Cloning of cDNA,expression, and chromosomal location of genes encoding the three types of subunits of the barley tetrameric inhibitor of insect α-amylase

Three cDNA clones from barley developing endosperm, corresponding to proteins BTAI-CMa, BTAI-CMb and BTAI-CMd, which are the three types of subunits of the tetrameric inhibitor of insect -amylases, have been identified and sequenced. The deduced amino acid sequence of BTAI-CMb corresponds to the CM16/CM17 type of subunit in wheat (92/90% identical residues) and has one putative N-glycosylation site (NLT) and a possible kinase-C phosphorylation site (SCR). The BTAI-CMa sequence differs at four amino acid residues from a previously reported one from cv. Bomi and the sequence deduced for BTAI-CMd completes (11 N-terminal residues) and confirms previously available data. The gene for BTAI-CMa (Iat1) is located in the arm of barley chromosome 7H (syn.1), while genes for both BTAI-CMb (Iat2) and BTAI-CMd (Iat3) are in the long arm of chromosome 4H. The three genes are expressed in endosperm and their mRNAs are not detected in the other tissues tested, except Iat1, which seems to be expressed at a low level in coleoptile and roots, where it is switched off by 50 M methyl jasmonate.     

In vivo and in vitro synthesis of CM-proteins (A-hordeins) from barley (Hordeum vulgare L.)

CM-proteins from barley endosperm (CMa, CMb, CMc, CMd), which are the main components of the A-hordein fraction, are synthesized most actively 10 to 30 d after anthesis (maximum at 15–20 d). They are synthesized by membranebound polysomes as precursors of higher apparent molecular weight (13,000–21,000) than the mature proteins (12,000–16,000). The largest in vitro product (21,000) is the putative precursor of protein CMd (16,000), as it is selected with anti-CMd monospecific IgG's, and is coded by an mRNA of greater sedimentation coefficient (9 S) than those encoding the other three proteins (7.5 S). CM-proteins always appear in the soluble fraction, following different homogenization and subcellular fractionation procedures, indicating that these proteins are transferred to the soluble fraction after processing.     

Endosperm sterol phenotype and germination in wheat

Free and conjugated sterols of endosperm, coats, scutellum, coleoptile and roots have been analysed at different germination stages in two wheat cultivars with different endosperm sterol phenotypes. It seems that sterol metabolism of the developing tissues, namely coleoptile and roots, is not affected by the sterol conjugation profile of the endosperm. Enough sterol is present in the mature embryo to supply the germinating axis during the observation period (144 hr at 16°). The data suggest that sterol is transferred from scutellum to coleoptile and roots during germination.     

The Spatial Distribution of Sucrose Synthase Isozymes in Barley

The sucrose (Suc) synthase enzyme purified from barley (Hordeum vulgare L.) roots is a homotetramer that is composed of 90-kD type 1 Suc synthase (SS1) subunits. Km values for Suc and UDP were 30 mM and 5 [mu]M, respectively. This enzyme can also utilize ADP at 25% of the UDP rate. Anti-SS1 polyclonal antibodies, which recognized both SS1 and type 2 Suc synthase (SS2) (88-kD) subunits, and antibodies raised against a synthetic peptide, LANGSTDNNFV, which were specific for SS2, were used to study the spatial distribution of these subunits by immunoblot analysis and immunolocalization. Both SS1 and SS2 were abundantly expressed in endosperm, where they polymerize to form the five possible homo- and heterotetramers. Only SS1 homotetramers were detected in young leaves, where they appeared exclusively in phloem cells, and in roots, where expression was associated with cap cells and the vascular bundle. In the seed both SS1 and SS2 were present in endosperm, but only SS1 was apparent in the chalazal region, the nucellar projection, and the vascular bundle. The physiological implications for the difference in expression patterns observed are discussed with respect to the maize (Zea mays L.) model.     

Sharp divergence between wheat and barley at loci encoding novel members of the trypsin/α-amylase inhibitors family

Amino acid sequences for three members (CMx1, CMx2, and CMx3) of a new subfamily of trypsin/-amylase inhibitors in wheat have been deduced from the nucleotide sequences of the corresponding cDNAs. A cDNA clone encoding CMx1 was selected from a wheat developing endosperm library using a probe that encoded barley trypsin inhibitor BTI-CMe at low stringency. Sequences corresponding to CMx2 and CMx3 were obtained from cDNA amplified by the polymerase chain reaction. The three CMx sequences contain a premature stop codon after 363 nt, as well as a second stop codon at the same position as in BTI-CMe (nt 439–441). Southern analysis of DNAs from diploid, tetraploid, and hexaploid wheats, as well as from aneuploid lines, indicate that there is a single CMx locus in each of the three genomes of hexaploid wheat, respectively associated with chromosomal arms 4AS, 4BS, and 4DL. These genes are expressed early during endosperm development and not expressed at detectable levels in other tissues. Evolutionary implications are discussed.