These comprise 6% of the total yeast genes encoding 173 unknown proteins and 208 proteins related to folding, glycosyla-tion⁄ modification, translocation, protein degradation, Keywords e
Trang 1stress in Arabidopsis thaliana
Shinya Kamauchi, Hiromi Nakatani, Chiharu Nakano and Reiko Urade
Graduate School of Agriculture, Kyoto University, Uji, Japan
A nascent polypeptide synthesized on the rough
endoplasmic reticulum (ER) is translocated and
folded with the assistance of molecular chaperones
and other folding factors such as glycosylation⁄
modi-fication enzymes and disulfide oxidoreductases within
the ER However, the folding of nascent
polypep-tides occasionally does not occur, resulting in the
accumulation of unfolded or misfolded proteins in
the ER (ER stress) To solve this problem, eukaryotic
cells sense ER stress and induce a set of genes called
unfolded protein response (UPR) genes In the
bud-ding yeast Saccharomyces cerevisiae, ER
transmem-brane protein kinase⁄ riboendonuclease Ire 1p is activated by ER stress [1,2], and nonconventionally splices mRNA of basic leucine zipper transcription factor Hac 1p [3–5] Hac 1p is translated from the spliced mRNA and induces the UPR genes, having a UPR cis-acting regulatory element [6–8] On DNA microarray analysis, 381 genes have been identified
as UPR ones induced by both tunicamycin (TM) and dithiothreitol [9] These comprise 6% of the total yeast genes encoding 173 unknown proteins and 208 proteins related to folding, glycosyla-tion⁄ modification, translocation, protein degradation,
Keywords
endoplasmic reticulum; fluid microarray;
gene expression; tunicamycin; unfolded
protein response
Correspondence
R Urade, Graduate School of Agriculture,
Kyoto University, Uji, Kyoto 611-0011, Japan
Fax: +81 774 38 3758
Tel: +81 774 38 3757
E-mail: urade@kais.kyoto-u.ac.jp
Database
The nucleotide sequence data for soybean
SEL-1L are available in the DDBJ ⁄
EMBL ⁄ GenBank databases under accession
number AB197676.
(Received 15 March 2005, revised 11 May
2005, accepted 16 May 2005)
doi:10.1111/j.1742-4658.2005.04770.x
Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) In this case, so-called unfolded protein response (UPR) genes are induced We determined the transcriptional expression of Arabidopsis thaliana UPR genes by fluid microarray analysis
of tunicamycin-treated plantlets Two hundred and fifteen up-regulated genes and 17 down-regulated ones were identified These genes were reana-lyzed with functional DNA microarrays, using DNA fragments cloned through fluid microarray analysis Finally, 36 up-regulated and two down-regulated genes were recognized as UPR genes Among them, the up-regu-lation of genes related to protein degradation (HRD1, SEL-1L⁄ HRD3 and DER1), regulation of translation (P58IPK), and apoptosis (BAX inhibitor-1) was reconfirmed by real-time reverse transcriptase-PCR The induction of SEL-1L protein in an Arabidopsis membrane fraction on tunicamycin-treat-ment was demonstrated Phosphorylation of initiation factor-2a, which was inhibited by P58IPK, was decreased in tunicamycin-treated plantlets How-ever, regulatory changes in translation caused by ER stress were not detec-ted in Arabidopsis Plant cells appeared to have a strategy for overcoming
ER stress through enhancement of protein folding activity, degradation of unfolded proteins, and regulation of apoptosis, but not regulation of trans-lation
Abbreviations
AARE, amino acid response element; ATF6, activating transcription factor 6; AZC, L -azetidine-2-carboxylic acid; BI-1, Bax inhibitor-1; eIF2a, initiation factor-2a; Endo H, endoglycosidase H; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; ERSE, ER stress response element; MS, Murashige and Skoog medium; PDI, protein disulfide isomerase; PKR, double stranded RNA-activated protein kinase; P-UPRE, plant-specific UPR element; RAMP4, ribosomal-associated membrane protein 4; TM, tunicamycin; UPR, unfolded protein response; UPRE, UPR cis-acting regulatory element; XBP-1, X-box binding factor.
Trang 2vesicle trafficking⁄ transport, vacuolar protein sorting,
cell wall biogenesis, and lipid⁄ inositol metabolism
In comparison with those of yeast, the UPR genes
of mammalian cells are induced through a much more
complicated mechanism, which has been shown to be
triggered by at least three transcription factors, X-box
binding factor (XBP-1), activating transcription
fac-tor 6 (ATF6), and ATF4 [10] The mammalian paralog
of yeast Ire 1p is activated by ER stress and splices
the invalid mRNA into mature mRNA encoding
371-amino acid XBP-1 [11,12] XBP-1 translated from the
spliced mRNA is translocated to the nucleus [13],
where it binds to its target sequence in the regulatory
regions of the P58IPK, ERdj4, HEDJ, EDEM, protein
disulfide isomerase (PDI)-P5, ribosomal-associated
membrane protein 4 (RAMP4), DnaJ⁄ HSP40-like
genes, etc [14] ATF6 is an ER transmembrane protein
that senses ER stress through its luminal domain, and
then moves to Golgi bodies to be cleaved by site-1 and
site-2 proteases [15–17] The cleaved ATF6 cytoplasmic
domain is released from Golgi membranes into the
nucleus, where it induces, in the presence of nuclear
factor Y, ER chaperone genes including BIP, GRP94,
Calreticulin and ORP150, which have an ER stress
response element (ERSE) in their regulatory regions
[18,19] PERK is an interferone-induced double
stran-ded RNA-activated protein kinase (PKR)-related
pro-tein that senses ER stress through its luminal domain
and then phosphorylates initiation factor-2a (eIF2a),
resulting in inhibition of bulk protein translation
[20,21] and stimulation of translation of ATF4 [22]
ATF4 is a basic leucine zipper transcription factor that
induces the transcription of many amino acid synthetic
enzymes and amino acid transporters by binding to
the amino acid response element (AARE) in the
regu-latory regions of these genes [23] ATF4 has also been
shown to stimulate the transcription of CHOP, which
is important for apoptotic cell death [24]
In contrast to the UPR mechanism(s) in yeast and
animal cells, that of plant cells is not well understood
Putative plant paralogs of yeast Ire1p have been found
in Arabidopsis thaliana and Oryza sativa [25,26] Their
N-terminal luminal domains have each been shown to
function as a sensor for ER stress in yeast However,
neither target mRNAs of transcription factors for
plant Ire1p nor target genes induced by this system
have been identified On the other hand, the mRNAs
of BiP, calreticulin, calnexin and PDI have been shown
to be induced on treatment with TM and dithiothreitol
in Arabidopsis, Zea mays, Phaseolus vulgalis, Glycine
max and Nicotiana tabacum on northern analysis [27–
31] The 21 UPR genes up-regulated by the stress
induced by both TM and dithiothreitol have been
identified among 8297 genes of the 27 000 protein-coding genes of Arabidopsis with an Affimetrix Gene-Chips [32]
In this paper, we present a list of the UPR genes of Arabidopsis identified among all the protein-coding genes In order to increase the accuracy of the list, the genes selected on fluid microarray analysis were reana-lyzed by functional DNA microarray analysis In addi-tion to the genes related to protein folding and degradation, genes related to protein translation and apoptosis are also included in the list
Results
Fluid microarray analysis of gene expression
on TM-treatment
To identify UPR genes among all the genes expressed
in Arabidopsis, we adopted the fluid microarray method, by which target genes can be cloned from selected fluid microarray beads The fluid microarray beads and probes for array analysis were prepared using the mRNA from plantlets treated with or with-out TM for 6 h BiP mRNA, a representative UPR gene, in TM-treated plantlets, was shown to increase 5.7 times compared to the level in untreated plantlets
on real-time RT-PCR analysis For the control experi-ment, competitive hybridization and sorting of the beads with a cell sorter were performed on 4· 104 beads with a 1 : 1 mixture of the probes, which had been prepared from noninduced plantlets, and differen-tially labeled with Cy5 and fluorescein In the control experiment, almost all of the beads after the control hybridization were sorted in the diagonal line region, the fluorescence intensities for fluorescein and Cy5 being the same (Fig 1A) Based on the distribution of beads in this experiment, we set three gates to collect beads, i.e., for ones more heavily labeled with Cy5 (U1 and U2) and fluorescein (D) For differential gene expression analysis, probes from TM-treated plantlets were labeled with Cy5 Probes from nontreated plant-lets were labeled with fluorescein Then 4· 105 beads were hybridized with a 1 : 1 mixture of the two types
of probes For analysis of differential gene expression,
1473 and 1703 beads were collected in fractions U1 and U2 of the up-regulated genes, and 3550 beads in fraction D of the down-regulated genes (Figs 1B and 2A) The DNA fragments on beads in these fractions were amplified by PCR and then sequenced In the up-regulated fractions, 215 genes (Table S1) were found as clusters of clones, which were identified on more than two beads, and 412 as singlet clones, which were identified on single beads (Table S2) For the
Trang 3down-regulated fraction, 10% of the total beads were
analyzed to reveal 17 genes as clusters of clones
(Table 1) and 34 as singlet clones (Table S2)
Analysis with functional DNA microarrays
In order to increase the accuracy of the list of UPR
genes, we reanalyzed the genes selected on fluid
micro-array analysis with functional DNA micromicro-arrays The
functional DNA microarrays were prepared by
spot-ting PCR fragments from the 215 up-regulated cluster
genes (Table S1) and the 17 down-regulated cluster
genes (Table 1) cloned on fluid microarray analysis
Singlet genes were omitted from the functional DNA
microarray analysis, because the list of singlet genes
was predicted to contain missorted non-UPR genes
at a high frequency Functional DNA microarray
analyses were performed with mRNA preparations
from plantlets treated with or without TM,
dithio-threitol or l-azetidine-2-carboxylic acid (AZC) AZC
is a proline analog that is incorporated in nascent
polypeptides instead of proline and prevents the
fold-ing of the polypeptides [33] Induction of BiP mRNA
by dithiothreitol- or AZC-treatment (3 h or 17 h,
respectively) was confirmed to be 3.4 or 22-times
higher than that in untreated plantlets on real-time
RT-PCR analysis To identify the up-regulated UPR
genes, it was required that candidate UPR genes
show a mean fold variation of greater than 1.2-fold
with all the treatments with TM, dithiothreitol and
AZC In addition, from the list, we eliminated the
genes in which the degree of variation was lower
than the sum of the background variation and twice
the standard deviation The degree of background
variation was obtained by means of a self⁄ self hybridi-zation experiment with Cy5 or Cy3-labeled target DNA fragments prepared from nontreated plantlet mRNA Thus, the expression difference between selec-ted genes was regarded as being significant below a probability of error of 5% Thirty-six genes were con-firmed to be induced under the three different induct-ive conditions, because these genes satisfied this criterion (Fig 2B and Table 2) These genes com-prised 30 for which some functional information was available and six for which no information was avail-able Among them, 27 genes were putative paralogs that have been reported to be UPR genes in yeast and⁄ or mammalian cells The functional categories comprise protein folding (13 genes), translocation (six genes), ER-associated protein degradation (ERAD) (three genes; HRD1-like, SEL-1L⁄ HRD3-like, and DER1-like), protein glycosylation and modification (two genes), regulation of translation (P58IPK) [34], and vesicle trafficking (two genes) The induction of HRD1-like, SEL-1L⁄ HRD3-like, DER1-like, and P58IPK mRNA was confirmed by real-time RT-PCR analysis (Fig 3) In addition, we found that an anti-apoptosis protein, Bax inhibitor-1 (BI-1) [35,36], was also included in the list of up-regulated UPR genes Induction of this paralog by ER stress in organisms other than plants has not been reported The induc-tion of BI-1 mRNA by ER stress in Arabidopsis was confirmed by real-time RT-PCR analysis (Fig 3) Furthermore, the induction (1.5-fold variation) of Homo sapiens BI-1 by TM-treatment for 24 h was confirmed in Hep G2 cells, a cell line derived from a human hepatoma, by real-time RT-PCR (data not shown)
Fig 1 Competitive hybridization on fluid microarrays (A) Control hybridization: 4 · 10 4
beads were hybridized with a 1 : 1 mixture of differ-entially labeled probes from noninduced plantlets (B) Competitive hybridization: 4 · 10 5 beads were hybridized with a 1 : 1 mixture of cDNA probes prepared from induced (Cy5) and noninduced plantlets (fluorescein) as described under Experimental procedures After hybridization, beads that went to gates U1, U2 and D were collected and subjected to gene analysis as described under Experimental procedures.
Trang 4To identify the down-regulated UPR genes, we
required that candidate UPR genes show a mean fold
variation of lower than 0.8-fold with all the treatments
with TM, dithiothreitol and AZC Two genes encoding
vegetative storage proteins, Vsp1 and Vsp2 [37,38],
sat-isfied this criterion Vsp2-beads comprised 58% of the
beads collected and were analyzed at gate D
Putative cis-acting regulatory element
of UPR genes
In yeast, ER stress activates Ire1p, which triggers the nonconventional splicing of HAC1 mRNA [3–5] Hac1p produced from the spliced mRNA induces the transcription of UPR genes by binding to their UPR cis-acting regulatory element (UPRE), CAGCGTG [6–8] In mammals, four kinds of cis-acting regulatory elements, which respond to ER stress, are known Mammalian UPRE (TGACGTG-T⁄ G) has been shown to be the specific cis-acting regulatory element for XBP1 and is referred to as the XBP1 binding site [39,40] ERSE (CCAAT-N9-CCACG) has been found
to be recognized by both ATF6 and XBP1 in vitro [41] ERSEII (ATTGG-N-CCACG) has also been demonstrated to be a target of ATF6 [42] Binding of ATF6 to these cis-acting regulatory elements occurs in collaboration with general transcription factor nuclear factor-Y [43,44] AARE (C⁄ EBT-ATF) (TT-G⁄ T-CATCA), which was discovered in the CHOP pro-moter, is recognized by ATF4, translation of which is accelerated by ER stress [24] In plants, a plant-specific UPR element (P-UPRE) (ATTGGTCCACGTCATC), which contains two mammalian UPR cis-acting regula-tory elements such as ERSEII and XBP1 binding sequences, was found in the 5¢ upstream regions of the BiPand calnexin genes [45] Furthermore, complement-ary sequences to the mammalian ERSE and XBP1 binding sequences have been found in the 5¢ upstream regions of several genes that are induced by TM- or dithiothreitol-treatment [32,45] Therefore, we searched for P-UPRE, the XBP1 binding sequence, ERSE, AARE, or complementary sequences in the 5¢ upstream regions (up to 1000 nucleotides) of the UPR genes Sin-gle or plural putative cis-acting regulatory elements were found in the 5¢ upstream regions of 28 of the 36 up-regulated genes (Fig 2C and Table 3) No cis-acting regulatory element sequence was found in the 5¢ upstream regions of the two down-regulated genes
Increase in putative SEL-1L due to ER stress
in Arabidopsis
In yeast and mammalian cells, the HRD1⁄ HRD3 (SEL-1L) ubiquitination system coupled to protein degradation by 26S proteasomes is known to be induced to remove unfolded proteins under ER stress [9,46] Plant paralogs of these genes have not been identified yet In this study, the transcriptional induc-tion of genes homologous to mammalian HRD1 and SEL-1L [47–49] was observed (Fig 3) Then, HRD1-and SEL-1L-like cDNAs were cloned with mRNA of
Fig 2 Overview of the fluid microarray and functional microarray
analyses (A) Gene selection by fluid microarray analysis Gates,
U1, U2 and D were set as shown in Fig 1B Singlet, a gene
identi-fied on a single bead Cluster, a gene identiidenti-fied on more than two
beads (B) Analysis with functional DNA microarrays The genes
selected in (A) were analyzed Two hundred and thirty-two genes
(215 up-regulated cluster and 17 down-regulated cluster genes)
were spotted on functional DNA microarrays The functional DNA
microarray analysis was carried out with target DNA fragments
pre-pared from the mRNA of control plantlets or plantlets treated with
tunicamycin (TM), dithiothreitol (DTT) or L -azetidine-2-carboxylic acid
(AZC) as described under Experimental procedures The numbers
are the numbers of genes that showed an expression difference
between control plantlets and plantlets treated with TM, DTT or
AZC The numbers in the ‘Overlap’ row are the numbers of
overlap-ping up-regulated genes or down-regulated genes upon treatments
with the three reagents (C) Venn diagram of the numbers of
over-lapping and nonoverover-lapping putative UPR cis-acting regulatory
ele-ments of the 36 up-regulated genes selected in (B) The numbers
in parentheses are the numbers of genes that have a cis-acting
reg-ulatory element Bold letters are the numbers of overlapping genes.
ERSE, CCAAT-N9-(A ⁄ C)CACG; XbpI, TGACGTG(G ⁄ T); P-UPRE,
ATTGG(T ⁄ G)CCACGTCAT; AARE, TT(G ⁄ T)CATCA.
Trang 5Arabidopsis plantlets by RT-PCR Their nucleotide
sequences coincided with those presented in the
data-base of ‘The Arabidopsis Information Resource’
(http://www.arabidopsis.org/) The putative amino acid
sequence of an HRD1-like protein contained an
N-ter-minal signal sequence and five membrane-spanning
regions (data not shown) The recombinant luminal
domain of the HRD1-like protein was expressed in
Escherichia coliand purified Unfortunately,
autoubiq-uitination activity was not detected for the
recombin-ant HRD1-like protein On the other hand, the
putative amino acid sequence of Arabidopsis SEL-1L
(At SEL-1L) contained an N-terminal signal sequence
(Met1–Glu20), two N-glycosylation consensus
seq-uences, and a membrane-spanning region (Phe623–
Arg643) near the C-terminus (data not shown) The
amino acid sequence of a soybean paralog of SEL-1L,
which was deduced from the nucleotide sequence of
cDNA cloned from young leaves by RT-PCR, was
clo-sely similar to Arabidopsis ones (data not shown)
Anti-(At SEL-1L) serum was prepared with the
recom-binant luminal domain (Phe21–Val622) of At SEL-1L,
which was expressed in E coli and isolated The
anti-serum only immunoreacted with a 74 kDa protein of
control plantlets on western blotting analysis (Fig 4A)
With TM-treatment, the 74 kDa protein gradually
decreased and a 70 kDa band began to appear at 4 h
after the treatment During the next 24 h, the 70 kDa
band significantly increased The size of the 74 kDa
band decreased to 70 kDa on endoglycosidase H (Endo H) digestion On the other hand, the 70 kDa band was insensitive to Endo H (Fig 4B) From these results, the 70 kDa protein was thought to be a non-glycosylated form of At SEL-1L On cell fractionation,
At SEL-1L was assumed to be a membrane protein, as judging from the existence of a putative membrane spanning region (Fig 4C) The 70 kDa band of plant-lets treated with TM for 24 or 48 h was denser than the 74 kDa band of the control plantlets (Fig 4B) Thus, it was suggested that At SEL-1L polypeptides were synthesized from the At SEL-1L mRNA induced
by ER stress, but that N-glycosylation of newly syn-thesized At SEL-1L molecules was inhibited by TM
ER stress and phosphorylation of eIF2a
In this study, we found that the mRNA of P58IPKwas induced by ER stress (Table 2 and Fig 3) P58IPKwas first identified as an inhibitor of interferon-induced PKR in mammalian cells [50] The PKR family responds to different stress signals and attenuates translation by phosphorylating the specific serine resi-due of eIF2a [51] to protect cells from the stress P58IPK inhibits PKR-mediated translational arrest by inactivating the kinase by binding to the domain of PKR family members In mammals, ER stress also causes translational arrest through phosphorylation
of eIF2a by PKR-like ER kinase, PERK [20,52]
Table 1 Genes recovered at gate D and functional DNA microarray analysis of them Tunicamycin (TM), dithiothreitol (DTT) and L -azetidine-2-carboxylic acid (AZC) values are means for six experiments Control ratio obtained on competitive hybridization with Cy5- and Cy3-labeled control mRNA; values are means for six experiments SD, standard deviation; n.d., not determined.
AGI gene Description
Fluid microbead array (number of beads)
Functional DNA microarray (fold variation)
At3g04120 Glyceraldehyde-3-phosphate dehydrogenase C subunit 0 0 2 0.68 0.99 0.98 1.01 (0.03)
Trang 6Table 2 Genes up-regulated by ER stress Tunicamycin (TM), dithiothreitol (DTT) and L -azetidine-2-carboxylic acid (AZC) values are means for six experiments Control ratio obtained on competitive hybridization with Cy5- and Cy3-labeled control mRNA; values are means for six experiments SD, standard deviation.
AGI gene Description
Fluid microbead array (number of beads) Functional DNA microarray (fold variation)
PROTEIN FOLDING
GLYCOSYLATION ⁄ MODIFICATION
At2g02810 UDP-glucose ⁄ UDP-galactose
transporter a,b
At2g41490 UDP-GlcNac:dolichol phosphate
N-acetyl-glucosamine-1-phosphate
transferase a
TRANSLOCATION
PROTEIN DEGRADATION
TRANSLATION
VESICLE TRAFFICKING
ANTI-APOPTOSIS
UNCLASSIFIED
At5g09410 Similar to anther ethylene-up-regulated
calmodulin-binding protein ER1
UNKNOWN
a Gene identified by Martı`nez and Chrispeels [32] b Genes identified by Noh et al [54].
Trang 7Mammalian P58IPKhas been shown to be induced at a
later phase of ER stress [53] Deletion of P58IPK has
been reported to result in an increase in
phosphory-lated eIF2a Hence P58IPK is thought to function as a
feedback regulator for translational regulation in the
later phase of ER stress The phosphorylated Ser51 of
eIF2a in plantlets was examined during ER stress by
western blot analysis (Fig 5A) The level of
phosphor-ylated eIF2a (Ser51) in the plantlets treated with TM
was lower than that in untreated plantlets The
phos-phorylated eIF2a increased again on removal of TM
from the medium after 6 h of treatment However, the
protein synthesis in plantlets, which was assayed as the
incorporation of [35S]-labeled Met and Cys into
nas-cent proteins, was not affected by TM-treatment
(Fig 5B)
Discussion
In this study, we tried to make a list of the UPR genes
in Arabidopsis In total, 215 up-regulated and 17
down-regulated cluster genes were cloned from mRNA
of Arabidopsis plantlets treated with TM on fluid
microarray analysis A functional DNA array was
pre-pared by using the cloned gene fragments, and then
used for analysis Among the 215 up-regulated cluster
genes, only 63 showed statistically positive signals on
functional DNA array analysis, showing differences in
the expression of target mRNA of the plantlets treated
with or without TM Because the fluid microarray
beads included a large number with highly expressed housekeeping genes, some of them might be missorted
at the gates, which would expand the list of genes Of the beads collected at gates U1 and D on fluid micro-array analysis, 89 and 87% were regarded as up-regu-lated and down-reguup-regu-lated genes on functional DNA microarray analysis, respectively On the other hand, 38% of the beads collected at gate U2 were regarded
as up-regulated genes on functional DNA microarray analysis This suggests that the discrepancy between the values obtained in the two analyses is mainly due
to the beads missorted at gate U2 However, of the rest, the 50 genes that showed no up-regulated signal for the plantlets treated with TM showed an up-regula-ted signal in the plantlets treaup-regula-ted with dithiothreitol and⁄ or AZC In addition, 23 of the genes that showed
no difference in expression on DNA microarray analy-sis between plantlets treated and untreated with TM had putative UPR cis-acting regulatory elements in their upstream regions Furthermore, 27 of the 63 genes were eliminated on functional DNA microarray analysis from the list by setting some criteria There-fore, the remaining 36 genes, which satisfied these criteria, were considered to be reliable up-regulated UPR genes Among these 36 genes, 12 coincided with up-regulated UPR genes previously identified on ana-lysis with an Affimetrix GeneChips loaded with 8297 Arabidopsis probe sets [32,54] Two down-regulated genes, Vsp1 and Vsp2, which satisfied all the criteria, are known to be for temporary nitrogen-storage pro-teins [38], and are subject to regulation by sugars, light, phosphates, nitrogen, wounding, auxins, jasmo-nates and oxidative-stress [55] The down-regulation of Vsp1 and Vsp2 may result in an increase in the intra-cellular amino acid pool, which may play an important role in the recovery from ER stress In mammalian cells, ER stress affects cellular amino acid metabolism via the PERK⁄ ATF4-mediated signaling pathway, which induces some amino acid synthesis- and trans-port-related genes [23] No putative UPR cis-acting regulatory element was found in the 5¢ upstream regions of Arabidopsis Vsp1 and Vsp2 Therefore, it is not clear whether these genes are directly regulated by the UPR system or down-regulated by a metabolic dis-order caused by ER stress
Thirteen genes, which encode six protein families responsible for protein folding, are included in the UPRgene list Among them, BiP (three genes),
calnex-in(two genes), calreticulin (two genes), and AtHSP
90-7 (one gene) have been shown to be induced by ER stress on northern blotting [25,54] Four genes enco-ding PDI families are also included in the list PDI and its family members are characterized by the
pres-Fig 3 Confirmation of transcriptional induction of six genes by
real-time RT-PCR analysis The amounts of actin, BiP, HRD1,
SEL-1L, DER1, p58 IPK and BI-1 mRNAs in total RNA from Arabidopsis
plantlets treated with TM (black bars), DTT (hatched bars), or AZC
(white bars) for 2 h were determined by real-time RT-PCR as
des-cribed under Experimental procedures The value for each mRNA
was standardized to the value for actin mRNA in the corresponding
total RNA preparation Fold expression change was calculated as
the ratio of mRNA in the plantlets treated and untreated with a
stress reagent Each value represents the mean for two
experi-ments.
Trang 8Table 3 Putative cis-acting regulatory elements of genes up-regulated by ER stress Position designated from the 5¢ terminus of the ATG initiation codon Lowercase letters in sequences correspond to N9 in ERSE-like sequence CCAAT-N9-(A/C) CACG.
AGI gene Description
cis-Acting regulatory element
ERSE-like CGTGTctgcttgtgATTGG(220–202)
Xbp1 binding-like TGACGTGG(240–233)
Xbp1 binding-like CCACGTCA(253–246)
Xbp1 binding-like TGACGTGT(131–124)
ERSE-like CCAATtacaattgtACACG(134–116) At2g02810 Similar to UDP-glucose ⁄ UDP-galactose transporter –
At2g41490 UDP-GlcNac:dolichol phosphate
–
Xbp1 binding-like TGACGTGT(322–315)
ERSE-like CGTGTaataatataATTGG(146–128)
ERSE-like CGTGTttaattatcATTGG(304–286)
Xbp1 binding-like TGACGTGG(477–470) Xbp1 binding-like ACACGTCA(609–602) At4g21730 Similar to NEM-sensitive fusion protein –
At5g09410 Similar to anther ethylene-up-regulated
Xbp1 binding-like TGACGTGG(396–389)
Trang 9ence of one or two thioredoxin homologous motifs per
molecule Yeast and mammalian PDIs are known as
multifunctional folding catalysts and molecular
chaper-ones, which catalyze the formation and rearrangement
of disulfide bonds between correct pairs of cysteine
residues in nascent polypeptide chains in the ER [56]
Mammalian PDI functions not only as a catalytic
enzyme but also as a subunit of microsome
triacylglyc-erol transfer protein [57] and prolylhydroxylase [58]
Mammalian PDI family ER-60⁄ ERp57, which also
exhibits protein oxidoreductase activity, interacts and
cooperates with calnexin or calreticulin for oxidative
folding of N-glycosylated proteins [59–61] The genes
of these PDI families are UPR genes [41] In the
Ara-bidopsisgenome, 13 genes encoding putative
PDI-rela-ted proteins, i.e At1g04980 (NP 171990), At1g07960
(NP172274), At1g15020 (NP 172955), At1g35620 (NP
564462), At1g21750 (NP 173594), At1g52260
(NP 175636), At1g77510 (NP 177875), At2g01270 (NP
565258), At2g32920 (NP 180851), At2g47470
(NP182269), At3g54960 (NP 191056), At3g16110 (NP
188232), and At5g60640 (NP 568926), were found
Identification and characterization of these PDI family
proteins were not carried out However, they were
sup-posed to play important roles in protein folding, as
four PDI-related genes among the above 13 genes were
confirmed to be induced by ER stress A gene enco-ding cyclophilin family protein ATCYP20-1 was identi-fied as a UPR gene Twenty-nine genes encoding cyclophilin family members were found in the Arabi-dopsis genome [62] Among them, five gene products are assumed to be targeted to the ER lumen with N-terminal signal peptides Among them, ATCYP20-1 has the amino acid sequence RFWH, which is an essential sequence for peptidyl prolyl cis, trans iso-merase activity Hence, it is suggested that ATCYP20-1 may participate in the folding of proteins in the ER The genes of six translocation-related proteins were found to be induced In mammalian cells and yeast, translocon subunit proteins are thought to be induced
to enhance retrotranslocation of unfolded proteins from the ER to the cytosol [63] The retrotranslocated proteins are degraded by 26S proteasomes Recently,
in tobacco, a GFP-fusion protein containing the P region of calreticulin, which is a model of a misfolded
A
B
C
Fig 4 Increase in At SEL-1L in the membranes of Arabidopsis
plantlets on TM-treatment (A) Plantlets were incubated in the
pres-ence (lanes 7–12) or abspres-ence of TM (lanes 1–6) for the indicated
times Proteins were extracted and then subjected to SDS ⁄ PAGE.
At SEL-1L was stained by western blotting with antiserum as
des-cribed under Experimental procedures (B) Plantlets were incubated
in the presence (lanes 3 and 4) or absence of TM (lanes 1 and 2)
for 48 h Proteins were extracted, digested with (lanes 2 and 4) or
without (lanes 1 and 3) Endo H, and then subjected to SDS ⁄ PAGE.
At SEL-1L was stained by western blotting with antiserum as
des-cribed under Experimental procedures (C) The total (lane 1),
super-natant (lane 2), and membrane (lane 3) fractions obtained from the
plantlets treated with TM for 48 h on centrifugation at 100 000 g
were subjected to SDS ⁄ PAGE, and At SEL-1L was stained by
western blotting with antiserum as described under Experimental
procedures.
A
B
Fig 5 Effect of TM-treatment on phosphorylation of eIF2a Plant-lets were incubated in the medium with TM for 6 h (lane 2), 7 h (lane 3), or 9 h (lane 4), or without TM for 9 h (lane 1) as described under Experimental procedures In other experiments, plantlets were incubated in the medium with TM for 6 h and then incubated
in the medium without TM for an additional 1 h (lane 5) or 2 h (lane 6) (A) After the incubation, the proteins were extracted from the plantlets and subjected to SDS ⁄ PAGE Phosphorylated Ser51 of eIF2a was determined by western blot analysis as described under Experimental procedures (B) After the incubation, proteins of the plantlets were metabolically labeled with [ 35 S]Met and [ 35 S]Cys for
20 min at 25 C Then, the proteins were extracted and subjected
to SDS ⁄ PAGE Labeled proteins were determined by fluorography
as described under Experimental procedures.
Trang 10protein in the ER, was shown to be retrotranslocated
to the cytosol, ubiquitinated, and then degraded [64]
The induction of translocon subunits by ER stress in
Arabidopsis suggests that an ERAD system similar to
those of yeast or mammalian cells may remove
mis-folded proteins produced in the ER of plant cells This
is supported by our finding that the genes encoding
putative plant DER1, HRD1 and SEL-1L⁄ HRD3 were
also induced by ER stress DER1 is a hydrophobic
protein that is localized to the ER In yeast, deletion
of DER1 prevents degradation of unfolded proteins,
suggesting that the function of DER1 may be
specific-ally required for ERAD [65] Yeast HRD1 is an
ER-membrane-anchored ubiquitin ligase, which is required
for the degradation and ubiquitination of several
ERAD substrates, and is associated with relevant
ubiquitin-conjugating enzymes [46] At HRD1, which
has the same nucleotide sequence as that registered in
‘The Arabidopsis Information Resource’, was cloned
by RT-PCR with mRNA from Arabidopsis Six
trans-membrane regions and a RING-H2 domain of
Arabi-dopsis HRD1 (At HRD1) showed high sequence
homology with those of yeast and human HRD1s
Unfortunately, it is unclear whether or not At HRD1
functions as an ubiquitin ligase, as the cytosolic
domain of At HRD1, which was expressed in E coli
and isolated, showed no self-ubiquitination activity
with an in vitro assay system involving commercial
human E1 and yeast E2 (UbCH5c) Yeast HRD3 is an
ER-resident glycoprotein with a single span near the
C-terminus, which stabilizes HRD1 and regulates the
cytosolic HRD1 RING-H2 domain through
interac-tion with the HRD1 transmembrane domain [66] We
showed that At SEL-1L was a membrane-anchored
glycoprotein and that it increased under ER stress In
order to clarify the details of the mechanism of plant
ERAD, functional characterization of these proteins
must be performed
In mammalian cells, ER stress responses are
com-posed of three steps, i.e., enhancement of the refolding
and degradation of unfolded proteins, attenuation of
translation [20,21], and apoptosis [24] ER stress has
not been found to cause attenuation of translation in
plants In this study, we found that the P58IPK gene
was up-regulated by ER stress Mammalian P58IPK is
induced at a later phase of ER stress and inhibits
PKR-mediated translational arrest by binding to the
kinase domain of the PKR family [53] However, bulk
protein translation of Arabidopsis was not affected by
ER stress, even though the phosphorylation of eIF2a
(Ser51) was partially inhibited by ER stress The
phos-phorylation of eIF2a (Ser51) increases the translational
efficiency of yeast GCN4 mRNA and mammalian
ATF4 mRNA, which have four and two upstream open reading frames in the 5¢ noncoding portion, respectively [67,68] Induction of Arabidopsis P58IPK followed by a decrease in the phosphorylation of eIF2a (Ser51) may increase the translational efficiency for unidentified gene(s)
It is unclear whether apoptosis may function as a UPR in plants, although inhibition of ER-type IIA
Ca2+-pumps has been reported to induce ER stress and apoptosis in soybean cells [69] In this study, we identified apoptosis-related gene BI-1 as a UPR gene BI-1 is an evolutionarily conserved integral membrane protein localized in the ER [35,36] In mammalian cells, BI-1 affords protection from apoptosis induced
by ER stress by inhibiting BAX activation and translo-cation to mitochondria, by preserving the mitochond-rial membrane potential, and by suppressing caspase activation [70] BAX and Bcl2, and their relatives were not found in plants However, in rice and barley, BI-1 has been shown to suppress fungal elicitor-induced apoptosis [71,72]
Experimental procedures
Plant materials and treatments Sterile seeds of Arabidopsis thaliana (Columbia) were germi-nated in 0.5· Murashige and Skoog medium [73] containing 1% (w⁄ v) sucrose (MS), and cultured for two weeks To prepare a cDNA tagged library and probes for transcrip-tome analysis with fluid microarrays or functional DNA microarrays, whole plantlets were treated by immersing
their roots in MS containing 5 lgÆmL)1TM, 1 mm dithio-threitol or 50 mm AZC for the indicated times For the control experiment, plantlets were treated with MS without stress reagents For relative quantification of mRNA by real-time RT-PCR, and pulse-labeling experiments with [35S]Met and [35S]Cys, the upper parts of plants were cut off from their roots and immersed in MS with a stress rea-gent
Real-time RT-PCR analysis Total RNA was isolated with an RNeasy Plant Mini kit (Qiagen, Valencia, CA) from plant tissues treated with or without TM for 6 h Relative quantification of mRNA was carried out by the real-time RT-PCR method with an ABI PRISM 7000 Sequence Detection System (Applied Biosys-tems, Foster City, CA) Forward primers, 5¢-AAGTCGT TGCACCTCCTGAGA-3¢, 5¢-TCAAGGACGCTGTTGT CACTGT-3¢, 5¢-ACACGGCAAATAACGTTCATCTCTA-3¢, 5¢-GGACTGCTTTCATCTGGCTTGT-3¢, 5¢-TCTCT GTTGGGTTTATCTCTTTGGTT-3¢, 5¢-TGATGGAAGA AGCAGTGGATGA-3¢ and 5¢-CGTAGAAGAGTGGTA