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Crop Science 43:1340-1348 (2003)
© 2003 Crop Science Society of America

CROP PHYSIOLOGY & METABOLISM

Autumn Defoliation Effects on Alfalfa Winter Survival, Root Physiology, and Gene Expression

D. M. Haagenson, S. M. Cunningham, B. C. Joern and J. J. Volenec*

Dep. of Agronomy, Purdue Univ., West Lafayette, IN 47907-1150 USA

* Corresponding author (jvolenec{at}purdue.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Harvesting alfalfa (Medicago sativa L.) after mid-September in the North-Central USA often reduces plant winter survival, but the physiological mechanisms associated with poor winter survival are not understood. Our objective was to determine how autumn harvesting affects alfalfa root physiology, gene expression, and plant winter survival. In Exp. 1, seven fall harvest dates were used to identify 1 to 15 October as a critical interval where significant changes in alfalfa winter survival and root physiology occur in Indiana. In Exp. 2, rows of six alfalfa cultivars possessing contrasting fall dormancy (FD) were established in May. Plants in one-half of each row were defoliated in mid-October, and roots were sampled at this defoliation and again in December. Winter injury was determined in mid-April. Shoot removal in mid-October increased winter injury and reduced plant vigor in spring. As expected, the October defoliation reduced root protein and starch concentrations in December, but unexpectedly increased root sugar concentrations. In addition, defoliation did not reduce the steady state transcript levels of several cold-acclimation responsive (car) genes that are associated with genetic variation in winter survival. Although positively associated with genetic differences in winter hardiness, factors other than root sugar accumulation and expression of these car genes regulate defoliation-induced changes in winter survival of these alfalfa cultivars.

Abbreviations: car, cold-acclimation response gene • FD, fall dormancy • HPLC, high performance liquid chromatography • RFO, raffinose family oligosaccharides • TNC, total nonstructural carbohydrate • VSP, vegetative storage protein


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PRODUCERS IN THE NORTH-CENTRAL USA sow fall dormant alfalfa cultivars because of the positive association of FD with winter survival. One negative consequence of growing fall dormant cultivars is their reduced shoot elongation and leaf area expansion rates after forage harvest in summer (Volenec, 1985). This limits the number of annual cuttings, and subsequently reduces seasonal forage yield potential. When compared with dormant cultivars, those with intermediate FD [5 to 7, a relative measure as described by Teuber et al. (1998)] may provide enough forage for additional harvests each year. If less fall dormant cultivars were made available to northern production areas, benefits could include increased yield potential. However, a potential risk of raising cultivars possessing intermediate FD is their lower winter hardiness, a problem exacerbated by improper fall harvest management.

Past research indicates that autumn defoliation may decrease winter survival and stand persistence (Silkett et al., 1937; Grandfield, 1943). Smith (1972) reported that a period of 4 to 6 weeks of uninterrupted growth before a killing freeze was imperative for winter survival and plant persistence. Cutting during cold hardening lowered both plant persistence and root total nonstructural carbohydrate (TNC) concentrations. However, other research has shown that cutting during this critical fall rest period may not impact persistence or subsequent yield (Tesar and Yager, 1985; Sheaffer et al., 1986; Edmisten et al., 1988; Bélanger et al., 1992). In Virginia, harvesting in late rather than early fall generally increased shoot growth the following spring (Edmisten et al., 1988). Bélanger et al. (1992) used growing degree days instead of calendar dates to show that winter injury was reduced if the interval between the final harvest in late autumn and the previous harvest was 500 growing-degree-days or greater. Improved plant persistence obtained by delaying the final fall harvest is generally believed to result from increased root TNC levels (Tesar and Yager, 1985; Sheaffer et al., 1986), but this hypothesis has not been rigorously tested. Edmisten and Wolf (1988) speculated that an extended period of slow growth, low dark respiration rates, and high rates of photosynthesis during autumn months stimulates TNC accumulation and improves winter hardiness. Recently, Dhont et al. (2002) reported that mass of TNC in roots in autumn had a stronger correlation with shoot growth in spring than does the root TNC concentration.

The physiological mechanisms for reduced persistence and vigor in spring resulting from autumn defoliation are not clearly understood. In addition, it is not known how autumn defoliation alters the expression of specific cold hardiness genes whose expression has been consistently associated with genetic differences in alfalfa winter hardiness (Cunningham et al., 2001). The goal of this research was to determine the effects of autumn defoliation on root physiology and winter survival of alfalfa. This objective was achieved through two experiments: (i) assessment of several autumn cutting dates on the accumulation of reserves in alfalfa roots; (ii) evaluating the effects of autumn defoliation on root physiology and winter survival of six alfalfa cultivars possessing contrasting FD and winter hardiness. We expected that autumn defoliation would prevent accumulation of sugars, starch, and protein in roots, and be accompanied by reduced winter survival. We also expected untimely autumn defoliation to reduce the expression of specific car genes whose expression is closely associated with winter survival. We hypothesized that autumn defoliation would have the largest impact on winter survival of cultivars possessing intermediate FD (FD = 5, 6, and 7). Left uncut, plants with intermediate dormancy might survive winter, but these plants would regrow in autumn when defoliated, consuming their root reserves, and this would prevent these plants from cold acclimating.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Culture and Sampling
Experiment 1
This experiment was conducted at the Agronomy Research Center, Purdue University, West Lafayette, IN, during the 1993 to 1994 growing season. Fall cutting treatments and root sampling dates are summarized in Table 1. An established stand of ‘Resistar’ alfalfa (FD = 4) was initially defoliated on 7 September and at weekly intervals beginning 1 October and ending 12 November. Roots were excavated 23 November, 10 March, and 21 April to assess the effects of these fall cutting dates on root nonstructural carbohydrate, protein, and amino N concentrations.


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  		Table 1. Dates of herbage removal and root harvests, Exp. 1.

 
Experiment 2
Plant materials used in this experiment included the dormant cultivars Pioneer 53Q60 FD = 3), Pioneer 54H69 (FD = 4), three semidormant experimental germplasms from Pioneer Hi-Bred International, Z57NO2, 96P51PSI, and 96P55PS1 (FD of 5 to 7), and the nondormant cultivar Pioneer 5939 (FD = 9). Seedlings were established at the Agronomy Research Center, Purdue University, West Lafayette, IN, in early May of 1998 and 1999. Seeds were sown in 3-m-long rows spaced 92 cm apart in a randomized complete block with four replicates. Resultant plant populations were {approx}60 plants m-1 of linear row. The soil was a Starks-Fincastle silt loam (fine-silty, mixed, superactive, mesic, Aeric Endoaqualfs and Epiaqualfs) that was fertilized and limed according to soil test specifications for high alfalfa yield. Seeds were inoculated with Sinorhizobium meliloti (Liphatech Corp., Milwaukee, WI) before planting. Plots were hand-weeded, and insects controlled as needed. Planting and root sampling dates are summarized in Table 2. Plant heights were measured in mid-October at eight randomly selected positions within each subplot, and the average used to estimate FD. Shoots on one-half of each plot were removed, leaving a 5-cm stubble in mid-October. Plant survival was determined when shoot growth of winter hardy cultivars resumed in April by excavating 15 to 30 plants and scoring the winter injury of each plant using the following scale: 1 = uninjured; 2 = injured; and 3 = dead. A weighted average (by count and injury category) was calculated for each plot and used for statistical analysis. Plant heights were measured in April at eight randomly selected positions within each subplot, and the average used as an estimate of spring vigor.


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  		Table 2. Timetable of crop management activities, Exp. 2.

 
Root tissues for laboratory analyses were collected 12 October (at defoliation) and in early December. Roots dug to a soil depth of {approx}20 cm were washed free of soil using cold water. The uppermost 5 cm of each taproot was removed, diced into small pieces, and packed with solid CO2 (for protein and carbohydrate analyses) or immersed in liquid N2 (for RNA analysis). The upper 5 cm of taproots were selected for analysis because this was a well-defined tissue region that minimized variation due to rooting depth, degree of branching, and other root morphological differences that could confound results obtained by analyzing the entire root system. Tissues for protein and carbohydrate analyses were lyophilized, ground to pass a 1-mm screen, and stored at -20°C. Tissues for RNA analysis were stored at -80°C.

Biochemical Analysis of Root Reserve Pools
Starch and total soluble sugars were assayed by the methods described by Li et al. (1996). Sucrose, raffinose, and stachyose oligosaccharides were separated and quantified by high performance liquid chromatography (HPLC; Dionex BioLC, Sunnyvale, CA). Sugars were eluted isocratically using a CarboPac PA1 column (1.4 x 25 cm; Dionex Corp.) at room temperature with 200 mM sodium hydroxide at a flow rate of 1 mL min-1. Peak identity and sugar quantity were determined by comparison with standards.

Protein was assayed by the Bradford method as reported previously (Cunningham et al., 1998). For sodium dodecylsulfate polyacrylamide gel electrophoresis analysis, proteins were separated in 0.75-mm thick gels containing 12% (w/v) acrylamide (Laemmli, 1970), and stained with Coomassie Brilliant Blue R-250. Protein gel blots were probed with antisera raised to the 15 and 32 kDa alfalfa vegetative storage proteins (VSPs) as described by Cunningham and Volenec (1996). Amino N was determined using ninhydrin with glycine as a standard (Rosen, 1957).

RNA Isolation and Northern Blot Hybridization Analysis
Total RNA was isolated using hot phenol, and RNA (20 µg) was separated on a 1.5% (w/v) agarose-formaldehyde gel as described previously (Gana et al., 1998). Three cold-acclimation responsive cDNA clones from alfalfa were labeled with 32P-deoxycytidine triphosphate using random priming (Feinberg and Vogelstein, 1983). The three cDNA probes included: RootCAR1 [GenBank Accession AF072932, similar to cas15a (GenBank Accession AAA16927) and cas15b (GenBank Accession AAA16926)]; bN-1 12a3, similar to cas17 (GenBank Accession L13415) and cas18 (GenBank Accession L07516); and bC2E 40a, similar to cas18. Hybridization and washing of membranes were done as described by Gana et al. (1997).

Statistical Analysis
Experiment 1
The experimental design was a randomized complete block with four replicates arranged as a split-plot. Autumn cutting date was designated as the whole plot and root sampling dates as subplots. Analysis of variance was used to partition variation into replicate, autumn cutting date, and root sampling date effects and interactions. Where the F-test was significant, an LSD was calculated to compare means. Twice the standard error of the mean is presented for individual means to provide a 95% confidence interval around the mean (Snedecor and Cochran, 1980).

Experiment 2
The experimental design was a randomized complete block arranged as a split-split plot. Whole plots included the six cultivars or germplasms, subplots were the two defoliation treatments (October cut and uncut), and the sub-subplots were the respective root sampling dates (October and December). The experiment was replicated four times in each of 2 yr, 1998 to 1999, and 1999 to 2000. Years were treated as random effects and genotypes, defoliation, and harvest treatments were treated as fixed effects. Variances across years were found to be homogenous using Bartlett's test (Steel and Torrie, 1980); therefore, data are presented as means averaged across both years. Fall dormancy, winter injury, and spring vigor data were analyzed using ANOVA (SAS Institute, 1999). Root composition data were analyzed using the general linear models (GLM) procedure. An F-protected LSD (P ≤ 0.05) was calculated for mean comparisons of treatment main effects. Significant differences (P ≤ 0.05) within two-way and higher-order interactions were determined as twice the SE (Snedecor and Cochran, 1980).


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1
Influence of Autumn Defoliation Date on Root Carbohydrate Concentrations
Root sugar and starch concentrations were affected by autumn defoliation, root sampling date, and the interaction of the two variables. Averaged across cutting treatments, root starch concentrations were highest in November (183 mg g-1), while March and April had the lowest root starch concentrations of 81 and 53 mg g-1, respectively (data not shown). Defoliation on 2 October and 8 October decreased November root starch concentrations by 30% when compared with root starch concentrations of control plants defoliated 7 September (Fig. 1A). Averaged across autumn cutting treatments, root sugar concentrations were higher on 10 March (181 mg g-1) and 23 November (135 mg g-1) when compared with that of roots sampled on 21 April (103 mg g-1) (data not shown). Defoliation on 2 and 8 October increased 23 November root sugar concentrations by 15% when compared with roots of control plants cut on 7 September (Fig. 1B). Roots sampled on 21 April also had higher root sugar concentrations in response to early October defoliation, but defoliation in October did not alter sugar concentrations of roots sampled in 10 March (data not shown).



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  		Fig. 1. Effect of autumn defoliation on (A) starch and (B) sugar concentrations of alfalfa roots sampled in late November. Alfalfa was cut on 7 September (control) and at weekly intervals from 2 October through 12 November. Error bars represent one standard error of the mean (n = 4). Exp. 1.

 
Influence of Autumn Defoliation Date on Root Amino N and Soluble Protein Concentrations
Root amino N concentrations were affected by autumn defoliation and root sampling date. When compared with the control plants defoliated 7 September, amino N concentrations were lower on 23 November in roots of plants defoliated 2 October, 6 November, and 12 November (Fig. 2A). Defoliation did not influence root amino N concentrations in March, but in April amino N concentrations in roots of plants defoliated 8 October (0.16 mmol g-1) and 15 October (0.16 mmol g-1) were significantly lower than those from roots of control plants defoliated 7 September (0.25 mmol g-1) (data not shown).



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  		Fig. 2. Effect of autumn defoliation on (A) amino N and (B) buffer-soluble protein concentrations from alfalfa roots harvested in late November. Alfalfa was cut on 7 September (control) and at weekly intervals from 2 October through 12 November. Error bars represent one standard error of the mean (n = 4). Exp. 1.

 
Root buffer-soluble protein concentrations were affected by defoliation treatment and sample date, but these variables did not interact. Averaged across cutting dates, root protein concentrations were highest in March (45 mg g-1), and lowest in April (27 mg g-1). Defoliation on 2 October significantly reduced buffer-soluble protein concentrations in roots sampled in November (Fig. 2B); however, defoliation on 2 October had no effect on protein concentrations of roots sampled later in March or April (data not shown). Sodium dodecylsulfate polyacrylamide gel electrophoresis analysis of proteins in roots sampled in November revealed a decrease in certain polypeptides, specifically those with molecular masses of 15, 17, 19, 26, and 32 kDa from plants defoliated 2 and 8 October (Fig. 3A). Immunoblot analysis of protein gel blots using antibodies raised against the 15 and 32 kDa alfalfa VSPs also revealed that defoliation on 2 and 8 October decreased VSP concentrations (Fig. 3B).



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  		Fig. 3. Sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblot analyses of buffer-soluble proteins extracted from alfalfa taproots in late November. (A) Buffer-soluble protein (25 µg per lane) stained with Coomassie Brilliant Blue R-250. Molecular mass standards (kDa) are listed to the left. (B) Protein gel blot containing 5 µg protein per lane was probed using antisera raised to the 15 and 32 kDa vegetative storage proteins. Samples were loaded in lanes according to their respective cutting dates: Lane 1, 7 September; Lane 2, 2 October; Lane 3, 8 October; Lane 4, 15 October; Lane 5, 22 October; Lane 6, 29 October; Lane 7, 6 November; Lane 8, 12 November. Exp. 1.

 
Experiment 2
Plant Height, Winter Injury, and Spring Vigor
Soil and air temperatures during both years of the study at the Agronomy Research Center are shown in Fig. 4. A killing freeze (defined as ambient air temperature of -4°C or 25°F) occurred on 5 Nov. 1998 and on 15 Nov. 1999. Winter injury patterns of these germplasms were similar both years. Plant height (used to estimate FD) of the six germplasms differed in the expected manner when measured on the day of defoliation on 12 October (Table 3). The height of the fall dormant, winter hardy cultivars 53Q60 and 54H69 was 21.5 and 20.5 cm, respectively, while height of the nondormant, non-winter-hardy 5939 approached 35 cm. Height of the semidormant germplasms 96P51PSI, Z57NO2, and 96P55PSI was intermediate.



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  		Fig. 4. Minimum air and soil (10-cm depth) temperatures at the Agronomy Research Center, West Lafayette, IN. Dates of the first killing freeze (-4°C) were 5 Nov. 1998 and 15 Nov. 1999. Exp. 2.

 

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  		Table 3. Influence of cultivar or germplasm source on fall height, winter injury, and spring height. Data were averaged across defoliation treatments and two years, Exp. 2.

 
Plant height in October was associated with winter injury. Averaged across defoliation treatments, fall dormant cultivars 53Q60 and 54H69 had significantly less injury than the semidormant germplasms 96P51PSI, Z57NO2, and 96P55PSI, and all five of these alfalfa sources exhibited less winter injury than the nondormant cultivar 5939 (Table 3). As expected, defoliation in early October increased alfalfa winter injury of most germplasms (Fig. 5A). Early October defoliation increased winter injury of 54H69, 96P51PSI, Z57NO2, and 5939, but did not significantly enhance injury of 53Q60 and 96P55PSI.



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  		Fig. 5. Effect of October defoliation on alfalfa (A) winter injury and (B) spring height. One half of plants in each plot were defoliated in October. Winter injury and spring height were assessed in April. Winter injury ratings are weighted means based on mid-April observations of 20 to 30 plants using the following scoring scale: 1 = no injury, 2 = injured plant, and 3 = dead plant. Included in the study were the dormant cultivars ‘53Q60’ (Q60) and ‘54H69’ (H69), the nondormant cultivar ‘5939’, and three semidormant germplasms: 96P51PSI (P51), Z57NO2 (NO2), and 96P55PSI (P55). Error bars represent one standard error of the mean (n = 8). Exp. 2.

 
Germplasm and autumn defoliation significantly influenced spring vigor as determined by shoot heights in April (Table 3, Fig. 5B). April shoot heights of the dormant cultivars 53Q60 and 54H69 were low because of slow initiation of shoot growth in spring typical of dormant alfalfas. Averaged across cutting treatments, the semidormant germplasms 96P51PSI and Z57NO2 were tallest in April. Shoot heights of the semidormant germplasm 96P55PSI and the nondormant 5939 were reduced when compared with 96P51PSI and Z57NO2. Compared with intact plants, mid-October defoliation significantly decreased spring vigor of all germplasms (Fig. 5B).

Influence of Autumn Defoliation on Carbohydrate Concentrations
Root starch concentration increased with decreased FD and was associated with enhanced winter injury. In undefoliated plants sampled in December, the nondormant, nonhardy 5939 had the highest root starch concentration (415 mg g-1), whereas the dormant 53Q60 had the lowest root starch concentration (304 mg g-1) (Fig. 6A). Defoliation in October significantly reduced root starch concentrations of all cultivars in December. When compared with uncut plants sampled in December, defoliation decreased root starch levels by an average of 35%.



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  		Fig. 6. Effect of October defoliation on starch (A) and soluble sugar (B) concentrations in alfalfa taproots. One half of plants in each plot were defoliated in October. Roots were sampled in October at the time of defoliation, and in December. Included in the study were the dormant cultivars ‘53Q60’ (Q60) and ‘54H69’ (H69), the nondormant cultivar ‘5939’, and three semidormant germplasms: 96P51PSI (P51), Z57NO2 (NO2), and 96P55PSI (P55). Error bars represent one standard error of the mean (n = 7). Exp. 2.

 
As expected, root sugar concentrations of uncut plants in December increased with increased FD. Fall dormant 53Q60 and 54H69 had the highest sugar concentrations (137 and 138 mg g-1, respectively), whereas roots of the nondormant, nonhardy 5939 had the lowest root sugar concentration in December (57 mg g-1) (Fig. 6B). Contrary to our expectations, however, defoliation in October significantly and consistently increased root sugar concentrations of all germplasms in December (Fig. 6B). Root sugar concentrations from defoliated plants sampled in December increased an average of 46% over that of corresponding uncut plants. The largest defoliation-induced increase in root sugar concentration was observed in the nondormant 5939 where December root sugar levels increased from 57 mg g-1 in uncut plants to 114 mg g-1 in roots of plants defoliated in October.

Because the response of root sugar concentrations in December were contrary to our expectations, the composition of root sugar pools, with particular emphasis on sucrose and raffinose family oligosaccharides (RFO), was determined using HPLC. Trends in sucrose, raffinose, and stachyose concentrations were similar to those of total root sugar concentrations (Fig. 7). For each sugar, the fall dormant cultivars contained the highest concentration and the nondormant the lowest, with the semidormant containing intermediate levels. Averaged across cultivars and germplasms, defoliation in October resulted in a 26% increase in root sucrose concentrations in December. This increase in sucrose concentration due to defoliation was less in fall dormant cultivars (16%) when compared with the nondormant 5939 (68%) (Fig. 7A). Defoliation in October did not significantly increase raffinose concentrations in December (Fig. 7B), but the October defoliation did significantly increase stachyose concentrations in the fall dormant 53Q60 and the semidormant 96P51PSI and 96P55PSI (Fig. 7C).



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  		Fig. 7. Effect of October defoliation on (A) sucrose, (B) raffinose, and (C) stachyose concentrations in alfalfa taproots sampled in December. One half of plants in each plot were defoliated in October. Included in the study were the dormant cultivars ‘53Q60’ (Q60) and ‘54H69’ (H69), the nondormant cultivar ‘5939’, and three semidormant germplasms: 96P51PSI (P51), Z57NO2 (NO2), and 96P55PSI (P55). Error bars represent one standard error of the mean (n = 7). Exp. 2.

 
Influence of Autumn Defoliation on Amino N and Buffer-Soluble Protein Concentrations
Amino N concentrations increased between October and December in roots of all germplasms (Fig. 8A). Fall dormancy was not associated with root amino N concentration, but defoliation in October significantly reduced amino N concentrations in roots of 53Q60 and 96P55PSI in December (Fig. 8A).



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  		Fig. 8. Effect of October defoliation on (A) amino N and (B) buffer soluble protein concentrations in alfalfa taproots. One half of plants in each plot were defoliated in October. Roots were sampled in October at the time of defoliation, and in December. Included in the study were the dormant cultivars ‘53Q60’ (Q60) and ‘54H69’ (H69), the nondormant cultivar ‘5939’, and three semidormant germplasms: 96P51PSI (P51), Z57NO2 (NO2), and 96P55PSI (P55). Error bars represent one standard error of the mean (n = 7). Exp. 2.

 
Defoliation in October prevented accumulation of buffer-soluble protein in roots of all germplasms in December, and in the case of 5939, significantly reduced root protein concentrations when compared with root protein concentrations in October (Fig. 8B). Buffer-soluble protein in roots of uncut plants increased significantly between October and December for all cultivars and germplasms. The fall dormant 53Q60 and 54H69, and the semidormant germplasm 96P51PSI had the highest protein concentrations in December, followed by the semidormant 96P55PSI and Z57NO2. As expected, the nondormant, nonhardy 5939 had the lowest root buffer-soluble protein concentrations.

Protein composition was influenced by defoliation in October (Fig. 9A). Roots of intact plants of 53Q60, 96P51PSI, Z57NO2, and 96P55PSI contained an abundance of 25 and 26 kDa polypeptides in December that was decreased in roots of plants defoliated in October. Abundance of a 27-kDa polypeptide in roots was decreased in December of 53Q60, 54H69, 96P51PSI, and Z57NO2 plants that were defoliated in October. Roots of 53Q60, 96P51PSI, and Z57NO2 defoliated in October had decreased abundance of 15, 19, and 32 kDa polypeptides in December. In addition, the abundance of a 31 kDa polypeptide increased in roots of defoliated 54H69 and Z57NO2 plants. Immunoblotting revealed that defoliation in October decreased the abundance of the 15 and 32 kDa VSPs in 53Q60, 54H69, and 96P55PSI, while no differences in relative VSP abundance were detected in 96P51PSI, Z57NO2, and 5939 (Fig. 9B).



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  		Fig. 9. Sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblot analysis of buffer-soluble proteins extracted from alfalfa taproots in early December. Twenty-five micrograms of soluble protein were analyzed per lane, and proteins were stained with Coomassie Brilliant Blue R-250 (A). Molecular mass standards (kDa) are listed to the left. Protein gel blots containing 5 µg protein per lane were analyzed using antisera from the 15 and 32 kDa vegetative storage proteins (B). Alfalfa plants were cut in October (C) or uncut (U). Included in the study were the dormant cultivars ‘53Q60’ (Q60) and ‘54H69’ (H69), the nondormant cultivar ‘5939’, and three semidormant germplasms: 96P51PSI (P51), Z57NO2 (NO2), and 96P55PSI (P55). Exp. 2.

 
Expression of Cold-Acclimation Responsive Genes
When compared with transcript levels in roots in October, plants of the fall dormant cultivars, and semidormant germplasms exhibited the expected increase in steady state levels for all three car genes (Fig. 10). In contrast, steady state transcript levels for these genes in roots of the nondormant, nonhardy 5939 were low in October and did not increase in roots in December. Contrary to our expectations, defoliation did not reduce steady state transcript level for any of these car genes. To our surprise, defoliation in October even increased steady state transcript levels for RootCar1 in roots of 54H69 and 5939, and transcript abundance of bC2E 40a in roots of 96P51PSI and 5939 in December.



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  		Fig. 10. RNA blot analysis of root cold hardiness gene expression of six alfalfa cultivars with contrasting fall dormancy and winter hardiness. Twenty micrograms of total RNA was loaded per lane and blots were hybridized with three 32P-labeled cold-acclimation responsive cDNAs: bN-1 12a3, RootCar1, and bC2E 40a. Alfalfa plants were cut in October (C) or uncut (U). Roots were sampled at the time of defoliation in October or after a period of cold acclimation in December. Included in the study were the dormant cultivars ‘53Q60’ (Q60) and ‘54H69’ (H69), the nondormant cultivar ‘5939’, and three semidormant germplasms: 96P51PSI (P51), Z57NO2 (NO2), and 96P55PSI (P55).

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In Exp. 1, seven fall cutting dates were imposed on Resistar alfalfa to alleviate spatial constraints of field-testing multiple cutting dates on several alfalfa cultivars simultaneously. Although considerable climatic variation may exist on an annual basis, the results for this experiment were used to identify the time interval where fall cutting caused considerable change in alfalfa root physiology in West-Central Indiana: 1 October to 15 October. Defoliation in this window was used in Exp. 2 to determine the impact of fall harvest on root physiology of several alfalfa cultivars and germplasms possessing contrasting FD and winter survival.

In Exp. 2, defoliation in early October increased winter injury and reduced spring vigor of six alfalfa cultivars chosen for variation in FD. As expected, shoot removal in October significantly reduced root starch concentrations of all cultivars and germplasms in December (Fig. 6A), and confirms the general belief that root TNC (mostly starch) accumulation is essential for alfalfa winter survival. However, when root starch (and TNC) concentrations of cultivars differing in FD are evaluated, this relationship is reversed because nondormant, nonhardy alfalfa cultivars consistently have the highest root starch concentrations in autumn (Bula et al., 1956; Volenec, 1985; Castonguay et al., 1995; Cunningham and Volenec, 1998). This has led us to reevaluate the relationship between root TNC concentrations, C partitioning between starch and sugar pools in roots, and alfalfa winter hardiness. Recently, we and others have shown that root sugar concentrations are consistently and positively associated with cultivar differences in winter hardiness (Castonguay et al., 1995; Cunningham and Volenec, 1998; Cunningham et al., 1998, 2001). This led us to our current hypothesis that autumn defoliation results in low root sugar concentrations, which in turn, causes plants to die during winter.

In agreement with past studies, root sugar concentrations increased with increased FD and were associated with the good winter hardiness of 53Q60 and 54H69. We expected defoliation in October to prevent sugar accumulation in roots and result in the enhanced winter injury we observed. Contrary to our hypothesis, autumn cutting increased root sugar concentrations in December by an average of 45% when compared with those of uncut plants (Fig. 6B). The enhanced winter injury despite the accumulation of sugar caused by autumn defoliation agrees with a recent study where alfalfa root sugar levels were increased by fall cutting (Dhont et al., 2002). The increased root sugar concentrations may be due to several things, including extensive defoliation-induced starch hydrolysis, releasing sugars that accumulate in roots, and/or reduced sugar utilization due to cold temperature-induced reductions in dark respiration and shoot growth rates.

The unexpected positive association between root soluble sugar concentrations in December and winter injury due to untimely fall defoliation raised questions regarding potential changes in sugar pool composition. Recent research has shown a close association between RFO accumulation in alfalfa roots during autumn and winter survival (Castonguay et al., 1995; Cunningham et al., 2003). Our hypothesis was that defoliation in October would decrease RFO concentrations in roots in December and result in greater winter injury of these autumn-harvested plants. Although a positive association between root RFO concentrations and genetic differences in winter hardiness were observed in undefoliated plants, defoliation-induced trends in sucrose, raffinose, and stachyose concentrations were generally similar to those of total root sugar concentrations (Fig. 6B and 7). Defoliation in October increased sucrose and stachyose concentrations in December, whereas root raffinose concentrations were unaffected by defoliation in October. There was no evidence to support our hypothesis that autumn defoliation would reduce concentrations of sucrose and RFOs in alfalfa roots in December, and with it, winter hardiness. The defoliation-induced increase in root sugar concentrations, including RFOs, from these germplasms possessing contrasting FD is consistent with recent results where the impact of fall cutting date on root sugar composition and concentrations were evaluated using two winter hardy alfalfa cultivars (Dhont et al., 2002). Clearly, the physiological basis for increased winter injury resulting from untimely autumn defoliation is not associated with root sugar or RFO concentrations in these germplasms.

In addition to monitoring root carbohydrate concentrations, root amino N and buffer-soluble protein concentrations were quantified because root N reserves are important for alfalfa regrowth and stress tolerance (Volenec et al., 1996). Amino N concentrations were not consistently influenced by October defoliation (Fig. 8A), but defoliation in October reduced root protein concentrations in December, and changed the relative abundance of several specific proteins (Fig. 8B and 9A). While the function of most of these proteins is not known, October defoliation decreased the abundance of alfalfa root VSPs. These VSPs accumulate in roots in autumn and are utilized during spring when shoot growth is initiated and during regrowth following defoliation in summer (Hendershot and Volenec, 1993; Avice et al., 1996; Barber et al., 1996; Cunningham and Volenec, 1996; Noquet et al., 2001). Reduced root VSP levels in December would be expected to decrease plant vigor and shoot growth in spring.

The expression of cold hardiness genes in roots of undefoliated plants was consistent with genetic variation in alfalfa winter hardiness (Fig. 10) (Mohapatra et al., 1989; Monroy et al., 1993; Wolfraim et al., 1993; Cunningham et al., 1998, 2001). When compared with October transcript levels, roots from uncut plants of the fall dormant cultivars and semidormant germplasms had increased car gene expression in December, whereas the nondormant, nonhardy 5939 had very low steady state transcript levels for car genes. Contrary to our hypothesis, defoliation in mid-October did not reduce steady state transcript levels of these car genes in December. However, the fact that defoliation did not alter the transcript abundance of these cold hardiness genes may not be that surprising given that little is known about the function of their protein products in planta. Nevertheless, it is an oversimplification to assume a cause–effect relationship between transcript abundance of these (or others yet to be identified) genes and alfalfa winter hardiness per se. Future research to assign function to the protein products from these genes is key to understanding the physiological or biochemical processes altered by untimely autumn defoliation and the role these genes may play in alfalfa cold acclimation.


   ACKNOWLEDGMENTS
 
The assistance of Drs. N.C. Carpita and M.A. Madson with the HPLC analysis of sugar pools is gratefully acknowledged. This work was supported, in part, by USDA-IFAFS grant number 00-52100-9611.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contribution from the Purdue Univ. Agric. Exp. Stn., Journal Series No. 16820.

Received for publication July 4, 2002.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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