A pectate lyase from Zinnia elegans is auxin inducible.
The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans-differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate. In a search for genes involved in modifications to cell-wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72-h cDNA library with probes from mRNA at time-points of 24 h and 72 h was done revealing a number of transcripts up-regulated between these times. One of these cDNAs shows homology to pectate lyase, a pectin-degrading enzyme. The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N-terminal signal peptide of about 2 kDa, and one potential N-glycosylation site. Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium. In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia. Recombinant protein made in Escherichia coli possesses calcium-dependent pectate lyase activity. Pectate lyase activity is detected in elongating and differentiating in vitro cell populations. The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed.
Department of Cell Biology, John Innes Centre, Colney, Norwich, UK.
C
C
Domingo
K
K
Roberts
N J
NJ
Stacey
I
I
Connerton
F
F
Ruíz-Teran
M C
MC
McCann
D000595:Amino Acid Sequence; D001483:Base Sequence; D002454:Cell Differentiation; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017403:In Situ Hybridization; D007210:Indoleacetic Acids / Q000494:pharmacology*; D008969:Molecular Sequence Data; D059828:Plant Cells; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D007210:Indoleacetic Acids; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase