ACTA PHYSIOLOGIAE PLANTARUM Vol. 22. No. 2.2000:143-149
Does the lack of vernalization requirement interfere with winter survival of oilseed rape plants? Marcin Rapacz, Ewa Chilmonik Department of Plant Physiology, Faculty of Agriculture, Agricultural University o f Krak6w, Podtuzna 3, 30-239 Krak6w, Poland tel. +4812 4253301, fax. +4812 4253202, E-mail (M.Rapacz):
[email protected]
Key words: Brassica napus (L.) var. oleifera, cold acclimation, c h l o r o p h y l l fluorescence, g r o w t h cessation, oilseed rape, p r e h a r d e n i n g , vernalization, winter survival
Abstract Recent studies (Rapaez 1999) have shown that cultivars of spring-type oilseed rape are able to cold-acclimate to the level comparable with winter cultivars, but only after prehardening which results both in the increase of photosynthetic activity and in growth cessation. It is commonly known that under field conditions spring-type cultivars could not survive winter. Present studies were undertaken to explain the reasons for low winter hardiness of spring type rape plants. Six cultivars of spring and two of winter rape were sown in the open-air vegetation room at the end of August. The obtained results indicate that the degree of frost damage in spring-type plants increased in the course of winter and this increase was parallel to elongation of generative shoots observed after periods of warming. Each spring cultivar was completely killed by frost just after its generative shoot reached 15-20 cm, irrespective of its frost resistance level, determined previously under laboratory conditions. In the case of winter cultivars survival rate was consistent with laboratory-estimated frost resistance. It is suggested that spring rape could not survive winter because of its limited ability to prevent shoot elongation during winter at temperatures slightly above 0 °C. It was also found that less efficient photosynthetic electron transport in autumn was observed in these spring cultivars in which the elongation
of generative shoots was observed already during the first warm break in winter.
List of abbreviations: F0 - f l u o r e s c e n c e w h e n all P S I I reaction centres are o p e n e d in d a r k - a d a p t e d leaves, Fm - f l u o r e s c e n c e w h e n all P S I I reaction centres are c l o s e d in d a r k - a d a p t e d leaves, Fv - variable f l u o r e s c e n c e after dark acclimation, Fv/Fm a p p a r e n t q u a n t u m y i e l d o f P S I I , tl/2 - half rise time f r o m F0 to Fm.
Introduction W i n t e r - t y p e cultivars o f different plants (e.g. oilseed rape) are a s s u m e d to h a v e h i g h e r frost resistance level than s p r i n g - t y p e plants. R a p e s e e d cultivars are classified as winter or spring types b a s e d on their v e r n a l i z a t i o n r e q u i r e m e n t s for f l o w e r i n g ( A n d e r s s o n and O l s s o n 1961) and not on their levels o f their frost resistance or w i n t e r survival. O n e o f the possible r e a s o n s for a l o w e r level o f a c c l i m a tion abilities in s p r i n g - t y p e plants c a n be their limited capability to c e a s e g r o w t h d u r i n g cold a c c l i m a tion and, in c o n s e q u e n c e , the l o w e r a m o u n t o f ene r g y accessible f o r p r o c e s s e s c o n n e c t e d with acclimation.
143
M. RAPACZ & E. C H I L M O N I K
The hypothesis that limited ability to cease growth during the cold acclimation period can be decisive for low resistance of spring type plants was confirmed partially in recent studies concerning prehardening of spring and winter-type cultivars of oilseed rape (Rapacz 1999). Prehardening results in cessation of elongation growth and increases the photosynthetic rate during cold acclimation (Rapacz 1998b, Rapacz and Janowiak 1998). As a result, prehardened (under both natural and artificial conditions) spring-type plants are able to cold acclimate to the level comparable with winter cultivars. It was concluded that prehardening enabled proper cold acclimation of plants irrespective of their vernalization requirement for flowering (Rapacz 1999). Also, during field studies, it was confirmed that some spring cultivars are able to survive winter better than some winter forms (Teutonico et al. 1993). However other observations show that spring-type cultivars cannot survive winter under field conditions, irrespective of the fact that they were prehardened.
under laboratory conditions (Rapacz 1999). Spok, Pactol and Semu 86/223 belong to frost hardy cultivars and their frost resistance is higher than that of winter cv. Gdrczafiski. Frost resistance of cv. Star is close to that of cv. Grrczafiski. Mtochowski and SCHP 004 were frost susceptible. Cultivar Mtochowski is more resistant (slightly hardier than winter cv. Idol). Conditions o f the e x p e r i m e n t
Plants were sown in the open-air vegetation room at the end of August. The conditions applied (roof made of glass) excluded the presence of snow cover during winter. The temperature course during the experiment is shown in Fig. 1. The degree of plant damage and their growth characteristics were screened before winter, after each period of heavy ?^
The studies were undertaken to explain the reason for low winter hardiness of spring-type rape.
Material and Methods Nov.
Dec.
Jan.
Feb.
Mar.
Apr
P l a n t material
Six cultivars of spring oilseed rape (Mtochowski ZH Mtochdw, Poland; Pactol, Spok and SEMU 86/223 - Semences Cargil, France; SCHP 004 and Star - DLF Trifolium, Denmark and two cultivars of winter oilseed rape (G6rczafiski - MHRiNR Krakdw, Poland and Idol - Semences Cargil, France) were used in the experiments. All the cultivats are double zero forms, except for Gdrczafiski and Mtochowski, which are a high erucic acid cultivat. Winter cultivars Idol and G6rczariski differ in their cold acclimation ability and vernalization requirement (Rapacz 1999, Rapacz and Markowski 1999). G6rczafiski is more frost resistant (but in comparison with other winter cultivars it is moderate hardy) and has high vernalization requirement supplemented with photoperiodic control of shoot elongation and flowering. Cultivar Idol is characterized by low frost resistance and low vernalization requirement. Spring cultivars used in the experiment differ in their frost resistance determined
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Fig. 1. Daily mean and minimal temperatures during winter of the experiment (1995/96). Vertical grid lines indicate the dates of analyses excluding % of plant survival, which were counted after 7 days of subsequent growth at +22 °C. frosts during winter and in the spring. Dates of measurements are indicated in Fig. 1. At these terms a part of plants (1 pot with 10 plants from each cultivar) was transferred into a glasshouse (22 °C) where elongation of shoots and the degree of frost damage were analysed. Frost damage was estimated by 3 different methods. 1) Leaf discs were cut to determine membrane injury triggered by frost occurring under field conditions. To this end electrolyte leakage was measured as described elsewhere (Rapacz 1999). 2) The damage of photosynthetic apparatus was determined by means of apparent quantum yield of PSII (Fv/Fm) by the method announced below. 3) Percent of plant survival was counted after 7 days of growth at 22 °C.
WINTER SURVIVAL OF SPRING-TYPE OILSEED RAPE
Measurements o f chlorophyll fluorescence
Fluorescence induction parameters were measured at +22 °C in dark-adapted young, fully expanded leaves using Plant Stress Meter (PSM; BioMonitor, Umea, Sweden). All measurements were made in 10 replications (see below). Measured parameters were used for characterisation of photosynthetic efficiency in the autumn on the 3 rd of November and also for assessment of foliar frost damage during and after winter. For estimation of frost damage only the measurements in which Fv values were higher than 0 were taken into consideration. The decrease in F0 reflects damage in PSII antenna systems (Rutten and Santarius 1992). Fv/F m - as a measure of current photochemical capacity (apparent quantum yield) of PSII is constant in unstressed leaves of diverse species and decreases under many kinds of stresses (Schreiber and Bilger 1993). The parameter tl/2 is the function of the rate of the photochemical reaction and the pool size of electron acceptors on the reducing side of PSII, including the plastoquinone pool (Oquist and Wass 1988). Higher values of this parameter indicate more efficient photosynthetic electron transport and thus higher efficiency of photosynthesis. Results Frost damage and
elongation of generative shoots
All the viability tests used (Fv/Fm, electrolyte leakage and % of frost-killed plants) indicated collectively increase in the level of frost damage during the winter period. N o plant of spring-cultivars survived winter (Fig. 2). Frost which appeared in the second half of November (minimal temperature reached -14 °C; Fig. 1) did not cause serious damage estimated on the 4 th of December. Only in the case of two cultivars having the lowest cold-acclimation abilities (SCHP 004 and Mtochowski; see Material and Methods) foliar frost damages were visible as the decrease in Fv/F m (Fig. 2A, B). Probably the warmer weather in the middle December (mean temperatures even close to 5 °C; Fig. 1) resulted in promotion of generative shoot elongation in five cultivars (all except cv. Mtochowski). Diverse shoot length was measured on the 9 th of January
(Fig. 2). The longest shoots were observed in cv. Pactol (about 20 cm; Fig. 3E). In cultivar Spok generative shoots was present but their length did not exceed 5 cm (Fig. 2F). In the second half of December the 4-day long period of hard frosts were also observed. The mean temperature dropped down to -18 °C, and the minimal temperature reached -23 °C. This decrease in temperature resulted in increase in frost damage. On the 9 th of January frostkilled plants wereobserved for the first time and foliar damage increased dramatically (Fig. 2). The decrease in frost damage was the highest not in cultivars of lower frost resistance (SCHP 004 and Mtochowski) but in cultivars which had elongated their generative shoots to the highest degree, especially in frost-resistant cultivar Pactol (Fig. 2E). On the 9 th of January differences in frost damage between winter cultivars were noticed for the first time (Fig. 3). Electrolyte leakage test showed the higher damage in the more frost sensitive cv. Idol. In the middle of January another period of warm weather followed by heavy frost at the turn of January and February and during February was observed (Fig. 1). During assessment performed on the 23 rd of February the continuation of generative shoots elongation was noticed for all spring cultivars (Fig. 2). Cuitivars which started to elongate their shoots during December were entirely frost killed and the resistance of cv. Mtochowski fell suddenly which was accompanied by the beginning of shoot elongation (Fig. 2B). The differences in hardiness among winter cultivars were visible in the case of all parameters of frost injury (Fig. 3). In March, after another warm break in the second half of February, frosts reaching -15 °C was also noticed. The winter was very long and even in the beginning of April mean day temperatures were below 0 °C. The final estimation of freezing injuries was determined on the 30 th of April. Spring cultivar Mtochowski did not managed survive winter (Fig. 2B) and the more resistant winter cultivar Gdrczafiski survived in the better condition than Idol (Fig. 3). T h e results showed that the degree of frost damage in spring-type plants increased in the course of winter and this increase was parallel to progression in the growth of generative shoots observed after each warmer period of winter (Fig. 2). Each spring cultivar was completely killed by frost just after its gen-
145
M, R A P A C Z & E. C H I L M O N I K
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Fig. 2. Frost damage (frost killed plants, electrolyte leakage from leaves - EL, apparent quantum yield o f PSII - Fv/Fm) and the length of flower shoots in spring cultivars of oilseed rape occurred under field conditions during winter 1995/96 (for details concerning temperatures see Fig. 1). Vertical bars denote SD. Values marked with different letter differ statistically from those noted for other cultivars at P = 0.05, according to D u n c a n ' s multiple range test.
erative shoot reached 15-20 cm, irrespective of its frost resistance level determined under laboratory conditions (Rapacz 1999). Winter-type cultivars in which no elongation of generative shoots took place during winter survived winter time (Fig. 3). In this case differences in survival rates were consistent with those in laboratory-estimated frost resistance.
146
Properties of photosynthetic apparatus in autumn
The results of measurements of chlorophyll fluorescence induction parameters in autumn are presented in Table. No statistically significant differences in apparent quantum yield of PSII (Fv/Fm) and (excluding cv. Spok) in F0 were noticed. However values of other parameters varied between cultivars. The highest values of F m and Fv were observed in winter-type cultivars and also in spring cultivar Mlochowski - the cultivar which main-
WINTER SURVIVAL OF SPRING-TYPE OILSEED R A P E
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than other spring cultivars. The results concerning fluorescence characteristics during winter (with exception to Fv/Fm which was used as the indicator of foliar frost d a m a g e ) were not s h o w n and discussed. The same changes m a y h a v e been interpreted as the result o f s o m e internal changes resulting in (or triggered by) increase in elongation growth rate or as the result o f frost injuries.
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Fig. 3. Frost damage (frost killed plants, electrolyte leakage from leaves - EL, apparent quantum yield of PSII- Fv/Fm) and the length of flower shoots in winter cultivars of oilseed rape occurred under field conditions during winter 1995/96 (for details concerning temperatures see Fig. 1). Vertical bars denote SD. Values marked with different letter differ statistically from those noted for other cultivars at P = 0.05, according to Duncan's multiple range test. tained the cessation of elongation growth for the longest time during winter (Fig. 2B). The highest values of tl/2 were noticed also for winter-type cultivars as well as for spring cultivars M l o c h o w s k i and Spok, which elongated generative shoots later
T h e results o f N e u n e r and Buchner (1998) s h o w e d that the use o f Fv/F m in estimation o f frost d a m ages m a y entail overestimation o f frost resistance (even b y 5 °C) in c o m p a r i s o n to electrolyte leakage test. According to these authors it is the result o f underestimation o f frost d a m a g e on partially frostinjured leaves. In the presented investigations a good coincidence b e t w e e n changes in Fv/Fm and in other indicators of frost d a m a g e was noticed. It is suggested that in the present w o r k the risk of m a k ing errors was prevented, since the m e a s u r e m e n t s of chlorophyll fluorescence and electrolyte leakage were m e a s u r e d on the s a m e section o f the s a m e leaf. Thus chlorophyll fluorescence seems to be a sufficiently g o o d p a r a m e t e r in a s s e s s m e n t of foliar frost damage. T h e results obtained suggest that the vernalization requirement does not s e e m to be necessary for cold acclimation of oilseed rape, but usually (under norm a l winter conditions) it is essential to maintain a high frost resistance level throughout the winter. A c c o r d i n g to Levitt (1972), in winter annuals the decrease in g r o w t h rate is required for proper cold
Table. Fluorescence induction parameters of spring and winter rape cultivars in autumn measured on the 3rd of November. Values marked with the same letter did not differ statistically at P = 0.05, according to Duncan's multiple range test. Cultivar spring-type SCHP004 Mlochowski Star Semu86/223 Pactol Spok winter-type Idol G6rczafiski
Fluorescence parameters F0 Fm,
Fv
Fv/Frn
t]r~
0.322 a 0.291 ab 0.318 a 0.271 ab 0.305 a 0.234 b
1.006 1.323 1.097 1.042 1.017 0.867
b a b b b c
0.684 1.032 0.779 0.771 0.712 0.633
b a ab ab b b
0.68 a 0.78 a 0.71 a 0.74 a 0.70 a 0.73 a
71 98 56 56 70 84
0.311 0.317
1.244 a 1.219 a
0.933 0.902
a a
0.75 0.74
84 ab 87 ab
a a
a a
b a c c b ab
147
M. RAPACZ & E. CHILMONIK
acclimation. Only on this condition the use of the photosynthetic products in cold acclimation is possible (e.g. it refers to accumulation of soluble sugars). Consequently, promotion of the elongation growth brings about consumption of accumulated photosynthetic products and loss of freezing resistance. Hardiness also varies inversely with the size characteristics of the plant, e.g. height (Levitt 1956). This relationship can be expected to hold true only if the measured characters are the result of development during both the hardening (Levitt 1972) and prehardening periods (Rapacz 1998b). For instance prehardened plants of oilseed rape, which form a rosette and slowly grow during hardening are less frost sensitive than non-prehardened plants that were characterized by the upright growth (Rapacz 1998a, Rapacz 1998b). The results obtained in the presented paper, indicate that spring rape can not survive winter because of its limited ability to maintain ceased growth even when the temperature rises slightly above 0 °C. Probably high winter survival of spring rape reported previously (Teutonico et al. 1993) could not have been possible if periods of warming would have occurred during winter (unfortunately no data indicating the temperatures during winter were included). The results of another investigation indicated that the lack of vernalization requirements might prevent the cold acclimation of winter cereals (Fowler and Carles 1979). A limited ability of spring cultivars to acclimate in cold was demonstrated during field studies of winter and spring cultivars of barley, oat, rye and wheat. Even in October spring plants were not able to reach the resistance level comparable to that observed in winter plants. In both spring and winter cultivars dry matter accumulated at the rate which would suggest that the energy production during the acclimation period was not a limiting factor for cold acclimation under normal field conditions in autumn. But the distribution of the energy accumulated varied between spring and winter cultivars. Spring cultivars showed only limited abilities to decrease crown water content during October and also in 3 out of 4 cases to cease shoot growth (under natural prehardening conditions). Those two observations may explain the low level of their frost resistance. Hence the limited ability to cease growth during autumn (prehardening phase) may be the reason for low winter sur-
148
vival of spring-type cereals. But it does not refer to spring type oilseed-rape plants, which are able to cease growth during prehardening in autumn (Rapacz 1999). T h e presented results also showed that a more efficient photosynthetic electron transport in autumn was observed in cultivars, which maintained prehardening-induced growth cessation for a longer time, e.g. in winter and in some spring cultivars (Mlochowski and Spok). It may suggest that the relationship between two main effects of prehardening (stimulation of photosynthesis and retardation of elongation growth during cold acclimation) may be more closely related than previously expected. If we assume that during prehardening low temperature of the day results in overexcitation of PSII (Gray et al. 1997) we can speculate that it may lead both to retardation of plant growth and to the adaptation of photosynthetic apparatus to functioning under conditions of high "excitation pressure" at low temperature. Such adaptation consisted in an increase in the activity of major photosynthetic enzymes (Hurry et al. 1995, Gray et al. 1996, Strand et al. 1999), changes in phosphorylation of D1 protein (Salonen et al. 1998) as well as in the increased plastoquinone pool (Gray et al. 1998). The later is reflected by increased tl/2 values observed, for instance, after prehardening (Rapacz and Janowiak 1998). The results presented showed that lower values of tl/2 was characteristic to cultivars which earlier started elongation of generative shoots. It was observed prior (over one month) to elongation of shoots. Two hypotheses were possible: 1) Some differences in functioning of photosynthetic apparatus, which is opposite to changes observed during growth under elevated PSII excitation pressure, may be favourable to acceleration of growth rate during short periods of warmth. In spring-type plants acceleration of growth rate results in elongation of generative shoots that is not under control of vernalization requirement. 2) Changes in elongation growth rate, which was not evidenced in the presented experiment, occurred already before chlorophyll fluorescence was measured on the 3 rd of November. Under conditions of warm weather in the middle of November and December it escalated and this resulted in elongation of generative shoots. It may suggest that promotion of elongation growth can trigger changes in
WINTER SURVIVAL OF SPRING-TYPE OILSEED RAPE
photosynthetic apparatus. Unfortunately there is no experimental evidence in what way.
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Rapacz M. 1998. The after-effects of temperature and irradiance during early growth of winter oilseed rape (Brassica napus L. var. oleifera cv. G6rczafiski) seedlings on the progress of their cold acclimation. Acta Physiol. Plant. 20, 73-78. Rapacz M. 1998b. Physiological effects of winter oilseed-rape (Brassica napus vat. oleifera) prehardening to frost. II. Growth, energy partitioning and water status during cold acclimation. J. Agron. Crop Sci. 181, 81-87. Rapacz M. 1999. Frost resistance and cold acclimation abilities of spring-type oilseed rape. Plant Sci. 147, 5564. Rapacz M., Janowiak F. 1998. Physiological effects of winter oilseed-rape (Brassica napus var. oleifera) prehardening to frost. I. Frost resistance and photosynthesis during cold acclimation. J. Agron. Crop Sci. 181, 13-20.
Rapacz M., Markowski A. 1999. Winter hardiness, frost resistance and vernalization requirement of European winter oilseed rape (Brassica napus vat. oleifera) cultivars within the last 20 years. J. Agron. Crop. Sci. 183,243-253. Rutten D., Santarius K.A. 1992. Age-related differences in frost sensitivity of the photosynthesis apparatus of two plagiomnium species. Planta 187, 224-229. Salonen M., Aro E.M., Rintamaki E., 1998. Reversible phosphorylation and turnover of the D 1 protein under various redox states of Photosystem II induced by low temperature photoinhibition. Photosynth. Res. 58, 143-151. Schreiber U., Bilger W. 1993. Progress in chlorophyll fluorescence research: major development during the past years in retrospect. Progr. Bot. 54, 151-173. Strand A., Hurry V., Henkes S., Huner N., Gustafsson P., Gardestrom P., Stitt M. 1999. Acclimation of Arabidopsis leaves developing at low temperatures. Increasing cytoplasmic volume accompanies increased activities of enzymes in the Calvin cycle and in the sucrose-biosynthesis pathway. Plant Physiol. 119, 13871397. Teutonico R.A., Palta J.W., Osborn T.C. 1993. In vitro freezing tolerance in relation to winter survival of rapeseed cultivars. Crop Sci. 33: 103-107.
Received December 07, 1999; accepted March 17, 2000
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