Expression of β-Amylase from Alfalfa Taproots1

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

Genetic Manipulation of Condensed Tannins in Higher Plants1 : II. Analysis of Birdsfoot Trefoil Plants Harboring Antisense Dihydroflavonol Reductase Constructs

We have produced and analyzed transgenic birdsfoot trefoil (Lotus corniculatus L.) plants harboring antisense dihydroflavonol reductase (AS-DFR) sequences. In initial experiments the effect of introducing three different antisense Antirrhinum majus L. DFR constructs into a single recipient genotype (S50) was assessed. There were no obvious effects on plant biomass, but levels of condensed tannins showed a statistical reduction in leaf, stem, and root tissues of some of the antisense lines. Transformation events were also found, which resulted in increased levels of condensed tannins. In subsequent experiments a detailed study of AS-DFR phenotypes was carried out in genotype S33 using pMAJ2 (an antisense construct comprising the 5′ half of the A. majus cDNA). In this case, reduced tannin levels were found in leaf and stem tissues and in juvenile shoot tissues. Analysis of soluble flavonoids and isoflavonoids in tannin down-regulated shoot tissues indicated few obvious default products. When two S33 AS-DFR lines were outcrossed, there was an underrepresentation of transgene sequences in progeny plants and no examples of inheritance of an antisense phenotype were observed. To our knowledge, this is the first report of the genetic manipulation of condensed tannin biosynthesis in higher plants.

Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment.

Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.