Plant Molecular Biology 36: 565–571, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Analysis of the expression of two thiolprotease genes from daylily
(Hemerocallis spp.) during ﬂower senescence
, Mercedes de la Calle
, Michael S. Reid
& Victoriano Valpuesta
Departamento de Bioqu
imica y Biolog
ia Molecular, Universidad de M
alaga, 29071 M
alaga, Spain (
Department of Environmental Horticulture, University of California, Davis, CA 95616, USA
Received 3 June 1997; accepted in revised form 11 October 1997
Key words: ﬂower senescence, daylily, thiolprotease, cDNA, gibberellic acid
A cDNA clone encoding a daylily (Hemerocallis spp.) thiolprotease (SEN11), whose expression is strongly up-
regulated in ﬂower tepal senescence, has been isolated. The amino acid sequence, deduced from the nucleotide
sequence, showed highest similarity to plant thiolproteases of Vigna mungo, Phaseolus vulgaris and Hemerocallis
(SEN102), and contains a putative ER retention signal that has been described in Vigna mungo. SEN102 and
SEN11 transcripts were not detectable in ﬂower buds at the opening stage, but two peaks of transcripts were seen
after 9 h and 19 h, in both petals and sepals, when wilting symptoms were apparent. The pattern of protease
activity migrating on a 26.3 kDa protein was similar to the SEN102 and SEN11 transcript proﬁles. These two
genes were also expressed in stamens and leaves, but their transcripts were undetectable in carpels and rhizomes.
The expression of SEN102 was lower in the senescent leaf than in the green leaf. The pattern of expression of
these genes suggests their involvement in the protein hydrolysis occurring in tepals at the late senescence stage,
whereas in leaves they could be involved in the constitutive protein turnovermachinery.Exogenous gibberellic acid
application to cut ﬂowers increased transcripts of both genes.
Most studies on ﬂower senescence have been per-
formed inclimactericspeciessuch as carnation, morn-
ingglory, petunia, and orchidwhere the plant hormone
ethylene plays a major role in the initiation and onset
of this ordered physiological process. The increased
respiration, increased activities of catabolic enzymes,
degradationofmacromolecules, and loss of membrane
al senescence , are also observed in ﬂowers where
senescence is ethylene independent , the so-called
non-climacteric ﬂowers. Daylily (Hemerocallis spp.)
is a convenient model ﬂower to study the molecular
basis of non-climacteric ﬂowers.
Daylilies carry ephemeral ﬂowers whose life from
budopeningto tissue senescence is a scant 36 h. Under
long photoperiods opening commences at midnight,
The nucleotide sequence data reported for SEN11 appears in
the GenBank under the accession number U12637.
and the ﬂowers are fully expanded 9 h later. After
13 h some loss of turgor is observed in the borders
of tepals, and the tepals are visibly wilted after 20 h.
At 36 h after bud opening the ﬂower has completely
collapsed. The onset of wilting is accompanied by a
small respiratory peak, a loss of sugars and ions, and
an accumulation of phospholipid precursors . Only
traces of ethylene are produced by the ﬂower during
wilting, and neither exposure to external ethylene nor
the application of inhibitors of ethylene biosynthesis
or action modify the timing or pattern of senescence
. During the 24 h after bud opening the ﬂower
loses most of its dry weight and soluble carbohydrates
in both the complete ﬂower and the petals [6, 24].
A 75% reduction in protein content is accompanied
by a marked reduction in high-molecular-weightpoly-
peptides in the electrophoretic protein pattern. When
cut ﬂower buds are treated during opening with an
inhibitor of eukaryotic protein synthesis, senescence