The Plant Journal - 2005 - Buchanan Wollaston - Comparative Transcriptome Analysis Reveals Significant Differences in Gene

The Plant Journal (2005) 42, 567–585 doi: 10.1111/j.1365-313X.2005.02399.
Comparative transcriptome analysis reveals significant

differences in gene expression and signalling pathways
between developmental and dark/starvation-induced
senescence in Arabidopsis
Vicky Buchanan-Wollaston1,*, Tania Page1, Elizabeth Harrison1, Emily Breeze1, Pyung Ok Lim2,3, Hong Gil Nam2, Ji-Feng Lin4,
Shu-Hsing Wu4, Jodi Swidzinski5,6, Kimitsune Ishizaki5 and Christopher J. Leaver5
1
Warwick HRI, University of Warwick, Wellesbourne CV35 9EF, UK,
2
Division of Molecular and Life Sciences, POSTECH, Pohang 790-784, South Korea,
3
Department of Science Education, Cheju National University, Cheju 690-756 South Korea,
4
Institute of Botany, Academia Sinica, Taipei 11529, Taiwan,
5
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK, and
6
Department of Botany, University of Toronto, Toronto, ON M5S 3B2, Canada
Received 22 December 2004; revised 14 February 2005; accepted 18 February 2005.

*
For correspondence (fax 02476 574500; e-mail vicky.b-wollaston@warwick.ac.uk).
Summary
An analysis of changes in global gene expression patterns during developmental leaf senescence in
Arabidopsis has identified more than 800 genes that show a reproducible increase in transcript abundance.
This extensive change illustrates the dramatic alterations in cell metabolism that underpin the developmental
transition from a photosynthetically active leaf to a senescing organ which functions as a source of mobilizable
nutrients. Comparison of changes in gene expression patterns during natural leaf senescence with those
identified, when senescence is artificially induced in leaves induced to senesce by darkness or during sucrose
starvation-induced senescence in cell suspension cultures, has shown not only similarities but also
considerable differences. The data suggest that alternative pathways for essential metabolic processes such
as nitrogen mobilization are used in different senescent systems. Gene expression patterns in the senescent
cell suspension cultures are more similar to those for dark-induced senescence and this may be a consequence
of sugar starvation in both tissues. Gene expression analysis in senescing leaves of plant lines defective in
signalling pathways involving salicylic acid (SA), jasmonic acid (JA) and ethylene has shown that these three
pathways are all required for expression of many genes during developmental senescence. The JA/ethylene
pathways also appear to operate in regulating gene expression in dark-induced and cell suspension
senescence whereas the SA pathway is not involved. The importance of the SA pathway in the senescence
process is illustrated by the discovery that developmental leaf senescence, but not dark-induced senescence, is
delayed in plants defective in the SA pathway.
Keywords: senescence, development, dark-induced, cell suspension , signalling pathways, salicylic acid.
Introduction
Developmental senescence in ageing leaves is a complex Thus, the study of senescence has been complicated by the
and highly organized process resulting in many changes in lack of coordinated development of the cells within an
gene expression and metabolic processes. Within a individual leaf and various methods have been used to
senescing leaf, the individual cells are usually at many dif- artificially induce senescence to obtain a synchronous
ferent stages of senescence with the distal parts of the leaf process. For example, dark-induced senescence has been
senescing first and the cells surrounding the veins tending to used frequently as a useful method to induce synchronous
remain active longer to maximize transport from the leaf. senescence as many typical senescence symptoms such as
ª 2005 Blackwell Publishing Ltd 567

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568 Vicky Buchanan-Wollaston et al.
chlorophyll degradation and loss of protein occur. various aspects of development including senescence. PCD
Also, suspension cultures can be used to synchronize in plants exhibits many different phenotypes but is usually
developmental events and senescence-like symptoms are characterized by controlled vacuolar collapse and DNA
induced in ageing cell suspensions. In this paper, we com- laddering (Jones, 2001). DNA laddering has been seen very
pare gene expression in three different types of senescence rarely in leaf senescence and vacuolar collapse may only
and our results indicate that, while dark-induced or sus- occur in the very final stages (Lee and Chen, 2002). However,
pension culture senescence have many features common to senescing cells eventually die and the mechanism of this
developmental senescence and could be useful model may have processes in common with other PCD events.
systems for certain aspects, there are also considerable dif- In this paper, we report the use of an Affymetrix GeneChip
ferences in other key processes that take place. system to identify genes that are upregulated in develop-
Leaf ageing in plants is accompanied by a genetically mental senescence in Arabidopsis. Gene expression data
programmed senescence process, in which nutrients are from the dark-induced senescence experiments described
mobilized from the dying leaves to support active growth in by Lin and Wu (2004) and also data from Arabidopsis cell
the younger parts of the plant (Hörtensteiner and Feller, suspension cultures undergoing starvation-induced senes-
2002). The metabolic changes occurring during senescence cence and showing clear symptoms of PCD (Swidzinski
are essential to support plant growth and reproduction and et al., 2002) were used to compare and contrast gene
the cells of the senescing leaf must remain viable until the expression changes that occur in developmental senescence
mobilization of salvaged nutrients is complete. Senes- with those taking place in these two other types of senes-
cence in plants is highly regulated and depends cence. Exploitation of the global expression patterns
upon the modulated expression of many different genes obtained in these three separate data sets has allowed us
(Buchanan-Wollaston et al., 2003; Gepstein et al., 2003). The to show clear differences between the three types of
identification of genes that are up- or downregulated during senescence.
leaf senescence has been a focus of a considerable amount Gene expression during senescence is under the control
of research and in recent years the putative functions of of a complex combination of signalling pathways, some of
senescence-related genes has increased our understanding which have key roles in other plant processes such as in
of the regulation and metabolic basis of this important response to environmental stresses. For example, the ethy-
developmental process. lene signalling pathway has a role in modulating the rate of
Several different groups have identified genes that show leaf senescence, mutants defective in this pathway show
increased expression during the onset of senescence in delayed senescence (Grbic and Bleecker, 1995). Also the
several plant species although the majority of experiments jasmonic acid (JA) pathway is involved in senescence,
have been conducted with Arabidopsis (reviewed in treatment with JA induces premature senescence and
Buchanan-Wollaston et al., 2003). The current number of various senescence-enhanced genes are expressed in
such genes is approximately 150 (Gepstein et al., 2003) but response to JA treatment (He et al., 2002). However, mutants
is certainly not a complete picture. Guo et al. (2004) have affected in JA signalling do not show obvious alteration in
identified a collection of over 2000 expressed sequence tag senescence phenotype. Salicylic acid (SA), a key signalling
clones that represent genes that are expressed in senescing molecule in plant pathogen responses, has also been shown
leaves of Arabidopsis. However, the proportion of these that to be required for expression of some senescence-enhanced
show senescence-enhanced expression has not been deter- genes (Morris et al., 2000). The elucidation of key senes-
mined. Microarray analysis has been used to investigate the cence-specific pathways is complicated by the crosstalk
expression patterns of several thousand genes during between these non-specific stress-induced pathways. We
autumnal leaf senescence in poplar, and similar types of have extended the microarray analysis to examine the role
genes have been identified (Andersson et al., 2004). of the ethylene, JA and SA pathways on gene expression
Recently Lin and Wu (2004) have used microarray analysis during senescence. Comparisons made with expression of
to identify a large group of genes that show differences in these genes in the other types of senescence or PCD has
transcript abundance in response to dark treatment of allowed us to determine the role of these pathways in dark-
Arabidopsis. induced and cell suspension senescence.
Swidzinski et al. (2002) showed that Arabidopsis suspen-
sion cultures undergoing natural ageing, or after heat
Results and discussion
treatment, expressed symptoms typical of programmed cell
death (PCD). They found that a number of senescence-
Genes showing increased transcript abundance in leaf
related genes were upregulated in these treated cells
senescence
indicating that there were common processes occurring.
Plant PCD occurs in a wide range of specialized situations The array data described in this paper are based on levels of
such as response to pathogen attack, waterlogging and in hybridization to Affymetrix GeneChip microarrays. This
ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Gene expression in different types of senescence 569
determines transcript abundance and may not necessarily

represent increased rates of transcription of individual Regulation
Degradation
genes as post-transcriptional regulation will vary between
different genes. Also, transcript levels do not necessarily Regulation
Degradation
reflect the amount of final active protein product. However,
for simplicity, increased transcript abundance is often
referred to as induced gene expression.
Arabidopsis Affymetrix GeneChip arrays were probed
with RNA from mature green leaves (before flowering) and Carbohydrate
Transcription Transport Protein and
from leaves in the mid senescent stage (5–15% visible Stress/defence N mobilisation
Protein binding
chlorosis). In the green leaf sample, expression of 8538 Kinase and
Degradation of Nucleic acid
macromolecules degradation
genes was detected, while in the senescent leaf sample, phosphatases Autophagy Lipid degradation
Signalling
expression of 9152 genes was detectable. This suggests Enzymes of Chl degradation
Calcium unknown function
that, of the 24 000 genes represented on the array over Unknown genes
Hormone
30% are expressed at a detectable level in each of the leaf Regulatory genes
pathways
Secondary metabolism
samples.
Genes showing increased abundance in leaf senescence Figure 1. Potential functions of senescence-enhanced genes.
were identified using array data from two independent The 827 genes that show at least threefold increase in transcript abundance
were sorted into putative functional groups according to their TIGR v. 5.0
experiments and genes that showed at least a threefold
annotation as shown in Table S1 and Table 1. These groups are illustrated by
upregulation were selected. This resulted in 827 upregu- the central pie chart with extra charts used to divide the genes encoding
lated genes (Table S1). This list of genes has been putative regulatory proteins and genes involved in macromolecule degrada-
tion into smaller functional groups.
generated by combination of the two different data sets,
which strengthens confidence in the data as the genes
selected show the same relative changes in abundance of transcription factors expressed during developmental
during senescence under two different growth regimes. senescence (Guo et al., 2004) and showing increased
Thus, an extensive new set of senescence-enhanced genes expression in dark-induced senescence (Lin and Wu, 2004)
has been identified. has been described previously.
The genes listed in Table S1 provide a valuable indica- A number of genes involved in potential ubiquitination
tion of the metabolic activities that are changing in the leaf pathways are upregulated and this indicates that protein
when senescence takes place. The genes have been sorted degradation via the 26S proteosome is active during senes-
depending on the likely role of each gene that is upreg- cence. Genes expressed encode a number of F-box proteins,
ulated in senescence, according to their annotation (TIGR C3HC4-type RING finger proteins and other members of the
v. 5.0). This confers a potential function for each encoded ubiquitin ligase complex such as the ASK1 protein and E2
protein but in many cases this is due to similarity to known conjugating enzymes. The presence of a RING finger domain
proteins and may not reflect their true activity. The range is a characteristic of RING-class E3 ubiquitin protein ligases
of gene functions represented in the 827 senescence- that act by transferring ubiquitin from an E2 enzyme to a
enhanced genes is illustrated in Figure 1 and summarized substrate protein that is targeted for degradation. F-box
in Table 1. Some of these groups are discussed in the next proteins are also a variable component of an ubiquitin ligase
section. complex and different E3 ligase complexes allow the
targeting of specific proteins for degradation (reviewed in
Regulatory genes. The massive shift in gene expression Smalle and Vierstra, 2004). The upregulation of a number of
patterns and metabolic functions that occurs during senes- these genes during senescence indicates that specific
cence is reflected in the substantial increase in transcript targeting of proteins for degradation may be an important
abundance of putative regulatory genes. Around 100 genes mechanism for the control of senescence. The importance of
encoding putative transcription factors show increased such genes in the control of senescence has been illustrated
expression. These include MYB factors, AP2 domain pro- by the discovery of the F-box gene ORE-9; mutation in this
teins, zinc finger proteins and a number of DNA- and RNA- gene results in a delayed senescence phenotype, indicating
binding proteins (listed in Table S1). In addition, 24 mem- that the target of this protein has an important role in
bers of the family of NAC domain proteins show enhanced senescence (Woo et al., 2001).
expression. In Arabidopsis, there are around 100 of these Many genes involved with protein modification, such as
genes, which contain a plant-specific, highly conserved protein kinases and phosphatases, are upregulated during
N-terminal domain (Ooka et al., 2003) and certain members senescence indicating that kinase-signalling cascades are
have been implicated in different developmental or stress likely to function during senescence. Expression of several
response processes (e.g. Fujita et al., 2004). A similar range genes involved with calcium regulation is increased

Table 1 Potential functional groups of genes showing increased transcript abundance in developmental leaf senescence (from Table S1)
Gene class Putative function Number Examples
Regulation Transcription factors 96 AP2 domain, bZIP, CCAAT binding, Leu zipper, Myb,
NAC domain, WRKY, zinc finger, other DNA binding
Protein:protein interaction 9 Armadillo, WD-40
Ubiquitination control 30 F-box, ubiquitin binding, RING finger
Protein kinase/phosphatase 66 Protein kinase, LRR domain, protein phosphatase
Signalling 17 RALF, RelA/SpoT, remorin
Calcium binding 13 C2 domain, EF hand, calmodulin binding
Hormone pathways 17 Auxin responsive, ethylene responsive, ABA signalling,
cytokinin oxidase
Macromolecule degradation Protein degradation 29 Aspartyl and cysteine proteases, serine carboxypeptidase,
and mobilization vacuolar processing
Amino acid degradation and 27 Glutamine synthetase, homogentisate 1,2-dioxygenase,
N mobilization lysine-ketoglutarate reductase, uricase
Nucleic acid degradation and 14 Nucleases, acid phosphatase
phosphate mobilization
Lipid degradation and mobilization 29 Lipase, a-dioxygenase, acyl-CoA dehydrogenase
Chlorophyll degradation 2 ELIP protein, Rieske domain protein
Sulphur metabolism 2 Allinase, ATP-sulfurylase
Carbohydrate metabolism 63 Amylase, b-1,3-glucanase, polygalacturonase, exotosin,
glucosyl hydrolases, invertase, pectinesterase,
glucosyl transferases
Lignin synthesis 3 Cinnamyl-alcohol dehydrogenase,
caffeoyl-CoA 3-O-methyltransferase
Transport 74 ABC transporters, amino acid permease, cation exchangers,
MATE efflux, sugar and peptide transporters
ATPases 7 AAA type, plasma membrane ATPase
Metal binding 10 Ferritin, copper chaperone, zinc binding
Stress related Antioxidants 11 Alternative oxidase, glutaredoxin, peroxidase
Stress and detoxification 17 DNAJ, heat shock, glutathione S transferase
Defence related 11 Chitinase, isochorismate synthase, major latex protein,
osmotin
Secondary metabolism Alkaloid biosynthesis 9 Strictosidine synthase, reticuline oxidase
Flavonoid/anthocyanin pathway 19 Chalcone synthase, dihydroflavonol reductase,
leucoanthocyanidin dioxygenase
Autophagy 5 Autophagy genes APG7, 8a, 8b, 8h, 9
Structural 4 Actin binding, myosin, tropinone reductase
Unclassified enzymes 110 Cytochrome p450s, copper amine oxidase, dehalogenases,
unknown role short-chain dehydrogenases
Unknown genes 132 Unknown proteins
including genes encoding calcium and calmodulin-binding have roles in plant development or stress responses (Haruta
proteins. Calcium signalling may be an important compo- and Constabel, 2003).
nent in the regulation of senescence processes. The involve-
ment of calcium in many types of cell death has been Macromolecule degradation. Many senescence upregulat-
described (Jones, 2001) and increased levels of calcium ions ed genes encode enzymes likely to be involved in degrada-
have been observed to correlate with senescence of parsley tion of macromolecules and mobilization of metabolites.
mesophyll cells (Huang et al., 1997). Many types of protease, including cysteine, serine, aspartyl
Two RelA/SpoT genes (RSH2 and RSH3) are upregulated; and vacuolar processing proteases are found. Also, genes
these proteins have been suggested to play a part in ppGpp encoding enzymes that degrade amino acids such as lac-
signalling in plant stress responses (van der Biezen et al., toylglutathione lyase which is involved in threonine degra-
2000). Also expressed are two RALF (rapid alkalinization dation and proline oxidase, involved in proline degradation
factor) and a phytosulphokine gene both of which have been are upregulated. Mobilization of nitrogen from the senesc-
postulated as polypeptide hormones (Ryan et al., 2002). ing cell to other parts of the plant is likely to occur via amino
RALF proteins respond to pH changes in cells and appear to acid transport in the phloem. Several genes encoding

enzymes involved in glutamate/glutamine metabolism are metal transport of Hþ exchange. The upregulation of a range
upregulated including two glutamate decarboxylase genes, of transporters, especially those involved in sugars, amino
two glutamate receptor proteins and three cytosolic gluta- acids and peptides, reflects the new function of a senescing
mine synthetases. It has been previously reported that much leaf, as it becomes a source tissue for the mobilization of
of the N transported from senescing leaves is in the form of nutrients to the rest of the plant.
glutamine (Finnemann and Schjoerring, 2000). Many genes
encoding amino acid permeases (eight genes) and peptide Antioxidant genes. During senescence, maintaining pro-
transporters (seven genes) are upregulated indicating in- tection against oxidative stress is important to protect the
creased transport from the leaf cells. cells from premature death and delayed senescence mu-
Three genes that have a role in ureide metabolism tants have been found to have increased tolerance to oxi-
(uricase, xanthine dehydrogenase and allantoinase) show dative stress (Woo et al., 2004). Two genes encoding
increased expression. This may indicate a role for allantoin alternative oxidases (AOX) are upregulated in senescing
or other ureides as alternative N-rich molecules that could leaves. The role of the AOX proteins is thought to limit ROS
be transported from the senescing leaf. Alternatively, or formation by reducing the activity of the electron transport
additionally, these enzymes could be involved in mobiliza- chain thereby reducing oxidative stress in the mitochondria
tion of N from nucleic acid degradation. Massive degrada- (Millenaar et al., 1998). Overexpression of AOX in tobacco
tion of RNA occurs during senescence and several nuclease cell cultures was shown to reduce the overall ROS levels
genes are upregulated. Nucleic acid breakdown is not only a (Maxwell et al., 1999). In addition, three different glutare-
source of nitrogen but also an important source of phos- doxin genes are upregulated during senescence as well as
phorous. Four different acid phosphatase genes are upreg- two peroxidase genes (Table S1). Also, genes encoding en-
ulated; the role of these proteins in plants is not clear but it zymes involved in the synthesis of tocopherol such as 4-
has been postulated that they may be involved in phosphate hydroxyphenylpyruvate dioxygenase and homogentisate
acquisition (Duff et al., 1994). Increased levels of these phytylprenyltransferase are upregulated. Tocopherol, a free
proteins in senescing leaves may indicate a role for these radical scavenger, has been previously implicated as an
proteins in the storage or mobilization of phosphate antioxidant in senescing leaves (Falk et al., 2003).
released by nucleic acid degradation. Phosphate transport
from the senescing leaf may be via the AtPT2 gene which is Autophagy. An interesting class of genes that are upregu-
also upregulated. AtPT2 was identified in phosphate starved lated in tandem during senescence is a group encoding
Arabidopsis leaves and shown to have a role in phosphate autophagy-related proteins. Autophagy is a regulated
transport (Muchhal et al., 1996). recycling process where double-membrane-bound vesicles,
Many of the genes involved in the chlorophyll degrada- known as autophagosomes, traffic cytosolic contents or or-
tion pathway are expressed constitutively in the leaf, imply- ganelles to the vacuole where they are broken down and
ing that the pathway is not under transcriptional control reallocated to essential processes. The role of autophagy in
(reviewed in Hörtensteiner, 2004). Recently, the acd1 gene senescence is not clear but it is likely that some of the mas-
(accelerated cell death 1) was shown to encode the enzyme sive degradation of cellular components could occur via this
pheophorbide a oxygenase (Pruzinská et al., 2003), a key mechanism. Disruption in Arabidopsis autophagy genes has
enzyme in chlorophyll degradation. This gene showed been shown to result in accelerated leaf senescence (Doel-
increased expression during leaf senescence (Table S1) ling et al., 2002; Hanaoka et al., 2002) and this may indicate
but enzyme activity was increased to a greater extent that controlled degradation via autophagy is necessary to
indicating the possibility of post-transcriptional control stabilize cellular viability and carry out efficient senescence.
(Pruzinská et al., 2003). The upregulated expression of the
gene encoding early light-induced protein (ELIP) may be
Gene expression in other types of senescence
linked to chlorophyll degradation. The ELIP proteins bind
free chlorophyll and have been postulated to have a role in Dark-induced senescence. One of the problems that is
protection from oxidative stress caused by the release of encountered when studying developmental senescence in
phototoxic free chlorophyll. This protein may have an Arabidopsis is the lack of consistency in the rates of senes-
important role in the initial stages of degradation and bind cence between leaves and also in different cells within the
the chlorophyll as it is released from the pigment protein same leaf. Many research groups have exploited the use of
complexes (Binyamin et al., 2001; Hutin et al., 2003). dark-induced senescence as a convenient method to syn-
chronize the senescence process as many of the events that
Transport. Many transporter genes show increased tran- occur in dark treated leaves are known to mirror those that
script levels during senescence including a number of ABC occur in developmental senescence. However, there have
transporters and several sugar, peptide, amino acid and been concerns expressed about the validity of this method
cation transporters, many of which are associated with as a direct comparison to natural senescence (e.g. Becker

and Apel, 1993). Weaver et al. (1998) showed that several

senescence-associated genes were differentially expressed Cell suspension Dark induced
senescence senescence
after stress treatments including darkness. Lin and Wu (2004)
recently carried out microarray analysis to characterize gene 308
expression during dark-induced senescence in Arabidopsis 776 1233
leaves and they identified many metabolic pathways that
were altered. The availability of global gene expression data
for developmental and dark-induced senescence has 229
allowed us to carry out a direct comparison of gene
expression in natural developmental senescence with that in
dark-induced senescence to clarify the different and com-
mon processes that occur in the two types of senescence.
We used the Affymetrix expression data to compare the 218
two types of senescence. Genes showing a threefold
expression change between untreated plants and 5-day
Developmental leaf senescence
dark-treated plants were identified and compared with the
gene list described above. Of the 827 senescence-enhanced
genes, 53% (437) were also at least threefold upregulated in Figure 2. Venn diagram to illustrate the number of genes upregulated in the
three different types of senescence.
the dark-treated leaves. However, perhaps surprisingly, 34%
Numbers of genes in each category are shown. All the lists represented in the
(277) of the genes were not upregulated at all in the dark (the developmental senescence groups can be determined from Table S1.
remaining 13% were upregulated two- to threefold). The The groups only include genes that show at least a threefold upregulation
in the relevant experiments.
effect of dark treatment on the expression of senescence-
enhanced genes can be seen in Table S1 and many differ-
ences are obvious. In addition, there were nearly 2000 genes
that were at least threefold upregulated in the dark treat- The expression of genes in dark-induced senescence
ment, meaning that around 1500 of these were not altered in was included in a combined analysis of the three types of
developmental senescence. Examination of the differences senescence (Figure 2). The majority (65%) of the genes that
in gene expression between these two types of senescence were common to developmental and cell culture senes-
offers a new insight into the events taking place. cence also showed increased expression during dark-
induced senescence. Thus, there is a group of 229 genes
that are expressed in all three types of senescence and a
Starvation-induced senescence in suspension culture cells
group of 218 genes only upregulated in leaf senescence.
Programmed cell death is observed in heterotrophic sus- Also of interest is a larger group of 308 genes that show
pension cultures that are starved of sucrose and allowed to increased expression only in dark-induced and cell culture
enter a senescent phase. The hallmark features of PCD such senescence and not in leaf senescence. This may imply
as DNA laddering and membrane shrinkage are observed in that there is more in common between dark- and starva-
these ageing and dying cultures (Swidzinski et al., 2002). An tion-induced senescence than with developmental leaf
Affymetrix GeneChip analysis was carried out using RNA senescence.
isolated from suspension culture cells that had been grow-
ing for 10 days without subculturing and gene expression
Comparative analysis of genes expressed in three types of
patterns were compared to control cultures that had ob-
senescence
tained the correct culturing regime with fresh sucrose (as
described in Swidzinski et al., 2002). Genes that showed at The gene expression data were analysed to identify groups
least a threefold increase in transcript abundance in the of genes that showed different expression patterns in the
starved cultures were identified and subjected to a similar three types of senescence. There is likely to be more noise in
analysis as that described above for the dark-induced sen- the dark-induced and cell suspension senescence experi-
escence experiment. Of the 827 senescence-enhanced ments compared with the leaf senescence data as this
genes, 326 also showed at least threefold upregulation in the comes from a combination of two independent experiments.
senescing suspension culture cells (data shown in Table S1). Therefore, the numbers of genes differentially expressed
However, this means that around 500 of the genes upregu- only in dark senescence or cell suspension senescence is
lated in developmental senescence were not upregulated in probably higher than if more replicates had been analysed.
senescing suspension cultures. Also, over 1000 genes However, in spite of this, a study of general trends can be
showing at least a threefold increased expression in the carried out and obvious differences between the three types
senescing cultures were not upregulated in leaf senescence. of senescence can be identified.

inducible genes such as the type A response regulators,

Differential expression in the different types of senescence
ARR4, ARR6, ARR7 and ARR9, that have key roles in
Hormone signalling pathways. Analysis of genes involved cytokinin signalling and two genes involved in cytokinin
in different hormone signalling pathways allows us to as- synthesis, an isopentenyl phosphotransferase and a cytoki-
sess their relative importance in different types of senes- nin synthase gene, show reduced transcript abundance in
cence. Abscisic acid (ABA) has been implicated in the senescence (Table 2). Parallel to that is an increase in a
regulation of stress-induced senescence (Yang et al., 2003) cytokinin oxidase transcript that may be involved in cytoki-
and expression of several senescence-enhanced genes is nin degradation.
induced on treatment with ABA (Weaver et al., 1998). The However, analysis of expression patterns of these genes
gene encoding the key enzyme in ABA biosynthesis, 9-cis- does indicate a difference between the three types of
epoxycarotenoid dioxygenase (NCED), and two aldehyde senescence (Table 2). Many of the cytokinin signalling genes
oxidase genes AAO1 and AAO3, which are involved in ABA that are downregulated in developmental senescence are
biosynthesis (Seo et al., 2000), show increased transcript not expressed at all in suspension culture cells (senescent or
abundance, indicating that ABA levels probably increase in control) and this indicates that loss of cytokinin is unlikely to
senescing leaves. Also, two protein phosphatase genes, be a signal for the senescence/cell death processes that are
implicated in early events of ABA signalling, ABI1 and ABA2 occurring in these cells. Cytokinin signalling appears to be
(Leung et al., 1997), are upregulated (Table 2). Investigation affected in dark-induced senescence as reduced expression
of the expression patterns of these genes in dark-induced of cytokinin synthase and one of the cytokinin-related
and cell suspension senescence shows that most of these response regulators is evident. Also, increased expression
genes are also upregulated (Table 2) which indicates that the of cytokinin oxidase occurs in dark-induced senescence.
ABA signalling pathway is active in all three types of sen- Interestingly, increased expression of an ARR1-like type B
escence. response regulator gene (represented by two probes on the
Cytokinin levels are reduced in senescing leaves and this array) is seen in both dark-induced and cell suspension
is thought to be one of the key signals for the initiation of senescence. Type B ARRs act as transcriptional activators of
senescence (Gan and Amasino, 1995). A number of cytokinin the type A ARR genes but their N-terminal domains may
Table 2 Expression changes in genes involved in ABA and cytokinin biosynthesis and signalling in different types of senescence
Leaf Dark Cell suspension

Locus Annotation (sen/MG) (dark/con) (sen/con)
ABA signalling
At3g14440 9-cis-Epoxycarotenoid dioxygenase, putative 5.79 9.22
At4g18350 Neoxanthin cleavage enzyme-like 16.87 10.19
At5g57050 ABI2 protein phosphatase 2C ABI2 (PP2C) 5.48 2.39 83.00
At4g26080a ABI1 protein phosphatase ABI1 4.88 4.34 13.55
At5g02810 ABI3-interacting protein 2.56 3.49 3.09
At2g36270a Abscisic acid insensitive 5 (ABI5) 7.78 61.67
At3g19290 Abscisic acid responsive element-binding factor 2.62 5.85 5.18
At1g49720 Abscisic acid responsive element-binding factor 2.80 8.57 1.64
At4g34000a Abscisic acid responsive element-binding factor(ABF3) 4.58 2.35 2.70
At5g13200 ABA-responsive protein-like 2.61 5.34 2.09
At5g59220 Protein phosphatase 2C-like ABA-induced protein 7.36 7.14 5.69
Cytokinin signalling
At1g75450 Cytokinin oxidase, putative 4.68 3.63 1.73
At3g63110 Cytokinin synthase (adenylate isopentenyltransferase), AtIPT3 0.33 0.15
At1g74890 Arabidopsis response regulators type A, ARR15 0.20 0.03
At1g10470 Arabidopsis response regulators type A, ARR4 0.30 1.09 6.25
At3g48100 Arabidopsis response regulators type A, ARR5 0.33 0.96 2.41
At3g16855 Arabidopsis response regulators type B, ARR1 1.03 6.65 13.41
At3g16857 Arabidopsis response regulators type B, ARR1 2.94 4.73 3.52
At1g27320 Cytokinin receptors, AHK3 2.63 5.88 2.32
At3g16360 Histidine phosphotransfer proteins, AHP4 22.50
Yellow boxes indicate over threefold increase in expression compared with the control (green leaf or untreated sample), blue boxes indicate over
threefold downregulation, grey indicate undetectable expression in that experiment.
a
Senescence-enhanced in ATGE experiment only.

negatively regulate this activation in the absence of cytoki- sis plants (Lam et al., 1995) and in maize seedlings
nin (reviewed in Hutchinson and Kieber, 2002). (Brouquisse et al., 1998).
To help in the investigation of the putative functions in the However, PPDK expression is induced in all three types of
different groups of genes, we made use of the MAPMAN senescence and as genes encoding glyoxylate cycle en-
programme recently described by Thimm et al. (2004). For zymes are not induced (see below), the role of this gene in
this programme, each Arabidopsis gene of known or developmental senescence may be to provide precursors for
predicted function has been assigned to a particular func- glutamine synthesis via PEP carboxylase and/or PEP carb-
tional compartment (bin). Entering the array data for a series oxykinase and the TCA cycle.
of genes allows the display of the relative changes in
expression of different genes on to diagrams of metabolic Lipid catabolism. Many lipid catabolism genes appear to be
pathways. upregulated in dark-induced senescence but many of these
do not show a parallel increase in leaf senescence (Fig-
Nitrogen mobilization. MAPMAN data for metabolic path- ure S1b). Lin and Wu (2004) used the lipid catabolism genes
ways for the genes expressed only in leaf senescence defined by Graham and Eastmond (2002) to examine their
showed few easily identifiable pathways (Figure S1a). expression in dark-induced senescence and concluded that
However, it does show that genes involved in ammonia many genes involved in b-oxidation of lipids were upregu-
assimilation, such as three cytosolic glutamine synthetase lated. We have now compared the expression of these genes
genes, are not expressed in dark-induced or suspension in all three types of senescence and large differences are
culture senescence (Table 3). These enzymes are thought to evident (Table 3). Interestingly there are many more lipid
have a key role in N mobilization from senescing leaves and catabolism genes upregulated in dark and cell suspension
this may imply that N mobilization does not occur in the dark senescence than in leaf senescence. The raw data for these
(or an alternative pathway may operate, see below). Two experiments show that these genes are expressed at a con-
genes encoding glutamate decarboxylase are also only up- siderably higher level in dark-induced senescence. Similarly,
regulated in leaf senescence. Glutamate decarboxylase is sucrose degradation genes are upregulated in dark-induced
involved in the conversion of glutamate to GABA (4-ami- senescence and to a lesser extent in cell suspension senes-
nobutyrate). The role of GABA in the plant is unclear but it cence (Table 3). The parallel regulation of these genes in
may have a role as a signalling molecule to coordinate the dark-induced and cell suspension senescence is likely to be
C:N balance in changing nutrient environments as is occur- related to carbohydrate starvation. Similar effects of sucrose
ring during senescence (reviewed in Bouche and Fromm, starvation have been seen in other studies on Arabidopsis
2004). It is thought that the C:N balance in plants could be plants (Thimm et al., 2004) and sucrose-starved suspension
controlled via a family of glutamate receptors (ATGLRs) cultures (Contento et al., 2004). Loss of photosynthetic fixed
(Kang and Turano, 2003) and two members of this family carbon, when plants are placed in the dark, results in rapid
show increased expression in senescence (Table S1). depletion of the sugar levels in the leaves (Brouquisse et al.,
Metabolism of GABA is via the GABA shunt, which involves 1998; Tcherkez et al., 2003). Sugar levels in the medium
three enzymes including the cytosolic glutamate decarb- supplying the senescent suspension culture cells fall to be-
oxylase and mitochondrial enzymes GABA transaminase low 20% of the original level (JS, unpublished data). Car-
and succinic semialdehyde dehydrogenase. This pathway bohydrate starvation has been shown to result in significant
has a potential role in reducing oxidative stress in the induction of b-oxidation activity (Graham and Eastmond,
mitochondria (Bouche et al., 2003) and it is possible that this 2002) and this is reflected by the observed upregulation of
could also have an important protective role in senescence. many genes in dark-induced and cell suspension senes-
In contrast, two genes encoding glutamate dehydroge- cence. In developmental leaf senescence, endogenous sugar
nase, and the genes encoding aspartate amino transferase levels tend to increase which would explain why there is no
and asparagine synthase are upregulated in dark-induced extensive induction of b-oxidation genes. However, there is
and suspension culture senescence but not in developmen- some suggestion that senescence in petals could be induced
tal senescence (Figure S1b; Table 3). Lin and Wu (2004) by sugar starvation as external application of sugar can
proposed a novel pathway involving these genes for the delay senescence (van Doorn, 2004).
generation of asparagine for export from dark-treated Interestingly, genes involved in the glyoxylate cycle, such
leaves. It may be that glutamine is the N metabolite as malate synthase and isocitrate lyase do not show any
mobilized in developmental senescence, while asparagine increase in expression in any of the types of senescence.
is primarily mobilized in dark-induced senescence. Lin and These genes have been shown to be upregulated in senesc-
Wu (2004) showed an increase in asparagine levels in the ing tissues of other plants such as cucumber (reviewed in
dark-treated Arabidopsis leaves used for this analysis. An Graham and Eastmond, 2002) and are required for the
increase in asparagine levels in parallel with a decrease in conversion of lipid to sugar via gluconeogenesis. This
glutamine levels has been shown in dark-treated Arabidop- implies that the acetyl CoA released from the b-oxidation

Table 3 Metabolic genes that show differential expression in the three types of senescence
Leaf Dark Cell suspension

Locus Annotation (sen/MG) (dark/con) (sen/con)
N mobilization and transport?

At5g17330 Glutamate decarboxylase 1 10.82 0.65 0.26
At2g02010; At2g02000 Glutamate decarboxylase, putative 21.43 1.32 0.24
At2g41220 Glutamate synthase, chloroplast (GLU2) 3.46 1.63 1.65
At3g17820 Glutamine synthetase (GS1) 4.58 1.64 0.71
At5g16570 Glutamine synthetase, putative 25.19 0.54 2.00
At5g37600 Glutamine synthetase, putative 5.68 2.25 3.48
At5g18170 Glutamate dehydrogenase 1 (GDH1) 0.68 6.58 1.01
At5g07440 Glutamate dehydrogenase 2 (GDH2) 2.30 12.19 6.20
At3g47340 Glutamine-dependent asparagine synthetase 1 0.74 7.43 199.16
At5g11520 Aspartate aminotransferase 1.83 11.50 5.51
At2g38400 Alanine–glyoxylate aminotransferase 2.31 114.85 19.44
Pyruvate orthophosphate dikinase
At4g15530 Pyruvate phosphate dikinase 5.14 9.33 131.77
Genes involved in fatty acid catabolism (as defined in Graham and Eastmond, 2002)
Fatty acid activation and import into the peroxisome
At3g05970 Long-chain-fatty-acid–CoA ligase 2.38 3.45 1.40
At3g16910 AMP-dependent synthetase and ligase 1.42 1.25 3.96
At5g16340 AMP-binding protein, putative 2.17 4.23 14.75
At4g39850 Peroxisomal ABC transporter (PXA1) 3.48 4.20 2.07
Peroxisomal straight chain saturated and unsaturated fatty acid b-oxidation
At4g16760 Acyl-CoA oxidase (ACX1) 2.40 2.89 1.83
At3g51840 Short-chain acyl-CoA oxidase 1.93 4.01 8.97
At2g35690 Acyl-CoA oxidase, putative 1.21 2.34 3.42
At3g06810 acyl-CoA dehydrogenase-related 3.04 5.94 3.21
At3g06810 Acyl-CoA dehydrogenase-related 1.78 2.90 2.59
At4g29010 Fatty acid multifunctional protein (AIM1) 2.16 2.79 4.38
At3g06860 Fatty acid multifunctional protein (MFP2) 4.25 4.32 3.53
At3g15290 3-Hydroxybutyryl-CoA dehydrogenase 1.01 2.32 2.00
At2g33150 Acetyl-CoA C-acyltransferase 2.31 5.21 4.41
Branched chain fatty acid b-oxidation
At3g06850 Branched chain a-keto acid dehydrogenase E2 1.58 15.45 20.96
At3g13450 Branched-chain a-keto acid dehydrogenase E1 1.00 20.56 29.72
At1g55510 2-Oxoisovalerate dehydrogenase 1.48 14.90 17.82
At3g45300 Isovaleryl-CoA-dehydrogenase 2.08 11.15 34.34
At1g03090 Methylcrotonyl-CoA carboxylase 0.97 18.49 17.14
At3g60510 Enoyl-CoA hydratase/isomerase 0.66 2.83 3.63
Fatty acid a-oxidation
At3g01420 a-Dioxygenase 23.67 56.46 270.74
At1g73680 a-Dioxygenase 3.39 3.02 7.36
At1g54100 Aldehyde dehydrogenase 5.86 8.32 2.82
Sucrose catabolism
At1g35580 b-Fructofuranosidase/invertase, 0.90 3.63 4.74
At3g06500 b-Fructofuranosidase/invertase, 0.64 13.26 8.66
At3g13790 b-Fructosidase (BFRUCT1) 1.70 3.27 12.99
At1g12240 b-Fructosidase (BFRUCT4) 0.43 7.77 97.61
At5g20830 Sucrose synthase 2.61 10.63 4.45
At1g62660 b-Fructosidase (BFRUCT3) 1.86 5.01
At1g50460 Hexokinase 0.66 3.09 0.52
Trehalose metabolism
At1g23870 Trehalose-phosphatase 1.26 16.48 17.06
At4g24040 Trehalase, putative 1.77 12.21 15.66
Colour representation as in Table 2.

of fatty acids is respired directly in Arabidopsis and is not (Schluepmann et al., 2003) and there is evidence that tre-
used for gluconeogenesis. halose-6-phosphate is required for regulation of sugar
Two genes involved in branched chain amino acid (leu, ile metabolism (Eastmond et al., 2003). Genes encoding TPS
and val) catabolism, BCKDH subunits E1 and E2 are upreg- and TPP are upregulated in dark-induced senescence. The
ulated in dark-induced and cell suspension senescence but induction of this pathway has previously been reported in
not in developmental leaf senescence (Figure S1b; Table 3) plants exposed to a long night period, and in sugar-starved
(Fujiki et al., 2001). Branched chain amino acid catabolism suspension cultures by Thimm et al. (2004) and Contento
may be activated specifically in low carbohydrate conditions et al. (2004), respectively. It is likely that the induced
in order to provide an alternative carbohydrate source to the expression of this pathway is to try to redress the sugar
cell. b-Oxidation is required for complete degradation of the imbalance caused by the dark or starvation treatments and
ketoacids. thus this pathway is not required during developmental
Genes involved in a-oxidation of lipids show increased senescence.
transcript abundance in all three types of senescence
(Table 3) and may have a role in a response to stress (De Flavonoid biosynthesis. Another group of genes that stand
Leon et al., 2002; Hamberg et al., 1999). Lipid 2-hydroper- out as being differentially expressed in the three types of
oxides, which are produced by a-oxidation, may be precur- senescence are those genes involved with flavonoid bio-
sors of the oxylipin signalling molecules, including JA. The synthesis. MAPMAN analysis shows that approximately the
increased expression of these genes may be related to a same number of genes that have been classified in this
stress response rather than having a fatty acid degradation group are expressed in developmental and dark-induced
role. senescence (Figure S1a,b) but a closer examination of these
indicates that only one is common to both (Table 4). It ap-
Trehalose metabolism. Another pathway that shows altered pears that a different flavonoid biosynthesis pathway may
expression in dark-induced and cell suspension senescence operate in the absence of light. Flavonoid pathway enzymes
but not developmental senescence is that involved in tre- such as those encoded by chalcone synthase (At5g13930)
halose metabolism (Figure S1; Table 3). Trehalose is syn- and dihydroflavonoid reductase (At5g42800) were consid-
thesized from glucose by the action of trehalose-6- ered to be represented by single copy genes in Arabidopsis
phosphate synthase (TPS) and trehalose-6-phosphate as mutants in these cause a transparent testa phenotype and
phosphatase (TPP). The role of trehalose in plants is not flavonoids were absent in other parts of the plant
clear but the pathway is essential for development (reviewed in Winkel-Shirley, 2001). However, this flavonoid
Table 4 Genes that may have a role in flavonoid biosynthesis
Leaf Leaf Dark Dark Cell suspension Cell suspension

Locus Annotation (raw) (sen/MG) (raw) (dark/con) (raw) (sen/con)
At4g22870 Leucoanthocyanidin dioxygenase 2376 105.03 4 7

At3g29590 Anthocyanin 5-aromatic acyltransferase 313 48.10 7 5
At5g39090 Anthocyanin 5-aromatic acyltransferase 186 5.08 18 5
At5g39050 Anthocyanin 5-aromatic acyltransferase 526 5.82 162 2.37 54
At5g13930 Chalcone synthase 3014 9.65 2 8
At5g42800 Dihydroflavonol 4-reductase 2571 45.87 11 31
At4g12300 Flavonoid 3¢,5¢-hydroxylase 370 3.41 131 1.87 221 5.12
At5g07990 Flavonoid 3¢-hydroxylase (F3¢H) 511 9.24 26 25
At5g54060 Glycosyltransferase family protein 1630 49.67 5 16
At5g17050 UDP glucose:flavonoid 3-o-glucosyltransferase 919 9.82 15 23
At3g55970 Leucoanthocyanidin dioxygenase 259 10.67 628 35.92 2
At1g19540 Isoflavone reductase 25 1642 35.50 303 3.35
At2g38240 Flavonol synthase 139 2.05 368 36.34 30
At5g49690 UDP-glucoronosyl/UDP-glucosyl transferase 23 81 38.99 155 3.66
At2g39980 Transferase family 34 378 4.00 16
At1g02050 Chalcone and stilbene synthase family 17 70 7.38 10
At4g35420 Dihydroflavonol 4-reductase family 30 100 9.81 48
At5g24530 Flavanone 3-hydroxylase 1489 1.75 770 4.67 583 13.46
At3g50210 2-Oxoacid-dependent oxidase 431 2.78 180 2.95 152 2.36
At5g05600 Flavonol synthase 254 1.63 504 5.23 49
At4g16330 Flavonone-3-hydroxylase 429 1.04 618 4.17 456 1.67
Colour representation as in Table 2.

biosynthesis pathway is regulated in leaves by light in a The senescence-related expression of the remaining 575
diurnal fashion (Harmer et al., 2000) and therefore may not senescence-enhanced genes is therefore independent of
be activated in dark-induced senescence. This may indicate these signalling pathways although more replicate experi-
specific regulation of an alternative pathway in dark-induced ments would be needed to prove that there was no effect of
senescence with increased expression of potential alterna- the mutation. There are several genes that appear to be
tives to these genes, e.g. At1g02050 (chalcone and stilbene downregulated in two of the three mutants and a few that
synthase family, similar to rice chalcone synthase), require all three pathways for optimum expression. For
At4g35420 (dihydroflavonol reductase family) and two dif- example, 26 genes are downregulated in both coi1 and ein2
ferent flavonol synthase genes. In cell suspension senes- backgrounds and 25 genes show lower expression in NahG
cence this pathway appears not to be induced (Table 4). This and coi1. A small number of genes are upregulated in each
raises the question of the role of this pathway in senescence. mutant and this may indicate that the pathway that controls
In developmental leaf senescence, it is thought that in- these genes is suppressed by the presence of the other
creased light stress due to the degradation and release of signalling pathway. For example, several genes that require
chlorophyll increases the need for protective flavonoid and the JA and ethylene pathways for expression are upregu-
anthocyanin in the senescing tissues. Indeed this is why lated in the NahG background. It has been shown previously
leaves turn orange and red in the autumn (Feild et al., 2001). that the presence of the SA signalling pathway represses
In dark-induced senescence different stresses might be evi- gene expression induced by the JA/ethylene pathway sys-
dent, macromolecule degradation results in the release of tem during pathogen responses (Gupta et al., 2000) and the
ROS and increased flavonoid production might have a pro- same may be true in senescence.
tective role. However, the same stresses are present in the
cell suspension senescence but no similar pathway is Jasmonate and ethylene pathways. Examination of the
expressed. genes that are altered in each mutant background gives an
indication of which genes may be controlled by each
pathway. A large proportion of the genes altered by the
Role of signalling pathways in gene expression during
mutants in the JA and/or ethylene pathways encode
senescence
hydrolases (Figure 3) and many are present in the carbo-
The initiation and progression of plant senescence appears hydrate metabolism section of Table S1. Genes that are
to involve a complex combination of signalling pathways downregulated in both coi1 and ein2 mutants are shown in
with considerable crosstalk between other plant responses. Table 5 and have been divided into three groups. These
The elucidation of the role and importance of each of these genes mostly encode proteins such as polygalacturonase
pathways will help in the clarification of the genes and sig- and pectinesterase that may have a role in cell wall deg-
nals that are senescence-specific. As described above, three radation. Also two nuclease genes RNAse1 and BFN1 are
well-characterized stress response pathways (those invol- expressed at a lower level in the mutants. Therefore, some
ving ethylene, JA and SA signals) have been implicated in senescence-related degradation functions (carbohydrates
senescence (Grbic and Bleecker, 1995; He et al., 2002; Morris and nucleic acids) may depend on signalling pathways
et al., 2000). Levels of these signalling molecules increase involving JA and ethylene. The presence of the potential
during senescence and induce the expression of specific regulator, the NAC domain gene, NAC3, in this group
genes. We have used Affymetrix array experiments to indicates a potential role for this gene in controlling this
identify alterations in senescence-related gene expression in aspect of senescence. Expression of the alpha-DOX1
plants that are defective in each of these pathways. This has (At3g01420) is dependent on both JA and ethylene path-
allowed us to group the senescence-enhanced genes into ways and is also slightly downregulated in the NahG
classes that are dependent on one or other signalling path- plants. This gene (described above as being involved in
ways and identify the genes that are independent of these a-oxidation of fatty acids; Table 2) has been shown to have
stress-related signals. a role in oxylipin production protecting plants from oxi-
Of the 827 genes that are upregulated during senescence, dative stress and cell death (De Leon et al., 2002). In this
19% (165) show at least twofold reduced expression in the paper, expression of the alpha-DOX1 gene was shown to
NahG transgenic plant that is defective in the SA signalling require the presence of the SA pathway but JA and ethy-
pathway (Gaffney et al., 1993); 12% (103 genes) show lene signalling was not examined. The expression of this
reduced expression in the coi1 mutant defective in the JA gene, which probably leads to JA and other oxylipin bio-
signalling pathway (Xie et al., 1998) and 9% (73 genes) show synthesis is reduced in the absence of JA and ethylene
reduced expression in the ethylene signalling mutant ein2 signals and this may indicate a feedback control of this
(Alonso et al., 1999). The expression ratio for each of the signalling pathway.
senescence-enhanced genes in senescing leaves of each Some genes that depend on JA and ethylene show
mutant compared with the wild type is shown in Table S1. increased expression in the absence of the SA pathway.

that even within this small group there are three overlapping
indin g Coil but distinct pathways that control their expression.
Pro tein b
lar fu nction
s The majority of the genes that depend on JA and ethylene
m olecu
Other activ
ity for expression during leaf senescence show increased
ctivity rase vity
ctor a Trans
fe e acti
on fa Kinas abundance during both dark-induced and cell suspension
Tran scripti vity
e acti
enzym senescence (Table S1, Table 5). This indicates that these
y Other
ctivit
p orter a
Trans pathways have an active signalling role in these other two
types of senescence.
g
bindin
otide
Nucle
y Salicylic acid pathway. The majority of the genes that are
bindin
g activit
Other lase
Hydro dependent on the SA pathway encode kinases, transf-
unk...
nction erases and hydrolases (Figure 3; Table S1). The SA path-
ular fu
Molec
way has a key role in the disease resistance response and
tions
this may be reflected in the fact that there are many
r func
r mo lecula Ein2 putative disease resistance leucine-rich repeat genes
Othe
ity ctivity dependent on the SA pathway for expression during
activ rase a
tion factor Trans
fe
activit
y
crip Kinas
e
Trans senescence. Hydrolases that show dependence on the SA
ctivity activity
Trans
p orter a enzym
e pathway include a class IV chitinase and also the senes-
Other
cence-specific protease SAG12. This result is supported
by previous work in which we found that expression of
g
bindin
otide both these genes was downregulated in several mutants
Nucle
bindin
g defective in the SA pathway (Morris et al., 2000). Other
Other ctivity
lase a hydrolases downregulated in NahG include three different
k... Hydro
un
nction
ular fu AAA-type ATPases all of which are in close proximity in
Molec
the genome. AAA proteins act in a variety of cellular

RNA bi
nding functions, including cell cycle regulation, protein degra-
DNA or
NahG dation, organelle biogenesis and vesicular transport;
binding
Protein
hence, the origin of their name (ATPases associated with
nctions
ol ecular fu different activities). Dependence on the SA pathway for
Other m
ctiv ity
tivity rase a expression may implicate these genes in pathogen re-
ption factor ac Transfe
Transcri
ity
sponses or may indicate a senescence-specific role for the
ort er activ activity
Transp Kinase SA pathway. Similarly, several genes involved in calcium
signalling and a group of putative glucosyl and fucosyl
in g transferases do not show senescence-enhanced expres-
ide bind
Nucleot
ity sion in the NahG transgenic plant.
bindin
g e activ
Other enzym
Other Significantly, many of the genes that are the most reduced
un... lase activity
nction Hydro in expression (over fourfold) in the NahG senescent leaves
ular fu
Molec
do not show increased expression in dark-induced or in cell
Figure 3. Functional categories of genes dependent on different hormone suspension senescence (Table 6; Table S1). Genoud et al.
signalling pathways for senescence-enhanced expression. (2002) reported that the efficient activation of the SA
Each senescence-enhanced gene that was at least twofold downregulated in
any of the three hormone-deficient backgrounds was assigned a putative signalling pathways during pathogen responses was
molecular function according to their GO annotations (Gene Ontology dependent on light signals and both SA-induced gene
Consortium, 2000). Pie charts show the proportions of each function that expression and the hypersensitive response were strongly
was reduced in expression in the Coi1, Ein2 mutant and NahG transgenic
plants. reduced in tissues in the dark. The same is evident in dark-
induced senescence.
Senescence phenotype of NahG transgenic plants

Examples of such genes are a chitinase, a glucanase and an
osmotin-like gene (thaumatin-like) all of which have been As shown above, a group of genes that show enhanced
implicated in pathogen responses. There are also a few expression in developmental senescence but not in dark-
genes that appear to be dependent on all three signalling induced senescence are dependent on the SA pathway for
pathways for expression. This group includes a monooxyg- expression (Table 6). Therefore, the potential importance
enase, a putative subtilase and an ABC transporter. All genes of this group of genes in senescence could be asses-
shown in Table 5 are senescence-enhanced and it appears sed by comparing the progress of the two types of

Table 5 Senescence-enhanced genes showing reduced expression in Coi1 and Ein2 mutants
Identifier Annotation Sen/MG NahG Coi1 Ein2 Dark/light Cell death/control
At3g15500 No apical meristem (NAM) family (NAC3) 19.82 0.99 0.31 0.26 29.20 25.87
At1g61566 Rapid alkalinization factor (RALF) 11.73 0.84 0.24 0.38
At2g02990 Ribonuclease 1 (RNS1) 9.47 1.89 0.25 0.38 1.75 0.54
At1g11190 Bifunctional nuclease (BFN1) 14.09 0.7 0.26 0.43 22.85 97.90
At2g41850 Endo-polygalacturonase 13.17 1.77 0.03 0.11 44.08
At2g47040 Pectinesterase family 82.09 0.91 0.04 0.11
At3g57510 Endo-polygalacturonase (ADPG1) 22.32 0.8 0.18 0.14 5.18
At4g37990 Mannitol dehydrogenase (ELI3-2) 21.84 1.8 0.2 0.21 15.16 22.98
At3g60140 b-Glucosidase 68.13 1.05 0.21 0.24 61.57 155.61
At1g02790 Exopolygalacturonase (PGA3) 8.88 0.56 0.24 0.33
At1g62760 Invertase/pectin methylesterase inhibitor 73.71 1.12 0.33 0.38 30.04 14.72
At2g14620 Xyloglucan:xyloglucosyl transferase 8.06 0.54 0.35 0.3 6.65
At2g29470 Glutathione S-transferase 25.59 0.83 0.05 0.11 46.01 7.98
At3g01420 a-Dioxygenase 23.67 0.59 0.28 0.23 56.46 270.74
At4g37430 Cytochrome P450 81F1 20.20 0.8 0.17 0.36 19.48 57.69
At3g12500 Basic endochitinase 6.79 3.93 0.2 0.16 1.56 0.28
At4g16260 Glucan endo-1,3-b-glucosidase 11.97 5.29 0.2 0.14 19.50 116.15
At4g11650 Osmotin-like protein (OSM34) 56.82 6.77 0.21 0.3 9.36
At1g19250 Flavin-containing monooxygenase 15.38 0.1 0.21 0.24
At1g32960 Subtilase family protein 9.13 0.29 0.21 0.38 0.28
At1g15520 ABC transporter family protein 7.64 0.17 0.26 0.25 4.37
Colour representation as Table 2.
senescence in NahG plants. The third and fourth leaves loss of this protein has a strong influence on the delayed
were sampled at different times of development (for the senescence phenotype.
developmental senescence analysis) and after 0, 2 or
4 days in the dark for the dark-induced senescence ana-
Conclusions
lysis. Developmental senescence is delayed considerably
in the NahG transgenic plants, both by assessment of Three different types of senescence have been studied in
chlorophyll levels and by measurement of photosynthetic this paper and differences in gene expression have been
activity Fv/Fm (Figure 4). However, there is no difference characterized. The similarities and differences that have
in the rates of change of these two parameters in the been observed are summarized by the model in Figure 5. In
dark-induced senescence between the wild type and the all cases, the senescence results in dismantling of cellular
NahG transgenic plants. This is clear evidence that the SA constituents and degradation of macromolecules including
pathway has a very important specific role to play in proteins, lipids and nucleic acids. Thus it is likely that genes
developmental senescence. that we have found to be expressed in all three types of
Some of the genes that depend on the SA pathway for full senescence are involved in these common processes. In
expression in developmental senescence but are not addition, to extend viability in the senescing cells there are a
expressed in dark-induced senescence are shown in Table 6. number of stress pathways including those dependent on
Within this list there are likely to be some key genes that are JA and ethylene that are expressed in all three senescence
essential for the normal progression of senescence and processes.
functional analysis of these is an important next step to There are also considerable differences in processes that
define the importance of each gene. The list includes several take place in the three types of senescence. In developmen-
leucine-rich repeat genes similar to known disease resist- tal senescence, photosynthesis continues, although at a
ance genes and many of these are known to depend on the reduced rate, and this presumably provides some energy for
SA pathway for expression (Durner et al., 1997). Also there the process to take place. However, leaves undergoing
are a number of other kinase genes and two transcription senescence in the light have to contend with oxidative stress
factors that may have a regulatory role. The senescence- resulting from the degradation products of chlorophyll and
specific SAG12 gene appears on this list, this gene has been other macromolecules. Genes related to flavonoid synthesis
shown previously to be partly dependent on the SA pathway show increased expression in developmental senescence
for expression (Morris et al., 2000) but there is still some only although an alternative pathway of unknown function
gene expression in the NahG plants so it is unlikely that the may be involved in dark-induced senescence. In dark-

Table 6 Developmental senescence-enhanced genes dependent on SA pathway (at least fourfold downregulated in NahG), not upregulated in
the dark or in cell suspension senescence (less than twofold)
Locus identifier Annotation (putative function) Sen/MG NahG/WT Dark/light Cell suspension/con
At3g28580 AAA-type ATPase 9.78 0.08 0 0.40

At3g28540 AAA-type ATPase 5.17 0.15 0.07 0
At3g28510 AAA-type ATPase 19.33 0.02 0 0
At3g13100 ABC transporter 3.88 0.16 0 0
At3g47480 Calcium-binding EF hand 4.97 0.07 1.44 0.26
At5g39670 Calcium-binding EF hand 3.18 0.13 1.62 0.22
At1g33960 Avirulence-responsive (AIG1) 3.57 0.05 0 0
At2g33080 Leucine-rich repeat family protein 53.38 0.16 0 0
At3g24900 Leucine-rich repeat family protein 4.45 0.04 0.48 0
At4g28490 Leucine-rich repeat family protein 5.58 0.21 1.62 0
At1g74710 Isochorismate synthase 1 (ICS1) 3.30 0.10 1.33 0
At2g19190 Receptor-like kinase,(SIRK) 9.86 0.18 0 0
At4g04500 Protein kinase family protein 8.30 0.08 0 0
At4g23150 Protein kinase family protein 4.38 0.04 0 0
At1g21240 Wall-associated kinase, putative 5.83 0.06 0 0
At4g35580 No apical meristem (NAM) family 11.82 0.08 2.92 0
At2g26400 Acireductone dioxygenase (ARD/ARD¢) 7.42 0.02 0 0.26
At3g13620 Amino acid permease family protein 10.48 0.05 0 0
At4g10500 Oxidoreductase, 2OG-Fe(II) oxygenase 5.93 0.04 2.62 0
At1g19250 Flavin-containing monooxygenase 15.38 0.10 0 0
At3g07600 Heavy-metal-associated domain 9.92 0.12 0 0
At5g24550 Glycosyl hydrolase family 1 protein 6.38 0.07 0 0
At5g11920 Glycosyl hydrolase family 32 protein 4.36 0.21 0 0.65
At3g53150 UDP-glucosyl transferase 12.84 0.04 0 0
At1g35230 Arabinogalactan-protein (AGP5) 3.06 0.09 1.09 0
At1g14080 Xyloglucan fucosyltransferase (FUT6) 96.00 0.17 0 0
Protease genes partially downregulated in the NahG background
At5g45890 SAG12, cysteine proteinase 203.07 0.37 0 38.79
At1g44130 Nucellin protein, putative 5.70 0.35 2.45 0.23
At1g32960 Subtilase family protein 9.13 0.29 0 0.28
The value 0 indicates undetectable expression in any sample of that experiment.
induced and cell suspension senescence the main signal for senescence. Developmental senescence is delayed in plants
the process could be a rapid reduction in sugar levels, defective in SA signalling but dark-induced senescence
leading to a more significant switch to lipid degradation to progresses normally in these plants. This shows that one
supply an energy source. The differences in the C:N balance or more of the genes that depend on the SA pathway for
or the source of carbon skeletons in the light grown tissues expression during senescence has an important role in the
compared with the other two treatments may be the reason developmental senescence process.
why these cells appear to synthesis glutamine instead of
asparagine for export. In the senescing suspension culture
cells there are also clear indications of PCD occurring and Experimental procedures
this is likely to require a set of genes not expressed in
the senescing leaves, at least not at the time they were Plant material
harvested. Developmental senescence and mutant analysis. Arabidop-
Signalling pathways that are induced during senescence sis plants (wild types Col-0, Col-5, transgenic NahG, and mutants
have a downstream effect on transcription and genes that ein2 and coi1) were grown at 22C under 12 h day/12 h night
depend on the three stress response pathways, involving conditions. The two wild-type green leaf samples Col-0 and Col-5
were harvested at Boyes stage 3.9 (Boyes et al., 2001). Three to four
SA, JA and ethylene, for senescence-enhanced expression,
fully expanded leaves were harvested from approximately 15 indi-
have been identified. The SA pathway is not expressed in vidual plants. For the senescent samples, wild-type Col-0 and Col-5
dark-induced or cell suspension senescence while the JA and mutants in genes in the ethylene pathway (ein2) and the
and ethylene signals are clearly active in all three types of jasmonate pathway (coi1) and the NahG transgenic plant which is

Figure 4. Comparison of senescence param-

(a) (c)
eters in wild-type (Col) and NahG plants during
Chlorophyll content (%)

age-dependent senescence (a, b) and dark-
Chlorophyll content (%)

100 100
induced senescence (c, d).
Age-dependent senescence symptoms in the Col 80 80
and NahG transgenic line. Chlorophyll content 60 60
(a) and photochemical efficiency (Fv/Fm) of PSII 40 40
(b) was examined every 4 days from 20 to 44 Col Col
DAE. 20 20
NahG NahG
Dark-induced senescence symptoms in the Col 0 0
and nahG transgenic line. Chlorophyll content (c) 20 24 28 32 36 40 44 0 2 4 6
and photochemical efficiency (Fv/Fm) of PSII (d) DAE Days
was examined every 2 days during dark incuba-
tion. (b) (d)
Fv/Fm, maximum quantum yield of PSII electron
100 100
transport (maximum variable fluorescence/maxi-
80 80
Fv/Fm (%)
mum yield of fluorescence). Error bars indicate
Fv/Fm (%)
SD. DAE, days after emergence. 60 60
40 40
Col Col
20 20
NahG NahG
0 0
20 24 28 32 36 40 44 0 2 4 6
DAE Days
defective in the SA pathway were grown until the mid flowering arrays/specific/arab.affx) containing 22 810 probe sets. Two inde-
stage (Boyes stage 6.0–6.9; Boyes et al., 2001). Three to four partially pendent biological replicates were performed for all samples except
senescent leaves, showing 0–10% yellowing were harvested from the dark control sample which was carried out on one slide only.
approximately 15 plants at this stage. The leaves from the NahG The array experiments for the developmental senescence and the
plants were harvested when they were at the same senescent stage mutant analysis (VBW) and the suspension culture cell death ana-
as the wild type and other mutants. Because of the delayed senes- lysis (JS and CL) were carried out at Nottingham Arabidopsis Stock
cence in the NahG plants, the harvested leaves were likely to be Centre (NASC) using Affymetrix recommended protocols for cDNA
older than the wild-type leaves. RNA was isolated by the method of synthesis, array hybridization, chip scanning and data accumulation
Logemann et al. (1987) and purified using RNAeasy columns using the Affymetrix Microarray Suite version 5.0. The ATGenEx-
(Qiagen, Valencia, CA, USA). Two independent biological replicates press data were from experiments carried out by the European
were used for each mutant. For the AtGenExpress experiments leaf consortium coordinated by Lutz Nover (Frankfurt), Thomas Altmann
6 or 8 were used from 17-day-old plants for the green leaf samples (Potsdam) and Detlef Weigel (Tübingen) (http://web.uni-
and leaves 6–8 were used from 35-day-old plants for the senescent frankfurt.de/fb15/botanik/mcb/AFGN/atgenex.htm). These data
leaf sample. were from triplicate slides for each time point. The raw data files for
the NASC experiments and the AtGenExpress data are publicly
Dark-induced senescence. The methodology and data used in available and can be downloaded from NASC (http://affyme
this paper were originally described in Lin and Wu (2004). Arabid- trix.arabidopsis.info/narrays/experimentbrowse.pl).
opsis thaliana ecotype Col-0 was grown at 22C under short-day Raw data from each experiment were scaled to an average of 100.
conditions (8-h L/16-h D) with 100 lmol m)2 sec)1 white light. For Data were normalized in GeneSpring (Silicon Genetics, Redwood
dark treatments, Arabidopsis plants at stage 1.10 (10 true leaves; City, CA, USA) using the default one colour normalization procedure
approximately 40–50 days after germination) were placed in the (each measurement was divided by the 50th percentile of all
same growth chamber (22C) without light (light switched off at measurements in that sample, each gene was divided by the
dusk of the day prior to treatment). The plant materials for control median of its measurements in all samples). GeneSpring was used
(0 day), 1 day and 5 days were harvested at the days indicated. At to filter out unwanted genes and to identify up- and downregulated
each time point, 12 plants were harvested for RNA isolation and genes for further analysis. In each experiment, genes that were
subsequent microarray hybridization. flagged absent in all samples and any genes that had a raw value of
Arabidopsis total RNA samples were isolated as described <50 on all slides were filtered out and not analysed further.
previously (Chang et al., 1993). The population of mRNA was then Gene lists to identify genes up- or downregulated in each
isolated from total RNA with use of the Oligotex mRNA kit (Qiagen). experiment were generated as follows.
Affymetrix ATH1 Genome Array hybridization and data acquisition
were performed as described previously (Lin and Wu, 2004). Developmental senescence. The four slides at NASC are:
Buchanan-Wollaston_A-1-bwoll-C0G, Buchanan-Wollaston_A-2-
Suspension cultures. Suspension culture cells were grown, bwoll-C5G_SLD, Buchanan-Wollaston_A-3-bwoll-C0S_SLD, and
harvested and RNA isolated as described in Swidzinski et al. (2002). Buchanan-Wollaston_A-4-bwoll-C5S_SLD, and the nine slides from
ATGE are: ATGE_14 _A, ATGE_14_B, ATGE_14_C. ATGE_15_ A,
ATGE_15_B, ATGE_15_C, ATGE_25_ A, ATGE_25_B, and AT-
Microarray analysis GE_25_C. To determine genes showing up- or downregulation in
senescence the two experiments were analysed separately. For the
All the array experiments were carried out using the ATH1 Arabid- NASC experiment, the COG and C5G were used in GeneSpring as
opsis GeneChip microarray (http://www.affymetrix.com/products/ replicates for green leaf and the COS and C5S data as replicates for

showing a twofold increase in expression were listed from the

NASC and from the ATGE experiment and then genes common to
Dark these two lists were identified using a Venn diagram analysis in
Starvation Development GeneSpring. The average ratio for the two experiments were cal-
culated for each gene and 827 genes showing an average threefold
increase in the two experiments were selected. This gene list is
shown in Table S1.
Sugar depletion Loss of cytokinin
Mutant analysis. Two slides were generated for senescing leaves
ABA from each mutant. NahG (Buchanan-Wollaston_A-5-bwoll-NG1_S,
Buchanan-Wollaston_A-6-bwoll-NG2_SLD), ein2 (Buchanan-
SA
JA Ethylene Wollaston_A-7-bwoll-Ei1_SLD, Buchanan-Wollaston_A-8-bwoll-
Ei2_SLD), and coiI1 (Buchanan-Wollaston_A-9-bwoll-Co1_SLD,
Buchanan-Wollaston_A-10-bwoll-Co2_SLD).
Senescence is induced For the mutant analysis, genes were filtered as described above
and genes showing at least a twofold difference in expression in
senescent leaves of the mutant compared with the wild type were
selected. Venn diagram analysis in GeneSpring was then used to
identify the senescence-enhanced genes that were altered in
Flavonoid
Alternative synthesis expression in the different mutant backgrounds. The ratio data for
flavonoids?
Hydrolases the senescence-enhanced genes are shown in Table S1.
Glutamine
Asparagine mobilisation
mobilisation Common
degradation Dark-induced senescence. Affymetrix GeneChip ATH1 slides
genes were used as described in Lin and Wu (2004). Two slides for the dark
FA degradation e.g.Fatty acid Kinases
sucrose/trehalose alpha oxidation pathogen genes treatment and one slide for the control were analysed in Gene-
metabolism SAG12 Spring. Genes showing an absent or very low signal were filtered
out as described above. Two comparisons, each using one of the
dark treatment slides and the same control slide were used and ratio
Figure 5. Model illustrating the similar pathways and alternative pathways
that operate in the three types of senescence. data were averaged to find genes showing an expression change
Genes that are expressed in the different types of senescence are illustrated in between the treatments. These data were used in Tables 2–6 and
coloured boxes, green for developmental senescence only, blue for the Table S1 to calculate dark-related expression changes for genes of
starvation-induced senescence that is caused by both dark treatment and in interest.
the ageing suspension cultures and orange for the genes that are expressed in
all three types of senescence.
Developmental senescence only is controlled at least partially by reduction in
Cell death. Two replicate slides were analysed for each treatment,
cytokinin levels and results in the differential expression of genes for control and senescent (cell death) (NASC data: Swidzinski Control
flavonoid biosynthesis and glutamine metabolism. In addition, the SA ATH1 Replicate 1, Swidzinski Control ATH1 Replicate 2, Swidzinski
pathway is only active in developmental senescence resulting in the Senescence ATH1 Replicate 1, Swidzinski Senescence ATH1 Repli-
expression of a number of putative pathogen-related genes and kinases. cate 2). Genes that were flagged absent in all samples and any
Starvation-induced senescence, which probably results from sugar depletion, genes that had a raw value of <50 on all slides were filtered out of
induces the expression of genes involved in asparagine metabolism, fatty the analysis. Normalized data were used to calculate a senescent/
acid degradation and a putative alternative flavonoid pathway. ABA response control ratio for each gene and genes showing a threefold upregu-
genes are expressed in all three types of senescence implicating this hormone
lation in the senescing cells were identified.
in senescence regulation. However, an absolute requirement for this signal
for any genes in senescence has not been determined, hence the dotted line.
Also, certain genes that depend on JA and ethylene for expression are Measurement of senescence parameters in NahG plants.
expressed in all three types of senescence, including a number of hydrolases Plants were grown in an environmentally controlled growth room at
and carbohydrate metabolism enzymes. A number of other groups of genes 22C with a 16-h light/8-h dark cycle with moderate light intensity
including those required for fatty acid a-oxidation are expressed in all three
(150 lM m)2 sec)1). Chlorophyll content and photochemical effi-
types of senescence. Some but not all of these are dependent on JA and/or
ciency were examined at several developmental ages of leaves
ethylene signalling.
in planta (for age-dependent experiment) or at the given times after
incubating detached leaves in darkness (for dark-induced leaf sen-
escence experiment), using the third and fourth leaves of wild type
(Col) and nahG transgenic plants. Chlorophyll was extracted from
the senescent leaf. This helped to ensure that any gene expression individual leaves by heating the leaves in 95% ethanol at 80C.
differences obtained were general in these two variants of the Chlorophyll concentration per fresh weight of leaf was calculated as
Columbia accession. Normalized data for each gene were used to described by Lichtenthaler (1987). The photochemical efficiency of
generate the senescent/green ratio which was used to identify those photosystem II (PSII) was deduced from the characteristics of
genes showing at least a twofold change in expression. For the chlorophyll fluorescence (Oh et al., 1996) using a portable plant
AtGenExpress data two experiments were carried out in Gene- efficiency analyzer (Hansatech Instruments, Norfolk, UK). The ratio
Spring with either the slide 14 data (leaf 6) or the slide 15 data (leaf 8) of maximum variable fluorescence to maximum yield of fluores-
compared with the senescent leaf data (slide 25). The normalized cence, which corresponds to the potential quantum yield of the
senescent/green ratios for these were averaged and any genes photochemical reactions of PSII, was used as the measure of the
showing a twofold increase in expression was generated. Genes photochemical efficiency of PSII (Oh et al., 1996).

Acknowledgements Bouche, N. and Fromm, H. (2004) GABA implants: just a metabolite?

Trends Plant Sci. 9, 110–115.
We thank the Nottingham Arabidopsis Stock Centre (NASC) for Bouche, N., Fait, A., Bouchez, D., Moller, S.G. and Fromm, H. (2003)
carrying out the Affymetrix array experiments and the BBSRC for Mitochondrial succinic-semialdehyde dehydrogenase of the
providing funds to subsidize the cost of these experiments under gamma-aminobutyrate shunt is required to restrict levels of
the Investigating Gene Function Initiative. We acknowledge the use reactive oxygen intermediates in plants. Proc. Natl Acad. Sci.
of microarray data produced by the AtGenExpress project, which is USA, 100, 6843–6848.
coordinated by Lutz Nover (Frankfurt), Thomas Altmann (Potsdam) Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E.,
and Detlef Weigel (Tübingen), and supported by funds from the DFG Davis, K.R. and Gorlach, J. (2001) Growth stage-based phenotypic
and the Max Planck Society. The leaf data were generated by Jan analysis of Arabidopsis: a model for high throughput functional
Lohmann and Markus Schmid (MPI Tübingen). We thank Prof. John genomics in plants. Plant Cell, 13, 1499–1510.
Turner from University of East Anglia for the gift of Coi1 mutant Brouquisse, R., Gaudillère, J.-P. and Raymond, P. (1998) Induction
seed. VBW, EH, TP, EB and CJL thank the BBSRC for their financial of a carbon-starvation-related proteolysis in whole maize plants
support. We thank Prof. Brian Thomas for his comments on the submitted to light/dark cycles and to extended darkness. Plant
manuscript. Physiol. 117, 1281–1291.
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The Plant Journal - 2005 - Buchanan Wollaston - Comparative Transcriptome Analysis Reveals Significant Differences in Gene

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The Plant Journal (2005) 42, 567–585 doi: 10.1111/j.1365-313X.2005.02399.

Comparative transcriptome analysis reveals significant

Received 22 December 2004; revised 14 February 2005; accepted 18 February 2005.

ª 2005 Blackwell Publishing Ltd 567

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

determines transcript abundance and may not necessarily

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Gene class Putative function Number Examples

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

and Apel, 1993). Weaver et al. (1998) showed that several

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

inducible genes such as the type A response regulators,

Leaf Dark Cell suspension

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Leaf Dark Cell suspension

N mobilization and transport?

Colour representation as in Table 2.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Table 4 Genes that may have a role in flavonoid biosynthesis

Leaf Leaf Dark Dark Cell suspension Cell suspension

At4g22870 Leucoanthocyanidin dioxygenase 2376 105.03 4 7

Colour representation as in Table 2.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

the genome. AAA proteins act in a variety of cellular

Senescence phenotype of NahG transgenic plants

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Identifier Annotation Sen/MG NahG Coi1 Ein2 Dark/light Cell death/control

Colour representation as Table 2.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

At3g28580 AAA-type ATPase 9.78 0.08 0 0.40

The value 0 indicates undetectable expression in any sample of that experiment.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Figure 4. Comparison of senescence param-

Chlorophyll content (%)

Chlorophyll content (%)

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

showing a twofold increase in expression were listed from the

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

Acknowledgements Bouche, N. and Fromm, H. (2004) GABA implants: just a metabolite?

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 567–585

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