Freezing and high temperature thresholds of photosystem 2 compared to ice nucleation, frost and heat damage in evergreen subalpine plants

Freezing and high temperature thresholds of photosystem 2 (PS2), ice formation and frost and heat damage were measured in leaves of evergreen subalpine plants under conditions of naturally low (winter) to high (summer) PS2 efficiencies (F_V/F_M). The temperature‐dependent change in basic Chl fluorescence (F₀) (T‐F₀) technique that is usually used to assess the high temperature threshold of PS2 in a new approach was applied to test freezing temperature thresholds of PS2. T‐F₀ curves (+5 °C to ?10 °C at 2 K h^?1) revealed a significant, sudden increase in F₀ on extracellular ice formation (?4.0 or ?5.5 °C). The rise in F₀ was recorded 0.3–0.6 K below ice nucleation (10–20 min later) and was produced by freeze dehydration of cells. The rise in F₀ was not caused by frost damage, as during winter LT₅₀ was lower than ?27 °C and not by formation of ice on the leaf surface. Hence, F₀ measurements during freezing are a useful tool to distinguish between surface ice and extracellular ice inside the leaf tissue which cannot be differentiated by other ice‐detecting methods. PS2 efficiency significantly affected the shape of the high temperature T‐F₀ curves (20–65 °C at 1 K min^?1). Under F_V/F_M >0.6, two F₀ maxima were recorded. The fast rise phase to the first F₀ maximum corresponded with tissue heat damage (LT₅₀: 46.9–54.3 °C). The second F₀ maximum occurred at leaf temperatures between 55 and 60 °C. Under F_V/F_M <0.2 only, the second F₀ maximum was detectable. Lack of awareness of the missing F₀ maximum would lead to an overestimation of the PS2 high temperature threshold by >10 K; hence, under low F_V/F_M, it cannot be determined by the T‐F₀ technique.     

Effects of prolonged freezing stress on the photosynthetic apparatus of moderately hardy leaves as assayed by chlorophyll fluorescence kinetics

Abstract Moderately frost-hardy leaves of the wintergreen broadleaf woody shrubs Pyracantha coccinea and Ligustrum ovalifolium and the winter annual herb Spinacia oleracea were subjected to extended freezing stress up to 15 d at temperatures 2–8°C above the mean lethal temperature (LT₅₀). After thawing, the fast kinetics of in vivo chlorophyll fluorescence of photosystem II (PSII) and the potential of linear photosynthetic electron transport of isolated thylakoid membranes was measured at room temperature. The lower the minimum freezing temperature and the longer the time of exposure, the greater was the suppression of the fluorescence signals of the leaves and decrease of the electron transport capacity of the thylakoid membranes. The pattern of inactivation of PSII -mediated electron flow, i.e. inhibition of photoreaction to photochemistry and/or electron donation to the photochemical reaction, during long-term freezing at temperatures somewhat above the LT₅₀ of the leaves was similar to that observed earlier after relatively brief exposure of leaves and isolated thylakoid membranes to more severe freezing stress. As injury occurred during freezing in complete darkness, it is likely that prolonged winter stress under natural environmental conditions causes changes in the photosynthetic apparatus of moderately hardy leaves which are not due to photoinhibition.     

Application of trehalose ameliorates heat stress and promotes recovery of winter wheat seedlings

Trehalose was supplied to wheat (Triticum aestivum L.) seedlings just before a high temperature (40 °C) treatment and some physiological parameters were measured during the heat stress and recovery. The application of trehalose decreased the net photosynthetic rate (P_N) of wheat seedlings under the heat stress, but to a small extent increased the dry mass (DM) and leaf water content (LWC) after recovery from the heat stress. The trehalose-induced decrease in PN under the heat stress was not associated with a stomatal response. The heat stress slightly decreased the maximal efficiency of photosystem II (PS II) photochemistry (the variable to maximum chlorophyll a fluorescence ratio, F_v/F_m) similarly in the trehalose treated or non-treated plants. Under the heat stress, the actual efficiency of PS II photochemistry (Φ_PSII) and the efficiency of excitation energy capture by open reaction centers (F_v′/F_m′) were lower in the trehalose-pretreated seedlings, whereas they were higher after the recovery. The patterns of changes in nonphotochemical quenching (NPQ) were contrary to those of ?_{PS II} and F_v′/F_m′. The chlorophyll content was lower, whereas the β-carotene content and the degree of de-epoxidation (DEPS) of xanthophyll cycle pigments were higher in the trehalose-pretreated wheat seedlings under the heat stress. These results suggest that exogenous trehalose partially promotes recovery of wheat by the increase of NPQ, β-carotene content, and DEPS.     

Effects of dual stress (high salt and high temperature) on the photochemical efficiency of wheat leaves (Triticum aestivum)

In this study, we have focused on those components of Photosystem (PS) II which are significantly affected by dual stress (high salt and temperature) on wheat as measured by Plant Efficiency Analyser (PEA). It was observed that some of the chlorophyll a fluorescence parameters were temperature dominated, while some other parameters were salt dominated. We have also observed additive effects for parameters like antenna size heterogeneity. An important observation was that in high temperature alone, the K-step was observed at 40 °C, while in case of dual stress, the K-step was observed at 45 °C, while the Chl a fluorescence transient of 40 °C?+?0.5 M?NaCl was quite similar to 35 °C transient curve. In the presence of salt, K-step was observed at higher temperature suggesting a protection of OEC by salt. Plants are under dual stress, but effect of temperature stress is less severe in presence of salt stress. Thus, we can say that salt stress caused partial prevention from high temperature stress but it did not cause complete protection of PS II.     

Induced expression of the class II chitinase gene during cold acclimation and dehydration of bermudagrass (Cynodon sp.)

Bermudagrass cultivars vary greatly in their ability to survive freezing temperatures as a result of a differential ability to cold acclimate (CA) at temperatures slightly above 0°C. Little information exists on the genetic and physiological mechanisms associated with the cold acclimation process in bermudagrass. Experiments were conducted to study the changes in chitinase gene expression during cold acclimation of freeze-tolerant bermudagrass cultivars. A chitinase gene (CynCHT1) was isolated from ’Midiron’ bermudagrass. Because the hydrophilic protein putatively encoded by the gene lacked an N-terminal cysteine-rich domain and a hydrophobic C-terminal extension, it was classified a class II chitinase. The expression patterns of this and related chitinase genes in response to CA, drought, and ABA were investigated in freeze-tolerant ’MSU’ (LT₅₀=?11°C), Midiron (LT₅₀=?10°C) and ’Uganda’ (LT₅₀=?8°C) bermudagrasses. Northern-blot analysis indicated expression in the crown tissues induced by CA at 8°C/2°C day/night temperature cycles. Induction of gene expression was evident in tissues sampled at 2 and 28 days after initiating CA. Expression after 2-days de-acclimation at 28°C/24°C was similar to control levels. Significantly higher levels of CA-induced chitinase gene expression were observed in MSU and Midiron, compared to Uganda. Similar expression patterns were observed among the cultivars in responses to drought and ABA. These results suggest that chitinases have important roles in bermudagrass response to low temperature and dehydration stresses.     

Cold-induced changes of enzymes,proline, carbohydrates,and chlorophyll in wheat

Quantitative changes in total leaf soluble proteins, proline, carbohydrate content, chlorophyll fluorescence, guaiacol peroxidase (POD) and catalase (CAT) activities were determined in a less cold-hardy (LCH) spring cv. Kohdasht (LT₅₀ = −6°C), a semi cold-hardy (SCH) facultative cv. Azar 2 (LT₅₀ = −15°C), and a cold-hardy (CH) winter cv. Norstar (LT₅₀ = −26°C) of wheat (Triticum aestivum L.) exposed to 4°C for 9 weeks. Seedlings were grown in a controlled growth room for 14 days at 20°C and then transferred to 4°C (experimental day 0) for 63 days (cold treatment); otherwise they were maintained continuously at 20°C (control treatment). The samples were harvested 0, 2, 21, 28, 42, and 63 days after exposure to 4°C. The results showed significant low temperature (LT)-induced accumulation of total soluble proteins, proline, and carbohydrates and elevation in activities of CAT and POD in leaves of SCH and CH winter cultivars rather than in LCH spring cultivar. In contrast, the chlorophyll fluorescence (F _v/F _m) declined during LT treatment irrespective of cultivar. The results suggest that developmental traits such as vernalization requirement of wheat affects on cold-tolerance expression system of plants.     

Freeze avoidance: a dehydrating moss gathers no ice

Using cryo‐SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water‐filled hydroid cells cavitated and were embolized at ?4 °C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ～20% of their original volume at a nadir temperature of ?20 °C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from ?20 °C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ～?12 °C while desiccated moss showed no evidence of freezing with lowering of nadir temperature to ?20 °C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.     

Effects of high temperature on photosynthesis, chlorophyll fluorescence, chloroplast ultrastructure, and antioxidant activities in fingered citron

Effects of heat stress on the photosynthesis system and antioxidant activities in Fingered citron (Citrus medica var. sarcodactylis Swingle) were investigated. Two-year-old Fingered citron plants were exposed to different temperature (28, 35, 40, and 45°C) for 6 h; then the photosynthetic capacity, chlorophyll fluorescence, chloroplast ultrastructure, and antioxidant activities in the leaves were evaluated. Exposure to 40 and 45°C for 6 h resulted in a significant decrease in the photosynthetic rate (P _n), carboxylation efficiency (CE), the maximal photochemical efficiency of photosystem II, and the light-saturated photosynthetic rate, which were related to the reduction of CO₂ assimilation, inactivation of photosystem II and photosynthetic electron transport. Moreover, transmission electron microscopy showed chloroplast ultrastructural alterations, including their swelling, matrix zone expanding, and lamella structure loosening. Furthermore, heat stress, especially at 45°C, caused oxidative damage resulted from ROS accumulation in Fingered citron leaves accompanied by increases in activities of superoxide dismutase, peroxidase, and catalase. However, exposure to 35°C for 6 h or 40°C for 4 h had no significant influence on the photosynthetic capacity at all. The results suggest that Fingered citron plants show no heat injury when temperature is below 40°C.     

Evergreen alpine shrubs have high freezing resistance in spring,irrespective of snowmelt timing and exposure to frost: an investigation from the Snowy Mountains,Australia

Over winter, alpine plants are protected from low-temperature extremes by a blanket of snow. Climate change predictions indicate an overall reduction in snowpack and an earlier thaw; a situation which could expose the tips of shrubs which extend above the snowpack to freezing events in early spring, and cause foliar frost damage during the onset of physiological activity. We assessed the photosynthetic responses of freezing-damaged shrub leaves from an assay of freezing temperatures in the Snowy Mountains in south-eastern Australia, using chlorophyll fluorometery ex situ. We sampled leaves that were exposed early during the spring thaw and leaves that were buried in snow for up to two extra weeks, from four evergreen shrub species at monthly intervals following the period of snowmelt. Freezing resistance (estimated from LT₅₀) was poorest at the earliest spring sampling time, in both exposed above-snow and protected below-snow foliage in all species. Protected foliage in early spring had lower freezing resistance than exposed foliage, but not significantly so. By the third sampling time, freezing resistance was significantly better in the lower protected foliage (LT₅₀ of ? 14) compared with the upper exposed foliage (LT₅₀ of ? 10) in one species. Over the course of spring, freezing resistance improved significantly in all species, with LT₅₀ values of between ? 10 and ? 15 °C by the third sampling time, which is lower than the minimum air temperatures recorded at that time (> ? 5 °C). The results indicate that the dominant evergreen shrub species in this area may only be susceptible to freezing events very early in spring, before a period of frost-hardening occurs after snowmelt. Later in spring, these alpine shrubs appear frost hardy, thus further perpetuating the positive feedbacks surrounding shrub expansion in alpine areas.     

Effects of frost hardening, dehardening and freezing stress on in vivo chlorophyll fluorescence of seedlings of Scots pine (Pinus sylvestris L.)

Abstract. The kinetics of in vivo chlorophyll fluorescence of photosystem II (PS II) was measured at room temperature and 77 K during frost hardening of seedlings of Scots pine (Pinus sylvestris L.), and after exposure of frost-hardened shoots to sub-freezing temperatures. A more pronounced decrease in variable fluorescence yield for the upper exposed than for the lower shaded surface of the needles suggested that some photoinhibition occurred during prolonged frost hardening at 50 μmol photons m^?2 s^?1 and 4°C. Reversible inhibition of photosynthesis after exposure to sub-freezing temperatures was initially manifested as an increase of steady-state energy-dependent fluorescence quenching (q_E) and a reduction in the rate of O₂ evolution. Further inhibition after treatment at still lower temperatures caused a progressive decline of steady-state photochemical quenching (q_Q) and the rate of O₂ evolution, whereas q_E remained high. This implies an inactivation of enzymes in the photosynthetic carbon reduction cycle decreasing the consumption of ATP and NADPH, which is likely to cause an increase of membrane energization and a reduction of the primary electron acceptor (Q_A) of PS II. Alternatively, the changes in q_Q and q_E might be attributed to an inhibition of photophosphorylation. Severe, irreversible damage to photosynthesis resulted in a suppression of q_E and of variable fluorescence yield, probably because the photochemical efficiency of PS II was impaired. Changes in the fast fluorescence kinetics at room temperature after severe freezing damage were interpreted as an inhibition of the electron flow from Q_A to the plastoquinone pool. It is suggested that irreversible freezing injury to needles of frost-hardened P. sylvestris causes damage to the Q_B,-protein.     

Heat stress induces in leaves an increase of the minimum level of chlorophyll fluorescence, Fo: A time-resolved analysis

A time-resolved study of the effects of heat stress (23 to 50°C) on F_o level of chlorophyll fluorescence of leaves having different antenna content has been performed in order to elucidate the causes of heat induced increase of F_o in vivo. The multi-exponential deconvolution of the decays after a picosecond flash at F_o have shown that the best fit in both wild-type and the mutant chlorina F2 of barley leaves is obtained with three components in the temperature range utilized (100, 400 and 1200 ps at 23°C). In intermittent light greened pea leaves, a fourth long lifetime component (4 ns at 23°C) is needed. The comparison of the three types of leaves at 23°C shows that the content of the LHCII b complex does not affect the lifetimes of the two main components (100 and 400 ps) and affects their preexponential factors. This result suggests that in the PS II unit the exciton transfer from LHC IIb to the rest of the antenna is irreversible. The effects of heat stress on individual lifetime components, T_i, included several changes. Utilizing for PS II unit an extended ‘Reversible Radical Pair’ model, having three compartments, to interpret the variations of T_i and A_i induced by temperature increases, it can be inferred that heat determines: (i) an irreversible disconnection of a monor antenna complex which is not the LHC IIb complex, this effect is induced by temperatures higher than 40°C; (ii) a decrease of the quantum efficiency of Photosystem II photochemistry which is due to several effects: a decrease of the rate of charge separation, an increase of P⁺I^- recombination rate constant and a decrease of the stabilization of charges. These effects on Photosystem II photochemistry start to occur above 30°C and are partially reversible.     

Temperature-induced changes of malondialdehyde, heat-shock proteins in relation to chlorophyll fluorescence and photosynthesis in Conocarpus lancifolius (Engl.)

The effect of variable temperatures (10–50 °C) on photosynthesis and chlorophyll fluorescence in Conocarpus lancifolius was evaluated. Additionally, the ability of the species to synthesize heat-shock proteins (HSPs) to protect against high temperatures, and malondialdehyde (MDA) as a by-product of lipid peroxidation was investigated. Plants at 10 °C showed virtually no measurable growth, leaf discoloration and a few brown lesions, while high temperatures (40 and 50 °C) promoted growth and lateral branch development. Chlorophyll content index, photochemical efficiency (F _v/F _m) of PS II, electron transport rate and photosynthetic rate declined with decreasing temperature but increased significantly at higher temperatures. Heat-shock protein (HSP 70 kDa) was produced at temperatures 30–50 °C and an additional 90 kDa protein was also produced at 50 °C. Increase in the efficiency of excitation energy captured by the open PS II reaction centers (F _v/F _m) increased linearly (P ≤ 0.05) with the accumulation of HSP 70 at higher temperatures. However, at low temperatures the concentration of MDA increased significantly, indicating lipid peroxidation due to oxidative stress. The production and accumulation of HSP 70 and 90 kDa coupled with increased electron transport rate and photochemical efficiency can be used to assess survival, growth capacity and to some extent the tolerance of C. lancifolius to elevated temperatures.     

Apparent absence of viruses in most symptomless field-grown sweet potato in Uganda

Heat tolerance of groundnut (Arachis hypogaea L.) genotypes was evaluated by solute leakage and chlorophyll fluorescence techniques in heat-hardened and non-hardened plants. To determine the appropriate hardening treatment, 1-month-old plants of two groundnut genotypes, ICGV 86707 and Chico were conditioned at five combinations of hardening (37°C) and non-hardening (30°C) air temperatures over a 5-day period. Heat injury, was assessed through measurements of electrolyte leakage after stressing leaf discs to 55°C for 15 min. The relative injury was significantly influenced by the conditioning temperatures and by the temperature during 24 h prior to measurement if those involved non-hardening conditions. Relative injury and chlorophyll fluorescence were measured after stressing leaves of six genotypes at a range of temperatures between 49°C and 55°C. Significant genotype × hardening treatment interactions were observed in relative injury and chlorophyll fluorescence. Chico was susceptible to heat stress, the relative injury test identified ICGV 86707 as tolerant, and the chlorophyll fluorescence test identified ICGV 86707 as tolerant under hardened conditions and ICGV 87358 as tolerant when non-hardened. When expressed as percentage of control values, the relative injury and chlorophyll fluorescence measurements over the 49–53°C stress temperature range were strongly correlated. Chlorophyll concentrations were increased by hardening in all genotypes except Chico. In Chico, chl_b concentration was decreased and the chl_a/b ratio increased by hardening, and chlorophyll concentrations were correlated with chlorophyll fluorescence parameters. Chlorophyll concentration may therefore provide an alternative means of screening for heat tolerance.     

Cryoproective role of ribitol in Xanthoparmelia somloensis

Thalli of Xanthoparmelia somloensis with natural content of polyols (control) and polyol-free thalli (acetone-rinsed) were used to study ribitol effects at low temperatures. Thalli segments were cultivated in ribitol concentration of 32 or 50 mM for 168 h at temperatures +5, 0, and ?5 °C. The chlorophyll fluorescence parameters (potential yield of photochemical reactions in PS 2 (variable to maximum fluorescence ratio, F_v/F_m), effective quantum yield of photochemical reactions in PS 2 (Φ_PS2), and non-photochemical quenching (NPQ) were monitored in 24-h intervals using an imaging system. The effect of 32 mM ribitol on F_v/F_m and Φ_PS2 was apparent only at ?5 °C, however, the effect was seen throughout the whole exposure. Surprisingly, 50 mM ribitol concentration treatment led to a decrease in F_v/F_m and Φ_PS2 and to an increase in NPQ values at ?5 °C, while no change was observed at 0 °C and +5 °C. Acetone-rinsing caused decrease of F_v/F_m, Φ_PS2 and NPQ.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

Photosynthesis in cold acclimated leaves of plants with various degrees of freezing tolerance

The objective of this study was to compare the photosynthetic changes during cold acclimation in various plant types able to acquire different degrees of freezing tolerance. Four herbaceous and six woody plants were hardened under natural or artificial conditions and – after determination of their frost resistance (LT₅₀) – the net photosynthetic rate at an ambient CO₂ of 33 Pa (P_n33), the dependencies of P_n to light and to CO₂ and the room temperature chlorophyll a fluorescence were recorded under optimal conditions. Herbaceous plants acquired freezing tolerances to temperatures between ?10 and ?15°C when hardened at temperatures around 0°C. Most leaves fully developed prior to frost hardening exhibited typical symptoms of senescence after frost hardening. In non-senescing leaves P_n33 was reduced by 15 to 50% mainly due to a reduced stomatal conductance. After hardening at temperatures around ?10°C Brassica survived down to ?24°C, but P_n33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C P_n33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to ?36°C: P_n33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at ?10°C induced a tolerance of ?32 to n33 was almost totally blocked, but at 20/15°C it returned to the values of the plants hardened at 0°C within a few days. In woody plants disturbances were invariably localized in the chloroplasts. Thus, conifers, and especially Pinus cembra, can survive much lower temperatures than herbaceous plants and, at the same level of freezing tolerance, exhibit appreciably less restriction in relative P_n33.     

Photochemical heat-shock response in common bean leaves as affected by previous water deficit

The heat sensitivity of photochemical processes was evaluated in the common bean (Phaseolus vulgaris) cultivars A222, A320, and Carioca grown under well-watered conditions during the entire plant cycle (control treatment) or subjected to a temporal moderate water deficit at the preflowering stage (PWD). The responses of chlorophyll fluorescence to temperature were evaluated in leaf discs excised from control and PWD plants seven days after the complete recovery of plant shoot hydration. Heat treatment was done in the dark (5 min) at the ambient CO₂ concentration. Chlorophyll fluorescence was assessed under both dark and light conditions at 25, 35, and 45°C. In the dark, a decline of the potential quantum efficiency of photosystem II (PSII) and an increase in minimum chlorophyll fluorescence were observed in all genotypes at 45°C, but these responses were affected by PWD. In the light, the apparent electron transport rate and the effective quantum efficiency of PSII were reduced by heat stress (45°C), but no change due to PWD was demonstrated. Interestingly, only the A222 cultivar subjected to PWD showed a significant increase in nonphotochemical fluorescence quenching at 45°C. The common bean cultivars had different photochemical sensitivities to heat stress altered by a previous water deficit period. Increased thermal tolerance due to PWD was genotype-dependent and associated with an increase in potential quantum efficiency of PSII at high temperature. Under such conditions, the genotype responsive to PWD treatment enhanced its protective capacity against excessive light energy via increased nonphotochemical quenching.     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

Post rapid freezing growth of Antarctic strain of Heterococcus sp. monitored by cell viability and chlorophyll fluorescence

The soil microalgae of the genus Heterococcus are found in cold environments and have been reported for the terrestrial ecosystems of several Sub-Antarctic and Antarctic Islands. This study focused on resistance of Heterococcus sp. to sub-zero temperature. Heterococcus sp. was isolated from soil samples from James Ross Island, Antarctica. Culture of Heterococcus sp. grown in liquid medium were used to study ribitol effects at sub-zero temperatures on the species resistance to rapid freezing (RF, immersion of a sample into liquid nitrogen) and consequent cultivation on agar. Before the experiment, Heterococcus sp. was cultured in liquid medium for 11 months and then treated in ribitol concentrations of 32 or 50 mM for 2 h. Then, 1 ml samples were frozen to −196 °C in liquid nitrogen (day 0) and inoculated on BBM agar after thawing. Number of living and dead cells was evaluated and the cell viability (P_ν) was calculated repeatedly using the optical microscopy approach. The addition of ribitol caused a noticable increase in P_ν on days 9, 12, 14 (with a P_ν of 25–45% in ribitol-treated samples compared to 10% in the untreated control). In the following period (d 16–19), the positive effect of ribitol on P_ν was less pronounced but still statistically significant. To evaluate the negative effects of RF on chlorophyll fluorescence parameters, the potential yield of photochemical reactions in PS II (F_V/F_M), and the effective quantum yield of photochemical reactions in PS II (Ф_PSII) were measured immediately before and after RF. Consequently, F_V/F_M and Ф_PSII of agar inoculates were measured repeatedly for 30 d cultivation in 3 d interval. Both the 32 and the 50 mM addition of ribitol caused earlier detection of the parameters (d 16) compared to the control measurements (d 23) as well as reaching the maximum values of the chlorophyll fluorescence parameters earlier (d 23 in ribitol-treated samples compared to d 25 in control samples). Heterococcus sp. proved to be a species resistant to rapid freezing. The ability may help the species to survive in harsh Antarctic environments typified by rapid fluctuations in temperature that may bring a rapid freezing of the alga.