Exploiting Secondary Growth in Arabidopsis. Construction of Xylem and Bark cDNA Libraries and Cloning of Three Xylem Endopeptidases

• Published in: Plant physiology (Bethesda) 2000-07, Vol.123 (3), p.1185-1196
• Main Authors: CHENGSONG ZHAO, JOHNSON, B. J, KOSITSUP, B, BEERS, E. P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antisense Elements (Genetics); Arabidopsis - enzymology; Arabidopsis - growth & development; Arabidopsis - metabolism; Bark; Biological and medical sciences; CDNA libraries; Cloning; Cloning, Molecular; Complementary DNA; Cysteine Endopeptidases - metabolism; Development and Hormone Action; E coli; Enzyme Activation; Enzymes; Fundamental and applied biological sciences. Psychology; Gels; Gene Library; Genes. Genome; Hydrogen-Ion Concentration; Hypocotyl - enzymology; Hypocotyl - growth & development; Hypocotyl - metabolism; Immunoblotting; Messenger RNA; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Phloem; Plant growth. Development of the storage organs; Plant physiology and development; Plant Stems - enzymology; Plant Stems - growth & development; Plant Stems - metabolism; Plant Structures - enzymology; Plant Structures - growth & development; Plant Structures - metabolism; Plant tissues; Plants; Population density; Protein Processing, Post-Translational; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger - analysis; Sequence Homology, Amino Acid; Serine Endopeptidases - metabolism; Vascular tissues; Vegetative apparatus, growth and morphogenesis. Senescence; Xylem
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.123.3.1185

The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.