Regrowth of White Clover after chilling: Assimilate Partitioning and Vegetative Storage Proteins

G. Corbel*, Ch. Robin, B.E. Frankow-Lindberg, A. Ourry, and A. Guckert

Gwénola Corbel, Ch. Robin and A. Guckert, Unité associée "Agronomie et Environnement". E.N.S.A.I.A.- INRA. BP 172. 54505 Vandoeuvre-lès-Nancy. Cedex. France.
B.E. Frankow-Lindberg, Sveriges lantbruksuniversitet, Institutionen för växtodlingslära, Box 7043, S-750 07 Uppsala, Sweden. 
A. Ourry, Unité Associée INRA "Physiologie et Biochimie Végétales" IRBA Université F-14032 Caen Cedex, France.
Received___________*. Corresponding author (corbel@ensaia.inpl-nancy.fr)

Abbreviations- DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; RSA, relative specific activity; SSW, specific stolon weight; VSP, vegetative storage proteins 

Abstract 
Under temperate climates, grassland species are subjected to overwintering which may significantly influence their early spring growth capacity. In white clover (Trifolium repens L.), it is acknowledged that overwintering capacity can differ among cultivars. Ability of this forage legume to recover from winter damage will, therefore, have a great influence on its persistence in grass/clover associations. Experiments were undertaken with two different white clover cultivars (Huia and AberHerald). Leaf appearance rate, dry matter distribution, 14C assimilate partitioning and vegetative storage protein accumulation were determined in plants subjected to a 4-wk chilling period (5°C/0°C, day/night) and subsequent warmer temperatures (15°C/10°C), and compared to control plants (20°C/15°C). Chilling treatment decreased leaf appearance rate, with AberHerald producing more leaves than Huia. This can be considered as a major aspect of cold adaptation strategy, because leaf appearance rate controlled carbon acquisition. Low temperature increased dry matter partitioning to below-ground tissues. AberHerald allocated more assimilates to stolons than Huia. Accumulation of a 17.3 kDa protein, believed to act as a vegetative storage protein, also increased after the chilling treatment. Regrowth was characterized by rapid mobilization of the 17.3 kDa protein in stolons and by preferential carbon allocation to stolon apices. AberHerald showed a higher regrowth potential than Huia in view of its morphological and physiological characters which include carbon acquisition and assimilate partitioning patterns, favoring shoot regrowth and acquisition of stolon reserves.

In temperate climates, the effects of overwintering on perennial cultivated plants deserves attention since the climatic events occurring during the winter greatly influence spring regrowth. This is particularly true in the case of white clover grown in association with grasse because clover is susceptible to winter damage (Harris et al., 1983; Frame and Newbould, 1986; Woledge et al., 1990). The principal consequences of low temperatures are losses of dry matter and stolon and bud death which can reduce spring regrowth potential. Competition between species is, therefore, exacerbated in early spring. As a consequence, the persistence of the sward is reduced. This is often verified because the legume usually has a poor competitive ability with the associated grass. Therefore, clovers which are tolerant to low winter temperatures and have a good regrowth potential in spring are needed to improve the persistence of the pasture and reliability of yield from year to year.
Breeding programs have developed cold-tolerant cultivars from ecotypes collected in cool areas. Collins et al. (1991) have shown a considerable degree of genetic variation in the ability of white clover to overwinter and regrow in spring. White clover cv. AberHerald, bred at IGER Aberystwyth from lines collected in the Zurich Oberland of the Swiss Alps, is known to be tolerant to low temperature and shows good spring regrowth potential (Rhodes and Fothergill, 1992; Collins et al. 1996; Caradus and Woodfield 1997). However, we still do not clearly understand the plant characters and physiological mechanisms explaining the differential responses of cultivars to cold hardiness. 
It is important to determine the morpho-physiological characters involved in order to identify more winter-hardy genotypes able to produce more biomass in early spring.
We decided to investigate parameters related to carbon acquisition and partitioning (stolon morphogenesis, biomass and partitioning of recently fixed carbon) and the fate of N reserves, as these factors are engaged in regrowth after defoliation (Corre et al., 1996). We assumed that these processes are also involved in regrowth after previous exposure to cold temperatures. The involvement of starch and soluble sugars has already been investigated in overwintering and spring growth (Vez, 1961; Lüscher and Nösberger, 1992; Guinchard et al., 1997). Shoot recovery after a cold period involves starch mobilization from the remaining source tissues to regrowing sink organs (i.e. roots and stolons acting as source organs while stolon meristems act as sink tissues).
Therefore, our objective was to assess the morphological and physiological traits involved in the response of two diverse cultivars to low temperatures. The study involved two cultivars, AberHerald mentioned above and Huia, selected from clover lines from New Zealand, characterized by its maritime climate. Because rapid regrowth in spring is more desirable than increased production in winter (Davies, 1998), our investigations centered on regrowth as a consequence of events occurring during acclimation and cold periods. 

Materials and methods

Plant material and growth conditions
The experiment included two white clover cultivars, Grasslands Huia and AberHerald. Grasslands Huia originated from New Zealand whereas AberHerald was developed from material collected in the Zurich Oberland of the Swiss Alps. AberHerald is characterized by winter hardiness and good spring growth (Rhodes and Fothergill, 1992). Sixty stolon tips of each cultivar were planted individually in a potting mixture (30% sand and 70% peat v/v) in (10 x 10 x 10 cm) plastic containers. The stolon tips had an apical bud and two nodes on which the youngest expanding leaf was kept. Plants were first grown in a greenhouse for 3 wk to allow the cuttings to establish and then they were grown in a controlled environment, with a 12h photoperiod, at 20/15°C (day/night) and 80% relative humidity, for 3 wk before the experiment. Light was provided by 10 HQI 400W Osram mercury vapor lamps (Osram, France), delivering a fluorescence rate of 290 mmol m-2s-1 of photosynthetically active radiation. The plants were watered regularly at the end of each photoperiod and fertilized once a week with a nitrogen-free nutrient solution (Robin et al., 1989).
After being planted, the cuttings underwent a clipping regime, that simulated the morphology of stolon tips growing within a canopy. The fourth unfolded leaf (counted from the apical bud) was clipped regularly (corresponding to four days for control plants and ten days for chilled plants), when a new leaf had reached developmental stage 1.0 (Carlson, 1966).

Experimental design and temperature treatments
All plants (6 wk old) were cold acclimated at 10/4°C (day/night) with a 10-h photoperiod for 3 wk before the temperature treatments were applied. 
The following two temperature treatments were 20/15 °C (control) and 5/0 °C (chilling) (day/night) for 4 wk with a photoperiod of 10h. Plant containers were rotated in the growth rooms once per day throughout the experiment.
After 4 wk, the temperature of the chilling treatment was increased by 2°C per day to reach 15/10°C (day/night) after 5 d. This temperature was maintained for 9 d and these 2 wk were defined as the regrowth period. Control plants were maintained at 20/15°C. Different regrowth temperatures were chosen for chilled and control plants, for the two following reasons: first the 15/10°C (day/night) temperature simulated early spring regrowth under a temperate climate and second drastic and rapid changes of air temperature were avoided for chilled plants. irty plants of each cultivar were assigned randomly to each temperature treatment.

Plant measurements and harvesting
Leaf appearance rate was recorded daily and expressed as the number of leaves produced per week. Plants were harvested three times after the beginning of treatments: (H1) at the end of the 4-wk temperature treatment, (H2) 5 d after the beginning of regrowth and (H3) at the end of regrowth. At each harvest, six plants of each cultivar were sampled and separated into leaves, stolon and roots. Leaves were used for measurements of osmotic potential (data not shown). The material (stolons and roots) was freeze-dried, weighed, and ground into a fine powder (5 mm) for further analyses. The root/stolon ratio was calculated from the weight of root and stolon (expressed in g plant-1).

14C labeling experiment.
The second unfolded leaf (counted from the apex) of the main stolon was selected for 14C labeling. Labeling was done the day before H2 according to Robin et al. (1987). The target leaf was inserted into a labeling chamber which remained sealed during the labeling procedure. Before labeling, the leaf was acclimated to a gas mixture of 380 mL CO2 L-1. A pulse of 14CO2 from a bottle of gas mixture (200 mL O2 L-1, 800 mL N2 L-1, 380 ml CO2 L-1) was delivered for two minutes with an open flow regulated at 120 mL min-1 by a mass flow meter (specific activity: 4.3 MBq mg-1 C; activity = 0.21 MBq leaf -1). Thus there was no significant modification of the leaf microenvironment during labeling. After labeling, an open flow of air was circulated through the leaf cuvette. The light environment during labeling was 300 mmol m-2s-1 of photosynthetically active radiation. Immediately after the labeling, the plants were returned to their respective temperature treatment. Twelve plants of each cultivar and each temperature treatment were labeled.
Labeled plants were washed free of potting mixture 24h after labeling and were dissected into three sections: (i) the apex (two developing nodes plus the apical bud), the youngest unfolded leaf and the labeled leaf (including its subtending petiole), (ii) the main stolon and the third unfolded leaf and, (iii) the roots. Immediately after harvest this material was frozen in liquid nitrogen and freeze-dried, weighed and ground into a fine powder (5 mm).
Carbon and nitrogen contents of the organs were determined by the combustion of dry subsamples in a C and N autoanalyzer (Carlo-Erba Na 1500, Carlo-Erba, Italy). The 14CO2 contained in plant material was first digested by sodium hypochlorite (60 mL L-1) and a liquid scintillation cocktail (Hionic fluor, Packard Bioscience BV, The Netherlands) was added and then counted in a Tri-Carb liquid scintillation spectrometer (TRI-CARB 2100TR, Packard Instruments, Meriden, CT). The radioactivity of whole organs (whole organ Bq) was calculated from the following formula:
	whole organ Bq = subsample Bq x whole organ mass / subsample mass
Carbon 14 assimilate partitioning was expressed as a percentage of total radioactivity recovered in the plant excluding the labeled leaf. The radioactivity recovered indicated that 14C distribution was a result of both size and metabolic activity of the compartments (apex, stolon and roots). Comparing compartments on the basis of their metabolic activity required that 14C-partitioning be expressed relative to compartment size as expressed by distribution of C. This was achieved by using Relative Specific Activity (RSA) as described by Cralle and Heichel (1985) and Robin et al. (1987).

Vegetative storage proteins
	Extraction of soluble proteins.
Soluble proteins were extracted from 200-g freeze-dried samples at 4°C with 5 mL of 28 mM TRIS-HCl buffer (pH 7.5) containing 2mM phenyl-methyl-sulfonyl fluoride (PMSF), 22 mM TRIS-Base, 10 mM leupeptin and 100 mM dithiothreitol (DTT). The extracts were centrifuged at 1200 x g (15min), the supernatant was filtered and the nucleic acids were then precipitated using protamine sulfate (1 mg mL-1) for 15 min. After centrifugation (1800 x g, 10 min, 4°C), the supernatant was precipitated with acetone (1:84, v:v) at 4°C. The proteins precipitated in the pellet were centrifuged (18000 x g, 5min, 4°C) and then dissolved in 1.5 mL of extraction buffer. The soluble proteins were separated into four subsamples and a further precipitation was achieved using the deoxycholate/trichloroacetic acid method described by Peterson (1983). After centrifugation (18000 x g, 5min, 4°C) the pellet was resuspended with 1 mL of 50 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid)-NAOH buffer (pH 7.5) and used for determination of soluble protein concentration by the method of Bradford (1976).
	SDS-PAGE of soluble proteins
SDS-PAGE electrophoresis used a 15 g L-1 duracryl running gel with a stacking gel containing 5.5 g L-1 acrylamide in a Millipore Electrophoresis System (Investigator, Millipore, Saint-Quentin-en-Yvelines, France). One pellet of soluble proteins was resuspended in Laemmli (1970) buffer denaturated 5 min at 100°C and then centrifuged (12000 x g, 2min). Samples of proteins (25ml) corresponding to proteins extracted from 200 mg DW were loaded onto wells. Two wells were used for loading known molecular weight proteins. The gels were run for 5 h at a constant 500V.
	2-D electrophoresis of soluble proteins
One pellet of soluble proteins resuspended in O’Farrell (1975) buffer was used to run 2-D SDS-PAGE gels, according to a modified procedure (Corre et al., 1996) from O’Farrell (1975). First-dimension isoelectric focusing was run on 4.1g L-1 acrylamide tube gel containing 9.5 M urea, 20 mL L-1 Triton X-100, 5 mM (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulphonate), and 20 mL L-1 Millipore 2D optimized carrier ampholytes (pH 3-10). After 2.5 h of prefocusing to 1500 V with current limited to 110 mA per tube, 20 ml protein samples, corresponding to proteins extracted from 200 mg DW were loaded onto the basic end of the gel tube and focusing with 2000 V for17.5 h. The gels were then removed and equilibrated in a 0,375 M buffer containing 30 g L-1 SDS and 50 mM DTT. The second dimension (which was as the one-dimensional gels) separated the isoelectro-focused proteins.
	Analysis of gels
Following electrophoresis, gels were silver stained as described by Lopez et al. (1991). Gels were scanned and then quantitatively analyzed using the Millipore Bioimage Computerized Image Analysis System. The individual staining intensity of each polypeptide was expressed as a percentage of total gel staining intensity. Electrophoresis was performed on samples from each harvest (H1, H2 and H3).

Statistical analysis
The data was analyzed by ANOVA (Systat) as a completely randomized design. Before ANOVA, data concerning ratios (root/stolon) and percentages (export of 14C) were square-root or log transformed, respectively. Statistics presented in tables are from transformed data. Values are means of 6 replicates.

Results

Leaf appearance, dry matter yield and partitioning 
Chilling significantly decreased the leaf appearance rate in both cultivars (Table 1). The two cultivars differed significantly during the chilling period but not during the regrowth period. At the end of the experiment, the mean number of leaves per plant was higher for AberHerald (P=0.06) than for Huia.
Chilling significantly influenced dry matter partitioning of both cultivars at each sampling (Table 2). AberHerald always partitioned significantly more dry matter above ground (P<0.001, see root/stolon) compared to Huia. Irrespective of cultivar, chilled plants partitioned significantly more dry matter to roots and less to shoots (P<0.01) than control plants, both after chilling (H1) and during regrowth (H2 and H3). The specific stolon weight (SSW) increased under low temperatures at each harvest (Fig. 1). At the end of the regrowth period, the SSW of AberHerald was greater than that of Huia irrespective of the temperature treatments.

The 14C assimilate partitioning
The proportion of assimilated 14C transported to the shoot (including the stolon with the apical bud) was large in comparison to that transported to roots (Table 3). The distribution patterns of the 14C in shoots and roots varied significantly with temperature. A higher proportion of 14C was exported to the shoots of control plants, while more 14C was exported to the roots at low temperatures. AberHerald exported more 14C to the stolon than to the roots compared to Huia (P<0.05) (Table 3).
The apical part of the stolon had the highest relative specific activity (RSA) compared to stolons and roots.The values of chilled plants were 1.42 and 1.76 for the apex, 0.75 and 0.82 for the stolon and 0.96 and 0.89 for the roots for Huia and AberHerald, respectively. The RSA of roots was significantly increased by chilling for both cultivars (0.75 and 0.63 for Huia and AberHerald control and 0.96 and 0.89 for Huia and AberHerald chilled; P<0.01).

Changes in relative contents of Vegetative Storage Proteins
A two-dimensional electrophoresis analysis showed the prominence of putative VSP in stolons and roots of white clover. The staining intensity identified one prominent polypeptide with a molecular mass of 17.3 kDa and an isoelectric point of 5.3 in the stolon (Fig. 2A). The same polypeptide of 17.3 kDa with an isoelectric point of 5.3 and two prominent proteins with a molecular mass of 15 kDa, but whose isoelectric points differed (6.6 and 7.0, respectively) were identified in the roots (Fig. 2B). 
Chilling increased the relative 17.3 kDa protein content of stolons (Table 4), but had no effect on its proportion in roots. Conversely, after chilling the relative 15-kDa polypeptide content of roots was decreased in chilled plants (Table 4). During the five first days of the regrowth period (H2), the 17.3-kDa protein in the stolon was partly hydrolyzed. Nine days later (H3), the staining intensity of this 17.3kDa protein remained the same as at H2. The staining intensity of the 15kDa protein in the roots increased during the regrowth period (H2 and H3) in chilled and control plants. The 17.3kDa protein in the roots remained at all temperatures.

Discussion
Under controlled conditions we measured the regrowth of white clover following a cold period in order to identify the morpho-physiological characters involved in responses to temperature. We compared two cultivars with a contrasting overwintering behavior (Collins et al., 1991; Frankow-Lindberg, 1997). This is the first attempt to assess the fate of vegetative storage proteins in forage plants under chilled temperatures (5/0°C day/night) and simulated regrowth conditions (15/10°C day/night). 
Although low temperature caused a marked decline in leaf appearance rate, AberHerald produced more leaves than Huia under low temperature. This can be considered a major aspect of cold adaptation strategy, because this morphogenetic character coupled with greater leaf area determine carbon acquisition by the plant. This finding could explain the fact that AberHerald exhibits a faster regrowth in the field than Huia when temperature increases (Robin et al., 1999).
Chilled plants proportionally partitioned more dry matter below-ground which was also observed by Boller and Nösberger (1983) and Frankow-Lindberg (1997). AberHerald allocated more assimilates than Huia to the stolons at the expense of the roots. This was confirmed by the smaller root/shoot ratio in AberHerald and higher stolon thickness deduced from the specific stolon weight. Collins and Rhodes (1995) suggested a relationship between stolon weight per unit length and stolon carbohydrate content. Stolons function as the site of reserve storage and mobilization, and ensure regrowth from buds either through the remobilization of reserves, or from recent photosynthates. It is likely that reserve carbohydrates are required by white clover during the winter to develop cold hardiness (Smith, 1964; Guinchard et al., 1997) and to meet respiratory requirements when photosynthetic activity is reduced by low temperatures (Harris et al., 1983). However, more recent studies have pointed out the involvement of N reserves (Rong et al., 1996) suggesting that C reserves are not the only requirement for winter survival.
Soluble proteins, which are accumulated in vegetative tissues of forage legume species during winter hardening, are depleted when shoot growth resumes in spring (Volenec et al., 1996). However, their precise contribution to overwintering and regrowth is still unclear.
We found three prominent polypeptides in white clover: one 17.3-kDa polypeptide in stolons and roots, and two 15-kDa polypeptides in roots which were recently identified in white clover as VSPs (Corre et al., 1996; Bouchart et al., 1998), because they are involved in N storage and mobilization during regrowth after defoliation (Corre et al., 1996). The 17.3-kDa VSP is accumulated to a large extent during winter (Bouchart et al., 1998). In different species, it has been suggested that VSPs may play a role in cold acclimation and freezing tolerance (Cyr and Bewley, 1990) as they show a seasonal pattern of accumulation with a peak at the beginning of winter (Staswick, 1994). In our conditions, VSPs showed the same pattern during the experiment for both cultivars: an accumulation during the cold period followed by a large mobilization when temperatures increased (Table 4). However, both cultivars showed the same increase in the 17.3-kDa VSP content of stolons despite a different cold hardening capacity. Therefore, we cannot assume that cold hardiness is related to a different capacity in terms of the accumulation of this VSP.
Mono-dimensional electrophoresis used to study pattern during chilling and regrowth does not distinguish the two isoforms of 15-kDa protein. So we considered the combination of the two isoforms. A significant depletion of the 15kDa VSP in roots occurred at low temperature, in contrast to the increase of the 17.3-kDa VSP. Further analysis using polyclonal antibodies raised against clover leghaemoglobin (data not shown) revealed that the root 15-kDa VSP reacts with this antibody. This suggests that the 15-kDa VSP is leghaemoglobin, hydrolyzed during chilling as suggested by Gordon et al. (1990). Furthermore, this explains why the relative 17.3-kDa VSP content of the roots was increased under chilling treatment while the relative leghaemoglobin content of nodulated roots was decreased.
We evaluated the importance of recently assimilated carbon in the regrowth potential of cultivars. The relative sink strength of the apex (expressed by RSA) was the highest observed amongst organs, with a high metabolic activity of regrowing tissues 5 days after the beginning of regrowth, parallel to the hydrolysis of VSPs. During regrowth, AberHerald partitioned more assimilates to the stolons and less to the roots than Huia did (Table 2). Moreover, the sink strength of the apex of AberHerald (expressed by RSA) tended to be higher, irrespective of the temperature treatment. Therefore, AberHerald was able to mobilize more recent C than Huia to the shoots including the growing apex.

Conclusion
The screening of genotypes which differ in their regrowth ability over the seasons is of major importance to improve the persistence and yield of white clover in pastures (spring competitiveness). AberHerald showed a higher regrowth potential than Huia through morphological and physiological characters including carbon acquisition and assimilate partitioning patterns which favored shoot regrowth and the acquisition of stolon reserves. Storage protein accumulation patterns suggested that they were involved in winter hardening and regrowth. Further work is needed to determine whether the availability of N reserves at the beginning of regrowth can explain the differential response of cultivars to temperature.

Acknowledgments.
The authors gratefully acknowledge the financial support given to B. Frankow-Lindberg by the COST 814 action "Crop development for cool and wet regions of Europe" for a short-term visit to Nancy (France) and the technical assistance of P. Vion and M. Meyer.

References
Bradford, M.M. 1976. A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Boller, B. C., and J. Nösberger. 1983. Effects of temperature and photoperiod on stolon characteristics, dry matter partitioning, and nonstructural carbohydrate concentration of two white clover ecotypes. Crop Sci. 23: 1057-1062.
Bouchart, V., J.H. Macduff, A. Ourry, M.M. Svenning, A.P. Gay, J.C. Simon, and J. Boucaud. 1998. Seasonal pattern of accumulation and effects of low temperatures on storage compounds in Trifolium repens. Physiol. Plant. 104:65-74.
Caradus, J.R., and D.R. Woodfield. 1997. World checklist of white clover varieties II. N. Z. J. Agric. Res. 40:115-206.
Carlson, G.E. 1966. Growth of clover leaves - developmental morphology and parameters at ten stages. Crop Sci. 6:293-294.
Collins, R.P., J. Connolly, M. Fothergill, B.E. Frankow-Lindberg, A. Guckert, M.P. Guinchard, A. Lüscher, J. Nösberger, I. Rhodes, Ch. Robin, B. Staehli, and S. Stoffel. 1996. Variation in the overwintering of white clover cultivars in cool wet areas of Europe. p. 201-204. In G. Parente et al. (ed.) Grassland and Land Use systems-Proceedings XVI European Grass and Forage Meeting, Grado, Italy. 
Collins, R.P., M.J. Glendinning, and I. Rhodes. 1991. the relationships between stolon characteristics, winter survival and annual yields in white clover (Trifolium repens L.). Grass For. Sci. 4:51-61.
Collins, R.P., and I. Rhodes. 1995. Stolon characteristics related to winter survival in white clover. J. Agric. Sci., Cambridge 124:11-16.
Corre, N., V. Bouchart, A. Ourry, and J. Boucaud. 1996.  Mobilization of nitrogen reserves during regrowth of defoliated Trifolium repens L. and identification of potential vegetative storage proteins. J. Exp. Bot. 47, 301:1111-1118.
Cralle, H.T., and G.H. Heichel. 1985. Interorgan photosynthate partitioning in alfalfa. Plant Physiol. 79:381-385.
Cyr, D.R., and J.D. Bewley. 1990. Proteins in the roots of the perennial weeds chicory (Chicorium intybus L.) and dandelion (Taraxacum offinale) are associated with overwintering. Planta 182:370-374.
Davies, A. 1998. Pasture research in the U.K: present knowledge and future prospects. Can. J. Plant Sci. 78: 211-216.
Frame, J., and P. Newbould. 1986. Agronomy of white clover. Adv. Agron. 40: 1-88.
Frankow-Lindberg, B.E. 1997. Assimilate partitioning in three white clover cultivars in the autumn, and the effect of defoliation. Ann. Bot. 79:83-87. 
Gordon, A.J., W. Kessler, and F.R. Michin. 1990. Defoliation-induced stress in nodules of white clover. I. changes in physiological parameters and protein synthesis. J. Exp. Bot. 41:1245-1253.
Guinchard, M.P., Ch. Robin, P. Grieu, and A. Guckert. 1997. Cold acclimation in white clover subjected to chilling and frost: changes in water and carbohydrates status. Eur. J. Agron. 6:225-233.
Harris, W., I. Rhodes, and S.S. Mee. 1983. Observations on environmental and genotypic influences on the overwintering of white clover. J. Appl. Ecol. 20:609-624.
Laemmli, U.K. 1970. Cleavage of structural proteins of the head of bacteriophage T4. Nature 227:680-685.
Lopez, M.F., W.F. Patton, B.L. Utterback, N. Chung-Welch, P. Barry, W.M. Skea, and RP. Cambria. 1991. Effect of various detergents on protein in the second dimension of two dimension gels. Ann. of Biochem. 199:35-44.
Lüscher, A., and J. Nösberger. 1992. Overwintering and spring growth of white clover. p. 167-171. In Proceedings of the 14th General meeting of the European Grassland Federation. Lahti, Finland.
O'Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021.
Peterson, G.L. 1983. Determination of total protein. Methods in Enzymology. 91:95-119.
Rhodes, I., and M. Fothergill. 1992. Plant Genetics and Breeding. p. 33-34. In White clover breeding. Institute of Grassland and Environmental Research, AFRC Dyfed, UK.
Robin, Ch., T. Chone, and A. Guckert. 1987. Rôle de la position de la feuille dans l'assimilation et le transport du carbone chez le trèfle blanc (Trifolium repens L.). Agronomie 7:599-605.
Robin, Ch., G. Corbel, M.P. Guinchard, C. Bazard, and A. Guckert. 1999. Comportement hivernal et repousse printanière du trèfle blanc : importance du cultivar. Fourrages 157:21-32.
Robin, Ch., L. Shamsun-Noor, and A. Guckert. 1989. Effect of potassium on the tolerance to PEG-induced water stress of two white clover varieties (Trifolium repens L.). Plant Soil 120:153-158.
Rong L., J.J. Volenec, B.C. Joern, and S.M. Cunningham. 1996. Seasonal changes in nonstructural carbohydrates, protein and macronutrients in roots of alfalfa, red clover, sweet clover and birdsfoot trefoil. Crop Sci. 36:617-623.
Smith, D. 1964. Winter injury and the survival of forage plants. Herb. Abst. 34:203-209.
Staswick, PE. 1994. Storage proteins of vegetative plant tissues. Ann.Rev. Plant Physiol. Plant Mol. Biol. 45:303-322.
Vez, A. 1961. Etude du développement du trèfle blanc en relation avec les fluctuations de quelques substances organiques. Ber. Schweiz. Bot. Ges. 71:118-173.
Volenec, J.J., A. Ourry, and B.C. Joern. 1996. A role for nitrogen reserves in forage regrowth and stress tolerance. Physiol. Plant. 97:185-193.
Woledge, J., V. Tewson, and I.A. Davidson. 1990. Growth of grass/clover mixtures during winter. Grass For. Sci. 45:191-201. 

Table 1. Leaf appearance rate at stolon tip during acclimation, chilling and regrowth and mean number of leaves per plant.

	Chilling after acclimation (10/4 °C)

nkjop

	Regrowth

	number of leaves
 appeared	
	                            leaves/week		
Control	20/15 °C	20/15 °C		
Huia 	0.171	0.314	11.6	
AberHerald	0.202	0.310	12.5	
				
Chilling	5/0 °C	15 /10 °C		
 Huia	0.032	0.190	 4.2	
AberHerald	0.056	0.190	 5.0	
				
Cultivar	*	NS	+	
Temperature	***	***	***	
C*T	NS	NS	NS	
*,**,*** Significant at the 0.05, 0.01 and 0.001 probability levels, respectively, NS = not significant. +, main effect of cultivar significant at P = 0.06.

Table 2. Dry matter partitioning above and below ground and root-stolon ratio at three harvests. Control plants were at a constant temperature (20/15 °C) throughout the experiment, except during the three weeks acclimation period (10/4 °C).

	End of chilling
H1		beginning of regrowth
H2		End of regrowth
H3	
	Stolon	Roots	root/stolon		Stolon	Roots	root/stolon		Stolon	Roots	root/stolon	
	g/plant			g/plant			g/plant		
Control												
Huia	0.31	0.19	0.63		0.42	0.26	0.62		0.38	0.22	0.63	
AberHerald	0.45	0.17	0.39		0.45	0.21	0.48		0.60	0.22	0.38	
												
Chilling												
Huia	0.22	0.31	1.42		0.22	0.29	1.40		0.27	0.34	1.26	
AberHerald	0.27	0.26	0.99		0.26	0.30	1.17		0.35	0.33	0.99	
												
Cultivar	*	NS	***		NS	NS	***		*	NS	***	
Temperature	**	***	***		***	*	**		*	**	**	
C*T	NS	NS	NS		NS	NS	NS		NS	NS	NS	
*,**,*** Significant at the 0.05, 0.01 and 0.001 probability levels, respectively, NS = not significant.

Table 3. 14C assimilate partitioning to the plant organs 5 d after the beginning of the regrowth period. Plants are harvested 24h after the 14C labeling of the second unfolded leaf. Values are means of 6 replicates. The temperature was 20/15 °C for Control plants and 15/10 °C for Chilling plants.

Treatment	Cultivar	apex	stolon	root	
		% of total exported activity	
Control	Huia	35	43	22	
	AberHerald	38	47	15	
					
Chilling	Huia	32	22	46	
	AberHerald	31	34	35	
					
	Cultivar	NS	*	*	
	Temperature	NS	***	***	
	C*T	NS	NS	NS	
	*,**,*** Significant at the 0.05, 0.01 and 0.001 probability levels, respectively, NS = not significant.

Table 4. Changes in Vegetative Storage Protein relative content of stolon and roots. The relative staining intensity of the 17.3-kDa and 15-kDa VSPs was quantified by image analysis and expressed as % of the total integrated intensity.

	End of chilling
H1		Beginning of regrowth
H2 		End of regrowth
H3	
Stolon	17.3kDa			17.3kDa			17.3kDa		
									
Control									
Huia	12.3			 6.2			 9.8		
AberHerald	 9.1			 9.6			 6.3		
Chilling									
Huia	20.4			12.8			11.3		
AberHerald	22.2			12.8			11.2		
									
Roots	17.3kDa	15kDa		17.3kDa	15kDa		17.3kDa	15kDa	
									
Control									
Huia	 9.5	15.2		 8.7	16.4		 8.5	17.3	
AberHerald	 9.1	12.7		 7.3	14.0		 8.1	20.5	
Chilling									
Huia	 9.4	10.4		 7.5	11.8		 7.7	16.3	
AberHerald	 8.7	 8.9		 7.8	12.8		 9.0	18.0	

Figure captions 
Figure 1. Effect of chilling temperature on Specific Stolon Weight (g m-1) at three harvests. *, **, *** Significant at the 0.05, 0.01 and 0.001 probability levels, NS = not significant.

Figure 2. Two-dimensional gel electrophoresis of soluble proteins of Trifolium repens L. (cultivar Huia) stolons (A) and roots (B) harvested at the end of chilling. Vegetative Storage Proteins are identified by arrows.