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a Dep. of Agronomy, Purdue Univ., West Lafayette, IN 47907-1150 USA
b Station de Recherches, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd., Sainte-Foy, QC, Canada G1V 2J3
c Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd., Sainte-Foy, QC, Canada G1V 2J3
* Corresponding author (jvolenec{at}purdue.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: cDNA, complementary DNA • FD, fall dormancy • GaS, galactinol synthase • HPLC, high pressure liquid chromatography • LSD, least significant difference • mRNA, messenger RNA • RFO, raffinose family oligosaccharides
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several physiological processes also have been associated with improved winter hardiness of alfalfa. For decades, the accumulation of starch and soluble sugars in roots has been the focus of research (Graber et al., 1927; Grandfield, 1943; Smith, 1964). Initially, it was believed that the accumulation of total nonstructural carbohydrates (TNC, sum of sugar and starch concentrations) in roots was critical to successful overwintering and subsequent spring growth of this perennial species. Later, it was shown that soluble sugars accumulated in alfalfa roots and crowns as plants hardened for winter (Bula et al., 1956; Ruelke and Smith, 1956), but how sugar accumulation affected genetic differences in winter hardiness was not studied. Recently, we have shown that sugar concentrations are consistently lower in roots of nondormant alfalfa cultivars when compared with fall dormant, winter hardy alfalfa cultivars (Cunningham and Volenec, 1998).
Castonguay et al. (1995) reported that sucrose, stachyose, and raffinose accumulated in alfalfa roots, while concentrations of glucose, fructose, and starch declined during alfalfa cold acclimation. Further, differences in the maximum level of freezing tolerance between nonhardy and winter hardy cultivars were better related to the capacity of the plants to accumulate stachyose and raffinose than to accumulate sucrose. To understand mechanisms controlling fall dormancy and winter hardiness better, we have studied alfalfa populations selected for contrasting fall dormancy. These populations also differ in winter hardiness and several other traits including root sugar concentrations (Cunningham et al., 1998, 2001). They permit study of discrete changes in physiology and gene expression associated with selection for contrasting fall dormancy and winter hardiness in a manner not possible using traditional cultivars that differ for many characteristics. We do not know if changes in sugar composition occurred as a result of genetic selection for contrasting fall dormancy in these germplasms, and if expression of genes for key enzymes involved in RFO synthesis, such as galactinol synthase, are associated with RFO accumulation and improved winter survival. Our objectives were to determine how selection for contrasting fall dormancy influenced winter survival, the RFO concentrations, and steady state mRNA levels for GaS in alfalfa roots.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiment 1—Controlled Environment Conditions
Plants of the fall dormant, cold tolerant cv. Rambler (FD = 1) and Apica, and the nondormant, cold-sensitive cv. CUF 101 (FD = 9) were grown from seed in 15-cm pots filled with a mixture (9:3, v/v) of top soil/peat moss. Plants were grown for 5 wk in an environmentally controlled chamber set to a 21/17°C (day/night) temperature regimen under a 16-h photoperiod of approximately 250 µmol m-2 s-1 of photosynthetic photon flux density. Plants were kept well watered and fertilized once a week with 1 g L-1 of a commercial fertilizer (20-20-20, Plant-Prod, Brampton, ON). Plants were subsequently cold acclimated at a constant 2°C temperature and an 8-h photoperiod of 150 µmol photons m-2 s-1. After 2 wk at 2°C, a group of plants was transferred to a freezer set to -2°C, and kept in the dark for an additional 2 wk.
Total RNA was extracted as described by De Vries et al. (1988) and quantified by UV absorption at 260 nm. Ten micrograms of total RNA was denatured in formaldehyde and was size fractionated on a 1% (w/v) formaldehyde agarose gel (Fourney et al., 1988). The RNA was transferred to nylon membranes (Hybond N+, Pharmacia Biotech., Oakville, ON) and hybridized overnight at 68°C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.25% (w/v) low-fat powder milk with the [32P]dCTP-labeled GaS probe prepared from the purified cDNA insert (Sambrook et al., 1989). Membranes were exposed to Kodak X-Omat AR5 X-ray film at -80°C.
Experiment 2—Unheated Glasshouse
Plants were grown from seed at the end of the summer 1994 under the environmentally controlled conditions described in Exp. 1, and transferred to an unheated greenhouse in mid-October for low temperature acclimation as described in Castonguay and Nadeau (1998). Variation in air and soil temperatures during the overwintering period can be found in Castonguay and Nadeau (1998). The experiment was conducted as a completely randomized factorial design with cultivars and dates of samplings as the main factors. The sampling dates were 10 October, 24 October, 7 November, 29 November, 13 December, 5 January, 24 January, 14 February, 3 March, and 23 March. Crown tissues were ground to a fine powder in liquid N2. Total RNA was extracted as described by De Vries et al. (1988) and quantified by UV absorption at 260 nm. Five micrograms of total RNA were vacuum transferred to a nylon membrane (Hybond N+) with a Bio-Dot apparatus (Bio-Rad, Mississauga, ON). Membranes were hybridized overnight at 68°C in 2x SSC, 0.25% (w/v) low-fat powder milk, and 1% (w/v) SDS with the [32P]dCTP-labeled probe prepared from the purified insert of GaS (Sambrook et al., 1989). Membranes were exposed to Kodak X-Omat AR5 X-ray film at -80°C, and transcript levels were quantified by densitometry analysis of autoradiographs by OneD Scan software (Scanalytics Inc., Billerica, MA). Densitometry means and standard errors were based on results obtained from three independent observations per treatment.
Experiment 3—Fall Dormancy/Winter Hardiness Selection
Three cycles of selection for contrasting fall dormancy from ‘Norseman’ [highly fall dormant (FD); fall dormancy rating of 1], ‘Lahontan’ (FD = 6), ‘CUF 101’ (nondormant, FD = 9) and ‘Wadi Qurayat’ (very nondormant, FD = 11) were used to create populations with less fall growth (more fall dormant, designated as "L") and high levels of fall growth (less fall dormant, designated "H") from each of these cultivars (parental cultivar designated as "O"). These cultivars were selected for study because they represent the range of fall dormancy observed in alfalfa (Teuber et al., 1998). Details of plant culture and tissue sampling used in Exp. 3 have been described previously (Cunningham et al., 1998). Seeds were sown in rows spaced 92 cm apart in a randomized complete block with three replicates in each of two years at the Agronomy Research Center, Purdue University, West Lafayette, IN. Resultant plant populations were approximately 40 plants m-1 of linear row. Plant height was measured in October at eight randomly selected positions within each row, and the average used as an indication of fall dormancy for that plot as described by Teuber et al. (1998). Plant survival was determined in March the year after seeding when shoot growth had resumed. Plants were excavated, surviving plants counted, and percent survival calculated. Temperature data for both winters of the study has been reported previously (Cunningham et al., 1998).
Root samples were taken to a soil depth of approximately 25 cm, and were washed free of soil under a stream of cold water. Crowns were severed from roots and roots were separated into the uppermost 5 cm ("root tops") and the remainder of the root system. Root tissues were chopped into 1-cm segments. Tissues for sugar analysis were frozen on solid CO2, lyophilized, and were ground to pass a 1-mm screen and stored at -20°C. Root top tissues for RNA gel blot analysis were immersed in liquid N2 and stored at -80°C.
Soluble sugars were extracted in methanol-chloroform-water (12:5:3, v/v/v) as previously described (Castonguay et al., 1995). The aqueous phase was collected, and a 1-mL subsample was evaporated to dryness and resolubilized in water containing ethylenediaminetetraacetic acid (EDTA) (Na+, Ca2+, 0.13 mM). Sucrose, raffinose, and stachyose were separated by HPLC (Waters Scientific, Milford, MA) on a Sugar-Pak column (6.5 x 300 mm) eluted isocratically with water containing 0.13 mM EDTA (Ca2+, Na+) at 85°C and quantitated with a differential refractometer.
Total RNA was isolated by means of phenol as described previously (Gana et al., 1998). Total RNA (20 µg) was separated on 1.5% agarose-formaldehyde gels (Lehrach et al., 1997) and the RNA was transferred to Zeta-probe membranes (Bio-Rad) after electrophoresis. The membranes were prehybridized for 4 h at 42°C with slow shaking. Hybridization and washing of membranes were done as described by Gana et al. (1997). Membranes were exposed to X-ray film at –80°C.
Experiment 3 was replicated three times in each of two years. Populations (H, O, and L) were nested within cultivars for analysis. Data were analyzed as a split-plot design with repeated sampling of plants from within rows through time. Data were analyzed by SAS (SAS Institute, Cary, NC). Where F-tests were significant (P 
0.05), an LSD was calculated for mean comparisons. Rank correlations were used to relate sucrose and RFO concentrations to winter survival of these populations because intermediate winter survival values (10–75%) were not obtained in this study (Table 1) making it inappropriate to use regression to examine these relationships. Instead, populations were ordered from 1 (highest) to 12 (lowest) root sucrose (or RFO) concentration in December (Fig. 1 and 2) and winter survival (Table 1), and simple linear regression used to correlate the rankings.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Exp. 2, steady state transcript levels for GaS increased rapidly in crowns of the cold-tolerant cultivars Apica and Rambler on transfer to unheated greenhouse conditions on October 10 (Fig. 4). By comparison, steady state transcript levels in crowns of CUF 101 did not change significantly during this study. Transcript levels declined slowly between December and February in crowns of Apica and Rambler. Transcript levels increased a second time in early March in crowns of the winter-hardy cultivars. These variations in GaS transcript level during the overwintering period, and in particular the rise in transcript level in late winter, can be related to previously reported temperature changes and their impact on GaS transcript abundance (Castonguay and Nadeau, 1998).
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Trends in fall dormancy were positively associated with winter hardiness (Table 1). Norseman-O and selections from it exhibited excellent winter survival (>92%), while Lahontan-O and selections from it exhibited good winter survival (>75%). Winter survival of the very nondormant populations was poor. CUF 101-O and -H, and all Wadi Qurayat populations were severely injured and died during both winters. In contrast, selection for fall dormancy producing the CUF 101-L population resulted in plants with excellent winter survival (93%).
Raffinose and stachyose concentrations were very low in roots of all populations when sampled in September and October of both years (Fig. 1). By November, raffinose and stachyose concentrations increased in roots of all three Norseman and Lahontan selections, and the winter hardy population CUF 101-L. By comparison, roots of CUF 101-O and -H, and all three Wadi Qurayat populations did not accumulate significant concentrations of stachyose and raffinose in November. By December, roots of winter hardy plants (all Norseman and Lahontan populations and CUF 101-L) had accumulated raffinose and stachyose concentrations of 3.5 g kg-1 dry wt. or greater, whereas concentrations of these sugars were 2.5 g kg-1 dry wt. or less in roots of the nonhardy populations. Selection for less fall dormancy in the very dormant Norseman cultivar slightly reduced the raffinose and stachyose concentrations in roots of Norseman-H when compared with Norseman-O.
Sucrose concentrations also were influenced by cold acclimation both years (Fig. 2). Sucrose concentrations increased from approximately 20 g kg-1 dry wt. in roots of all populations sampled in September to over 120 g kg-1 dry wt. in roots of the very winter hardy Norseman-L. Sucrose accumulation began in October in the Norseman populations. By November, roots of all Norseman and Lahontan populations, and the more fall dormant CUF 101-L contained higher sucrose concentrations as compared with the nonhardy CUF 101-O and–H and the Wadi Qurayat populations. Extensive sucrose accumulation occurred in December in roots of CUF 101-O and -H and all the Wadi Qurayat populations. However, differences in root sucrose concentrations among CUF 101 populations in December was small relative to the three-fold difference in raffinose and stachyose concentrations (Fig. 1), and was not associated with winter hardiness differences of these populations.
Linear regression of winter survival ranking (Table 1) versus the ranking of root sucrose or raffinose-family oligosaccharide (RFO, sum of raffinose + stachyose concentrations) concentrations in December (Fig. 1 and 2) was used to evaluate the relationship between accumulation of these sugars and winter hardiness. Rank correlations were used to relate sucrose and RFO concentrations to winter survival of these populations because intermediate winter survival values (10–75%) were not obtained in this study (Table 1) making regressions of sugar concentrations versus percent survival difficult to interpret. In addition, predicting rank of winter hardiness is one likely use of the root sucrose and RFO data by plant breeders who wish to identify the best (or worse) plants in a population irrespective of the concentrations per se. While accumulation of both sugar pools was positively associated with winter survival (Fig. 5), RFO rankings were better related to the ranking for winter survival (r2 = 0.92) than was sucrose (r2 = 0.48). A relatively close association existed between sucrose and winter survival rankings for the alfalfa populations with high values for both (Rankings 1 to 4), but the relationship was less precise as sucrose concentrations continued to decline and winter survival became variable.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, accumulation of sugars has been shown to influence alfalfa winter survival. We recently reported a close association between cold acclimation-induced accumulation of both protein and sugar in alfalfa roots and genetic variation in alfalfa winter survival (Cunningham et al., 2001). Winter injury declined markedly as root sugar concentrations increased from 50 to 100 mg g-1 dry wt. Castonguay et al. (1995) reported that crowns of fall-dormant, winter hardy alfalfa cultivars accumulated higher concentrations of raffinose and stachyose when compared with nondormant, nonhardy alfalfa cultivars. Differences in the maximum level of freezing tolerance between nonhardy and winter-hardy cultivars were better related to the capacity of the plants to accumulate RFO than to accumulate sucrose. This agrees with our results that clearly show a closer association between RFO accumulation and winter survival than between sucrose accumulation and winter survival. In this experiment, plants with the highest root sucrose concentrations (Rankings 1 to 4) also ranked high for winter survival (Fig. 5), but the relationship between these two characteristics was not close for plants with intermediate and low rankings for root sucrose concentrations (Rankings 6 to 12). By comparison, the relationship between ranking of root RFO concentration and survival was very consistent (high r2) with a slope near unity. However, it remains unclear whether the close relationship between RFO and winter survival is attributable to differences in dormancy as postulated for seeds (Foley, 1996), or to differences in freezing tolerance.
Accumulation of raffinose and stachyose during cold acclimation has been reported for other plants, especially woody perennial species. Ashworth et al. (1993) reported that raffinose accumulation occurred in stem tissues during cold acclimation of red osier dogwood (Cornus sericea L.). Raffinose was barely detectable in summer and early fall, but increased in January to one-fifth and one-third of the total soluble sugar pool in bark and wood tissues, respectively. Imanishi et al. (1998) examined seasonal changes in the freezing tolerance, water content and soluble sugar composition of shoot apices of Lonicera caerulea L. Raffinose and stachyose accumulated rapidly from September to November, while the levels of total soluble sugars and sucrose gradually increased from June to September, preceding the substantial decrease in the temperature required to kill 50% of the plants. They concluded that RFO are involved in acquisition of freezing tolerance of the shoot apices of this species. Cox and Stushnoff (2001) also reported that bud RFO concentrations increased as temperatures declined in winter and diminished as temperatures rose in spring in trembling aspen (Populus tremuloides Michx.). As in our alfalfa study, RFO concentrations were highly correlated to low temperature survival of buds. A similar pattern of RFO accumulation and enhanced cold tolerance was observed in vegetative buds of Norway spruce (Picea abies L.) (Lipavska et al., 2000).
Galactinol synthase catalyzes the first committed step in RFO biosynthesis and could play a key regulatory role in the carbon partitioning between sucrose and RFO in overwintering organs. Changes in RFO concentrations in alfalfa roots were positively associated with timely increases in GaS transcript levels. Steady state levels of GaS in alfalfa crowns increased within 8 h of exposure to cold, nonfreezing temperatures, but expression was greatly enhanced if these tissues were exposed to –2°C for 2 wk (Fig. 3). Induction of GaS transcript also occurred in an unheated glasshouse (Exp. 2) and in the field (Exp. 3) in November in roots and crowns of winter hardy cultivars and populations. In Exp. 3, the nonhardy CUF 101-O and -H both accumulated GaS transcript in December (Fig. 6), but this did not result in extensive RFO accumulation in roots (Fig. 1). This underscores the importance of timely GaS expression in autumn thereby allowing sufficient time for RFOs to accumulate to levels that are effective in enhancing cold tolerance.
Cold-induction of GaS has been observed in other plant species. Sprenger and Keller (2000) identified two cDNAs for GaS in Ajuga reptans L., a frost-hardy evergreen labiate. Both isoforms were cold inducible and expression of the source leaf-specific galactinol synthase-1 expression was correlated positively with GolS activity. They concluded that galactinol synthase-1 was mainly involved in the synthesis of storage RFOs and that the other GaS isoform, galactinol synthase-2, was involved in synthesis of transport RFOs. Liu et al. (1998) reported that cold temperatures increased GaS mRNA levels in the vegetative tissues of Arabidopsis thaliana (L.) Heynh., and these GaS transcripts declined on exposure to room temperature. Recently, Taji et al. (2002) reported that GaS gene expression in A. thaliana was stress specific. Expression of two of seven GaS genes was up-regulated by drought and salinity, whereas a third GaS gene was induced specifically by cold stress.
In alfalfa crowns, Castonguay and Nadeau (1998) reported a close relationship between activities of sucrose-phosphate synthase and GaS and the levels of sucrose and RFO, respectively. A delay of approximately 2 wk was observed between the rise in GaS activity and RFO accumulation, an observation that could explain why high GaS transcript levels in December (Fig. 6) did not immediately result in elevated RFO concentrations in December (Fig. 1). As we observed with GaS transcript levels (Fig. 6), they found that GaS enzyme activity increased earlier in autumn and reached higher levels in winter-hardy cultivars when compared to a nonhardy cultivar. Clearly, both hardy and nonhardy alfalfa cultivars possess GaS genes and can accumulate both GaS transcripts and RFO. The subtle mechanisms regulating expression of GaS genes, transcript accumulation, and RFO accumulation represent important steps in our future efforts to improve winter survival of alfalfa genetically.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication April 24, 2002.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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