Plant Molecular Biology 37: 455–469, 1998.

455

c 1998 Kluwer Academic Publishers. Printed in Belgium.

A comparison of the expression patterns of several senescence-associated

genes in response to stress and hormone treatment

Louis M. Weaver, Susheng Gan1 , Betania Quirino and Richard M. Amasino

Dept. of Biochemistry, University of Wisconsin at Madison, 420 Henry Mall, Madison, WI 53706, USA ( author
for correspondence); 1 current address: Tobacco and Health Research Institute and Dept. of Agronomy, University
of Kentucky, Lexington, KY 40546, USA

Received 10 September 1997; accepted in revised form 6 January 1998

Key words: gene expression, phytohormone, Lea, leaf senescence, senescence-associated gene, stress

Abstract

The expression of several Arabidopsis thaliana senescence-associated genes (SAGs) in attached and/or detached
leaves was compared in response to age, dehydration, darkness, abscisic acid, cytokinin, and ethylene treatments.
Most of the SAGs responded to most of the treatments in a similar fashion. Detachment in darkness and ethylene
were the strongest inducers of both SAGs and visible yellowing. Detachment in light was also a strong inducer
of SAGs, but not of visible yellowing. The other treatments varied more in their effects on individual SAGs.
Responses were examined in both older and younger leaves, and generally were much stronger in the older ones.
Individual SAGs differed from the norms in different ways, however, suggesting that their gene products play a role
in overlapping but not identical circumstances. Some SAGs responded quickly to treatments, which may indicate
a direct response. Others responded more slowly, which may indicate an indirect response via treatment-induced
senescence. Four new SAGs were isolated as part of this work, one of which shows strong similarity to late
embryogenesis-abundant (Lea) genes.

Introduction 44]). Such genes are often referred to as senescence-

Leaf senescence is an orderly, active process in which
nutrients in a leaf are reclaimed and mobilized to other
parts of the plant. It is often referred to as the final stage
of leaf development, to emphasize that it is both highly
regulated and genetically programmed. The changes
most closely associated with senescence are declines
in total protein and RNA levels and chloroplast break-
down. Its most visible symptom is chlorophyll loss
and concomitant leaf yellowing. It has been known for
some time that transcription and translation are neces-
sary for senescence to progress [6], and in the past
few years many genes have been cloned for which the
corresponding messenger RNAs are upregulated dur-
ing the process (for recent reviews see [5, 8, 32, 35,
The Nucleotide sequence data reported will appear in the EMBL
and GenBank Nucleotide Sequence Databases under the accession
numbers AF053063, AF053064, AF053065.
associated genes, or SAGs.
Senescence occurs in response to aging, and under
constant environmental conditions is relatively con-
stant and predictable [19]. Certain stresses and hor-
mones, however, are able to hasten or repress sen-
escence. Drought, darkness, detachment, and the hor-
mones abscisic acid (ABA) and ethylene can all induce
visible yellowing (see for example [4, 29, 34, 36]). The
hormone cytokinin is generally regarded as an inhibit-
or of senescence (e.g. [14, 34]). It is also known that
certain stresses and hormones are able to induce genes
directly responsive to those stresses and hormones. For
instance, several genes are known to be inducible by
ABA (e.g. [37, 45]) , drought (e.g. [3, 23, 47]), and
darkness (e.g. [43]).
What is less clear is the overlap between the SAGs
and the stress- and hormone-response genes. That there
is such an overlap is clear from the fact that five of the
11 genes cloned as SAGs and examined in this paper
456
are similar or identical to genes isolated independently
based on their responses to stress (see below). There
are other examples of such overlap (e.g. [2, 9, 20, 41]).
All stress-responsive genes are certainly not SAGs,
however, nor can it be assumed that all SAGs will also
respond to senescence-inducing stresses, because it
cannot be assumed that senescence induced by stresses
or hormones is identical to age-mediated senescence.
There is in fact some evidence that such induced sen-
escence is not identical, and there is similar evidence
that hormones that appear to repress senescence may
not in fact repress all aspects of it [4, 36]. It cannot
even be assumed that all genes cloned as SAGs are
in fact directly involved in the regulation or the cata-
bolic processes of the senescence syndrome, because
the senescence syndrome is generally thought to stress
the cell, and some SAGs might function primarily in
protecting the senescing cell from that stress.
Most currently cloned SAGs were identified by dif-
ferential cDNA cloning approaches, and little is known
of them beyond the fact that their mRNA levels increase
during natural (age-mediated) senescence. The func-
tions of some of these genes can be inferred from their
deduced amino acid sequences, but for others that is not
the case. It is hoped that by examining other circum-
stances in which these genes are induced something
can be learned about their roles. If a SAG is shown to
be induced solely in response to natural senescence, for
instance, then it is very likely that it functions directly
in the senescence program. If, on the other hand, a
‘SAG’ is shown to be induced more quickly and more
strongly by a particular stress than by aging, its role
in senescence may be secondary. It is also hoped that
studying the expression of various genes in response
to stresses and hormones and senescence will provide
some insight into the relationships among those pro-
cesses. Becker and Apel, for instance, have shown
that of three genes differentially expressed in detached
barley leaves left in the dark only one was similarly
upregulated during natural senescence, suggesting that
the two processes are not identical [4]. Here we expand
such work by examining the leaf expression of a relat-
ively large number of SAGs in response to age, dehyd-
ration, detachment, darkness, ABA, cytokinin, and
ethylene. We examine these responses in leaves of at
least two different developmental stages, to determ-
ine whether they are age-dependent. We also exam-
ine expression as a function of time after treatment to
determine the rapidity of the response.
This work addresses the question of whether a gene
cloned as a SAG is in fact primarily a SAG, or wheth-
er it responds more strongly to one or several oth-
er factors, and only indirectly to age. The goal is to
determine the relationship between genes identified as
SAGs and the various factors that can induce senes-
cence. A related goal is to identify which genes might
be the best molecular markers of senescence, by identi-
fying those which respond to the fewest other stimuli.
Materials and methods
Plant material and treatments
Seeds of Arabidopsis thaliana ecotype Landsberg
erecta were originally obtained from the Arabidop-
sis stock center at Ohio State University. Plants were
grown on Fafard germination mix (Conrad Fafard,
Agawam, MA) under continuous cool white fluores-
cent light (120 mol m,2 s,1 ) in growth chambers.
Under these conditions plants flowered after forming
ca. 8 rosette leaves (cotyledons were not counted).
The last four leaves (leaves 5 through 8) were used
in the experiments. All plants used in a given experi-
ment were taken from a single synchronously growing
population. Only identically aged leaves were pooled
(i.e. leaves 5, 6, 7 and 8 were each pooled separately).
Those leaves all had an adult morphology [31, 42],
and it is likely that any difference in response between
them is a result of age and not that some were juvenile
and others adult.
Abscisic acid (ABA) as mixed isomers and
cytokinin (N6 -benzyladenine, BA) were purchased
from Sigma. The ABA solution sprayed on plants
was 0.1 mM ABA and 0.02% Tween-20. The con-
trol solution was 0.02% Tween-20. The BA solution
was at 0.1 M. In detached leaf experiments leaves
were removed and submerged completely in either BA
or water for ca. 2 min prior to being placed upright
(with petioles submerged) in the appropriate solution.
Control leaves (0 days of treatment) were handled
similarly but placed only in water and frozen within
20 min of detachment. Ethylene gassing experiments
were carried out on whole plants in Plexiglas cham-
bers with ethylene at 1 ppm. Dehydration treatments
consisted of cutting plants at their base and placing the
severed rosettes on filter paper to dry in dim light (20
to 40 mol m,2 s,1 ) at relative humidity ca. 65%. In
the attached leaf/darkness experiments whole plants in
their pots were transferred to darkness.

RNA extraction and blotting
Total RNA was extracted as described by Puissant and
Houdebrine (1990). In some experiments RNA Isol-
ator (Genosys Biotechnologies, The Woodlands, TX),
which is a commercial reagent based on that meth-
od, was used. 15 g of RNA was size-fractionated
by electrophoresis on 1% formaldehyde-agarose gels
and transferred onto nylon membranes by capillary
blotting. Probes were 32 P labeled by random-priming
(Prime-a-Gene kit, Promega, Madison, WI). Hybridiz-
ation was done at 65  C overnight and membranes
were washed 2 times for 45 min each in 0.04 M
NaH2 PO4 pH 7.2, 1% SDS, 1 mM EDTA. Probe
hybridization was visualized with a Phosophorim-
ager using ImageQuant software (Molecular Dynam-
ics, Sunnyvale, CA).
Measurement of chlorophyll content
Ground, frozen plant tissue (from the same samples
used for RNA extraction) was extracted in 2.2 ml
96% ethanol and chlorophyll quantified photometric-
ally using the method of Wintermans and DeMots
[46]. In the ABA experiment measurements were
repeated three times and the results averaged. Most of
the detached leaf/leaf 7 measurements were repeated
twice. All other measurements were performed only
once due to limited tissue. Error bars are shown on
the graphs when measurements were performed more
than once. Chlorophyll content is presented relative to
the control time point of the youngest leaf in a given
experiment.
Differential screening
SAG18 was isolated by differentially screening a
cDNA library made from mid-senescent leaves, fol-
lowing the procedure of Lohman et al. [28]. RD21
was isolated using a modified version of Represent-
ational Difference Analysis, a PCR-based subtract-
ive technique [26] (PCR-select cDNA Subtraction kit,
Clontech, Palo Alto, CA). SAGs 20 and 21 were isol-
ated by randomly picking clones from a cDNA library
derived from fully senescent leaves and using them to
probe RNA blots to identify ones that were SAGs.
457
Sequence analysis
The newly isolated SAGs were compared with
sequences in the NCBI non-redundant database using
the BLASTX program [1, 15].
Results
Experimental design
We define senescence-associated genes (SAGs) as
those genes with steady-state mRNA levels that
increase during age-mediated (natural) senescence. We
define visible senescence as a characteristically pat-
terned leaf yellowing that generally begins at a leaf’s
edges and reaches the veins last. Many stresses and hor-
mones are known to induce visible senescence. This
implies that many stresses and hormones will also in
the course of time induce SAGs as well, at least inso-
far as induced senescence is biochemically similar to
age-mediated senescence. Brief stress and hormone
treatments, however, are unlikely to quickly induce
senescence, so genes which respond quickly to them
are likely responding directly to the treatments them-
selves. We present time courses of induction, to address
both whether a SAG can respond to a given treatment
and how quickly it can respond. An important consid-
eration when examining the response to senescence-
promoting factors is the age of the leaf examined,
because of the possibility that leaves able to respond
to a given treatment at one developmental stage might
be unable to do so at another. An annual plant can, in
a sense, be regarded as a colony of leaves of a variety
of ages, all of which follow a similar but temporally
staggered pathway to senescence [19]. For this reas-
on it is critical when looking for treatment-induced
changes in gene expression not to pool leaves across
developmental stages, but rather to separately examine
specific developmental stages.
To address these concerns we have done two types
of experiments. In the first type, referred to as a
constant induction/variable final age experiment (Fig-
ure 1A), a constant induction is used, for example 3 h
of desiccation, which is applied to leaves at a wide
range of developmental stages, from very young to
completely yellow. This type of experiment gives a
broad view of how a leaf’s ability to respond to a given
stress changes with age. In the second type of experi-
ment, referred to as a variable induction/constant final
age experiment (Figure 1B), plants are subjected to
458
stresses at staggered intervals and all are harvested at
the same time and age. This answers the question of
how quickly a leaf can respond to a given stress at a
given developmental stage. For each treatment we did
two such experiments, one using older leaves and one
younger.
Isolation of SAGs
The purpose of this work was both to study the
factors that affect expression of individual SAGs and
to determine whether patterns emerge when looking
at relatively large numbers of them. In these studies
we examined 11 SAGs. Five of these (SAGs12, 13,
14, 17 and ERD1) were previously described by Loh-
man et al. [28] (ERD1 under the name SAG15), and
one (SAG2) by Hensel et al. [19]. SEN1 was isolated
by Oh et al. [36] and shown by them to respond to
a variety of factors in addition to senescence. Two of
these genes (SAG2 and SAG12), and one described
below (RD21), are likely cysteine proteases, a relat-
ively common type of SAG to emerge from differential
screenings (e.g. [9, 11, 19, 28, 39]). Two (SAGs 14
and 17) are metal-binding proteins, which may also be
a common category (e.g. [7, 9, 20, 28]). Two of these
genes were independently isolated as responding to
factors other than senescence (SAG14 to darkness [43]
and ERD1 to dehydration [22]), while SEN1 is simil-
ar to a darkness-induced gene [36]. For this study we
isolated four additional SAGs by differentially screen-
ing cDNA libraries in a variety of ways (see Materials
and methods). One of these proved identical to RD21,
isolated by others as a drought-induced gene [47]. The
three others, SAGs 18, 20, and 21, are novel. Similarity
searches using the partial sequences of SAGs 18 and
20 found no strong similarities to other sequences in
the databases, although SAG20 does show weak sim-
ilarity to a potato wound-induced gene (wun1 [27]).
SAG21 shows strong similarity (up to 58% amino acid
identity across the partial sequence) to members of the
late embryogenesis abundant, or Lea, gene family. The
best characterized members of this family are thought
to be involved in protecting the developing seed from
dehydration stress, but others have been found in the
adult plant, including recent examples that respond to
one or more stresses and hormones including drought,
salt, cold, heat, wounding, ethylene, ABA, gibberellin,
and auxin. [12, 16, 33, 40, 49]. SAG21 is most sim-
ilar to potato clone 32B, for which the corresponding
mRNA is upregulated in both the leaves of tuberizing
potato plants and in green tomato fruits shortly before
they ripen [21]. Tuberization is often accompanied by
leaf senescence, and so might be expected to induce
the expression of senescence-associated genes. Fruit
ripening is thought to share many similarities with leaf
senescence (e.g. see [10]), and it is worth noting that
the expression pattern of 32B in tomato fruits is simil-
ar to that of SAG21 in Arabidopsis leaves: its message
level peaks shortly before ripening (or senescence), and
then declines (see next section and Figure 2). To our
knowledge this is the first implication of LEA genes in
senescence. DNA blot analyses of SAGs 18, 20, and 21
suggest that all are single copy genes (data not shown).
Effects of various factors on SAG expression
Age
All SAGs are, by definition, upregulated during age-
mediated (natural) senescence. This study was inten-
ded to answer the question of when exactly each
SAG, at the level of steady-state mRNA abundance,
is induced with respect to the other SAGs and to leaf
development, and also the degree and specificity of
that induction. In this experiment only the fifth true
leaf was used.
Most of the SAGs examined had some degree of
basal expression in non-senescent leaves, suggesting
that their gene products play a role in non-senescent
tissue as well (Figure 2). Many of the SAGs were
induced relatively gradually as the leaf aged, with
that induction preceding visible yellowing. Two genes
were notable exceptions. SAG13 only showed clearly
detectable expression shortly (2 days) before visible
senescence began, while SAG12 showed no detectable
expression until after significant visible yellowing (as
presented in [28]). SAGs 12 and 13 are also expressed
at unusually high levels (data not shown). SAG21 is
notable for reaching peak expression before full sen-
escence, and then declining in abundance. None of
the genes examined show elevated expression in both
young and old leaves, as is the case for one known
class of SAGs (e.g. see [8, 17, 38]).
Detached leaves in darkness
It has long been known that in many species leaves
detached from the plant and placed in darkness will
quickly senesce, and such ‘artificial senescence’ has
been used in senescence research. Becker and Apel,
however, have shown that genes induced during dark
detachment are not necessarily induced during natural
senescence, which demonstrates that the two processes
 459

Figure 1. Schematic representation of the two types of experiments done in the paper. In the constant induction/variable final age experiments
(panel A) leaves are harvested over a period of days following a constant treatment (or no treatment, in the case of the age-only experiment).
This yields a developmental series of leaves. In the variable induction/constant final age experiments (panel B) leaves are subjected to treatments
at staggered intervals, and all are harvested on the same day and at the same age. Two variable induction/constant final age experiments were
done per treatment, one on older leaves and one on younger leaves.

are not identical [4]. Here we examine the situation in
reverse, to determine if genes induced during natur-
al senescence are also induced during artificial sen-
escence. It is also well known that cytokinins are fre-
quently able to block visible senescence, although they
do not necessarily block the expression of all SAGs [4,
36]. For this reason we also examined the ability of
cytokinin to affect this dark/detachment-induced sen-
escence.
This study was carried out by removing the fifth
and seventh leaves and placing them upright with peti-
oles submerged in either water or a cytokinin solution,
and placing them in the dark for the indicated periods.
The experiment was terminated and all samples collec-
ted on the same day, and therefore those leaves which
received the longest treatments began them when they
were the youngest (see Figure 1B). Undetached control
leaves 5 (the 0 day time point in Figure 3) were about
10% yellow on the day that all the leaves were harves-
ted, while undetached leaves 7 were completely green.
Leaves incubated in the dark in water did show clearly
visible yellowing, in patterns typical of age-induced
senescence, although leaves incubated in cytokinin did
not (data not shown, but see chlorophyll measurements,
Figure 3). Leaf five data is only shown to three days
darkness, because after that time the tissue without
cytokinin had degenerated. Leaf 7 data are shown up
to 8 days because it remained intact throughout that
period.
All the SAGs except one were induced in older
leaves (leaf 5) in response to detachment, and all but
two were induced in younger leaves (leaf 7) as well
(Figure 3). The exceptions were SAG21, which was
not induced by the treatment in either older or younger
460

Figure 2. Age-dependent expression of the SAGs. The fifth leaves

were harvested from a synchronously growing population of Ara-
bidopsis at the indicated day after germination, and total RNA extrac-
ted and transfered to membranes. The photographs at the top of the
figure indicate the developmental stage of the tissue comprising each
time point. DAG is days after germination. Blots were probed with
the indicated gene. CAB (chlorophyll a/b binding protein gene) is
used to indicate the progression of senescence. 18S ribosomal RNA
(rRNA) is a loading control. Exposure times varied for each gene
examined, and so band intensities cannot be compared across genes.

leaves, and SAG12, which responded relatively weakly

in older leaves and not in younger ones. For most
SAGs the induction was stronger and/or faster in the
older leaves, indicating that developmental factors are
important in this response.
Cytokinin treatment, which repressed visible yel-
lowing (data not shown), also repressed the expression
of most of the SAGs. That repression was generally
Figure 3. Expression of SAGs in detached leaves incubated in the
darkness, with and without cytokinin. The indicated leaves were
detached from the plant and incubated in the darkness in either
water or BA (cytokinin) solution. Control leaves (0 days) remained
on the plant throughout the course of the experiment. Chlorophyll
levels are given relative to that in leaf 7 prior to treatment (leaf 7 day
0). Control leaves 5 (leaf 5 day 0) were ca. 10% yellow when the
experiment was terminated. Control leaves 7 were fully green. ‘ND’
means that a given lane was not run.
 461

stronger in the younger leaves, which were presum-

ably not yet developmentally ready to senesce, than
it was in the older leaves which would have begun to
senesce even in the absence of detachment. The one
SAG that cytokinin did not clearly repress was SEN1,
although in the younger leaves it may have had some
effect. This lack of cytokinin inhibition is consistent
with what Oh et al. have reported [36].

Detached leaves in light

The work described above demonstrates that the incub-
ation of detached leaves in darkness induces both vis-
ible yellowing and SAGs. To determine to what degree
that induction was the result of detachment itself, we
examined detached leaves that were incubated in the
light rather than the darkness (Figure 4). Again we
used leaves 5 and 7, detached at the same time and
from the same lot of plants as in the dark experiment.
This treatment did not induce obvious visible yellow-
ing, although chlorophyll levels did decline in the older
leaves, and in the younger ones by five days. The older
leaves, which yellowed in the non-detached controls,
continued to yellow normally after detachment, and
over all the detached and control leaves appeared sim-
ilar. As in the dark detachment experiment, however,
the older leaves detached for the longest times began to
lose integrity and RNA was not isolated from them. On
average, these older leaves remained intact two days
longer in the light than in the darkness. Cytokinin was
again able to inhibit both visible yellowing and tissue
degradation.
All the SAGs were induced by this treatment
(although SAG12 only weakly), but most were induced
only in the older leaves, which had already begun to
senesce (Figure 4). Cytokinin was generally effective
in inhibiting SAG expression in the younger leaves, as
was the case in the previous experiment, and also in the
older ones. Under these conditions SEN1 behaved like
the other SAGs, and cytokinin did inhibit its expres-
sion. It is notable that SAG21, which alone was not
induced during the dark incubation above, was induced
by detachment alone. This implies that in detached
leaves darkness actually inhibits SAG21 expression.

Whole plants in darkness

These experiments were prompted in part by the fact
that one of the SAGs (SAG14) was independently isol-
ated as a dark-induced gene [43], while another, SEN1,
was cloned by similarity with one [36]. To determine
whether other SAGs are induced by darkness, SAG
Figure 4. Expression of SAGs in detached leaves incubated in the
light, with and without cytokinin. The experiment was conducted as
in Figure 3, but the leaves were incubated in the light rather than the
darkness. No chlorophyll measurement was taken for the leaf 5/1
day/BA timepoint.
462

mRNA levels in whole plants placed in the dark for

varying periods were determined. As in the previous
experiments, the plants treated the longest were placed
in the dark the earliest, to allow them all to be harvested
on the same day after germination (Figure 1B). Obvi-
ous growth stopped after placement in the dark, with
the result that the leaves treated longest were much
smaller and less expanded than those of the controls
(they looked much as they had when first placed in
the darkness). This treatment produced no visible yel-
lowing, although the leaves that endured it the longest
were pale, and chlorophyll levels did slowly decline.
Control leaves 6 had just begun to yellow, while control
leaves 8 were fully green.
Of the 11 SAGs examined, five (SAGs 14, 20, 21,
ERD1 and SEN1) were significantly upregulated by
darkness, two (SAGs 13 and 17) were quickly (within
one day) down-regulated by it, and five (SAGs 2, 12, 18
and RD21) were only slightly affected (Figure 5). Most
SAGs that responded positively to darkness respon-
ded both in younger and older leaves (SAG15 is the
exception), although the response was stronger and
more notable in the older ones in which expression
was already higher. This suggests that for such SAGs
the dark response and the age-mediated response are
additive. SAG21 responded quickly and strongly in
both younger and older leaves, and was unusual in that
its mRNA level was highest after only one day of dark-
ness. This response was also notable in that SAG21
was unique in not responding to darkness in detached
leaves. Consistent with what Oh et al. have reported
[36], we observed SEN1 to be strongly induced by
darkness, in both younger and older leaves. SAG12
again showed little or no response to this treatment,
while SAGs 13 and 17 in contrary fashion to most of
the other genes showed a strong negative response.
Almost all the SAGs showed a decline by five days
in darkness, including those which were significantly Figure 5. Expression of SAGs in whole plants placed in darkness.
Whole plants were placed in the dark for the indicated periods, and
stimulated by shorter treatments. This may be because
leaves 6 and 8 harvested. All leaves were harvested on the same
such prolonged dark treatments inhibited the develop- day and at the same age, such that the only difference between the
ment of the relatively young leaves that first received samples is how long the plants spent in the darkness. Chlorophyll
them, to the point that they were effectively too young levels are given relative to the leaf 7 control, which was fully green.
Leaf 5 controls were just beginning to visibly yellow. No chloropyll
to either begin to senesce or to respond to the darkness measurement was obtained for the leaf 8/8 day timepoint.
with SAG induction. Prolonged darkness might also
cause a general decline in mRNA levels.

ABA treatment
Abscisic acid (ABA) is commonly known to induce
senescence. Many studies of it in this context have
used detached leaves floating or immersed in an ABA
solution (e.g. [4, 36]). Because detachment itself can
induce many senescence genes (see above) we pre-
ferred instead to spray whole plants with ABA. This
treatment has been shown sufficient to induce known
ABA-responsive genes (e.g. [45, 47]). Under our con-
 463

ditions it induced older, but not younger, leaves to

visibly yellow after 24 to 48 hs. The rapid increase in
mRNA levels of Cor15, an ABA-responsive gene, also
indicate that the topical ABA treatment was effective
(Figure 6).
We chose leaf 6 as the older of the two leaves used
in this experiment. In controls it was just beginning to
visibly yellow at the time of harvest, and ABA treat-
ment caused a relatively slight enhancement of that
yellowing. Leaves 5 and older yellowed more strongly
in response to ABA (data not shown). We chose leaf
6 because we thought it would be more informative
to determine the affect of ABA on younger leaves just
beginning to respond. Leaf 8, used as the younger of
the two leaves, showed no visible response to the ABA.
About half the SAGs (SAGs 12, 13, 17, ERD1 and
RD21) responded to ABA, and as in the previous exper-
iments that response was generally much greater in the
older leaves than in the younger (Figure 6). This was
consistent with the induction of yellowing, and sug-
gests that ABA can cause leaves that are already begin-
ning to senesce to do so faster, but is not very effective
at inducing leaves that have not begun to senesce to do
so. ERD1 is a good example of this. There were some
exceptions to the lack of ABA response in younger
leaves. SAG13 responded quickly (in 4 h) and strongly
to ABA in both younger and older leaves. Its response
pattern in younger leaves (a fast, transient spike) in fact
resembled that of Cor15, the ABA-responsive posit-
ive control used in this experiment. This suggests that
SAG13 may respond directly to ABA, rather than only
indirectly to ABA-induced senescence. We observed
a weak transient spike of RD21 expression in leaf 8
four hours after treatment as well. Yamaguchi et al.
have reported that RD21 is not ABA-responsive, but
the earliest time point they show after treatment is 10 h,
so our result is not necessarily inconsistent with theirs
[25, 47]. SAG12, which responded slightly or not at all
to all previous treatments, responded very strongly to
ABA, although only in older leaves. SAG12 responded
more slowly than SAG13, however, and its response
correlated more closely with visible yellowing.
Three genes, SAG20, SAG21, and SEN1, did
not respond to ABA. The SEN1 result is particularly Figure 6. Expression of SAGs in plants sprayed with ABA. This
surprising because Oh et al. have reported a strong experiment was carried out similarly to that in Figure 5, except
response in detached leaves incubated with ABA [36]. plants were sprayed with 0.1 mM ABA in 0.02% Tween-20 at the
Above we showed that SAG21 responded very dif- indicated time rather than placed in the darkness. Control plants were
sprayed with surfactant 12 h before harvest. Control leaves 6 were
ferently to darkness in attached vs. detached leaves. just beginning to visibly yellow, while control leaves 8 were fully
Evidently, SEN1 shows a similar disparity in its ABA green. The ABA-responsive gene Cor15 was added as a positive
response. SAG2 and SAG14 responded only slightly to control.
464

ABA, in a manner consistent with the slight yellowing

that ABA induced.

Ethylene
Ethylene is generally known to induce senescence [30].
Our treatment consisted of leaving plants continuously
in an ethylene chamber for the indicated times. Under
these conditions ethylene treatment clearly accelerated
visible yellowing in leaf 6, which under control condi-
tions was just beginning to senesce. Leaf 8, which in the
control was fully green, responded visibly only slightly
to ethylene, if at all, and then only after prolonged treat-
ment (data not shown). Chlorophyll levels declined in
both leaves 6 and leaves 8, but more strongly in the
former (Figure 7).
The response of most of the genes tested correlated
with the visible yellowing: increasing incubation times
gave increasing SAG expression, and that increase was
strongest in the older leaves (Figure 7). Some of the
SAGs (SAGs 12, 17, and 18) showed no response at
all in younger leaves. Others (SAGs 13, 14, 20 and
ERD1) showed a clear but much reduced response in
younger leaves. SEN1 responded strongly to ethylene
in both younger and older leaves. SAG21 responded
more strongly in the younger leaves than in the older
ones, which is in contrast with the yellowing and the
behavior of the other SAGs. SAG12 gave a strong and
clear response only in the older leaves after ethylene
had caused visible yellowing.

Dehydration
This experiment was prompted in part by the fact that
two of the SAGs in our grouping were independently
cloned as dehydration-response genes (ERD1, also
published as SAG15 [22, 28], and RD21 [47]). A
constant induction/variable final age experiment (Fig-
ure 1A) was done to assess the dehydration response
of these and the other SAGs, in which leaves at a vari-
ety of developmental stages, from quite young to very
old, were subjected to a constant three hour dehydra- Figure 7. Expression of SAGs in plants treated with ethylene. This
tion. Plants were severely wilted after treatment, and experiment was carried out similarly to those in Figures 5 and 6,
no obvious yellowing had occurred (data not shown). except plants were incubated in Plexiglas chambers with ethylene at
1 ppm. Control plants were left in growth chambers. Control leaves
About half the SAGs (SAGs 14, 17, 20, 21 and 6 were just beginning to visibly yellow, while control leaves 8 were
ERD1) responded to this dehydration to a greater or fully green. SEN1 was previously reported as responding strongly
lesser degree (Figure 8). Some of the SAGs, in fact, to ethephon and served as a positive control.
exhibited a stronger response to it under these condi-
tions than others previously reported as dehydration-
inducible (e.g. SAG14, cloned by one group as a SAG12 was again not induced by this treatment, and
darkness-induced gene, responded more strongly than may even have been slightly repressed by it. RD21,
ERD1, cloned by another as a dehydration gene). cloned by others as a dehydration-response gene [47]
and by us as a SAG, also did not respond to dehydra-

Figure 8. Expression of SAGs in plants subjected to dehydration.
Over a period of 11 days plants were taken from a synchronously
growing population, cut at the base, and the severed rosettes placed
on filter paper to dry for 3 h. Total RNA was isolated from leaves 5
and probed with the indicated gene. ERD10 was used as a dehydra-
tion positive control. Most leaves had begun to senesce by day 31,
and were fully senescent by day 35. Note that this was a separate
experiment from that in Figure 2, and so the results are not directly
comparable.
tion under these conditions. This may be because our
treatment was harsher and more rapid than the method
used by Yamaguchi et al. [47] (see Materials and meth-
ods). Two different dehydration-responsive genes used
as controls, rd29a [48] (data not shown) and ERD10
[23], did respond strongly to this treatment.
465
Discussion
The genes examined here have been cloned as SAGs –
genes with mRNA levels that increase during natural
senescence. Three of them, however, were independ-
ently cloned as genes that respond to other factors, and
two are related to such genes, and this and similar cases
in the literature (e.g. [2, 9, 20, 41]) suggest an over-
lap between SAGs and stress and hormone-response
genes. One goal of this work was to evaluate the extent
of this overlap. To this end we have examined the
response of a number of SAGs to age, dehydration,
darkness, detachment, abscisic acid, cytokinin, and
ethylene.
Leaves at different developmental stages may
respond to identical treatments differently. It has been
shown, for example, that older leaves nearing senes-
cence can respond to at least one inducer of senes-
cence (ethylene) while younger leaves do not [18]. We
have attempted to control for such affects by beginning
with synchronously growing plants and pooling only
identical leaves (e.g. pooling all the fifth leaves separ-
ately from the others). If a given SAG responds in an
older leaf to a certain treatment, it is possible it may be
responding primarily to treatment-induced senescence,
rather than to the treatment itself. If response occurs in
a younger leaf, however, it is more likely to be direct.
The kinetics of SAG induction is similarly important
when trying to determine the true elicitor of a response.
A stress or hormone treatment is unlikely immediately
to induce senescence, and so genes responding quickly
are more likely to be responding directly to the treat-
ment, rather than indirectly to treatment-induced sen-
escence.
Most of the genes examined responded in a broadly
similar fashion to most of the treatments. In general the
treatments that induced the strongest visible yellowing
also induced the largest numbers of SAGs, which is
consistent with the simplistic notion that yellowing
is a good marker of senescence (although evidence
against this notion has been presented; e.g. see [36]).
The SAGs by no means behaved identically in the vari-
ous experiments, however. Figure 9 groups the SAGs
based on the results of most of these experiments, and
graphically displays their overlapping responses.
The treatment that was most effective in indu-
cing SAGs, in both younger and older leaves, was
incubating detached leaves in darkness. This treat-
ment also induced leaf yellowing and chlorophyll loss
that paralleled the SAG mRNA induction. Every SAG
examined except one (SAG21) was induced by this
466
treatment, and, of those that were, all but one (SAG12)
was induced in both older and younger leaves, though
generally more strongly in the older. This broad ability
to induce SAGs in both older and younger leaves was
unique among the treatments tested, and implies that
detached darkness is a very strong inducer of senes-
cence. The work of Becker and Apel, however, sug-
gests that it is an inducer of a good deal more, as
some genes associated with it are not in fact induced
during natural senescence [4]. The genes induced by
dark detachment-induced senescence might thus be a
superset of those induced by age-mediated senescence,
with all or most of the SAGs induced and other genes
besides.
Cytokinin, a hormone generally regarded as an
inhibitor of senescence, inhibited SAG expression in
these dark detachment experiments. This inhibition
was generally strongest in the younger leaves, where
it frequently inhibited the treatment-mediated SAG
increases entirely. Cytokinin was generally less effect-
ive, or not effective at all, in preventing SAG mRNA
induction in the older Arabidopsis leaves. One way to
view this is that cytokinin allowed presenescent leaves
to remain presenescent, but was much less effective in
halting senescence in leaves in which it had already
begun.
Detaching leaves and incubating them in the light
was also a general inducer of SAGs, although a weak-
er one than dark detachment. Unlike dark detachment,
it did not induce obvious visible yellowing, however,
although chlorophyll levels declined by the later time
points (Figure 7). One possible explanation for the dis-
parity between chlorophyll measurements and visible
yellowing is that in dark detachment chlorophyll was
actively broken down, yielding the patterned yellow-
ing characteristic of senescence, while in light detach-
ment a different process occurred, producing a uni-
formly pale (but not yellow) leaf. Light detachment
was also not very effective at inducing SAGs in young-
er leaves. However, nine of ten SAGs were signific-
antly induced in older leaves. Cytokinin was able to
repress this induction for most SAGs in both older
and younger leaves. In other words, under these more
weakly senescence-inducing conditions cytokinin was
able to make both older and younger leaves behave
like younger ones. Detachment may deprive the leaf
of its source of cytokinin, which could explain the
resultant induction of SAGs. Darkness might be a sep-
arate inducer of senescence, which acts cumulatively
with the drop in cytokinin levels caused by detachment
alone. External cytokinin would then be expected to
inhibit the detachment-induced SAG induction, but not
necessarily the darkness-induced increases.
Ethylene was a strong inducer of visible yellow-
ing and SAGs, although not as strong as dark detach-
ment. Visible yellowing was clearly induced in the
older leaves, as were almost all the SAGs. Visible
yellowing was induced to a much smaller degree in
younger leaves, however, and was paralleled by more
modest rises in only about half the SAGs. These results
are largely consistent with those of Grbic and Bleeck-
er, who have proposed that ethylene functions by con-
trolling when leaves developmentally primed to sen-
esce will do so, but has little effect on younger leaves
not so primed [18]. We show, however, that at least
some SAGs respond in younger leaves.
ABA spraying clearly induced visible yellowing in
the oldest leaves of the plant (in our case leaves 5 and
older, data not shown). In the oldest leaves used in the
experiments, however (leaves 6, most of which were
just beginning to yellow), visible response was only
slight. The youngest leaves, leaves 8, did not respond
visibly at all. This was qualitatively similar to the case
with ethylene, although ethylene had a much stronger
effect, and can be interpreted as indicating that ABA
functions to make leaves that are already senescing
do so faster. In the oldest leaves tested ABA induced
the expression of about half the SAGs. Because this
induction preceded significant yellowing, it may be
that these genes are involved in an early stage of the
senescence syndrome.
The results of the experiments in which whole
plants, as opposed to detached leaves, were put in
the dark are more varied still. This treatment did not
produce visible yellowing, although leaves did become
pale and chlorophyll levels declined. This discrepancy
between visible yellowing and chlorophyll loss is sim-
ilar to that which occurred during light detachment.
About half the SAGs were induced, particularly in
older leaves. SAGs 13 and 17, however, were signi-
ficantly down-regulated by the treatment (and both in
contrary fashion were up-regulated in detached leaves
subjected to darkness). This suggests that the biology
of dark-incubated attached and detached leaves is dif-
ferent, and that dark incubation of whole plants may not
be a good model of senescence. As was the case with
the other treatments, most genes responded poorly or
not at all in the younger leaves. Three genes, however,
were an exception to that (SAG14, SAG21, and SEN1).
Those genes may be responding directly to darkness,
rather than indirectly to any darkness-induced senes-
 467

Figure 9. A graphical depiction of the overlap between age-mediated and stress and hormone-mediated gene expression, in older (senescent,
panel A) and younger (non-senescent, panel B) leaves. The central circle represents age-mediated gene induction, and SAGs with basal levels
of expression in younger leaves are drawn within it in panel B. The triangles which intersect the circle represent the various treatment-mediated
gene inductions. The size of the triangles roughly correlates with the number of SAGs induced by the corresponding treatment. Genes are listed
as induced when that induction seems significant. Note how few genes are specific to age-mediated induction, and the different patterns that
emerge when looking at induction in older and younger leaves.

cence (see also [24] for a discussion of attached dark-
ness versus natural senescence).
About half the SAGs surveyed responded positively
to the relatively quick and severe dehydration inflicted
on them. This response did not follow the pattern estab-
lished in most of the other experiments, however, in
that it was almost always strongest in the youngest
leaves, which would ordinarily be expressing the least
amounts of the message. This, and the fact that the
response occurred in just three hours, suggests that it
was direct. Four of the five genes that were induced
by drought also were induced by darkness in attached
leaves (the fifth, SAG17, was repressed). This might
suggest that they function in a relatively broad stress
response, defending the cell from stresses that may be
common to darkness and drought and senescence.
Certain individual SAGs behaved uniquely relative
to the others. SEN1, cloned by homology to a dark-
induced gene [36], was unusual in many ways. It was
induced by dark detachment more strongly in younger
leaves than in older ones, and was not significantly
repressed under those circumstances by cytokinin, as
were most of the other SAGs. It was however repressed
by cytokinin in detached leaves left in the light. It was
also induced very strongly in both young and old dark-
incubated attached leaves. These results indicate that
the dark-induced component of its response pattern is
very strong. Its ethylene response differed from most
of the other SAGs as well, in that it responded strongly
in both younger and older leaves. Oh et al. describe
many of these responses [36]. Here we show that they
are not typical of the SAGs we examined. These results
might suggest that the role of SEN1 in the leaf is wider
than senescence.
SAG21 is also notable. In untreated plants its
abundance peaks at or shortly before visible senes-
cence begins, and declines thereafter, which in and of
itself would be consistent with a postulated class of
senescence regulatory genes [38]. Its other responses,
however, do not suggest a primary involvement in
senescence. It was the only gene tested that was not
induced by dark detachment, despite the fact that it
was induced strongly in both dark-incubated attached
leaves and light-incubated detached leaves. Its ethylene
response was also very unusual in that younger leaves
clearly responded more strongly than older ones. As
468
mentioned earlier, closely related genes have recently
been isolated that respond to one or more of a vari-
ety of stresses and hormones [12, 16, 33, 40, 49].
SAG21 therefore appears to belong to a gene family
with individual members that respond to a wide vari-
ety of sometimes overlapping factors, presumably per-
forming a similar biochemical function in each. The
senescence response of these other members has not
been reported.
SAG13 is notable for its lack of basal expression in
untreated younger leaves, which is unusual both among
the SAGs examined here and those reported elsewhere,
and tends to suggest a senescence-specific function. It
is induced strongly shortly before visible yellowing,
when the first molecular events of the senescence syn-
drome are likely to occur, and may be a good molecular
marker of early senescence. SAG13 responds rapidly
to ABA, however, in both older and younger leaves,
and this apparently direct response suggests that its
gene product may also play a role in processes other
than senescence. The negative response of SAG13 to
attached darkness was unusual.
SAG12 emerges from these experiments as the gene
which comes closest to specificity for natural senes-
cence. It has no detectable expression in young leaves
and is only induced to an appreciable degree in older
leaves after they have become about 20% yellow. It is
not induced significantly by any treatment that does not
also cause visible yellowing, with the possible excep-
tion of detachment in light (although in the case of
ABA it was induced in only slightly yellow leaves).
SAG12 appears to us to be the best molecular mark-
er of senescence identified to date. The fact that its
promoter, when fused to a gene that drives cytokinin
production and put into tobacco, gives otherwise nor-
mal plants that exhibit delayed leaf senescence also
indicates highly specific expression [13]. One possible
explanation for this high degree of specificity is that
the gene product of the highly expressed SAG12, a
cysteine protease, may be deleterious to the cell, and
so may need to be restricted to late senescence.
The SAGs examined here show a variety of expres-
sion patterns in response to factors other than senes-
cence. It is hoped that these expression patterns, and
those of other SAGs not yet examined, will provide
clues to both the roles of the individual SAGs and
the relationship between senescence and the various
factors that affect it.
Acknowledgments
We thank Allison Wilson and Tony Bleecker for
help with ethylene treatments and for the SAG2
clone. Tomohiro Kiyosue and Kazuo Shinozaki kindly
provided the ERD10 and rd29a clones, and Sarah
Gilmour and Michael Thomashow the Cor15 clone.
This work was supported through the Consorti-
um for Plant Biotechnology Research (DE-FG02-
97ER20280), Northrup King and the Graduate School
of the University of Wisconsin. L.M.W. and B.Q. were
partially supported by a Cell and Molecular Biology
training grant.
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