Klaus-Peter Häger

Evolution of legumin genes: loss of an ancestral intron at the beginning of angiosperm diversification

The polymerase chain reaction was used to survey gymnosperm legumin genes. Characterization of 46 cloned amplificates, differing in sequence and size (1.2–1.6 kb), revealed the ubiquitous occurrence of legumin genes and their organization in small subfamilies in the 22 species investigated. The 3′ portions of the genes, coding for the legumin β‐polypeptides, show a highly conserved intron/exon structure divergent from those of angiosperms: an additional intron (intron IV) uniformly interrupts the region coding for the C‐terminal part of the β‐polypeptides. Phylogenetic analysis of the respective coding sequences as well as the organization of the Magnolia B14 legumin gene also investigated here both indicate that intron IV is ancestral and was lost during early angiosperm evolution. Taking into account the intronlexon structures from all legumin genes known, our results suggest that legumin genes evolved by subsequent loss of introns, providing also further evidence for a common origin of legumins and vicilins.

Legumin encoding sequences from the Redwood family (Taxodiaceae) reveal precursors lacking the conserved Asn-Gly processing site

We have cloned and sequenced two different cDNAs encoding legumins from Japanese red cedar (Cryptomeria japonica, Taxodiaceae). The derived amino acid sequences show between 34% and 55% identity when compared with legumins from angiosperms and from Pinaceae, respectively. The predicted precursors are unusual in that they contain potential glycosylation signals, and we have found the corresponding β‐polypeptides actually to be glycosylated. As most outstanding feature one of the precursors is lacking the Asn‐Gly processing site which has been assumed to be highly conserved in legumin gene evolution. Legumin encoding sequences amplified from genomic DNA suggest that these unusual precursors are widespread if not ubiquitous in the Taxodiaceae family. From previous reports on legumin precursors with divergent processing sites, on the proteases involved in legumin precursor processing and from the results presented here it is concluded that the Asn‐Gly processing site has been acquired rather than conserved during legumin gene evolution.

The N-terminal amino acid sequence of the β-subunit of the legumin-like protein from seeds of Ginkgo biloba

The sequence of the first 52 amino acids at the N-terminus of the beta-subunit of a legumin-like protein from seeds of the gymnosperm Ginkgo biloba were determined by automated sequencing and DABITC/PITC microsequence analyses of peptides derived from the protein by enzymatic digestions and chemical cleavage with CNBr. The protein from Ginkgo exhibits sequence homologies (32-49% identities) with the 11S globulins and legume-like proteins from seeds of various angiosperm monocotyledons and dicotyledons.

Seed storage proteins of Cupressaceae are homologous to legumins from angiosperms: Molecular characterization of cDNAs from incense cedar (Calocedrus decurrens [Torr.] Florin)

Abstract Legumins are shown to be the predominant storage proteins present in the detergent soluble fractions of seeds from incense cedar and from all other Cupressaceae investigated. The subunits of these proteins are made up of α- and β-polypeptides with molecular weights in the range of 30–34 kDa and 23–25 kDa, respectively. Unusual for legumins, those from incense cedar are glycosylated. An incense cedar endosperm cDNA library was constructed and three different legumin cDNAs were characterized; they represent two divergent legumin subfamilies with nucleotide similarities of about 71%. Comparisons with legumins from gymnosperms and angiosperms reveal between 57% and 34% identity on the amino acid level. The deduced amino acid sequences suggest that incense cedar legumins are synthesized as precursors to be processed similarly as is known from those of angiosperms. However, none of them contains the post-translational cleavage site, Asn-Gly, which is assumed to be highly conserved in angiosperm legumin precursors. A phylogenetic analysis suggests that both legumin subfamilies described here originated relatively soon after divergence of the genes derived from the common ancestor of Pinaceae and Cupressaceae legumins. Moreover, legumins prove to be reliable molecular markers to elucidate seed plant relationships.

The major β-amylase isoforms of wheat leaves correspond to one of two ubiquitously expressed β-amylase genes

Abstract Leaves of wheat ( Triticum aestivum L.), cv. Star) exhibit five distinguishable isoforms of a β-amylase (EC 3.2.1.2) considered to represent the tissue-‘ubiquitous’ type of exohydrolase common to all cereals. The object of this study was to determine whether the multiple leaf isoforms originate from different genes or reflect post-translational processing of an isoform first expressed in juvenile leaf tissue. Two different cDNAs encoding for β-amylase were isolated from leaves and each produced an active β-amylase protein upon heterologous expression in Escherichia coli . Transcripts of these two genes were detected in tissues of wheat leaves, roots, flowers and seeds. However, only one of the two heterologously expressed β-amylases appeared to correspond to the β-amylase isoforms detectable in non-endosperm wheat tissues. It exhibited specific sequence identities with, and electrophoretic mobility under non-denaturing conditions similar to, the initially expressed leaf β-amylase isoforms. As does the initially in vivo expressed leaf isoform, the heterologously expressed β-amylase was converted by a β-amylase-free wheat leaf extract into secondary isoforms which closely resemble β-amylase isoforms appearing in vivo upon the maturation of leaf tissue. The molecular masses and the N-terminal amino acid sequences of the heterologously expressed β-amylase, its secondary conversion products and the extractable leaf β-amylases indicate that at least the major components of wheat leaf β-amylase polymorphism reflect C-terminal proteolytic processing of a single β-amylase translation product.

Molecular characterization and evolution of the cytosolic class II 17.0 kDa small heat-shock protein gene family from Picea abies (L.) Karst

Abstract In order to investigate the pattern of small heat-shock protein (sHSP) expression in Norway spruce, seedlings were exposed to thermal stress. [35S]methionine in vivo labelling revealed a set of eight predominant sHSPs with molecular weights ranging from 16.0 to 27.0 kDa, comprising a 17.0 kDa cytosolic class II sHSP which is already predicted from a previously cloned cDNA sequence (Forest Genetics 4 (1997) 131). Western blot analysis showed no significant amounts of the 17.0 kDa sHSP in non-stressed vegetative tissues, and upon heat shock it accumulates to levels comparable to those constitutively found in embryo tissues from mature seeds. The Picea abies 17.0 kDa sHSP is encoded by a gene family. Six functional genes and two pseudogenes were obtained from PCR-based cloning, indicating a high degree of sequence conservation with nucleotide identities between 88 and 99%. There is evidence that the gene family is subjected to gene conversion, preferentially homogenizing the 5′ moieties of the genes. The genes code for two distinct polypeptides with molecular weights of 16910 and 16870 Da, the former contains a putative phosphorylation site RXXS. Unlike angiosperm sHSP genes, those from P. abies contain two introns, located in the 5′ un-translated and coding region, respectively. Homologous introns exist in sHSP genes from Picea glauca, and Funaria hygrometrica, suggesting the presence of introns as a retained primitive condition of plant sHSP gene evolution.