Plant Physiol. (1998) 118: 1495-1506

Expression of -Amylase from Alfalfa Taproots¹

Joyce A. Gana, Newton E. Kalengamaliro, Suzanne M. Cunningham, and Jeffrey J. Volenec^*

Department of Agronomy, Purdue University, West Lafayette, Indiana 47907-1150

	ABSTRACT

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Alfalfa (Medicago sativa L.) roots contain large quantities of -amylase, but little is known about its role in vivo. We studied this by isolating a -amylase cDNA and by examining signals that affect its expression. The -amylase cDNA encoded a 55.95-kD polypeptide with a deduced amino acid sequence showing high similarity to other plant -amylases. Starch concentrations, -amylase activities, and -amylase mRNA levels were measured in roots of alfalfa after defoliation, in suspension-cultured cells incubated in sucrose-rich or -deprived media, and in roots of cold-acclimated germ plasms. Starch levels, -amylase activities, and -amylase transcripts were reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. -Amylase transcript was high in roots of intact plants but could not be detected 2 to 8 d after defoliation. -Amylase transcript levels increased in roots between September and October and then declined 10-fold in November and December after shoots were killed by frost. Alfalfa roots contain greater -amylase transcript levels compared with roots of sweetclover (Melilotus officinalis L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.). Southern analysis indicated that -amylase is present as a multigene family in alfalfa. Our results show no clear association between -amylase activity or transcript abundance and starch hydrolysis in alfalfa roots. The great abundance of -amylase and its unexpected patterns of gene expression and protein accumulation support our current belief that this protein serves a storage function in roots of this perennial species.

	INTRODUCTION

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-Amylase catalyzes the hydrolysis of -1,4-glucosidic linkages from the nonreducing ends of starch molecules releasing maltose and producing -limit dextrin (Thomas et al., 1971). It is abundant in seeds and roots of certain species (Doehlert et al., 1982; Yoshida and Nakamura, 1991; Boyce and Volenec, 1992a) and is also present in other vegetative tissues (Beck and Ziegler, 1989). Because of its high abundance and its perceived role in starch metabolism, -amylase has been the focus of several physiological and molecular studies. Molecular analyses of plant -amylase have been conducted using cDNAs or genomic sequences from both dicots (Monroe et al., 1991; Yoshida and Nakamura, 1991; Totsuka and Fukazawa, 1993) and monocots (Sadowski et al., 1993; Yoshigi et al., 1994; Wagner et al., 1996; Wang et al., 1997). Encoded amino acid sequences for these plant -amylases are highly conserved, with amino acid similarity ranging from 60% to 96%. An endosperm-specific -amylase has been described for rye (Rorat et al., 1991) and barley (Yoshigi et al., 1994) that contains Gly-rich repetitive sequences in the carboxyl terminus of the protein.

The mode of regulation of plant -amylase genes appears complex and at times contradictory. In many plant systems -amylase transcript accumulation is regulated by sugars. Arabidopsis -amylase mRNA levels increased in rosette leaves when plants or excised, fully expanded leaves were supplied with Suc, Glc, and Fru but were not affected by mannitol or sorbitol (Mita et al., 1995). Exposure of Arabidopsis plants to light was essential for the accumulation of the -amylase transcript. Light also induced accumulation of the -amylase transcript in mustard cotyledons (Sharma and Shopfer, 1987). Sweet potato -amylase gene expression occurs in darkness if leaf-petiole cuttings are supplied with Suc (Nakamura et al., 1991). Dipping sweet potato leaf-petiole cuttings in polygalacturonic acid or chitosan also induced -amylase mRNA accumulation, whereas mechanical wounding of leaves only occasionally induced -amylase gene expression (Ohto et al., 1992). ABA induced the expression of sweet potato -amylase in leaf-petiole cuttings within 12 h of treatment (Ohto et al., 1992). However, in rice aleurone cells, ABA inhibited de novo synthesis of -amylase and reduced -amylase transcript levels (Wang et al., 1996). In most systems studied to date increases in -amylase activity and accumulation of -amylase transcripts were associated with starch deposition in tissue. This raises questions regarding the in vivo role of plant -amylase as a starch hydrolase.

Alfalfa (Medicago sativa L.) is an excellent system in which to study mechanisms of starch utilization and accumulation. In agricultural ecosystems alfalfa is completely defoliated at approximately 30-d intervals. Rapid herbage regrowth after defoliation has been positively associated with quantities of carbon and nitrogen reserves in taproots, including starch (Graber et al., 1927; Smith, 1962; Hendershot and Volenec, 1993b). Previous reports showed that defoliation results in a decline in both root amylase activity (>99% -amylase) and starch concentration (Volenec and Brown, 1988; Volenec et al., 1991). Roots of other forage legumes such as sweetclover (Melilotus officinalis L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.) contain about one-half (red clover) or less of the -amylase activity levels found in alfalfa roots (Li et al., 1996). Like alfalfa, roots of sweetclover and red clover exhibit a decline in -amylase activity that parallels a decline in root starch concentration after defoliation (Li et al., 1996). Very low total amylase specific activity was observed in roots of birdsfoot trefoil, even though root starch was depleted in a manner similar to that of alfalfa after defoliation (Boyce et al., 1992). Clearly, the pattern of decreased -amylase activity coincident with large declines in root starch concentration are inconsistent with the perceived role of -amylase as a starch hydrolase in roots of alfalfa and the other forage legumes.

Nitrogen-containing compounds in alfalfa roots (such as amino acids and proteins) have been shown to be positively associated with the rate of herbage regrowth after defoliation (Kim et al., 1991; Hendershot and Volenec, 1993b; Ourry et al., 1994; Barber et al., 1996; Volenec et al., 1996) and in the spring when shoot growth resumes (Volenec et al., 1991; Hendershot and Volenec, 1993a; Li et al., 1996). Three polypeptides constituting approximately 40% of the root's soluble protein pool have been isolated and characterized (Cunningham and Volenec, 1996). We believe that these polypeptides are VSPs because they accumulate in alfalfa taproots in early autumn and disappear in the spring and after plants are defoliated, in a manner that is consistent with functions assigned to VSPs (Cyr and Bewley, 1990). Boyce and Volenec (1992b) purified a 57.5-kD -amylase protein from alfalfa taproots and found that it constituted 8% of root-soluble protein. The seasonal pattern of -amylase activity followed the trends in concentration of root VSPs, increasing in autumn and declining markedly in spring (Volenec et al., 1991; Hendershot and Volenec, 1993a; Li et al., 1996). Because defoliation reduces nitrogenase activity (Vance et al., 1979) and uptake of nitrogen from the soil (Kim et al., 1993), we speculate that -amylase, like VSPs, may be hydrolyzed to its constituent amino acids, which are then transported to regrowing shoots to provide some of the nitrogen needed for herbage growth in spring and regrowth after defoliation in the summer (Boyce and Volenec, 1992b).

In view of the great abundance of -amylase in alfalfa roots and its unexpected pattern of activity during root starch loss, it is important to understand how -amylase gene expression is regulated by environmental cues such as defoliation and cold temperatures. Our objectives were: (a) to isolate a cDNA for alfalfa root -amylase; (b) to determine tissue-specific expression of the -amylase gene; (c) to examine the extent to which roots of other perennial forage legumes accumulate -amylase mRNA; (d) to determine genomic organization of the -amylase gene; and (e) to determine how defoliation, Suc deprivation in cell-suspension cultures, and winter hardening influence -amylase transcript levels, -amylase activity, and root starch concentrations.

	MATERIALS AND METHODS

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Plant Material Used for Isolation of -Amylase cDNA

Total RNA Extraction

Isolation and Identification of Alfalfa -Amylase cDNA

DNA Sequencing and Analysis

Tissue- and Species-Specific Expression of -Amylase

2

1

Effects of Defoliation on Starch Accumulation and -Amylase Expression

Influence of Suc Deprivation on Starch Accumulation and -Amylase Expression in Cell Suspensions

2

1

1

1

1

Effects of Fall Hardening on Starch Utilization and -Amylase Expression in Contrasting Alfalfa Germ Plasms

Northern Hybridization Analysis

Genomic Southern Analysis

Starch Analysis

-Amylase Assay

1

	RESULTS

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Isolation and Characterization of Alfalfa -Amylase

The complete nucleotide sequence was 1671 bp long, and the longest open reading frame was 1553 bp. We designated this cDNA pMSBA1 and the sequence was deposited in GenBank (accession no. AF026217). The open reading frame encodes a protein of 496 amino acid residues, with a calculated molecular mass of 55,950 D and a predicted pI of 5.31. The first ATG codon is found in position 4 of the nucleotide sequence and is presumed to be the translation initiation codon after comparison with the soybean and white clover -amylases. Three potential polyadenylation signals (Joshi, 1987) were found in the 141-bp 3-untranslated region.

The MSBA1-encoded protein contains the two -amylase signature motifs predicted by the PROSITE program (Bairoch, 1994). Alignment of the amino acid sequences of -amylase from several plant species (Fig. 1A) shows that the encoded alfalfa -amylase contains the eight highly conserved motifs identified by Pujadas et al. (1996). These authors indicated that these motifs define structural and/or functional elements of -amylases. The predicted amino acid sequence of MSAB1 is nearly identical with those of white clover (96%) and soybean (92%). Phylogenetic dendrogram analysis reveals that MSBA1 is more closely related to white clover and soybean -amylases than with other plant -amylases (Fig. 1B). The dendrogram shows that MSBA1 is clustered with -amylases from dicots and is clearly separated from monocots. The MSBA1 lacks the Gly-rich repeated motif found in the COOH terminus of rye and barley endosperm -amylases (Fig. 1A).

View larger version (51K): [in this window] [in a new window]   Figure 1. A, Deduced amino acid sequence of MSBA1 aligned with sequences of some selected plant -amylases obtained from GenBank. The multiple alignment was done with the PILEUP program of the DNA sequence analysis package from Genetics Computer Group. The eight highly conserved regions of all -amylases identified by Pujadas et al. (1997) are boxed. B, Phylogenetic tree of plant -amylases. -Amylase protein sequences of different species were obtained from GenBank. The protein sequences were aligned using PILEUP. DISTANCE and GROWTREE programs (Genetics Computer Group) were used to analyze the evolutionary distances and create the phylogenetic dendrogram. Genome Analysis of the -Amylase Gene To determine distribution of the -amylase gene in the tetraploid alfalfa genome, genomic DNA was digested with various restriction enzymes (Fig. 2). Two strong hybridizing bands were seen in the lanes where DNA was digested with HindIII, EcoRV, and NdeI. Three hybridizing bands were obtained for BglII and one strong band with several weak bands for EcoRI. Because pMSBA1 does not have NdeI or BglII sites, we expected to observe one hybridizing band if -amylase were present as a single gene (except for interruptions by an intron), but instead these enzymes gave more than one band. We expected two bands from the HindIII and EcoRV digests, since both have a single restriction site in the pMSBA1 cDNA. Because of the hybridization patterns we obtained, it was difficult to tell whether -amylase was present as a single gene. We then made a probe to only one of the HindIII fragments from pMSBA1 and reprobed the Southern blots. We obtained the same hybridization pattern (Fig. 2) as when the entire cDNA was used as a probe. Our conclusion was that -amylase was encoded by more than one gene.

Tissue- and Species-Specific Expression of -Amylase

View larger version (39K): [in this window] [in a new window]   Figure 3. Tissue- and species-specific expression of the -amylase gene. A, Total RNA from leaves, stems, and roots of alfalfa. B, Total RNA from roots of alfalfa (ALF), white clover (WC), sweetclover (SC), red clover (RC), and birdsfoot trefoil (BFT) at the time of flowering. Northern blots were hybridized with the 32P-labeled pMSBA1 -amylase insert.

Defoliation-Induced Changes in Starch Concentrations, -Amylase Activity, and -Amylase Transcript Levels

View larger version (26K): [in this window] [in a new window]   Figure 4. Changes in starch concentration, -amylase activity, and -amylase transcript levels as influenced by defoliation. Greenhouse-grown plants were defoliated at flowering. Roots were sampled immediately (0 h), at 3, 6, and 12 h, and at 1, 2, 4, 8, 12, 16, 20, 24, and 28 d after defoliation and analyzed for starch (A) and for -amylase activity (B). In B, U is units. The LSD is shown at the 5% probability level. C, Total RNA (20 µg) was analyzed by northern analysis using radiolabeled -amylase cDNA, and the membrane was stripped and reprobed with a 32P-labeled alfalfa 18S ribosomal cDNA. C, Cut (defoliated) plants; U, uncut, intact control plants. Fr. wt., Fresh weight.

Northern analysis was used to determine how defoliation affected -amylase transcript abundance and its relationship to defoliation-induced declines in -amylase activity. -Amylase mRNA abundance declined within 12 h after defoliation, attaining very low levels on d 2, 4, and 8 (Fig. 4C). Reaccumulation of -amylase transcript began on d 12 and reached levels similar to that observed in roots of intact plants by d 28. The -amylase transcript levels did not change in roots of intact plants during this period. The decline in -amylase transcript levels was more rapid than the decline in -amylase activity (Fig. 4, B and C). This lag in the loss of -amylase activity may be due to the relative slow turnover and stability of the -amylase polypeptide. -Amylase from mustard was also shown to be relatively stable, with a slow turnover rate (Subbaramaiah and Sharma, 1989).

Levels of Starch, -Amylase Activity, and mRNA in Cells Grown in Suc-Rich and Suc-Deprived Medium

View larger version (44K): [in this window] [in a new window]   Figure 5. Levels of starch (A), -amylase activity (B), and -amylase transcript (C) during incubation of alfalfa cell cultures in Suc-containing and Suc-deprived B5g medium. Cell cultures were sampled at the time of inoculation (Inoc; 0 h) and 24, 48, and 72 h after cultures were transferred to Suc-free medium, and then starch, -amylase, and -amylase mRNA were measured. U, Units.

Effect of Fall Hardening on Starch Concentrations, -Amylase Activity, and -Amylase Transcript Levels

View larger version (68K): [in this window] [in a new window]   Figure 6. Changes in starch concentration, -amylase activity, and -amylase transcript abundance in roots of alfalfa germ plasms exhibiting contrasting fall dormancy during winter hardening in autumn. Roots were sampled from field plots in September, October, November, and December. Starch levels (A) and -amylase activity (B) were measured. The LSD is shown at the 5% probability level. U, Units. C, Total RNA (20 µg) was analyzed by northern analysis using radiolabeled -amylase cDNA and the membrane was stripped and reprobed with a 32-P-labeled alfalfa 18S ribosomal cDNA.

When expressed on a protein basis, the harvest by germ plasm interaction was significant for -amylase (Fig. 6B). -Amylase activity declined from September to December in roots of cv CUF 101-L but did not change in cv CUF 101-O, whereas -amylase activity increased in cv CUF 101-H roots from September to November and then declined in December. -Amylase activity increased from September to October in roots of cv Norseman-L and cv Norseman-O and then, like root starch concentrations, decreased after October. In contrast, -amylase activity decreased from September to October and then increased in November in roots of cv Norseman-H. This consistent association of high -amylase activity with high root starch concentrations suggests that declines in root starch concentrations were not caused by higher -amylase activity. Northern analysis showed that -amylase transcript levels increased from September to October in all germ plasms except Norseman-H (Fig. 6C). The high transcript levels for -amylase in September and October declined markedly by November and December for all germ plasms. Although large declines in -amylase mRNA occurred between October and December, -amylase activity remained high in roots of all populations, suggesting high stability in planta for the -amylase polypeptide.

	DISCUSSION

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We have isolated and sequenced a -amylase cDNA that we believe encodes the -amylase polypeptide previously described (Boyce and Volenec, 1992b) for several reasons. First, the molecular mass of the encoded -amylase polypeptide (approximately 55,950 D) is similar to the 57-kD estimate for the -amylase polypeptide obtained previously using SDS-PAGE (Boyce and Volenec, 1992b). Second, tissue distribution of the -amylase transcript and -amylase activities agree with both being much more abundant in roots than in leaves or stems. Third, -amylase transcript levels are very abundant in roots of alfalfa. Lesser amounts were detected in roots of red clover, sweet- clover, and white clover, and amounts were below our detection limit in roots of birdsfoot trefoil. -Amylase activity was also found to be more prevalent in roots of alfalfa than red clover, with virtually no activity in roots of birdsfoot trefoil (Boyce et al., 1992; Li et al., 1996). The -amylase polypeptide constitutes 8% of the total soluble protein fraction in roots (Boyce and Volenec, 1992b). Similarly, we found large quantities of -amylase mRNA in alfalfa roots, as indicated by the short exposure time (less than 5 min) needed for obtaining signals on radiographic films.

The physiological role of -amylase in alfalfa roots remains obscure. In this study we examined how treatments known to reduce tissue starch concentrations (defoliation, Suc-deprivation of suspension-cultured cells, and cold acclimation) influence -amylase activity and transcript abundance. Taproots of uncut alfalfa plants showed high starch concentrations, high -amylase activities, and abundance of the -amylase transcript (Fig. 4). Defoliation reduced root starch levels but, contrary to our expectations for starch hydrolase, also resulted in lower -amylase activity and transcript levels. We have also shown that suspension-cultured cells grown in Suc-rich medium maintain high starch levels, elevated -amylase activity, and higher quantities of the -amylase transcript compared with cells deprived of Suc, which are forced to use starch as a carbon source (Fig. 5).

In agreement with previous studies (Volenec et al., 1991; Li et al., 1996), we found that cold acclimation of alfalfa stimulated starch degradation in autumn in winter-hardy germ plasms but without a concomitant increase in -amylase activity or transcript level (Fig. 6). Roots of birdsfoot trefoil contain very low -amylase activity (Li et al., 1996), and the -amylase transcript was not detectable; yet starch degradation occurs in a similar manner in roots of birdsfoot trefoil and alfalfa after defoliation (Boyce and Volenec, 1992b) and during cold acclimation (Li et al., 1996). The amount of -amylase mRNA in alfalfa, red clover, and birdsfoot trefoil correlates to the specific activity of total amylase activities observed previously for these forage legumes (Li et al., 1996). Our data suggest that -amylase is not a key enzyme in starch degradation in roots of these forage legumes, because accumulation of the transcript and enzymatic activity did not coincide with the loss of root starch.

Large quantities of -amylase are found in starch-storing organs (Doehlert et al., 1982; Yoshida and Nakamura, 1991; Boyce and Volenec, 1992a). We found high -amylase mRNA levels in stems of alfalfa even though this tissue contains little starch. Wang et al. (1995) isolated a phloem-specific -amylase from Streptanthus tortuosus (Brassicaceae). Because monoclonal antibodies made to this -amylase cross-reacted with -amylase from Arabidopsis, it was suggested that the major form of Arabidopsis -amylase could be a phloem-specific enzyme. These authors speculated that the phloem-specific -amylase may function to hydrolyze maltodextrins in sieve elements to prevent the accumulation of large polysaccharides that could impede carbon transport (Wang et al., 1995). Pea epicotyl -amylase was shown to hydrolyze maltodextrins with few Glc molecules (Lizotte et al., 1990). Whether alfalfa -amylase has a similar role in the hydrolysis of maltodextrin in phloem tissue of alfalfa stems is not known.

Since the function of alfalfa -amylase is not clear, perhaps signals that enhance or repress expression of the -amylase gene may lend some clue as to its function. Sugar-inducible expression of the -amylase gene is well documented in Arabidopsis and sweet potato (Nakamura et al., 1991; Ohto et al., 1992; Mita et al., 1995, 1997). In agreement, cell cultures grown in Suc-free medium reduced -amylase mRNA, activity, and starch concentrations (Fig. 5). Ironically, roots of field-grown alfalfa accumulate a significant quantity of soluble sugars in November and December (Cunningham et al., 1998), and yet during this period levels of -amylase mRNA are very low compared with the high mRNA levels in September and October (Fig. 6), when sugar concentrations are low. Mita et al. (1997) proposed that the gene for Arabidopsis -amylase is regulated by a combination of both positive and negative signals that are dependent on the level of sugars. Southern analysis shows that -amylase is encoded by a small gene family (Fig. 2), members of which may be regulated by different sugars or sugar concentrations. Three -amylase isozymes reported previously from alfalfa taproots (Doehlert et al., 1982; Habben and Volenec, 1991) may each be encoded by a different member of the -amylase gene family. In contrast, -amylase from Arabidopsis and maize is encoded by a single gene (Mita et al., 1995; Wang et al., 1997).

View larger version (27K): [in this window] [in a new window]   Figure 2. Southern analysis of the -amylase gene. Genomic DNA (10 µg) was digested separately with restriction enzymes, as indicated in each lane, and analyzed by hybridization with the radiolabeled -amylase probe.

Although the in vivo starch hydrolase function of alfalfa -amylase is not clear, it has been proposed that -amylase may function as a VSP, providing the nitrogen needed for shoot regrowth (Hendershot and Volenec, 1993b; Li et al., 1996). This is an attractive hypothesis because -amylase conforms to the definition of VSPs based on the perceived physiological function described by Cyr and Bewley (1990). VSPs are preferentially synthesized in storage organs and exhibit depletion during reactivation of meristems, and their abundance greatly exceeds that of other proteins in perenniating organs. -Amylase conforms to these definitions of a VSP because it is more abundant in alfalfa roots than in stems and leaves. Defoliation reactivates meristems on crowns for shoot regrowth and substantially reduces -amylase activity and -amylase transcripts.

Eight percent of the soluble protein in alfalfa taproots is -amylase (Boyce and Volenec, 1992a). Defoliation reduces nitrogenase activity and nitrogen fixation (Vance et al., 1979). Alfalfa root -amylase shows patterns of activity similar to three abundant VSPs we have characterized from alfalfa roots (Cunningham and Volenec, 1996). These four polypeptides (including -amylase) accumulate in alfalfa taproots in early autumn and disappear in spring, when shoot growth resumes, and during shoot regrowth when plants are defoliated. As shown by ¹⁵N labeling, significant movement of nitrogen occurs from this VSP-enriched protein pool to shoots after defoliation (Avice et al., 1996; Barber et al., 1996). Our data support the view that -amylase from alfalfa roots functions as a VSP.

We have presented new information concerning the mode of regulation of the -amylase gene by winter hardening and defoliation stress in alfalfa roots. Further work will concentrate on enhancing our understanding of the function of -amylase using antisense DNA transformation studies. Work is also under way to clone and characterize the cDNAs for the three alfalfa taproot VSPs that we described previously (Cunningham and Volenec, 1996). We hope that understanding the structural and biological features of these VSPs will reveal the reasons that -amylase serves a similar role in the roots of this species.

	FOOTNOTES

   Received July 20, 1998; accepted September 18, 1998.

	ABBREVIATIONS

Abbreviation: VSP, vegetative storage protein.

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