Expression of beta-amylase from alfalfa taproots

Abstract

Alfalfa (Medicago sativa L.) roots contain large quantities of beta-amylase, but little is known about its role in vivo. We studied this by isolating a beta-amylase cDNA and by examining signals that affect its expression. The beta-amylase cDNA encoded a 55.95-kD polypeptide with a deduced amino acid sequence showing high similarity to other plant beta-amylases. Starch concentrations, beta-amylase activities, and beta-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, beta-amylase activities, and beta-amylase transcripts were reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. beta-Amylase transcript was high in roots of intact plants but could not be detected 2 to 8 d after defoliation. beta-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 beta-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 beta-amylase is present as a multigene family in alfalfa. Our results show no clear association between beta-amylase activity or transcript abundance and starch hydrolysis in alfalfa roots. The great abundance of beta-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.

Figure 1

A, Deduced amino acid sequence of MSBA1 aligned with sequences of some selected plant β-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 β-amylases identified by Pujadas et al. (1997) are boxed. B, Phylogenetic tree of plant β-amylases. β-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 β-Amylase Gene To determine distribution of the β-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 β-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 β-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 β-amylase was encoded by more than one gene.

Figure 3

Tissue- and species-specific expression of the β-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 β-amylase insert.

Figure 4

Changes in starch concentration, β-amylase activity, and β-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 β-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 β-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.

Figure 5

Levels of starch (A), β-amylase activity (B), and β-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, β-amylase, and β-amylase mRNA were measured. U, Units.

Figure 6

Changes in starch concentration, β-amylase activity, and β-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 β-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 β-amylase cDNA and the membrane was stripped and reprobed with a ^32-P-labeled alfalfa 18S ribosomal cDNA.

Figure 2

Southern analysis of the β-amylase gene. Genomic DNA (10 μg) was digested separately with restriction enzymes, as indicated in each lane, and analyzed by hybridization with the radiolabeled β-amylase probe.