Full text of DOI:10.1016/S0953-7562(08)61993-0

Mycol. Res. 105 (10): 1223–1230 (October 2001). Printed in the United Kingdom.
1223
Genes with increased transcript levels following harvest of the
sporophore of Agaricus bisporus have multiple physiological
roles
Daniel C. EASTWOOD*, Crawford S. KINGSNORTH†, Helen E. JONES and Kerry S. BURTON
Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK.
E-mail: daniel.eastwood!hri.ac.uk
Received 23 December 2000; accepted 27 June 2001.
We screened a cDNA library generated from harvested and stored sporophores of Agaricus bisporus and identified 19 genes with
higher transcript levels than at the time of harvest. Five of these genes had no detectable mRNA levels prior to detachment from
the mycelium. Sequence analysis of ten clones revealed significant similarities to known genes, these code for proteins involved in
polymer breakdown and metabolism, cell wall synthesis, stress tolerance, cytochrome P450 activity and DNA binding. The diversity
of functions of these genes suggests the changes in the sporophore after harvest involve several different physiological processes.
natural senescence observed in the uncut sporophore (Fig. 1);
tissue discolouration, loss of texture, localised cellular collapse
and cell emptying (Evered & Burton 1995, McGarry & Burton
INTRODUCTION
Morphogenesis of the homobasidiomycete Agaricus bisporus
involves the differentiation of a vegetative mycelium into a
multicellular sporophore, or mushroom. The sporophore
consists of the stipe (stalk) and the pileus (cap) which is
covered with a ‘skin’ layer. The underside of the cap is
differentiated into gill tissue that bears basidiospores. During
development the stipe grows longitudinally and the cap
expands, exposing the gills ready for spore dispersal. The
resources required for sporophore and basidiospore production
are supplied by the vegetative mycelium, which is in contact
with the substrate, i.e. leaf litter in natural habitats or compost
if cultivated. After sporulation the sporophore senesces and
dies, presumably to avoid over-commitment of resources to
the sporophore at the expense of the mycelium. The regulation
of this final stage of development is not known. Characteristics
of natural mushroom senescence include darkening tissue
colour, loss of texture and cellular disruption and collapse
1
994, Umar & van Griensven 1997). However, other workers
suggest the harvested sporophore does not senesce, but
instead is subject to a ‘post-harvest stress disorder’ in
response to water and nutritional limitation (Moore 1998,
Umar & van Griensven 1997).
Detached sporophores continue to have high rates of
respiration linked to a switch from nutrient import to the
breakdown of storage compounds (Hammond & Nichols
1
975). Mannitol, trehalose, glycogen and soluble protein
levels decrease (Burton et al. 1997, Hammond & Nichols 1975)
while levels of the components of the cell wall and of urea
increase (Hammond 1979). Compositional changes in the
detached A. bisporus sporophore are not uniform throughout
the different tissues of the mushroom (Burton et al. 1997,
Donker & Braaksma 1999, Hammond & Nichols 1975). There
is additional evidence of the mass movement of resources
from the stipe to the cap and gill tissues during development
in the harvested sporophore (Donker & van As 1999,
Hammond & Nichols 1975). These post-harvest metabolic
changes are the result of existing enzymes and pathways that
are able to operate reversibly, for example mannitol
dehydrogenase (Morton, Dickenson & Hammond 1985), the
de novo production of new proteins and the down-regulation
of other proteins. The only described example of de novo
protein synthesis occurring in the A. bisporus sporophore
postharvest is that of serine proteinase (Burton et al. 1997) and
is controlled at the level of transcription (Kingsnorth, Eastwood
(
McGarry & Burton 1994, Umar & van Griensven 1997).
Cultivated A. bisporus sporophores are often harvested at
an immature stage prior to sporulation. The harvest is both a
wounding event and a massive disruption to the metabolism
of the sporophore, since the sporophore is detached abruptly
from its supply of nutrients and water. However, despite the
damage, the sporophore continues to develop similarly to the
non-harvested mushroom (Hammond 1979); the stipe grows
longitudinally, the cap continues to expand, gill formation
occurs and spores are produced (Braaksma et al. 1998).
Prolonged storage results in tissue changes comparable with
&
Burton 2001). Serine proteinase levels also increase during
natural senescence in A. bisporus (Burton et al. 1997). However,
little additional information is available on the biochemistry
and genetic regulation of the harvested mushroom.
*
Corresponding author.
Current address: IACR-Broom’s Barn, Higham, Bury St Edmunds,
†
Suffolk IP28 6NP, UK.

Differential gene expression in harvested mushrooms
1224
Day 0
Day 3
Fig. 1. Postharvest changes in the mushroom Agaricus bisporus, observed at harvest (day 0) and after three days storage (day 3) at
18 mC, and 90–95% relative humidity.
We are interested in the postharvest biology of the RNA isolation
mushroom because its high commercial value can be lost after
harvest due to quality loss. In this study we aim to gain an
insight into the biochemistry and genetic regulation in
sporophores postharvest, by identifying genes with higher
transcript levels after harvest. cDNA derived from sporophores
stored for 2 d was compared with cDNA from freshly
harvested sporophores by differential screening. Genes with
higher transcript levels 2 d after harvest were selected,
sequenced and characterised.
RNA was isolated from fresh mushroom tissues and from
tissues stored for 2 d following harvest. RNA isolation was
carried out according to established protocols (Sambrook,
Fritsch & Maniatis 1989). Absorbance measurements at 260
and 280 nm were used to assess RNA concentration and
purity (Sambrook et al. 1989). RNA integrity was determined
by formaldehyde agarose gel electrophoresis (Rosen & Villa-
+
Kormaroff 1993). Poly(A) RNA was isolated from the total
RNA using affinity chromatography oligo d(T) cellulose
mRNA purification kit (Amersham Pharmacia Biotech).
MATERIALS AND METHODS
cDNA library construction
Strains
TM
A cDNA library was constructed with the λ-ZAP Express
Agaricus bisporus strain U3 (Sylvan, UK) was used throughout
the study. Mushrooms were grown according to commercial
practice at the Horticulture Research International mushroom
cropping unit. During cropping, mushroom sporophores are
produced in synchronous weekly flushes; sporophores used
experimentally were from the second flush only. Sporophores
were harvested and either frozen immediately in liquid
+
cDNA synthesis kit (Stratagene) from 5 µg poly(A) RNA
extracted from day 2 sporophores. The primary library of
approximately 10& clones was amplified according to
Stratagene protocols.
Preparation of an ordered cDNA library
nitrogen (termed day 0), or stored for 2 d in a controlled Phagemid clones (pBK-CMV) were excised from the λ-ZAP
environment, 18 mC and 90–95% relative humidity (day 2), cDNA library and cloned as described in the manufacturer’s
before freezing in liquid nitrogen. Frozen samples were stored instructions (Stratagene) and selected on Luria-Bertani (LB)
−
agar medium in Petri plates containing 50 µg ml kanamycin;
"
at k80 mC.
The Escherichia coli strain XL1-Blue (Stratagene) was used each plate contained 0n8 mg X-gal and 0n8 mg IPTG. White
for plasmid maintenance and cloning. The pBluescriptSK colonies were isolated and inoculated into wells of a microtitre
−
dish containing 200 µl of ‘media 96’ (tryptone 10 g l , yeast
"
(
Stratagene) vector was used for routine cloning.

D. C. Eastwood and others
1225
−
"
−"
−"
extract 5 g l , NaCl 5 g l , KH PO 6n3 g l , sodium citrate Accession numbers
#
%
−
"
−"
−"
0
5
n45 g l , MgSO 90 mg l , (NH ) SO 0n9 g l , glycerol
%
% #
%
The sequences identified in this study were submitted to the
EMBL database (accession nos AJ271690 to AJ271708).
−
"
% (v\v)) and 50 µg ml kanamycin. The microtitre plates
were incubated overnight at 37 m, frozen and stored at k80 m.
RESULTS AND DISCUSSION
Differential screening
Isolation of differentially expressed cDNA
Differential screening was carried out according to established
protocols (Sambrook et al. 1989). Bacteria from the ordered We screened 2992 arbitrarily selected clones from a cDNA
library were transferred onto duplicate nylon membranes library, constructed from the mRNA of Agaricus bisporus
(
Roche Diagnostics) and prehybridised separately. sporophores that had been stored for two days following
Differential probes were prepared with cDNA created from harvest. We initially identified 154 clones that hybridised to
+
5 µg poly(A) RNA extracted from day 0 and day 2 cDNA derived from sporophores harvested and stored for
sporophores. Synthesis of cDNA was achieved through two days (day 2), and not to cDNA from freshly harvested
+
reverse transcription of poly(A) RNA with the Ready-To-Go sporophores (day 0). A second round screening, using RNA
T-primed cDNA synthesis kit (Amersham Pharmacia Biotech). extracts from different day 0 and day 2 sporophores, reduced
The cDNA was purified with the QIAquick nucleotide the number of clones to 40. The tertiary screen (using the
removal kit (Qiagen) prior to being used as a template for cDNAs as probes) reduced the number of clones with higher
random priming, incorporating [α-$#P]dCTP (Rediprime kit, transcript levels two days after harvest to 38. These 38 clones
Amersham Pharmacia Biotech). The duplicate membranes were sequenced fully and compared. Clones with very similar
were hybridised with denatured cDNA probe from either day sequences. i.e. over 98% sequence similarity were grouped
0
or day 2 sporophores. Putative, differentially expressed together in gene families. From the 38 clones, 19 gene families
cDNA clones were identified and the corresponding phagemid were identified, and all families contained potential open
colonies re-screened with cDNA probes extracted from reading frames.
different day 0 and day 2 sporophores. The inserts of the
phagemid clones which showed a greater transcript level at
Identification of genes with higher transcript levels
day 2 were used as probes in a Northern blot tertiary screen.
Total RNA from day 0 and day 2 sporophores were separated The nucleic acid and protein sequences of the 19 genes\gene
with formaldehyde agarose gel electrophoresis prior to families were compared with other known sequences from the
Northern blot analysis (Sambrook et al. 1989).
databases. Whole sequences, motifs and structural character-
Agaricus bisporus hemicellulase (hem1) and 28S rRNA genes istics were analysed. Ten of the 19 gene families had strong
were used as controls. hem1 is constitutively expressed at a similarities to the sequences of known proteins; another two
constant level under a range of conditions in Aspergillus showed no overall similarity, but their derived protein
nidulans (Bradshaw et al. 1993) and had been previously used sequences contained motifs with which functions have been
as a loading control with A. bisporus (Yagu
%
8S rRNA gene has also been used as a loading control for with proteins of known function. Five of the genes (SHS 1, 3,
e et al. 1997). The associated (Table 1). The remaining seven had no similarities
2
total RNA samples (de Groot et al. 1998).
7, 8 and 14) had no detectable expression at day 0 (Fig. 2). The
remaining genes were transcribed at low levels prior to
harvest and up-regulated greatly following detachment and 2
Nucleic acid sequence determination analysis
days’ storage (Fig. 2).
Some of the genes with sequences similar to proteins with
BigDye terminator cycle sequencing kit (Perkin– known biochemical functions could be considered according
DNA sequencing reactions were carried out using the ABI
TM
Prism
Elmer). Electrophoresis and acquisition of sequence data were to probable cellular function, e.g. nitrogen metabolism and
carried out by the DNA Sequencing Unit at the University of mobilisation, polysaccharide degradation, cell wall synthesis,
Durham.
stress tolerance, and cytochrome P450. The genes were
Sequences were analysed with the DNAStar program identified in this study by comparing RNA from freshly
DNAStar, USA). Homology studies on both nucleic acid and harvested sporophores with RNA from harvested and stored
(
amino acid sequences were carried out using the FASTA, sporophores, and are therefore referred to as sporophore
BlastX and BlastP algorithms available on the European harvested and stored (SHS) genes. This does not imply
Bioinformatics Institute (EBI) (www.ebi.ac.uk) homepage and however that their increased transcription is due specifically to
the
OWL
(www.biochem.ucl.ac.uk\bsm\dbbrowser\ the harvest, as other processes are also occurring e.g. growth
OWL\OWL) composite protein sequence database. Database and development.
searches were carried out using the default settings. In
addition, the encoded proteins were also analysed using links
Nitrogen metabolism and mobilisation
available through the ExPASy (www.expasy.com) homepage.
Each encoded protein was analysed for conserved domains, Genes encoding two enzymes involved in nitrogen metab-
motifs and fingerprints; protein structural analyses; profile and olism (leucine aminopeptidase and argininosuccinate lyase)
pattern searches; protein localisation markers and trans- had higher transcript levels following sporophore harvest and
membrane spanning regions.
storage. Leucine aminopeptidase (SHS 1) catalyses the release

Differential gene expression in harvested mushrooms
1226
Table 1. Characteristics of cDNA clones from Agaricus bisporus sporophores which show elevated transcript levels after harvest and storage at 18 mC,
0–95% relative humidity.
9
BLASTP Search Results
Similarity
Clone
EMBL No)
No of clones
in library
Size of cDNA
Insert (kb)
Size of
mRNA (kb)
(
Putative Gene Product
Probability*
%
Ratio†
SHS 1 (AJ271690)
SHS 2 (AJ271691)
1
1
1n2
0n7
1n4
1n3
Leucine aminopeptidase
EC 3n4.11n10
Argininosuccinate lyase
1n9e−&$
55
80
197\357
1n9e−()
190\235
EC 4n3.2n1
SHS 3 (AJ271692)
SHS 4 (AJ271693)
SHS 5 (AJ271694)
5
1
1
0n7
0n4
0n5
1n6
1n6
1n1
Glucuronyl hydrolase
β(1-6) Glucan synthase
Superoxide dismutase
EC 1n15n1.1
2n5e−"(
4n5e−!&
4n0e−&$
49
42
84
173\353
107\251
111\131
SHS 6 (AJ271695)
SHS 7 (AJ271707)
1
1
0n4
1n2
0n8
1n7
Metallothionein
Cytochrome P450 I
EC 1n14n14n1
2n1e−!(
5n3e−'(
56
69
14\25
173\250
SHS 8 (AJ271708)
SHS 9 (AJ271696)
SHS 10 (AJ271697)
SHS 11 (AJ271698)
SHS 12 (AJ271699)
SHS 13 (AJ271700)
1
1
2
1
1
8
1n3
0n6
0n7
2n3
0n5
0n6
1n7
0n6
1n0
2n5
1n1
1n0
Cytochrome P450 II
EC 1n14n14n1
2n4e−%'
3n0e−#&
6n9e−"!
NA
63
143\224
74\97
35\64
NA
Cruciform DNA
binding protein
Riboflavin aldehyde-
forming enzyme
Contains leucine
zipper motif
76
54
NA
NA
NA
Contains glycine-
proline rich motif
Contains glycine\
serine-rich motif
Unknown
NA
NA
NA
NA
SHS 14 (AJ271702)
SHS 15 (AJ271701)
SHS 16 (AJ271703)
SHS 17 (AJ271704)
SHS 18 (AJ271705)
SHS 19 (AJ271706)
2
3
3
1
3
1
1n2
1n3
0n6
0n4
0n8
0n5
1n6
1n3
0n7
1n5
1n3
0n6
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Unknown
Transmembrane protein
Unknown
Unknown
Unknown
*
The probability of the database match occurring randomly.
Ratio of similar amino acids of A.bisporus protein .
†
region compared of best fitting protein
of amino acids from the N-terminus of peptides. The soluble enzymes have conserved catalytic domains, yet utilise a wide
protein levels decline in the Agaricus bisporus sporophore after range of substrates (Henrissat et al. 1995). Unsaturated
harvest while the level and activity of serine proteinase glucuronyl hydrolase is an exoglycanase which releases
increases (Burton et al. 1997). However serine proteinase has glucuronyl monosaccharides from di-, tri- and tetrasaccharides
a narrow substrate specificity and the final products are short formed during polysaccharide degradation by polysaccharide
chain peptides upon which leucine aminopeptidase may act lyases (Hashimoto et al. 1999). The substrates of the previously
(
Burton et al. 1993). Argininosuccinate lyase (SHS 2) is an described unsaturated glucuronyl hydrolase are glucuronic
integral enzyme of the urea cycle. The level of urea increases acid-containing degradation products of mucopolysaccharides,
fold in the sporophore between day 0 and day 4 post- e.g. chondroitin and hyaluronate, which are often associated
9
harvest storage (Hammond 1979). The urea cycle is probably with the extracellular matrix (Hashimoto et al. 1999). However,
linked to the citric acid cycle via argininosuccinate lyase so the substrate for this enzyme is not known in the harvested A.
that amino acids released during proteolysis are de-aminated, bisporus sporophore. Interestingly, Umar & van Griensven
nitrogen is accumulated as urea and carbon skeletons are (1997) observed by electron microscopy a loss of extracellular
decarboxylated, leading to the generation of high energy matrix in mushrooms after harvest.
phosphate bonds.
Cell wall synthesis
Polysaccharide breakdown
The transcript level of a gene encoding β(1-6) glucan synthase
Five clones identified in the tertiary screen have the same (SHS 4) increases after harvest and storage. The upper stipe
derived protein sequence which is similar (49%) to unsaturated region of A. bisporus elongates greatly during postharvest
glucuronyl hydrolase (SHS 3) identified from a Bacillus sp. growth and development (Braaksma et al. 1998). Since (1-6)β-
(
GenBank AP001514). The protein probably belongs to the linked glucan side branches increase in the cell wall of the
family of glycosyl hydrolase enzymes (EC 3n2.1–3n2.3). These upper stipe during rapid elongation (Mol & Wessels 1990),

D. C. Eastwood and others
1227
Sequence
Northern Analysis
Days from Harvest)
Sequence
Northern Analysis
(Days from Harvest)
(
0
0
2
2
0
0
2
2
HEM 1
Control
SHS 10
SHS 11
2
8s rRNA
Control
SHS 1
SHS 2
SHS 12
SHS 13
SHS 14
SHS 3
SHS 4
SHS 5
SHS 15
SHS 16
SHS 6
SHS 7
SHS 17
SHS 18
SHS 19
SHS 8
SHS 9
Fig. 2. Northern analysis of gene transcripts with increased levels after harvest and storage from Agaricus bisporus sporophores. Two
independent RNA extractions were analysed for each time point; day 0 represents extracts from sporophores immediately after harvest,
day 2 sporophores were stored for 2 d after harvest.
the β(1-6) glucan synthase may possibly be the enzyme activity of NADPH oxidase increases in the mushroom
responsible for the changes in cell wall composition.
postharvest (Hammond 1978). The product of superoxide
dismutase catalysis, hydrogen peroxide, is also potentially
highly reactive and damaging, however the activity of the
hydrogen peroxide-scavenging enzyme, catalase, is high in
Stress tolerance
Two genes often associated with stress tolerance had increased the mushroom sporophore (data not shown).
transcription levels following harvest, a superoxide dismutase Metallothioneins are low molecular weight, cysteine-rich
SHS 5) and a class II metallothionein-like protein (SHS 6). metal binding proteins often associated with stress responses
(
Superoxide dismutase catalyses the reaction of superoxide and developmental processes (Borghesi & Lyres 1996). The
radicals with protons to produce hydrogen peroxide (Nelson metallothionein identified in this study contained an open
et al. 1996). Two distinct families of superoxide dismutase reading frame of 71 amino acids with a region containing 14
$
enzymes have been described and they are considered to have metal-binding cysteine residues. Previously, Munger & Lerch
separate evolutions (Smith & Doolittle 1992). The superoxide (1985) described a 25 amino acid peptide fragment from A.
dismutases from Agaricus bisporus characterised here belongs bisporus as a metallothionein protein, but its protein sequence
to the iron\manganese-type of superoxide dismutases, which was distinct from the derived protein sequence of the gene
are usually located in the mitochondrial matrix (Natvig et al. identified here. The precise function of the metallothioneins
1
996). Superoxide radicals are highly reactive, non-reduced has not been fully established, but they appear to play a role
forms of oxygen which can cause severe disruption to cell in binding metal ions which can be toxic to a cell at high
function and to membranes (Farr & Kogoma 1991). Superoxide concentrations (Robinson et al. 1993). In addition to the
radicals can be produced enzymatically by NADPH oxidase, presence of heavy metal cations, metallothioneins may be
NADH dehydrogenase, succinate dehydrogenase, lactate induced by starvation or the presence of free radicals,
dehydrogenase, glutathione reductase and cytochrome P450; although the induction by free radicals appears to be mediated
and non-enzymatically by the autoxidation of cellular by secondary events (Borghesi & Lyres 1996). The level of
components including ubiquitols, catechols, thiols, flavins and soluble protein falls in the detached A. bisporus sporophore by
quinones (Farr & Kogoma 1991, Kawasaki et al. 1999). The 90% in the stipe and 80% in the cap (Burton et al. 1993).

Differential gene expression in harvested mushrooms
1228
Therefore, the function of metallothionein may be to bind free motif (L-X(6)-L-X(6)-L-X(6)-L), which is a DNA binding motif
metal ions released from the degradation of metal-binding commonly associated with transcription factors and with
proteins.
enzymes that undergo protein\protein interactions
Landschulz, Johnson & McKnight 1988, O’Shea, Rutkowski
(
&
Kim 1989).
SHS 12 has similiarities (expressed as Blast P probability
Cytochrome P450
The genes for two separate cytochrome P450 (SHS 7 and 8) and percentage similarity) with a number of proteins of
−
genes had higher transcript levels in the mushroom after unknown function from Arabidopsis thaliana (5n8e , 53%,
"'
−
""
harvest. Cytochrome P450 (CYP) enzymes are a group of GenBank O80818), Sporobolus stapfianus (1n0e , 50%,
−
""
proteins involved in the oxidative metabolism of a large GenBank O04820) and Saccharomyces cereviseae (7n4e , 50%,
number of compounds, they have been classed into 74 families GenBank P38216). SHS 13 was a serine and glycine-rich
(
Nelson et al. 1996, van den Brink et al. 1998) and have diverse protein. SHS 14, SHS 15, SHS 16, SHS 17, SHS 18 and SHS
roles in many aspects of cell function (Nelson et al. 1996). 19 had novel sequences and similarly to other proteins could
Previous work identified a cytochrome P450 (CYPA) not be determined. However, SHS 16 contained a number of
associated with the early stages of sporophore development potential transmembrane domains.
in A. bisporus and was described as a flavonoid 3h,5h
hydroxylase (de Groot et al. 1997). The cytochrome P450 I
and II described here were similar to CYPA but were clearly
DISCUSSION AND CONCLUSIONS
different proteins with similarities to CYPA of 69% and 57% We identified 19 genes\gene families with elevated transcript
respectively. Similarity and motif searching with cytochrome levels by differential screening of cDNA derived from freshly
P450 enzymes of known catalytic function suggested that harvested (day 0) and detached and stored mushrooms (day
these cytochrome P450s belong to the class of mono- 2). The identities of ten of these genes\gene families were
oxygenase, oxidoreductase enzymes (EC 1n14n14n1), respon- determined by sequencing and database comparison. Pre-
sible for the metabolism of a wide range of substrates, viously, only one protein, serine proteinase, was known to
including fatty acids, steroids and vitamins. Cytochrome P450 increase after harvest or during natural senescence (Burton et
II was most similar to a cytochrome P450 isolated from al. 1993). Serine proteinase is considered to have a nutritional
‘
dumpy’ mutants of the ink cap mushroom, Coprinus cinereus function in the harvested mushroom. The serine proteinase
Muraguchi & Kamada 2000). Stipe elongation is blocked in gene is abundantly transcribed in stored Agaricus bisporus
(
the ‘dumpy’ mutant as a consequence of changes to the eln2 mushrooms (Kingsnorth et al. 2001) and we expected to
gene encoding the cytochrome P450 enzyme.
identify it in this study. However, failure to identify the gene
may have been due to the lethal nature of the gene when
cloned into E. coli cells (Kingsnorth et al. 2001).
DNA binding
Consideration of the ten identified up-regulated genes
Following harvest, increased transcript levels were found of a suggests that the detached and stored sporophore is
gene, (SHS 9) encoding a protein which has similarity to the undergoing numerous diverse physiological functions sim-
non-histone cruciform DNA binding proteins HMP1 and ultaneously, e.g. nutritional (polymer degradation and metab-
Gv1, from Ustilago maydis and Glomus versiforme respectively olism), stress tolerance and cell wall synthesis (polymer
(
Dutta et al. 1997, Burleigh & Harrison 1998). Three conserved synthesis). These diverse activities are consistent with the
regions are common to each protein sequence, probably harvested mushroom being nutritionally isolated, wound
involved in DNA binding function (data not shown). This damaged, continuing to develop and composed of distinct
class of protein has been suggested to be involved in inducing tissues with different physiologies. Within a tissue, different
or maintaining the DNA architecture rather than in a specific physiologies also occur, for example, cell death has been
role associated with recombination (Dutta et al. 1997).
observed to be localised and discrete, with dying cells
adjacent to growing cells (Evered & Burton 1995, Umar & van
Griensven 1997). Studies of natural senescence in other
organisms also have identified an array of ‘up-regulated’
Riboflavin aldehyde-forming enzyme
Two clones were similar to a riboflavin aldehyde-forming genes. In Brassica napus, genes with increased transcription
enzyme (SHS 10) from Schizophyllum commune (EMBL AF levels include those involved in protein, nucleic acid and
0
05405). In S. commune, the enzyme catalyses the formation of chlorophyll breakdown, lipid and nitrogen mobilisation,
the 5h-aldehyde and 5h-acid of riboflavin, both of which are cytochrome P450s and genes of unknown function (Buchanan-
present in sporophores of Agaricus bisporus (Chen & Wollaston 1997). Six senescence-associated genes have been
McCormick 1997a, Tachibana & Murakami 1983). The identified from daylily (Hemerocallis hybrid) petals, aspartic
physiological role of the enzyme in S. commune or A. bisporus proteinase, nuclease, fatty acid elongase, a cytochrome P450-
has yet to be determined (Chen & McCormick 1997b).
associated fatty acid hydroxylase and a second cytochrome
P450 (Panavas et al. 1999). It is not known how many of the
1
9 genes\gene families identified in this paper have increased
Other sequences
transcription due specifically to harvest\post-harvest pro-
Two further sequences of unknown function showed some cesses. Parallel work (in progress) has shown that at least two
homology to known genes. SHS 11 contained a leucine zipper of the genes (SHS 10 and SHS 14) also have increased

D. C. Eastwood and others
1229
transcription levels during the natural, non-harvest devel-
opment of sporophores (data not shown).
It is not clear how gene responses may have evolved in
relation to harvesting. There may be a strategy evolved
5h-aldehyde-forming enzyme from Schizophyllum commune. Journal of
Biological Chemistry 272: 20077–20081.
de Groot, P. W. J., Schaap, P. J., van Griensven, L. J. L. D. & Visser, J. (1997)
Isolation of developmentally regulated genes from the edible mushroom
Agaricus bisporus. Microbiology 143: 1993–2001.
in direct response to sporophore detachment by external Donker, H. C. W. & Braaksma, A. (1999) Changes in metabolite concentrations
factors, e.g. wild animals, wind. However, it is more likely the
expression of these genes evolved independently in response
to a number of different stimuli. Many of these stimulus-
response relationships would have probably arisen in mycelial
cells early in their evolution. Therefore, the nutritional genes
detected by "$C-NMR in the senescing mushroom (Agaricus bisporus).
Postharvest Biology and Technology 10: 127–134.
Donker, H. C. W. & van As, H. (1999) Cell water balance of white button
mushrooms (Agaricus bisporus) during its postharvest lifetime studied by
quantitative magnetic resonance imaging. Biochimica et Biophysica Acta
1427: 287–297.
have increased transcription in response to nutritional Dutta, S., Gerhold, D. L., Rice, M., Germann, M. & Kmiec, E. B. (1997) The
cloning and over expression of a cruciform binding protein from Ustilago
limitation, which is distinct from the signal-response relation-
ships of stress adaptation and cellular growth. The physiology
of the harvested and stored mushroom consists of a complex
of changes which presumably are co-ordinated but originate
maydis. Biochimica et Biophysica Acta 1352: 258–266.
Evered, C. E. & Burton, K. S. (1995) Cryo SEM study of changes in tissue
anatomy of harvested sporophores of Agaricus bisporus. Mushroom Science
14: 717–721.
from different stimuli. Further work is taking place to identify Farr, S. B. & Kogoma, T. (1991) Oxidative stress responses in Escherichia coli
which of these 19 genes\gene families show increased
transcription during natural senescence.
The genes with higher transcript levels represent only part
of the biology of the harvested mushroom. Genes with
and Salmonella typhimurium. Microbiological Review 55: 561–585.
Hammond, J. B. W. (1978) Carbohydrate catabolism in harvested mushrooms.
Phytochemistry (Oxford) 17: 1717–1719.
Hammond, J. B. W. (1979) Changes in composition of harvested mushrooms
(
Agaricus bisporus). Phytochemistry 18: 415–418.
reduced or unchanged transcription were not identified in this Hammond, J. B. W. & Nichols, R. (1975) Changes in respiration and
study, but these would also contribute to the biology of the
harvested sporophore. The gene encoding chitin synthase has
been shown to have decreased transcription following harvest
carbohydrates during the post-harvest storage of mushrooms (Agaricus
bisporus). Journal of the Science of Food and Agriculture 26: 835–842.
Hashimoto, W., Kobayashi, E., Nankai, H., Sato, N., Miya, T., Kawai, S. &
Murata, K. (1999) Unsaturated glucuronyl hydrolase of Bacillus sp. GL1:
Novel enzyme prerequisite for metabolism of unsaturated oligosaccharides
produced by polysaccharide lyases. Archives of Biochemistry and Biophysics
368: 367–374.
(
Sreenivasaprasad, Burton & Wood 2000). Furthermore, the
regulatory steps beyond transcription are also likely to exert
major influences on gene expression.
Henrissat, B., Callebaut, I., Fabregas, S., Lehn, P., Mornon, J.-P. & Davies, G.
(
1995) Conserved catalytic machinery and the prediction of a common fold
for several families of glycosyl hydrolases. Proceeding of the National
Academy of Sciences, USA 92: 7090–7094.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support of the Ministry of Agriculture,
Fisheries and Food (Project HH2117SMU), the Biotechnology and Biological
Sciences Research Council (Programme 207h) and the Commission of the
European Union (Programme AIR2-CT093–0953).
Kawasaki, T., Henmi, K., Ono, E., Hatakeyama, S., Iwano, M., Satoh, H. &
Shimamoto, K. (1999) The small GTP-binding protein Rac is a regulator of
cell death in plants. Proceedings of the National Academy of Sciences, USA 96:
10922–10926.
Kingsnorth, C. S., Eastwood, D. C. & Burton, K. S. (2001) Cloning and post-
harvest expression of serine proteinase transcripts in the cultivated
mushroom Agaricus bisporus. Fungal Genetics and Biology 32: 135–144.
Landschulz, W. H., Johnson, P. F. & McKnight, S. L. (1988) The leucine
zipper – a hypothetical structure common to a new class of DNA-binding
proteins. Science 240: 1759–1764.
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Corresponding Editor: J. I. Lelley