The protein binds in a sequence specific manner to the CaMV 35S promoter which is down regulated when ABZ1 is coexpressed.. In the present work, a small basic leucine zipper bZIP transcri
Trang 1Functional dissection of a small anaerobically induced bZIP
transcription factor from tomato
Simone Sell and Reinhard Hehl
Institut fu¨r Genetik, Technische Universita¨t Braunschweig, Germany
A small anaerobically induced tomato transcription factor
was isolated from a subtractive library This factor,
desig-nated ABZ1 (anaerobic basic leucine zipper), is
anaerobi-cally induced in fruits, leaves and roots and encodes a
nuclear localized protein ABZ1 shares close structural and
sequence homology with the S-family of small basic leucine
zipper (bZIP) transcription factors that are implicated in
stress response Nuclear localization of ABZ1 is mediated by
the basic region and occurs under normoxic conditions
ABZ1 binds to G-box-like target sites as a dimer Binding
can be abolished by heterodimerization with a truncated
protein retaining the leucine zipper but lacking the DNA binding domain The protein binds in a sequence specific manner to the CaMV 35S promoter which is down regulated when ABZ1 is coexpressed This correlates with the anaer-obic down regulation of the 35S promoter in tomato and tobacco These results may suggest that small bZIP proteins are involved in the negative regulation of gene expression under anaerobic conditions
Keywords: anaerobiosis; bZIP; DNA binding; Lycopersicon esculentum; transcription factor
Plant survival under adverse environmental situations is
largely dependent on their adaptation strategies
Anaero-biosis or low oxygen conditions occur when plants are
subjected to flooding or to waterlogging of the soil Under
these conditions oxygen is rapidly consumed by
micro-organisms and plant roots [1] Plants react to these
conditions with a variety of responses To compensate the
decrease in energy production and the lack of NADH
regeneration, the rate of glycolysis is increased and
fermen-tative pathways are induced [1] Furthermore, plants can
respond to flooding with the induction of aerenchyma in the
root cortex and hyponastic growth to push their vital organs
above water level [2,3]
The reactions of plants towards a low oxygen
environ-ment entail a significant reprogramming of gene expression
which comprises transcriptional induction and selective
translation of mRNAs for anaerobic proteins [4] Plants
probably sense the lack of oxygen as an electron acceptor in
the mitochondria Mitochondria are implicated in an early
response because they release calcium to the cytosol in
response to anaerobiosis [5] The complete signal
transduc-tion pathway has yet to be elucidated Recent data suggest that O2deprivation stimulates a G-protein signal transduc-tion pathway that results in the inductransduc-tion of alcohol dehydrogenase (ADH) expression [6] Other components of the signal transduction pathway may comprise 14-3-3 proteins, calcium dependent kinases and several transcrip-tion factors [7–9] ADH, one of the most extensively studied genes that is induced during oxygen deprivation, is probably induced by the transcription factor AtMYB2 in Arabidopsis thaliana[10]
A comprehensive analysis of low oxygen regulated gene expression was recently reported for A thaliana [11] In a microarray containing 3500 cDNA clones, 210 differentially expressed genes were identified Among these were 21 nonredundant down regulated genes In contrast to tran-scriptional induction and post-trantran-scriptional regulation, little is known about low oxygen mediated down regulation
of gene expression
In the present work, a small basic leucine zipper (bZIP) transcription factor (TF) designated ABZ1, was isolated from a tomato cDNA library enriched for anaerobically induced genes This TF was studied in detail In addition to binding site specificity, nuclear localization, identification of DNA– and protein–protein binding domains it is shown that efficient DNA binding of a heterodimer requires two DNA binding domains in the interacting proteins The putative role of ABZ1 in the anaerobic response pathway is discussed
Materials and methods Tomato cDNA library construction and screening
A cDNA library from tomato, Lycopersicon esculentum cv Micro-Tom [12], was generated in plasmid pSport1 using the Invitrogen (Karlsruhe, Germany) SuperScriptTM Plas-mid System with Gateway Technology for cDNA
Correspondence to R Hehl, Institut fu¨r Genetik Technische
Universita¨t Braunschweig Spielmannstr 7, D-38106 Braunschweig,
Germany Fax: +49 531 391 5765, Tel.: +49 531 391 5772,
E-mail: R.Hehl@tu-braunschweig.de
Abbreviations: ADH, alcohol dehydrogenase; as-1, activation
sequence-1; bZIP, basic leucine zipper; DAPI,
4¢-6-diamidino-2-phenylindole; EMSA, electrophoretic mobility shift assay; GUS,
b-glucuronidase; LUC, luciferase; NLS, nuclear localization signal;
RBSS, random binding site selection.
Note: A website is available at http://www.tu-braunschweig.de/ifg/ag/
hehl
Note: The EMBL/GenBank accession number of ABZ1 is AJ715788.
(Received 9 July 2004, revised 30 September 2004,
accepted 4 October 2004)
Trang 2Synthesis and Cloning according to the protocol of the
supplier Poly(A)+ RNA (5 lg) isolated from fruits, roots,
leaves, and stems of anaerobically induced tomato plants
was employed for cDNA synthesis The plants were grown
in a greenhouse and were 3 months old Anaerobic
incubations were carried out in an airtight glass container
(Merck, Darmstadt, Germany) together with Anaerocult A
(Merck) for 20 h in a light chamber (12 : 12 h light/
darkness) Screening of the library and all other
recombin-ant DNA work was done according to standard protocols
[13] Sequence analysis of cDNA clones and other in vitro
constructs used in this work was performed by Seqlab
Company, Go¨ttingen, Germany The sequences were
ana-lysed for subcellular localization signals using PSORT at
http://psort.nibb.ac.jp/form.html Sequence comparisons
and alignments were performed at http://www.ncbi.nlm
nih.gov/BLAST and http://www.ch.embnet.org/software/
ClustalW.html The phylogenetic tree was generated using
CLUSTALX1.81 [14]
RNA isolation and Northern blot hybridizations
Total RNA was isolated according to a previously
published procedure [15] To isolate poly(A)+ RNA,
1 mg total RNA was used with the OligotexTM mRNA
Midi Kit (Qiagen, Hilden, Germany) according to the
manufacturer’s protocol Northern blots were performed
according to standard protocols [13] Total RNA (10 lg)
was used for RNA gel electrophoresis and radioactive
probes were generated using the HexaLabelTM DNA
Labeling Kit from MBI Fermentas (St Leon-Rot,
Germany)
Recombinant protein production and purification
For the expression in and purification of recombinant
proteins from Escherichia coli the QIAexpressionistTM
system from Qiagen was used The ABZ1 coding region
and two truncated derivatives from ABZ1 were PCR
amplified from a full size cDNA clone using the following
primer pairs Full size, ABZ1(1–138): 5¢-TATAGGA
TCCATGTCACCTTTAAGGCAGAG-3¢ and 5¢-ATAT
N-ter-minal deletion, ABZ1(47–138): 5¢-TATGGATCCATGC
TTCTGCAAGATTTGACAGG-3¢ and 5¢-ATATCCC
deletion, ABZ1(1–100): 5¢-TATAGGATCCATGTCACC
TTTAAGGCAGAG-3¢ and 5¢-ATACCCGGGTTATAA
ATACCTGAGCCTATCAGTC-3¢
The BamHI and SmaI sites within the primers
(under-lined) were used to directionally clone the amplified DNA
fragments into plasmid pQE-30 Prior to this, the amplified
DNA fragments were cloned into pCR2.1 (Invitrogen)
and sequenced Recombinant pQE-30 plasmids were
transformed together with the repressor plasmid pREP4
(Qiagen) in BL21-CodonPlus(DE3)-RIL E coli cells
(Stratagene, Amsterdam, the Netherlands) Induction of
protein expression and purification over Ni-nitrilotriacetic
acid columns were performed according to The
QIAex-pressionistTM manual Because the recombinant proteins
were mainly localized in the insoluble fraction, a previously
reported method that includes steps for de- and re-naturing
of the protein was employed [16] Protein concentrations were determined according to Bradford [17]
Electrophoretic mobility shift assays and random binding site selection
Electrophoretic mobility shift assays (EMSA) were carried out according to Ausubel et al [18] The four probes used for EMSA, RBSS1, 1.1, 5 and 6.1 are shown in Table 1 These probes were used because they represent different classes of binding sites RBSS1-Mu, containing a mutation
in the ACGT core sequence (GGTTGATTAGGGAA), was used as a nonspecific competitor All fragments are bordered by primer binding sites 5¢-CAGGTCAGT TCAGCGGATCCTGTCG-3¢ and 5¢-GCTGCAGTTG CACTGAATTCGCCTC-3¢ that were also used for PCR amplifications during the random binding site selection (RBSS) assay Random binding site selection was per-formed according to Ausubel et al [18] Oligonucleotides consisted of five random nucleotides 5¢ and 3¢ of the ACGT core sequence bordered by the above mentioned primer binding sites Oligonucleotides were amplified in the pres-ence of [32P]dCTP[aP] and incubated with recombinant ABZ1 After electrophoretic separation the bound oligo-nucleotides were eluted from the gel and subjected to another round of PCR amplification, ABZ1 binding, and EMSA Following five rounds of selection the amplified fragments were cloned into pCR2.1 and sequenced Another fragment used for EMSA was a 100 bp fragment from the CaMV 35S promoter that was amplified
by PCR using the primers 5¢-TATGTCGACCGAG GAACATAGTGGAAAAAG-3¢ and 5¢-ATAGTCGACT GGGATTGTGCGTCATCCCTT-3¢
Fragments for EMSA were amplified by PCR from recombinant pCR2.1 (RBSS1, 1.1, 5 6.1, and RBSS1-Mu) and pRT103-GUS (b-glucuronidase) [19] in the presence of [32P]dCTP[aP] Binding reactions were carried out in 15 lL
10 mMTris/HCl pH 7.5; 40 mMNaCl; 1 mMEDTA; 4% (v/v) glycerol; 10 mM 2-mercaptoethanol; 10 mM dithio-threitol; 5 mMphenylmethanesulfonyl fluoride; 2 lg/15 lL poly(dI-dC) [20] for 30 min at room temperature The amounts of recombinant protein, radioactively labelled
Table 1 Sequences obtained from a random binding site selection (RBSS) assay with ABZ1 RBSS1, 1.1, 5 and 6.1 are among the 17 selected binding sites Nucleotide frequency at each position is shown relative to the center of the palindromic core K ¼ G/T, N ¼ A/C/G/
T Below the sequences the frequency of the nucleotides A, C, G and T
at each position of the 17 selected binding sites is shown A consensus sequence was derived from these frequencies.
–5 )4 )3 )2 )1 Core +1 +2 +3 +4 +5
Trang 3fragment and competitor DNA is given in the relevant
figure legends After addition of 3 lL nondenaturing 5·
loading dye [50 mM EDTA pH 8.0; 50 mM Tris/HCl
pH 8.0; 50% (v/v) glycerol; 12.5 mg/10 mL
bromphenol-blue; 12.5 mg/10 mL xylencyanol] the binding assay was
loaded on a native polyacrylamide gel (5–9%) After
electrophoresis in 1· Tris/glycine the gel was dried under
vacuum and exposed to X-ray films
Nuclear localization assays
Plasmid constructs for the analysis of nuclear localization
were generated by fusing full size ABZ1 and truncated
derivatives of ABZ1 in-frame upstream to the amino
terminal end of the uidA gene in pRT103-GUS [19] The
ABZ1 coding region and three truncated derivatives from
ABZ1 were PCR amplified from a full size cDNA clone
using the following primer pairs Abz1(1–138): 5¢-TA
5¢-ATATCCATGGAAAATTTAAACAATCCTGATG-3¢
GCAGAG-3¢ and 5¢-ATATCCATGGGCTTCTTCATC
CTCGATCGC-3¢ Abz1(1–24): 5¢-TATACTCGAGAT
GTCACCTTTAAGGCAGAG-3¢ and 5¢-ATATCCATG
GTCTCATCCATTCCTGCATAC-3¢ Abz1(45–138):
5¢-TATACTCGAGATGCAGAAGCTTCTGCAAGATTT
GAC-3¢ and 5¢-ATATCCATGGAAAATTTAAACAA
TCCTGATG-3¢
The XhoI and NcoI sites within the primers (underlined)
were used to directionally clone the amplified DNA
fragments into plasmid pRT103-GUS The resulting clones
were subsequently sequenced
Layers of onion epidermal cells were placed on
Mur-ashige–Skoog media containing 3% (w/v) sucrose and
transformed by particle bombardment with recombinant
ABZ1-GUS constructs Loading of the gold particles was
performed according to the CaCl2/spermidin protocol [21]
Particle bombardment of epidermal cell layers was
per-formed with 600–700 p.s.i using the Du Pont PDS-1000
particle delivery system [22] After bombardment the tissue
was incubated at 24C for 24 h in a light chamber
(12 : 12 h light/darkness) The histochemical GUS assay
was performed by incubating the tissue in 1 lgÆmL)1
5-bromo-4-chloro-3-indolyl-b-glucuronic acid (X-Gluc) in
50 mM NaPO4 pH 7.0; 1 mM EDTA; 0.1% (v/v) Triton
X-100; 1 mMK-ferrocyanid; 1 mMK-ferricyanid for 4 h at
37C [23] For microscopy, tissues were transferred into
50 mM NaPO4 pH 7.0 For staining of the nuclei, tissues
were treated by adding 1 lgÆmL)1
4¢-6-diamidino-2-phen-ylindole (DAPI) and incubated for 15 min at room
temperature Light microscopy was carried out with a
fluorescent microscope (Axioplan 2, Zeiss, Jena, Germany)
using a 360–370 nm filter for DAPI stained tissue
Transient gene expression analysis in tobacco and
tomato leaves
For transient gene expression analysis four different effector
plasmids were constructed
All constructs are based on plasmid pVKH-35S-pA1
(kindly provided by D Großkopf,
Max-Delbru¨ck-Laboratorium, Ko¨ln, Germany) pVKH-35S-pA1 harbours
a CaMV 35S promoter and a poly(A)+ signal separated by
a cloning linker The coding region of ABZ1 was released with BamHI and SmaI from a recombinant pCR2.1 plasmid that harboured the complete ABZ1 coding region (see above) This fragment was subcloned into pVKH-35S-pA1 which was digested with BamHI and HindIII in which the HindIII site has been filled in This resulted in effector construct designated pVKH-35S-ABZ1-pA1
To generate pVKH-35S-AD-ABZ1-pA1 the activation domain of GAL4 was amplified from pGADT7-Rec (Clon-tech, Heidelberg, Germany) The following primers were used to amplify the activation domain: 5¢-TATAGGATCC ATGGCCAATTTTAATCAAAGTGGGA-3¢ and 5¢-AT ATGGATCCCTCTTTTTTTGGGTTTGGTGGGGT-3¢ The amplified fragment was cut with BamHI (restriction site is underlined in the primers) and cloned into BamHI digested pVKH-35S-ABZ1-pA1 The orientation of the insert and the integrity of the construct was analysed by restriction digest and sequencing
The two effector plasmids pVKH-C1-ABZ1-pA1 and pVKH-C1-AD-ABZ1-pA1 were generated by replacing the 35S promoter with the C1 promoter from the sugar beet cab11gene The 1097 bp long C1 promoter was released with HindIII and BamHI from plasmid pC1L-1097 (kindly provided by D Stahl, Planta GmbH, Einbeck, Germany) After HindIII digestion, the ends were filled in so that the released fragment harbours one blunt and one BamHI end This allowed the directional cloning of the C1 promoter into pVKH-35S-ABZ1-pA1 and pVKH-35S-AD-ABZ1-pA1 from which the 35S promoter was released with SacI and BamHI and in which the SacI site was filled in to generate a blunt end
As a reporter, a 35S-uidA construct was generated by removing the TATA box from plasmid pBT10-TATA-GUS [24] with NcoI and PstI and replacing it with a 500 bp NcoI/PstI CaMV 35S promoter fragment The resulting plasmid is designated pBT10-35S-GUS
For transformation controls, a luciferase gene was used that was either expressed from the 35S promoter in pRT101-LUC [19] or from the C1 promoter in pC1L-1097 For transformation, leaf discs with a diameter of 4 cm were cut from tobacco leaves and placed on wet 3MM Whatman paper Equimolar amounts of effector, reporter and control plasmids were loaded onto gold particles according to standard protocols [21] Particle bombardment was performed with 1100 p.s.i using the Du Pont PDS-1000 particle delivery system [22] After bombardment the tissue was incubated at 24C for 24 h in a light chamber (12 : 12 h light/darkness)
For protein extraction 0.3 g of tissue was homogenized with liquid nitrogen and by adding of 100 lL extraction buffer (0.1MNaH2PO4pH 7.8; 1 mMdithiothreitol) After centrifugation for 10 min with 25 500 g at 4C the supernatant was used for quantitative GUS and luciferase assays Protein concentrations were determined according
to Bradford [17] The determination of GUS activity was performed according to Jefferson & Jefferson et al [25,26] Four hundred and fifty microliters of GUS-reaction buffer [1 mM 4-methyl-umbelliferyl-b-D-glucuronide;
50 mM NaPO4 pH 7.0; 10 mMEDTA; 0.1% (v/v) Triton X-100; 0.1% (v/v) N-laurylsarcosine; 10 m
Trang 42-mercapto-ethanol] were prewarmed to 37C and 50 lL protein
extract was added to start the reaction A blank value was
determined by transferring 50 lL of the reaction after 1 min
into 950 lL stop-buffer (0.2M Na2CO3) Additional
ali-quots were transferred into stop-buffer after 20, 40 and
60 min, respectively The extinction generated by the
reaction product 4-MU was measured in a spectral
photometer (Kontron Instruments, Eching, Germany,
SFM 25; excitation 365 nm; emission 455 nm) to determine
the relative fluorescence units of the generated 4-MU per
min and lg protein These values were corrected with the
values obtained for the transformation control plasmid
expressing the luciferase gene [27] For luciferase assays,
50 lL protein extract was transferred to 350 lL luciferase
buffer (25 mM glycylglycine; 15 mM MgSO4; 1 mM ATP
pH 7.8) After injecting 150 lL substrate solution (0.2 mM
luciferin in 25 mMgycylglycine pH 7.8) the emitted photons
were measured in a luminometer in a time interval of 10 s
(Berthold Lumat 9501; Bad Wildbad, Germany)
The determination of the relative GUS activity using the
luciferase values were performed as described previously
[24,28] The resulting values were used to display the GUS
activity of the different transformations relative to the GUS
activity obtained without effector plasmids which was set to
100% (see below)
For the determination of the relative expression strength
of the CaMV 35S promoter under aerobic and anaerobic
conditions in tobacco and tomato, a previously reported
approach was employed [29] As a reporter a 35S-uidA
construct was generated by removing the TATA box from
plasmid pBT10-TATA-GUS [24] with NcoI and PstI and
replacing it with a 500 bp NcoI/PstI CaMV 35S promoter
fragment The resulting plasmid is designated
pBT10-35S-GUS After bombardment of leaf discs with equimolar
amounts of pBT10-35S-GUS and pRT101-LUC [19], two
of the leaf discs from the same bombardment were incubated aerobically and the other two were incubated anaerobically in an airtight glass container (Merck) together with Anaerocult A (Merck) Incubation was carried out for
24 h in a light chamber (12 : 12 h light/darkness) Luci-ferase activity, b-glucuronidase activity, and the determin-ation of the relative b-glucuronidase activity were performed as described [29]
Results ABZ1 belongs to a family of small bZIP transcription factors and is anaerobically induced in fruits, roots, and leaves
Suppression subtractive hybridization was employed for the isolation of cDNA fragments for anaerobically induced genes from tomato cv Micro-Tom (S Sell & R Hehl, unpublished observations) A full size clone was isolated for
a cDNA fragment that is homologous to bZIP transcription factors The cDNA is 1216 base pairs long, which includes a poly(A) tail of 20 base pairs Figure 1 shows the cDNA that contains a 596 bp leader encoding a short 30 amino acid long peptide reminiscent of upstream open reading frames found in other TFs [30,31] The 596 bp leader is followed by
414 bp coding region and 186 bp 3¢ untranslated sequence The 414 bp coding region translates into a 138 amino acids long protein with a proposed molecular mass of 15.4 kDa Figure 1 shows that the protein harbours a basic region followed by a leucin zipper consisting of five leucines and one isoleucine that are spaced exactly by six amino acids The basic region also harbours a putative nuclear localiza-tion signal (see below) The gene for the small bZIP protein from tomato was designated ABZ1 for anaerobic basic leucine bZIP
Fig 1 cDNA sequence and deduced amino
acid sequence of the ABZ1 gene The138 amino
acid long sequence of ABZ1 (nucleotide
posi-tions 597–1010) harbours a basic leucine
zip-per domain (bold) that contains a putative
bipartite nuclear localization signal (double
underlined) The single amino acids that are
part of the leucine zipper are underlined The
30 amino acid long sequence encoded by the
upstream open reading frame (nucleotide
positions 358–447) harbours several amino
acids (bold) that are conserved in other
up-stream open reading frames.
Trang 5To investigate the spatial expression of ABZ1, RNA blot
hybridizations were carried out Total RNA from
aerobi-cally and anaerobiaerobi-cally treated organs from tomato was
hybridized with the ABZ1 cDNA fragment isolated in the
differential screen for anaerobically induced genes Figure 2
shows that the gene is anaerobically induced in fruits, leaves,
and roots of tomato The ubiquitous nature of the anaerobic
induction of ABZ1 may suggest a more general role in
regulating anaerobic gene expression
Phylogenetic analysis using the basic and leucine zipper
domains of 16 bZIP transcription factors shown in Fig 3
indicates that ABZ1 is most closely related to BZI-4 from
tobacco [32], and belongs to a family of small bZIP proteins
that are often induced upon environmental stress [31] For
example, maize LIP15 and rice LIP19 are low temperature
induced bZIP transcription factors that share 68.9%
sequence identity at the amino acid level [33,34] BZI-4
from tobacco is transcribed specifically in the stamen, the
petals and the pistils of the tobacco flower [32]
The basic domain confers nuclear localization of ABZ1
For gene expression regulation ABZ1 needs to be imported
into the nucleus Using bioinformatic tools it was found that
ABZ1 harbours a putative bipartite nuclear localization
signal (NLS) in its basic region between amino acids 25 and
44 (Fig 1) To determine whether this region harbours a
functional NLS, nuclear localization was investigated with
fusion proteins using the b-glucuronidase (uidA) reporter
gene Fusion constructs were made with the whole 138
amino acid long protein, with the 24 and 44 amino terminal
and 94 carboxy terminal amino acids, respectively These
fusion constructs were transformed by particle
bombard-ment into onion epidermal cells Figure 4 shows that the
majority of the GUS protein that is fused with the complete
138 or the amino terminal 44 amino acids of ABZ1 localizes
to the nucleus while the GUS protein alone or fused either
with the 24 amino terminal or with the 94 carboxy terminal
amino acids of ABZ1 does not localize to the nucleus This
indicates that ABZ1 harbours the signal for nuclear
localization and that a functional NLS is localized within
the basic region between amino acids 25 and 44 Further-more, nuclear localization was achieved under aerobic conditions
ABZ1 binding specificity and dimer formation Transcription factors of the bZIP family are known to bind
to G-box like sequences [35] To investigate the binding specificity of ABZ1, the protein was expressed in E coli as a His-tag fusion protein The purified protein was employed
in a binding site selection experiment in which 17 putative binding sites were identified Table 1 shows the sequence of four binding sites and the frequency of the nucleotides at each position of the 17 selected binding sites Figure 5A shows EMSA for the four individual binding sites RBSS1, 1.1, 5 and 6.1 (Table 1) These four sites are efficiently and specifically bound by ABZ1 while the sequence GGTTGATTAGGGAA that harbours a mutation in the
Fig 2 Anaerobiosis specific expression of ABZ1, in tomato Total
RNA from fruits (lanes 1 and 2), leaves (lanes 3 and 4), and roots (lanes
5 and 6) that were either prepared from aerobic organs (lanes 1, 3, and
5) or anaerobically incubated organs (lanes 2, 4, and 6), were
hybrid-ized with a cDNA fragment from the ABZ1 gene A single 1.2 kb
transcript hybridizes with the probe Staining of the gels prior to
blotting indicates equal loading of the RNA.
Fig 3 Phylogenetic relationship of ABZ1 with other bZIP proteins A phylogenetic tree was constructed by the neighbor joining method with the basic leucine zipper region of 16 different bZIP proteins Zm, Zea mays; Os, Oryza sativa; Nt, Nicotiana tabacum; Le, Lycopersicon esculentum; Am, Antirrhinum majus; Pc, Petroselinum crispus; At, Arabidopsis thaliana The bZIP proteins compared are BZI-4, BZI-3, and BZI-2 [32], tbz17 [53], bZIP911 and bZIP910 [30], CPRF6 [54], mLIP15 [33], LIP19 [34], BZI-1 [55] The five most similar Arabidopsis proteins were also included and the Arabidopsis genome identification number provided At3g62420 corresponds to AtbZIP53, At1g75390 to AtbZIP44, At2g18160 to AtbZIP02, and At4g34590 to AtbZIP11 [31] ABZ1 is underlined The scale represents the frequency of amino acid changes Bootstrap values are indicated.
Trang 6G-box core sequence (RBSS1-Mu) and was used as an
unspecific competitor in EMSAs is not bound by ABZ1
(Fig 5A; u Cmp) EMSAs with RBSS1 were subsequently
used to analyse specific binding conditions
To confirm that the DNA binding domain resides in the
basic region, an N-terminally deleted protein was expressed
in E coli The deleted protein comprises amino acids 47–
138 Figure 5B shows that the wild type ABZ1 binds
efficiently to the RBSS1 sequence while the amino
termin-ally deleted protein does not bind This shows that the
binding domain of ABZ1 resides in the amino terminal 46
amino acids
Because both proteins harbour the leucine zipper domain,
it was analysed if they interact with each other If interaction
of the full size and the amino terminally deleted protein leads
to a heterodimer that binds DNA, a faster migrating
complex would be expected Surprisingly, addition of the
truncated ABZ1 protein abolishs binding of full size ABZ1 in
a concentration dependent manner (Fig 5B) This indicates
that in vitro binding of a dimer requires the presence of a
DNA binding domain in each interacting protein
This was further investigated using a carboxy terminal
deletion of ABZ1 This protein harbours the first 100 amino
acids including basic and leucine zipper domains Figure 5C
shows that binding of this protein yields a faster migrating
complex (C2) compared to the full size ABZ1 (C1) When
both proteins are added in equimolar concentrations a third
complex is observed that shows an intermediate migrating
behaviour (Fig 5C; C1+2) This complex is interpreted to
be caused by heterodimer formation between full size ABZ1
and the carboxy terminally deleted ABZ1
To summarize, ABZ1 binds to RBSS1 as a dimer and
efficient DNA binding requires a DNA binding domain in
both interacting proteins This result may have important
implications for the regulatory properties of small bZIP
transcription factors
ABZ1 binds to the CaMV 35S promoter which is
anaerobically down regulated in tobacco and tomato
One well known target of bZIP transcription factors is the
CaMV 35S promoter [36] Figure 6A,B shows that
recom-binant ABZ1 binds to a 100 bp fragment from the CaMV
35S promoter which harbours three potential binding sites
for ABZ1 One of the three putative binding sites within this fragment is the activation sequence-1 (as-1) between positions )65 and )85 that consists of two imperfect palindromes, with the palindromic centers spaced by 12 bp and which is known to be bound by different tobacco bZIP TFs [37] The random binding site selection experiment indicates a high similarity between RBSS6.1 and the second imperfect palindrome of as-1
EMSA analysis with recombinant ABZ1 revealed three shifted complexes of which two complexes can be com-pletely competed with RBSS1 (Fig 6A) The three shifted complexes observed may be due to differential occupation
of ABZ1 binding sites and may represent different numbers
of ABZ1 proteins bound to the promoter fragment These results show that ABZ1 can also bind to the CaMV 35S promoter
The binding of the anaerobically induced ABZ1 tran-scription factor to the CaMV 35S promoter indicates that the 35S promoter may be regulated under anaerobic conditions To investigate this proposal, transient gene expression analyses were performed by transforming a CaMV 35S promoter uidA reporter gene construct into tobacco and tomato leaves Subsequent to particle bom-bardment, leaves were incubated under aerobic and anaer-obic conditions followed by a quantitative GUS assay As shown in Fig 6C, in both host tissues, expression of the 35S promoter is significantly lower under anaerobiosis when compared with expression under aerobic conditions In tobacco, anaerobic expression is only 14% relative to aerobic expression while in tomato the difference between anaerobic and aerobic expression is less stringent (52%, Fig 6C)
ABZ1 down regulates the CaMV 35S promoter
in a transient gene expression assay The effect of ABZ1 on gene expression of the CaMV 35S promoter was analysed with transient expression assays conducted by coexpressing the ABZ1 protein together with the CaMV 35S driven uidA (GUS) gene in tobacco leaves
As a transformation standard, a luciferase gene under the control of the sugar beet cab11 promoter was employed This promoter does not harbour G-box binding sites and confers reporter gene expression in tobacco leaves (D Stahl,
Fig 4 The basic region of ABZ1 is required for nuclear localization Fusion gene constructs expressing parts of or the whole ABZ1 protein fused to the b-glucuronidase (uidA) reporter gene were transformed by particle bombardment into onion epidermal cells As indicated, the constructs express amino acids 1–24, 1–44, 1–138, and 45–138 from the ABZ1 protein fused in-frame with the uidA gene Transformed cells were subjected to histochemical GUS staining and to a DAPI staining of the nucleus.
Trang 7personal communication) Figure 7 shows that the
coex-pression of ABZ1 with the 35S-uidA construct leads to
down regulation of GUS expression compared to the
35S-uidA construct alone (compare 35S-35S-uidA with 35S-ABZ1/
35S-uidA, Fig 7A) Expression of the 35S promoter is
about 40% reduced in the presence of ABZ1 than without
When a fusion construct between ABZ1 and the activation
domain of GAL4 is coexpressed with the 35S-uidA
construct, expression is higher than observed with ABZ1
(compare 35S-ABZ1/35S-uidA with
35S-AD-ABZ1/35S-uidA, Fig 7A)
To minimize possible autoregulatory effects of ABZ1 on its own expression this experiment was repeated by expres-sing ABZ1 with the sugar beet cab11 promoter which is void
of putative ABZ1 binding sites Figure 7B shows that similar results were obtained compared to ABZ1 expression with the 35S promoter Coexpression of ABZ1 with the 35S-uidA construct leads to down regulation of GUS expression compared to the uidA construct alone (compare 35S-uidA with C1-ABZ1/35S-35S-uidA, Fig 7B) Expression of the 35S promoter is again about 40% reduced in the presence of ABZ1 than without When a fusion construct between
A
Fig 5 ABZ1 binding specificity and dimer formation Electrophoretic mobility shift assays with recombinant full size ABZ1(1–138) and two truncated derivatives harbouring amino acids 47–138 and 1–100, respectively Shifted complexes (C) and free probe (P) are indicated (A) Four sequences (probes) derived from a random binding site selection assay (Table 1) were radioactively labelled, and 0.1 ng (4 · 10 3 c.p.m.) were either incubated with (+) or without (–) ABZ1 As indicated (+/–) specific (RBSS1) or unspecific (u Cmp) competitor was added in a 1 · 10 5 molar excess and separated on a nondenaturing polyacrylamide gel (10%) (B) Increasing amounts of truncated derivative of ABZ1 harbouring amino acids 47–138 interferes with DNA binding of full size ABZ1 One microgram of protein (+) and increasing amounts of truncated ABZ1 (1.5 lg, 2.1 lg, and 2.5 lg, designated by the elongated triangle) was incubated with 0.05 ng (2 · 10 3 c.p.m.) radioactively labeled RBSS1 fragment (P) and separated on a nondenaturing polyacrylamide gel (9%) The complex C1 decreases when truncated ABZ1 lacking the first 46 amino acids is incubated in increasing concentrations together with full size ABZ1 (C) Dimer formation between full size ABZ1 and a truncated derivative of ABZ1 harbouring amino acids 1–100 One microgram of ABZ1 (+) and 770 ng truncated ABZ1 (+) was incubated with 0.1 ng (4 · 10 3 c.p.m.) radioactively labeled RBSS1 fragment and separated on a nondenaturing polyacrylamide gel (9%) A novel complex (C1+2) is observed when truncated and full size ABZ1 are incubated simultaneously with the radioactive probe.
Trang 8ABZ1 and the activation domain of GAL4 is coexpressed
with the 35S-uidA construct, expression is higher than
observed with ABZ1 (compare 35S-ABZ1/35S-uidA with
C1-AD-ABZ1/35S-uidA, Fig 7B)
In summary, these two independent sets of experiments
support the notion that the anaerobically induced ABZ1
transcription factor contributes to the anaerobic down
regulation of the 35S CaMV promoter
Discussion
Anaerobic gene expression regulation
The primary plant stress in flooded or compressed soils is
conferred by oxygen limitation that is most apparent in
below ground tissue Plants respond to oxygen limitation
with a significant reprogramming of gene expression These
responses usually permit a prolonged survival under these
adverse conditions Gene expression regulation involves
various mechanisms Many genes are induced by
transcription factors In Arabidopsis thaliana for example, this is achieved by the low oxygen induction of the AtMYB2 transcription factor which leads to the enhanced expression
of the ADH1 gene [10] The extent of post-transcriptional regulation is best illustrated when the limited number of anaerobic proteins detected is compared to the large number
of genes that are still transcribed under low oxygen conditions [4,11,38,39] It has long been observed that low oxygen conditions suppress the translation of the majority
of mRNAs and increase translation of a particular subset corresponding to anaerobic proteins This may be related to impaired ribosomal RNA transcription and ribosomal protein synthesis under oxygen deprivation [40–42] The analysis of ribosome loading patterns indicated that trans-lational control of anaerobic genes occurs at the initiation and postinitiation phases in a message-specific manner [43] Another post-transcriptional mechanism of low oxygen regulated gene expression is the increased splicing efficiency
of specific introns [44,45]
In the present study a small anaerobically induced bZIP transcription factor designated ABZ1 was identified from tomato ABZ1 binds to the CaMV 35S promoter (Fig 6A)
C
Fig 6 Binding of ABZ1 to the CaMV 35S promoter and down
regu-lation under anaerobic conditions (A) An electrophoretic mobility shift
assay with a radioactively labeled 100 bp fragment from the CaMV
35S promoter is shown Lanes 1–4 harbour the radioactive probe (P)
while in lanes 2–4, 500 ng recombinant ABZ1 was added to the probe
(0.3 ng, 2 · 10 3 c.p.m.) resulting in three shifted complexes (C1, C2,
and C3) Specific competition was achieved with a 4 · 10 3 molar excess
of fragment RBSS1 (lane 3) The unspecific competitor in which the
ACGT core sequence of fragment RBSS1 was altered to ATTA did
not compete for binding when added in a 4 · 10 3 molar excess (lane 4).
(B) The sequence of the 100 bp fragment from the 35S promoter used
for EMSA is shown Putative ABZ1 binding sites are underlined The
as-1 element between positions )65 and )85 is indicated (C)
Expres-sion of a 35S-uidA promoter reporter gene construct after transient
bombardment of tobacco and tomato leaves under aerobic and
anaerobic conditions The expression strength under anaerobic
con-ditions is displayed relative to the expression under aerobic concon-ditions
(100%) The mean values were derived from seven (tobacco) and six
(tomato) measurements.
Fig 7 Transient gene expression analysis using reporter and effector gene constructs in particle bombardments on tobacco leaves Reporter construct 35S-uidA harbours the b-glucuronidase gene under the control of the CaMV 35S promoter (A) Relative expression strength
of the 35S promoter in the presence and absence of effector constructs under the control of the 35S promoter Expression strength is shown relative to the 35S-uidA expression (100%) The mean value was derived from 10 uidA), 10 uidA + 35S-ABZ1), and 12 (35S-uidA + 35S-AD-ABZ1) measurements, respectively (B) Relative expression strength of the 35S promoter in the presence and absence of effector constructs under the control of the C1 promoter Expression strength is shown relative to the 35S-uidA expression (100%) The mean value was derived from eight (35S-uidA), seven (35S-uidA + C1-ABZ1), and eight (35S-uidA + C1-AD-ABZ1) measurements, respectively.
Trang 9The promoter activity is reduced under anaerobiosis in
tobacco and tomato (Fig 6C) Although many bZIP
transcription factors have been isolated from plants, their
role in gene expression regulation under low oxygen
conditions has not been analysed extensively Previously,
de Vetten & Ferl isolated a G-box binding protein from
maize, GBF1, which is anaerobically induced [46] The main
structural differences between GBF1 and ABZ1 are the size
(377 amino acids for GBF1 vs 138 for ABZ1) and a proline
rich region at the N terminus of GBF1 The proline rich
region of GBF1 may indicate that this protein is a
transcriptional activator [47] A second anaerobically
induced G-box binding factor from maize, mLIP15, is
structurally more similar to ABZ1 because it is 135 amino
acids long and also lacks a proline rich region at its N
terminus [33] Both maize G-box binding factors were
shown to interact with the maize ADH1 promoter, which is
anaerobically induced [33,46] It may be conceivable that in
maize GBF1 acts as a transcriptional activator while
expression of mLIP15 may modulate or repress anaerobic
expression by competing or interacting with GBF1 Small
bZIP proteins may be one of the components of the cellular
machinery that contribute to the low oxygen mediated
down regulation of gene expression
Functional dissection of ABZ1
The ABZ1 transcription factor isolated in this study was
extensively analysed using biochemical approaches
Although the basic and leucine zipper domain are often
assumed to be the DNA binding and dimerization domains,
the present study confirms this experimentally (Fig 5) The
nuclear localization signal resides within the basic domain
required for DNA binding Some bZIP factors are regulated
by a subcellular localization mechanism in response to
environmental cues For example nuclear import of the
parsley bZIP factor CPRF2 is light mediated [48]
Cyto-plasmatic retention of the bZIP factor RSG is mediated by a
14-3-3 protein which has been suggested to modulate the
endogenous amounts of gibberellins through the control of
a gibberellic acid biosynthetic enzyme [49] In the study
presented here no evidence for cytoplasmatic retention of
ABZ1 was found for the full size ABZ1 and the protein is
readily detected in the nucleus under aerobic conditions
(Fig 4)
Interestingly, the binding of ABZ1 to its target sequence
can be abolished with increasing amounts of a truncated
ABZ1 that lacks the DNA binding domain (Fig 5B)
Therefore, efficient DNA binding requires that the
dimeri-zation occurs with another bZIP factor harbouring a basic
DNA binding domain Whether heterodimerization of
ABZ1 to other bZIP factors occurs, has not been analysed
directly However, because its closest relative BZI-4
hetero-dimerizes with BZI-1 [32] it may be conceivable that ABZ1
can also form heterodimers Remarkably, no other bZIP
transcription factor was isolated in a yeast two hybrid screen
(S Sell & R Hehl, unpublished observations) This may
either relate to an insufficient number of primary clones or to
the fact that the mRNA used for constructing the prey library
was isolated from anaerobic tissue and may not contain
transcripts for other bZIP factors because their expression
may be down regulated under anaerobic conditions
Because ABZ1 is able to bind to the 35S promoter which
is down regulated under anaerobic conditions and in the presence of ABZ1 in cobombardment analyses, this may suggest that ABZ1 either competes with other bZIP factors for the same binding sites or that heterodimerization also down regulates target gene expression In mammalian systems heterodimer formation of a bZIP factor with another leucine zipper containing transcription factor results in down regulation of target gene expression [50]
To date several attempts to generate transgenic tomato or tobacco plants that overexpress ABZ1 have failed Reverse genetic approaches to analyse the role of small bZIP proteins in anaerobic gene expression may be more readily carried out in A thaliana
Therefore, a screen for A thaliana homologs was performed usingTAIR BLAST [51] ABZ1 is closely related
to the four bZIP transcription factors AtbZIP53 (At3g62420; 53% identity), AtbZIP44 (At1g75390; 42% identity), AtbZIP02 (At2g18160; 50% identity), and Atb-ZIP11 (At4g34590; 58% identity) Recently, data on AtbZIP02 and AtbZIP11 suggested that these small bZIP factors bind to the sequence ACTCAT and may act as transcriptional activators under hypoosmotic conditions [52] It may be very interesting to learn how small bZIP proteins are involved in transcriptional activation This may relate to the position of the cis-regulatory element in the promoter or to the presence of interacting proteins that contribute a transcription activation domain
Acknowledgements
This work was supported by a grant through the Forschungsschwer-punkt Agrarbiotechnologie des Landes Niedersachsen (VW-Vorab).
We are grateful to Ralf R Mendel for critical reading of the manuscript and to Robert Ha¨nsch for advice using the particle delivery system We would like to thank Jo¨rn Petersen for help with the phylogenetic analysis.
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