Plant Molecular Biology 42: 439–450, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Chlorophyll breakdown in Chlorella protothecoides: characterization of
degreening and cloning of degreening-related genes
, Joanne Chinner
, Philippe Matile
, Howard Thomas
and Iain S.
Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland (
author for cor-
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion,
SY23 3EB, UK
Received 17 May 1999; accepted in revised form 9 November 1999
Key words: cDNA, Chlorella protothecoides, chlorophyll breakdown, degreening, differential screening, senes-
Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM−N) in the dark rapidly de-
graded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth
conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites
as did the addition to BM−N of cycloheximide or a chelator, 2,2
-bipyridyl. In contrast, chloramphenicol or the
protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation
of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase.
During growth in BM–N, protease activity increased and proteins immunologically detectable with an antibody
against a senescence-enhanced cysteine protease accumulated. cDNAs from BM−NandBM+N cells were used
for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expres-
sion (dee)inC. protothecoides. Several different dees were identiﬁed with different patterns of expression during
Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4wasmost
abundant and its expression was exclusive in BM–N cultures. Analysis of the dee sequences showed that they
encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a
-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.
Chlorophyll breakdown is the most conspicuous
symptom of leaf senescence and fruit ripening and has,
therefore, been widely used as a marker to monitor
senescence. It is not only important for the under-
standing of a fundamental biological process, but also
has substantial economic impact (Brown et al., 1991).
For example, in the food industry, inhibition of Chl
The nucleotide sequence data reported will appear in the EMBL
database under the accession numbers AJ238625 (dee8), AJ238626
(dee10), AJ238627 (dee112), AJ238628 (dee188), AJ238629
(dee189), AJ238630 (dee138), AJ238631 (dee22), AJ238632
(dee76), AJ238633 (dee165), AJ238634 (dee25) and AJ238635
breakdown during the processing of ‘green vegeta-
bles’ is desired (Heaton and Marangoni, 1996). On
the other hand, degreening is an essential part of nor-
mal ripening in many fruits and interference with the
process can lead to practical problems. For instance,
in banana peel ripening, which is accompanied by de-
sirable degreening, elevated temperatures inhibit Chl
breakdown so that harvested fruits require shipping
and storage at below 24
C (Seymour et al., 1987).
Chl catabolism can also have value for tourism. For
example, the autumnal colour change of deciduous
trees during the ‘Indian summer’ brings many visi-
tors to the northeast of the USA every year (Hendry
et al., 1987). Conversely, the inhibition of breakdown
is important in the maintenance of green lawns in the