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Fig. 2A, B. Daily rhythm of chilling-induced desiccation of cotton seedlings under low or high RH. Seedlings grown at 33 ~ C, 85 % RH

were exposed at 6-h intervals during the LDC to 5~ C under 85% RH for 1.5 d (A) or 100% RH for 3 d (B). At the end of the chilling exposure, cotyledon water content was measured, r----3 Light period; ~ dark period; 9 non-chilled; 9 chilled reached cotyledon or shoot FW below 0.5 g and a cotyledon chlorophyll content below 0.45 mg/cotyledon ultimately died before completing their life cycle. Chilling under 85% RH resulted in a differential water loss from the cotyledons depending on the time in the LDC when exposure had begun. The magnitude of changes in water loss inversely correlated with the magnitude of the daily changes in CR. Thus, chilling started at the middle of the light period, when CR is the lowest, resulted in a substantial water loss, but chilling started at the middle of the dark period, when CR is the highest, did not result in a significant water loss (Fig. 2A). Chilling under 100% RH starting at any time during the LDC did not result in any water loss (Fig. 2B). Nevertheless, return of the chilled seedlings to standard growth conditions caused differential damage, depending on the time in the LDC when chilling had begun (Fig. 1D-F).

Effect of R H before chilling on the daily rhythm of CR. In addition to daily changes in CR the cotton seedlings showed daily changes in cotyledon water content. Under 33 ~ C, 85% RH, cotyledon water content started to decline from the beginning and reached the lowest level at the end of the light period. Then, water content started to increase at the beginning and reached maximal level at the end of the dark period (Fig. 3D). The daily changes in CR and in cotyledon water content are affected by low-temperature-induced acclimation and the RH before chilling (Fig. 3A-F). Acclimation by 26 ~ C, 85% RH for 24 h induced CR that persisted throughout the LDC. The magnitude of the CR induced by the low temperature was similar to that reached at the daily resistant phase (Fig. 3A). Water content of acclimated seedlings was always lower than that of non-acclimated ones at all times during the LDC (Fig. 3D). Transfer of non-acclimated and acclimated seedlings to 100% RH prior to chilling changed their CR and water content. Non-acclimated seedlings treated for 6 h at 100% RH lost their CR at the beginning of the dark period, thus shortening the resistant phase to only the middle of the dark period. Similar treatment to accli-

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Fig. 3A-F. Effect of RH before chilling on cotyledon water content and on the daily rhythm of CR of cotton seedlings. Seedlings grown at 33 ~ C, 85% R H were chill-acclimated by a treatment of 26 ~ C, 85% R H for 24 h. Then, at 6-h intervals during the LDC seedlings were transferred to 100% R H under the previous growth temperature for 0 h (A, D), 6 h (B, E), or 12 h (C, F). At the end of these treatments, cotyledon water content was determined (D-F). Afterwards, the seedlings were exposed to 5~ C, 85% RH for 1.5 d. After chilling the seedlings were returned to 33 ~ C, 85% R H for 5 d and at the end of this period shoot FW was measured (A-C). r-'--3 Light period; ~ dark period; 9 non-acclimated; A acclimated

mated seedlings eliminated the CR at the beginning and middle of the light period and created daily changes like those of non-acclimated seedlings under 85% RH (Fig. 3B). Water content of non-acclimated and acclimated seedlings under 100% RH for 6 h increased, but water content in acclimated seedlings remained lower than in the non-acclimated seedlings (Fig. 3E). A longer treatment of 100% RH for 12 h before chilling further decreased CR and increased water content of non-acclimated and acclimated seedlings. However, the pattern of daily changes of CR of those non-acclimated and acclimated seedlings remained similar to that of seedlings treated with 100% RH for 6 h before chilling exposure (Fig. 3C). Water content of non-acclimated and acclimated seedlings under 100% RH for 12 h increased and no daily changes in water content were noticed. Water content of acclimated seedlings was lower than that of non-acclimated seedlings (Fig. 3F).

The daily rhythm of CR as affected by low temperatures under low or high RH. The effects of 6-h and 12-h treatments with low temperatures under low or high RH on subsequent CR in cotton seedlings was examined at dif-

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ferent times during the LDC. In the following description of the results the reference points of "sensitive phase" and "resistant phase" are those occurring under the standard conditions (33 ~ C, 85% RH). Treatment of seedlings grown under standard conditions with 26 ~ C, 85% for 6 h or 12 h starting at various times during the LDC generally resulted in increased CR. Such treatments that ended during the sensitive phase induced CR to approximately the magnitude reached in the resistant phase. Treatments that ended at the time of the resistant phases did not induce additional CR (Fig. 4A). Treatments with 26~ under 100% RH for 6 h or 12 h, unlike treatments under 85% RH, did not induce CR at any time during the LDC and generally resulted in decreased CR. Such treatments that ended during the sensitive phase did not change the CR. Treatments ending at the beginning of the resistant phase substantially reduced CR to almost the level of the sensitive phase. Treatments ending at the end of the resistant phase also reduced CR. However, the level of that CR was still higher than that reached at the sensitive phase (Fig. 4D). Treatments of 19~ C for 6 h or 12 h under 85% or 100% RH starting at various times during the LDC affected CR similarly to comparable treatments of 26~ (data not shown). The effects of treatment of 12~ C for 6 h or 12 h under 85% or 100% RH starting at various times during the LDC on CR differed from comparable treatments of 26 ~ C and 19~ C. A treatment of 12~ C, 85% RH for 6 h started at the beginning of the sensitive phase did not induce CR and the seedlings remained sensitive even after 12 h when the resistant phase was reached. Treatments of 12~ C, 85% RH started at the middle of the light period slightly reduced CR 6 h or 12 h later when the resistant phase was reached. Seedlings treated with 12~ C,

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experiment, at 6-h intervals, cotyledon movement was monitored and seedlings were exposed to chilling of 5~ C, 85% RH for 1.5 d. After chilling, the seedlings were returned to 33 ~ C, 85% RH for 5 d and at the end of this period shoot FW was determined, v--q Light period; ~ dark period; ~ subjective night; 9 shoot FW; 9 angle; , 3 3 ~ 85% R H ; ,5~ 100% RH

A. Rikin: Temporal organization of chilling resistance 85 % R H starting at the beginning or middle o f the resistant phase remained resistant 6 h or 12 h later when the sensitive phase was reached (Fig. 4B). Treatments o f 12~ under 100% R H started at the beginning and the middle o f the sensitive phase and at the beginning o f the resistant phase affected C R similarly to comparable treatments under 85% RH. Treatments o f 12 ~ C, 100% R H started at the middle o f the resistant phase resulted in increased C R in the following sensitive phase 6 h later, but 12 h later at the end o f the sensitive phase, the CR declined (Fig. 4E). Treatments of 5 ~ C, 85% R H given anytime during the L D C arrested the immediate CR. Thus, treatments started at the sensitive or the resistant phase resulted in a sensitive or a resistant level, respectively, from that time on (Fig. 4C). Treatments of 5 ~ C under 100% R H given at various times o f the L D C affected CR similarly to comparable treatments under 85% R H except the treatment that started at the middle o f the sensitive phase. This treatment did not immediately arrest the sensitive phase and the resistant phase was reached 6 h later (Fig. 4F).

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100% R H affects the temporal organization of C R in cotton seedlings by inducing a phase shifting in the circadian system was tested. A treatment of 5 ~ C, 100% R H for 6 h was given during the last half of the light period or the last half o f the dark period. Then, the seedlings were maintained for several cycles under continuous light and phase shifts in CR and cotyledon movement were examined. A treatment o f 5~ C, 100% R H during the last half of the light period caused a phase delay o f 6 h in cotyledon movement and in the reset phase the vertical position was reached at the end of the subjective night. Also, this low-temperature treatment caused a phase delay of 6 h in CR and in the reset phase; CR developed in the middle of the subjective night. However, at the end of the low-temperature treatment which corresponded to the end of the light period, in a transient phase shifting, the seedlings reached their maximal CR similar to seedlings kept all the time under the standard conditions (Fig. 5A). A treatment o f 5~ C, 100% R H during the last half of the dark period did not cause phase shifting o f cotyledon movement or CR. However, at the end of the low-temperature treatment which corresponded to the end of the dark period, a transient phase shifting occurred in cotyledon movement and CR. Thus, at the end of the dark period, the cotyledons remained in vertical position and C R stayed at its peak (Fig. 5B). Discussion Environmental factors such as ambient R H and temperature affect the temporal organization of CR on two levels. Firstly, they intervene with the functioning of the circadian clock, i.e., inducing transient and stable phase shifts or arresting the clock movement. Secondly, they modify the overt expression of the rhythm, i.e., changing

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the degree of C R without changing the basic timing mechanism. The main effects of temperature and R H on the circadian clock and C R in cotton seedlings are summarized in Fig. 6. Low temperature s down to 26 ~ C do not induce any phase shifting. A lower temperature of 5 ~ C under 85% R H arrests the circadian clock at any time during the L D C (Rikin 1991). Thus, the rhythm of C R to 5 ~ C, 85% R H reflects the actual resistance at the time of the beginning o f the chilling. In this rhythm the seedlings are sensitive during most of the light period and resistant during most of the dark period. A temperature of 5~ C under 100 % arrests the circadian clock only at the begin-

522 ning o f the light and the beginning of the dark period. When such a temperature starts at the middle of the dark period it does not cause a stable phase shifting but in a transient phase shifting, at the end of the dark period the seedlings are resistant. When it starts at the middle of the light period it causes a stable phase delay but in a transient phase shifting, at the end of the light period the seedlings attain CR. Because of these effects, the resistant phase to 5~ C, 100% R H is longer than to 5~ C, 85% RH, starting already at the middle of the light period. Low temperatures down to 15~ under 85% R H induce CR in cotton seedlings. Usually, a 6-h exposure to the low acclimating temperature induces maximal CR (McMillan and Rikin 1990; Rikin 1991). Thus, a decrease from the standard growing temperature of 33 ~ C under 85% R H to 26 ~ C or 19~ C under 85% R H for 6 h or 12 h induces C R and the seedlings are resistant throughout the L D C irrespective of the circadian phase. Changes in R H prior to chilling affect the temporal organization of CR probably through changes in water content. A R H of 100% at 33 ~ C reduces CR. Also, low temperatures of 26 ~ C or 19 ~ C under 100% RH, unlike similar temperatures under 85% RH, do not induce acclimation at any time during the LDC. Rather, such conditions reduce CR during the resistant phase. In acclimated seedlings that are resistant throughout the LDC, 100% R H before chilling reinstates the sensitive phase and thus restores the rhythm. The persistence of the rhythm under 100 % RH, although with lower magnitude of CR, indicates that changes in water content before chilling exposure do not affect the circadian time mechanism but only the overt expression of the rhythm. One o f the pronounced symptoms o f chilling injury in many plant species is desiccation (Levitt 1980). Therefore, it is plausible that daily changes in chillinginduced desiccation may account at least in part for the daily changes in CR. However, it has been shown that in tomato seedlings the daily changes in CR are not related to daily change in stomatal conductance or to daily changes in root functioning (King and Reid 1987). In cotton seedlings chilled under 85% R H the highest or lowest water loss occurs during the time in the LDC when the seedlings are the least or the most resistant, respectively. These daily changes in chilling-induced water loss are probably affected by the known changes in root hydraulic conductance at low temperatures and the daily changes in stomate opening (Boyer 1985). Under 100% RH, no chilling-induced water loss occurs but the daily rhythm of CR still exists. Therefore, the daily changes in chilling-induced desiccation are not the sole cause for the daily changes in CR. Circadian rhythms are an adaptation to daily changes in environmental conditions such as light, dark and ambient temperatures (Biinning 1973). The circadian nature of the rhythm of C R ensures that resistance develops toward the end of the day when temperatures start to decrease. Then, if the night is cold, down to a certain temperature, the plants acquire CR toward the end of the

A. Rikin: Temporal organization of chilling resistance night and the beginning of the day by a fast acclimation mechanism. When lower temperatures are reached or start at the beginning or the middle of the night and continue into the day, the circadian rhythm is arrested at the resistant phase. A transient phase-advance caused by chilling of 5 ~ C, 100% R H starting at noon results in a resistant phase when otherwise a sensitive phase occurs, thus extending the resistant phase. This temporal organization in the response to chilling characterized in cotton seedlings renders an adaptive advantage to cotton and probably to other chilling-sensitive plants.

References Anderson, C.M., Wilkins, M.B. (1989) Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi. Planta 177, 456-469 Boyer, J.S. (1985) Water transport. Annu. Rev. Plant Physiol. 36, 473-516 B/inning, E. (1973) The physiological clock. Springer-Verlag, Berlin Couderchet, M., Koukkari, W.L. (1987) Daily variations in the sensitivity of soybean seedlings to low temperature. Chronobiol. Int. 4, 537-541 Edmunds, L.N., Jr. (1988) Cellular and molecular bases of biological clocks. Models and mechanisms for circadian timekeeping. Springer-Verlag, New York Graham, D., Patterson, B.D. (1982) Responses of plants to low, nonfreezing temperature: proteins, metabolism, and acclimation. Annu. Rev. Plant Physiol. 33, 347-372 Guinn, G. (1971) Chilling injury in cotton seedlings: Changes in permeability in cotyledons. Crop Sci. 11, 101-102 Inskeep, W.P., Bloom, P.R. (1985) Extinction coefficient of chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol. 77, 483-485 King, A.I., Reid, M.S. (1987) Diurnal sensitivity and desiccation in seedlings of tomato. J. Am. Soc. Hort. Sci. 112, 821-824 Levitt J. (1980) Responses of plants to environmental stresses, vol. I: Chilling, freezing and high temperature stresses. Academic Press, New York McMillan, K.D., Rikin, A. (1990) Relationships between circadian rhythm of chilling resistance and acclimation to chilling in cotton seedlings. Planta 182, 455-460 Njus, D., McMurry, L., Hastings, J.W. (1977) Conditionality of circadian rhythmicity: synergistic action of light and temperature. J. Comp. Physiol. 117, 335-344 Oltmanns, O. (1960) ~)ber den Einflul3 der Temperatur auf die endogene Tagesrhythmik und die Blfihinduktion bei der Kurtztagpflanze KalanchoY blossfeldiana. Planta 54, 233-264 Rikin, A. (1991) Temperature-induced phase shifting of circadian rhythms in cotton seedlings as affected by variations in chilling resistance. Planta 185, 407-414 Sweeney, B.M., Hastings, J.W. (1960) Effects of temperatures upon diurnal rhythms. Cold Spring Harbor Symp. Quant. Biol. 25, 87-104 Wheaton, T.A., Morris, L.L. (1967) Modification of chilling sensitivity by temperature conditining. Proc. Am. Soc. Hortic. Sci. 41, 529-533 Wilson, J.M. (1976) The mechanism of chill- and drought-hardening of Phaseolus vulgaris leaves. New Phytol. 76, 257-270 Wilson, J.M., Crawford, R.M.M. (1974) Leaf fatty-acid content in relation to hardening and chilling injury. J. Exp. Bot. 25, 121-131