A Genomic Cluster Containing Four Differentially Regulated Subtilisin-like Processing Protease Genes Is in Tomato Plants

Abstract

Screening of a genomic library from tomato plants (Lycopersicon esculentum) with a cDNA probe encoding a subtilisin-like protease (PR-P69) that is induced at the transcriptional level following pathogen attack (Tornero, P., Conejero, V., and Vera, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6332-6337) resulted in the isolation of a cluster of genomic clones that comprise a tandem of four different subtilisin-like protease genes (P69A, P69B, P69C, and P69D). Sequence analyses and comparison of the encoded proteins revealed that all are closely related (79 to 88% identity), suggesting that all are derived from a common ancestral gene. mRNA expression analysis as well as studies of transgenic plants transformed with promoter-␤-glucuronidase fusions for each of these genes revealed that the four genes exhibit differential transcriptional regulation and expression patterns. P69A and P69D are expressed constitutively, but with different expression profiles during development, whereas the P69B and P69C genes show expression following infection with Pseudomonas syringae and are also up-regulated by salicylic acid. We propose that these four P69-like proteases, as members of a complex gene family of plant subtilisin-like proteases, may be involved in a number of specific proteolytic events that occur in the plant during development and/or pathogenesis.Proteolysis is fundamental for the normal functioning of multicellular organisms and plays key roles in a variety of processes such as development, physiology, defense and stress responses, and adaptation to the changing environment. In plants, despite the importance of all these processes and involvement of different classes of proteinases (Refs. 1-5, and references therein), it still remains to be defined more precisely what components and molecular mechanisms are responsible for regulating specific aspects of protein degradation/processing. A major task for research will be to determine which pathway of proteolysis is responsible for the degradation of particular proteins.The serine proteases are one of the best characterized groups of proteolytic enzymes in higher organisms. They can be grouped in six clans, of which one of the largest is the subtilisin-like clan (EC 3.4.21.14) that includes over 200 different members. Despite this wealth of knowledge, very little is know about subtilisin-like proteases in plants. Recently, we and others have shown the existence of members of this clan in plants, including Arabidopsis (6), tomato (7, 8), melon (9), and Lilium plants (10). According to a recent classification (11), the subtilisin-like proteases from plants can be grouped within the Pyrolysin subfamily, which is highly related to the Kexin subfamily of proteases involved in the posttranslational processing of peptide hormones (12, 13). Comparative molecular, biochemical, and cellular studies indicate that the subgroup of plant subtilisin-like enzymes are characterized by the presence of a large polypeptide sequence insertion preceding the reactive Ser residue and/or lon...

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“…Thus, metalloproteinases might be involved in proteolysis of foreign proteins in the plant extracellular matrix. In addition, members of the family of the plant subtilisin-like proteases were transcriptionally activated by pathogen attack in tomato (25,26) and by treatment with methyl jasmonate that mediates wounding and pathogen responses in plants (21).…”

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Purification and Characterization of Serine Proteases That Exhibit Caspase-Like Activity and Are Associated with Programmed Cell Death in Avena sativa

“…Gene families of subtilisin-like Ser proteases have been identified in several plant species. Lycopersicon esculentum (tomato) has had 10 genes for subtilisin-like Ser proteases thus far cloned and characterized (Vera and Conejero, 1988;Tornero et al, 1997;Jordá et al, 1999Jordá et al, , 2000Meichtry et al, 1999;Janzik et al, 2000;Riggs et al, 2001). Whereas Arabidopsis (Arabidopsis thaliana) has had only four genes characterized (Neuteboom et al, 1999;Berger and Altmann, 2000;Tanaka et al, 2001;Hamilton et al, 2003), at least 10 more genes have been annotated as being subtilisin-like Ser proteases.…”