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A Comparison of Leaf and Petal Senescence in Wallflower
Reveals Common and Distinct Patterns of Gene
Expression and Physiology1[W]
Anna Marie Price2, Danilo F. Aros Orellana, Faezah Mohd Salleh, Ryan Stevens, Rosemary Acock,
Vicky Buchanan-Wollaston, Anthony D. Stead, and Hilary J. Rogers*
Cardiff School of Biosciences, Cardiff CF10 3TL, United Kingdom (A.M.P., D.F.A.O., F.M.S., R.S., R.A., H.J.R.);
Warwick HRI, University of Warwick, Wellesbourne, Warwick, Warwickshire CV35 9EF, United Kingdom
(V.B.-W.); and School of Biological Sciences, Royal Holloway, University of London Egham, Surrey TW20 0EX,
United Kingdom (A.D.S.)
Petals and leaves share common evolutionary origins but perform very different functions. However, few studies have
compared leaf and petal senescence within the same species. Wallflower (Erysimum linifolium), an ornamental species closely
related to Arabidopsis (Arabidopsis thaliana), provide a good species in which to study these processes. Physiological
parameters were used to define stages of development and senescence in leaves and petals and to align these stages in the two
organs. Treatment with silver thiosulfate confirmed that petal senescence in wallflower is ethylene dependent, and treatment
with exogenous cytokinin and 6-methyl purine, an inhibitor of cytokinin oxidase, suggests a role for cytokinins in this process.
Subtractive libraries were created, enriched for wallflower genes whose expression is up-regulated during leaf or petal
senescence, and used to create a microarray, together with 91 senescence-related Arabidopsis probes. Several microarray
hybridization classes were observed demonstrating similarities and differences in gene expression profiles of these two organs.
Putative functions were ascribed to 170 sequenced DNA fragments from the libraries. Notable similarities between leaf and
petal senescence include a large proportion of remobilization-related genes, such as the cysteine protease gene SENESCENCEASSOCIATED GENE12 that was up-regulated in both tissues with age. Interesting differences included the up-regulation of
chitinase and glutathione S-transferase genes in senescing petals while their expression remained constant or fell with age in
leaves. Semiquantitative reverse transcription-polymerase chain reaction of selected genes from the suppression subtractive
hybridization libraries revealed more complex patterns of expression compared with the array data.
Both leaves and flowers have a finite life span, and
since it is thought that all floral organs, including
petals, evolved from leaves (Friedman et al., 2004), we
might expect commonality in their senescence mechanisms. Both in leaves and petals, a key feature of
senescence is remobilization of resources; in both organs, this has been demonstrated experimentally using
isotope labeling (Nichols and Ho, 1975; Mae et al., 1985;
Bieleski, 1995) or pigment transport (Erdelská and
Ovečka, 2004). This is reflected in some of the major
classes of genes whose expression is up-regulated in
1
This work was supported by grants from the Biotechnology and
Biological Sciences Research Council (to A.M.P.), the Chilean Government, Ministry of Agriculture (to D.F.A.O.), and the Malaysian
Government (to F.M.S.).
2
Present address: Centre for Molecular Oncology, Institute of
Cancer, Barts, and the London School of Medicine and Dentistry,
John Vane Science Centre, Charterhouse Square, London EC1M
6BQ, UK.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Hilary J. Rogers ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.120402
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both these tissues during senescence. These include
genes encoding proteases, nucleases, and enzymes
involved in lipid and carbohydrate metabolism
(Buchanan-Wollaston, 1997; Wagstaff et al., 2002). In
both organs, remobilization requires a carefully orchestrated dismantling of the cellular machinery to
avoid cell death until remobilization is complete. In
leaves, senescence-associated genes (SAGs) have been
classified into two expression types: those exclusively
expressed during senescence (class I) and those whose
expression increases during senescence from a basal
level (class II; Gan and Amasino, 1997). However,
within these classes, there are diverse expression
patterns (Smart, 1994; Buchanan-Wollaston, 1997),
indicating different regulatory pathways. Levels of
reactive oxygen species (ROS) rise in both petals and
leaves during senescence (Borochov and Woodson,
1989; Merzlyak and Hendry, 1994), maybe as a result of
macromolecule degradation. This is accompanied by
up-regulation of genes involved in protection against
ROS, such as catalase in leaves (Buchanan-Wollaston and
Ainsworth, 1997; Zimmermann et al., 2006) and superoxide dismutase in petals (Panavas and Rubinstein,
1998).
The roles of petals and leaves are very different, as
are their development and the signaling mechanisms
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A Comparison of Leaf and Petal Senescence in Wallflower
that trigger their senescence. An early step in petal
development is the conversion of chloroplasts to chromoplasts (Thomson and Whatley, 1980), and this has
been compared with the transformation of chloroplasts into gerontoplasts that occurs during leaf senescence (Thomas et al., 2003), implying similarities
between developing (nonsenescent) petals and senescent leaves. This would suggest that senescenceassociated events in petals may occur at an earlier
stage compared with leaves and that cellular degradation accompanied by the expression of some genes
that are highly up-regulated in senescent petals is
already evident while the petals are in the early stages
of development (Wagstaff et al., 2002, 2003). The
primary function of petals is to attract pollinators, so
they are frequently highly pigmented and scented and
a sink rather than a source of photosynthates. Floral
life span is closely linked to pollination in some
species, which triggers rapid floral deterioration
(Stead and van Doorn, 1994). However, even in the
absence of pollination, floral life-span is finite. Although a few environmental factors such as temperature and drought can affect floral longevity, senescence
is irreversible in the majority of species and there is
tight species-specific control over the maximum duration of a flower (Primack, 1985). In contrast, leaves are
sources of photosynthate for most of their life span,
and their longevity is strongly influenced by nutrient
status, light, and other environmental factors. Fertilization does accelerate leaf senescence in some species
(Hayati et al., 1995) but not in others, such as Arabidopsis (Arabidopsis thaliana; Hensel et al., 1993). However, as in petals, expression of some genes associated
with leaf senescence is also detected before visible
signs of deterioration (Buchanan-Wollaston, 1997), indicating that in both petals and leaves senescence
processes may be initiated early.
Two classes of plant growth regulators, ethylene and
cytokinins, are definitely involved in both petal and
leaf senescence in some species. The sensitivity of petal
senescence to endogenously produced, or exogenously applied, ethylene is species specific, and species can be broadly divided into those in which petal
senescence is ethylene sensitive and those in which it
is not (Rogers, 2006). In carnation (Dianthus caryophyllus), an ethylene-sensitive species, ethylene production and ethylene biosynthetic genes are both
up-regulated in petals late during the vase life of the
flower (ten Have and Woltering 1997). In leaves, as in
flowers, ethylene sensitivity is related to the age of the
organ; however, in general, the role of ethylene in leaf
senescence is less central (Grbić and Bleecker, 1995).
Up-regulation of cytokinins delays both leaf (Gan and
Amasino, 1995) and petal (Chang et al., 2003) senescence, and it has been suggested that a fall in cytokinins may trigger an increase in ethylene sensitivity
during petal senescence in unpollinated ethylenesensitive species (van Doorn and Woltering, 2008).
Other plant growth regulators are probably also involved, but the signaling pathways and their cross talk
are poorly understood. Transcriptional regulation of
senescence in both leaves and petals is also complex
and as yet not fully understood. Transcription factors
that are up-regulated during leaf senescence, such as
WRKY53 (Hinderhofer and Zentgraf, 2001) and many
others, have been identified (Buchanan Wollaston
et al., 2005), but as yet their interactions have not
been fully elucidated. Similarly, transcription factors
up-regulated during petal senescence have been identified in several genera (Alstroemeria [Breeze et al.,
2004] and Iris [van Doorn et al., 2003]) but not fully
characterized.
Global transcriptomic and EST analyses have probed
senescence independently in leaves in Arabidopsis
(Gepstein et al., 2003; Buchanan-Wollaston et al., 2005)
and petals (in Alstroemeria [Breeze et al., 2004], Iris [van
Doorn et al., 2003], and Rosa [Channeliere et al., 2002]);
however, to date there is a lack of comparisons of leaf
and petal senescence transcriptomes in the same species. Wallflower (Erysimum linifolium) is a useful ornamental species in which to compare leaf and petal
senescence. It is closely related taxonomically to Arabidopsis (Stevens, 2001) but has larger pigmented flowers whose development and senescence are easily
staged. Thus, in the study presented here, the objectives were (1) to use microarray analysis of subtractive
libraries from wallflower leaves and petals to compare
the global gene expression changes occurring during
senescence in these two tissues and relate these to
changes in the physiology of the two organs during
senescence and (2) to take advantage of the close
taxonomic relationship between wallflower and Arabidopsis to compare and contrast expression patterns
between the two species in the two tissues and reveal
species-specific or tissue-specific differences in the
senescence program.
RESULTS
Physiology of Leaf and Petal Senescence in Wallflower
One flower on the wallflower raceme opened each
day, taking 7 d to complete its development from bud
opening to full abscission of the calyx, corolla, and
androecium (Fig. 1). Thus, eight stages of development
were assigned based on number of days after opening.
Stage 0 was defined as the lowest unopened bud;
additional early bud stages were designated stages
21 and 22. No difference in morphology or in rate of
development was noted for the flowers at different
times of year. Stage 4 was the stage at which the first
signs of visible petal deterioration became evident.
Leaves could be characterized within one whorl and
were assigned to seven developmental groups based
on relative size and chlorophyll content (Fig. 2). At
stage 5, leaves showed the first signs of yellowing,
indicating senescence, and this corresponded with a
20% reduction in chlorophyll levels. Dry weight-fresh
weight ratio and total protein content were also deter-
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Price et al.
Figure 1. Stages of wallflower flower development. Stage 22 and stage 21, Two sequential buds below the lowest unopened
bud on the raceme. Sepals completely cover petals. Stage 0, Lowest unopened bud on raceme. Petals are dark purple in color,
tightly curled within sepals. Stage 1, Flower fully opened. Petals are pale purple, with sepals folded back midway along their
length. Stigma is yellow and fuzzy in appearance, four of six anthers are visible, all undehisced, positioned close to the stigma
with the tips curled over the stigma. Stage 2, As stage 1, but petals are darker in color. All six anthers are visible, two newly
emerged anthers are dehisced and curled back from the stigma. Stage 3, The flower is not as tightly held together as previously.
Petals are wilting slightly and darker again in color. Fuzz on stigma is not as fine as previously. All six anthers are dehisced and
curled back from the stigma. Stage 4, The flower is loosely held together. Petals are limp and curled over at the tips. Flower
appearance has deteriorated. Stage 5, As stage 4, but more extreme. Petals are wilted, stigma is discolored with dark purple areas.
Stage 6, Sepals, petals, and stamens are beginning to abscise. Remaining petals look withered and dry. Stage 7, All sepals, petals,
and stamens are abscised; only the stigma remains. Bar 5 10 mm.
mined for each developmental stage of petals and
leaves (Figs. 3 and 4). There was a sharp reduction in
dry weight-fresh weight ratio between petal stages 0
and 1, coinciding with flower opening, followed by a
rise starting from stage 4 as petals lost turgor. Protein
loss started after stage 3, coincident with the first signs
of petal deterioration. In leaves, the dry weight-fresh
weight ratio started to rise after stage 5, while protein
loss started after stage 4, again preceding the start of
visual signs of senescence.
Due to the close taxonomic relationship between
wallflower and Arabidopsis, it seemed likely that
ethylene would be an important regulator of petal
senescence in this species too. In ethylene-sensitive
species, treatment with a pulse of an ethylene inhibitor
such as silver thiosulfate (STS) delays flower senes-
cence (Serek et al., 1995). In wallflower, detached
flowers harvested at stage 1 and held in water senesced over the same period as attached flowers, with
full abscission on day 7. However, when pulsed for 1 h
with STS on the day of harvest, abscission was delayed
by 2 d. STS-pulsed flowers also senesced more slowly,
taking 4 d to progress from stage 3 to stage 5, instead of
2 d when held in water. Given that in ethylenesensitive species, such as carnation, cytokinins are
also implicated in petal senescence (Taverner et al.,
2000), the role of cytokinins in wallflower was tested.
Treatment with either 0.1 or 1.0 mM kinetin or with
0.1 mM 6-methyl purine (an inhibitor of cytokinin
oxidase) delayed senescence and abscission of flowers harvested at stage 1 by 2 d (Supplemental Figs. S1
and S2).
Figure 2. Stages of wallflower leaf development. Stage 1, Very young leaves, less than 50% expanded. Stage 2, Very young
leaves, 50% to 75% expanded. Stage 3, Young leaves, 75% to 100% expanded. Stage 4, Mature green leaves. Stage 5, Older
mature leaves, green with signs of yellowing on the tip. Stage 6, Old leaves, up to 50% of leaf area yellow. Stage 7, Very old
leaves, mostly or all yellow. Below each image is the total chlorophyll for that leaf stage expressed as a percentage of maximum.
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A Comparison of Leaf and Petal Senescence in Wallflower
Figure 3. Fresh weight (FW), dry weight (DW), and ratio of dry weight
to fresh weight during petal (A) and leaf (B) development and senescence. Dry weight was determined by drying 20 to 100 petals or leaves
at 60°C for 5 d. Error bars represent 6 SE (n 5 3).
Construction of Wallflower Petal and Leaf Subtracted
Libraries and Screening by Microarray Analysis
Based on the physiological characterization of leaf
and petal senescence, subtracted libraries were constructed for use in transcriptomic analysis to identify
genes whose expression is up-regulated during the
senescence of these two organs. For this purpose,
petals from stages 22, 21, and 0 (early to mature
buds) were combined to represent young petals, and
petals from stages 3, 4, and 5 (early to late visible signs
of petal wilting) were combined to represent old
petals. Leaf stage 3 (75%–100% expansion, 80% chlorophyll) was used to represent young leaves that had
not yet reached their full photosynthetic capability,
and stages 5 and 6 (early to later stages of leaf yellowing, in which chlorophyll levels had fallen to 81% and
44% of maximum, respectively) were combined to
represent old leaves. A total of 1,018 and 614 clones for
leaves and petals, respectively, were obtained from the
subtraction. PCR-amplified inserts from all 1,632
clones from the subtracted libraries were used to
generate a cDNA microarray, and 431 probes showed
a consistent expression pattern with both pairs of
labeled RNA when analyzed using GeneSpring software. The results from the microarray analysis are
summarized in Supplemental Table S1. Two fragments
representing known genes WLS63 and WPC11A were
spotted in three replicate dilutions (36 data points) and
showed very similar changes in expression with low
variability between replicates (for WLS63, leaves down,
1.1 6 0.2 [values are mean fold 6 SE], petals up, 3.8 6
0.4; for WPC11A, leaves up, 10 6 1.0, petals up, 136 6
27), indicating the reproducibility of the array results.
Six of the possible nine classes of expression (i.e. upregulated in both old petals and old leaves compared
with the young tissue, up-regulated in petals but
unchanging in leaves, up-regulated in petals but
down-regulated in leaves, etc.) were represented in
the microarrays (Table I). Of the 427 probes (excluding
the replicates described above), expression of 305
probes was up-regulated reproducibly in old petals
compared with young petals. Of these, the expression
of 232 probes was up-regulated in both old organs,
while the expression of 61 probes was up-regulated in
old petals but remained stable in leaves, and the
expression of a further 12 probes was up-regulated
in petals with age but was down-regulated in old
leaves. As expected from the enrichment of the genes
by suppression subtractive hybridization (SSH), the
majority of probes on the array indicated up-regulated
expression with senescence in the tissue from which
they were derived, confirming that the subtraction of
the SSH libraries was effective. Of 164 probes from the
petal cDNA library, whose expression could be reliably determined in both tissues, the expression of 98%
was up-regulated with age in petals. For 263 probes
derived from the leaf cDNA library, 52% showed upregulated expression with age in leaves, although
larger numbers of leaf-derived probes on the array
were stable in expression with leaf senescence (47%;
Supplemental Table S1).
Sequence Analysis of Wallflower Genes from the
SSH Libraries
Following microarray analysis, fragments representing selected probes on the array were chosen for
Figure 4. Total protein content of petals (A) and leaves (B). Error bars
represent 6 SE (n 5 15).
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Price et al.
Table I. Expression classes from microarray analysis
Petal
Petal
Petal
Unchanged Down-Regulated Up-Regulated
Leaf unchanged
Leaf down-regulated
Leaf up-regulated
103
1
18
0
0
0
61
12
232
sequencing to represent the different classes of gene
expression presented in Table I. In addition, a random
selection of clones from the SSH libraries were also
sequenced. Once poor and short sequences had been
removed, 210 ESTs were obtained (GenBank accession
numbers are listed in Supplemental Table S1) and 127
of the sequences clustered into 27 contigs (WC1–
WC27), with the largest contig containing 28 sequences (Table II). The remaining 83 sequences were
singletons (i.e. represented only once). Thus, the redundancy (number of sequences clustered divided by
the total number of sequences; Breeze et al., 2004) of
the EST collection was 60% (which means that the
chance of finding the same sequence again in any new
clones sequenced is 60%). However, there may be
further redundancy due to nonoverlapping fragments
of the same gene. Genes within contigs were given
codes: WLC (Wallflower Leaf Contig) and WPC (Wallflower Petal Contig), and singletons were denoted
WLS and WPS. Contigs are hereafter referred to in the
form WC1 (Supplemental Table S1). Putative gene
functions were assigned based on a BLAST search, and
in most cases the closest match was to Arabidopsis genes,
as wallflower is of the same subfamily (Brassicoideae;
Stevens, 2001). In total, 193 wallflower sequences
could be assigned to a closely matching Arabidopsis
gene, and 73 Arabidopsis genes were identified as the
closest match. Analysis of gene functions revealed that
three contigs (WC4, WC5, and WC26, comprising
altogether 26 sequences) and four further sequences
that did not overlap the contigs, amounting to 14% of
the sequences, matched SAG12. Three contigs (WC1,
WC2, and WC16, comprising 25 sequences) matched
nonoverlapping regions of the same chitinase gene
(At2g43570), and a fourth contig (WC3 of three sequences)
matched most closely a different chitinase gene
(At4g19810). Thus, 13% of the sequences represented
chitinase-like genes. A further 7% of the sequences
matched glutathione S-transferases (GSTs), 7% matched
metallothioneins, and 4% matched a lipid transfer
protein.
Representation of the Functional Categories in the
Different Expression Classes
There was a striking difference in the representation
of putative functional categories between the different
gene expression classes on the microarray (Fig. 5).
Sequences were obtained for 75% of the probes on the
microarray whose expression was up-regulated in
senescent petals and was either unchanged or downregulated in senescent leaves. Over one-third of these
sequences (40%) were related to chitinases. The majority of these chitinase-related sequences (20) were
most closely related to an Arabidopsis class IV chitinase (At2g43570; contigs WC1 and WC2), while one
was more closely related to an Arabidopsis family 18
glycosyl hydrolase (At4g19810; contig WC3); both Arabidopsis genes are putatively involved in cell wall metabolism.
A further 23% of the sequences from this array
expression class (up-regulated in old petals, either
unchanged or down-regulated in senescent leaves)
showed homology to GSTs. All of the 10 sequenced
probes that were up-regulated in senescent petals but
down-regulated in senescent leaves showed closest
homology to the f class of GSTs (Wagner et al., 2002),
and nine were assigned to one contig (WC10). All the
WC10 sequences were closest to AtGSTF3, while the
singleton sequence was closer to AtGSTF7. However,
as all of the clones were partial, it is difficult to assign
the sequences unambiguously to an Arabidopsis homolog, as a key diagnostic triplet of amino acids at
positions 66 to 68 relative to AtGSTF2 (Wagner et al.,
2002) was not included in the wallflower clones and, in
addition, AtGSTF3 and AtGSTF2 were 95% identical.
All of the GST-related probes in this expression class
showed similar expression patterns on the microarray
(leaf, 0.35 6 0.02 [values are mean fold 6 SE]; petal,
15.3 6 1.69). The expression of two further probes on
the microarray whose sequence showed homology to
GSTs was up-regulated in senescent petals but was
stable in senescent leaves (leaf, 1.5 6 0.23; petal, 4.3 6
0.09). These sequences formed a separate contig
(WC21) showing closest homology to AtGSTZ1. The
remainder of the sequenced probes on the microarray,
for which putative functions could be ascribed and
whose expression was up-regulated in senescent
petals but stable in senescent leaves, represented
metal-binding proteins (one probe), proteins associated with ROS/stress (five probes) or signaling (five
probes), proteins involved in remobilization/metabolism (three probes), and one gene involved in mRNA
stability. The metal-binding protein was a putative
copper chaperone most closely homologous to CCH/
ATX1 that is thought to play a role in remobilization of
copper from metalloprotein degradation (Himelblau
and Amasino, 2000) and was 4-fold up-regulated in
petals. ROS/stress-related proteins include a PR5-like
protein (petals, up-regulated by 6.3-fold), a cytosolic
thioredoxin (petals, up-regulated by 3.0-fold), SAG21
(petals, up-regulated by 5.7-fold), and a cytochrome
P450 family protein (petals, up-regulated by 23.3-fold).
Signaling proteins include a rhodopsin-like receptor
(petals, up-regulated by 2.9-fold), a Rab acceptor
(petals, up-regulated by 7.5-fold), and a Rab subfamily
GTPase (petals, up-regulated by 2.3-fold). Finding
genes encoding proteins involved in remobilization
is not surprising, although genes whose role may be
specific to remobilization in petals and not leaves may
be significant in defining the difference between remobilization in the two organs. The three up-regulated
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A Comparison of Leaf and Petal Senescence in Wallflower
Table II. Most abundant sequenced transcripts from the SSH libraries
Arabidopsis
Genome Initiative
Code for Closest
Arabidopsis Match
Wallflower
Contig
No. of
Clones
Putative Function/Closest
Arabidopsis Homolog
Functional
Class
At2g43570
At5g45890
At1g07600
At2g02930
At3g22600
At2g43570
At1g32450
At3g22600
At5g01600
At2g23790
At2g45570
At1g73260
–
At4g19810
At1g11190
At4g02520
At5g02040
At5g01220
At1g05560
At2g02390
At5g40690
At2g45220
At5g45890
–
At2g43570
At1g07600
At5g45890
WC1
WC4
WC24
WC10
WC6
WC2
WC11
WC7
WC17
WC8
WC14
WC15
WC18
WC3
WC9
WC10
WC13
WC19
WC20
WC21
WC22
WC23
WC5
WC12
WC16
WC25
WC26
28
26
13
10
9
9
5
5
4
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Chitinase class IV
SAG12
Metallothionein
ATGSTF3
Lipid transfer protein
Chitinase class IV
Peptide transporter
Lipid transfer protein
Ferretin
Unknown protein
Cytochrome P450
Endopeptidase inhibitor
No hits
Chitinase
Bifunctional nuclease
ATGSTF2
Rab acceptor
Unknown protein
UDP glycosyl transferase
ATGSTZ1
Unknown protein
Pectin esterase inhibitor
SAG12
No hits
Chitinase class IV
Metallothionein
SAG12
Stress/defense
Remobilization
Metal binding
Defense
Remobilization
Stress/defense
Remobilization
Remobilization
Metal binding
Unknown
ROS/stress
Remobilization
–
Stress/defense
Remobilization
Defense
Signaling
Unknown
ROS/stress
Defense
Unknown
Remobilization
Remobilization
–
Stress/defense
Metal binding
Remobilization
genes identified here were a lipid transfer protein
(leaves, 1.59-fold; petals, 5.14-fold), a thiol protease
(leaves, 1.41-fold; petals, 2.77-fold), and a AAA-type
ATPase family protein (leaves, 1.51-fold; petals, 4.95fold). Only one gene involved in the regulation of gene
expression and up-regulated only in petal senescence
was identified, and it showed homology to CCR4related proteins (WLS63, three replicates on the array;
leaf, 0.88 6 0.15; petal, 3.8 6 0.43). CCR4-NOT proteins
in yeast are involved in the regulation of gene expression via mRNA stability (Chen et al., 2002).
In contrast, of those probes that were up-regulated
in both senescent leaves and petals and for which
meaningful sequence was obtained, the highest proportion (23%) was represented by SAG12, while chitinase genes represented only 5% and no GST genes
were up-regulated in both tissues (Fig. 5). The expression of all of the SAG12 probes was reliably determined from the microarray, and all were up-regulated
in both leaves and petals, although more strongly in
petals (leaves, 95 6 17; petals, 216 6 35). A lower
proportion of the sequences in this expression class,
compared with those that were only up-regulated in
petals with age, related to signaling and included three
genes with putative functions in auxin responses, one
in cytokinin responses, and one in ethylene synthesis.
The expression of all of these genes with putative roles
in signaling was more highly up-regulated with age in
petals than in leaves. Three sequences were homologous to transcriptional regulators, and the expression
of these genes was also more highly up-regulated in
aging petals compared with leaves: a WRKY75 transcription factor (At5g13080) and two members of the
plant-specific NAC family of transcription factors
(At2g33480 and At5g64530).
The expression of only a few probes (18) was upregulated in senescent leaves while remaining unchanged in petals. Sequences were obtained for seven
of these: four were putative ferretin genes (leaf, 3.6 6
1.7), while the rest were of unknown function (Supplemental Table S1).
Semiquantitative Reverse Transcription-PCR of
Selected Wallflower Genes
Genes were selected for semiquantitative reverse
transcription (RT)-PCR based on their putative function and results from the microarray experiments, to
confirm the validity of the arrays and also to determine
more precise timing of expression for selected genes of
interest. SAG12 was selected as it represented a high proportion of probes whose expression was up-regulated
in both old leaves and petals (Supplemental Table S1).
Semiquantitative RT-PCR (Fig. 6) showed that the
expression of SAG12 remained low in leaves until
stage 6, at which point chlorophyll levels were re-
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complex pattern than was evident from the arrays in
which pooled tissue stages were used (Fig. 6). Thus,
although WLS63 expression was low in stage 3 leaves,
selected to represent young tissue, and remained low
as leaves aged, the highest levels of expression were
early in leaf development at stage 1. In petals, expression reached a maximum before the final stages of
petal senescence, at stage 4. WC11 expression fluctuated during leaf age, being already high in young
leaves and reaching a peak at stage 4, when fresh
weight, dry weight, protein levels, and chlorophyll
levels were maximal, falling thereafter. Its expression
in petals was very low in young buds (stages 22 and
21) but increased already to 60% of maximum from
stage 0, when the buds were not yet open. It reached a
maximum expression level at stage 1 (young open
flowers) and remained high until it dropped slightly in
late senescence (stage 5).
Use of Arabidopsis Gene Probes in Cross-Species
Microarray Analysis and Comparisons with
Arabidopsis Gene Chip Data
Figure 5. Putative functional classes of wallflower genes from different microarray expression classes. Comparison of putative functional
classes of genes represented in two expression categories from the
microarray analysis. A, Up-regulated in senescent petal but either
stably expressed or down-regulated in senescent leaf. B, Up-regulated
in both senescent petal and senescent leaf. Functional classes were
derived from Gene Ontogeny annotations and from putative functions
based on sequence homology.
duced to 44% of maximum and protein levels to 35% of
maximum. Expression then increased significantly in
stage 6 leaves, reaching a maximum at the oldest stage
used in the RT-PCR, stage 7. At this stage, both the
protein and chlorophyll levels had decreased to less
than one-quarter of their maximum. In petals, however, although SAG12 expression was very low in buds
and young open flowers, it was already substantially
up-regulated in mature, stage 2 flowers, at which time
protein levels, fresh weight, and dry weight were at or
close to their maximum. Thereafter, SAG12 levels in
petals fell until by stage 5 they were less than 20% of
the maximum value.
Two additional genes were selected: first, the CCR4like protein (WLS63), and second, a gene with a
putative role in remobilization, a peptide transporter
(WC11). On the array, expression of WLS63 was upregulated only in petals with age, while the expression
of WC11 was up-regulated in both, although to a much
greater extent in petals. In both cases, the expression
pattern from semiquantitative RT-PCR was consistent
with the array data, but a better resolution was
obtained from the RT-PCR due to the larger number
of separate tissue stages used. This revealed a more
In addition to the wallflower probes, 91 Arabidopsis
probes were also printed onto the arrays. Many of
these Arabidopsis sequences were selected as genes
whose expression was already known to change with
leaf senescence in Arabidopsis. Expression patterns of
52 of these genes in wallflower petals and leaves were
reliably detected on the arrays for both tissues. Gene
expression patterns in Arabidopsis mature green
leaves (MG, analogous to wallflower stage 4 leaves)
and two stages of leaf senescence (S1, between stage 4
and stage 5, and S2, between stage 5 and stage 6 of wall-
Figure 6. RT-PCR of selected genes from the SSH libraries. Semiquantitative RT-PCR over petal (A) and leaf (B), young (Y) and old (O) stages
as defined in the text, expressed as percentage of maximum value 6 SE
(n $ 3) for SAG12, WLS63, and WC11. Note that data for WLS63 and
WC11 expression levels for stage 7 leaves were not determined.
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A Comparison of Leaf and Petal Senescence in Wallflower
flower leaf senescence) of 10 of these genes were verified
by northern analysis (Fig. 7), showing a range of expression patterns. Data on the expression of all of the Arabidopsis genes was also obtained from AtGenExpress
(Supplemental Table S2). Leaf and petal stages were
chosen to resemble most closely the stages used for the
wallflower SSH and arrays. All four data sets for
young/old leaves and petals were obtained from the
Weigel laboratory experiments (Schmid et al., 2005).
Senescing leaves (from 6-week-old Arabidopsis plants),
corresponding to stage 5 to 6 wallflower leaves, were
compared with leaf 8 (from 4-week-old Arabidopsis
plants), which is not fully expanded and thus resembles wallflower leaf stage 3. For petals, petal stage 15
(Smyth et al., 1990), which equates to stage 3 wallflower petals, was compared with stage 12 petals
(unopened bud, nondehisced), comparable to stage 0
in wallflower. Comparing the northern expression
data with the Weigel laboratory array data indicated
that the senescing leaf material used in the arrays
included leaves at the same stage as the S2 of the
northern blots, since in Arabidopsis, SAG12 expression was only detected late in senescence. Expression
patterns of nine of the 10 genes for which northern
data are presented here were detected on the Affymetrix Weigel arrays, and eight of them showed increased
gene expression in senescent leaves by both methods.
In the case of LSC141 (At5g49360), Affymetrix array
expression was strong in both leaf stages (means, stage
8, 513; senescing, 799); however, the increase in expression (leaf, 1.6-fold) was below a 2-fold threshold.
For 47 Arabidopsis genes, data were available from
both the Affymetrix Arabidopsis arrays and the wallflower arrays. Of these, 81% (38 genes) showed the
same pattern of expression in at least one tissue in both
species and 38% (18 genes) showed the same pattern in
both tissues in both species. The expression of five
genes, 1-aminocyclopropane-1-carboxylic acid (ACC)
oxidase, catalase, blue copper-binding protein, SAG21,
and an unknown protein that is strongly induced by
brassinolide (At2g38640), was unchanged in leaves
but up-regulated in senescent petals of both species.
Ferretin was up-regulated in senescing leaf but not
petal tissue of both species, while six genes, histone
H1-3, a hydrolase, a Cys protease, an RNase, SAG12,
and xyloglucan transferase, were up-regulated in both
petals and leaves of both species. The remainder were
unchanged with age in both tissues of both species.
Ten genes were up-regulated in both petals and leaves
with age in Arabidopsis but only in petals in wallflower.
These were cytochrome P450, copper homeostasis
factor, POP dikinase, NADPH-dependent oxidoreductase, Gln synthetase, two Cys proteases, alcohol
dehydrogenases, ERD1, and an unknown protein.
Conversely, xylosidase and b-glucosidase were upregulated in both wallflower tissues with age but only
in one of the two tissues in Arabidopsis.
Affymetrix data, from the Weigel data sets (Schmid
et al., 2005) described above, were available for 61 of
the 73 genes identified as the closest hits to the
sequenced wallflower genes on the wallflower array
(Supplemental Table S2). Of these, six genes were
included in the 52 Arabidopsis genes discussed above.
Thus, data were available from all three combinations,
enabling a three-way comparison of the expression of
the Arabidopsis gene and wallflower gene when
probed with the wallflower transcripts and the Arabidopsis gene expression pattern on the Arabidopsis
Affymetrix arrays (the At codes for these genes are
shown in boldface in Supplemental Table S2). For
three genes (SAG12, up-regulated in both petals and
leaves; ferretin, up-regulated only in leaf; SAG21, upregulated only in petals) there was exact correspondence; for another two genes (a cytochrome P450 and a
copper homeostasis factor gene) the expression pattern was in broad agreement, although with the wallflower probe the leaf signal was below the threshold
for an up-regulated response; finally, a xylosidase gene
(At5g49360) was up-regulated only in petals on the
Affymetrix array while it was stable in leaves. This
result, however, contrasts with the northern analysis for
this gene (Fig. 7), which showed a clear up-regulation
of expression in the later stages of leaf senescence. The
data from the Arabidopsis gene on the wallflower
array hybridized to wallflower transcripts, and for the
wallflower homolog WLS27, were in better agreement
with the northern data, showing up-regulation of
expression with age in both tissues.
Of the 61 sequenced wallflower probes that matched
Arabidopsis genes and for which Affymetrix expression data were available for senescent leaves and
petals from the Weigel data, 85% shared the same
expression pattern with their Arabidopsis homolog in
at least one of the two tissues and 53% shared the same
expression pattern in both tissues. However, there
were some notable differences in those genes that were
particularly abundant in the wallflower array or that
are of interest because of potential roles in signaling or
regulation (Supplemental Table S2). Thus, expression
of the major class of chitinase genes (WC1/2/16 in
Supplemental Table S2, which is the mean of contigs
WC1, WC2, and WC16) on the wallflower array was
strongly up-regulated with age in wallflower petals
(mean, 36-fold) but not in leaves. However, in Arabidopsis, expression of the homolog (At2g43570) on the
Affymetrix arrays was strongly up-regulated in both
tissues (leaves, 10.1-fold; petals, 4.8-fold). Expression
of the largest group of wallflower GST sequences
(WC10, mean of contig WC10 on Supplemental Table
S2) homologous to Arabidopsis AtGSTF3 (At2g02930)
was strongly up-regulated in senescent wallflower
petals (16.5-fold) but down-regulated in senescent wallflower leaves. Expression of Arabidopsis AtGSTF3 on
the Affymetrix arrays showed a similar pattern, with
up-regulation in petals with age (2.7-fold) but no
change in leaves. However, two of the wallflower
sequences (WC21, mean of contig WC21 in Supplemental Table S2) showed closest homology to AtGSTZ1
(At2g02390). Expression of these wallflower probes
was up-regulated strongly in senescent wallflower
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Price et al.
Figure 7. Northern analysis of 10 Arabidopsis genes
represented on the microarrays. MG represents mature Arabidopsis green leaf with maximum chlorophyll levels (100%); S1 and S2 are stages of
Arabidopsis leaf senescence with 98% and 60% chlorophyll levels, respectively (Buchanan-Wollaston and
Ainsworth, 1997).
petals (4.3-fold) but was only very mildly up-regulated
in leaves (1.5-fold). Expression of Arabidopsis AtGSTZ1
on the Affymetrix arrays was strongly up-regulated in
both leaves (4.9-fold) and petals (6-fold).
Four genes with potential roles in signaling differed
in expression patterns between Arabidopsis and wallflower. Although expression of the three genes relating
to auxin signaling (WPS46, WPS103, and WPS53 in
Supplemental Table S2) was up-regulated with age in
both tissues of both species, expression of a putative
cytokinin oxidase (wallflower probe, WPS96; Arabidopsis gene, At1g75450) was strongly up-regulated in
Arabidopsis leaf (8.5-fold) but only very weakly in
petals (1.5-fold) on the Affymetrix arrays. In contrast,
the wallflower homolog (WPS96 in Supplemental
Table S2) was strongly up-regulated in both wallflower
tissues (leaves, 5.8-fold; petals, 8.9-fold). Two sequences
relating to Rab signaling were identified from the
wallflower libraries. Expression of a wallflower Rab
acceptor homolog, WLC13A (Supplemental Table S2),
was strongly up-regulated in old wallflower petals
(7.5-fold) but only very weakly in old wallflower leaves
(1.6-fold). In contrast, expression of the closest Arabidopsis homolog, At5g02040, was up-regulated in old
leaves (2.3-fold) but was stable with age in petals on
the Affymetrix arrays. Expression of the second Rabrelated wallflower sequence (WPS55 in Supplemental
Table S2), a putative member of the Rab small GTPases,
was weakly up-regulated in old wallflower petals (2.3fold) but was stable in wallflower leaves. However,
the Arabidopsis homolog (At1g49300) was stable with
age in both Arabidopsis petals and leaves on the
Affymetrix arrays. Finally, a putative rhodopsin-like
receptor gene also differed in expression pattern in the
two species. Expression of the Arabidopsis gene
(At1g12810) was up-regulated in both tissues (leaves,
2.8-fold; petals, 2.1-fold) on the Affymetrix arrays,
while the wallflower homolog (WPS95 in Supplemental Table S2) was only up-regulated in old wallflower
petals (2.9-fold) but not in old leaves.
Four transcription factors were also identified on the
wallflower arrays. Expression of a WRKY75 homolog
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A Comparison of Leaf and Petal Senescence in Wallflower
(WLS67) and two members of the No Apical Meristem
(NAM) family (WPS52 and WLS62) was up-regulated
in both tissues in both species (Supplemental Table S2).
Expression of the Arabidopsis homolog of the WRKY75
transcription factor (At5g13080) was up-regulated
much more strongly in old leaves compared with old
petals (leaves, 202-fold; petals, 13.8-fold) on the Affymetrix arrays, whereas the expression pattern of its wallflower homolog, WLS67, was reversed, with much
stronger up-regulation in old wallflower petals (67-fold)
compared with old wallflower leaves (7-fold). There
was a similar contrast in pattern for one of the NAM
family transcription factors (At5g64530/WLS62 in
Supplemental Table S2). Expression of this gene was
much more highly up-regulated in old petals compared with old leaves in wallflower (leaves, 2.2-fold;
petals, 22-fold), while on the Arabidopsis Affymetrix
arrays the pattern was reversed (leaves, 4.3-fold;
petals, 2-fold). Finally, expression of a CCR4 family
protein (WLS63 in Supplemental Table S2) was upregulated in both aging Arabidopsis leaves and petals
(leaves, 2.8-fold; petals, 4.0-fold) on the Affymetrix
arrays, while in wallflower it was only up-regulated in
petals with age (3.7-fold) and stable in leaves.
DISCUSSION
Remobilization during Petal and Leaf Senescence
in Wallflower
Species can be broadly divided into those in which
petals wilt before abscission and those in which petals
abscise at full turgor (van Doorn and Stead, 1997).
Generally, the longer the petals persist, the more
remobilization of nutrients is likely to occur. Patterns
of dry weight-fresh weight ratio changes during wallflower petal senescence are consistent with data from
other genera, such as Alstroemeria and Tulipa (Collier,
1997), Hemerocallis (Lay Yee et al., 1992), Digitalis
(Stead and Moore, 1977), and Sandersonia (Eason and
Webster, 1995), in which some wilting occurs before
abscission. However, in wallflower, the magnitude of
change between the maximal values of open flowers
and heavily wilted flowers is quite low (at stage 5,
fresh weight and dry weight are 41% and 67%, respectively, of the maximum) compared with Hemerocallis,
in which fresh weight decreases to 2% of maximum
and dry weight decreases to 33% of maximum. The
change, however, is greater than in Digitalis (dry
weight remains at 88% of maximum) or Alstroemeria
(dry weight remains at 80% and fresh weight at 40% of
maximum). Thus, wallflower petals appear to be more
similar to Alstroemeria in their loss of fresh weight (40%
of maximum in Alstroemeria) but closer to Tulipa in the
loss of dry weight (60% of maximum in Tulipa). This
indicates a flower in which there is substantial, but not
extreme, wilting before petal abscission and in which
some remobilization is probably taking place. This is
supported by the array results: a large proportion of
the sequenced genes that were up-regulated in senescent wallflower petals related to remobilization (38%
overall). This is in agreement with transcriptomic
studies of petals from other species in which wilting
occurs (Alstroemeria [Breeze et al., 2004] and Iris [van
Doorn et al., 2003]). The vast majority of genes related
to remobilization were up-regulated in both senescent
petals and leaves, and the largest proportion of the
genes whose expression was up-regulated with age in
both tissues were putatively involved in remobilization. Including SAG12, these represent just over half of
the genes in this category. Again, this agrees with other
transcriptomic studies of leaves (Guo et al., 2004;
Buchanan-Wollaston et al., 2005) and petals (van
Doorn et al., 2003; Breeze et al., 2004). All of the
SAG12 targets belonged to this expression class as
expected, and metal-binding proteins were also well
represented, again reflecting other studies discussed
above. However, three genes related to remobilization
were specifically up-regulated in wallflower petals
and not leaves, a AAA-type ATPase family protein, a
lipid transferase, and a thiol protease. These may be
interesting genes to study further.
While the process of remobilization, and many of
the genes involved, are shared between petals and
leaves in wallflower, the timing of both physiological
events and gene expression in the two organs differs.
Whereas in petals the dry weight-fresh weight ratio
was rising well before any visible signs of wilting, in
leaves the first signs of visible senescence, and the
drop from maximal chlorophyll levels, coincided with
the start of the rise in dry weight-fresh weight ratio.
The loss of both fresh weight and dry weight was
comparable between petals and leaves; however, the
extent of protein breakdown differed, with 65% of the
maximal level of protein remaining in petals by stage 5
compared with only 5% in stage 7 leaves. The fall in
leaf protein coincided with chlorophyll degradation,
reflecting the fact that the majority of remobilized
protein from leaves is from chloroplasts (Thomas and
Donnison, 2000). The precise timing of SAG12 expression also differed between the two organs when examined more closely by RT-PCR. The leaf data are in
agreement with data from Arabidopsis (Lohman et al.,
1994), with up-regulation of SAG12 late in senescence.
However, in petals, SAG12 is already substantially upregulated in mature nonsenescent flowers and falls to
less than 20% of maximal levels by late senescence.
SAG12 encodes a papain-like Cys protease located in
senescence-associated vacuoles. It is presumed to play
a role in proteolysis; however, sag12 knockouts are not
perturbed in their leaf senescence (Otegui et al., 2005).
Assuming that the role of SAG12 is equivalent in
petals and leaves, the different expression programs
could reflect different patterns of cellular degradation.
Electron microscopy of petals reveals very early cellular death in much of the mesophyll while the epidermal cells remain intact (Weston and Pyke, 1999;
Wagstaff et al., 2003). Hence, perhaps the majority of
SAG12 activity is already complete in many petal cells
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Price et al.
at a relatively earlier stage of organ senescence. The
temporal difference in expression patterns in different
cell types is something that array experiments often
overlook, and it is only with laser dissection microscopy, or other single cell-based PCR techniques, that
these differences will be elucidated.
The expression of another gene with a presumed
role in remobilization, WC11, encoding a putative
peptide transporter, was also examined by RT-PCR,
and the expression pattern of this gene also differs
between the two organs. Although WC11 expression is
strongly up-regulated according to the array during
both petal and leaf senescence, RT-PCR shows that the
pattern is more complex. It was expressed from young
leaves through to mature leaves, with increased expression early in senescence. In contrast in petals,
there is a clear up-regulation that precedes other signs
of senescence, and expression remains high. Peptide
transporters form a superfamily of structurally related
membrane proteins (Chiang et al., 2004). Different
members of the Arabidopsis gene family show tissuespecific expression. The closest Arabidopsis gene to
WC11, At1g32450, is part of the PTR family, transporting dipeptides and tripeptides (Waterworth and
Bray, 2006), although it does not fall into one of the
major subfamilies. The Arabidopsis gene is expressed
in mature tissues and is strongly up-regulated in leaf
senescence (Affymetrix data from Genevestigator).
Hence, this gene may have a function both during
leaf development and during the remobilization occurring during leaf senescence. In petals, the role may
be different, in that expression during early development is very low and there is a far greater up-regulation
during senescence, indicating a more specific role in
senescence-associated remobilization.
nescence in wallflower may be a reduction in cytokinin
levels via cytokinin oxidase. In carnation (Dianthus
caryophyllus) petals, sensitivity to ethylene of excised
petals was reduced by exogenous application of cytokinin (Taverner et al., 2000), indicating cross talk between these two growth regulators, which is worthy of
further investigation in wallflower. The Arabidopsis
cytokinin oxidase gene, At1g75450, was only very
weakly up-regulated in petals (1.5-fold) on the Affymetrix arrays, whereas up-regulation in leaves was
much more significant (8.5-fold). This could indicate
either temporal or species-specific differences in the
role of this enzyme in petal senescence.
One aim of this study was to identify genes that
might indicate differences in the regulation of petal
and leaf senescence. Only one wallflower sequence
related to transcriptional regulation was identified in
the class of probes from the wallflower microarray that
were up-regulated in petals but not in leaves; this was
WLS63, a CCR4-related gene. In yeast, the CCR4
protein forms part of the CCR4-NOT complex, which
acts as an RNA deadenylase, and is involved in
nutrient and stress sensing (Collart, 2003). The role
of these genes in plants has not been fully investigated.
RT-PCR showed that expression patterns of WLS63 in
wallflower are very different between leaves and
petals. WLS63 peaks in expression in petals relatively
late, at stage 4, when petals are already showing
visible signs of senescence, after the peak in SAG12
expression. This suggests that it may be important in
mRNA stability late in senescence, perhaps targeting
specific transcripts for degradation. In leaves, WLS63
transcripts are at their highest levels in young leaves
and fall thereafter to lower levels of expression. This
could imply either that it is not involved in leaf
senescence in this species or, alternatively, that its
down-regulation stabilizes specific transcripts.
Regulation of Wallflower Petal and Leaf Senescence
Pulse treatment of cut flowers with STS indicated
that ethylene is involved in both petal senescence and
abscission in this species. It was a surprise, therefore,
not to find more genes related to ethylene biosynthesis
or responses in the petal SSH library. In fact, only one
ACC oxidase-like gene was found. This gene, however, was strongly up-regulated in both senescent
leaves and petals, as expected. In addition, Arabidopsis ACC oxidase on the array was up-regulated 3-fold
when hybridized to messages from wallflower petals.
Many of the SSH library genes represented 3# untranslated region sequences and were thus difficult to annotate;
therefore, it seems likely that further ethylene-related
genes are up-regulated in both leaf and petal wallflower
senescence but were not identified as such.
Treatment with cytokinin (kinetin) delayed both petal
senescence and abscission, as did treatment with the
inhibitor of cytokinin oxidase, 6-methyl purine. A cytokinin oxidase gene (At1g75450, WPS96), was strongly
up-regulated in old petals in wallflower (9-fold). Thus,
part of the mechanism for the regulation of petal se-
Shared and Petal-Specific Gene Expression
The high prevalence of SAG12 clones (14% overall;
8% of petal clones and 21% of leaf clones) is expected
due to the close taxonomic relationship to Arabidopsis
and Brassica, in which SAG12 is a highly abundant
transcript in senescent leaves (Lohman et al., 1994;
Guo et al., 2004). Although metallothioneins were
represented in both libraries (6% in petal and 7% in
leaf), the levels were not as high as those found in
other EST studies of petal senescence, in which they
were present at levels of 19% in Alstroemeria (Breeze
et al., 2004) and 11% in Rosa (Channeliere et al., 2002),
indicating species-specific differences in the expression of these genes and perhaps in their role in petal
senescence. Metallothioneins have been found in other
studies of leaf ESTs, although not at such high levels as
in wallflower (e.g. rice [Oryza sativa] mature leaves, 3%
[Gibbings et al., 2003]; senescent Arabidopsis leaves,
3% [Guo et al., 2004]).
Two genes were found at unexpectedly high frequency in the array class up-regulated in senescent
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A Comparison of Leaf and Petal Senescence in Wallflower
petals but not leaves: chitinases and GSTs. The very
high abundance of chitinase genes in the wallflower
petal libraries (23%) was a surprise, and there was a
clear interorgan difference, with only 2% of the genes
found in the leaf library identified as chitinase. Although chitinase transcripts have been reported as
senescence enhanced in other species in leaves in
Brassica (Guerrero et al., 1990; Hanfrey et al., 1996)
and petals in Alstroemeria (Breeze et al., 2004), EST
studies of senescent petals or leaves have not revealed
the high abundance found here. The major role of
chitinases was usually thought to be in pathogen
defense, and they are classed as pathogenesis-related
proteins. However, it is becoming clear that chitinases
may also have roles in signaling and programmed cell
death (Kasprzewska, 2003).
GSTs are up-regulated in petal senescence in other
species, such as carnation (Meyer et al., 1991). Although the role of most plant GSTs is unclear (Wagner
et al., 2002), some, including those of the Arabidopsis
f class, may act as glutathione peroxidises, protecting
cells from ROS damage, while others may have roles in
hormone metabolism. Two wallflower targets were
most closely homologous to AtGSTZ1, which is involved in Tyr metabolism (Dixon et al., 2000). The
other wallflower clones were closest in amino acid
sequence to f class GSTs from Arabidopsis: AtGSTF2
and AtGSTF3. AtGSTF2 is membrane associated (Zettl
et al., 1994) and ethylene responsive; both AtGSTF2
and AtGSTF3 have a putative ethylene-responsive
enhancer element in their promoter sequences similar
to that found in the petal senescence-enhanced carnation GST (Itzhaki et al., 1994) and are also up-regulated
by salicylic acid (Wagner et al., 2002). AtGSTZ1 transcription is not induced by ethylene but is induced by
methyl jasmonate, and both AtGSTF2 and AtGSTZ1
are induced by the auxin analog 2,4-dichlorophenoxyacetic acid (Wagner et al., 2002). Thus, GSTs are clearly
involved in processes related to senescence, and their
up-regulation in wallflower petals but not in leaves
may reflect important differences in the regulation of
senescence by plant growth regulators or in the fine
control of senescence progression in these two tissues.
Clearly, the role of GSTs in wallflower petal senescence
is also worthy of further investigation.
high level of agreement. Thus, although caution must
be exercised in the interpretation of data from crossspecies experiments, due to the complications of gene
families and inherent difficulties in precisely assigning
stages of development, these data strongly support the
use of this approach here.
Due to the close taxonomic relationship between the
two species, floral architecture in wallflower and
Arabidopsis is similar, and in both species leaves
form sequentially in a spiral. However, wallflower
petals differ from Arabidopsis petals in their purple
pigmentation and much slower development and
senescence. Differences in leaf senescence strategy
might also occur due to the diverse life cycles in the
two species: perennial in wallflower and ephemeral
Arabidopsis. Genes that share expression patterns
between the two species thus reflect perhaps the
underlying evolutionary conservation, while those
with differing patterns may reflect species-specific
strategies. Over one-third (38%) of the Arabidopsis
genes on the array and 53% of the wallflower genes
shared gene expression patterns in the two species,
indicating a conservation of a significant portion of the
gene expression profile. However, a number of genes
differed in expression pattern between the two species. These include both the AtGSTZ1 gene and the
Arabidopsis chitinase gene (At2g43570), which were
up-regulated with senescence in Arabidopsis leaves
while the wallflower homologues were not. These
differences may reflect a divergence of senescence
strategies in the two species and, again, would be
interesting for future studies.
CONCLUSION
This study has revealed considerable differences in
gene expression during senescence both between
petals and leaves and between two closely related
species. Further work to understand petal and leaf
senescence in these species will exploit the advantages
of wallflower for biochemical studies and the myriad
resources for forward and reverse genetics available
for Arabidopsis.
MATERIALS AND METHODS
Cross-Species and Cross-Tissue Comparisons of
Expression Patterns
Overall, 57% of the Arabidopsis genes on the array
gave consistent results when hybridized to the wallflower transcripts. This compares favorably with other
studies using species taxonomically related to Arabidopsis (e.g. in Thlaspi arvense arrays, only 31% of
probes cross-hybridized to Arabidopsis [Sharma et al.,
2007]). Likewise, for the six wallflower genes on the
array, for which the closest Arabidopsis homolog was
also included on the array and data were available
from the Affymetrix experiments, there was a very
Plant Material
Leaves and petals were collected from wallflower (Erysimum linifolium
‘Bowles Mauve’) and staged (Figs. 1 and 2). Material for RNA extraction was
immediately frozen in liquid nitrogen and stored at 280°C until required.
Cut Flower Treatments
Individual flowers were detached from the raceme at stage 1, and the
pedicel was immediately submerged in water. Flowers were held at 20°C and
16 h of light either in water or in solutions of kinetin (1.0 or 0.1 mM) or 6-methyl
purine (0.1 mM; Sigma-Aldrich). For ethylene inhibitor treatment, flowers
were held in STS (4 mM AgNO3:32 mM NaS2O3) for 1 h and then transferred to
water. Each experiment consisted of 10 replicate flowers, which were monitored daily to record senescence stage and day of petal abscission.
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Price et al.
Chlorophyll Extraction
(DNAstar; Lasergene). Contig assembly was performed using DNAstar
(Lasergene), setting a threshold match of 80%.
Chlorophyll was extracted from leaves using 70% acetone, and absorbance
was measured at 645, 652, and 663 nm using a Cecil Instruments Visible/UV
spectrophotometer. Chlorophyll concentrations (in micrograms of chlorophyll
per milliliter of extract) were calculated according to Bruinsma (1963) and
normalized to fresh weight.
Protein Content
Proteins were extracted by grinding 16 petals or one leaf from each
developmental stage in 200 mL of 100 mM Tris-HCl, pH 8.0, 20% glycerol, and
30 mM dithiothreitol at 4°C. Following centrifugation for 30 min at 12,000g and
4°C, supernatants were stored at 280°C until required. Protein content was
quantified using the method described by Bradford (1976) using Bradford
reagent (Sigma-Aldrich) and bovine serum albumin as a standard, quantified
spectrophotometrically at 590 nm in a Molecular Devices Emax precision
microplate reader, using the Molecular Devices SOFTmax Pro software
(version 3; Molecular Devices). Each sample and standard was in triplicate
on each plate, and each assay was repeated on five different extracts made
from independently harvested material.
RNA Extraction
Extractions from 0.2 g of both leaves and petals were performed using
2 mL of TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol,
following grinding to a powder in liquid nitrogen using a mortar and pestle.
For larger scale extractions, 10 mL of TRI reagent was added to 1 g of ground
tissue and homogenized using an IKA Labortechnik T25 basic polytron
motorized homogenizer. Extractions followed the manufacturer’s protocol for
TRI reagent except that the chloroform phase was back-extracted with 1 mL of
sterile, distilled water and RNA was isopropanol precipitated before extraction with isoamyl alcohol:phenol:chloroform (1:25:24) until the interface was
clear. Finally, the extracts were chloroform extracted, ethanol precipitated,
ethanol washed, and resuspended in sterile distilled water. RNA was treated
with RQ1 DNase (Promega) and, for use in making probes, was further
purified using an RNeasy purification column (Qiagen).
Subtracted Library Construction
Two subtracted libraries were made to enrich for genes that are upregulated during senescence: one from petals and one from leaves. Equal
amounts of RNA were combined to make cDNA from young petals (stages
22, 21, and 0), which was subtracted from that from old petals (stages 3, 4,
and 5). The leaf library was constructed from young leaves (stage 3) subtracted
from old leaves (combined stages 5 and 6). First-strand cDNA was synthesized
from 1 mg of total RNA using the Smart cDNA synthesis kit (Clontech). PCR
cycle number was optimized for second-strand synthesis to ensure that
amplification was in the exponential phase. Old leaf template required 19
cycles, while 17 cycles were used for the other templates. Ligation efficiency
was tested using degenerate primers designed to the SAG12 gene from
Arabidopsis (Arabidopsis thaliana) and Brassica napus: SAG12F, 5#-TTGCCGGTTTCTGTTGAYTGG-3#; SAG12R, 5#-TGGTGTGCCACTGCYTTCAT-3#,
where Y 5 C/T. Subtraction of the Smart double-stranded cDNA was performed using PCR-Select cDNA subtraction (Clontech) according to the manufacturer’s protocol. The final amplification step was carried out over 12 cycles.
The SAG12 primers were also used to test the efficiency of the subtraction.
PCR products were cleaned using the Qiaquick PCR purification kit (Qiagen),
concentrated by ethanol precipitation, and cloned into pGEM-T easy (Promega). Individual colonies were grown as liquid cultures on 96-well plates
and stored as glycerol stocks at 280°C.
For sequencing, inserts were PCR amplified using M13 forward and
reverse primers from the glycerol stocks or isolated using a Qiaprep Miniprep
kit (Qiagen). PCR products were purified using Millipore MANU 03050 plates
or Qiaquick PCR cleanup kits (Qiagen). All sequencing was performed using
M13 forward and reverse primers with BigDye version 2 (Applied Biosystems) and analyzed on an Applied Biosystems 3100 sequencer. Database
searches were carried out using the BLAST network service (National Center
for Biotechnology Information), The Arabidopsis Information Resource, and
AtGenExpress. A match was assessed using a combination of low E value and
the length of the homology in tBLASTx. Alignments of proteins and sequences
were performed using BIOEDIT version 7.0.1 (Hall, 1999) and Seqman
Microarrays
PCR products were cleaned using the Whatman 96-well PCR cleanup kit
and checked by gel electrophoresis. Fifty to 100 ng of each PCR product in 2.5
mL was added to 2.5 mL of dimethyl sulfoxide on 384-well plates using a
Perkin-Elmer multiprobe liquid-handling robot, then spotted as a 4 3 12
metagrid of 12 3 12 subgrids onto UltraGAPS II (Gamma Amino Propyl
Silane; Corning)-coated slides using 150-mm solid pins on a Genomic Solutions Flexys workstation. Each slide carried three replicates of each target
DNA. Slides were air dried for 12 h, baked at 80°C for 2 h, and UV cross-linked
with an Autocrosslink cross linker (Stratagene). They were stored with
desiccant in the dark at room temperature until required.
Labeling of RNA for Hybridization to the Arrays
mRNA was amplified using the MessageAmp aRNA kit (Ambion) according to the manufacturer’s protocol from 5 mg of extracted total RNA from the
same combined tissue stages used to generate the SSH libraries (petals stages
22, 21, and 0 representing young petals; stages 3, 4, and 5 representing old
petals; leaf stage 3 representing young leaves; and leaf stages 5 and 6
representing old leaves). Material was derived from six clonal plants, and
RNA used for labeling was combined from different batches of petals and
leaves collected on different dates to ensure the randomization of any possible
bias in the material or in the RNA extraction. Different RNA extracts were
used in the construction of the SSH libraries and for labeling the RNA to be
hybridized to the arrays. The CyScribe Post-Labeling kit (Amersham Biosciences) was used to label the aRNA, with either Cy3 or Cy5 according to the
manufacturers’ instructions, using 1 mg of aRNA.
Each labeled RNA pair was lyophilized using an Edwards Freeze Dryer
Modulyo Pirani 501 and resuspended in 50 mL of hybridization buffer
containing 25% formamide, 53 SSC, 0.1% SDS, 0.5 mg mL21 poly(dA), and
0.5 mg mL21 yeast tRNA.
Slides were prehybridized for 45 min in a 53 SSC, 0.1% SDS, 1% bovine
serum albumin solution preheated to 42°C, washed five times in milliQ water
and twice in isopropanol, and air dried. For hybridization, the labeled RNA
was heated to 95°C for 5 min and applied to the microarray slide surface. A
second microarray slide was lowered over the labeled RNA and the slides
were hybridized back to back overnight in a humid chamber at 42°C.
Hybridizations were carried out four times with dye swapping. Following
hybridization, the slides were separated by immersion in 23 SSC, 0.1% SDS at
42°C and washed in the same solution for 5 min. The slides were further
washed in a solution of 0.13 SSC, 0.1% SDS for 10 min at room temperature,
followed by four changes of 0.13 SSC for 1 min each at room temperature. The
slides were rinsed in isopropanol and dried by centrifugation for 1 min at
2,000g in a MSE Mistral 2000 centrifuge.
Analysis of Microarray Slides
The slides were scanned using an Affymetrix 428 array scanner with the
supplied software (Affymetrix) at 532 nm (Cy3) and 633 nm (Cy5). Scanned
images were quantified using Imagene version 5 software (Biodiscovery). Spot
quality labeling (flags) was defined for empty spots with a signal strength
threshold of 1 and for shape regularity with a threshold of 0.4. The median
signal intensity across each spot and the median background intensity were
calculated in both channels, and these data were exported into GeneSpring
version 6 (Agilent). Background intensity was subtracted from spot intensity
for both channels, giving the background-corrected spot intensity. Each slide
carried three replicates of each gene, and four slides were used in the
experiment, including a dye swap for each probe pair. The scores for the 12
data points per spot were averaged in GeneSpring, and threshold ratios of 2
and 0.5 were set. Genes with a P value of .0.05 and not passing the Benjamini
and Hochberg false discovery rate were excluded. A list was generated
containing all genes whose expression changed in at least one tissue and
whose expression could be reliably determined in both tissues.
Northern Hybridization
Northern blotting and hybridization were performed using RNA extracted
from Arabidopsis leaves as described by Buchanan-Wollaston and Ainsworth
1910
Plant Physiol. Vol. 147, 2008
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Copyright © 2008 American Society of Plant Biologists. All rights reserved.
A Comparison of Leaf and Petal Senescence in Wallflower
(1997). For making probes, inserts from Arabidopsis cDNA clones were PCR
amplified and labeled with [32P]dCTP using the RediPrime random priming
labeling kit (Amersham Biosciences). Ribulose bisphosphate carboxylase
small chain was included as a probe to verify the loading.
Semiquantitative RT-PCR
Specific PCR primers were designed to the EST sequences obtained
from the wallflower SSH libraries to SAG12 as detailed above, clone WLS63
(LP H9 F, 5#-GTTTGGACCGGGTTGCTC-3#; LP H9 R, 5#-ACTCCGGCGTGTTTCACC-3#), which amplify a 130-bp fragment of the gene, and
clone WC11A (P1 F4 F, 5#-AGAGCTTCGGAAGCGCTCTG-3#; P1 F4 R,
5#-AGGTACCACTTTGCACATGC-3#), which amplify a 219-bp gene fragment.
Normalization controls were performed using primers PUV2 (5#-TTCCATGCTAATGTATTCAGAG-3#) and PUV4 (5#-ATGGTGGTGACGGGTGAC-3#;
Dempster et al., 1999), which amplify a 488-bp fragment of 18S rRNA.
Reactions were cycled in a Perkin-Elmer 2700 thermocycler or an MJ Research
PTC100, using 15 min at 95°C for Hotstar Taq polymerase (Qiagen), 1 min at
95°C for standard Taq polymerase, followed by cycles of 95°C for 10 s, 65°C for
30 s, and 68°C for 2.5 min for the SAG12, WLS63, and WC11 primers on the
Perkin-Elmer 2700 and 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min on
the PTC100. Replicates for each primer set were all amplified on the same
machine to avoid any variability due to the machine parameters. Annealing
temperature for the PUV and GST primers was 50°C on both machines.
Products were analyzed by agarose gels, and PCR products were quantified
using the Gene Genius bioimaging system and GeneSnap software (Syngene,
Synoptics). Product quantitation from the 18S target was used to normalize
results for all other primer sets. To ensure that the reactions were in the
exponential phase and therefore product quantitation could be considered
semiquantitative in relation to message abundance, cycles were optimized
and limited for each primer set and cDNA synthesis batch combination.
Specific cycle number is not reported here, as it was optimized independently
for each batch of cDNA. Dilution series of the cDNA were included in every
PCR run, and results were only accepted when a linear response was obtained.
This methodology has been used successfully to obtain semiquantitative RTPCR data for a range of experimental systems (Wagstaff et al., 2002, 2003, 2005;
Parfitt et al., 2004; Orchard et al., 2005).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the accession numbers provided in the text and in Supplemental Table S1.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Effects on abscission time of cut wallflowers
treated with cytokinin (A; 1.0 or 0.1 mM kinetin), 6-methyl purine (B; 0.1
mM), and STS (C), compared with water control (n 5 10 flowers per
treatment).
Supplemental Figure S2. Effects on progression of petal development and
senescence in cut wallflowers treated with water (A), 1.0 mM kinetin
(B), 0.1 mM kinetin (C), 0.1 mM 6-methyl purine (D), and STS (E; n 5 10
flowers per treatment).
Supplemental Table S1. List of wallflower and Arabidopsis microarray
probes for which reliable data from both wallflower petal and leaf
hybridizations could be obtained.
Supplemental Table S2. Comparison between expression of genes represented by probes on the wallflower array, hybridized to senescent and
young wallflower petal and leaf transcripts, and the expression of the
nearest Arabidopsis homolog in Arabidopsis senescent and young
petal and leaf from Affymetrix data.
ACKNOWLEDGMENTS
We are grateful to Gareth Lewis, Steven Hope (School of Biosciences
Cardiff), and Rachel Edwards and Jeanette Selby (Warwick) for sequencing
and to Lyndon Tuck (Cardiff) for plant maintenance.
Received April 2, 2008; accepted June 2, 2008; published June 6, 2008.
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1912
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