Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant ts 11.
To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell line ts11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number of ts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stage ts11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active on ts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.
Kragh
K M
KM
Danisco Biotechnology, Copenhagen, Denmark.
Hendriks
T
T
de Jong
A J
AJ
Lo Schiavo
F
F
Bucherna
N
N
Højrup
P
P
Mikkelsen
J D
JD
de Vries
S C
SC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D018552:Daucus carota / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D007887:Fabaceae / Q000201:enzymology; D014644:Genetic Variation; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D002688:Chitinases