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Developmental Regulation of Low-temperature Tolerance in Winter Wheat
Annals of Botany 87: 751±757, 2001 doi:10.1006/anbo.2001.1403, available online at http://www.idealibrary.com on
Developmental Regulation of Low-temperature Tolerance in Winter Wheat S . M A H FO O Z I , A . E . L I M I N and D . B . FOW L E R * Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5A8 Received: 15 December 2000 Returned for revision: 23 January 2001 Accepted: 9 February 2001 Published electronically: 5 April 2001 Vernalization and photoperiod genes have wide-ranging eects on the timing of gene expression in plants. The objectives of this study were to (1) determine if expression of low-temperature (LT) tolerance genes is developmentally regulated and (2) establish the interrelationships among the developmental stages and LT tolerance gene expression. LT response curves were determined for three photoperiod-sensitive LT tolerant winter wheat (Triticum aestivum L. em Thell) genotypes acclimated at 4 8C under 8 h short-day (SD) and 20 h long-day (LD) photoperiods from 0 to 112 d. Also, three de-acclimation and re-acclimation cycles were used that bridged the vegetative/reproductive transition point for each LD and SD photoperiod treatment. A vernalization period of 49 d at 4 8C was sucient for all genotypes to reach vernalization saturation as measured by minimum ®nal leaf number (FLN) and con®rmed by examination of shoot apices dissected from crowns that had been de-acclimated at 20 8C LD. Before the vegetative/reproductive transition, both the LD- and SD-treated plants were able to re-acclimate to similar LT50 (temperature at which 50 % of the plants are killed by LT stress) levels following de-acclimation at 20 8C. De-acclimation of LD plants after vernalization saturation resulted in rapid progression to the reproductive phase and limited ability to re-acclimate. The comparative development of the SD (non-¯owering-inductive photoperiod) de-acclimated plants was greatly delayed relative to LD plants, and this delay in development was re¯ected in the ability of SD plants to re-acclimate to a lower temperature. These observations con®rm the hypothesis that the point of transition to the reproductive stage is pivotal in the expression of LT tolerance genes, and the level and duration of LT acclimation are related to the stage of phenological development as regulated by vernalization # 2001 Annals of Botany Company and photoperiod requirements. Key words: Triticum aestivum L., wheat, low-temperature tolerance, vernalization, photoperiod, phenological development.
I N T RO D U C T I O N Photoperiod and vernalization requirements have a major in¯uence on plant development. Cereals are normally longday (LD) plants (Thomas and Vince-Prue, 1997) in which day length aects apical morphogenesis, leaf production and other developmental processes (Kirby, 1969). Shortday (SD) conditions extend the length of the vegetative phase by increasing the number of leaves and delaying the reproductive phase visualized by `double ridge' formation (Mahfoozi et al., 2000, 2001). LDs accelerate ¯oral initiation and heading by reducing the number of leaves. Vernalization is the acceleration of ¯owering by a cold treatment (Chouard, 1960). Exposure to temperature in the vernalization range (0±10 8C) shortens the vegetative phase by decreasing the number of leaves in cereals with a vernalization requirement (Wang et al., 1995). Low-temperature (LT) acclimation in cereals is a cumulative process that is initiated at approx. 10 8C (Fowler et al., 1999). The exposure of hardened plants to warm temperature results in rapid de-acclimation. The process of LT acclimation can then be restarted by exposure to temperatures within the LT acclimation range. Winter cereal plants gradually lose their ability to tolerate belowfreezing temperatures when they are maintained for long periods of time (i.e. over winter) at temperatures in the * For correspondence. Fax 001 306 966 5015, e-mail brian.fowler@ usask.ca
0305-7364/01/060751+07 $35.00/00
optimum range for LT acclimation. This over-winter decline in LT response is due to an inability of cereals to maintain LT tolerance genes in an up-regulated state once vernalization (Fowler et al., 1996a, b) and photoperiod (Mahfoozi et al., 2000, 2001) requirements have been satis®ed. Based on these observations, it is likely that any factor which in¯uences the length of the vegetative growth stage aects the expression of LT tolerance genes in cereals exposed to acclimating temperatures (Fowler et al., 1996b). Consequently, timing of the transition from the vegetative to reproductive phase is of fundamental interest not only in terms of ¯owering time but also in the regulation of LT gene expression (Fowler et al., 1999). The objectives of this study were to investigate the expression of LT tolerance in relation to plant development both before and after transition from the vegetative to the reproductive phase and to determine the interrelationships among photoperiod and vernalization requirements as they aect phenological development and LT tolerance gene expression. M AT E R I A L S A N D M E T H O D S The photoperiod sensitivity of the three LT tolerant winter wheat genotypes (`Cheyenne', `Norstar' and `Warrior') used in this study was veri®ed using ®nal leaf number (FLN) measurements on vernalized plants (49 d at 4 8C) that were moved to 20 8C LDs ( ¯oral-induction conditions) and SDs. The basic LT tolerance curves were established by subjecting # 2001 Annals of Botany Company
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Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance
the winter wheat genotypes to two photoperiod treatments (8 and 20 h day lengths) and nine acclimation periods at a constant 4 8C. Patterns of phenological development were established over ®ve vernalization times (0, 21, 49, 91 and 112 d). SD and LD treatments were used to accelerate or delay phenological development of the three genotypes during acclimation, de-acclimation and then re-acclimation before and after the vegetative/reproductive transition point as determined by FLN and stage of shoot apex development. LT tolerance Experimental design for the LT tolerance study was a three (genotypes) two ( photoperiods) 16 (acclimation, de-acclimation periods) factorial in a three replicate randomized complete block (RCBD). Seeds of `Cheyenne', `Norstar' and `Warrior' were placed on moist ®lter paper in petri dishes and imbibed in the dark at 4 8C for 48 h. Seeds were then germinated at a constant temperature of 20 8C in the dark for 24 h. Actively germinating seeds were transferred, embryo down, to plexiglass trays with holes backed by a 1.6 mm mesh screen (Fowler et al., 1983) and returned to germinating conditions until their roots were 1± 2 cm long. They were then placed in hydroponic tanks ®lled with continuously aerated half strength modi®ed Hoagland's solution (Brule-Babel and Fowler, 1988). Germinated seeds were then grown at 20 8C under 8 h (SD) or 20 h (LD) day lengths at a light intensity of 320 mmol m ÿ2 s ÿ1 for 14 d before being exposed to corresponding LD or SD conditions at 4 8C for LT acclimation. The procedure outlined by Limin and Fowler (1988) was used to determine LT50 (the temperature at which 50 % of the plants are killed by LT stress) of each hydroponically grown genotype at the end of each LT acclimation period in SD and LD treatments. Crowns were covered in moist sand in aluminium weighing cans and placed in a programmable freezer that was held at ÿ3 8C for 12 h. After 12 h, they were cooled at a rate of 2 8C h ÿ1 down to ±17 8C, then at a rate of 8 8C h ÿ1. Five crowns were removed from the freezer at 2 8C intervals for each of ®ve test temperatures selected for each genotype in each treatment. Samples were thawed overnight at 4 8C. Thawed crowns were transplanted into ¯ats containing `Redi-earth' for re-growth. The ¯ats were placed in a growth room maintained at 20 8C with a 20 h day/4 h night. Plant recovery was rated (alive vs. dead) after 3 weeks and LT50 was calculated for each sample. LT50 was determined for the three genotypes for nine 4 8C acclimation periods (0, 21, 35, 49, 56, 70, 77, 91 and 112 d) under LD and SD photoperiods. Three additional de-acclimation and re-acclimation cycles were also included for each LD and SD photoperiod. After 21, 56 and 77 d of LT acclimation, plants were de-acclimated at 20 8C for 14 d and then re-acclimated for 21 d at 4 8C under the same photoperiods used for LT acclimation. LT50 was determined after 14 d de-acclimation and 21 d re-acclimation following the 21 and 77 d acclimation treatments. LT50 was determined after 14 d de-acclimation and 7 and 21 d
re-acclimation following the 56 d acclimation treatment (Fig. 4). Phenological development Seeds were placed on moist ®lter paper in Petri dishes and imbibed in the dark at 4 8C for 48 h. Seeds were then germinated at a constant temperature of 20 8C in the dark for 24 h. Germinated seeds of the three genotypes were grown at 20 8C in 8 h (SD) or 20 h (LD) photoperiods at a light intensity of 320 mmol m ÿ2 s ÿ1 for 14 d in 15 cm pots (two plants per pot) ®lled with `Redi-earth'. They were then exposed to 4 8C LD or SD conditions for ®ve LT acclimation periods (0, 21, 49, 91 and 112 d) at a light intensity of 220 mmol m ÿ2 s ÿ1. The tops of pots were wrapped in aluminium foil to help prevent radiant heat absorption from the lights, and the plants were uniformly fertilized with `Osmocote' sustained-release fertilizer and a nutrient-complete (`Tune-up'2) water-soluble solution as required. Experimental design was a three (genotype) two ( photoperiod) ®ve (vernalization time) factorial in a three replicate RCBD. Also, two plants per pot from each genotype were grown under similar conditions to the FLN method (described below) to determine SD sensitivity. Plants were vernalized at 4 8C for 49 d and then moved to 20 8C under LD ( ¯oral-induction) and SD (non-inductive) conditions until the ®nal leaf emerged on the main shoot. FLN of LD and SD plants was compared. Two methods were used to determine the stage of phenological development. In the ®rst method, the time to vernalization saturation was estimated using the ®nal leaf number (FLN) procedure described by Wang et al. (1995). Both LD and SD vernalized plants were moved to LDs at 20 8C with a light intensity of 320 mmol m ÿ2 s ÿ1 after 0, 21, 49, 91 and 112 d acclimation. Leaves on the main shoot were numbered with a permanent marker until the ¯ag leaf emerged and FLN could be determined. Vernalization saturation was considered to have occurred when plants reached their minimum leaf number. In the second method, the stage of shoot apex development was determined on LD and SD crown samples of plants grown under the conditions for LT acclimation described above. A minimum of two hydroponically grown plants from each cultivar were sampled for dissection at each freezing test time in the acclimation and de-acclimation treatments to determine the phenological growth stages and identify the transition to the reproductive phase as indicated by `double ridge' formation in the shoot apex (Kirby and Appleyard, 1987; McMaster, 1997). Data analyses Analyses of variance were conducted to determine the level of signi®cance of dierences due to main eects and their interactions in each experiment. Where signi®cant dierences were identi®ed, regression analyses of treatment means were used to plot curves that best described the shape and behavior of the responses. The exponential decay linear combination equation was employed to describe the
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance relationship between FLN (y) and days of acclimation at 4 8C (x) for the 7 to 112 d periods. y y0 ae ÿbx cx
24
1
22
where a, b and c are constants. The peak four parameter Weibull equation was used to describe the relationship between LT50 (y) and days of acclimation at 4 8C (x).
h e
ÿ
ic 1 xÿx0 c ÿ1 c b c
2
cÿ1 c
Non-linear regression procedures outlined by SigmaPlot (2000) were used to provide least squares estimates of the regression coecients in these equations. R E S U LT S A N D D I S C U S S I O N Phenological development Analysis of variance for FLN indicated that genotypes, acclimation periods and the genotype acclimation period and photoperiod (PP) acclimation period interactions were signi®cant (P 5 0.01). The PP genotype interaction was not signi®cant for FLN indicating that `Cheyenne', `Warrior' and `Norstar' respond similarly to changes in photoperiod. Exposure to temperatures in the vernalization range shortens the vegetative phase by decreasing the FLN in genotypes with a vernalization response (Wang et al., 1995). In the present study, plants were moved to 20 8C LDs ( ¯oral-induction conditions) at the end of each acclimation (vernalization) period to determine the minimum FLN and response to vernalization. When grown at 4 8C for 49 d, the average leaf number for `Cheyenne', `Norstar' and `Warrior' was reduced from 21.0 to 11.9, 21.8 to 12.2 and 19 to 11.4, respectively (Fig. 1), indicating that these are winterhabit genotypes with a vernalization requirement. When vernalized plants (49 d at 4 8C) of the three winter wheat genotypes were moved to 20 8C under LD ( ¯oral-induction) and SD (non-inductive) conditions they produced an average of 12.5 leaves under LDs and 17.0 leaves under SDs indicating sensitivity to SD photoperiods. This con®rms earlier reports that `Cheyenne' (Scarth and Law, 1983), `Warrior' (Keim et al., 1973) and `Norstar' (Mahfoozi et al., 2001) are SD sensitive. The interaction between photoperiod and vernalization response determines the FLN on the main stem. FLN measurements indicated that vernalization saturation occurred at approximately the same time under both LDs and SDs (49 d) in the present study. LD plants did not increase their FLN following vernalization saturation indicating that they had received the signal to switch from the vegetative to the reproductive phase (Fig. 2). In contrast, a signi®cant (P 5 0.05) increase in FLN from 11.6 to 13.5 leaves between 49 and 112 d indicated that the non-inductive SD condition held back developmental
20 Final leaf number
1 ÿc " 1 #cÿ1 c ÿ 1 c x ÿ x0 cÿ1 c y a b c c
753
18
16
14
Cheyenne
Norstar
12
Warrior 10
0
14
70 84 28 42 56 Days of vernalization at 4 oC
98
112
F I G . 1. Mean ®nal leaf number [ pooled for long (LD 20 h) and short (SD 8 h) day treatments] of `Cheyenne', `Norstar' and `Warrior' winter wheat vernalized at 4 8C for 0±112 d and then moved to 20 8C LD ¯oral-induction conditions. s.e. 0.36. See Table 1 for regression coecients [eqn (1)].
T A B L E 1. Estimated regression coecients (exponential decay linear combination equation [eqn (1)] for 21±112 d) for ®nal leaf numbers of `Cheyenne', `Warrior' and `Norstar' wheat* (see Fig. 1) acclimated at 4 8C under both long (LD 20 h) and short (SD 8 h) days{ for 0±112 d and then moved to 20 8C LD (see Fig. 2) Regression coecients Treatment
a
b
c
y0
R2
`Cheyenne' `Warrior' `Norstar' LD SD
18.03 16.21 19.32 15.19 17.27
0.042 0.034 0.071 0.051 0.058
0.051 0.055 0.032 0.017 0.046
7.25 5.92 9.71 10.00 8.58
0.99 0.99 0.99 0.99 0.99
* Mean of LD and SD treatments. { Mean of `Cheyenne', `Warrior' and `Norstar'.
growth by delaying the reproductive transition of vernalized plants. This observation is consistent with earlier reports from similar studies on SD sensitive winter-habit wheat and barley genotypes (Mahfoozi et al., 2001). FLN measurements indicated that winter wheat plants did not reach vernalization saturation until approx. 49 d at 4 8C (Fig. 1). Dissection of de-acclimated (20 8C) LD
754
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance
22
20
Final leaf number
18
16
LD
14 SD 12
10
0
14
28
70 42 56 Days at 4 oC
84
98
112
F I G . 2. Mean ®nal leaf number ( pooled for `Cheyenne', `Norstar' and `Warrior') of winter wheat grown at 4 8C for 0±112 d under long (LD 20 h) and short (SD 8 h) days and then moved to 20 8C LD conditions. s.e. 0.30. See Table 1 for regression coecients [eqn (1)].
( ¯oral-inductive condition) shoot apices con®rmed that 21 d acclimated plants grown under both LDs and SDs were still in the vegetative phase (Fig. 3). When 56 d LDacclimated plants were de-acclimated, `double ridge' formation began within 9 d and the apices were fully reproductive within 2 weeks (56 d LD de-acclimated plants in Fig. 3). In contrast, 56 d SD-acclimated plants deacclimated for 2 weeks in 20 8C SD non-inductive conditions (Fig. 3) did not form `double ridges' within the 2 week de-acclimation period. However, when the SDacclimated plants were de-acclimated in 20 8C LDs ( ¯oral induction conditions) the shoot apices responded like those of the LD-treated plants and entered the reproductive phase within 2 weeks (data not shown). Therefore, plants grown under SDs were fully capable of reproductive transition but the SD photoperiod continued to delay development. Similarly, 77 d SD-acclimated plants only approached the reproductive phase as indicated by shoot elongation when de-acclimated for 2 weeks under SDs (Fig. 3) while those deacclimated under LDs had advanced to near the `terminal spikelet stage' (Kirby and Appleyard, 1987).
LT tolerance Analysis of variance of LT50 values of plants acclimated under LDs and SDs indicated that genotypes, acclimation periods, PP, and the PP acclimation period interaction
were highly signi®cant (P 5 0.01). The genotype PP interaction was not signi®cant indicating that the LT acclimation responses of `Cheyenne', `Norstar' and `Warrior' were similar under LD and SD conditions. Plants grown at 4 8C started to acclimate at a rapid rate under both LD and SD conditions (Fig. 4). As reported by Fowler et al. (1996b), the rate of change in LT tolerance then gradually slowed and eventually started to decline once vernalization saturation (transition from the vegetative to the reproductive phase) was achieved. Also, as previously shown (Mahfoozi et al., 2001), developmental dierences under LD and SD conditions were re¯ected in the LT tolerance of plants acclimated at a constant 4 8C. The dierences between LD and SD LT tolerance curves became progressively greater over time up to 112 d. The average LT50 for the three genotypes was ÿ17.3 and ÿ18.0 8C after 21 d of LT acclimation at 4 8C under LDs and SDs, respectively (Fig. 4). The LT50 of 21 d acclimated plants that were de-acclimated for 14 d at 20 8C was reduced to ÿ3.0 and ÿ7.0 8C under SDs and LDs, respectively. Both SD and LD plants were still in the vegetative phase (Figs 1 and 3) and were able to reacclimate to ÿ18.0 and ÿ17.5 8C, respectively, when grown at 4 8C for 21 d. LT50 of the re-acclimated plants was very similar to that of plants that had remained at 4 8C during the entire period (Fig. 4) indicating that plants which have not reached vegetative/reproductive transition retain their full ability to re-acclimate. The average LT50 for the three genotypes was ÿ18.0 and ÿ18.7 8C after 56 d of cold acclimation at 4 8C under LDs and SDs, respectively. The LT50 of 56 d acclimated plants that were de-acclimated for 14 d at 20 8C was reduced to ÿ2.3 and ÿ2.6 8C under SDs and LDs, respectively (Fig. 4). LD plants re-acclimated under LD conditions for 21 d at 4 8C had advanced to the reproductive phase and were only able to re-acclimate to ÿ7.0 8C, which is similar to the LT50 expected for a spring-habit wheat acclimated at 4 8C under LDs (Fowler et al., 1996b). In contrast, SD plants were still in the vegetative phase (56 d de-acclimated plants, Fig. 3) and were able to re-acclimate to ÿ13.7 8C, an LT50 similar to LD plants that had been maintained at 4 8C for the entire period (Fig. 4). The SD plants reacclimated from an LT50 of ÿ2.5 to ÿ11.9 8C in the ®rst 7 d they were held at 4 8C. The re-acclimation rate of the SD plants then slowed dramatically and an additional 14 d at 4 8C was required to decrease their LT50 by a further 2.1 8C, indicating that they were very near their minimum achievable LT50 after 3 weeks of re-acclimation. The 77 d LD- and SD-acclimated plants de-acclimated to an LT50 of ÿ2.0 8C after 14 d exposure to 20 8C. The LD plants were in the reproductive phase (Fig. 3) and only reacclimated to ÿ3.5 8C after 21 d at 4 8C. In contrast, the SD plants, which were only approaching the reproductive phase (elongated shoot apex, Fig. 3), were able to reacclimate to ÿ11.5 8C (Fig. 4). Photoperiod gene activity is higher at warmer temperatures (Rahman and Wilson, 1978; Yan and Wallace, 1996) and, as expected, plant responsiveness to both SD and LD photoperiods was much greater at the 20 8C de-acclimation temperature than at a constant 4 8C (Fig. 3; Mahfoozi et al.,
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance
755
F I G . 3. Apical development of 14 d de-acclimated `Norstar' winter wheat, before and after vernalization saturation time (49 d). The plants were acclimated at 4 8C under SD (8 h) and LD (20 h) photoperiods and de-acclimation treatments began 21, 56 and 77 d later under the same photoperiods at 20 8C.
2001). Plants that were de-acclimated before the transition to the reproductive phase were able to re-acclimate to similar LT50s under both LDs and SDs (Figs 3 and 4). In contrast, LD winter wheat plants had a decreasing ability to re-acclimate with advancing stages of development after vernalization saturation (de-acclimated at 20 8C after 56 and 77 d acclimation). The phenological development of the SD de-acclimated plants was greatly delayed compared to LD plants (Fig. 3). The SD photoperiod-inhibited development maintained the plants in a vegetative state and allowed re-acclimation levels to approach those of plants held at a constant 4 8C even after vernalization saturation. SD de-acclimated plants were phenologically more advanced than those held at a constant 4 8C SD because of a greater temperature-dependent development rate at 20 8C. This was coincidentally re¯ected in their LT tolerance being approximately equal to that of plants held at a constant 4 8C under LD conditions, which was slightly
more advanced than at constant 4 8C SD. These observations once again demonstrate that the stage of phenological development has a strong in¯uence on the level and duration of LT tolerance gene expression, and transition from the vegetative to the reproductive phase results in a down-regulation of the expression of LT tolerance genes. Developmental regulation of low-temperature tolerance Spring-habit photoperiod-sensitive genotypes grown under ¯oral-inductive conditions (LDs) reach their reproductive phase very quickly and are unable to develop a high level of LT tolerance (Fowler et al., 1996a, b). In contrast, there is an increased level and longer retention of LT tolerance due to a delay in the transition from the vegetative to the reproductive phase when photoperiod-sensitive cereals without a vernalization requirement (spring-habit genotypes) are grown under non-inductive (SD) ¯owering
756
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance 0 2 4 6
LT50
8 10 12 14 16 18
SD LD
20 0
14
70 84 28 42 56 Days of acclimation at 4 oC
98
112
F I G . 4. Mean low-temperature tolerance ( pooled for `Cheyenne', `Norstar' and `Warrior') of winter wheat acclimated under both long (LD 20 h; d, r, j, m) and short (SD 8 h; s, e, h, n) days at a constant 4 8C for 0±112 d (d, s). Plants were de-acclimated at 20 8C for 14 d after 21 (r, e), 56 (j, h) and 77 d (m, n) and then reacclimated at 4 8C for 21 d. An additional 7 d re-acclimation treatment was also used for 56 d de-acclimation/re-acclimation treatment. s.e. 0.50. See Table 2 for regression coecients [eqn (2)].
T A B L E 2. Estimated regression coecients {peak fourparameter Weibull equation [eqn (2)]} for average LT50 of `Cheyenne', `Warrior' and `Norstar' wheat acclimated at 4 8C under both long (LD 20 h) and short (SD 8 h) days for 0±112 d (see Fig. 4) Regression coecients Day length LD SD
a
b
c
x0
R2
ÿ18.56 ÿ19.19
98.5 110.1
1.40 1.37
40.49 42.51
0.99 0.99
conditions (Mahfoozi et al., 2000). A vernalization requirement in winter wheat extends the vegetative phase (Fig. 1) and allows the expression of LT tolerance genes for an extended period prior to the onset of severe LT stress (Figs 1 and 4; Fowler et al., 1996a; Mahfoozi et al., 2001). An understanding of this relationship has allowed for the construction of a ®eld-validated winter survival model that successfully simulates the over-winter changes in LT tolerance of a wide range of genotypes (Fowler et al., 1998, 1999). Results of our current experiments support the hypothesis that both Vrn/vrn and Ppd/ppd alleles regulate the expression of LT tolerance genes through their eect on the
rate of phenological development. They explain Stelmakh's (1998) ®eld observations which suggest that Vrn and Ppd genes accelerate developmental growth rates and reduce LT tolerance. This relationship also makes all LT tolerance associated characters or genes appear to be associated with developmental genes (Fowler et al., 1999) and explains the pleiotropic eects (growth habit and LT tolerance) attributed to developmental genes like vrn1 (Brule-Babel and Fowler, 1988; Sutka and Snape, 1989; Roberts, 1990). The vernalization saturation point was unaected by daylength in the present studies. However, non-inductive ¯owering conditions (SDs) delayed the development in plants exposed to LT by increasing FLN (Fig. 2). This delay, as indicated by shoot apex development under warm temperatures (Fig. 3), was re¯ected in longer retention of LT tolerance (Fig. 4; Mahfoozi et al., 2001). The greater LT tolerance of SD-treated winter wheat plants also indicates that the loss of LT tolerance of plants held at temperatures in the acclimation range is not normally due to day-length dependent photosynthate accumulation (Kohn and Levitt, 1965), depletion of energy reserves after over wintering (Levitt, 1972), or metabolic disturbances (Olien, 1967). Winter cereals have a vernalization requirement conditioned by the vrn alleles and acclimate upon exposure to LT. They continue to acclimate to LT until vernalization saturation, after which there is a loss of LT tolerance (Fowler et al., 1996b). The linkage of LT tolerance expression to phenological development adapts the plant to the environment for which it was selected or in which it evolved. For example, a high level of LT tolerance is no longer required after the onset of warm conditions in the spring when rapid growth and reproduction begin. Consequently, satisfaction of the vernalization requirement results in a decline in LT tolerance of over-wintering cereals (reviewed by Fowler et al., 1999). Dierences in the stage of phenological development provide an explanation for the observations that partially vernalized plants collected from the ®eld in the autumn have the ability to re-acclimate (Vincent, 1972; Gusta and Fowler, 1976a) while overwintered cereals ( presumably fully vernalized) are unable to re-acclimate in the spring (Gusta and Fowler, 1976b; Fowler and Gusta, 1977). A genotype (such as Dicktoo barley) with high sensitivity to SD photoperiods, but no vernalization requirement, can also achieve the maximum LT tolerance found in that species (Mahfoozi et al., 2000). In winter-habit genotypes, photoperiod sensitivity in¯uences LT tolerance gene expression even before vernalization saturation (Mahfoozi et al., 2001). This implies that the vernalization process is progressive and plant development can be in¯uenced by photoperiod during the vernalization process. Tolerance to LT stress is dependent upon the stage of phenological development and the point of transition to the reproductive stage is pivotal in the expression of LT tolerance genes (Figs 1, 3 and 4). However, the actual point of reproductive transition is not always clearly visible in the shoot apex morphology of plants grown at LTs (Fig. 3; Mahfoozi et al., 2001). The close association found between time to reproductive transition and loss of LT tolerance (Figs 1 and 3) supports the hypothesis (Fowler et al., 1996b) that plant response to LT acclimation is a function of the
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance degree and duration of expression of LT tolerance genes and developmental genes are the key factors responsible for the duration of expression of LT regulated genes. AC K N OW L E D GE M E N T S Financial support from the Agricultural Research, Education, and Extension Organization (AREEO) of the Agricultural Ministry of Iran is gratefully acknowledged. The excellent technical assistance of Garcia Schellhorn is greatly appreciated. L I T E R AT U R E C I T E D Brule-Babel AL, Fowler DB. 1988. Genetic control of cold hardiness and vernalization requirement in winter wheat. Crop Science 28: 879±884. Chouard P. 1960. Vernalization and its relation to dormancy. Annual Review of Plant Physiology 11: 191±238. Fowler DB, Gusta LV. 1977. The in¯uence of fall growth and development on cold tolerance of rye and wheat. Canadian Journal of Plant Science 57: 751±755. Fowler DB, Limin AE, Gusta LV. 1983. Breeding for winter hardiness in wheat. In: Fowler DB, Gusta LV, Slinkard AE, Hobin BA, eds. New frontiers in winter wheat production. Saskatoon, Canada: University of Saskatchewan. Printing Services. Fowler DB, Limin AE, Ritchie JT. 1998. Low-temperature tolerance in cereals. Vol. 1. Proceedings of 9th International Wheat Genetics Symposium. Saskatoon, Canada: University Extension Press. Fowler DB, Limin AE, Ritchie JT. 1999. Low-temperature tolerance in cereals: Model and genetic interpretation. Crop Science 39: 626±633. Fowler DB, Chauvin LP, Limin AE, Sarhan F. 1996a. The regulatory role of vernalization in the expression of low-temperature-induced genes in wheat and rye. Theoretical and Applied Genetics 93: 554±559. Fowler DB, Limin AE, Wang SY, Ward RW. 1996b. Relationship between low-temperature tolerance and vernalization in wheat and rye. Canadian Journal of Plant Science 76: 37±42. Gusta LV, Fowler DB. 1976a. Eects of temperature on dehardening and rehardening of winter cereals. Canadian Journal of Plant Science 56: 673±678. Gusta LV, Fowler DB. 1976b. Dehardening and rehardening of springcollected winter wheat and a winter rye. Canadian Journal of Plant Science 56: 775±779. Keim DL, Welsh JR, McConnell RL. 1973. Inheritance of photoperiodic heading response in winter and spring cultivars of bread wheat. Canadian Journal of Plant Science 53: 247±350. Kirby EJM. 1969. The eect of daylength upon the development and growth of wheat, barley and oats. Field Crop Abstracts 22: 1±7.
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Kirby EJM, Appleyard M. 1987. Cereal development guide. 2nd edn. Stoneleigh, Kenilworth, England: Arable Unit, National Agricultural Centre. Kohn H, Levitt J. 1965. Frost hardiness studies on cabbage grown under controlled conditions. Plant Physiology 40: 476±480. Levitt J. 1972. The response of the plants to environmental stresses. New York: Academic Press Inc. Limin AE, Fowler DB. 1988. Cold hardiness expression in interspeci®c hybrids and amphiploids of Triticeae. Genome 30: 361±365. McMaster GS. 1997. Phenology, development, and growth of wheat (Triticum aestivum L.) shoot apex: A review. Advances in Agronomy 59: 63±118. Mahfoozi S, Limin AE, Fowler DB. 2001. In¯uence of vernalization and photoperiod responses on cold hardiness in winter cereals. Crop Science (in press). Mahfoozi S, Limin AE, Hayes PM, Hucl P, Fowler DB. 2000. In¯uence of photoperiod response on the expression of cold hardiness in cereals. Canadian Journal of Plant Science 80: 721±724. Olien CR. 1967. Freezing stress and survival. Annual Review of Plant Physiology 18: 387±408. Rahman MS, Wilson JH. 1978. Determination of spikelet number in wheat. III. Eect of varying temperature on ear development. Australian Journal of Agricultural Research 29: 495±467. Roberts DWA. 1990. Identi®cation of loci on chromosome 5A of wheat involved in control of cold hardiness, vernalization, leaf length, rosette growth habit, and height of hardened plants. Genome 33: 247±259. Scarth R, Law CN. 1983. The control of day-length response in wheat by the group 2 chromosomes. Zeitschrift fuÈr P¯anzenzuchtung 92: 140±150. SigmaPlot. 2000. Regression equation library, SigmaPlot 2000 programming guide. Chicago, IL: SPSS Inc. Stelmakh AF. 1998. Regulation of ¯owering response genetic system aects the stress tolerance in bread wheat. Vol. 4. Proceedings of 9th International Wheat Genetics Symposium. Saskatoon, Canada: University Extension Press. Sutka J, Snape JW. 1989. Location of a gene for frost resistance on chromosome 5A of wheat. Euphytica 42: 41±44. Thomas B, Vince-Prue D. 1997. Photoperiodism in Plants. San Diego: Academic Press. Vincent A. 1972. Relationship between vernalization, hardening and phytochrome. In: Rajki S, ed. Proceedings of a colloquium on the winter hardiness in cereals. Marton-vaÂsaÂr, Hungary: Agricultural Research Institute, Hungarian Academy of Sciences. Wang SY, Ward RW, Ritchie JT, Fisher RA, Schulthess U. 1995. Vernalization in wheat I. A model based on the interchangeability of plant age and vernalization duration. Field Crop Research 41: 91±100. Yan W, Wallace DH. 1996. A model of photoperiod temperature interaction eects on plant development. Critical Reviews in Plant Sciences 15: 63±96.
Developmental Regulation of Low-temperature Tolerance in Winter Wheat S . M A H FO O Z I , A . E . L I M I N and D . B . FOW L E R * Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5A8 Received: 15 December 2000 Returned for revision: 23 January 2001 Accepted: 9 February 2001 Published electronically: 5 April 2001 Vernalization and photoperiod genes have wide-ranging eects on the timing of gene expression in plants. The objectives of this study were to (1) determine if expression of low-temperature (LT) tolerance genes is developmentally regulated and (2) establish the interrelationships among the developmental stages and LT tolerance gene expression. LT response curves were determined for three photoperiod-sensitive LT tolerant winter wheat (Triticum aestivum L. em Thell) genotypes acclimated at 4 8C under 8 h short-day (SD) and 20 h long-day (LD) photoperiods from 0 to 112 d. Also, three de-acclimation and re-acclimation cycles were used that bridged the vegetative/reproductive transition point for each LD and SD photoperiod treatment. A vernalization period of 49 d at 4 8C was sucient for all genotypes to reach vernalization saturation as measured by minimum ®nal leaf number (FLN) and con®rmed by examination of shoot apices dissected from crowns that had been de-acclimated at 20 8C LD. Before the vegetative/reproductive transition, both the LD- and SD-treated plants were able to re-acclimate to similar LT50 (temperature at which 50 % of the plants are killed by LT stress) levels following de-acclimation at 20 8C. De-acclimation of LD plants after vernalization saturation resulted in rapid progression to the reproductive phase and limited ability to re-acclimate. The comparative development of the SD (non-¯owering-inductive photoperiod) de-acclimated plants was greatly delayed relative to LD plants, and this delay in development was re¯ected in the ability of SD plants to re-acclimate to a lower temperature. These observations con®rm the hypothesis that the point of transition to the reproductive stage is pivotal in the expression of LT tolerance genes, and the level and duration of LT acclimation are related to the stage of phenological development as regulated by vernalization # 2001 Annals of Botany Company and photoperiod requirements. Key words: Triticum aestivum L., wheat, low-temperature tolerance, vernalization, photoperiod, phenological development.
I N T RO D U C T I O N Photoperiod and vernalization requirements have a major in¯uence on plant development. Cereals are normally longday (LD) plants (Thomas and Vince-Prue, 1997) in which day length aects apical morphogenesis, leaf production and other developmental processes (Kirby, 1969). Shortday (SD) conditions extend the length of the vegetative phase by increasing the number of leaves and delaying the reproductive phase visualized by `double ridge' formation (Mahfoozi et al., 2000, 2001). LDs accelerate ¯oral initiation and heading by reducing the number of leaves. Vernalization is the acceleration of ¯owering by a cold treatment (Chouard, 1960). Exposure to temperature in the vernalization range (0±10 8C) shortens the vegetative phase by decreasing the number of leaves in cereals with a vernalization requirement (Wang et al., 1995). Low-temperature (LT) acclimation in cereals is a cumulative process that is initiated at approx. 10 8C (Fowler et al., 1999). The exposure of hardened plants to warm temperature results in rapid de-acclimation. The process of LT acclimation can then be restarted by exposure to temperatures within the LT acclimation range. Winter cereal plants gradually lose their ability to tolerate belowfreezing temperatures when they are maintained for long periods of time (i.e. over winter) at temperatures in the * For correspondence. Fax 001 306 966 5015, e-mail brian.fowler@ usask.ca
0305-7364/01/060751+07 $35.00/00
optimum range for LT acclimation. This over-winter decline in LT response is due to an inability of cereals to maintain LT tolerance genes in an up-regulated state once vernalization (Fowler et al., 1996a, b) and photoperiod (Mahfoozi et al., 2000, 2001) requirements have been satis®ed. Based on these observations, it is likely that any factor which in¯uences the length of the vegetative growth stage aects the expression of LT tolerance genes in cereals exposed to acclimating temperatures (Fowler et al., 1996b). Consequently, timing of the transition from the vegetative to reproductive phase is of fundamental interest not only in terms of ¯owering time but also in the regulation of LT gene expression (Fowler et al., 1999). The objectives of this study were to investigate the expression of LT tolerance in relation to plant development both before and after transition from the vegetative to the reproductive phase and to determine the interrelationships among photoperiod and vernalization requirements as they aect phenological development and LT tolerance gene expression. M AT E R I A L S A N D M E T H O D S The photoperiod sensitivity of the three LT tolerant winter wheat genotypes (`Cheyenne', `Norstar' and `Warrior') used in this study was veri®ed using ®nal leaf number (FLN) measurements on vernalized plants (49 d at 4 8C) that were moved to 20 8C LDs ( ¯oral-induction conditions) and SDs. The basic LT tolerance curves were established by subjecting # 2001 Annals of Botany Company
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Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance
the winter wheat genotypes to two photoperiod treatments (8 and 20 h day lengths) and nine acclimation periods at a constant 4 8C. Patterns of phenological development were established over ®ve vernalization times (0, 21, 49, 91 and 112 d). SD and LD treatments were used to accelerate or delay phenological development of the three genotypes during acclimation, de-acclimation and then re-acclimation before and after the vegetative/reproductive transition point as determined by FLN and stage of shoot apex development. LT tolerance Experimental design for the LT tolerance study was a three (genotypes) two ( photoperiods) 16 (acclimation, de-acclimation periods) factorial in a three replicate randomized complete block (RCBD). Seeds of `Cheyenne', `Norstar' and `Warrior' were placed on moist ®lter paper in petri dishes and imbibed in the dark at 4 8C for 48 h. Seeds were then germinated at a constant temperature of 20 8C in the dark for 24 h. Actively germinating seeds were transferred, embryo down, to plexiglass trays with holes backed by a 1.6 mm mesh screen (Fowler et al., 1983) and returned to germinating conditions until their roots were 1± 2 cm long. They were then placed in hydroponic tanks ®lled with continuously aerated half strength modi®ed Hoagland's solution (Brule-Babel and Fowler, 1988). Germinated seeds were then grown at 20 8C under 8 h (SD) or 20 h (LD) day lengths at a light intensity of 320 mmol m ÿ2 s ÿ1 for 14 d before being exposed to corresponding LD or SD conditions at 4 8C for LT acclimation. The procedure outlined by Limin and Fowler (1988) was used to determine LT50 (the temperature at which 50 % of the plants are killed by LT stress) of each hydroponically grown genotype at the end of each LT acclimation period in SD and LD treatments. Crowns were covered in moist sand in aluminium weighing cans and placed in a programmable freezer that was held at ÿ3 8C for 12 h. After 12 h, they were cooled at a rate of 2 8C h ÿ1 down to ±17 8C, then at a rate of 8 8C h ÿ1. Five crowns were removed from the freezer at 2 8C intervals for each of ®ve test temperatures selected for each genotype in each treatment. Samples were thawed overnight at 4 8C. Thawed crowns were transplanted into ¯ats containing `Redi-earth' for re-growth. The ¯ats were placed in a growth room maintained at 20 8C with a 20 h day/4 h night. Plant recovery was rated (alive vs. dead) after 3 weeks and LT50 was calculated for each sample. LT50 was determined for the three genotypes for nine 4 8C acclimation periods (0, 21, 35, 49, 56, 70, 77, 91 and 112 d) under LD and SD photoperiods. Three additional de-acclimation and re-acclimation cycles were also included for each LD and SD photoperiod. After 21, 56 and 77 d of LT acclimation, plants were de-acclimated at 20 8C for 14 d and then re-acclimated for 21 d at 4 8C under the same photoperiods used for LT acclimation. LT50 was determined after 14 d de-acclimation and 21 d re-acclimation following the 21 and 77 d acclimation treatments. LT50 was determined after 14 d de-acclimation and 7 and 21 d
re-acclimation following the 56 d acclimation treatment (Fig. 4). Phenological development Seeds were placed on moist ®lter paper in Petri dishes and imbibed in the dark at 4 8C for 48 h. Seeds were then germinated at a constant temperature of 20 8C in the dark for 24 h. Germinated seeds of the three genotypes were grown at 20 8C in 8 h (SD) or 20 h (LD) photoperiods at a light intensity of 320 mmol m ÿ2 s ÿ1 for 14 d in 15 cm pots (two plants per pot) ®lled with `Redi-earth'. They were then exposed to 4 8C LD or SD conditions for ®ve LT acclimation periods (0, 21, 49, 91 and 112 d) at a light intensity of 220 mmol m ÿ2 s ÿ1. The tops of pots were wrapped in aluminium foil to help prevent radiant heat absorption from the lights, and the plants were uniformly fertilized with `Osmocote' sustained-release fertilizer and a nutrient-complete (`Tune-up'2) water-soluble solution as required. Experimental design was a three (genotype) two ( photoperiod) ®ve (vernalization time) factorial in a three replicate RCBD. Also, two plants per pot from each genotype were grown under similar conditions to the FLN method (described below) to determine SD sensitivity. Plants were vernalized at 4 8C for 49 d and then moved to 20 8C under LD ( ¯oral-induction) and SD (non-inductive) conditions until the ®nal leaf emerged on the main shoot. FLN of LD and SD plants was compared. Two methods were used to determine the stage of phenological development. In the ®rst method, the time to vernalization saturation was estimated using the ®nal leaf number (FLN) procedure described by Wang et al. (1995). Both LD and SD vernalized plants were moved to LDs at 20 8C with a light intensity of 320 mmol m ÿ2 s ÿ1 after 0, 21, 49, 91 and 112 d acclimation. Leaves on the main shoot were numbered with a permanent marker until the ¯ag leaf emerged and FLN could be determined. Vernalization saturation was considered to have occurred when plants reached their minimum leaf number. In the second method, the stage of shoot apex development was determined on LD and SD crown samples of plants grown under the conditions for LT acclimation described above. A minimum of two hydroponically grown plants from each cultivar were sampled for dissection at each freezing test time in the acclimation and de-acclimation treatments to determine the phenological growth stages and identify the transition to the reproductive phase as indicated by `double ridge' formation in the shoot apex (Kirby and Appleyard, 1987; McMaster, 1997). Data analyses Analyses of variance were conducted to determine the level of signi®cance of dierences due to main eects and their interactions in each experiment. Where signi®cant dierences were identi®ed, regression analyses of treatment means were used to plot curves that best described the shape and behavior of the responses. The exponential decay linear combination equation was employed to describe the
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance relationship between FLN (y) and days of acclimation at 4 8C (x) for the 7 to 112 d periods. y y0 ae ÿbx cx
24
1
22
where a, b and c are constants. The peak four parameter Weibull equation was used to describe the relationship between LT50 (y) and days of acclimation at 4 8C (x).
h e
ÿ
ic 1 xÿx0 c ÿ1 c b c
2
cÿ1 c
Non-linear regression procedures outlined by SigmaPlot (2000) were used to provide least squares estimates of the regression coecients in these equations. R E S U LT S A N D D I S C U S S I O N Phenological development Analysis of variance for FLN indicated that genotypes, acclimation periods and the genotype acclimation period and photoperiod (PP) acclimation period interactions were signi®cant (P 5 0.01). The PP genotype interaction was not signi®cant for FLN indicating that `Cheyenne', `Warrior' and `Norstar' respond similarly to changes in photoperiod. Exposure to temperatures in the vernalization range shortens the vegetative phase by decreasing the FLN in genotypes with a vernalization response (Wang et al., 1995). In the present study, plants were moved to 20 8C LDs ( ¯oral-induction conditions) at the end of each acclimation (vernalization) period to determine the minimum FLN and response to vernalization. When grown at 4 8C for 49 d, the average leaf number for `Cheyenne', `Norstar' and `Warrior' was reduced from 21.0 to 11.9, 21.8 to 12.2 and 19 to 11.4, respectively (Fig. 1), indicating that these are winterhabit genotypes with a vernalization requirement. When vernalized plants (49 d at 4 8C) of the three winter wheat genotypes were moved to 20 8C under LD ( ¯oral-induction) and SD (non-inductive) conditions they produced an average of 12.5 leaves under LDs and 17.0 leaves under SDs indicating sensitivity to SD photoperiods. This con®rms earlier reports that `Cheyenne' (Scarth and Law, 1983), `Warrior' (Keim et al., 1973) and `Norstar' (Mahfoozi et al., 2001) are SD sensitive. The interaction between photoperiod and vernalization response determines the FLN on the main stem. FLN measurements indicated that vernalization saturation occurred at approximately the same time under both LDs and SDs (49 d) in the present study. LD plants did not increase their FLN following vernalization saturation indicating that they had received the signal to switch from the vegetative to the reproductive phase (Fig. 2). In contrast, a signi®cant (P 5 0.05) increase in FLN from 11.6 to 13.5 leaves between 49 and 112 d indicated that the non-inductive SD condition held back developmental
20 Final leaf number
1 ÿc " 1 #cÿ1 c ÿ 1 c x ÿ x0 cÿ1 c y a b c c
753
18
16
14
Cheyenne
Norstar
12
Warrior 10
0
14
70 84 28 42 56 Days of vernalization at 4 oC
98
112
F I G . 1. Mean ®nal leaf number [ pooled for long (LD 20 h) and short (SD 8 h) day treatments] of `Cheyenne', `Norstar' and `Warrior' winter wheat vernalized at 4 8C for 0±112 d and then moved to 20 8C LD ¯oral-induction conditions. s.e. 0.36. See Table 1 for regression coecients [eqn (1)].
T A B L E 1. Estimated regression coecients (exponential decay linear combination equation [eqn (1)] for 21±112 d) for ®nal leaf numbers of `Cheyenne', `Warrior' and `Norstar' wheat* (see Fig. 1) acclimated at 4 8C under both long (LD 20 h) and short (SD 8 h) days{ for 0±112 d and then moved to 20 8C LD (see Fig. 2) Regression coecients Treatment
a
b
c
y0
R2
`Cheyenne' `Warrior' `Norstar' LD SD
18.03 16.21 19.32 15.19 17.27
0.042 0.034 0.071 0.051 0.058
0.051 0.055 0.032 0.017 0.046
7.25 5.92 9.71 10.00 8.58
0.99 0.99 0.99 0.99 0.99
* Mean of LD and SD treatments. { Mean of `Cheyenne', `Warrior' and `Norstar'.
growth by delaying the reproductive transition of vernalized plants. This observation is consistent with earlier reports from similar studies on SD sensitive winter-habit wheat and barley genotypes (Mahfoozi et al., 2001). FLN measurements indicated that winter wheat plants did not reach vernalization saturation until approx. 49 d at 4 8C (Fig. 1). Dissection of de-acclimated (20 8C) LD
754
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance
22
20
Final leaf number
18
16
LD
14 SD 12
10
0
14
28
70 42 56 Days at 4 oC
84
98
112
F I G . 2. Mean ®nal leaf number ( pooled for `Cheyenne', `Norstar' and `Warrior') of winter wheat grown at 4 8C for 0±112 d under long (LD 20 h) and short (SD 8 h) days and then moved to 20 8C LD conditions. s.e. 0.30. See Table 1 for regression coecients [eqn (1)].
( ¯oral-inductive condition) shoot apices con®rmed that 21 d acclimated plants grown under both LDs and SDs were still in the vegetative phase (Fig. 3). When 56 d LDacclimated plants were de-acclimated, `double ridge' formation began within 9 d and the apices were fully reproductive within 2 weeks (56 d LD de-acclimated plants in Fig. 3). In contrast, 56 d SD-acclimated plants deacclimated for 2 weeks in 20 8C SD non-inductive conditions (Fig. 3) did not form `double ridges' within the 2 week de-acclimation period. However, when the SDacclimated plants were de-acclimated in 20 8C LDs ( ¯oral induction conditions) the shoot apices responded like those of the LD-treated plants and entered the reproductive phase within 2 weeks (data not shown). Therefore, plants grown under SDs were fully capable of reproductive transition but the SD photoperiod continued to delay development. Similarly, 77 d SD-acclimated plants only approached the reproductive phase as indicated by shoot elongation when de-acclimated for 2 weeks under SDs (Fig. 3) while those deacclimated under LDs had advanced to near the `terminal spikelet stage' (Kirby and Appleyard, 1987).
LT tolerance Analysis of variance of LT50 values of plants acclimated under LDs and SDs indicated that genotypes, acclimation periods, PP, and the PP acclimation period interaction
were highly signi®cant (P 5 0.01). The genotype PP interaction was not signi®cant indicating that the LT acclimation responses of `Cheyenne', `Norstar' and `Warrior' were similar under LD and SD conditions. Plants grown at 4 8C started to acclimate at a rapid rate under both LD and SD conditions (Fig. 4). As reported by Fowler et al. (1996b), the rate of change in LT tolerance then gradually slowed and eventually started to decline once vernalization saturation (transition from the vegetative to the reproductive phase) was achieved. Also, as previously shown (Mahfoozi et al., 2001), developmental dierences under LD and SD conditions were re¯ected in the LT tolerance of plants acclimated at a constant 4 8C. The dierences between LD and SD LT tolerance curves became progressively greater over time up to 112 d. The average LT50 for the three genotypes was ÿ17.3 and ÿ18.0 8C after 21 d of LT acclimation at 4 8C under LDs and SDs, respectively (Fig. 4). The LT50 of 21 d acclimated plants that were de-acclimated for 14 d at 20 8C was reduced to ÿ3.0 and ÿ7.0 8C under SDs and LDs, respectively. Both SD and LD plants were still in the vegetative phase (Figs 1 and 3) and were able to reacclimate to ÿ18.0 and ÿ17.5 8C, respectively, when grown at 4 8C for 21 d. LT50 of the re-acclimated plants was very similar to that of plants that had remained at 4 8C during the entire period (Fig. 4) indicating that plants which have not reached vegetative/reproductive transition retain their full ability to re-acclimate. The average LT50 for the three genotypes was ÿ18.0 and ÿ18.7 8C after 56 d of cold acclimation at 4 8C under LDs and SDs, respectively. The LT50 of 56 d acclimated plants that were de-acclimated for 14 d at 20 8C was reduced to ÿ2.3 and ÿ2.6 8C under SDs and LDs, respectively (Fig. 4). LD plants re-acclimated under LD conditions for 21 d at 4 8C had advanced to the reproductive phase and were only able to re-acclimate to ÿ7.0 8C, which is similar to the LT50 expected for a spring-habit wheat acclimated at 4 8C under LDs (Fowler et al., 1996b). In contrast, SD plants were still in the vegetative phase (56 d de-acclimated plants, Fig. 3) and were able to re-acclimate to ÿ13.7 8C, an LT50 similar to LD plants that had been maintained at 4 8C for the entire period (Fig. 4). The SD plants reacclimated from an LT50 of ÿ2.5 to ÿ11.9 8C in the ®rst 7 d they were held at 4 8C. The re-acclimation rate of the SD plants then slowed dramatically and an additional 14 d at 4 8C was required to decrease their LT50 by a further 2.1 8C, indicating that they were very near their minimum achievable LT50 after 3 weeks of re-acclimation. The 77 d LD- and SD-acclimated plants de-acclimated to an LT50 of ÿ2.0 8C after 14 d exposure to 20 8C. The LD plants were in the reproductive phase (Fig. 3) and only reacclimated to ÿ3.5 8C after 21 d at 4 8C. In contrast, the SD plants, which were only approaching the reproductive phase (elongated shoot apex, Fig. 3), were able to reacclimate to ÿ11.5 8C (Fig. 4). Photoperiod gene activity is higher at warmer temperatures (Rahman and Wilson, 1978; Yan and Wallace, 1996) and, as expected, plant responsiveness to both SD and LD photoperiods was much greater at the 20 8C de-acclimation temperature than at a constant 4 8C (Fig. 3; Mahfoozi et al.,
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance
755
F I G . 3. Apical development of 14 d de-acclimated `Norstar' winter wheat, before and after vernalization saturation time (49 d). The plants were acclimated at 4 8C under SD (8 h) and LD (20 h) photoperiods and de-acclimation treatments began 21, 56 and 77 d later under the same photoperiods at 20 8C.
2001). Plants that were de-acclimated before the transition to the reproductive phase were able to re-acclimate to similar LT50s under both LDs and SDs (Figs 3 and 4). In contrast, LD winter wheat plants had a decreasing ability to re-acclimate with advancing stages of development after vernalization saturation (de-acclimated at 20 8C after 56 and 77 d acclimation). The phenological development of the SD de-acclimated plants was greatly delayed compared to LD plants (Fig. 3). The SD photoperiod-inhibited development maintained the plants in a vegetative state and allowed re-acclimation levels to approach those of plants held at a constant 4 8C even after vernalization saturation. SD de-acclimated plants were phenologically more advanced than those held at a constant 4 8C SD because of a greater temperature-dependent development rate at 20 8C. This was coincidentally re¯ected in their LT tolerance being approximately equal to that of plants held at a constant 4 8C under LD conditions, which was slightly
more advanced than at constant 4 8C SD. These observations once again demonstrate that the stage of phenological development has a strong in¯uence on the level and duration of LT tolerance gene expression, and transition from the vegetative to the reproductive phase results in a down-regulation of the expression of LT tolerance genes. Developmental regulation of low-temperature tolerance Spring-habit photoperiod-sensitive genotypes grown under ¯oral-inductive conditions (LDs) reach their reproductive phase very quickly and are unable to develop a high level of LT tolerance (Fowler et al., 1996a, b). In contrast, there is an increased level and longer retention of LT tolerance due to a delay in the transition from the vegetative to the reproductive phase when photoperiod-sensitive cereals without a vernalization requirement (spring-habit genotypes) are grown under non-inductive (SD) ¯owering
756
Mahfoozi et al.ÐDevelopmental Regulation of Low-temperature Tolerance 0 2 4 6
LT50
8 10 12 14 16 18
SD LD
20 0
14
70 84 28 42 56 Days of acclimation at 4 oC
98
112
F I G . 4. Mean low-temperature tolerance ( pooled for `Cheyenne', `Norstar' and `Warrior') of winter wheat acclimated under both long (LD 20 h; d, r, j, m) and short (SD 8 h; s, e, h, n) days at a constant 4 8C for 0±112 d (d, s). Plants were de-acclimated at 20 8C for 14 d after 21 (r, e), 56 (j, h) and 77 d (m, n) and then reacclimated at 4 8C for 21 d. An additional 7 d re-acclimation treatment was also used for 56 d de-acclimation/re-acclimation treatment. s.e. 0.50. See Table 2 for regression coecients [eqn (2)].
T A B L E 2. Estimated regression coecients {peak fourparameter Weibull equation [eqn (2)]} for average LT50 of `Cheyenne', `Warrior' and `Norstar' wheat acclimated at 4 8C under both long (LD 20 h) and short (SD 8 h) days for 0±112 d (see Fig. 4) Regression coecients Day length LD SD
a
b
c
x0
R2
ÿ18.56 ÿ19.19
98.5 110.1
1.40 1.37
40.49 42.51
0.99 0.99
conditions (Mahfoozi et al., 2000). A vernalization requirement in winter wheat extends the vegetative phase (Fig. 1) and allows the expression of LT tolerance genes for an extended period prior to the onset of severe LT stress (Figs 1 and 4; Fowler et al., 1996a; Mahfoozi et al., 2001). An understanding of this relationship has allowed for the construction of a ®eld-validated winter survival model that successfully simulates the over-winter changes in LT tolerance of a wide range of genotypes (Fowler et al., 1998, 1999). Results of our current experiments support the hypothesis that both Vrn/vrn and Ppd/ppd alleles regulate the expression of LT tolerance genes through their eect on the
rate of phenological development. They explain Stelmakh's (1998) ®eld observations which suggest that Vrn and Ppd genes accelerate developmental growth rates and reduce LT tolerance. This relationship also makes all LT tolerance associated characters or genes appear to be associated with developmental genes (Fowler et al., 1999) and explains the pleiotropic eects (growth habit and LT tolerance) attributed to developmental genes like vrn1 (Brule-Babel and Fowler, 1988; Sutka and Snape, 1989; Roberts, 1990). The vernalization saturation point was unaected by daylength in the present studies. However, non-inductive ¯owering conditions (SDs) delayed the development in plants exposed to LT by increasing FLN (Fig. 2). This delay, as indicated by shoot apex development under warm temperatures (Fig. 3), was re¯ected in longer retention of LT tolerance (Fig. 4; Mahfoozi et al., 2001). The greater LT tolerance of SD-treated winter wheat plants also indicates that the loss of LT tolerance of plants held at temperatures in the acclimation range is not normally due to day-length dependent photosynthate accumulation (Kohn and Levitt, 1965), depletion of energy reserves after over wintering (Levitt, 1972), or metabolic disturbances (Olien, 1967). Winter cereals have a vernalization requirement conditioned by the vrn alleles and acclimate upon exposure to LT. They continue to acclimate to LT until vernalization saturation, after which there is a loss of LT tolerance (Fowler et al., 1996b). The linkage of LT tolerance expression to phenological development adapts the plant to the environment for which it was selected or in which it evolved. For example, a high level of LT tolerance is no longer required after the onset of warm conditions in the spring when rapid growth and reproduction begin. Consequently, satisfaction of the vernalization requirement results in a decline in LT tolerance of over-wintering cereals (reviewed by Fowler et al., 1999). Dierences in the stage of phenological development provide an explanation for the observations that partially vernalized plants collected from the ®eld in the autumn have the ability to re-acclimate (Vincent, 1972; Gusta and Fowler, 1976a) while overwintered cereals ( presumably fully vernalized) are unable to re-acclimate in the spring (Gusta and Fowler, 1976b; Fowler and Gusta, 1977). A genotype (such as Dicktoo barley) with high sensitivity to SD photoperiods, but no vernalization requirement, can also achieve the maximum LT tolerance found in that species (Mahfoozi et al., 2000). In winter-habit genotypes, photoperiod sensitivity in¯uences LT tolerance gene expression even before vernalization saturation (Mahfoozi et al., 2001). This implies that the vernalization process is progressive and plant development can be in¯uenced by photoperiod during the vernalization process. Tolerance to LT stress is dependent upon the stage of phenological development and the point of transition to the reproductive stage is pivotal in the expression of LT tolerance genes (Figs 1, 3 and 4). However, the actual point of reproductive transition is not always clearly visible in the shoot apex morphology of plants grown at LTs (Fig. 3; Mahfoozi et al., 2001). The close association found between time to reproductive transition and loss of LT tolerance (Figs 1 and 3) supports the hypothesis (Fowler et al., 1996b) that plant response to LT acclimation is a function of the
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