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SummaryThe N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosomeinactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticuIocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly Inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.

ABSTRACTcDNA clones encoding three additional serine carboxypeptidases (Ser-CPs) have been isolated from a gibberellic acid-induced barley aleurone cDNA library. The three deduced Ser-CPs belong to the two-chain subfamily of Ser-CPs; they are synthesized as precursors with a putative signal peptide, propeptide, and linker peptide between the A and B chains. Their identification provides the proof for the existence of more than three Ser-CPs in cereal grains, and, based on their sequences, they may exhibit new substrate specificities. The expression of these and of the three previously isolated Ser-CPs from barley grains (CP-MI, CP-MII, and CP-MIII) has been investigated by Northern and Western analysis and RNA PCR. CP-MII is the only Ser-CP to be expressed and accumulate in the developing grain and is stored in its active form in the mature grain. All six Ser-CPs are expressed de novo in the germinating grain, in the scutellum, and/or in the aleurone. Furthermore, at least CP-MI, CP-MII, and CP-MIII are secreted into the endosperm. In addition, all Ser-CPs (except CP-MI) are also expressed in the roots and shoots of the growing seedling. This enzyme family thus appears to be ubiquitous in the barley plant, which suggests that Ser-CPs play additional roles besides their participation in the mobilization of storage proteins.During germination of cereal seeds, the scutellum and the aleurone layer, surrounding the starchy endosperm, synthesize hydrolases in response to the phytohormone gibberellic acid (GA3) released by the embryo. Many of these enzymes are secreted into the endosperm and mobilize storage macromolecules to support the growth of the seedling (1). In particular, the breakdown of storage proteins is effected by the concerted action of endoproteases and exoproteases. Serine carboxypeptidases (Ser-CPs) are the major exopeptidases found in germinating cereal seeds. Enzymatic assays with various N-blocked dipeptide substrates reveal that Ser-CP activity is present in the resting barley grain and increases during germination. Chromatographic separation of protein extracts from germinating barley (2) or wheat (3) seeds yields five fractions exhibiting different Ser-CP activities, suggesting the existence of five Ser-CPs. However, only two two-chain barley (CP-MI and CP-MII), one single-chain barley (CP-MIII), and one two-chain wheat (CP-WII) Ser-CPs have been isolated and characterized from germinating seeds (4-6) and have partially different substrate preferences.Hammerton and Ho (7) established that aleurone layers isolated from mature barley seeds synthesize a Ser-CP. In response to GA3, the synthesis is enhanced and the protein is secreted. This Ser-CP is assumed to be CP-MIII, based on substrate preference and inhibition by mercuric ions. Two genes encoding Ser-CPs homologous to CP-MIII, isolated from wheat (8) and rice (9), are also expressed in the aleurone during germination. In contrast, expression of CP-MI has only been detected in the scutellum of germinating barley seeds (10).We have endeavored t...

Keywords: E n d o s p e r m , storage protein, H o r d e u m vulgare, gene family, heterogeneity, m o l e c u l a r c l o n i n gThe 873 base pair coding region ofa Hor-2 gene of barley and the adjacent 550 base pair upstream and 413 base pair downstream regions were sequenced. The gene is devoid of introns and encodes a 271 amino acid long B 1 hordein polypeptide containing a putative 19 amino acid signal peptide. The remaining part of the coding sequence can be divided into three parts. In the 53 residue amino-terminal region there are 9 glutamine-proline blocks with a preferred core sequence PQQP, separated by one or two other residues giving a giutamine proline content of 78%. The second part encodes 164 amino acids, 41%of which are glutamine + proline organised in scattered blocks. Seven cysteine residues are coded for by this portion of the gene. The last part encodes the carboxyterminal 35 amino acids none of which is glutamine.In the 550 base pair upstream region the sequence TATAAATA is found at -71 base pairs from the initiator methionine. In the 3' non-coding region three putative polyadenylation signals, AATAAA, are present.Comparison of the gene with 3 partial cDNA clones indicates that the charge polymorphism in the B 1 polypeptide group is due to point mutations in the part of the gene corresponding to the carboxy terminal half of the polypeptide. Comparison with the sequence of a second B hordein gene suggests that insertions or deletions ofglutamine-proline blocks in the amino-terminal domain are a major source of size polymorphisms in the B hordein family.Abbreviations: bp = base pair; kbp = kilo base pair Springer-Verlag

The wild ancestor of cultivated barley, Hordeum vulgare subsp. spontaneum (K. Koch) A. & Gr. (H. spontaneum), is a source of wide genetic diversity, including traits that are important for malting quality. A high ␤-amylase trait was previously identified in H. spontaneum strains from Israel, and transferred into the backcross progeny of a cross with the domesticated barley cv Adorra. We have used Southern-blot analysis and ␤-amy1 gene characterization to demonstrate that the high ␤-amylase trait in the backcross line is co-inherited with the ␤-amy1 gene from the H. spontaneum parent. We have analyzed the ␤-amy1 gene organization in various domesticated and wild-type barley strains and identified three distinct ␤-amy1 alleles. Two of these ␤-amy1 alleles were present in modern barley, one of which was specifically found in good malting barley cultivars. The third allele, linked with high grain ␤-amylase activity, was found only in a H. spontaneum strain from the Judean foothills in Israel. The sequences of three isolated ␤-amy1 alleles are compared. The involvement of specific intron III sequences, in particular a 126-bp palindromic insertion, in the allele-dependent expression of ␤-amylase activity in barley grain is proposed.