Planta (1985)164:512-516

Planta 9 Springer-Verlag 1985

Injury to potato leaves exposed to subzero temperatures in the absence of freezing* O.M. Lindstrom and J.V. Carter Laboratory of Plant Hardiness, University of Minnesota, Department of Horticultural Science and Landscape Architecture, 305 Alderman Hall, St. Paul, MN 55108, USA

Abstract. Electrolyte leakage was measured in " h a r d e n e d " and " n o n h a r d e n e d " leaves of three potato species, Solanum tuberosum L., S. acaule Bitt. and S. commersonii Dun., and one interspecific cross, "Alaska Frostless" (S. acaule x S. tuberosum) when exposed to various subzero temperatures. The leaves were undercooled (no ice present) from 0 ~ to - 1 2 . 5 ~ for 4 5 m i n and to -4~ for up to 10 d. Regardless of the degree of undercooling no injury was observed in any of the potatoes, " h a r d e n e d " or " n o n h a r d e n e d " , for up to 12 h. After 5 d, however, electrolyte leakage was observed in " h a r d e n e d " S. tuberosum, S. acaule and S. commersonii, and in " n o n h a r d e n e d " "Alaska Frostless". After 10 d exposure all potatoes, " h a r d e n e d " and " n o n h a r d e n e d " , showed a significant amount of electrolyte leakage as compared to their controls kept at 0 ~ C for 10 d.

Key words: Chilling injury - Solanum - Temperature (chilling) - Undercooling.

Introduction

Since freezing of nonhardy plants results in their death, an increase in undercooling (supercooling) of tissue water in plants can enable them to survive exposure to lower temperatures. For example, Arny et al. (1976) reported that the degree of undercooling can be enhanced in corn leaves by reducing the population of epiphytic bacteria, Pseudomonas syringae, an efficient ice nucleator. However, if steps are taken to increase the undercooling of plant tissue water to avoid death by freezing, the question arises as to whether the expo* Scientific Journal Series Paper No. 13842 of the Minnesota Agricultural Experiment Station, St. Paul, Minn

sure to lower temperatures in the absence of ice will in itself be injurious. Many plants are injured by temperatures above 0~ Molisch (1897; see English translation, p. 377) suggested the term "Erk/iltung" (death by cooling) for low-temperature damage in the absence of freezing. The temperature range at which chilling injury has been reported varies from 15 ~ C in rice (Adir 1968) and sugarcane (Tsunoda et al. 1968) to below the freezing point in the absence of freezing in some fungi (Linder 1915; Onoda 1937). Levitt (1980, p. 23) states that " f o r the vast majority of plants, however, a chilling stress refers to any temperature below 10-15 ~ C, and down to 0 ~ C . " In almost all cases chilling injury has been studied at temperatures above 0 ~ C. In the work reported here, however, we have examined injury caused by low temperatures in undercooled tissues. Therefore, we are defining chilling injury as injury to a plant or plant part at any temperature below 10-15 ~ C, as long as no ice is present. This includes temperatures below the freezing point of the tissue water. Chen and Li (1980) and Chen et al. (/976) have reported hardiness levels of several potato species and crosses. Solanum tuberosum and the hybrid cultivar "Alaska Frostless" cannot survive ice formation in their tissues and subsequent cooling below - 2 . 5 ~ in either the hardened or nonhardened state. Nonhardened S. acaule can survive ice formation and subsequent cooling to - 6 ~ C while hardened plants survive to - 9 ~ C; S. commersonii is hardy to - 4 . 5 ~ C when grown under nonhardening conditions, and to - 1 1 . 5 ~ C when grown under hardening conditions. In the work reported here, hardened and nonhardened potato leaves were assessed for chilling injury when exposed to subzero temperatures in the absence of ice formation.

O.M. Lindstrom and J.V. Carter: Subzero chilling injury in potatoes Materials

and methods

Plant material. Three potato species, Solanum acaule Bitt., S. eommersonii Dun., S. tuberosum L., and one interspecifie cross, " A l a s k a Frostless" (S. tuberosum - S. aeaule), were studied. Solanum tuberosum and " A l a s k a Frostless" were propagated from tubers while S. acaule and S. commersonii were propagated either from stem cuttings or by division of the parent plant. The stem cuttings were placed in a mist chamber for one m o n t h to root. Rooted cuttings, divided plants, and tuber sections were planted in 20-cm pots containing a 3 : 2 : 2 (by vol.) mixture of soil, sand and peat, and then placed in growth chambers. Plants received a 14-h photoperiod with 20/15~ day/night temperatures. A photon-flux density of 450 gmol s - 1 m 2 photosynthetically active radiation was supplied to the plants. The plants were watered as needed and a nutrient solution (20:20:20) was applied to the soil once a week. Plants grown under the above conditions are termed " n o n h a r d e n e d " . After one m o n t h some of the nonhardened plants were exposed to a constant 2 ~ C temperature treatment. All other environmental factors remained the same. After two weeks under these conditions the plants are termed "cold hardened". Not all species grown under the low-temperature regime gained cold tolerance compared with plants grown under the nonhardening conditions; however, all plants grown under the low-temperature regime will be referred to as "cold hardened" material. Analysis o f cold hardiness. The hardiness level of the plants in this study was determined using the method described by Chen et al. (1976). In all these tests, ice formation was induced at the freezing point of the tissue water. Solanum tuberosum and " A l a s k a Frostless" did not survive freezing at temperatures below - 2 . 5 ~ C in either the hardened or nonhardened state. Nonhardened S. acaule survived exposure to - 6 ~ C while hardened plants survived freezing to - 9 ~ C. Solanum commersonii was hardy to - 4 ~ C when grown under non-hardening conditions, and to - 1 1 . 5 ~ C after hardening. Preparation o f samples. Whole leaves or leaflets were excised and placed in a Differential Thermal Analysis (DTA) apparatus or floated in vials containing water for their chilling treatment. To examine the effects of chilling at subzero temperatures only plant tissue that was not frozen was used since the formation of ice crystals would have confounded the effects of chilling. The D T A apparatus used was similiar to that described by George et al. (1974) and Quamme et al. (1974). Heat released by the freezing of tissue water is detected by the apparatus so if heat is not detected during the chilling treatment the tissue water is assumed not to be frozen. Chilling experiments from

513 0 ~ C to - 12.5 ~ C for up to 12 h duration were accomplished using the D T A apparatus. Floating leaf disks were also used to monitor the physical state of water in the leaves. The leaf disks were floated on 6 ml of deionized, double-distilled water. Since the leaf is in direct contact with this water, if either the leaf or the water in the container freezes it is assumed that one will nucleate the other, resulting in the freezing of both. To verify this point, unhardened S. tuberosum leaf tissue was placed on the surface of water in vials, and the vials placed in a temperature bath set at - 5 ~ C. After thermal equilibrium was achieved a metal rod, previously cooled to below 0 ~ C, was used to initiate freezing in parts of the leaf samples above the water surface. Extremely gentle contact between metal rod and leaf tissue caused almost instant nucleation of ice in the surrounding water (after 1 s or less). Thus, if the container water remained unfrozen for the duration of the chilling treatment, the plant tissue was also unfrozen during the chilling treatment. The floating-leaf method was used for chilling treatments of 5 and 10 d duration. Analysis o f chilling injury. Conductivity, as described by Dexter et al. (1932) and Aronsson and Eliasson (1970), was used to determine the extent of injury. After the chilling treatment the leaves were taken from the D T A apparatus and placed in 6 ml of deionized, double-distilled water. The samples chilled in the temperature bath were kept in the same water in which they were chilled. The samples were then placed at 4 ~ for 24 h. After this they were brought to room temperature, vacuuminfiltrated with water (15 rain), placed on a shaker (125 rpm) for 1 h, and the conductivity of the container water was measured. The samples were then killed by placing them in a - 7 0 ~ C freezer for approx. 12 h, after which they were thawed, brought to room temperature, placed on a shaker for 1 h, and the conductivity of the container water was again measured. Electrolyte leakage of a treated leaf is reported as percent of the total electrolytes released from the - 7 0 ~ C-treated tissue. The treatments using the D T A were replicated a minimum of ten times, while the treatments using the floating leaves were replicated at least 20 times. These treatments consisted only of unfrozen undercooled leaves. In no case were any frozen leaves used. The controls received the same treatment as the test leaves except that they were held at 0 ~ for the length of time that the test leaves were subjected to sub-zero chilling.

Results

Electrolyte leakage from potato leaves chilled for 45 min at temperatures ranging from - 2 . 5 ~ C to - 12.5 ~ C is shown in Table 1. Hardened and non-

Table 1. Potato leaves were exposed to various subzero temperatures for 45 min. In no case was any of the tissue frozen. Electrolyte

leakage (mean • SE) of the treated samples is expressed as a percent of the total electrolytes in the same sample Chilling temperature

S. acaule

Alaska Frostless

S. commersonii

S. tuberosum

(o c)

Hardened

Nonhardened

Hardened

Nonhardened

Hardened

Nonhardened

Hardened

Nonhardened

Control(0~ -2.5 -4.0 -6.5 -8.0 -12.5

8.4• 8.0• 7.9• 9.7• 13.1• 10.9•

15.7• 14.3• 17.4• 17.8• 27.9• nodata

8.6• 7.2• 7.7• 11.6• 9.9• 11.2•

9.3• 8.2• 11.6• 9.2• 12.6• nodata

9.2• 8.2• 5.4• 6.4• 10.9• 11.9•

10.1• 9.1• 12.0• 9.5• 9.8• 15.5•

8.0• 5.7• 7.2• 6.4• 7.4• 9.6•

12.6• 11.4• 8.9• 8.3• nodata nodata

514

O.M. Lindstrom and J.V. Carter: Subzero chilling injury in potatoes

Table 2. Potato leaves were chilled at - 4 ~ for 0, 2, 4 and 12 h. In no case was any of the leaf tissue frozen. Electrolyte leakage (mean • SE) of the treated samples is expressed as a percent of the total electrolytes in the same sample Time of chilling (h)

S. acaule

Control 2 4 12

Alaska Frostless

S. eommersonii

S. tuberosum

Hardened

Nonhardened

Hardened

Nonhardened

Hardened

Nonhardened

Hardened

Nonhardened

11.6• 10.1• 12.5• 9.8•

7.7• 8.2• 12.4• 8.4•

6.2• 8.5• 12.6• 9.8•

13.2• 14.6• 14.1• 15.9•

9.6• 8.2• 7.7• 10.1•

8.2• 9.2• 11.9• 11.1•

10.3• 9.6• 11.1• 12.6•

7.8• 5.1• 7.1• 6.9•

100-

I00-

90-

90-

8070.

g

m

:..:

60.

:,::

_~ 50.

/

/ //

/ / /

40

_-.. :.:

30-

--..

/

"i

r

%,.

20.

~ ~

80hardened

-]O~

control

// /

g 60o

B non-hardened

T

hardened control

//

--I ~

50

4030. //

~..:

20I0.

I0. 0

70-

non-hordened

5

10 Chilling time (Days)

Fig. l. Electrolyte leakage ( m e a n • from S. tuberosum leaves chilled for 5, 10 d at - 4 ~ C. In all cases the leaf tissue remained unfrozen. Electrolyte leakage of the treated samples is expressed as a percent of the total electrolytes in the same sample

0

5

10 Chilling

time (Days)

Fig. 2. Electrolyte leakage from S. acau/e leaves chilled for 5, 10 d at - 4 ~ C. Otherwise as in Fig. I I00, 9080-

hardened plants of all four species were given chilling treatments. There was, in most cases, a greater amount of electrolyte leakage from the nonhardened leaves compared with the hardened ones. However, since the hardened and nonhardened plants were grown under different conditions for varying lengths of time, caution should be used when comparing the hardened and nonhardened leaves directly. A better comparison is with the control of each treatment. The controls were treated identically, except they were kept at 0 ~ for 45 min. Electrolyte leakage from potato leaves chilled at - 4 ~ C for 0 (control), 2, 4, and 12 h is shown in Table 2. Again, there was no large amount of electrolyte leakage in any species, hardened or nonhardened, from the chilling treatments used. Some plants, chilled at - 4 ~ C for 5 and 10 d, did show high levels of electrolyte leakage (Figs. 14). After chilling for 5 d at - 4 ~ C, nonhardened S. tuberosum leaves had electrolyte leakage indistinguishable from the control treatment (0~ for 5 d), while hardened S. tuberosum leaves exhibited

~ non-hardened

70.

go 6o-

~ hardened

_a 5040-

- 7 0 ~ control

30-

S /

20-

/

10. 0

S 5 I0 Chilling time (Days)

Fig. 3. Electrolyte leakage from S. commersonii leaves chilled for 5, 10 d at - 4 ~ C. Otherwise as in Fig. 1

a significant degree of leakage (Fig. 1). After 10 d of chilling at - 4 ~ C, leaves from both hardened and nonhardened S. tuberosum exhibited high levels of electrolyte leakage (Fig. 1). In S. acaule and S. commersonii leaves, similar results were found (Figs. 2, 3). Leaves of "Alaska Frostless" chilled for 5 d (Fig. 4) showed more electrolyte leakage from the nonhardened material than from the hardened material. After 10 d of chilling both the har-

O.M. Lindstrom and J.V. Carter: Subzero chilling injury in potatoes IO0 90 80 70

~/~ non-hardened

/ / / // / /

8, 6O

o 5o

_1

4O

~

hardened

i[--~i 0 ~

control

30 2o I0 0

5

IO Chilling time (Days)

Fig. 4. Electrolyte leakage from "Alaska Frostless" leaves chilled for 5, 10 d at - 4 ~ C. Otherwise as in Fig. 1

dened and nonhardened "Alaska Frostless" leaves showed high levels of electrolyte leakage. The control leaves in the above experiment were kept at 0 ~ for 5 d and for 10 d to compare with the leaves chilled for 5 and 10 d, respectively. Discussion

Cell injury results in a loss of electrolytes from the cell sap, the greater the injury the greater the conductivity of the extract (Levitt 1980, p. 134). The level of electrolyte leakage that corresponds to lethal injury of a plant varies greatly. Palta et al. (1977) have shown that as high as 76% leakage occurred in onion bulbs which ultimately survived. Even though considerable injury occurred it was reversible when brought to favorable temperatures. We did not determine whether the injury we measured could be reversed if samples were returned to non-chilling temperatures. The electrolyte leakage we measured is, however, well above that of non-chilled controls and it can be assumed that injury has occurred and an increase in temperature would be required to reverse this injury. In hardiness tests described by Sukumaran and Weiser (1972), ice was always present to provide nucleators so extracellular tissue water was frozen at its freezing point, with no undercooling. In the case where the plant was injured, the injury could be the consequence of either the freezing process or the low temperatures. In our experiments, in which no freezing occurred, we have shown that exposure to temperatures ranging from 0 ~ to - 12.5 ~ C and for at least 12 h did not induce injury in leaf tissue of any of the potatoes tested. Therefore, it can be assumed that injury sustained by potato leaves exposed to low temperatures for

515

up to 12 h results from the freezing process and not from low-tempereature exposure. As chilling time increases, low temperature alone can cause injury to both hardened and nonhardened potato leaves. After 10 d of chilling, for example, both hardened and nonhardened leaves of all the potatoes tested were injured. Undercooling of water in plant tissues is common in plants (see review by Burke et al. 1976). It has been shown to occur in many different species and the extent of undercooling also has been shown to vary widely. Potato leaves have been reported to survive undercooling to - 6 ~ C (Hudson and Idle 1962), while potato sprouts were shown to undercool for 18 h at - 5 . 5 ~ and for 4 h at -7.5~ (Asahina 1954). There is some evidence that the extent of undercooling can be increased in herbaceous plants if populations of bacterial ice nucleators are reduced (Lindow et al. 1978). Plants such as S. tuberosum and "Alaska Frostless" cannot survive freezing at any temperature below 2.5 ~ C. One way to increase the cold hardiness of these species would be to increase the degree of undercooling. This would keep tissue water unfrozen at lower temperatures. For plants to survice by undercooling they would, however, have to tolerate exposure to the low temperatures per se. Our experiments indicate that potato leaves are able to tolerate exposure to subzero temperatures (in the absence of freezing) for time periods of several hours or even days. Thus, it appears that development of a greater degree of undercooling could increase the survival range of potato species that have no freezing tolerance. For potatoes that have freezing tolerance, for example S. acaule and S. commersonii, an increase in undercooling may, however, be harmful (Lindstrom and Carter 1980, 1983). All potatoes in this study, hardened and nonhardened, showed severe injury after being chilled at - 4 ~ C for 10 d. After chilling for only 5 d, however, differences were found between hardened and nonhardened leaves within a species, as well as between species. In each case the chilling of hardened and nonhardened leaves was compared with hardened and nonhardened controls, respectively, which were treated identically except for being held at 0 ~ for the duration of each treatment. Solanum tuberosum, S. acaule, and S. commersonii all exhibited injury in the hardened leaves while the nonhardened leaves were not different from their control. "Alaska Frostless" on the other hand showed no injury in the hardened leaves but did exhibit injury in the nonhardened leaves. Although a clear understanding of this phenomenon has not

516

O.M. Lindstrom and J.V. Carter: Subzero chilling injury in potatoes

been developed, it is possible that chilling injury is a cumulative process resulting from prolonged exposure to sub-optimal temperatures, and that the chilling stress begins when nonhardened plants are placed under hardening conditions (2 ~ C day/ night temperatures). Consequently, hardened plants were already partially chilled and predisposed to injury when exposed to further subzero chilling treatment because chilling-stress effects were additive. In any case the chilling injury was accelerated by the subzero chilling treatment, since the hardened controls placed at 0 ~ C for 5 d and 10 d showed no chilling injury. The fact that "Alaska Frostless" showed no chilling injury of the nonhardened leaves after the 5 d treatment may indicate that it has developed more chilling resistance than the other three potato species in the study. In conclusion, chilling injury does occur in higher plants at subzero temperatures. In agreement with Lyons et al. (1979) the injury is proportional to the duration of exposure. Chilling injury (above 0 ~ C) can be minimized by warming to above a threshold temperature (different for different plants), indicating that chilling injury is at least somewhat reversible (Levitt 1980, p. 46-49). This may also be true for subzero chilling injury; however, our experiments did not address this point. Thus, potato species exposed to subzero temperature may be able to recover, provided the temperature is periodically raised above their threshold chilling temperature. It is not known to what extent potatoes may undercool in the field. Soil, for example, is a good ice nucleator and may prevent intact plants from undercooling enough to enhance survival (Schnell 1974). Burton (1981) reports that the average commercial daily increment for potato growth is 400 kg ha-1, so extending the growing season just a few days at each end could greatly increase average potato yield. Therefore, investigating the ability of frost-sensitive potatoes to undercool seems to be a valid approach in attempting to expand the growing range of cultivated potatoes into colder climates. References Adir, C.R. (1968) Testing rice seedlings for cold water tolerance. Crop Sci. 8, 264-265 Arny, C.D., Lindow, S.E., Upper, C.D. (1976) Frost sensitivity of Zea mays increased by application of Pseudomonas syringae. Nature 262, 282-284 Aronsson, A., Eliasson, L. (1970) Frost hardiness in Scots pine. I. Conditions for test on hardy plant tissues and for evaluation of injuries by conductivity measurements. Stud. For. Suec. 77, 1-30

Asahina, E. (1954) A process of injury induced by the formation of frost on potato sprout. Low Temp. Sci. B 11, 13-21 Burke, M.J., Gusta, L.V., Quamme, H.A., Weiser, C.J., Li, P.H. (1976) Freezing and injury in plants. Annu. Rev. Plant Physiol. 27, 507-528 Burton, W.G. (1981) Challenges for stress physiology in potato. Am. Potato J. 58, 3-14 Chen H.H., Li, P.H. (1980) Characteristics of cold acclimation and deacclimation in tuber bearing Solanum species. Plant Physiol. 65, 1146-1148 Chen, P.M., Burke, M.J., Li, P.H. (1976) The frost hardiness of several Solanum species in relation to the freezing of water, melting point depression, and tissue water content. Bot. Gaz. 137, 313-317 Dexter, S.T., Tottingham, W.E., Graber, L.F. (1932) Investigations of hardiness of plants by measurement of electrical conductivity. Plant Physiol. 7, 63-78 George, M.F., Burke, M.J., Weisert, C.J. (1974) Supercooling in overwintering azalea flower buds. Plant Physiol. 54, 29-35 Hudson, M.A., Idle, D.B. (1962) The formation of ice in plant tissues. Planta 57, 718-730 Levitt, J. (1980) Responses of plants to environmental stresses. Academic Press, New York Linder, J. (1915) Uber den EinfluB gtinstiger Temperaturen auf gefrorene Schimmelpilze (Zur Kenntnis der Kfilteresistenz von Aspergillus niger). Jahrb. Wiss. Bot. 55, 1-52 Lindow, S.E., Arny, D.C., Upper, C.D., Barchet, W.R. (1978) The role of bacterial ice nuclei in frost injury to sensitive plants. In: Plant cold hardiness and freezing stress, pp. 249263, Li, P.H., Sakai, A , eds. Academic Press, New York Lindstrom, O.M., Carter, J.V. (1980) Assessing the freezing injury of cold hardened supercooled potato leaves. (Abstr.) Plant Physiol. 65, Suppl., 46 Lindstrom, O.M., Carter, J.V. (1983) Assessment of freezing injury of cold-hardened undercooled leaves of Solanum eommersonii. Cryo-Lett. 4, 361-370 Lyons, J.M., Raisson, J.K., Steponkus, P.L. (1979) Adaptations to chilling: Survival, germination, respiration and protoplasmic dynamics. In: Low temperature stress in crop plants: The Role of the membrane, pp. 565, Lyons, J.M., Graham, D., Raison, J.K., ed. Academic Press, New York Molisch, H. (1897) Untersuchungen fiber das Erfrieren der Pflanzen. G. Fischer, Jena. Engl. transln, in Cryo-Lett. 3, 331-390 (1982) Onoda, N, (1937) Mikroskopische Beobachtungen fiber das Gefrieren einiger Pflanzenzellen in flfissigem Paraffin. Bot. Inst. Kais. Univ. Kyoto, Bot. Zool. 5, 1845-2188 Palta, J.P., Levitt, J., Stadelmann, E.J. (1977) Freezing injury in onion bulb cells. II. Post-thawing injury or recovery. Plant Physiol. 60, 398-401 Quamme, H.A., Evert, D.R., Stushnoff, C,, Weiser, C.J. (1972) A versatile temperature control system for cooling and freezing biological materials. Hort. Science 7, 24-25 Schnell, R.C. (1974) Biogenic sources of atmospheric ice nuclei. Report No. A R I l l , Dept. of Atmosph. Resources, Coll. of Eng., University of Wyoming, Laramie, USA Sukumaran, N.P., Weiser, C.J. (1972) Freezing injury in potato leaves. Plant Physiol. g0, 564-567 Tsunoda, K., Fujimura, K., Nakahori, T., Oyomodo, Z. (1968) Studies on the testing method for cooling tolerance in rice plants. I. An improved method by means of short term treatments with cool and deep water. Jpn. J. Breed. 18, 33-40 Received 26 March; accepted 17 December 1984

Injury to potato leaves exposed to subzero temperatures in the absence of freezing.

Electrolyte leakage was measured in "hardened" and "nonhardened" leaves of three potato species, Solanum tuberosum L., S. acaule Bitt. and S. commerso...
521KB Sizes 0 Downloads 0 Views