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a Département de Phytologie, Université Laval, Sainte-Foy, QC, Canada, G1K 7P4
b Agriculture et Agroalimentaire Canada, Sainte-Foy, QC, Canada, G1V 2J3
* Corresponding author (castonguayy{at}agr.gc.ca)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: ARG, arginine • ASN, asparagine • ASP, aspartic acid • C, carbon • DW, dry weight • FW, fresh weight • GDD, growing degree days • GLU, glutamic acid • HIS, histidine • HPLC, high performance liquid chromatography • IOD, integrated optical density • kDa, kilodalton • N, nitrogen • PRO, proline • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • TNC, total nonstructural carbohydrates • VSP, vegetative storage protein
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies pointed out the role of N reserves in winter stress tolerance and in vigor of spring regrowth (Ourry et al., 1994; Volenec et al., 1996). Hendershot and Volenec (1992)(1993) reported that amino acids and soluble proteins, which accumulate markedly in alfalfa taproots during cold acclimation decreased when growth resumed in early spring. Following defoliation, the major amino acids, aspartic acid and asparagine, and vegetative storage proteins (VSP) of 15, 19 and 32 kDa are mobilized as N sources for shoot regrowth (Cunningham and Volenec, 1996; Avice et al., 1997b). Furthermore, Ourry et al. (1994) have shown that the amounts of N available in crowns and roots at the beginning of regrowth were closely related to the amount of N mobilized to new shoot tissues, and to alfalfa shoot yield at the end of the regrowth period. Under field conditions, Lemaire et al. (1992), in France, and Bélanger and Richards (2000), in eastern Canada, observed that the quantity of N in alfalfa taproots decreased during the first 2 to 3 wk of regrowth. In spite of its well documented impact on stand productivity and persistence, little information is available on the effect of fall harvest management of alfalfa on N reserves during the overwintering period. In the light of evidence regarding the role of N reserves in the determination of alfalfa regrowth, we investigated the changes in specific N reserve components (total N, amino acids, and soluble proteins) in roots of alfalfa, as affected by fall harvest treatments. The specific objective of this work was to assess the impact of fall harvest management on regrowth potential in relation to the quantitative changes in N reserves in alfalfa taproots throughout fall and winter.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Establishment
Plants were established under controlled environment conditions. Seeds of alfalfa cultivars AC Caribou (hardy) and WL 225 (less hardy) were sown on 9 May 1997 in pots filled with a mixture (1:1, v/v) of top soil/peat moss (Pro-mix BX, Premier Peat Moss, Rivière-du-Loup, QC, Canada). Seeds were inoculated with a commercial inoculant of Sinorhizobium meliloti (Zakhia and de Lajudie, 2001) (Liphatec Inc., Milwaukee, WI) at the time of planting. Seedlings were thinned to eight plants per pot after emergence. Plants were grown for 7 wk in an environmentally controlled chamber set to the following conditions: photoperiod, 16 h; day-time temperature, 20°C; night-time temperature 17°C; photosynthetic photon flux density, 250 µmol photons m-2 s-1 provided by a mixture of Cool White (VHO) fluorescent (GTE, Sylvania) and incandescent lamps. Plants were kept well watered and fertilized once a week with 1 g L-1 solution of a commercial fertilizer (20-20-20, Plant-Prod, Brampton, ON, Canada) containing micronutrients. Plants were harvested at the early flower stage of development on 2 July 1997 (first harvest) and on 7 Aug. 1997 (second harvest).
Transfer to Outdoor Conditions
Immediately after the second harvest (7 Aug. 1997), pots were transferred to an experimental field near Québec City (46°49'15'' N; 71°12'00'' W; altitude 
45 m) and buried in the soil. Plants initiated their regrowth under natural irradiance and temperatures. Plants remained outdoor from 7 Aug. 1997 to 10 Nov. 1997, and were allowed to acclimate under the natural hardening conditions that prevail in late summer and early fall in eastern Canada. Fall harvest treatments were applied during this period as described below.
Fall Harvest Treatments
Four harvest treatments were applied in the fall: no additional harvest (two harvest system; second harvest on 7 Aug. 1997), or a third harvest taken 400, 500, or 600 growing degree days (GDD) accumulated after the second harvest, on 8 Sept. 1997, 17 Sept. 1997, or 7 Oct. 1997, respectively. The GDD were calculated by subtracting 5°C from the average of daily maximum and minimum air temperatures (Bélanger et al., 1992). Harvesting height was about 4 cm, and there was little leaf area remaining.
Transfer to Unheated Greenhouse
Pots were dug out on 10 Nov. 1997 and transferred to an unheated greenhouse for overwintering. Plants completed their acclimation to low temperatures in the unheated greenhouse, and remained there throughout the winter. Plants were not defoliated. The unheated greenhouse was continuously ventilated during daytime to maintain the inside temperature similar to that of the outside. When the air temperature inside remained permanently below freezing, plants were covered with a layer of insulating fiberglass wool to simulate snow cover. Soil temperatures were near freezing throughout the winter and remained at subzero levels (Dhont et al., 2002). Accordingly, no plant damage or mortality was noted during the overwintering period.
Tissue Sampling
Samples were collected at each harvest date for the corresponding harvest treatment: at the second harvest on 7 Aug. 1997 for plants harvested twice in the summer, at the third harvest on 8 Sept., 17 Sept., or 7 Oct. 1997 for plants harvested, respectively, at 400, 500, or 600 GDD after the second harvest. Samples were also collected on three other occasions during the overwintering period for all harvest treatments: mid-fall (10 Nov. 1997), midwinter (12 Jan. 1998), and early spring (11 Mar. 1998). There was no measurable shoot regrowth on the last sampling date (11 Mar. 1998). At each sampling date, plants were washed free of soil under a stream of cold water. Remaining shoots were removed, and crowns were separated from roots. Roots were weighed and subsequently cut into small segments (about 4–5 mm). A 1-g fresh weight (FW) subsample was immediately incubated in 8 mL of methanol-chloroform-water (MCW) (12:5:3; v:v:v) at 65°C for 30 min to inhibit enzymatic activities, and stored at -20°C until further extraction. A 5-g FW subsample was stored at -40°C until extraction of soluble proteins. A 5 to 10-g FW subsample was oven dried for 48 h at 55°C for dry matter determination.
Alfalfa Regrowth Potential
At each sampling during the overwintering period (10 Nov. 1997, 12 Jan. 1998, and 11 Mar. 1998), four pots of each harvest treatment were transferred to an environmentally controlled chamber, and plants were allowed to regrow under the initial establishment conditions described above. Shoots were harvested after a 3-wk regrowth period on 2 Dec. 1997, 4 Feb. 1998, and 2 Apr. 1998, respectively, and oven dried at 55°C until constant weight for dry weight determination. Data are presented in Dhont et al. (2002) and were used to assess correlations between shoot regrowth and components of N reserves.
Amino Acid Determination
Extraction of amino acids was conducted in 15 mL of MCW (12:5:3, v/v/v) as described previously in Dhont et al. (2002) for the soluble sugar extraction. The aqueous phase, resulting from phase separation with 4 mL of water and 1 mL of chloroform, was collected and kept frozen at -40°C until amino acid analysis by HPLC. The amino acid analysis system used Waters Millenium32 software, and consisted of two Model 510 pumps, a Model 717plus Autosampler and a Model 474 Scanning Fluorescence Detector (Waters Corp., Milford, MA). Amino acids were eluted by means of a Waters Radial-Pak RESOLVE-C18 column (5 µm; 8 by 100 mm) with a gradient of Buffer A [0.05 M Na2HPO4, 0.05 M NaCH3COO, 2% (v/v) methanol, 2% (v/v) tetrahydrofuran, pH 7.5) and mobile phase B (65% methanol) containing the following proportions of Buffer A: 70%, 0 to 2 min, flow rate 0.1 mL min-1; 70%, 2 to 2.5 min, flow rate increased from 0.1 to 2.5 mL min-1; 70%, 2.5 to 6.5 min; 70 to 38% linear, 6.5 to 14 min; 38 to 15% Waters curve #7, 14.0 to 19.0 min; 15 to 0% linear, 19.0 to 20.0 min; 0%, 20.0 to 23.0 min; 0 to 70% linear, 23.0 to 24.0 min; 70%, 24.0 to 29.5 min; 70%, 29.5 to 30.0 min, flow rate decreased to 0 mL min-1. Precolumn derivatization was made by the Autosampler by mixing 10 µL of sample with 10 µL of a solution of 5 mg mL-1 of orthophtalaldehyde (SIGMA-ALDRICH Canada Ltd, Oakville, ON, Canada) in 0.5 M K borate buffer, pH 10.0. Fluorometric detection excitation wavelength was 338 nm and emission wavelength was 425 nm. Peak identity and amino acid quantity were determined by comparison to standards. Nineteen amino acids were analyzed: aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, 
-amino butyric acid, 
-amino butyric acid, methionine, valine, phenylalanine, leucine, isoleucine, and lysine.
Free proline was quantified by spectrophotometry at 515 nm by means of a colorimetric reaction with ninhydrin modified by Paquin and Lechasseur (1979). Volumes of reactives were proportionally reduced by a factor of 10 to carry reactions in 1.5-mL microcentrifuge tubes as follows: 60 to 100 µL sample; 300 µL distilled water; 300 µL ninhydrin solution; 300 µL glacial acetic acid; 800 µL toluene. Proline concentrations were determined by comparison to a 0- to 14-µg proline standard curve and then expressed on a (µmol g-1) DW basis.
Total N Determination
Total N concentrations in roots were measured in the gas evolved from combustion of finely ground (100 µm) dry tissue with a LECO elemental analyzer, Model CNS-1000 (LECO Co., St-Joseph, MI) (Jimenez and Ladha, 1993).
Soluble Protein Extraction
At each sampling date, 20 g FW of root tissue was used for each cultivar x harvest treatment combination, by pooling 5-g FW subsamples from each replicate. Then, approximately 2 g FW of root tissue were ground on ice in 20 mL of 50 mM imidazole-HCl buffer (pH 6.5) containing 2 mM phenylmethylsulfonylfluoride (PMSF) with a Polytron homogenizer (Brinkmann Canada Ltd, Rexdale, ON, Canada). The homogenates were then adjusted to 10 mM ß-mercaptoethanol, 0.01% (v/v) Triton X-100 and 1% (w/w) polyvinylpolypyrrolidone (PVPP), agitated on ice for 15 min, filtered through cheesecloth and centrifuged at 15 000 x g for 20 min at 4°C. Supernatants were collected and a 500-µL aliquot was removed for soluble protein quantification (Lowry et al., 1951). The remaining supernatants were adjusted to 0.015% (w/w) deoxycholic acid and incubated for 10 min at room temperature. For both aliquots and remaining supernatants, proteins were precipitated in a final concentration of 66% (w/v) ammonium sulfate. The precipitates were collected by centrifugation at 15 000 x g for 20 min at 4°C. Supernatants were discarded, pellets were drained with Whatman paper (no. 4) and kept frozen at -40°C until protein quantification and electrophoresis. Pellets were solubilized in 0.1 M NaOH for soluble protein quantification (Lowry et al., 1951) and in loading buffer (O'Farrell, 1975) for SDS-PAGE.
Electrophoresis
SDS-PAGE was performed using a 4.5% (w/v) acrylamide stacking gel and a 12 to 18% acrylamide separation gel in a BRL electrophoresis apparatus (Model V16-2; GIBCO BRL, Burlington, ON, Canada). Protein samples were load adjusted to a constant dry weight of plant material of approximately 850 µg DW in each well. Prestained SDS-PAGE molecular weight standards (Broad range: 200, 116.25, 97.4, 66.2, 45, 31, 14.4, 6.5 kDa; BIO-RAD Laboratories Canada Ltd, Mississauga, ON, Canada) were used for molecular weight determination. Following electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 (SIGMA-ALDRICH Canada Ltd, Oakville, ON, Canada) according to the method of Laemmli (1970). Protein profiles were analyzed by densitometry with the One-Dscan Image Analysis System (Scanalytics Inc., Billerica, MA). Since electrophoretic patterns were similar in the two cultivars, only those for AC Caribou are presented.
Data Analysis
The experiment was conducted by means of a completely randomized design with four replicates. The experimental unit was a pot containing eight plants. Analyses of variance were performed on data from the harvest dates corresponding to the harvest treatments, and on data from each sampling date during the overwintering period. A priori contrasts were used for comparison of harvest, and cultivar x harvest means. Standard errors of the mean (SEM) were calculated for each sampling date. As soluble protein quantification was done on pooled samples, no replicate was available to undergo analysis of variance. Correlations between shoot regrowth and concentrations and amounts of N components at the onset of regrowth were calculated. Statistical significance was postulated at P < 0.05. Statistical analyses were performed by SAS statistical procedures (SAS Institute, 1996).
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total soluble protein concentrations increased during fall and winter and declined slightly by mid-March for all treatments (Fig. 1E and F). In November, January, and March in AC Caribou, harvesting at 400 GDD reduced soluble protein concentrations as compared with plants harvested twice; soluble protein concentrations measured in January were also reduced in AC Caribou harvested at 500 GDD. For both cultivars, in November and January, soluble protein concentrations in plants harvested at 600 GDD did not markedly differ from those in plants harvested twice whereas in March, these concentrations tended to be higher in alfalfa harvested at 600 GDD (Fig. 1E and F).
Amino Acid Concentrations
Concentrations of aspartic acid, glutamic acid, serine, alanine, and tyrosine were generally not affected by harvest treatments (data not presented). Therefore, the effects of fall harvests are only presented for asparagine, proline, arginine, and histidine (Fig. 2). Fall harvests did not significantly affect asparagine concentrations in fall and spring. There was a greater increase in asparagine concentration in January for WL 225 than for AC Caribou harvested at 500 GDD compared with the two harvest treatment (Fig. 2A and B; Table 2). In January, asparagine levels were significantly lower in plants harvested at 400 GDD than in plants harvested twice (Fig. 2A and B; Table 2).
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The 400 and 500 GDD harvesting treatments significantly reduced both arginine and histidine concentrations as compared with the plants harvested twice (Fig. 2E–H; Table 2). From November 1997 to March 1998, concentrations of both amino acids did not differ significantly between plants harvested at 600 GDD and plants harvested only twice in the summer. However, in November in AC Caribou, histidine concentrations were significantly reduced when fall harvest was delayed to 600 GDD when compared with plants harvested only twice in the summer (Fig. 2G and H; Table 2).
Effects of Fall Harvests on Total Pools of N Reserves in Alfalfa Roots
Total N, Total Amino Acid, and Total Soluble Protein Pools
To investigate further the effects of a fall harvest on N reserves, we expressed their levels in roots of alfalfa on a total amount basis. In alfalfa harvested twice in the summer, total N and total amino acid amounts markedly increased from mid-summer to mid-November, and declined progressively until spring (Fig. 3A–D). AC Caribou harvested at 600 GDD showed an increase in total N and total amino acid pools from the time of the third harvest (7 Oct. 1997) to mid-November 1997 sampling, whereas these pools decreased in WL 225 harvested at 600 GDD during the same period.
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The results for total soluble proteins amounts were almost similar to those observed for total N and total amino acid amounts. In November and January, total amounts of soluble proteins were reduced in plants harvested three times in the fall when compared with plants harvested twice, with a more negative effect of the fall harvests taken at 400 and 500 GDD in AC Caribou (Fig. 3E and F). Harvesting at 400 and 500 GDD also reduced markedly total soluble protein pools in March in both cultivars.
Amino Acid Pools
In November 1997 and January 1998, fall harvests reduced asparagine amounts in roots of both cultivars with a tendency for total asparagine to increase as the third harvest was delayed from 400 to 600 GDD in the fall (Fig. 4A and B; Table 2). In March 1998, asparagine amounts were reduced in both cultivars by a fall harvest taken at 400 or 500 GDD.
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Effects of Fall Harvests on Electrophoretic Profiles of Soluble Proteins
Proteins of 15, 19, 23, and 32 kDa were the most abundant in roots of alfalfa during fall and winter (Fig. 5), and their integrated optical densities (IOD) accounted for 40 to 75% of the total integrated densities (Table 3). The IOD of the 32-kDa protein alone accounted in some cases for 30 to 40% of total integrated densities on the gels. This protein accumulated from midsummer to mid-January in all plants (Fig. 5). In plants harvested twice and in plants harvested a third time at 600 GDD, the IOD of the 32-kDa protein started to decrease by mid-March (Fig. 5A and D; Table 3). In contrast, the level of the 32-kDa protein did not decrease until March in plants harvested at 400 or 500 GDD (Fig. 5B and C; Table 3). The accumulation of the 32-kDa protein was strongly reduced in plants harvested a third time in the fall at 400 or 500 GDD (Fig. 5B and C; Table 3).
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The IOD of the 15- and 19-kDa proteins accounted for 1 to 10% and 10 to 20% of total densities, respectively. The levels of these two proteins remained relatively stable throughout the overwintering season in plants harvested a third time at 400 or 500 GDD (Fig. 5B and C; Table 3). In contrast, the 15- and 19-kDa proteins accumulated markedly during fall, reached a maximum in January and decreased thereafter in plants harvested twice (Fig. 5A; Table 3), whereas their levels remained unchanged from August through March in plants harvested at 600 GDD (Fig. 5D; Table 3).
Correlations between Shoot Regrowth and N Reserves of Alfalfa
We assessed the relationship between shoot regrowth and organic reserves expressed on concentration and total amount bases prior to each regrowth period. In November, there were significant correlations between shoot regrowth and total N, total amino acid, arginine, and histidine concentrations in roots at the onset of the regrowth period for both cultivars (Table 4). However, no significant correlations were observed in November between shoot regrowth and asparagine or proline concentrations. In January, total N, total amino acid, arginine, and histidine concentrations were still positively related to shoot regrowth in WL 225, whereas no significant correlation was observed between the concentration of any N component and shoot regrowth of AC Caribou (Table 4). In March, shoot regrowth of AC Caribou was correlated to all N component concentrations, except asparagine. In WL 225, there was no significant correlation observed between shoot regrowth in March and concentrations of N components, except for total N.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in Amino Acids in Alfalfa Roots during the Overwintering Period
The concentrations of some of the major amino acids varied dramatically during the fall acclimation period (Table 1). Our results agree with previous reports indicating that total amino acid concentrations in alfalfa taproots increase markedly from late summer to midfall and remain at high levels throughout winter (Morot-Gaudry et al., 1987; Hendershot and Volenec, 1992; Barber et al., 1996). The prominence of asparagine observed in our study confirms data from Hendershot and Volenec (1993) indicating that asparagine accounts for 40 to 60% of the total amino acid concentration in alfalfa taproots (Table 1).
The marked accumulation of proline that occurred during fall acclimation in roots of alfalfa has long been documented (McKenzie et al., 1988). Proline is a major solute which accumulates in plants in response to several environmental stresses including salinity, water, and high temperature (Kavi Kishor et al., 1995; Franco and Melo, 2000). The increase of proline has been related to cold tolerance in Nothofagus dombeyi (Mirb.) Blume leaves (Meza-Basso et al., 1986), perennial weeds (Cichorium intybus L. and Taraxacum officinale Wigg.) (Cyr et al., 1990), and Arabidopsis thaliana (L.) Heynh. (Nanjo et al., 1999). In spite of reports of higher concentrations of free proline in alfalfa roots of cold tolerant compared to cold sensitive cultivars (McKenzie et al., 1988), conflicting evidence (Paquin, 1986) has kept the contribution of proline to overwintering of alfalfa unclear. Evidence has shown that proline accumulates as a result of stress exposure (Delauney and Verma, 1993; Hare and Cress, 1997) and it has been suggested that proline accumulation in cold acclimated plants is merely a consequence of, rather than the cause of cold acclimation (Bertrand and Paquin, 1991; Wanner and Junttila, 1999). Even though our study cannot clarify the role of proline in alfalfa roots over winter, our results nevertheless show that proline accumulation might contribute as a reservoir of organic nitrogen.
Both arginine and histidine are barely detectable in unacclimated roots of alfalfa in August, but then significantly accumulate during fall and winter to become 10 to 25% of the total amino acid content (Table 3). The increase in arginine to nearly 20% of the total amino acid pool in November contrasts with the observation of Hendershot and Volenec (1993) who reported that this amino acid comprised only 0.7 to 3% of the total pool of amino acids measured in the fall. In our study, arginine started to accumulate in alfalfa roots from mid-September to mid-November, with the lowering of air and soil temperatures and growth cessation. This result is consistent with its potential association with the cold acclimation process as suggested in pine (Pinus radiata D.) (Coker, 1991) and bilberry (Vaccinium myrtillus L.) (Lähdesmäki et al., 1990). In white clover (Trifolium repens L.), arginine concentrations were higher in hardier and frost tolerant populations (Svenning et al., 1997). It has been proposed that arginine may act as chelator of nitrate to prevent damages to membranes and ice formation (Lähdesmäki et al., 1990). Kuznetsov et al. (1999) also suggested that the enhanced synthesis of arginine under stress conditions may be related to the necessity of binding excess ammonia. In that perspective, the accumulation of the amino acids arginine, proline, and even asparagine, may be a common adaptive trait for plant tolerance to environmental stress.
Effects of Fall Harvest on Total N and Amino Acids in Alfalfa Roots over Winter
Recent studies have indicated that the amount of N in the regrowing shoots of alfalfa depends on the availability of N reserves in taproots at the onset of regrowth (Ourry et al., 1994; Skinner et al., 1999) and that any treatment that modifies N deposition in source organs will affect the vigor of subsequent shoot regrowth (Avice et al., 1997a). In our study, total N amounts and concentrations in alfalfa taproots were reduced by a third harvest in the fall, with shorter intervals between the second and the third harvest (400 and 500 GDD) having the most adverse effects (Fig. 1 and 3). The depleting effect of early fall harvests on N reserves may result from the interruption of shoot growth after the second harvest, or from reserve mobilization after the third harvest. The 6-wk regrowth period generally required to fully restore root N content (Lemaire et al., 1992) was not achieved for alfalfa harvested a third time at 400 or 500 GDD. On the other hand, a limited shoot regrowth after the third harvest that did not differ among harvest treatments (data not presented) suggests that there was no significant mobilization of N reserves for alfalfa shoot growth in the fall. The reduction of N reserves by early fall harvests has also to be considered in regard to root growth and storage of organic reserves that occur between September and November in alfalfa (Justes et al., 2002). We previously reported that root growth in the fall was restricted by harvest treatments, with shorter intervals (400 and 500 GDD) having the more limiting impact (Dhont et al., 2002). Therefore, it is likely that early fall harvests not only impeded the restoration of N reserves following the second harvest, but also reduced N reserve deposition during fall, leading to a depression of root N amounts available to sustain spring regrowth.
Total amino acid concentrations were not as markedly affected by fall harvests as the concentrations of some of the individual amino acids. This was mainly the result of the mitigated impact of fall harvest on the concentration of asparagine, the most abundant amino acid. In spite of its abundance in taproots, asparagine was poorly related to shoot regrowth (Table 4). Proline concentrations as well as total amounts of proline were strongly reduced by fall harvests, although the gradation typically observed with other N components (two harvests > 600 GDD > 500 GDD = 400 GDD) was not observed (Fig. 2 and 4). Considering the potential role of proline in cold tolerance and its contribution to shoot regrowth as a source of N, it seems likely that a harvest taken in the fall regardless of its timing could weaken winter survival and spring regrowth of alfalfa.
Arginine and histidine were markedly reduced by fall harvests, and decreasing the regrowth interval between the second and the third harvest (400 or 500 GDD) significantly affected both their concentrations and total amounts (Fig. 2 and 4, E- G). Seasonal accumulation of arginine, and the close relationship between its abundance in taproots and shoot regrowth suggest that this amino acid may play a determinant role as N reserve in overwintering alfalfa. Mobilization and translocation of arginine as a N storage compound has already been pointed out in tubers of purple nutsedge (Cyperus rotundus L.) (Fischer et al., 1995), in seedlings of loblolly pine (Pinus taeda L.) (King and Gifford, 1997), and in soybean [Glycine max (L.) Merr.] (Goldraij and Polacco, 1999). In tuberized roots of chicory (Cichorium intybus L.), arginine was found to account for 70% of the total amino acids by the end of vegetative growth (Améziane et al., 1997). Our results also bring new insights regarding the role of histidine in perennial plants. Even though this amino acid represents only 5 to 6% of total amino acids, the role of histidine as storage source of N should be considered, since its levels were correlated to shoot growth. Furthermore, the accumulation of histidine is up regulated during fall acclimation and winter, suggesting a potential association with cold tolerance in overwintering alfalfa. Under the prevailing growth conditions of our study, the absence of a third harvest offers the best opportunity to accumulate amino-N compounds, allowing plants to prepare for the overwintering period and eventual spring growth. The relationship between proline, arginine, and histidine accumulation and alfalfa winter hardiness and spring growth certainly warrants further investigation.
Effects of Fall Harvest on Soluble Proteins in Alfalfa Roots over Winter
Total soluble proteins increased in alfalfa taproots from late summer to midwinter and declined between January and mid-March (Fig. 1 and 3, E and F). Li et al. (1996) reported an increase of 50% in soluble protein concentration in alfalfa taproots between September and December, followed by a decrease between March and May. In the present study, the reduction of total soluble protein concentrations by fall harvests taken at 400 and 500 GDD was more obvious in AC Caribou than in WL 225. Among the soluble proteins, we observed that four proteins of 15, 19, 23 and 32 kDa accumulated in the fall in plants harvested twice in the summer, and were reduced by harvesting at 400 or 500 GDD. Several reports already point out the specific accumulation in alfalfa roots of three polypeptides of 15, 19, and 32 kDa that harbor typical characteristics of VSP (Cunningham and Volenec, 1996; Li et al., 1996). The synthesis and accumulation of VSPs were shown to be induced by low temperatures and short days in other species including poplars (Populus x canadensis Moench) (Van Cleve and Apel, 1993) and white clover (Bouchart et al., 1998; Corbel et al., 1999). Recently, Noquet et al. (2001) have reported that short days could also induce VSP accumulation in alfalfa taproots. Binnie et al. (1994) found that fall accumulation of VSPs in seedlings of spruce species [Picea glauca (Moench) Voss and Picea engelmanni Parris.] was highly correlated with an increase in freezing tolerance. These reports along with the marked accumulation of VSPs during fall and their depletion in the following spring suggest that these specific proteins could be involved in both tolerance to freezing temperatures and spring growth vigor in alfalfa. Cunningham et al. (1998) observed that a 23-kDa protein was present and accumulated at higher levels in fall dormant alfalfa, concluding that this protein may be associated to alfalfa winterhardiness. Fall harvests that induce modifications in protein composition in roots of alfalfa could not only affect the cold acclimation process, but also spring growth, and ultimately winterhardiness in alfalfa.
In the fall, photosynthetic assimilation of CO2 and growth decrease with the decline in temperatures and the reduction in daylength (Edminsten and Wolf, 1988). This alters source–sink relationships and cause a reallocation of organic reserves from shoots to roots (Bouchart et al., 1998), especially N reserves in alfalfa (Noquet et al., 2001). Fall harvest, that is the removal of source leaves, is likely to interfere with these physiological changes, and prevents plants from building up root organic reserves that are essential for the overwintering period and for mobilization in spring when growth resumes. We have recently reported that fall harvests strongly reduce the pool of carbohydrate reserves (Dhont et al., 2002). The present report points out that fall harvests also affect specific root N components, i.e., arginine, histidine, proline, and proteins, that are associated to regrowth and could be involved in cold acclimation and overwintering of alfalfa. Further studies under field conditions are necessary to document the relationship between these N components and alfalfa stand persistence and productivity under late fall management practices.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication February 15, 2002.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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