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Effects of Interactions Between Low and High Temperature Treatments on Flowering of Spring Rape (Brassica napusvar.annua)
Annals of Botany 81 : 609–617, 1998
Effects of Interactions Between Low and High Temperature Treatments on Flowering of Spring Rape (Brassica napus var. annua) S. R. D A H A N A Y A KE and N. W. G A L W E Y Department of Plant Sciences, Faculty of Agriculture, The Uniersity of Western Australia, Nedlands, W.A. 6907, Australia Received : 7 November 1997
Returned for revision : 10 December 1997
Accepted : 26 January 1998
Exposure to high temperature (30 °C) before or after exposure to low temperature (0, 4 or 8 weeks at 4 °C) consistently increased the number of leaf nodes at flowering and delayed flowering in a range of genotypes of spring rape (Brassica napus var. annua L.). Four days of prior exposure to high temperature had more effect than 2 d, and the effect of subsequent exposure to high temperature was maximized when exposure commenced 1 week after the end of the low-temperature treatment. In genotypes that showed a vernalization response (i.e. in which the number of leaf nodes at flowering was reduced or flowering was advanced by low temperature), this response was reduced or eliminated by either prior high-temperature treatment (antivernalization) or subsequent high-temperature treatment (devernalization). A biochemical model to account for these effects is proposed. # 1998 Annals of Botany Company Key words : Brassica napus var. annua, spring rape, antivernalization, devernalization, vernalization.
INTRODUCTION
MATERIALS AND METHODS
The delay or prevention of flower induction and floral development by exposure to high temperatures before the application of a low-temperature stimulus that would otherwise promote flowering (antivernalization) or after such a stimulus (devernalization) has been demonstrated in a number of plant species including Brassica pekinensis (Louv.) Rupr. L. (Elers and Wiebe, 1984), Brassica oleracea convar. acephala var. gongylodes L. (Wiebe, Habegger and Liebig, 1992), Arabidopsis thaliana L. (Napp-Zinn, 1969) and Secale cereale ‘ Petkus ’ (winter rye) (Purvis and Gregory, 1952). However, although flowering is known to be promoted by exposure to low temperatures during vegetative development in many genotypes of Brassica napus, including spring types (B. napus var. annua), the interaction between such vernalizing stimuli and prior or subsequent exposure to high temperatures has not been investigated in this species. In B. pekinensis (Louv.) Rupr. high temperature prior to vernalization caused a delay in bolting (the rapid extension of the flowering stalk) and an increase in the number of leaves at flowering (Elers and Wiebe, 1984). In A. thaliana, another member of the family Brassicaceae, both anti- and devernalization responses have been demonstrated, and even the ability to be re-vernalized after devernalization, suggesting the involvement of thermolabile and thermostable intermediate products in the vernalization process (Napp-Zinn, 1969). In this paper we explore the effect of high-temperature treatments prior and subsequent to the application of a lowtemperature treatment on a range of genotypes of B. napus var. annua that vary in the extent of their vernalization response, and adapt the biochemical models proposed for other species in order to explain these effects.
Five inbred-backcross genotypes of Brassica napus var. annua, designated TB 14, TB 16, TB 28, TB 29 and TB 33, were used in this study. These were derived from a cross between the B. napus var. annua cultivars ‘ Target ’ (vernalization insensitive) and ‘ Bronowski ’ (vernalization sensitive ; Thurling and Vijendra Das, 1977) through two backcrosses to ‘ Target ’, and 14 subsequent generations of selfing, and were chosen for the diversity of their vernalization responses (Table 1).
0305-7364}98}05060909 $25.00}0
Experiment to detect antiernalization The seeds of four genotypes (TB 33 was omitted as insufficient quantities were available) were imbibed in 1200 KPa (302±44 g l−") polyethylene glycol (PEG). This treatment allows germination to commence but, because of the osmotic potential of the medium, does not allow morphological development to proceed (Ells, 1963 ; Heydecker, 1977 ; Bewley and Black, 1985). While development is arrested, treatments that affect its subsequent course can be applied, and their effects can thus be observed unconfounded by development during the treatment period. The seeds were then exposed to a temperature of 30 °C in the dark for 0, 2 or 4 d. They were then exposed to a temperature of 4 °C in the dark for 0, 4 or 8 weeks, each combination of high- and low-temperature treatment being applied to 30 seeds of each genotype. The seeds were then allowed to germinate by transferring them from the PEG solution to sterile water and the seedlings were transplanted to 4 cmsquare plastic folders (‘ pots ’), one seedling to each pot.
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# 1998 Annals of Botany Company
610
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering T 1. Vernalization responses of the lines used in the present inestigation and their parent lines
Number of leaves Control Low-temperature treated Number of days to flower Control Low-temperature treated
Target*
Bronowski†
TB 14†
TB 16†
TB 28†
TB 29†
TB 33†
7 6
9 7
16±9 14±8
15±8 13±8
9±2 8±9
16±1 13±4
15±3 13±6
54 51
74 59
98±1 52±2
63±5 45±3
56±1 43±2
68±1 45±8
66±5 44±9
* Data adapted from Thurling and Vijendra Das (1977). Treated plants were exposed to 3³1 °C for 6 weeks and were subsequently grown at 15 °C in a 24 h photoperiod. † Unpub. res. Treated plants were exposed to 4 °C for 6 weeks and were subsequently grown at 15 °C in an 18 h photoperiod. The s.e. were as follows : numbers of leaves, between 0±5 and 0±39 ; numbers of days to flower, between 0±12 and 1±54.
T 2. Contrasts among leels of temperature treatment factors Low-temperature treatment (weeks)
Treatment factor Level Coefficient of contrast
0 ®2
4 1
8 1
Prior high-temperature treatment (d) 0 ®2
2 1
4 1
Interval before subsequent high-temperature treatment (weeks) 0 1
1 1
2 1
— ®3
Experiment to detect deernalization
Statistical methods
Seeds of all five genotypes were imbibed in 1200 KPa PEG and then exposed to a temperature of 4 °C in the dark as described above for 0, 4 or 8 weeks, after which they were allowed to germinate and the seedlings were transplanted to pots. They were then exposed to a temperature of 30 °C for 7 d either (a) immediately after low temperature treatment ; (b) 1 week after low temperature treatment ; (c) 2 weeks after low temperature treatment ; or (d) control : no heat treatment. Each combination of low temperature and high temperature treatment was applied to 30 seeds of each genotype. In both experiments, the plants were grown in controlled environment rooms under an 18 h photoperiod. The main light period of 10 h was provided by Na-vapour and metal halide lamps giving a radiation level of 105 µE m−# s−" at the pot surface. Saturating long photoperiods (18 h) were obtained with a 75 W incandescent lamp (7±5 µE m−# s−"). The post-vernalization temperature was maintained at 15 °C. A higher post-vernalization temperature and a shorter photoperiod have been shown to maximize the response to vernalization (Thurling and Vijendra Das, 1977), but it was thought that such an environment might nevertheless not produce a clear expression of the response to prior or subsequent high-temperature treatment. In each experiment the pots, supported in iron frames, were arranged in a splitplot design of three blocks with high- and low-temperature treatments as main plot factors and genotype as sub-plot factor. Within each sub-plot there were ten plants. The number of leaf nodes on the main stem at flowering and the number of days from germination (i.e. transfer from the PEG solution) to the opening of the first flower were recorded for each plant.
Analysis of variance was performed to determine the effect on each variable measured of prior (or subsequent) high-temperature treatment, low-temperature treatment, genotype and their interactions. If genotype¬temperature interactions occurred, separate analyses of variance were performed for each genotype in order to characterize individual responses to the high- and low-temperature stimuli. In each analysis of variance the effects of the highand low-temperature treatments were partitioned between the effects of the contrasts specified in Table 2 and the deviations from these effects. Interactions between these contrasts provide straightforward measures of the antivernalization and devernalization responses : that is, of the change in the magnitude of the response to a lowtemperature stimulus (regardless of the duration of the stimulus) caused by exposure to a high-temperature stimulus (regardless of the duration or timing of the stimulus).
RESULTS Experiment to detect antiernalization Analysis of variance for the number of leaf nodes at flowering revealed that there were highly significant differences in response to prior high-temperature treatments, low-temperature treatments and between genotypes. All the two-way interactions between these factors were highly significant, but the three-way interaction was non-significant (data not shown). Separate analyses of variance for each genotype (Table 3) showed highly significant effects of prior high temperature in all genotypes, and in all except TB 28 these were largely accounted for by the contrast between the
611
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
T 3. Analyses of ariance of the effects of prior high-temperature treatment and low-temperature treatment on number of leaf nodes at flowering in each genotype Mean squares Number of leaf nodes at flowering Source
d.f.
TB 14
TB 16
TB 28
TB 29
Prior high T (P) Contrast Deviation Low T (L) Contrast Deviation P¬L Contrast¬contrast Deviations Residual
2 1 1 2 1 1 4 1 3 18
244±54*** 472±27*** 16±81*** 14±06*** 24±92*** 3±20*** 3±75*** 13±56*** 0±48* 0±11
286±47*** 500±74*** 72±20*** 2±07 3±59* 0±56 1±19 1±27 1±17 0±64
15±24*** 13±07*** 17±42*** 0±54 1±07 0±02 0±24 0±41 0±18 0±40
27±13*** 46±23*** 8±02*** 5±56*** 9±87*** 1±25 0±77 2±05* 0±35 0±33
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
TB 14
17
15
15
13
13
11
11
= 0.1904
Number of leaf nodes
9
7
9
0
4
2 TB 28
17
7
17
15
15
13
13
11
11
9
9
7
TB 16
17
0
2
0
2
4
2
4
TB 29
7 4 0 Prior high temp. treatment (d)
F. 1. Effect of antivernalization on number of leaf nodes at flowering of Brassica napus var. annua genotypes. Bar represents s.e. of differences of means with different prior high temperature, low temperature combinations. E, No low temperature ; +, 4 weeks low temperature ; _, 8 weeks low temperature.
control and the other treatments. There were highly significant effects of low temperature in genotypes TB 14 and TB 29, and, although the overall effect of low temperature was not significant in TB 16, the contrast between
the control and the other treatments was. The prior high temperature¬low temperature interaction was highly significant in TB 14, and most of this interaction was accounted for by the interaction between the contrasts, that is, by a
612
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
T 4. Analyses of ariance of the effects of prior high-temperature treatment and low-temperature treatment on time to flowering in each genotype Mean squares Time to flowering (d) Source
d.f.
TB 14
TB 16
TB 28
TB 29
Prior high T (P) Contrast Deviation Low T (L) Contrast Deviation P¬L Contrast¬contrast Deviations Residual
2 1 1 2 1 1 4 1 3 18
2282±8*** 3557±4*** 1008±2** 337±2 660±0* 14±5 178±8 667±4* 16±0 118±7
610±0 968±0* 252±1 688±1 821±4 554±8 74±0 114±1 60±6 206±0
155±1 219±1 91±0 101±0 136±0 66±0 6±4 0±0 8±5 72±2
1811±0*** 1224±0*** 2398±1*** 105±7 138±0 73±5 13±3 50±7 0±8 39±7
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
straightforward antivernalization effect. In TB 29, although the overall interaction was not significant, the interaction between contrasts was. All genotypes responded to prior high-temperature treatment by producing more leaf nodes at flowering, regardless of the low-temperature treatment, with exposure to high temperature for 4 d having a greater effect than 2 d (Fig. 1). However, this trend was much more marked in TB 14 and TB 16 than in the other two genotypes, and in these genotypes the additional effect of 2 further days of exposure was relatively slight (corresponding to the small proportion of variance accounted for by deviation from the contrast in this term of the ANOVA). In TB 14, exposure to low temperature in the absence of prior high temperature clearly decreased the number of leaves, with exposure for 8 weeks having a slightly greater effect than 4 weeks. Prior high temperature largely, though perhaps not completely, eliminated this effect : that is, this genotype showed a clear antivernalization response. [Previous experiments with the same genotypes showed that TB 14 was amongst the most responsive to vernalization in terms of the number of leaf nodes at flowering, and the most responsive in terms of the time to flowering (Table 1).] In TB 29 the same pattern was observed, although not so strongly, and there was a nonsignificant suggestion of the same pattern in TB 16. The overall analysis of variance for time to flowering showed that there were highly significant differences in responses to prior high-temperature treatments, low-temperature treatments and between genotypes. The prior high temperature¬genotype interaction was highly significant, but the other interactions were non-significant (data not shown). Separate analysis of variance for each genotype (Table 4) showed that the effect of prior high temperature was highly significant in TB 14 and TB 29. In TB 16, although the overall effect of prior high temperature was not significant, the contrast between the control and the other treatments was. The overall effect of low temperature was not significant in any individual case, although in TB 14 the contrast between the control and the other treatments was. The only significant interaction term was the interaction between the contrasts in TB 14.
In all genotypes except TB 28, the prior high-temperature treatment increased the time to flowering, the longer treatment having a greater effect (Fig. 2). In TB 14, the time to flowering was reduced by low-temperature treatment. There was a non-significant suggestion of the same pattern in the other genotypes, with the exception of TB 28, corresponding to the highly significant main effect of lowtemperature treatment in the combined ANOVA of all genotypes. In TB 14, 8 weeks’ exposure to low temperature had an only slightly greater effect than 4 weeks. In this genotype, the effect of low temperature was almost completely overcome by 2 d of prior high-temperature treatment : that is, TB 14 again showed a clear antivernalization response.
Experiment to detect deernalization Analysis of variance for the number of leaf nodes at flowering revealed that there were highly significant differences in response to subsequent high-temperature treatments, low-temperature treatments, and between genotypes. The genotype¬subsequent high temperature interaction was highly significant, but neither the other two-way interactions nor the three-way interaction were significant (data not shown). Separate analysis of variance for each genotype (Table 5) showed a highly significant effect of subsequent high-temperature treatment in all genotypes except TB 28 (for which this effect was nearly significant : P ¯ 0±062), and in all cases this effect was largely due to the contrast between the control and the other treatments. The effect of low temperature was highly significant in TB 28, TB 29 and TB 33, and in all cases this effect was entirely attributable to the contrast between the control and the other treatments. This contrast was also significant in TB 14. In no case was the subsequent high temperature¬low temperature interaction significant. For all genotypes with the possible exception of TB 28, and for all low-temperature treatments, the subsequent high-temperature treatment increased the number of leaf nodes at flowering, and this effect reached a maximum when
613
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
Time taken for first open flower to appear (d)
TB 14
TB 16
75
75
65
65
55
55 = 1.8985
45
35
45
0
4
2
35
0
TB 28
75
65
65
55
55
45
45
0
4
2
4
TB 29
75
35
2
2
35 4 0 Prior high temp. treatment (d)
F. 2. Effect of antivernalization on time to flowering of Brassica napus var. annua genotypes. For legend see Fig. 1.
T 5. Analyses of ariance of the effects of subsequent high-temperature treatment and low-temperature treatment on number of leaf nodes at flowering in each genotype Mean squares Number of leaf nodes at flowering Source
d.f.
TB 14
TB 16
TB 28
TB 29
TB 33
Subsequent high T (S) Contrast Deviation Low T (L) Contrast Deviation S¬L Contrast¬contrast Deviations Residual
3 1 2 2 1 1 6 1 5 24
50±07*** 80±12*** 35±05*** 5±11 7±80* 2±41 1±20 4±16 0±61 1±77
36±92** 95±42*** 7±67 2±42 3±21 1±63 0±80 0±45 0±88 4±93
3±34 4±63 2±70 14±04*** 25±07*** 3±01 0±02 0±02 0±02 1±19
73±84*** 192±01*** 14±76** 10±47** 16±90** 4±03 0±47 0±63 0±43 1±67
70±55*** 181±42*** 15±12*** 7±07** 13±61** 0±53 0±42 0±09 0±49 1±22
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
it was applied 1 week after the end of low-temperature treatment (Fig. 3). In all genotypes except TB 16, low temperature reduced the number of leaf nodes at flowering and in TB 14 there was a suggestion that this effect was eliminated by all the high-temperature treatments, although, as noted, the corresponding contrast was non-significant.
Analysis of variance of time to flowering revealed highly significant differences between subsequent high-temperature treatments, low-temperature treatments and genotypes. The genotype¬subsequent high temperature and genotype¬low temperature interactions were highly significant, and the three-way interaction was also significant (data not
614
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering TB 14
TB 16
15
15
13
13
11
11 = 0.5364
9
7
9
0
2 3 (no high temp.)
1
7
0
Number of leaf nodes
TB 28 15
13
13
11
11
9
9
0
2 3 (no high temp.)
1
2 3 (no high temp.)
TB 29
15
7
1
2 3 (no high temp.)
1
7
0
TB 33 15
13
11
9
7
0 2 3 (no high temp.) 1 Interval before subsequent high temp. treatment (weeks) F. 3. Effect of devernalization on number of leaf nodes at flowering of Brassica napus var. annua genotypes. Bar represents s.e. of differences of means with different subsequent high temperature, low temperature combinations. For other symbols, see Fig. 1.
shown). Separate analysis of variance for each genotype (Table 6) showed significant effects of subsequent high temperature in all genotypes, and in TB 14, TB 28 and TB 33 these were largely accounted for by the contrast between the control and the other treatments. The effect of low temperature was highly significant in TB 14 and TB 33,
and was largely accounted for by the contrast between the control and the other treatments. This contrast was also significant in TB 16 and TB 28. In genotype TB 14 only, the low temperature¬subsequent high temperature interaction was highly significant. This interaction was entirely accounted for by the interaction between the contrasts.
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
615
T 6. Analyses of ariance of the effects of subsequent high-temperature treatment and low-temperature treatment on time to flowering in each genotype Mean squares Time to flowering (d) Source
d.f.
TB 14
TB 16
TB 28
TB 29
TB 33
Subsequent high T (S) Contrast Deviation Low T (L) Contrast Deviation S¬L Contrast¬contrast Deviations Residual
3 1 2 2 1 1 6 1 5 24
6227±8*** 16028±5*** 1327±5*** 1413±2*** 2565±3*** 261±1 322±6** 1412±3*** 104±7 71±4
1245±3*** 1557±2*** 1089±3*** 206±0 392±7* 19±2 13±4 37±0 8±7 88±4
897±8*** 1877±9*** 407±8*** 93±5 184±9* 2±1 21±5 10±4 23±8 37±3
998±8*** 992±3*** 1002±0*** 77±1 117±9 36±3 19±1 14±7 20±0 45±6
1423±8*** 3241±4*** 515±1*** 681±5*** 1314±8*** 48±1 56±5 30±0 61±8 28±3
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
The pattern of response for the time of flowering (Fig. 4) was similar to that for the number of leaf nodes at flowering. For all genotypes and low-temperature treatments, subsequent high-temperature treatment increased the time to flowering, and this effect reached a maximum when the high temperature was applied 1 week after the end of the lowtemperature treatment. Low-temperature treatment reduced the time to flowering in all genotypes with the possible exception of TB 29, and in TB 14 the high-temperature treatment overcame the effect of low-temperature treatment, indicating that this genotype can be devernalized. The responses of TB 14 deserve special attention, since this genotype showed a strong response to vernalization with regard to both variables studied, and the interaction of this response with heat treatment was significant in most cases. DISCUSSION High-temperature treatments either before or after the application of a low-temperature treatment consistently increased the number of leaf nodes at flowering and delayed flowering. Four days of prior high temperature had more effect than 2 d. The effect of the subsequent high-temperature treatment was less marked if it was implemented immediately after the low-temperature treatment rather than 1 week later, indicating that newly-germinated seedlings are less sensitive to high temperature than slightly older plants. However, 2 weeks after the end of low-temperature treatment, exposure to high temperature again became less effective, suggesting that the pattern of morphological development was by this time largely determined. These are general effects of high temperature, observed both in the presence and in the absence of low-temperature treatment and in all genotypes studied regardless of their vernalization response. However, in the genotype with the clearest vernalization response, TB 14, and possibly in other genotypes, both prior and subsequent heat treatment had the capacity to eliminate the effect of low-temperature treatment on the number of leaf nodes at flowering and the time to flowering. With one exception, the interaction between the two contrasts in the analysis of variance
entirely accounted for the interaction between high- and low-temperature treatments, indicating that all the hightemperature treatments were sufficient to overcome the vernalizing effect of any of the low-temperature treatments. The exception was the number of leaf nodes in TB 14, for which 4 d of prior high-temperature treatment had a greater antivernalizing effect than 2 d. There was a non-significant suggestion of a similar pattern with regard to devernalization in the time to flowering of TB 14 and TB 33. High temperature applied 1 week after the end of low-temperature treatment (i.e. when the main effect of subsequent high temperature was at a maximum) overcame vernalization more fully than when applied later. In winter rye (‘ Petkus ’) it was stated that high temperature (35 °C) applied to unvernalized plants had no effect on ultimate flowering (presumably meaning that this treatment did not delay flowering), but that this treatment prevented a complete vernalization response to a subsequent lowtemperature treatment (Friend, 1953, cited by Purvis, 1961). It was therefore postulated that when heat is applied before vernalization a product, H, is formed and that when vernalization commences a stable precursor, A, is converted to a thermolabile intermediate product A«, which is removed so long as any of the accumulated H remains. When H is depleted and if exposure to low temperature continues, effective vernalization will begin and will proceed as usual. Thus the effect of prior high temperature will simply be to delay the onset of vernalization, and to attain the same final effect a longer exposure to low temperature will be needed. Friend’s investigation into the effect of high temperature before vernalization was extended by Purvis and Saunders (unpub. res., cited by Purvis, 1961), who found that the effect reached a maximum at 2 weeks exposure to 30 °C, and that in response to longer exposures the effect either remained the same or even diminished. The high-temperature treatment delayed the beginning of vernalization by about 2 weeks. Heat treatment was found to be ineffective both before and after 8 weeks of vernalization. A somewhat similar model can be developed for B. napus var. annua genotypes which show an antivernalization response. Friend’s model is consistent with the finding of the
616
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering TB 14
85
75
75
65
65
55
= 2.688
45
Time taken for first open flower to appear (d)
35
55
45
0
3 2 (no high temp.)
1 TB 28
85
35
75
65
65
55
55
45
45
0
0
3 2 (no high temp.)
1
85
35
1
3 2 (no high temp.)
1
3 2 (no high temp.)
TB 29
85
75
35
TB 16
85
0
TB 33
75
65
55
45
35
0 3 (no high temp.) 1 2 Interval before subsequent high temp. treatment (weeks)
F. 4. Effect of devernalization on time to flowering of Brassica napus var. annua genotypes. Bar represents s.e. of differences of means with different subsequent high temperature, low temperature combinations. For other symbols, see Fig. 1.
present investigation that a 4 d exposure to high temperature produced a greater increase in the number of leaf nodes at flowering in vernalization-sensitive genotypes than exposure for 2 d. The model’s prediction that high temperature before vernalization should lead to a lag period before the onset of effective vernalization is also consistent with the observed responses, since even following 8 weeks of vernalization,
antivernalized plants had a distinctly larger number of leaf nodes than the control and flowered much later. However, in our experiment, antivernalization was not overcome by longer vernalization perhaps because B. napus var. annua genotypes needed more than 8 weeks of vernalization to overcome the antivernalization effect. Devernalization was also demonstrated, and in another experiment in which
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering Low temp. (4°C) vernalization Thermostable precursor 1
Intermediate temp. (15°C) Thermolabile intermediate product (a) High temp. (30°C) devernalization
High temp. (30°C) antivernalization Thermostable precursor 2
Thermostable product (b) (reactive with the product of vernalization)
Inactive product (c) (does not promote flowering)
617 Thermostable end product (e) (promotes flowering)
Inactive product (d) (does not promote flowering)
F. 5. Proposed model for the vernalization process in Brassica napus var. annua.
plants were exposed, after vernalization, to less extreme high temperatures (25³2 °C) for the rest of their growth cycle, a similar pattern was observed, although in this case the effect of vernalization was not completely eliminated (results not presented). In A. thaliana, exposure of seedlings to high temperature (30 °C) for 5 d delayed flowering of unvernalized plants, and the retardation was greater in terms of both the final number of leaves and time to flowering when the heat treatment was applied after 8 d at 20 °C than when it was applied after 2 d (Napp-Zinn, 1969). The following model was postulated. An intermediate thermolabile substance is formed from a thermostable precursor at low and intermediate temperatures. If low temperatures continue, this substance is converted to a thermostable intermediate substance and this leads to the formation of an end product which leads to flowering. However, at high temperatures the thermolabile substance is irreversibly converted to another product. In other species it has been found that once the plant’s cold requirement has been fully satisfied, devernalization is no longer possible (e.g. Henbane : Lang and Melchers, 1947, cited by Purvis, 1961 ; Winter rye ‘ Petkus ’ : Purvis and Gregory, 1952 ; A. thaliana : Napp-Zinn, 1969 ; Chintraruck and Ketellapper, 1969). However, the present investigation indicates that in B. napus var. annua the potential for devernalization had not ceased even after 8 weeks of vernalization. On the basis of results reported here, the following model can be proposed for the vernalization process in genotypes of spring rape that show this response (Fig. 5). At low temperature (4 °C), the thermostable precursor 1 is converted to a thermolabile intermediate product (a). At intermediate temperature (15 °C) this is converted to a thermostable end product (e) which leads to flowering. However, if high temperature (30 °C) is applied before vernalization, the thermostable precursor 2 is converted to another thermostable product (b) which reacts with the thermolabile intermediate product (a) to form an inactive
product (c), i.e. one which does not promote flowering. If high temperature (30 °C) is applied after the formation of the intermediate product (a), but before its conversion to the thermostable end product (e), it is converted to another inactive product (d). Our experimental results indicate that the conversion of (a) to (e) takes about 2 weeks. This model implies that antivernalization can be overcome by a sufficiently prolonged period of vernalization, and could therefore be tested by the imposition of a longer vernalization treatment. LITERATURE CITED Bewley JB, Black M. 1985. Seeds : physiology of deelopment and germination. New York and London : Plenum Press, 343–345. Chintraruck B, Ketellapper HJ. 1969. Interaction of vernalization, photoperiod and high temperature in flowering of Arabidopsis thaliana (L.) Heynh. Plant and Cell Physiology 10 : 271–276. Elers B, Wiebe HJ. 1984. Flower formation of Chinese cabbage. II. Anti-vernalization and short-day treatment. Scientia Horticulturae 22 : 327–332. Ells JE. 1963. The influence of treating tomato seed with nutrient solutions on emergence rate and seedling growth. Proceedings of American Society of Horticultural Science 83 : 684–687. Heydecker W. 1977. Stress and seed germination : an agronomic view. In : Khan AA, eds. The physiology and biochemistry of seed dormancy and germination. Amsterdam : North-Holland Publishing Company, 237–282. Napp-Zinn K. 1969. Arabidopsis thaliana (L.) Heynh. In : Evans LT, eds. The induction of flowering. Melbourne : Macmillan, 291–304. Purvis ON. 1961. The physiological analysis of vernalization. In : Ruhland W, eds. Encyclopaedia of plant physiology. Berlin : Springer-Verlag 16 : 76–122. Purvis ON, Gregory FG. 1952. Studies in vernalization. XII. The reversibility by high temperature of the vernalized condition in Petkus Winter Rye. Annals of Botany 16 : 1–21. Thurling N, Vijendra Das LD. 1977. Variation in the pre-anthesis development of spring rape (Brassica napus L.). Australian Journal of Agricultural Research 28 : 597–607. Wiebe HJ, Habegger R, Liebig HP. 1992. Quantification of vernalization and devernalization effects for kohlrabi (Brassica oleracea convar. acephala var. gongylodes L.). Scientia Horticulturae 50 : 11–20.
Effects of Interactions Between Low and High Temperature Treatments on Flowering of Spring Rape (Brassica napus var. annua) S. R. D A H A N A Y A KE and N. W. G A L W E Y Department of Plant Sciences, Faculty of Agriculture, The Uniersity of Western Australia, Nedlands, W.A. 6907, Australia Received : 7 November 1997
Returned for revision : 10 December 1997
Accepted : 26 January 1998
Exposure to high temperature (30 °C) before or after exposure to low temperature (0, 4 or 8 weeks at 4 °C) consistently increased the number of leaf nodes at flowering and delayed flowering in a range of genotypes of spring rape (Brassica napus var. annua L.). Four days of prior exposure to high temperature had more effect than 2 d, and the effect of subsequent exposure to high temperature was maximized when exposure commenced 1 week after the end of the low-temperature treatment. In genotypes that showed a vernalization response (i.e. in which the number of leaf nodes at flowering was reduced or flowering was advanced by low temperature), this response was reduced or eliminated by either prior high-temperature treatment (antivernalization) or subsequent high-temperature treatment (devernalization). A biochemical model to account for these effects is proposed. # 1998 Annals of Botany Company Key words : Brassica napus var. annua, spring rape, antivernalization, devernalization, vernalization.
INTRODUCTION
MATERIALS AND METHODS
The delay or prevention of flower induction and floral development by exposure to high temperatures before the application of a low-temperature stimulus that would otherwise promote flowering (antivernalization) or after such a stimulus (devernalization) has been demonstrated in a number of plant species including Brassica pekinensis (Louv.) Rupr. L. (Elers and Wiebe, 1984), Brassica oleracea convar. acephala var. gongylodes L. (Wiebe, Habegger and Liebig, 1992), Arabidopsis thaliana L. (Napp-Zinn, 1969) and Secale cereale ‘ Petkus ’ (winter rye) (Purvis and Gregory, 1952). However, although flowering is known to be promoted by exposure to low temperatures during vegetative development in many genotypes of Brassica napus, including spring types (B. napus var. annua), the interaction between such vernalizing stimuli and prior or subsequent exposure to high temperatures has not been investigated in this species. In B. pekinensis (Louv.) Rupr. high temperature prior to vernalization caused a delay in bolting (the rapid extension of the flowering stalk) and an increase in the number of leaves at flowering (Elers and Wiebe, 1984). In A. thaliana, another member of the family Brassicaceae, both anti- and devernalization responses have been demonstrated, and even the ability to be re-vernalized after devernalization, suggesting the involvement of thermolabile and thermostable intermediate products in the vernalization process (Napp-Zinn, 1969). In this paper we explore the effect of high-temperature treatments prior and subsequent to the application of a lowtemperature treatment on a range of genotypes of B. napus var. annua that vary in the extent of their vernalization response, and adapt the biochemical models proposed for other species in order to explain these effects.
Five inbred-backcross genotypes of Brassica napus var. annua, designated TB 14, TB 16, TB 28, TB 29 and TB 33, were used in this study. These were derived from a cross between the B. napus var. annua cultivars ‘ Target ’ (vernalization insensitive) and ‘ Bronowski ’ (vernalization sensitive ; Thurling and Vijendra Das, 1977) through two backcrosses to ‘ Target ’, and 14 subsequent generations of selfing, and were chosen for the diversity of their vernalization responses (Table 1).
0305-7364}98}05060909 $25.00}0
Experiment to detect antiernalization The seeds of four genotypes (TB 33 was omitted as insufficient quantities were available) were imbibed in 1200 KPa (302±44 g l−") polyethylene glycol (PEG). This treatment allows germination to commence but, because of the osmotic potential of the medium, does not allow morphological development to proceed (Ells, 1963 ; Heydecker, 1977 ; Bewley and Black, 1985). While development is arrested, treatments that affect its subsequent course can be applied, and their effects can thus be observed unconfounded by development during the treatment period. The seeds were then exposed to a temperature of 30 °C in the dark for 0, 2 or 4 d. They were then exposed to a temperature of 4 °C in the dark for 0, 4 or 8 weeks, each combination of high- and low-temperature treatment being applied to 30 seeds of each genotype. The seeds were then allowed to germinate by transferring them from the PEG solution to sterile water and the seedlings were transplanted to 4 cmsquare plastic folders (‘ pots ’), one seedling to each pot.
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# 1998 Annals of Botany Company
610
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering T 1. Vernalization responses of the lines used in the present inestigation and their parent lines
Number of leaves Control Low-temperature treated Number of days to flower Control Low-temperature treated
Target*
Bronowski†
TB 14†
TB 16†
TB 28†
TB 29†
TB 33†
7 6
9 7
16±9 14±8
15±8 13±8
9±2 8±9
16±1 13±4
15±3 13±6
54 51
74 59
98±1 52±2
63±5 45±3
56±1 43±2
68±1 45±8
66±5 44±9
* Data adapted from Thurling and Vijendra Das (1977). Treated plants were exposed to 3³1 °C for 6 weeks and were subsequently grown at 15 °C in a 24 h photoperiod. † Unpub. res. Treated plants were exposed to 4 °C for 6 weeks and were subsequently grown at 15 °C in an 18 h photoperiod. The s.e. were as follows : numbers of leaves, between 0±5 and 0±39 ; numbers of days to flower, between 0±12 and 1±54.
T 2. Contrasts among leels of temperature treatment factors Low-temperature treatment (weeks)
Treatment factor Level Coefficient of contrast
0 ®2
4 1
8 1
Prior high-temperature treatment (d) 0 ®2
2 1
4 1
Interval before subsequent high-temperature treatment (weeks) 0 1
1 1
2 1
— ®3
Experiment to detect deernalization
Statistical methods
Seeds of all five genotypes were imbibed in 1200 KPa PEG and then exposed to a temperature of 4 °C in the dark as described above for 0, 4 or 8 weeks, after which they were allowed to germinate and the seedlings were transplanted to pots. They were then exposed to a temperature of 30 °C for 7 d either (a) immediately after low temperature treatment ; (b) 1 week after low temperature treatment ; (c) 2 weeks after low temperature treatment ; or (d) control : no heat treatment. Each combination of low temperature and high temperature treatment was applied to 30 seeds of each genotype. In both experiments, the plants were grown in controlled environment rooms under an 18 h photoperiod. The main light period of 10 h was provided by Na-vapour and metal halide lamps giving a radiation level of 105 µE m−# s−" at the pot surface. Saturating long photoperiods (18 h) were obtained with a 75 W incandescent lamp (7±5 µE m−# s−"). The post-vernalization temperature was maintained at 15 °C. A higher post-vernalization temperature and a shorter photoperiod have been shown to maximize the response to vernalization (Thurling and Vijendra Das, 1977), but it was thought that such an environment might nevertheless not produce a clear expression of the response to prior or subsequent high-temperature treatment. In each experiment the pots, supported in iron frames, were arranged in a splitplot design of three blocks with high- and low-temperature treatments as main plot factors and genotype as sub-plot factor. Within each sub-plot there were ten plants. The number of leaf nodes on the main stem at flowering and the number of days from germination (i.e. transfer from the PEG solution) to the opening of the first flower were recorded for each plant.
Analysis of variance was performed to determine the effect on each variable measured of prior (or subsequent) high-temperature treatment, low-temperature treatment, genotype and their interactions. If genotype¬temperature interactions occurred, separate analyses of variance were performed for each genotype in order to characterize individual responses to the high- and low-temperature stimuli. In each analysis of variance the effects of the highand low-temperature treatments were partitioned between the effects of the contrasts specified in Table 2 and the deviations from these effects. Interactions between these contrasts provide straightforward measures of the antivernalization and devernalization responses : that is, of the change in the magnitude of the response to a lowtemperature stimulus (regardless of the duration of the stimulus) caused by exposure to a high-temperature stimulus (regardless of the duration or timing of the stimulus).
RESULTS Experiment to detect antiernalization Analysis of variance for the number of leaf nodes at flowering revealed that there were highly significant differences in response to prior high-temperature treatments, low-temperature treatments and between genotypes. All the two-way interactions between these factors were highly significant, but the three-way interaction was non-significant (data not shown). Separate analyses of variance for each genotype (Table 3) showed highly significant effects of prior high temperature in all genotypes, and in all except TB 28 these were largely accounted for by the contrast between the
611
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
T 3. Analyses of ariance of the effects of prior high-temperature treatment and low-temperature treatment on number of leaf nodes at flowering in each genotype Mean squares Number of leaf nodes at flowering Source
d.f.
TB 14
TB 16
TB 28
TB 29
Prior high T (P) Contrast Deviation Low T (L) Contrast Deviation P¬L Contrast¬contrast Deviations Residual
2 1 1 2 1 1 4 1 3 18
244±54*** 472±27*** 16±81*** 14±06*** 24±92*** 3±20*** 3±75*** 13±56*** 0±48* 0±11
286±47*** 500±74*** 72±20*** 2±07 3±59* 0±56 1±19 1±27 1±17 0±64
15±24*** 13±07*** 17±42*** 0±54 1±07 0±02 0±24 0±41 0±18 0±40
27±13*** 46±23*** 8±02*** 5±56*** 9±87*** 1±25 0±77 2±05* 0±35 0±33
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
TB 14
17
15
15
13
13
11
11
= 0.1904
Number of leaf nodes
9
7
9
0
4
2 TB 28
17
7
17
15
15
13
13
11
11
9
9
7
TB 16
17
0
2
0
2
4
2
4
TB 29
7 4 0 Prior high temp. treatment (d)
F. 1. Effect of antivernalization on number of leaf nodes at flowering of Brassica napus var. annua genotypes. Bar represents s.e. of differences of means with different prior high temperature, low temperature combinations. E, No low temperature ; +, 4 weeks low temperature ; _, 8 weeks low temperature.
control and the other treatments. There were highly significant effects of low temperature in genotypes TB 14 and TB 29, and, although the overall effect of low temperature was not significant in TB 16, the contrast between
the control and the other treatments was. The prior high temperature¬low temperature interaction was highly significant in TB 14, and most of this interaction was accounted for by the interaction between the contrasts, that is, by a
612
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
T 4. Analyses of ariance of the effects of prior high-temperature treatment and low-temperature treatment on time to flowering in each genotype Mean squares Time to flowering (d) Source
d.f.
TB 14
TB 16
TB 28
TB 29
Prior high T (P) Contrast Deviation Low T (L) Contrast Deviation P¬L Contrast¬contrast Deviations Residual
2 1 1 2 1 1 4 1 3 18
2282±8*** 3557±4*** 1008±2** 337±2 660±0* 14±5 178±8 667±4* 16±0 118±7
610±0 968±0* 252±1 688±1 821±4 554±8 74±0 114±1 60±6 206±0
155±1 219±1 91±0 101±0 136±0 66±0 6±4 0±0 8±5 72±2
1811±0*** 1224±0*** 2398±1*** 105±7 138±0 73±5 13±3 50±7 0±8 39±7
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
straightforward antivernalization effect. In TB 29, although the overall interaction was not significant, the interaction between contrasts was. All genotypes responded to prior high-temperature treatment by producing more leaf nodes at flowering, regardless of the low-temperature treatment, with exposure to high temperature for 4 d having a greater effect than 2 d (Fig. 1). However, this trend was much more marked in TB 14 and TB 16 than in the other two genotypes, and in these genotypes the additional effect of 2 further days of exposure was relatively slight (corresponding to the small proportion of variance accounted for by deviation from the contrast in this term of the ANOVA). In TB 14, exposure to low temperature in the absence of prior high temperature clearly decreased the number of leaves, with exposure for 8 weeks having a slightly greater effect than 4 weeks. Prior high temperature largely, though perhaps not completely, eliminated this effect : that is, this genotype showed a clear antivernalization response. [Previous experiments with the same genotypes showed that TB 14 was amongst the most responsive to vernalization in terms of the number of leaf nodes at flowering, and the most responsive in terms of the time to flowering (Table 1).] In TB 29 the same pattern was observed, although not so strongly, and there was a nonsignificant suggestion of the same pattern in TB 16. The overall analysis of variance for time to flowering showed that there were highly significant differences in responses to prior high-temperature treatments, low-temperature treatments and between genotypes. The prior high temperature¬genotype interaction was highly significant, but the other interactions were non-significant (data not shown). Separate analysis of variance for each genotype (Table 4) showed that the effect of prior high temperature was highly significant in TB 14 and TB 29. In TB 16, although the overall effect of prior high temperature was not significant, the contrast between the control and the other treatments was. The overall effect of low temperature was not significant in any individual case, although in TB 14 the contrast between the control and the other treatments was. The only significant interaction term was the interaction between the contrasts in TB 14.
In all genotypes except TB 28, the prior high-temperature treatment increased the time to flowering, the longer treatment having a greater effect (Fig. 2). In TB 14, the time to flowering was reduced by low-temperature treatment. There was a non-significant suggestion of the same pattern in the other genotypes, with the exception of TB 28, corresponding to the highly significant main effect of lowtemperature treatment in the combined ANOVA of all genotypes. In TB 14, 8 weeks’ exposure to low temperature had an only slightly greater effect than 4 weeks. In this genotype, the effect of low temperature was almost completely overcome by 2 d of prior high-temperature treatment : that is, TB 14 again showed a clear antivernalization response.
Experiment to detect deernalization Analysis of variance for the number of leaf nodes at flowering revealed that there were highly significant differences in response to subsequent high-temperature treatments, low-temperature treatments, and between genotypes. The genotype¬subsequent high temperature interaction was highly significant, but neither the other two-way interactions nor the three-way interaction were significant (data not shown). Separate analysis of variance for each genotype (Table 5) showed a highly significant effect of subsequent high-temperature treatment in all genotypes except TB 28 (for which this effect was nearly significant : P ¯ 0±062), and in all cases this effect was largely due to the contrast between the control and the other treatments. The effect of low temperature was highly significant in TB 28, TB 29 and TB 33, and in all cases this effect was entirely attributable to the contrast between the control and the other treatments. This contrast was also significant in TB 14. In no case was the subsequent high temperature¬low temperature interaction significant. For all genotypes with the possible exception of TB 28, and for all low-temperature treatments, the subsequent high-temperature treatment increased the number of leaf nodes at flowering, and this effect reached a maximum when
613
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
Time taken for first open flower to appear (d)
TB 14
TB 16
75
75
65
65
55
55 = 1.8985
45
35
45
0
4
2
35
0
TB 28
75
65
65
55
55
45
45
0
4
2
4
TB 29
75
35
2
2
35 4 0 Prior high temp. treatment (d)
F. 2. Effect of antivernalization on time to flowering of Brassica napus var. annua genotypes. For legend see Fig. 1.
T 5. Analyses of ariance of the effects of subsequent high-temperature treatment and low-temperature treatment on number of leaf nodes at flowering in each genotype Mean squares Number of leaf nodes at flowering Source
d.f.
TB 14
TB 16
TB 28
TB 29
TB 33
Subsequent high T (S) Contrast Deviation Low T (L) Contrast Deviation S¬L Contrast¬contrast Deviations Residual
3 1 2 2 1 1 6 1 5 24
50±07*** 80±12*** 35±05*** 5±11 7±80* 2±41 1±20 4±16 0±61 1±77
36±92** 95±42*** 7±67 2±42 3±21 1±63 0±80 0±45 0±88 4±93
3±34 4±63 2±70 14±04*** 25±07*** 3±01 0±02 0±02 0±02 1±19
73±84*** 192±01*** 14±76** 10±47** 16±90** 4±03 0±47 0±63 0±43 1±67
70±55*** 181±42*** 15±12*** 7±07** 13±61** 0±53 0±42 0±09 0±49 1±22
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
it was applied 1 week after the end of low-temperature treatment (Fig. 3). In all genotypes except TB 16, low temperature reduced the number of leaf nodes at flowering and in TB 14 there was a suggestion that this effect was eliminated by all the high-temperature treatments, although, as noted, the corresponding contrast was non-significant.
Analysis of variance of time to flowering revealed highly significant differences between subsequent high-temperature treatments, low-temperature treatments and genotypes. The genotype¬subsequent high temperature and genotype¬low temperature interactions were highly significant, and the three-way interaction was also significant (data not
614
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering TB 14
TB 16
15
15
13
13
11
11 = 0.5364
9
7
9
0
2 3 (no high temp.)
1
7
0
Number of leaf nodes
TB 28 15
13
13
11
11
9
9
0
2 3 (no high temp.)
1
2 3 (no high temp.)
TB 29
15
7
1
2 3 (no high temp.)
1
7
0
TB 33 15
13
11
9
7
0 2 3 (no high temp.) 1 Interval before subsequent high temp. treatment (weeks) F. 3. Effect of devernalization on number of leaf nodes at flowering of Brassica napus var. annua genotypes. Bar represents s.e. of differences of means with different subsequent high temperature, low temperature combinations. For other symbols, see Fig. 1.
shown). Separate analysis of variance for each genotype (Table 6) showed significant effects of subsequent high temperature in all genotypes, and in TB 14, TB 28 and TB 33 these were largely accounted for by the contrast between the control and the other treatments. The effect of low temperature was highly significant in TB 14 and TB 33,
and was largely accounted for by the contrast between the control and the other treatments. This contrast was also significant in TB 16 and TB 28. In genotype TB 14 only, the low temperature¬subsequent high temperature interaction was highly significant. This interaction was entirely accounted for by the interaction between the contrasts.
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering
615
T 6. Analyses of ariance of the effects of subsequent high-temperature treatment and low-temperature treatment on time to flowering in each genotype Mean squares Time to flowering (d) Source
d.f.
TB 14
TB 16
TB 28
TB 29
TB 33
Subsequent high T (S) Contrast Deviation Low T (L) Contrast Deviation S¬L Contrast¬contrast Deviations Residual
3 1 2 2 1 1 6 1 5 24
6227±8*** 16028±5*** 1327±5*** 1413±2*** 2565±3*** 261±1 322±6** 1412±3*** 104±7 71±4
1245±3*** 1557±2*** 1089±3*** 206±0 392±7* 19±2 13±4 37±0 8±7 88±4
897±8*** 1877±9*** 407±8*** 93±5 184±9* 2±1 21±5 10±4 23±8 37±3
998±8*** 992±3*** 1002±0*** 77±1 117±9 36±3 19±1 14±7 20±0 45±6
1423±8*** 3241±4*** 515±1*** 681±5*** 1314±8*** 48±1 56±5 30±0 61±8 28±3
*** P ! 0±001, ** P ! 0±01, * P ! 0±05.
The pattern of response for the time of flowering (Fig. 4) was similar to that for the number of leaf nodes at flowering. For all genotypes and low-temperature treatments, subsequent high-temperature treatment increased the time to flowering, and this effect reached a maximum when the high temperature was applied 1 week after the end of the lowtemperature treatment. Low-temperature treatment reduced the time to flowering in all genotypes with the possible exception of TB 29, and in TB 14 the high-temperature treatment overcame the effect of low-temperature treatment, indicating that this genotype can be devernalized. The responses of TB 14 deserve special attention, since this genotype showed a strong response to vernalization with regard to both variables studied, and the interaction of this response with heat treatment was significant in most cases. DISCUSSION High-temperature treatments either before or after the application of a low-temperature treatment consistently increased the number of leaf nodes at flowering and delayed flowering. Four days of prior high temperature had more effect than 2 d. The effect of the subsequent high-temperature treatment was less marked if it was implemented immediately after the low-temperature treatment rather than 1 week later, indicating that newly-germinated seedlings are less sensitive to high temperature than slightly older plants. However, 2 weeks after the end of low-temperature treatment, exposure to high temperature again became less effective, suggesting that the pattern of morphological development was by this time largely determined. These are general effects of high temperature, observed both in the presence and in the absence of low-temperature treatment and in all genotypes studied regardless of their vernalization response. However, in the genotype with the clearest vernalization response, TB 14, and possibly in other genotypes, both prior and subsequent heat treatment had the capacity to eliminate the effect of low-temperature treatment on the number of leaf nodes at flowering and the time to flowering. With one exception, the interaction between the two contrasts in the analysis of variance
entirely accounted for the interaction between high- and low-temperature treatments, indicating that all the hightemperature treatments were sufficient to overcome the vernalizing effect of any of the low-temperature treatments. The exception was the number of leaf nodes in TB 14, for which 4 d of prior high-temperature treatment had a greater antivernalizing effect than 2 d. There was a non-significant suggestion of a similar pattern with regard to devernalization in the time to flowering of TB 14 and TB 33. High temperature applied 1 week after the end of low-temperature treatment (i.e. when the main effect of subsequent high temperature was at a maximum) overcame vernalization more fully than when applied later. In winter rye (‘ Petkus ’) it was stated that high temperature (35 °C) applied to unvernalized plants had no effect on ultimate flowering (presumably meaning that this treatment did not delay flowering), but that this treatment prevented a complete vernalization response to a subsequent lowtemperature treatment (Friend, 1953, cited by Purvis, 1961). It was therefore postulated that when heat is applied before vernalization a product, H, is formed and that when vernalization commences a stable precursor, A, is converted to a thermolabile intermediate product A«, which is removed so long as any of the accumulated H remains. When H is depleted and if exposure to low temperature continues, effective vernalization will begin and will proceed as usual. Thus the effect of prior high temperature will simply be to delay the onset of vernalization, and to attain the same final effect a longer exposure to low temperature will be needed. Friend’s investigation into the effect of high temperature before vernalization was extended by Purvis and Saunders (unpub. res., cited by Purvis, 1961), who found that the effect reached a maximum at 2 weeks exposure to 30 °C, and that in response to longer exposures the effect either remained the same or even diminished. The high-temperature treatment delayed the beginning of vernalization by about 2 weeks. Heat treatment was found to be ineffective both before and after 8 weeks of vernalization. A somewhat similar model can be developed for B. napus var. annua genotypes which show an antivernalization response. Friend’s model is consistent with the finding of the
616
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering TB 14
85
75
75
65
65
55
= 2.688
45
Time taken for first open flower to appear (d)
35
55
45
0
3 2 (no high temp.)
1 TB 28
85
35
75
65
65
55
55
45
45
0
0
3 2 (no high temp.)
1
85
35
1
3 2 (no high temp.)
1
3 2 (no high temp.)
TB 29
85
75
35
TB 16
85
0
TB 33
75
65
55
45
35
0 3 (no high temp.) 1 2 Interval before subsequent high temp. treatment (weeks)
F. 4. Effect of devernalization on time to flowering of Brassica napus var. annua genotypes. Bar represents s.e. of differences of means with different subsequent high temperature, low temperature combinations. For other symbols, see Fig. 1.
present investigation that a 4 d exposure to high temperature produced a greater increase in the number of leaf nodes at flowering in vernalization-sensitive genotypes than exposure for 2 d. The model’s prediction that high temperature before vernalization should lead to a lag period before the onset of effective vernalization is also consistent with the observed responses, since even following 8 weeks of vernalization,
antivernalized plants had a distinctly larger number of leaf nodes than the control and flowered much later. However, in our experiment, antivernalization was not overcome by longer vernalization perhaps because B. napus var. annua genotypes needed more than 8 weeks of vernalization to overcome the antivernalization effect. Devernalization was also demonstrated, and in another experiment in which
Dahanayake and Galwey—Temperature Effects on Spring Rape Flowering Low temp. (4°C) vernalization Thermostable precursor 1
Intermediate temp. (15°C) Thermolabile intermediate product (a) High temp. (30°C) devernalization
High temp. (30°C) antivernalization Thermostable precursor 2
Thermostable product (b) (reactive with the product of vernalization)
Inactive product (c) (does not promote flowering)
617 Thermostable end product (e) (promotes flowering)
Inactive product (d) (does not promote flowering)
F. 5. Proposed model for the vernalization process in Brassica napus var. annua.
plants were exposed, after vernalization, to less extreme high temperatures (25³2 °C) for the rest of their growth cycle, a similar pattern was observed, although in this case the effect of vernalization was not completely eliminated (results not presented). In A. thaliana, exposure of seedlings to high temperature (30 °C) for 5 d delayed flowering of unvernalized plants, and the retardation was greater in terms of both the final number of leaves and time to flowering when the heat treatment was applied after 8 d at 20 °C than when it was applied after 2 d (Napp-Zinn, 1969). The following model was postulated. An intermediate thermolabile substance is formed from a thermostable precursor at low and intermediate temperatures. If low temperatures continue, this substance is converted to a thermostable intermediate substance and this leads to the formation of an end product which leads to flowering. However, at high temperatures the thermolabile substance is irreversibly converted to another product. In other species it has been found that once the plant’s cold requirement has been fully satisfied, devernalization is no longer possible (e.g. Henbane : Lang and Melchers, 1947, cited by Purvis, 1961 ; Winter rye ‘ Petkus ’ : Purvis and Gregory, 1952 ; A. thaliana : Napp-Zinn, 1969 ; Chintraruck and Ketellapper, 1969). However, the present investigation indicates that in B. napus var. annua the potential for devernalization had not ceased even after 8 weeks of vernalization. On the basis of results reported here, the following model can be proposed for the vernalization process in genotypes of spring rape that show this response (Fig. 5). At low temperature (4 °C), the thermostable precursor 1 is converted to a thermolabile intermediate product (a). At intermediate temperature (15 °C) this is converted to a thermostable end product (e) which leads to flowering. However, if high temperature (30 °C) is applied before vernalization, the thermostable precursor 2 is converted to another thermostable product (b) which reacts with the thermolabile intermediate product (a) to form an inactive
product (c), i.e. one which does not promote flowering. If high temperature (30 °C) is applied after the formation of the intermediate product (a), but before its conversion to the thermostable end product (e), it is converted to another inactive product (d). Our experimental results indicate that the conversion of (a) to (e) takes about 2 weeks. This model implies that antivernalization can be overcome by a sufficiently prolonged period of vernalization, and could therefore be tested by the imposition of a longer vernalization treatment. LITERATURE CITED Bewley JB, Black M. 1985. Seeds : physiology of deelopment and germination. New York and London : Plenum Press, 343–345. Chintraruck B, Ketellapper HJ. 1969. Interaction of vernalization, photoperiod and high temperature in flowering of Arabidopsis thaliana (L.) Heynh. Plant and Cell Physiology 10 : 271–276. Elers B, Wiebe HJ. 1984. Flower formation of Chinese cabbage. II. Anti-vernalization and short-day treatment. Scientia Horticulturae 22 : 327–332. Ells JE. 1963. The influence of treating tomato seed with nutrient solutions on emergence rate and seedling growth. Proceedings of American Society of Horticultural Science 83 : 684–687. Heydecker W. 1977. Stress and seed germination : an agronomic view. In : Khan AA, eds. The physiology and biochemistry of seed dormancy and germination. Amsterdam : North-Holland Publishing Company, 237–282. Napp-Zinn K. 1969. Arabidopsis thaliana (L.) Heynh. In : Evans LT, eds. The induction of flowering. Melbourne : Macmillan, 291–304. Purvis ON. 1961. The physiological analysis of vernalization. In : Ruhland W, eds. Encyclopaedia of plant physiology. Berlin : Springer-Verlag 16 : 76–122. Purvis ON, Gregory FG. 1952. Studies in vernalization. XII. The reversibility by high temperature of the vernalized condition in Petkus Winter Rye. Annals of Botany 16 : 1–21. Thurling N, Vijendra Das LD. 1977. Variation in the pre-anthesis development of spring rape (Brassica napus L.). Australian Journal of Agricultural Research 28 : 597–607. Wiebe HJ, Habegger R, Liebig HP. 1992. Quantification of vernalization and devernalization effects for kohlrabi (Brassica oleracea convar. acephala var. gongylodes L.). Scientia Horticulturae 50 : 11–20.