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ARS Home » Research » Publications at this Location » Publication #63024
Title: A DESCRIPTION OF SOME BASIC CHITINASES ISOLATED FROM SWEET ORANGE CALLUS TISSUE

Author
item Mayer, Richard
item McCollum, Thomas
item Niedz, Randall
item McDonald, Roy
item BERDIS, E
item Doostdar, Hamed

Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/16/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Plants possess a variety of defensive mechanisms that aid them in resisting attacks by insect and pathogen pests. Chitinases, lysozymes, and chitosanases are well known plant defensive enzymes. All of these enzymes digest large sugar molecules (polysaccharides) that comprise the cell walls of plant pathogens and the exoskeletons of insect pests. Seven citrus chitinases have been purified and characterized as to their substrate preference, reaction products, molecular weights, and other physical attributes. One of the enzymes was found to have a very wide substrate range and could function as a chitinase and a lysozyme; amino acid sequence analysis indicated that this was a class III chitinase. Amino acid analysis showed that another enzyme was very similar to proteins found in tomato and tobacco, i.e. osmotins and AP24; this chitinase was only active against one type of polysaccharide. This AP24/osmotin-like protein has not been found previously in citrus and could prove valuable in enhancing citrus resistance against foot rot (Phytophthora spp.). These studies suggest that plants have many chitinases with a wide range of substrate capabilities that enable them to attack plant pests with very different polysaccharide structures. This information will be used in plant breeding programs to develop resistant varieties.

Technical Abstract: Seven basic endochitinases (EC 3.2.1.14) were purified to homogeneity from nonembryogenic Citrus sinensis L. Osbeck cv 'Valencia' callus tissue. The proteins ranged in size from Mr 23,000 to 28,000 and pI's from 10.3 to 10.6. One of the proteins, BCLVC, exhibited lysozyme, chitinase and chitosanase (based on hydrolysis of 18% acetylated chitosan) activities and was shown by N-terminal amino acid sequencing t be a class III chitinase. BCLVC lysozyme activity was inhibited by histamine (50% inhibition at 2 mM), imidazole (50% inhibition at 5 mM), histidine (50% inhibition at 25 mM) and chitotriose (25% inhibition at 25 mM). A second chitinase (BCVC2) was found to have an N terminal amino acid sequence that was homologous to tomato and tobacco AP24 proteins. BCLVC had the widest range of activity being capable of hydrolyzing 13.8 to 100% acetylated chitosan substrates. BCVC2 hydrolyzed only 100% acetylated chitosan. The remaining proteins (BCVC 1, 3, 4, 5, 6) were chitinase/chitosanases and expressed varying degrees of hydrolytic capabilities. Cross reactivities of the purified proteins with antibodies for a potato leaf chitinase Ab PLC, BCLVC, basic citrus chitinase, and tomato AP24 indicates that these are separate and distinct proteins.