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Advances in horticultural science - 2000 - 1 - differential thermal analysis, supercooling and cell viability in organs of olea europaea at subzero temperatures

Differential thermal analysis, supercooling and cell
viability in organs of Olea europaea at subzero
P. Fiorino, S. Mancuso
Dipartimento di Ortoflorofrutticoltura, Università di Firenze, Via G. Donizetti 6, 50144 Firenze, Italy.
Key words: chilling tolerance, electrolyte leakage, freezing temperature, Olea europaea, supercooling, visual score, vital stain.
Abstract: Cold hardiness in different organs and tissues of four different olive (Olea europaea L.) cultivars
characterised in a previous work as different in frost tolerance, was assessed by four different methods (diffe-
rential thermal analysis, visual score, vital stain and electrolyte leakage) to determine which method is more
reliable for estimating freezing injury in olive. Results obtained with the different techniques consistently
agreed. Among the four different procedures utilised, DTA was by far the fastest, whereas the visual score
method the simplest, although not as quantitative as the other three methods. It would appear from the present
study that the order of sensitivity in the different organs of olive is secondary roots > primary roots > apical lea-
ves > basal leaves > shoots > vegetative buds. ‘Ascolana’ was the most chilling-tolerant variety, whereas
‘Coratina’ the most chilling-sensitive. The wide range of tolerance showed by olive (i.e. from –11.2 to –15.3°C
for the leaves) together with the recurrent danger of frost in many areas of cultivation, made this species an
ideal candidate to breeding for low-temperature-tolerant plants.

1. Introduction
ness in autumn and a decrease in spring (Weiser, 1970;Repo, 1992).
Temperature extremes are one of the most impor- Experiments with different tree species and prove- tant factors limiting plant distribution and producti- nances have shown that the changes in frost hardiness vity. For olive trees, low temperatures are more limi- at various stages during annual development are gene- ting than are high temperatures in both ecological and tically determined, as well as the minimum hardiness agronomic contexts. In particular, olive cultivars accli- level during the growing season (Sakai and Eiga, mated to high temperatures maintain 70-80% of their 1985; Sakai and Larcher, 1987; Toivonen et al., 1991).
photosynthetic rate at 40°C (Bongi et al., 1987) whe- Many woody angiosperm species survive winter reas the low temperature tolerance of most cultivars is temperatures by deep supercooling tissue solution to not particularly noteworthy, as they generally succumb temperatures as low as the homogeneous nucleation temperature of the aqueous solution (near -40°C for Olive plants exposed to low temperatures can sur- plant solution). The freezing of this fraction of water vive either by avoiding ice formation in their tissues can be observed as an exotherm during cooling at a (supercooling ability) or by developing a tolerance for constant rate by differential thermal analysis (Ishikawa, 1984; Mancuso, 2000). Supercooling as a Frost hardening is a genetically-controlled trait cold hardiness mechanism has been demonstrated in which is driven by three key environmental factors: various temperate plant tissues such as xylem ray temperature, photoperiod and water stress (Tumanov parenchyma, leaf buds and flower buds (George et al., and Krasavtsev, 1959; Christersson, 1978; Levitt 1974; Sakai, 1979; Hong and Sucoff, 1980). In other 1980). According to the prevalent theory, certain spe- words, supercooling is usually employed by limited cific genes are activated as a result of changes in these tissues (xylem, buds, seeds). Compared with the majo- factors. These changes induce a metabolic hardening rity of plants, olive seems rather particular as will be mechanism, which results in an increase in frost hardi- shown in the present paper that most of its organs,from leaf to roots, use supercooling as a mechanism ofcold hardiness.
Received for publication 20 February 2000.
2. Materials and Methods
ments per 4 hr and storing them at the desired tempe-ratures for one day. Injury was rated by image analysis Plant material and growth conditions after incubation at room temperature and by measu- One chilling-tolerant (‘Ascolana’), two chilling- ring the conductivity of the leachate as described sensitive (‘Coratina’ and ‘Frantoio’), and one interme- diate variety (‘Leccino’) of three-year-old olive trees, The relative electrolyte leakage (REL) was calcula- grown in 3-l pots containing a 50:50 (v/v) mixture of sandy gravel and peat, were used. Plants were grown in a field located in Pescia, Italy (43°54’N, 10°41’E.
where L1 is the first conductivity measurement, repre- 30 m asl) and were brought into a laboratory 3 hr senting the ion leakage caused by freezing damage, before the experiment. Samples of current year shoots also including background leakage, and L2 is the of similar length, diameter and internode length, lea- second conductivity measurement performed on heat- ves removed from the third node from the top, vegeta- killed samples representing the total electrolyte con- tive buds, woody and non-woody roots, were used for the experiments. All the experiments were conducted Cold hardiness was expressed as LT50 (lethal tem- in winter on cold acclimated plants.
perature at which 50% of the ion leakage occurs; or, inthe case of visual score, the lethal temperature at Differential thermal analysis (DTA) which 50% of the tissues are damaged) by fitting the Excised plant tissues were placed on one side of a response curves with the following logistic sigmoid thermopile plate (Peltier, Melcor CP 1.4-17-10L, USA) and a “dried sample of tissue” on the other usedas a reference to detect differential temperature chan- ges between the two samples during the freezing (Mancuso, 2000). A copper-constantan microthermo-couple (0.2 mm diameter) was attached to the thermo- where x = treatment temperature, b = slope at inflec- pile plate near the tissue fragment to measure ambient tion point c, a and d determine the asymptotes of the temperature and the whole plate was covered on both sides with a 2 mm thick layer of closed cell polystyre-ne tied to the plates with Parafilm to decrease loss of heat to the surroundings. The samples were then pla- Five cross sections, two to three cells thick, were cut ced in a freezing cabinet and cooled at the rates of 5°C from near the middle of the leaf in deionised water hr-1, usually down to -30°C. It had been ascertained in using a new razor blade. The sections were taken at each preliminary experiments that Olea europaea tissues temperature and placed for 20 min in 0.02% (by weight) did not produce exotherms between -28 and -50°C.
neutral red [3-amino-7-dimethylamino-2-methylphena- Initially temperature was kept at 20°C, as in the labo- zine (HCl)]. Samples were stored at 5°C for 24 hr to ratory. Three different plant samples were tested con- maximise stain uptake (Didden-Zopfy and Nobel, currently. The signals from the thermopiles were low- 1982). For each treatment temperature, about 100 cells pass filtered, amplified and connected via a multichan- were examined microscopically at a magnification of nel A-D convertor card (Lab-PC-1200 National 200x in each of the five tissue samples, leading to a total Instrument, USA) to a P133 personal computer. Fresh of about 500 cells examined under each condition. Stain tissues were used for each different rate of freezing.
uptake occurs only for living cells and is detected by theintracellular localisation of the red dye.
Determination of the lethal freeze temperature Cold hardiness of leaves was estimated by measu- ring the conductivity of the leachate of leaf discs and 3. Results and Discussion
by visual rating of injury after controlled freezingtreatments carried out in an air cooled chamber. The initial and final temperature was 20°C, the rate of coo- Table 1 summarises cold hardiness (LT50) of ling and warming 7°C hr-1 and the minimum tempera- various organs of Olea europaea, measured visually, ture was maintained for 4 hr. About 50 leaf discs of 1 by DTA or by electrolyte leakage test. The values are cm in diameter for each test temperature were set in a higher for the chilling-sensitive cultivars (Coratina and polyethylene bag. Thirty discs were incubated in 10 ml Frantoio) in comparison with chilling-tolerant of distilled H2O at 26°C for 6 hr to measure the con- ‘Ascolana’. Moreover, such differences remain in all ductivity of the leachate. The remaining 20 discs were organs studied (with the exception of roots which, incubated for one week at room temperature for deter- from this point of view, are the least meaningful) mination of injury performed by image analysis (Scion giving very similar LT50 values among the different Image). Cold hardiness of shoots, leaf buds and roots were also measured by cooling samples at 5°C decre- According to previous tests on the cold hardiness of Table 1 - Frost hardiness of olive trees estimated by relative electrolyte leakage (REL), by visual score (VS) and by differential thermal analysis (DTA), at the end of January in cold acclimated plants of four different cultivar Rate of cooling is 5°C hr-1. The results are reported as means ± SEM.
olive made by electrical resistance measurements hr treatment at 0°C was indistinguishable from that of (Mancuso, 2000), we found that different organs in the control at 25°C, whereas no cells took up stain at olive show different cold hardiness. It would appear -25°C in all the cultivars tested, indicating lack of from the present study that the order of sensitivity is secondary roots > primary roots > apical leaves >basal leaves > shoots > vegetative buds. Table 2 - Stain uptake at various subzero temperatures for mesophyll Generally, electrolyte leakage test and image analy- cells of four cultivars of Olea europaea sis determination are in good agreement for leaves and shoots (Fig. 1), whereas for roots electrolyte leakage test predicts a 1-2°C higher LT50 for all the cultivars Data are expressed as a percentage of the fraction of cells accumulating neutral red when treated at 25°C for 2 hr (control). Rate of cooling is 5 °C hr-1.
Typical DTA profiles of Olea europaea leaf are shown in figure 2a. When the leaf was moistened Fig. 1 - Assessment of cold hardiness by electrolyte leakage method in Olea europaea leaf cooled at 5°C hr-1. The inset shows thevisual rate of injury of Olea europaea leaf cooled at the samecondition as above. The lines show symmetric non-linearregression curves for the shoots.
As the air temperature was lowered from 5°C at - 20°C, the percentage of mesophyll cells of the leavestaking up the vital stain neutral red decreased, andagain the pattern varied with the cultivar (Table 2). Forinstance, for a 2 hr treatment at -10°C, 86% of cells Fig. 2 - DTA profiles of Olea europaea leaf cooled at the rate of 5°C exhibited stain uptake for leaves from ‘Ascolana’ hr-1 under different conditions. (a) Leaf without moistening plants whereas in ‘Frantoio’ and ‘Coratina’ leaves the (b) Leaf moistened with a little water on the surface prior toDTA. HTE is the High Temperature Exotherm, LTE percentage of vital cells accounted for an average of tes the temperature at which 50% of the Low Temperature 73% and 68%, respectively. Stain uptake following a 2 about 2 hr before DTA, the HTE (High Temperature HTE, which can indicate freezing of vascular and Exotherm) shifted to a slightly higher temperature and intercellular water, was not detected (Fig. 4) in shoots, became larger (Fig. 2b). HTE was not related to injury roots and vegetative buds in agreement with previous of the leaf and might arise from the freezing of the findings (Mancuso, 2000). This was probably because water in the intercellular space. On the contrary, LTE free water accounted for a minimum of total water.
(Low Temperature Exotherm) was not affected by The LTEm revealed by differential thermal analysis fit moistening and seems to be closely related to injury in very well with the lethal temperatures (LT50) calcula- ted from the electrolyte leakage measurements, thus All leaves showed sharp exotherms, however exci- confirming the presence of supercooling in leaves sed leaves of all four cultivars froze at exotherm tem- (Larcher, 1970) and other organs (Mancuso, 2000) of peratures significantly lower than leaves from intact plants (1.5-3°C lower). Ashworth and Davis (1984) Woody plants frequently possess various freezing and Andrew et al., (1986) found in peach and cherry strategies ranging from extracellular freezing to extraor- trees that there was a logarithmic increase in freezing gans freezing and supercooling depending on tissues temperature with an increase in specimen size.
(Levitt, 1980). Thus, the occurrence in olive of a mecha- Ashworth (1990) suggests that this persistent pattern nism of “freezing avoiding” by supercooling generalised of increasing freezing temperature with increasing in most tissues is an exception worthy of study.
specimen size may result from a greater probabilitythat large specimens have more ice nucleation sites.
Nevertheless, we found this true only if portions ofleaves were used, while no differences were foundusing intact leaves of different size (data not shown).
Consequently, we directed our attention to the relatedpossibility that the water in the petioles or in thexylem is more vulnerable to freezing than the water inthe leaf tissue. To test the existence of differential iceformation in the different tissues, exotherm temperatu-res were recorded concurrently in leaf, petiole andshoot of the same branch cooled at rate of temperaturechanges of 5°C hr-1. We found that freezing was initia-ted in petioles 2-4 s earlier than leaves of the samebranch (Fig. 3). This initial freezing of petiole watermay then rapidly propagate to leaves which freeze at ahigher temperature than when they were excised fromthe shoot. Our results are consistent with findings for avariety of fruit trees that plant organs (flowers, fruits),after detachment, freeze at a lower temperature thanequivalent organs that remain a part of intact plants(Proebsting et al., 1982; Andrew et al., 1986).
Fig. 3 - Representative result of a concurrent DTA in leaf, petiole and shoots of Olea europaea showing that freezing was initiated inpetioles 3 s earlier than leaves of the same branch. This initial Fig. 4 - Differential thermal analysis in different organs of freezing of petiole water may then rapidly propagate to leaves.
Olea europaea cooled at 5°C hr-1.
4. Conclusion
DIDDEN-ZOPFI B., NOBEL P.S., 1982 - High temperature tole- rance and heat acclimation of Opuntia bigelovii. - Oecologia, In conclusion, the results reported in the present GEORGE M.F., BURKE M.J., WEISER C.J., 1974 - study, in addition to determining which methods are Supercooling in overwintering azalea flower buds. - Plant more reliable for estimating freezing injury in olive, show differential response to subzero temperatures HONG S., SUCOFF E., 1980 - Units of freezing of deep super- among four cultivars characterised by different free- cooled water in woody xylem. - Plant Physiol., 66: 40-45.
ISHIKAWA M., 1984 - Deep supercooling in most tissues of win- tering Sasa senanensis and its mechanism in leaf blade tissues.
Results obtained with differential thermal analysis, visual score and relative electrolyte leakage consisten- LARCHER W., 1970 - Kalteresistenz und uberwinterungsvermo- tly agreed. Among the four different methods utilised, gen mediterraner Holzpflanzer. - Oecologia Plantarum, 5: DTA was by far the fastest procedure, whereas the visual score method the simplest, although is not as LEVITT J., 1980 - Responses of plants to environmental stresses.
Vol. I. Chilling, freezing and high temperature stresses. - quantitative as the other three methods.
Results were consistent with previous findings MANCUSO S., 2000 - Electrical resistance changes during expo- (Mancuso, 2000) and with anecdotal evidence that sure to low temperature measure chilling and freezing toleran- suggest that ‘Ascolana’ is one of the most tolerant ce in olive tree (Olea europaea L.) plants. - Plant Cell olive cultivar, whereas ‘Coratina’ is one of the most PROEBSTING E.L., ANDREW P.K., GROSS D.C., 1982 - sensitive. In contrast, to other woody species, results Supercooling young developing fruit and floral buds in deci- showed a generalised supercooling mechanism in duous orchards. - HortSci., 17: 67-68.
olive which make olive interesting for the study of REPO T., 1992 - Seasonal changes of frost hardiness in Picea biophysical and biochemical aspects of frost tolerance.
abies and Pinus sylvestris in Finland. - Can. J. For. Res., 22:1949-1957.
SAKAI A., 1979 - Freezing avoidance mechanism of primordial shoots of conifer buds. - Plant Cell Physiol., 20: 1381-1390.
SAKAI A., EIGA S., 1985 - Physiological and ecological aspects of cold adaptation of boreal conifers, pp. 157-170. - ANDREW P.K., PROEBSTING E.L., GROSS D.C., 1986 - Ice In: KAURIN A., O. JUNTILA, and J. NILSEN (eds.). Plant nucleation and supercooling in freeze-sensitive peach and production in the north. Norwegian University Press, sweet cherry tissues. - J. Amer. Soc. Hort. Sci., 111: 232-236.
ASHWORTH E.N., 1990 - The formation and distribution of ice SAKAI A., LARCHER W., 1987 - Frost survival of plants: within Forsythia flower buds. - Plant Physiol., 92: 718-725.
responses and adaptation to freezing stress. - Springer-Verlag, ASHWORTH E.N., DAVIS G.A., 1984 - Ice nucleation within peach trees. - J. Amer. Soc. Hort. Sci., 109: 198-201.
TOIVONEN A., RIKALA R., REPO T., SMOLANDER H., 1991 BONGI G., MENCUCCINI M., FONTANAZZA G., 1987 - - Autumn colouration of first year Pinus sylvestris seedlings Photosynthesis of olive leaves: effect of light flux density, leaf during frost hardening. - Scand. J. For. Res., 6: 31-39.
age, temperature, peltates, and H2O vapor pressure deficit on TUMANOV I.I., KRASAVTSEV O.A., 1959 - Hardening of gas exchange. - J. Amer. Soc. Hort. Sci., 112: 143-148.
northern woody plants by temperatures below zero. - Soviet CHRISTERSSON L., 1978 - The influence of photoperiod and tem- perature on the development of frost hardiness in seedlings of WEISER C.J., 1970 - Cold resistance and injury in woody plants.
Pinus sylvestris and Picea abies. - Physiol. Plant., 44: 288-294.


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