First published online March 7, 2002; 10.1104/pp.010813
Plant Physiol, April 2002, Vol. 128, pp. 1255-1263
Altering the Expression of the Chlorophyllase Gene
ATHCOR1 in Transgenic Arabidopsis Caused Changes in
the Chlorophyll-to-Chlorophyllide Ratio1
Celso Eduardo
Benedetti* and
Paulo
Arruda
Centro de Biologia Molecular Estrutural, Laboratório Nacional
de Luz Síncrotron, Campinas, SP, CP6192, CEP 13084-971, Brazil
(C.E.B.); and Centro de Biologia Molecular e Engenharia Genética,
and Depto de Genética e Evolução, Instituto de
Biologia, Universidade Estadual de Campinas, CP6010, CEP 13083-970,
Campinas, SP, Brazil (P.A.)
The Arabidopsis gene ATHCOR1, which encodes the
CORI1 (coronatine-induced) protein, was expressed in bacterial cells.
Soluble recombinant CORI1 was purified and shown to possess
chlorophyllase (Chlase) activity in vitro. To determine its activity in
vivo, wild-type Arabidopsis and coi1 mutant, which lacks
ATHCOR1 transcripts, were transformed with sense and
antisense forms of the gene. Wild-type and coi1 plants
overexpressing ATHCOR1 showed increased contents of
chlorophyllide (Chlide) without a substantial change in the total
amount of the extractable chlorophyll (Chl). These plants presented
high Chlide to Chl ratios in leaves, whereas antisense plants and
nontransformed coi1 mutant showed undetectable
ATHCOR1 mRNA and significantly lower Chlide to Chl
ratios, relative to wild-type control. Overexpression of
ATHCOR1 caused an increased breakdown of Chl
a, as revealed by the Chlide a to
b ratio, which was significantly higher in sense than
wild-type, coi1 mutant, and antisense plants. This
preferential activity of CORI1 toward Chl a was further
supported by in vitro analyses using the purified protein. Increased
Chlase activity was detected in developing flowers, which correlated to
the constitutive expression of ATHCOR1 in this organ.
Flowers of the antisense plant showed reduced Chlide to Chl ratio,
suggesting a role of CORI1 in Chl breakdown during flower senescence.
The results show that ATHCOR1 has Chlase activity in
vivo, however, because coi1 flowers have no detectable
ATHCOR1 mRNA and present Chlide to Chl ratios comparable
with the wild type, an additional Chlase is likely to be active in
Arabidopsis. In accordance, transcripts of a second Arabidopsis Chlase
gene, AtCLH2, were detected in both normal and mutant flowers.
1
This work was supported by a grant (no.
95/06662-5) and a long-term fellowship (no. 97/0917-7 to C.E.B.) from
the Fundação de Amparo à Pesquisa do Estado de
São Paulo.
*
Corresponding author; e-mail celso{at}lnls.br; fax
5519-32877110.
© 2002 American Society of Plant Physiologists
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