 7.0, in the absence or presence of either sorbitol or NaCl. Considerable accumulation of FFA in aged chloroplasts of CS plants: bean and maize line F7-RpIII was accompanied by a marked inactivation of oxygen evolution. This relation was not, however, found in chloroplasts of CR species: pea, wheat and maize line EP1-RpI, in which the accumulation of FFA upon aging was very low whereas the decline of the rate of oxygen evolution was pronounced. In contrast to changes observed at pH 5.5, the inactivation of oxygen evolution in chloroplasts of CR species aged at pH 7.0 was dependent on the composition of the medium, especially in wheat chloroplasts. Thus, for the evaluation of chilling sensitivity based on the measurements of oxygen evolution activity solely, either aging of chloroplasts at pH 5.5 or possibly at pH 7.0 with NaCl included into the incubation medium may be recommended. It is concluded that determination of both the extent of FFA accumulation and inactivation of oxygen evolution in aged chloroplasts might be applied as chilling tolerance indexes.

Comparison of galactolipase activity and free fatty acid levels in chloroplasts of chill-sensitive and chill-resistant plants.

Galactolipase activity in chloroplasts of several chill-resistant plants was found to be very low [0.02-0.13 mumol free fatty acid (FFA) liberated min-1 mg protein-1] or not detected. The same phenomenon was observed for soybean and members of the Cucurbitaceae such as cucumber, pumpkin, melon and squash. Since, following cold storage of cucumber leaves, the levels of monogalactosyl-diacylglycerol and digalactosyl-diacylglycerol in chloroplasts decrease while those of FFA accumulate it seems likely that in these typical chill-sensitive plants galactolipase is present but inactivated during isolation procedure. The low galactolipase activity in chloroplasts was accompanied by a relatively low FFA content ranging from 0.05 mumol to 0.30 mumol FFA mg chlorophyll (Chl)-1. However, both pea and horse bean chloroplasts (with low galactolipase activity) exhibit about 0.45 mumol FFA mg Chl-1. Elevated galactolipase activity was observed in chloroplasts of most chill-sensitive species (ranging from 0.31 mumol to 1.32 mumol FFA liberated min-1 mg protein-1) as well as in chloroplasts from broad bean, a member of a chill-resistant species (1.26 mumol FFA liberated min-1 mg protein-1). In addition in this latter group of plants FFA level in chloroplasts often did not fit the galactolipase activity. The results suggest that there exists a tendency for chilling tolerance of plants to decrease both galactolipase activity and FFA level. However, in some plant species with elevated galactolipase activity the chloroplast FFA level does not correlate with enzyme activity.

Isolation and characterization of an iron-containing superoxide dismutase from tomato leaves, Lycopersicon esculentum.

A cyanide-insensitive superoxide dismutase was purified from tomato leaves (Lycopersicon esculentum, Mill., var. Venture) to apparent homogeneity. The enzyme had twofold higher specific activity (about 4000 standard units) than ferric superoxide dismutases purified from Brassica campestris [Salin, M. L. and Bridges, S. M. (1980) Arch. Biochem. Biophys. 201, 369-374] and Nuphar luteum [Salin, M.L. and Bridges, S. M. (1982) Plant Physiol. 69, 161-165]. The protein had a relative molecular mass of about 42000 and was composed of two equal subunits noncovalently joined. It was negatively charged (pI = 4.6) and contained about 1.45 mol Fe/mol dimer and negligible amounts of Mn, Cu and Zn. Absorption spectrum and sensitivity to NaN3, H2O2 and temperature are also reminiscent of other ferric superoxide dismutases. Comparison of amino acid composition indicated, however, a closer relationship to the Mn-containing enzymes rather than to other Fe-containing superoxide dismutases. Two possible ways of Fe-containing superoxide dismutase acquisition by vascular plants were suggested.

Photosynthetic apparatus of chilling-sensitive plants. IX. The involvement of alpha-tocopherol in the electron transport chain and the anti-oxidizing system in chloroplasts of tomato leaves.

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied. 2. The cold and dark storage of leaves results in a loss of chloroplast alpha- and gamma-tocopherols of about 30-40% accompanied by an increase in chloroplast delta-tocopherol of about 40%. On illumination of stored leaves, an elevation of alpha- and gamma-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst delta-tocopherol content is not affected. 3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total alpha-tocopherol is functionally active in the electron transport of Photosystem II between the diphenylcarbazide (DPC) donation site and the inhibition site of DBMIB. 4. A small amount of alpha-tocopherol quinone (about 10% of alpha-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast alpha-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous alpha-tocopherol to alpha-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves. 5. The primary product ('alpha-tocopheroxide') formed during the alpha-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-alpha-tocopheron. 6. Exogenous alpha-tocopherol inhibits the lipid photoperoxidation by about 40-50% in chloroplasts from all three kinds of tomato leaf. 7. The results seem to suggest that chloroplast alpha-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.

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.

Photosynthetic apparatus in chilling-sensitive plants : V. Changes in protein fractions of leaves and isolated chloroplasts following cod and dark storage and illumination of tomato leaves.

Proteins of fresh, cold and dark-stored and illuminated tomato leaves were fractionated by SDS electrophoresis. The total soluble proteins extracted from fresh leaves were separated into 5 main fractions with MWs of 54,000, 45,000, 32,000, 23,000 and 14,000. The cold and dark storage of the leaves causes a marked reduction mainly in the fraction with MW of 45,000 which increased with the illumination of the cold and dark-storaged leaves. The polypeptides with MWs of 54,000 and 14,000 (probably large and small subunits of ribulose, bisphosphate carboxylase) were stable under these conditions. In contrast, the polypeptides with MWs of 54,000 and 14,000 are decreased following the storage of tomato leaves in the dark at room temperature. Chloroplast soluble proteins were seperated by SDS electrophoresis into fractions with MWs of 64,000, 54,000, 20,000 and 14,000. The same fractions in similar proportions were observed in soluble-chloroplast proteins from fresh as well as coold and dark-stored and illuminated leaves. No drastic changes in structural polypeptides were observed following cold and dark-storage and illumination of the leaves. The results indicated that the main protein fraction, which degradated following cold and dark storage of tomato leaves and synthetized during illumination, is the fraction of cytoplasmic protein which in SDS electrophoresis gives polypeptides of about 45,000 MW. The fractions of chloroplast proteins were stable under such conditions.

Photosynthetic apparatus in chilling-sensitive plants : VI. Cold and dark-induced changes in chloroplast superoxide dismutase activity in relation to loosely-bound manganese content.

Both total and CN-insensitive superoxide dismutase (SOD) activities in relation to the level of manganese were studied in chloroplasts and digitonin subchloroplast particles obtained from fresh, cold and dark-stored, and illuminated leaves of Licopersicon esculentum, Mill. Following cold and dark treatment of detached leaves and intact plants, both total SOD activity and Mn content were greatly diminished while illumination of the leaves (2 h, 8,000 lx, 25° C) resulted in a large increase of Mn content and a partial restoration, of SOD activity. When growing plants were kept for 3-4 days in the dark, either at 0° C or 25° C, a decrease in chloroplast SOD activity, Mn content, and Hill reaction was also observed. However, with a prolonged dark treatment either at 0° C or at 25° C, there was a slight increase in chloroplast Mn content accompanied by an enhancement of both SOD and Hill reaction activities, suggesting that even in the dark some translocation of Mn from cytosol into chloroplasts may occur. Hypotonically treated chloroplasts and digitonin subchloroplast particles contained almost exclusively CN-insensitive, SOD activity, while Tris-washed chloroplast preparations were completely deprived of SOD activity. All these data seem to indicate that a chloroplast loosely-bound Mn pool is involved in CN-insensitive SOD activity.

Photosynthetic apparatus in chilling-sensitive plants : IV. Changes in ATP and protein levels in cold and dark stored and illuminated tomato leaves in relation to Hill reaction activity.

Changes in the levels of both ATP and protein in relation to Hill reaction activity following cold and dark storage and illumination of leaves of Lycopersicon esculentum Mill. were studied. Loss of Hill reaction activity observed during cold and dark storage of leaves for 3-4 days was accompanied by about 50% decrease of both ATP and protein levels while the content of chlorophyll was not affected Illumination of cold and dark stored leaves (8000 lx for 2 h) resulted in almost a complete restoration of both ATP and protein levels as well as Hill reaction activity. The latter process proceeded, however with different kinetics than the former ones. The rate of Hill reaction activity increase very rapidly from the beginning of illumination while the ATP level diminished during the first hour of illumination. In addition there was a lag in the increases in protein content. By about two hours of illumination all these processes reached the maximum values. Following illumination of leaf dises stored in the cold and dark in the presence of either cycloheximide or DCMU, both ATP and proteins levels as well as Hill reaction activity were greatly diminished. These data seem to suggest that the lack of reactivation of Hill reaction activity in the presence of these two inhibitors is due to inhibition of ATP synthesis required primarily for manganese reincorporation into the thylakoid membrane and theraby restoration of Hill reaction activity (Kaniuga, Zabek and Sochanowicz, Planta 1978b). Contribution of cytoplasmic protein synthesis in this process appears to be of secondary importance, although the inactivation and reactivation of electron transport are accompanied by a large loss (as high as 50%) and the restoration of the initial protein content in leaves following illumination.

Photosynthetic apparatus in chilling-sensitive plants : III. Contribution of loosely bound manganese to the mechanism of reversible inactivation of hill reaction activity following cold and dark storage and illumination of Leaves.

The levels of both tightly and loosely bound Mn in chloroplasts from fresh, cold and dark stored as well as illuminated leaves of Lycopersicon esculentum Mill. were studied in relation to Hill reaction activity. The tightly bound Mn pool represents one third of the total Mn content in chloroplasts isolated from the fresh leaves, and its level does not change following cold storage and illumination of leaves. Upon cold and dark storage of leaves the loss from the chloroplasts of about 40%-50% of the total amount of Mn is accompanied by an almost complete inactivation of the Hill reactions, as studied with water as an electron donor, as well as by the appearance of an EPR signal characteristic of free Mn(2+) ions. Following illumination of such leaves, the restroration of Hill reaction activity is accompanied by an increase in the total Mn content in chloroplasts of up to 70%-80% of the Mn level measured in the fresh leaves and by disappearance of the EPR signal. In contrast, aging in the cold of isolated chloroplasts does not affect their Mn content. The addition of manganese does not result in the restoration of Hill reaction activity in chloroplasts from cold stored leaves but causes a restoration of this activity inhibited by linolenic acid. The data suggest that the loosely bound Mn pool (extractable with Tris) can be differentiated into two fractions: (1) one functionally inactive in electron transport and (2) one responsible for restoration of Hill reaction activity. Mn of the latter fraction (about 45% of the total Mn content) probably originates from the free Mn ions present in the interior of the chloroplasts following the cold and dark storage of leaves and from Mn reincorporated into chloroplasts from the cytoplasm. Incorporation of Mn from both these sources into thylakoid membrane to form a functionally active, loosely bound Mn pool proceeds during the illumination of leaves and results in the restoration of Hill reaction activity inhibited following the storage of leaves in dark and cold.

Photosynthetic apparatus in chilling-sensitive plants : I. Reactivation of hill reaction activity inhibited on the cold and dark storage of detached leaves and intact plants.

Chloroplast isolated from the detached leaves of chilling-sensitive plants-Phaseolus vulgaris L., Cucumis sativus L., and Lycopersicon esculentum Mill.-stored in the cold for 2-4 days in the dark exhibit an almost complete loss of Hill reaction activity, which on illumination of leaves is restored to almost the original level. In contrast, illumination of either chloroplast suspensions or homogenates from leaves stored in the cold in the dark does not cause restoration of electron transport. Cold and dark storage of leaves of chilling-sensitive plants affects the electron transport before the site of electron donation by diphenylcarbazide and results in an increased sensitivity of the Hill reaction of isolated chloroplasts to exogenous linolenic acid. Illumination of leaves reverses these processes. When tomato plants are exposed to 0°C in intermittent light, Hill reaction activity is not affected while dark storage either at 0°C or 25°C results in a significant decrease of Hill reaction activity after 2-3 days followed by the restoration of electron transport to the original level after 1 or 2 days of the prolonged dark storage of plants. When tomato plants are stored either at 0°C in intermittent light, at 0°C in dark, or at 25°C in dark the sensitivity of the Hill reaction to exogenous linolenic acid remains increased despite a significant restoration of this activity. In conclusion, both darkness and the detachment of leaves from the plant are more effective than cold treatment in damaging photosystem II whereas both light and intact structure of the cell are required for restoration of Hill reaction activity in chloroplasts following cold and dark storage of detached leaves.

Photosynthetic apparatus in chilling-sensitive plants : II. Changes in free fatty acid composition and photoperoxidation in chloroplasts following cold storage and illumination of leaves in relation to Hill reaction activity.

The composition of free fatty acids (FFA) in relation to Hill reaction activity and photoperoxidation of lipids was studied in chloroplasts isolated from fresh, cold and dark-stored as well as illuminated leaves of Lycopersicon esculentum Mill., Phaseolus vulgaris L. and Cucumis sativus L. Following the cold and dark-storage of leaves the loss of Hill reaction activity is accompanied by approximately a 5-fold increase in the amount of FFA and by an increase in the percentage of unsaturated FFA, particularly that of linolenic acid. Illumination of the cold- and dark-stored leaves restores both Hill reaction activity and the content and composition of chloroplast FFA. Following the second and third cycles of cold storage and illumination of leaves the percentage of unsaturated fatty acids in chloroplasts increases while that of saturated ones decreases despite of the significant restoration of Hill reaction activity. Since the illumination of cold-stored leaves results in peroxidation of inhibitory fatty acids it seems likely that this phenomenon could, at least partially, be responsible for the restoration of Hill reaction activity. Inhibition of Hill reaction activity by exogenous linolenic acid in chloroplasts of fresh, cold-stored as well as cold-stored and illuminated leaves could be reversed following the incubation of chloroplast suspension with BSA, however only to a value measured in the absence of unsaturated fatty acid. All these results indicate that the inhibition of Hill reaction activity due to the cold and dark storage of leaves is caused by both inhibitory FFA released from chloroplast lipids as well as by damage to the thylakoid structure affecting the electron transport within photosystem II.