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Alfalfa root carbohydrates and regrowth potential in response to fall harvests. (Crop Physiology & Metabolism).

THE HISTORICAL RECOMMENDATION of not harvesting alfalfa during a critical fall rest period has often been questioned (Tesar and Yager, 1985; Sheaffer et al., 1986, Belanger et al., 1992). In Minnesota, the interval between harvests or the number of harvests was as important as the final harvest date in determining alfalfa persistence and yield (Sheaffer et al., 1986; Brink and Marten, 1989). Fall harvest management could be based on the duration of the growth interval determined by the accumulation of GDD between the last summer harvest and a fall harvest (Belanger et al., 1992).

It has long been documented that defoliation reduces TNC concentrations in taproots of alfalfa (Graber et al., 1927) and that low concentrations of carbohydrates are associated with poor winter survival and reduced spring regrowth (Reynolds, 1971). However, mid-December TNC concentrations were correlated poorly to spring yields in a study in Virginia (Edminsten et al., 1988), suggesting that high TNC concentrations in taproots prior to the onset of winter acclimation might not be a prerequisite for alfalfa persistence and vigor of spring regrowth. Starch, a major nonstructural polysaccharide that accumulates in alfalfa taproots, is greatly reduced after a defoliation or when growth resumes in spring (Habben and Volenec, 1991; Kim et al., 1993). Defoliation also leads to a decrease in root concentrations of reducing sugars (Barta, 1988). Even though Habben and Volenec (1991) established a positive relationship between starch mobilization and initial vigor of alfalfa regrowth, Boyce and Volenec (1992) subsequently observed that alfalfa genotypes selected for low starch concentration in taproots consistently showed greater regrowth than genotypes selected for their capacity to maintain high starch concentration. Ourry et al. (1994) also noted that starch concentration in alfalfa roots was poorly related to shoot regrowth, with plants having low starch and high root N contents showing higher shoot dry matter yield than plants having high starch and low N content.

Fall accumulation of soluble sugars (mainly sucrose) in taproots is considered a determinant factor for cold tolerance and winter survival of alfalfa (MacKenzie et al., 1988). During late cold acclimation, Castonguay et al. (1995) noted that the fall accumulation in crowns of alfalfa of raffinose and stachyose, members of the raffinose family of oligosaccharides (RFO), was more closely related to the differences in alfalfa freezing tolerance than sucrose accumulation.

The timing of a fall harvest on C reserves and its impact on spring regrowth of alfalfa remains unclear. Additionally, the effect of fall harvest management on cryoprotective sugars including sucrose and RFO has not been investigated. The majority of results on C reserves in overwintering alfalfa are expressed on a concentration basis, and the total amount of carbohydrates available in roots for winter survival and spring regrowth has seldom been considered. A study of carbohydrate pools could provide valuable information on their role in the regrowth of alfalfa harvested in the fall. We investigated the effect of the timing of a fall harvest on quantitative changes, expressed on concentration and total amount bases, of specific C reserves in roots of alfalfa. The relationship between alfalfa regrowth potential and these specific C reserves was also studied.

MATERIALS AND METHODS

Plant Material

Establishment

Plants were established under a controlled environment to ensure uniformity and minimize uncontrolled sources of stress. Seeds of alfalfa cultivars AC Caribou and WL 225, recommended for production in Quebec, Canada, were sown on 9 May 1997 in 20-cm diam, 20-cm-deep plastic pots filled with a mixture (1:1, v/v) of top soil/peat moss (Pro-mix BX, Premier Peat Moss, Riviere-du-Loup, QC, Canada). The choice of the cultivars was based on observations of contrasting persistence under the harsh winter conditions of eastern Canada, with AC Caribou being hardier than WL 225 (R. Michaud, personal communication, Agriculture and Agri-Food Canada, Sainte-Foy, QC, Canada, 1996). Seeds were inoculated with a commercial inoculant of Sinorhizobium meliloti (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[degrees]C; night-time temperature 17[degrees]C; photosynthetic photon flux density, 250 [micro]mol photons [m.sup.-2] [s.sup.-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.sup.-1] solution of a commercial fertilizer (20-20-20, Plant-Prod, Brampton, ON). Micronutrient content of the fertilizer was 1 g [kg.sup.-1] Fe, 0.5 g [kg.sup.-1] Mn, 0.5 g [kg.sup.-1] Zn, 0.5 g [kg.sup.-1] Cu, 0.2 g [kg.sup-.1] B, and 0.005 g [kg.sup.-1] Mo. 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 Quebec City (46[degrees]49' 15" N; 71[degrees]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 Quebec. Fall harvest treatments were applied during this period as described below.

Fall Harvesting 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 either at 400, 500, or 600 growing degree days (GDD) cumulated after the second harvest; the third harvest occurred on 8 Sept. 1997, 17 Sept. 1997, or 7 Oct. 1997, respectively. The GDD were calculated by subtracting 5[degrees]C from the average of daily maximum and minimum air temperatures (Belanger et al., 1992). Harvesting height was about 4 cm, and there was little leaf area remaining.

Transfer to Unheated Greenhouse

Pots were dug 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. Air temperatures inside the unheated greenhouse and soil temperatures in pots were monitored and recorded from mid-October 1997 to mid-March 1998 (Fig. 1), as described in Castonguay et al. (1995). Data of air temperatures from mid-October to mid-November 1997 and of soil temperatures from mid-February to mid-March 1998 are missing. The first killing frost (< -5[degrees]C) occurred on 24 Oct. 1997. Soil temperatures were near freezing throughout the winter and remained at subzero levels (Fig. 1). Accordingly, no plant damage or mortality was noted during the overwintering period.

[FIGURE 1 OMITTED]

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: in mid-fall (10 Nov. 1997), mid-winter (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 (transition zone between shoots and roots) were separated from roots. Roots were weighed and subsequently cut into small segments (about 4-5 mm). A 1-g fresh weight subsample was immediately incubated in 8 mL of methanol-chloroform-water (MCW) (12:5:3; v:v:v:) at 65[degrees]C for 30 min to inhibit enzymatic activities, and stored at -20[degrees]C until further extraction. A 5- to 10-g fresh weight subsample was oven dried for 48 h at 55[degrees]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[degrees]C until constant weight for dry weight determination.

Tissue Extractions

Soluble sugars were extracted as described previously by Castonguay et al. (1995). Tissues were ground on ice in 8 mL of MCW (12:5:3, v/v/v) with a Polytron homogenizer (Brinkmann Instruments Canada Ltd, Rexdale, ON, Canada). Seven milliliters of MCW were used to rinse the homogenizer and then combined with the first 8 mL of homogenate. Tubes were centrifuged for 10 min at 1000 X g and the supernatant was collected. For phase separation, 4 mL of water and 1 mL of chloroform were added to the 15 mL of extract. After shaking, the tubes were centrifuged for 10 min at 1000 X g and the aqueous phase was collected. A 1-mL subsample was evaporated to dryness on a rotary evaporator, solubilized in ethylene-diaminetetraacetic acid (EDTA; [Na.sup.+], [Ca.sup.2+]; 50 mg [L.sup.-1]), and then kept frozen at -40[degrees]C until its analysis by HPLC. The nonsoluble residues left after extraction were washed twice with 10 mL of methanol and used for starch determination.

Carbohydrate Analyses

Mono-, di-, tri-, and tetrasaccharides were separated and quantified by HPLC (Waters, Milford, MA). The analytical system used Waters [Millenium.sup.32] Software, and consisted of a Model 510 pump, a Model [717.sup.plus] Autosampler, and Model 410 Differential Refractometer. A Waters Sugar-Pak I column (6.5 X 300 mm) was eluted isocratically at 85[degrees]C with EDTA ([Na.sup.+], [Ca.sup.2+]; 50 mg [L.sup.-1]) at a flow rate of 0.5 mL [min.sup.-1]. Peak identity and sugar quantity were determined by comparison to standards.

Starch was quantitated as glucose equivalents with the p-hydroxybenzoic acid hydrazide method of Blakeney and Mutton (1980) after gelatinization at 100[degrees]C and digestion for 90 rain with amyloglucosidase (Sigma A7255, Sigma Chemical Co, St. Louis, MO). Starch amounts were determined spectrophotometrically by reference to a glucose standard curve.

Results from carbohydrate analyses were expressed on a concentration basis (mg [g.sup.-1] DW) or on a total amount basis (mg [plant.sup.-1]) by multiplying for each pot the concentrations by the root dry weight (DW) per plant.

Data Analysis

The experimental unit was a pot containing eight plants. The experiment was conducted by means of a completely randomized design with four replicates. Analyses of variance were done on data from the harvest dates corresponding to the harvest treatments, and on data from each sampling date during the overwintering period (Table 1). A priori contrasts were used for comparison of harvest, and cultivar X harvest means (Table 1). Standard errors of the mean (SEM) were calculated for each sampling date. Correlations between shoot regrowth and root DW, concentrations and amounts of carbohydrates at the onset of regrowth were calculated (Table 2). Statistical significance was postulated at P < 0.05. Statistical analyses were performed by SAS statistical procedures (Statistical Analysis System Institute, Inc., 1996).

RESULTS

I. Root Biomass and Shoot Regrowth

Root DW increased from the second harvest (7 Aug. 1997) to early fall (600 GDD third harvest; 7 Oct. 1997), with a higher increase in WL 225 harvested at 600 GDD (Fig. 2A and B; Harvests). For all harvest treatments, root DW increased up to the first sampling date (10 Nov. 1997), and then declined progressively throughout the winter. This reduction was most noticeable in plants harvested only twice (2 harvests). Cultivars did not differ in root DW assessed at each sampling date (10 Nov. 1997; 12 Jan. 1998; 11 Mar. 1998, Table 1). A harvest taken in the fall at either 400, 500, or 600 GDD reduced root DW as compared with plants harvested only twice during the summer (Fig. 2A and B; Table 1). Root DW on all sampling dates tended to increase as the fall harvest was delayed from 400 to 600 GDD. In March, root DW in WL 225 was more reduced than in AC Caribou by harvesting at 400 GDD, in comparison with their respective root DW with the two-harvest treatment [Fig. 2A and B; cultivars X (2 harvests vs. 400), Table 1].

[FIGURE 2 OMITTED]

Shoot DW after a 3-wk regrowth period differed between the two cultivars in December and April (Fig. 2C and D; Table 1). Cultivar WL 225 produced higher shoot DW than AC Caribou in the fall and, conversely, AC Caribou produced more shoot DW in April than WL 225. Shoot regrowth was generally reduced by a fall harvest taken at 400 or 500 GDD as compared with plants harvested only twice. This reduction in shoot regrowth was generally greater for WL 225 in fall, and for AC Caribou in spring, as indicated by interactions (Table 1). Harvesting at 600 GDD did not reduce shoot regrowth as compared with plants harvested twice.

II. Concentrations of Carbohydrates

Soluble Sugar Concentrations

Raffinose was present on all harvest dates from 7 Aug. to 7 Oct. 1997 (Fig. 3A and B; Harvests) but stachyose was almost completely absent until the beginning of October (Harvest at 600 GDD) in both cultivars (Fig. 3C and D; Harvests). Raffinose accumulated to greater concentrations in AC Caribou than in WL 225 on all harvest and sampling dates (Fig. 3A and B; Table 1). In addition, the increase in raffinose concentration with an additional harvest taken at 400, 500, or 600 GDD was greater for AC Caribou than for WL 225 in November and January (Fig. 3A and B; cultivars X harvests contrasts, Table 1). In contrast, cultivars did not differ in stachyose concentrations on all harvest and sampling dates, except in March where stachyose dropped to a lower concentration in WL 225 than in AC Caribou (Fig. 3C and D; Table 1). Concentrations of raffinose and stachyose were higher in roots of plants harvested three times as compared with plants harvested only twice (Fig. 3 A-D; Table 1). In January and March, raffinose concentrations decreased as the previous fall harvest was delayed, whereas stachyose concentrations showed an opposite response.

[FIGURE 3 OMITTED]

Sucrose concentrations markedly increased in both cultivars, from approximately 50 mg [g.sup.-1] DW in late summer to nearly 200 mg [g.sup.-1] DW in mid-January (Fig. 3E and F). Sucrose concentrations on harvest dates and in the fall (10 Nov. 1997) were higher in AC Caribou than in WL 225, especially for the three-harvest treatments. Sucrose concentrations remained high until March at which time no differences were observed between cultivars. As noted with raffinose and stachyose concentrations, sucrose concentrations were higher in plants harvested in the fall than in plants harvested only twice (Fig. 3E and F; Table 1). Delaying the fall harvest did not noticeably affect sucrose concentrations.

Starch and Total Nonstructural Carbohydrate Concentrations

As fall harvest was delayed from 400 (8 Sept. 1997) to 600 GDD (7 Oct. 1997), there was a progressive accumulation of starch, primarily in AC Caribou (Fig. 4A and B; Harvests). Starch concentrations declined progressively from mid fall (10 Nov. 1997) to early spring (11 Mar. 1998) in both cultivars. This decline in starch concentration was more pronounced in plants harvested in the fall at 500 or 600 GDD for AC Caribou, and at 500 GDD for WL 225 (Fig. 4A and B). In contrast to soluble sugars, a fall harvest generally reduced starch concentrations at each sampling date, with the exception of November, when starch concentration in AC Caribou harvested at 500 or 600 GDD was higher than in plants harvested only twice (Fig. 4A; cultivars X (2 harvests vs. 500) and cultivars X (2 harvests vs. 600), Table 1).

[FIGURE 4 OMITTED]

As observed for starch, there was a progressive accumulation of TNC from the second harvest (7 Aug. 1997) to early fall (third harvest at 600 GDD; 7 Oct. 1997) in roots of AC Caribou (Fig. 4C; Harvests). The TNC concentrations remained stable throughout fall and winter in both cultivars (Fig. 4C and D). In November, TNC concentrations of AC Caribou harvested at 500 or 600 GDD were higher than those of plants defoliated twice (Fig. 4C and D; cultivars X (2 harvests vs 500) and cultivars X (2 harvests vs. 600), Table 1). In January and March, TNC concentrations were reduced by fall harvests in both cultivars, with a lesser effect for the 400 GDD treatment in January. As observed with starch, there was a tendency for TNC concentrations to decline with an increase in the interval between the second summer harvest and the fall harvest.

In fall, there was no significant correlation between shoot regrowth and root carbohydrate concentrations at the onset of the regrowth period, except in WL 225 for which root sucrose concentration was correlated negatively to shoot regrowth (Table 2). In March, the only significant positive correlation was observed in AC Caribou for starch concentration (Table 2). Correlations between root carbohydrate concentrations assessed on 12 Jan. 1998 and shoot regrowth subsequently measured on 4 Feb. 1998 were not significant (data not shown).

III. Pools of Carbon Reserves

Soluble Sugar Pools

Carbohydrate reserves were also expressed on the basis of their total accumulation in roots (Fig. 5 and 6). In both cultivars, raffinose markedly increased in roots from mid-summer (7 Aug. 1997) to mid-winter (12 Jan. 1998), and tended to decline thereafter in spring (Fig. 5A and B). The raffinose amounts were higher in roots of AC Caribou than in those of WL 225 on all harvest and sampling dates (Fig. 5A and B; Table 1). In November, a third harvest taken at 400 or 500 GDD reduced raffinose amounts. However, harvesting at 600 GDD caused a decrease in raffinose amounts in WL 225 but not in AC Caribou as compared with plants harvested twice [Fig. 5A and B; cultivars x (2 harvests vs. 600), Table 1]. Stachyose amounts started to increase later in fall than raffinose to reach similar values in both cultivars, and declined thereafter in March (Fig. 5C and D; Table 1). In November and January, fall harvests taken at 400 and 500 GDD strongly reduced the amount of stachyose per plant in both cultivars as compared with plants harvested twice (Fig. 5C and D; Table 1). Harvesting at 600 GDD had no effect on stachyose amounts as compared with plants defoliated only twice, except for AC Caribou in November [Fig. 5C and D; cultivars x (2 harvests vs. 600), Table 1]. Total sucrose amounts in roots of alfalfa increased during fall hardening and decreased in the spring. Sucrose amounts were generally reduced by fall harvest treatments. Fall harvesting at 400 or 500 GDD had the most adverse effects on sucrose amounts on all sampling dates (Fig. 5E and F; Table 1). However, in November, harvesting at 600 GDD caused a greater decrease in sucrose amounts in WL 225 than in AC Caribou [Fig. 5E and F; cultivars x (2 harvests vs. 600), Table 1]. In January, pools of sucrose were lower in roots of plants harvested at 600 GDD as compared with plants harvested twice.

[FIGURES 5-6 OMITTED]

Starch and TNC Pools

Starch and TNC amounts of both cultivars increased in late summer and early fall as the third harvest was delayed from 400 (8 Sept. 1997) to 600 (7 Oct. 1997) GDD (Fig. 6A-D; Harvests), and subsequently declined throughout winter in all treatments (Fig. 6A-D). A third harvest reduced total starch and TNC amounts as compared with alfalfa harvested only twice (Fig. 6A-D; Table 1). In mid-November, starch and TNC amounts were more severely reduced by harvesting at 600 GDD in WL 225 than in AC Caribou [Fig 6A-D; cultivars x (2 harvests vs. 600), Table 1]. Harvesting WL 225 at 400 GDD caused a more severe reduction in starch and TNC amounts than in AC Caribou in early spring (11 Mar. 1998) [Fig. 6 A-D; cultivars x (2 harvests vs 400) or cultivars x (2 harvests vs. 500), Table 1]. The cultivars did not differ in starch and TNC amounts, except in November when the accumulation of TNC was greater in AC Caribou than in WL 225 (Fig. 6C and D, Table 1).

In November, the two cultivars differed in their correlations between shoot regrowth and root carbohydrate amounts at the onset of the regrowth period. In WL 225, root DW and all carbohydrate components (raffinose, stachyose, sucrose, starch, and TNC) were correlated highly to shoot regrowth (Table 2). However, no significant correlation was observed in AC Caribou for root DW or any other component of the carbohydrate pool (Table 2). In spring, the raffinose and stachyose pools of both cultivars were not correlated significantly to shoot regrowth, but starch and TNC pools were positively related to shoot regrowth (Table 2). Differences between cultivars were observed for the relationship between shoot regrowth, and sucrose pool and root DW; significant correlations were found in WL 225 but not in AC Caribou (Table 2). Correlations assessed between root carbohydrate pools on 12 Jan. 1998 and shoot re growth subsequently measured on 4 Feb. 1998 were not significant (data not shown).

DISCUSSION

Even though fall harvesting management is a determinant factor for persistence and regrowth of alfalfa (Sheaffer et al., 1986; Brink and Marten, 1989), information remains scarce on the effects of the timing of fall harvests on the accumulation and composition of carbohydrate reserves. We investigated the timing of an additional harvest in the fall on alfalfa regrowth potential in relation to the evolution of carbohydrate components in roots throughout the overwintering period. Our study was conducted with pot-grown plants established under a controlled environment, but the plants were acclimated to winter conditions under natural temperature and photoperiod variations, outdoors, and in an unheated greenhouse. This approach allowed realistic overwintering conditions with natural variations in air and soil temperatures, without most of the constraints related to winter sampling under field conditions. Previous studies (Castonguay et al., 1995, 1998) have already confirmed that cold tolerance and carbohydrate composition of unheated greenhouse-acclimated plants were comparable to those observed in field studies (McKenzie et al., 1988).

Shoot Regrowth and Root DW

Our observation that shoot regrowth of alfalfa can be reduced by an additional harvest in the fall is in accordance with earlier observations by Sheaffer and Marten (1990) who showed that fall harvest management affects alfalfa yield in the following year. Increasing the length of the regrowth interval between the second summer harvest and the fall harvest mitigated the negative impact on shoot regrowth. Previous field studies also concluded that a reduction in the regrowth interval between the last two harvests reduced alfalfa yield in the following year (Edminsten et al., 1988; Brink and Marten, 1989; Sheaffer and Marten, 1990; Belanger et al., 1992). Using the accumulation of GDD since the last summer harvest for the timing of the fall harvest, Belanger et al. (1992) concluded that an accumulation of 500 GDD was sufficient to achieve maximum DM yield. In the current study, a fall harvest taken at 600 GDD was required to achieve a regrowth that did not differ from that of plants harvested only twice in the summer. Our results confirm the field observations of Belanger et al. (1999) in Atlantic Canada where first harvest yield and total seasonal yield of the subsequent year increased with the lengthening of the interval between the second summer harvest and a third harvest in the fall.

In our system, a root biomass of approximately 60 g [m.sup.-2] (0.9 g [plant.sup.-1]) at the time of the plant transfer to the field conditions (Second harvest, 7 August) was comparable to values of 50 to 100 g [m.sup.-2] measured at the same period in eastern Canada (4-9 August) with field-grown plants in their seeding year (Belanger and Richards, 2000). Our values of 120 g [m.sup.-2] (2 g [plant.sup.-1]) for root biomass in mid-September (500 GDD harvest date) were also close to those observed by Belanger and Richards (2000) during the same period (130-170 g [m.sup.-2]). Our results on root biomass changes from the time of the second harvest to mid-September are also in agreement with those of Khaiti and Lemaire (1992) in France, and Belanger and Richards (2000) in eastern Canada, who reported no or very limited increase in root biomass in the first 20 to 30 d following defoliation (essentially August) followed by a large increase in September. In our study, alfalfa root DW increased markedly in October and early November with the decline in temperature and photoperiod. In spite of several reports on the accumulation of carbohydrate reserves in October and November in alfalfa roots (Li et al., 1996, Cunningham et al., 2001), there are no reports in the literature on the concomitant alfalfa root growth during that period, especially in the seeding year. Our data indicate that TNC accumulation accounted for 40 to 50% of the root DW increase in the fall. The remaining portion of the root biomass increase could be attributable to an increase in structural DM and the accumulation of other organic components such as N or lipid reserves. Therefore, in our study, the transfer of pot-grown plants to outdoor conditions on 7 August and the subsequent exposure to field conditions led to root growth and TNC accumulation that are typical of field grown plants.

The root growth until mid-November of plants harvested twice was greater than that of plants harvested in the fall, even when the fall harvest was delayed until 600 GDD. Plants harvested for a third time in the fall had a root DW that was 30 to 60% lower than plants harvested only twice during the summer. Root DW varied with the interval between the second and a third harvest, the shorter regrowth intervals of 400 and 500 GDD having the most adverse effects. Avice et al. (1997a) noted that shorter regrowth intervals between harvests taken at the beginning of the growing season led to a reduction of root DW. Smaller root DW in plants harvested early in the fall (400 GDD) can be explained in part by the mobilization of existing C reserves to sustain initial shoot regrowth (Habben and Volenec, 1991), along with preferential allocation of newly assimilated C to the shoot to restore the imbalance created between sources and sinks (Frankow-Lindberg, 1997). A late fall harvest at 600 GDD had a lesser impact on root DW, as the period of accumulation of root organic reserves was increased and little regrowth occurred before the first killing frost.

The cultivars differed in their relationship between root DW and shoot regrowth. In fall and spring, root DW was related to subsequent shoot regrowth in WL 225 but not in AC Caribou, suggesting that cold adaptation of cultivars could affect the initiation of regrowth upon exposure to growing conditions. Even though the two cultivars are rated with similar levels of fall dormancy (Certified Alfalfa Seed Council, 1994; Michaud, personal communication, 1996), our results indicate differences in fall dormancy under our experimental conditions, with AC Caribou being more dormant than WL 225. Indeed, WL 225 presented superior regrowth in December than did AC Caribou. Furthermore, a greater accumulation of carbohydrates in roots of AC Caribou than in WL 225 in the fall agrees with Cunningham et al. (1998) who reported superior root soluble sugar accumulation in November and December in populations selected for higher fall dormancy.

Carbohydrate Concentrations

One of our objectives was to characterize the changes occurring during winter in soluble sugar and starch concentrations in alfalfa following different fall harvest treatments. As previously documented, concentrations of soluble sugars increased markedly during fall hardening (McKenzie et al., 1988; Li et al., 1996). Sucrose and the galactose-containing oligosaccharides, raffinose and stachyose, were the most abundant soluble sugars that accumulated in roots of cold acclimated alfalfa, in accordance with measurements made in crowns by Castonguay et al. (1995). Contrary to our expectations of a reduction in soluble sugars with a fall harvest, root concentrations of sucrose, stachyose, and raffinose in plants harvested in the fall were increased relative to plants harvested twice in the summer. The concentrations of these soluble sugars have been related to the acquisition of freezing tolerance in forage legumes (Volenec et al., 1991; Svenning et al., 1997; Castonguay et al. 1995, 1998) and therefore, it seems unlikely that fall harvesting impairs soluble sugar-based cryoprotection.

Differential accumulation of soluble sugars in taproots of alfalfa cultivars of contrasting hardiness was previously reported (Cunningham et al., 1998). In our study, changes in sucrose concentrations during cold acclimation and in response to fall harvest treatments were similar in both cultivars. However, raffinose accumulated to higher concentrations in roots of the hardier cultivar AC Caribou than in WL 225. Castonguay et al. (1995, 1998) reported that both stachyose and raffinose accumulated to higher levels in cultivars of superior winterhardiness, and related these oligosaccharides to the acquisition of cold tolerance. In contrast with raffinose accumulation, root concentrations of stachyose did not differ between cultivars.

Few studies have characterized the evolution of carbohydrate concentrations from early fall to spring in relationship with fall harvest management. It has been shown previously that TNC concentrations in the fall are maximized in a two-harvest system, and that the reductions in TNC concentration are minimized when a third harvest is delayed in the fall (Gervais and Bilodeau, 1985; Gervais and Girard, 1987). Edminsten et al. (1988) reported that TNC concentrations were higher if the regrowth interval between the last two harvests lasted for a minimum of 50 d. In contrast with those results, we observed that delaying fall harvest did not lead to higher TNC concentrations. Starch concentrations throughout the overwintering period were generally reduced by fall harvests while TNC concentrations were only slightly affected. The stability of TNC concentrations, irrespective of fall harvest treatments, was the result of a balance between a decrease in starch and a concomitant increase in soluble sugar concentrations. Our results agree with those of Sheaffer and Marten (1990) who observed that root TNC concentrations in November are not affected by fall harvests. Edminsten et al. (1988) also reported that in spite of differences in TNC concentrations prior to the onset of winter, there were no differences in TNC concentrations in mid-March before spring regrowth.

Root TNC and starch concentrations in fall and winter were not correlated to shoot regrowth. This agrees with Sheaffer and Marten (1990) who reported that root TNC concentrations in fall were not reliable predictors of alfalfa yields in the spring. Edminsten et al. (1988) also observed that December TNC concentrations were not related to spring yields. It is not known whether a carbohydrate concentration threshold in alfalfa taproots is required to sustain regrowth, but there are indications that modifications in the carbohydrate status will affect the initial rate of regrowth (Morvan-Bertrand et al., 1999; Skinner et al., 1999). The lack of consistent relationships between carbohydrate concentrations and shoot regrowth observed in our study does not preclude the importance of C reserves for regrowth as they make a contribution as a source of energy to sustain metabolic activity during heterotrophic periods, when respiration exceeds photosynthesis (Avice et al., 1997a,b).

Carbohydrate Pools

There is little information available on total amounts of carbohydrate reserves that accumulate in roots of alfalfa along with their fluctuations throughout winter as most of the published data have been expressed on a concentration basis. In contrast to our observations based on concentrations, a fall harvest strikingly depleted total amounts of starch and TNC in roots, with the fall harvest taken at 400 GDD having the greater effect (e.g., 200 mg TNC [plant.sup.-1] in 400 GDD vs. 800 mg TNC [plant.sup.-1] in 600 GDD in AC Caribou in November). Harvesting alfalfa early in the fall at 400 or 500 GDD interrupted root growth and the accumulation of carbohydrates. In contrast, plants harvested at 600 GDD accumulated higher amounts of carbohydrates in the fall before growth cessation. The reduction of carbohydrate amounts in response to a fall harvest was not likely due to their mobilization for regrowth, since fall regrowth after the third harvest did not differ among harvest treatments, with the exception for WL 225 harvested at 400 GDD which showed superior growth (data not shown).

The TNC amounts decreased throughout winter, especially in plants harvested twice, and this reduction followed a trend similar to that observed for root DW (e.g., 50% in AC Caribou, and 25% in WL 225 harvested twice). However, the minimum amounts of carbohydrates reached at the end of the winter were sufficient to maintain stable and consistent regrowth in early spring. Larger accumulation of root carbohydrates during the fall may provide some benefits to the plants harvested twice as compared with those harvested a third time in the fall, such as superior cold tolerance, or greater resistance to pathogens.

The reduction in TNC amounts represented about 35% of the decrease of root DW during winter. Hence losses of other root cell constituents occurred. Farrar and Jones (2000) indicated that in addition to C[O.sub.2], carbon losses may occur through rhizodeposition, which includes components such as mucilage, lactic acid, sugars, amino acids, and other organic components, as well as sloughed cells. It is not known how pools of constituents other than TNC are affected during overwintering in alfalfa subjected to different fall harvest managements.

Although all types of carbohydrates were correlated to shoot regrowth in fall in WL 225, no such relationship was observed in AC Caribou. Such differences in the relationship between the availability of C reserves and shoot regrowth between the two cultivars could be related to different fall acclimation response. Skinner et al. (1999) reported mat mere was no relationship between regrowth and the total TNC that accumulated in roots or that were remobilized. However, in spring, TNC amounts were related positively to the regrowth in both cultivars, indicating that total C reserves could be a determinant factor of alfalfa regrowth as plants come out of dormancy.

Our study showed that carbohydrate amounts were more affected by fall harvest and were better correlated to shoot regrowth than carbohydrate concentrations, underlining the importance of total availability of C reserves for shoot regrowth. Marked variations in the relationship between regrowth and total carbohydrates between cultivars and sampling dates clearly indicate that additional factors need to be considered for a more comprehensive understanding. Several reports have recently indicated that N reserves contribute to winter survival and shoot regrowth of alfalfa (Volenec et al., 1996; Avice et al., 1996, 1997b). Analyses of amino acids and soluble proteins accumulation currently underway will lead to a better understanding of the biochemical bases of the effects of fall harvest management on alfalfa regrowth.

Abbreviations: DW, dry weight; GDD, growing degree days; HPLC, high performance liquid chromatography; MCW, methanol chloroform water; RFO, raffinose family oligosaccharide; TNC, total non structural carbohydrates.
Table 1. Analyses of variance and a priori contrasts
for harvest dates and each sampling date for root and
shoot DW, and for all biochemical variables expressed
on a concentration basis as well as on a total pool basis.

                                    Root DW         Shoot DW
                                                   ([dagger])

                                               P > F

Cultivars                           <0.001              -
Harvest dates ([double dagger])     <0.001              -
 2 harvests vs. 400                  ns ([section])     -
 2 harvests vs. 500                 <0.001              -
 2 harvests vs. 600                 <0.001              -
Cultivars x harvest dates            0.005              -
 Cultivars x (2 harvests vs. 400)    ns                 -
 Cultivars x (2 harvests vs. 500)    ns                 -
 Cultivars x (2 harvests vs. 600)   0.001               -

Cultivars                            ns                0.007
Harvest                             <0.001            <0.001
 2 harvests vs. 400                 <0.001             0.003
 2 harvests vs. 500                 <0.001            <0.001
 2 harvests vs. 600                 <0.001             ns
Cultivars x harvests                 ns                0.046
 Cultivars x (2 harvests vs. 400)    ns                0.009
 Cultivars x (2 harvests vs. 500)    ns                0.029
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            ns                ns
Harvest                             <0.001             0.022
 2 harvests vs. 400                 <0.001             0.006
 2 harvests vs. 500                 <0.001             0.037
 2 harvests vs. 600                 <0.001             ns
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            ns                0.040
Harvests                            <0.001             0.006
 2 harvests vs. 400                 <0.001            <0.001
 2 harvests vs. 500                 <0.001             0.008
 2 harvests vs. 600                  ns                ns
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    0.012             ns
 Cultivars x (2 harvests vs. 500)    ns                0.012
 Cultivars x (2 harvests vs. 600)    ns                ns

                                          Concentrations

                                    Raffinose        Stachyose

                                                  P > F

                                            Harvest dates

Cultivars                           <0.001                ns
Harvest dates ([double dagger])      ns                  <0.001
 2 harvests vs. 400                  ns                   ns
 2 harvests vs. 500                  ns                   ns
 2 harvests vs. 600                  ns                  <0.001
Cultivars x harvest dates            0.012                0.052
 Cultivars x (2 harvests vs. 400)    ns                   ns
 Cultivars x (2 harvests vs. 500)    0.001                ns
 Cultivars x (2 harvests vs. 600)    ns                   0.020

                                             10 Nov. 1997

Cultivars                           <0.001                ns
Harvest                             <0.001               <0.001
 2 harvests vs. 400                  ns                   ns
 2 harvests vs. 500                  0.029                ns
 2 harvests vs. 600                 <0.001               <0.001
Cultivars x harvests                 0.019                ns
 Cultivars x (2 harvests vs. 400)    0.016                ns
 Cultivars x (2 harvests vs. 500)    0.012                ns
 Cultivars x (2 harvests vs. 600)    0.004                ns

                                            12 Jan. 1998

Cultivars                           <0.001                ns
Harvest                             <0.001                0.003
 2 harvests vs. 400                 <0.001                0.015
 2 harvests vs. 500                 <0.00                 0.002
 2 harvests vs. 600                  0.005               <0.001
Cultivars x harvests                 ns                   ns
 Cultivars x (2 harvests vs. 400)    ns                   ns
 Cultivars x (2 harvests vs. 500)    0.010                ns
 Cultivars x (2 harvests vs. 600)    0.043                ns

                                            11 Mar. 1998

Cultivars                           <0.001                0.002
Harvests                             0.016                0.002
 2 harvests vs. 400                  0.003                ns
 2 harvests vs. 500                  0.036                0.005
 2 harvests vs. 600                  0.016               <0.001
Cultivars x harvests                 ns                   ns
 Cultivars x (2 harvests vs. 400)    ns                   ns
 Cultivars x (2 harvests vs. 500)    ns                   ns
 Cultivars x (2 harvests vs. 600)    ns                   ns

                                          Concentrations

                                    Sucrose          Starch

                                   Harvest date

                                               P > F

Cultivars                            0.005             ns
Harvest dates ([double dagger])     <0.001            <0.001
 2 harvests vs. 400                  0.013             ns
 2 harvests vs. 500                 <0.001             0.002
 2 harvests vs. 600                 <0.001            <0.001
Cultivars x harvest dates            0.017            <0.001
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                0.003
 Cultivars x (2 harvests vs. 600)    0.003            <0.001

                                   10 Nov. 1997

Cultivars                           <0.001             0.005
Harvest                             <0.001            <0.001
 2 harvests vs. 400                 <0.001             0.001
 2 harvests vs. 500                 <0.001             ns
 2 harvests vs. 600                 <0.001             0.010
Cultivars x harvests                 ns               <0.001
 Cultivars x (2 harvests vs. 400)    0.024             ns
 Cultivars x (2 harvests vs. 500)    ns                0.010
 Cultivars x (2 harvests vs. 600)    0.033            <0.001

                                   12 Jan. 1997

Cultivars                            ns                ns
Harvest                              ns                0.002
 2 harvests vs. 400                  ns                ns
 2 harvests vs. 500                  0.041            <0.001
 2 harvests vs. 600                  0.013             0.003
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

                                   11 Mar. 1998
Cultivars                            ns               <0.001
Harvests                             ns               <0.001
 2 harvests vs. 400                  ns               <0.001
 2 harvests vs. 500                  ns               <0.001
 2 harvests vs. 600                  0.036            <0.001
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

                                   Concentrations     Pools

                                      TNC           Raffinose

                                              P > F

Cultivars                            0.042             0.001
Harvest dates ([double dagger])     <0.001            <0.001
 2 harvests vs. 400                  ns                ns
 2 harvests vs. 500                 <0.001             ns
 2 harvests vs. 600                 <0.001            <0.001
Cultivars x harvest dates           <0.001             ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    0.005             ns
 Cultivars x (2 harvests vs. 600)   <0.001             ns

Cultivars                           <0.001            <0.001
Harvest                             <0.001            <0.001
 2 harvests vs. 400                  0.045            <0.001
 2 harvests vs. 500                  0.002             ns
 2 harvests vs. 600                  ns                ns
Cultivars x harvests                <0.001             0.033
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    0.010             ns
 Cultivars x (2 harvests vs. 600)   <0.001             0.022

Cultivars                            ns               <0.001
Harvest                              ns                ns
 2 harvests vs. 400                  ns                ns
 2 harvests vs. 500                  0.045             ns
 2 harvests vs. 600                  ns                ns
Cultivars x harvests                 ns                0.033
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                0.033

Cultivars                            0.001            <0.001
Harvests                            <0.001             0.011
 2 harvests vs. 400                  ns                ns
 2 harvests vs. 500                  0.002             ns
 2 harvests vs. 600                 <0.001             ns
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

                                             Pools

                                   Stachyose         Sucrose

                                             P > F

Cultivars                            ns                ns
Harvest dates ([double dagger])     0.001             <0.001
 2 harvests vs. 400                  ns                ns
 2 harvests vs. 500                  ns               <0.001
 2 harvests vs. 600                 <0.001            <0.001
Cultivars x harvest dates            ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            0.011             0.002
Harvest                             <0.001            <0.001
 2 harvests vs. 400                 <0.001            <0.001
 2 harvests vs. 500                 <0.001            <0.001
 2 harvests vs. 600                  ns               <0.001
Cultivars x harvests                 ns                0.010
 Cultivars x (2 harvests vs. 400)    0.010             ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            ns                ns
Harvest                             <0.001            <0.001
 2 harvests vs. 400                  0.002            <0.001
 2 harvests vs. 500                  0.002            <0.001
 2 harvests vs. 600                  ns                0.031
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            ns                ns
Harvests                             0.001             0.002
 2 harvests vs. 400                  ns                0.006
 2 harvests vs. 500                  ns                0.001
 2 harvests vs. 600                  0.035             ns
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                0.046
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

                                             Pools

                                    Starch             TNC

                                             P > F

Cultivars                            ns                ns
Harvest dates ([double dagger])      ns               <0.001
 2 harvests vs. 400                  ns                ns
 2 harvests vs. 500                 <0.001            <0.001
 2 harvests vs. 600                 <0.001            <0.001
Cultivars x harvest dates            ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            ns                0.007
Harvest                             <0.001            <0.001
 2 harvests vs. 400                 <0.001            <0.001
 2 harvests vs. 500                 <0.001            <0.001
 2 harvests vs. 600                 <0.001            <0.001
Cultivars x harvests                <0.001            <0.001
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)   <0.001             0.004

Cultivars                            ns                ns
Harvest                             <0.001            <0.001
 2 harvests vs. 400                 <0.001            <0.001
 2 harvests vs. 500                 <0.001            <0.001
 2 harvests vs. 600                 <0.001            <0.001
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    ns                ns
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

Cultivars                            0.051             ns
Harvests                            <0.001            <0.001
 2 harvests vs. 400                 <0.001             0.001
 2 harvests vs. 500                 <0.001            <0.001
 2 harvests vs. 600                  0.009             ns
Cultivars x harvests                 ns                ns
 Cultivars x (2 harvests vs. 400)    0.033             0.026
 Cultivars x (2 harvests vs. 500)    ns                ns
 Cultivars x (2 harvests vs. 600)    ns                ns

([dagger]) Shoot regrowth was initiated on 10 Nov. 1997, 12 Jan. 1998,
and 11 Mar. 1998, and shoot DW was measured after three weeks on 2 Dec.
1997, 4 Feb. 1998, and 2 Apr. 1998, respectively.

([double dagger]) Harvest treatments were: 2nd harvest (7 Aug. 1997),
additional harvests in fall 400, 500, or 600 GDD after the second
harvest, on 8 Sept. 1997, 17 Sept. 1997, and 7 Oct. 1997, respectively.

([section]) Not significant at P < 0.05.
Table 2. Correlations between shoot DW after 3-wk regrowth
periods initiated on 10 Nov. 1997 and 11 Mar. 1998, and
carbohydrate components and root DW measured at the onset
of the regrowth periods for two alfalfa cultivars (WL 225
and AC Caribou). Carbohydrate components were expressed as
coacentrations (mg [g.sup.-1] DW) and pools (mg [plant.sup.-1]).

                  Carbohydrate concentrations    Carbohydrate pools

                 10 Nov.          11 Mar.       10 Nov.      11 Mar.
                  1997             1998          1997         1998

                    r                r             r            r

WL 225

Stachyose    0.01ns ([dagger])  -0.22ns         0.70 **      0.34ns
Raffinose    0.35ns             -0.42ns         0.66 **      0.25ns
Sucrose     -0.62 **            -0.02ns         0.70 **      0.55 *
Starch       0.00ns              0.35ns         0.59 **      0.54 *
TNC          0.14ns              0.36ns         0.63 **      0.55 *
Root DW                                         0.74 **      0.53 *

AC Caribou

Stachyose    0.20ns             -0.29ns         0.41ns       0.21ns
Raffinose    0.15ns             -0.40ns         0.27ns       0ns
Sucrose     -0.30ns             -0.37ns         0.33ns       0.39ns
Starch       0.07ns              0.56 *         0.32ns       0.61 *
TNC          0.20ns              0.35ns         0.33ns       0.56 *
Root DW                                         0.33ns       0.48ns

* Indicates significance at P = 0.05.

** Indicates significance at P = 0.01.

([dagger]) Not significant at P < 0.05.


ACKNOWLEDGMENTS

The technical assistance of Lucette Chouinard, Annie Tremblay, and Pierre Lechasseur is gratefully acknowledged.

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Catherine Dhont, Yves Castonguay, * Paul Nadeau, Gilles Belanger, and Francois-P. Chalifour

C. Dhont and F.-P. Chalifour, Departement de Phytologie, Universite Laval, Sainte-Foy, QC, Canada G1K 7P4; Y. Castonguay, P. Nadeau and G. Belanger, Agriculture and Agri-Food Canada, Sainte-Foy, QC, Canada G1V 2J3. This work was supported by a Research Grant from the Conseil des Recherches en Peche et en Agroalimentaire du Quebec (CORPAQ), and by a Research Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.-P. Chalifour. Contribution no. 723 of the Sainte-Foy Research Centre. Received 17 April 2001. * Corresponding author (castonguayy @em.agr.ca).
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