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CROP PHYSIOLOGY & METABOLISM

Regrowth of White Clover after Chilling

Assimilate Partitioning and Vegetative Storage Proteins

^a Unité associée "Agronomie et Environnement", E.N.S.A.I.A.-INRA, BP 172. 54505 Vandoeuvre-lès-Nancy, Cedex, France
^b Sveriges lantbruksuniversitet, Institutionen för växtodlingslära, Box 7043, S-750 07 Uppsala, Sweden
^c Unité Associée INRA "Physiologie et Biochimie Végétales" IRBA Université F-14032 Caen Cedex, France

corbel{at}ensaia.inpl-nancy.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
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 known 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 (5/0°C, day/night) and subsequent warmer temperatures (15/10°C), and compared with control plants (20/15°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.

Abbreviations: RSA, relative specific activity • SSW, specific stolon weight • VSP, vegetative storage proteins

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
IN TEMPERATE CLIMATES, the effects of overwintering on perennial cultivated plants deserve 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 grass 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. Indeed, legume usually has a poor competitive ability with the associated grass. As a consequence, the persistence of the sward is reduced. Therefore, clovers which are tolerant to low winter temperatures and have 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. AberHerald white clover, 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 that explain the differential responses of cultivars to cold hardiness.

It is important to determine the morpho-physiological characters involved 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 involved 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

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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 (300 g kg^-1 sand and 700 g kg^-1 peat) in 10- by 10- by 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 12-h photoperiod, at 20/15°C (day/night) and 80% relative humidity, for 3 wk before the experiment. Light was provided by 10 HQI 400 W Osram mercury vapor lamps (Osram, France), delivering 290 µmol m^-2 s^-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 4 d for control plants and 10 d 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 temperature treatments were 20/15°C (control) and 5/0°C (chilling) (day/night) for 4 wk with a photoperiod of 10 h. 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 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. Thirty 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 stolons and roots were freeze-dried, weighed, and ground into a fine powder (5 µm) for further analyses. The root/stolon ratio was calculated from the weight of roots 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 µL CO₂ L^-1. A pulse of ¹⁴CO₂ from a bottle of gas mixture (200 mL O₂ L^-1, 800 mL N₂ L^-1, 380 µL CO₂ L^-1) was delivered for 2 min 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 µmol m^-2 s^-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 24 h 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 µm).

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, Milan, 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, Groningen, 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:

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-mg freeze-dried samples at 4°C with 5 mL of 28 mM TRIS-HCl buffer (pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride (PMSF), 22 mM TRIS-Base, 10 µM leupeptin and 100 mM dithiothreitol (DTT). The extracts were centrifuged at 1200 x g (15 min), the supernatant was filtered and the nucleic acids were then precipitated with 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 (18 000 x g, 5 min, 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, 5 min, 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, 2 min). Samples of proteins (25 µL) corresponding to proteins extracted from 200 mg dry weight were loaded onto wells. Two wells were used for loading known molecular weight proteins. The gels were run for 5 h at a constant 500 V.

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 4.1 g 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 µA per tube, 20-µL protein samples, corresponding to proteins extracted from 200 mg dry weight were loaded onto the basic end of the gel tube and focusing with 2000 V for 17.5 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 the pooled sample of six replicates 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

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
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 than for Huia.

View larger version (44K): [in this window] [in a new window]   Fig. 1 Effect of chilling temperature on Specific Stolon Weight at three harvests. *, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels; NS = not significant

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).

View larger version (43K): [in this window] [in a new window]   Fig. 2 Two-dimensional gel electrophoresis of soluble proteins of white clover (cultivar Huia) stolons (A) and roots (B) harvested at the end of chilling. Vegetative Storage Proteins are identified by arrows

	Discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

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 determines 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 other 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 differential capacity for the accumulation of this VSP.

Mono-dimensional electrophoresis used to study changes in VSPs during chilling and regrowth does not distinguish the two isoforms of 15-kDa protein. So we considered the combined action of the two isoforms. A significant depletion of the 15-kDa VSP in roots occurred at low temperature, in contrast to the increase of the 17.3-kDa VSP. Further analysis with polyclonal antibodies raised against clover leghemoglobin (data not shown) revealed that the root 15-kDa VSP reacts with this antibody. This suggests that the 15-kDa VSP is leghemoglobin, 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 leghemoglobin content of nodulated roots was decreased.

We also 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 d 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 (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.

	Conclusions

TOP ABSTRACT INTRODUCTION Materials and methods Results Discussion Conclusions REFERENCES

 
The screening of genotypes which differ in their regrowth ability 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.

Received for publication October 19, 1998.

	REFERENCES

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