a novel wrky transcription factor, susiba2, participates in sugar

The Plant Cell, Vol. 15, 2076–2092, September 2003, www.plantcell.org © 2003 American Society of Plant Biologists

A Novel WRKY Transcription Factor, SUSIBA2, Participates in Sugar Signaling in Barley by Binding to the Sugar-Responsive Elements of the

iso1

Promoter

Chuanxin Sun, Sara Palmqvist, Helena Olsson, Mats Borén, Staffan Ahlandsberg, and Christer Jansson

1

Department of Plant Biology and Forestry Genetics, The Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden

SURE (sugar responsive) is a

cis

element in plant sugar signaling. The SURE element was reported first for potato, in whichit confers sugar responsiveness to the patatin promoter. A SURE binding transcription factor has not been isolated. Wehave isolated a transcription factor cDNA from barley and purified the corresponding protein. The transcription factor, SUSIBA2(sugar signaling in barley), belongs to the WRKY proteins and was shown to bind to SURE and W-box elements but not to theSP8a element in the

iso1

promoter. Nuclear localization of SUSIBA2 was demonstrated in a transient assay system with aSUSIBA2:green fluorescent protein fusion protein. Exploiting the novel transcription factor oligodeoxynucleotide decoystrategy with transformed barley endosperm provided experimental evidence for the importance of the SURE elements in

iso1

transcription. Antibodies against SUSIBA2 were produced, and the expression pattern for

susiba2

was determined atthe RNA and protein levels. It was found that

susiba2

is expressed in endosperm but not in leaves. Transcription of

susiba2

is sugar inducible, and ectopic

susiba2

expression was obtained in sugar-treated leaves. Likewise, binding to SURE elementswas observed for nuclear extracts from sugar-treated but not from control barley leaves. The temporal expression of

susiba2

in barley endosperm followed that of

iso1

and endogenous sucrose levels, with a peak at

�

12 days after pollination. Ourdata indicate that SUSIBA2 binds to the SURE elements in the barley

iso1

promoter as an activator. Furthermore, they showthat SUSIBA2 is a regulatory transcription factor in starch synthesis and demonstrate the involvement of a WRKY protein incarbohydrate anabolism. Orthologs to SUSIBA2 were isolated from rice and wheat endosperm.

INTRODUCTION

Development of the cereal seed is orchestrated by the coordi-nated activities of a large number of genes that encode metabolicand regulatory enzymes as well as other proteins (Olsen, 2001).This results in a triploid endosperm, the embryo, pericarp, seedcoat, and other tissues of the mature grain. The endosperm struc-ture consists of two tissues, the interior starch-filled endospermand the outer epidermal layer called the aleurone. Starch, whichis a mixture of amylopectin (a heavily branched polyglucan) andamylose (a mostly linear polyglucan), is deposited in the en-dosperm as granules. The synthesis and deposition of starch inthe endosperm depend on enzymes such as ADP–glucose py-rophosphorylase, starch synthases, starch-branching enzymes(SBEs), and starch-debranching enzymes. Most, if not all, of theseenzymes exist in two or more isoforms (for recent reviews onstarch biosynthesis, see Ball et al., 1998; Buléon et al., 1998; Myerset al., 2000; Smith, 2001; Nakamura, 2002). Starch-debranch-ing enzymes are grouped into two distinct classes, isoamylaseand pullulanase, each with different isoforms (Nakamura, 1996).Isoamylase (EC 3.2.1.68) is an essential enzyme in amylopectinsynthesis. However, the precise role of the enzyme in this pro-

cess is not clear, and different models have been proposed (Ballet al., 1998; Smith, 2001; Nakamura, 2002).

It has been reported that the expression of starch synthesisgenes, such as starch synthase in potato (Visser et al., 1991);ADP–glucose pyrophosphorylase in potato (Müller-Röber et al.,1990), sweet potato (Bae and Liu, 1997), Arabidopsis (Rook etal., 2001), and tomato (Li et al., 2002); SBE in potato (Kossmanet al., 1991), maize (Kim and Guiltinan, 1999), and Arabidopsis(Khoshnoodi et al., 1998); and isoamylase in barley (Sun et al.,1999), is sugar inducible. In contrast to the situation in bacteria,yeast, and mammals, in which sugar signaling cascades havebeen studied extensively, the sugar signaling transduction path-ways in plants are poorly understood (Rolland et al., 2002). Gen-erally, in higher plants, high sugar levels stimulate the expres-sion of genes involved in sink function, such as growth, storageof proteins, and the biosynthesis of starch and other carbohy-drates, whereas low sugar levels promote the photosynthesisand mobilization of energy reserves, such as the breakdown ofstorage starch or lipids.

Sugar signaling can be dissected into three steps: sugar sens-ing, signal transduction, and target gene expression. However,this division is confused by the dual function of sugars as nutri-ents and signaling molecules, and by the interaction (in plantsand animals) between sugar signaling and hormonal networks.In plants, this complexity is increased further by the vital role ofsugar production through photosynthesis. Hexoses, sucrose, andtrehalose might serve as elicitors of plant sugar signaling (Goddijnand Smeekens, 1998; Rolland et al., 2002). Hexokinase, sucrose,

1

To whom correspondence should be addressed. E-mail [email protected]; fax 46-18-673279.

Online version contains Web-only data.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.014597.

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Sugar Signaling in Barley 2077

glucose transporters, and various sugar receptors have beenproposed as components of the sugar-sensing machinery (Sheenet al., 1999; Smeekens, 2000; Rolland et al., 2002).

The Ser/Thr protein kinase Snf1 is a central participant in yeastsugar signaling (Carlson, 1999). Snf1 phosphorylates downstreamcomponents and also is itself activated by phosphorylation. Snf-related protein kinases (SnRKs) are found in yeast, mammals, andplants, in which they participate in a large number of regulatoryfunctions (Halford and Hardie, 1998; Hardie et al., 1998). Thereis evidence that some plant SnRKs are functionally homologouswith Snf1 in plant sugar signaling, although the exact nature oftheir responses to sugars remains to be clarified (Rolland etal., 2002). Other players implicated in sugar signaling transduc-tion pathways in plants are sugar metabolites, 14-3-3 pro-teins, trehalose-6-phosphate, and Ca

2

�

(Rolland et al., 2002).Little is known about the

cis

and

trans

factors that mediatethe final steps in plant sugar signaling. To date, five different typesof

cis

elements have been identified in sugar-regulated plant pro-moters: the SURE (sugar-responsive) (Grierson et al., 1994), SP8(Ishiguro and Nakamura, 1994), TGGACGG (Maeo et al., 2001),G-box (Giuliano et al., 1988), and B-box (Grierson et al., 1994;Zourelidou et al., 2002) elements. Only two putative transcrip-tion factors, SPF1 and STK, with relevance to plant sugar sig-naling have been isolated. SPF1 binds to the SP8 sequence, inwhich it functions as a repressor (Ishiguro and Nakamura, 1994),and STK binds to the B-box as an activator (Zourelidou et al.,2002).

We are interested in the endosperm-specific expression of the

sbeIIb

(encoding SBEIIb) and

iso1

(encoding isoamylase1) genesduring barley seed development. We have shown previously thatbarley

iso1

harbors an SP8a element and that it contributes to theendosperm specificity of

iso1

expression by recruiting a repressorin nonexpressing tissues (Sun et al., 1999). Similarly, the en-dosperm-specific expression of barley

sbeIIb

(Sun et al., 1998) isdetermined partly by a repressor binding to the B-box-like (Bbl)element in the second intron of the gene (Ahlandsberg et al.,2002b). In the work described here, we have isolated a tran-scription factor, SUSIBA2 (sugar signaling in barley), and exam-ined its interaction with the

iso1

promoter. We show that SUSIBA2represents a transcription factor involved in the regulation ofstarch synthesis.

RESULTS

Isolation and Characterization of the

susiba2

cDNA

The sugar responsiveness of the sporamin and

�

-amylase genesin sweet potato was reported more than a decade ago (Hattori etal., 1990), and two promoter sequences, SP8a and SP8b, wereimplicated as

cis

elements in the coordinated regulation of thesporamin multigene family and the

�

-amylase gene (Ishiguroand Nakamura, 1992). Subsequently, a cDNA that encodes atranscription factor named SPF1 was isolated from sweet po-tato, and it was reported that SPF1 binds to the SP8a and SP8bpromoter elements of the

�

-amylase and sporamin genes oftuberous roots (Ishiguro and Nakamura, 1994). Later, clones forSPF1-like proteins were isolated from cucumber (Kim et al.,1997) and parsley (Rushton et al., 1996).

To continue our studies of the sugar-mediated regulation ofthe barley

iso1

gene and the involvement of the SP8a element(Sun et al., 1999), we set out to isolate a cDNA clone for thebarley SPF1 ortholog. Based on the alignment of the sweet po-tato, cucumber, and parsley SPF1 cDNA sequences, primerswere designed for reverse transcriptase–mediated (RT) PCRamplification of barley cDNA using total RNA isolated from de-veloping barley endosperm at 9 days after pollination. Only onePCR product with the expected size was obtained (data notshown). The amplified product was used as a probe to screenan endosperm cDNA library from 10 days after pollination and aleaf library. From screening of 10

6

plaque-forming units, 14 pos-itive clones were recovered. All positive clones were subjectedto subcloning, restriction mapping, and sequence analysis. Re-striction mapping and sequence analysis revealed that the clonesgrouped into three distinct classes. They were designated

susiba1

,

susiba2

, and

susiba3

, respectively.From group 2, one representative clone was sequenced com-

pletely on both DNA strands. The length of the

susiba2

cDNA is2355 nucleotides, and the open reading frame starts at position247 and ends at position 1966 (Figure 1). The cDNA encodes a573–amino acid polypeptide, SUSIBA2, with a calculated mo-lecular mass of 62.2 kD, and it has 5

�

and 3

�

untranslatedregions and a poly(A

�

) tail sequence. These findings, togetherwith the results from RNA gel blot analyses (see below), suggestthat the

susiba2

cDNA is a full-length clone. One interesting fea-ture of

susiba2

is that an 18-nucleotide sequence between nu-cleotides 1051 and 1068 (Figure 1) is 100% complementary tothe gene for the human insulin-like growth factor1 (IGF1) recep-tor (nucleotides 173 to 190).

SUSIBA2 Belongs to the WRKY Superfamily of Transcription Factors

The deduced amino acid sequence of SUSIBA2 was used in aBLAST (Basic Local Alignment Search Tool) search of the Na-tional Center for Biotechnology Information (NCBI) databases.Alignment of the 9 top-scoring matches with the SUSIBA2 se-quence demonstrated that all 10 proteins are highly similar andthat SUSIBA2 belongs to group 1 of the WRKY superfamily ofplant transcription factors (Figure 2). The two WRKY domains(Eulgem et al., 2000) with their zinc finger motifs are highly con-served in SUSIBA2. Also, the N-terminal Ser- and Thr-rich re-gions that are present in many WRKY proteins are found inSUSIBA2. The function of these regions is not known but likelyinvolves regulation. Ser- and Thr-rich regions are characteristicof activation domains in transcription factors.

As would be expected from transcription factors, WRKY pro-teins contain nuclear localization signals (NLSs). The two mostcommon NLSs are monopartite signals, which are shortstretches enriched in basic amino acids, and bipartite signals,which are composed of two short basic stretches separated bya spacer (Merkle, 2001). The monopartite NLS depicted forWRKY proteins is conserved in SUSIBA2 (amino acids 328 to331; Figure 2). However, closer inspection of the sequencessuggests that the monopartite NLS in SUSIBA2 and otherWRKY proteins also might constitute a bipartite NLS close tothe consensus motif, KR-(24-74)-R..RK, for early auxin-induc-

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Figure 1. Structure of the susiba2 cDNA.

The 18-nucleotide sequence complementary to the human IGF1 recep-tor gene is underlined. The amino acid sequence of the tryptic fragmentobtained by microsequencing from the overproduced SUSIBA2 (seeFigure 3) is indicated with an open box.

ible proteins (Abel and Theologis, 1995). One putative bipartiteNLS in SUSIBA2 is KR-(66)-RK (amino acids 328 to 397). Theother possibility is KR-(37)-R.RK (amino acids 328 to 370) andinvolves the WRKYGQK sequence itself in the C-terminalWRKY domain. The function of the WRKYGQK heptapeptide isunknown, and we cannot exclude the possibility that it partici-pates in the nuclear localization process, although a bipartiteNLS arrangement for the N-terminal WRKYGQK sequencewas not found.

A different kind of NLS reported for the Arabidopsis WRKYprotein, AtWRKY6 (Robatzek and Somssich, 2001), is notpresent in SUSIBA2, although one of the Lys residues impli-cated in this motif is conserved in SUSIBA2 (corresponding toLys-344) and some other WRKY proteins (Figure 2). The mostdistinguishing feature of SUSIBA2 relative to other WRKY pro-teins is the 37–amino acid insertion after the C-terminal WRKYdomain (amino acids 435 to 471; Figure 2). A BLAST searchwith the insertion sequence did not show any matches in theNCBI databases (see below), suggesting that SUSIBA2 is anovel type of WRKY protein.

Overproduction of SUSIBA2 in

Escherichia coli

The

susiba2

cDNA was inserted into a pET expression vectorand overexpressed in

E. coli

after isopropylthio-

�

-galactosideinduction. The overproduced protein was purified by one-stepfast protein liquid chromatography on a His tag affinity column.Results from a typical overproduction and purification proce-dure are shown in Figure 3. The purified polypeptides were usedfor microsequencing, activity characterization, and antibodyproduction.

The purified protein fraction contained three polypeptides.They were separated by SDS-PAGE and subjected to in situtrypsin digestion. The digested fragments from each polypep-tide were separated by HPLC and applied to mass spectrome-try analysis. The upper two bands corresponded to polypep-tides with molecular masses of

�

70 and 68 kD and with theidentical sequence LAGGAVP. This sequence is found in the de-duced amino acid sequence of SUSIBA2 (Figure 1; amino acids235 to 241). The lower band corresponded to a 28-kD polypep-tide with the sequence EIAKQA. This sequence matches

E. coli

trehalose-6-phosphate synthase (amino acids 234 to 239). Thetwo SUSIBA2 polypeptides were extracted and used for the pro-duction of polyclonal antibodies.

The DNA Binding Activity of SUSIBA2

As an initial test of the DNA binding activity of SUSIBA2, weperformed electrophoretic mobility shift assays (EMSAs) withthree overlapping nucleotide fragments from the barley

iso1

promoter, all containing the SP8a sequence (Figures 4A and4B). The 822-bp SphI-HaeII fragment, encompassing SP8a andflanking sequences, and the 437-bp BspHI-HaeII fragment, con-taining SP8a and downstream flanking sequence, formed promi-nent DNA-protein complexes with the purified SUSIBA2 fraction.To our surprise, however, the 510-bp SphI-BsaI fragment, con-taining SP8a and upstream flanking sequence, bound verypoorly to SUSIBA2. We concluded that the target for SUSIBA2

in the

iso1

promoter is not SP8a but one or more sites down-stream of the SP8a element. From further analysis of the seg-ment downstream of SP8a in the BspHI-HaeII fragment, wefound that it harbors three A-rich sequence segments, 5

�

-AAAACTAAGAAAGACCGATGGAAAA-3

�

, 5

�

-AATACCAAAAAA-

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TAATAATAAAA-3

�

, and 5

�

-CGAAAAAATAAAGAAAATGAAAT-3

�

(nucleotides

�

597 to

�

573,

�

568 to

�

546, and

�

517 to

�

495 rel-ative to the transcriptional start site, respectively), each of whichshares a high degree of identity with the SURE element, as firstreported by Grierson et al. (1994) from work on the potatoclass-1 patatin promoter.

To follow up on this observation, we performed EMSAs witholigonucleotides consisting of the

iso1

SURE sequences, re-ferred to as SURE-a, SURE-b, and SURE-c for the upstream,middle, and downstream sequences, respectively (Figures 4Cand 4D). As is evident in Figure 4D, SUSIBA2 bound to all threeSURE probes, whereas, again, binding to the SP8a probe wasnegligible. The strongest binding activity was observed for theSURE-a sequence, which yielded a major DNA-protein com-plex of a low relative mobility (

M

r

). The same complex was ob-tained with the SURE-b and SURE-c probes, although only asa minor component; the major complex with the SURE-b andSURE-c probes had a higher

M

r

. This high

M

r

complex alsocould be discerned as a faint band with the SURE-a probe. Withthe SURE-b and SURE-c probes, additional complexes of inter-mediate

M

r

were seen. When the SURE-a probe was incubatedwith a nuclear extract from barley endosperm, a complex wasformed with the same

M

r

as with the overproduced SUSIBA2,suggesting that the complex produced with the endogenousSUSIBA2 was the same as that produced with the purified pro-tein. Because SUSIBA2 is a WRKY protein, it also would be ex-pected to bind to the W-box, the highly conserved binding sitefor WRKY proteins (Eulgem et al., 2000). A sequence identical tothe consensus sequence for the W-box, TGACT (nucleotides

�

400 to

�

396), was located in the

iso1

promoter, and the

iso1

W-box sequence was included in the binding assay (Figures 4Cand 4D). With the W-box probe, two DNA-SUSIBA2 complexescould be discerned. Thus, the results support the conclusionthat SUSIBA2 does not bind to the SP8a element and showthat, instead, it interacts with the SURE and W-box elements.

The observation that the SURE-SUSIBA2 complexes ap-peared with different

M

r

indicated that SUSIBA2 might exist intwo or more oligomeric states. This possibility was illustratedfurther by a titration EMSA in which the SURE-a probe was in-cubated with increasing amounts of SUSIBA2 protein (Figure5). As the concentration of SUSIBA2 increased from 0.01 to0.08

�

M, the migration of the formed complex shifted from highto low

M

r

. The same phenomenon was observed for the SURE-band SURE-c probes (data not shown).

Having demonstrated that SUSIBA2 binds to the SURE ele-ment, we were interested in further exploring the interaction be-tween SUSIBA2 and the SURE sequence. To this end, we con-structed engineered versions of the SURE-a element in thebarley

iso1

promoter and tested their affinity for the SUSIBA2protein (Figures 6A and 6B). Our first observation was that thebinding activity could be localized to the 20-nucleotide 3

�

endof the SURE-a sequence (SURE-a 3

�

). Subsequent modifica-tions of the 3

�

end revealed that, although the SURE element isan A-rich sequence, increasing the number of consecutive Anucleotides in the upstream portion of SURE-a 3

�

(SURE-a 3

�

m1 and SURE-a 3

�

m2) decreased its affinity for SUSIBA2.Converting most of SURE-a 3

�

to a poly(A) stretch (SURE-a 3

�

m4) resulted in the complete loss of binding activity. Interest-

ingly, when the number of consecutive A nucleotides in thedownstream region of SURE-a 3

�

was increased (SURE-a 3

�

m3), the binding activity was enhanced significantly and addi-tional complexes of higher

M

r

were produced. When the EMSAwas performed with nuclear extracts from barley endosperm(Figures 6A and 6C), the effects of the m1, m2, and m3 muta-tions in the SURE-a 3

�

probe were similar to those with the pu-rified SUSIBA2. One difference was that, with the nuclear ex-tract, the SURE-a 3

�

m3 probe also exhibited decreased bindingactivity compared with SURE-a 3

�

, albeit to a lower extent thandid the SURE-a 3

�

m1 and SURE-a 3

�

m2 probes. Contrary tothe situation with purified SUSIBA2, the nuclear extract displayedbinding activity to the 5

�

end of the SURE-a sequence and to theSURE-a 3

�

m4 probe.To further demonstrate that the complex formed between the

nuclear extracts and the SURE element contained SUSIBA2,we attempted an antibody supershift analysis. However, ourantiserum was raised against a denatured SUSIBA2 protein,and the antibodies reacted poorly with native SUSIBA2. As analternative means to address this issue, we examined the com-position of the DNA-protein complex after SDS-PAGE. A nar-row strip of the labeled complex between SURE-a 5

�

and anuclear extract on the polyacrylamide gel was excised. Theproteins were eluted and separated on an SDS gel, followed byprotein gel blot analysis with the SUSIBA2 antiserum and pre-immune serum. Because the overproduced SUSIBA2 proteinused for antibody production contained a His tag, we also in-cluded a control in which the protein gel blot was probed withHis tag antibodies. The SUSIBA2 antibodies produced a dou-ble band corresponding to 68 to 66 kD, the expected molecularmass for endogenous SUSIBA2 (Figure 7). No bands were de-tected with the preimmune serum, whereas the His tag anti-bodies reacted with a polypeptide of

�

96 kD.

Expression of

susiba2

and

iso1

Correlates withSucrose Levels

In previous investigations, we showed that the expression ofthe barley

iso1

gene is sugar inducible (Sun et al., 1999). In lightof different reports suggesting the involvement of the SURE el-ement in sugar signaling (Kim et al., 1991; Rhee and Staswick,1992; Grierson et al., 1994; Fu et al., 1995; Mita et al., 1995;Kim and Guiltinan, 1999; Li et al., 2002) and the results pre-sented here showing the SURE binding activity of the SUSIBA2transcription factor, it was of interest to study the expressionpattern of the

susiba2

gene. For the examination of temporalexpression, barley endosperms were collected at 7 to 27 daysafter pollination and total RNA was isolated. RNA gel blot analy-sis was performed with

susiba2

- or

iso1

-specific probes or witha PCR-amplified probe for the

paz1

gene, which encodes thestorage protein Z (Sørensen et al., 1989) and which was in-cluded for comparison. The expression level for

susiba2

waslow compared with that for

iso1

, but the patterns were similar,with a peak at

�

12 days after pollination (Figure 8A). A similartime course was obtained for the barley

sbeIIa

and

sbeIIb

genes(Sun et al., 1998). Expression of the

paz1

gene increased up toor beyond 22 days after pollination and then declined sharply,

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Figure 2. Analysis of the SUSIBA2 Primary Structure.

The amino acid sequence of SUSIBA2 was aligned with those of the nine closest matching proteins from a BLAST search. Identical amino acids areshown in black boxes, and similar amino acids are shown in gray boxes. The WRKYGQK peptide stretch is shown in pink. Putative nuclear localiza-tion signals are indicated with orange number signs (#) under the sequences. Ser- and Thr-rich regions are underlined in orange. The zinc finger–like

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Figure 2. (continued).

motifs in the two WRKY domains are indicated with green vertical arrows under the sequences. A. t. hyp, Arabidopsis hypothetical protein; A. t. put,Arabidopsis putative protein; A. t. unk, Arabidopsis unknown protein; A. t. wrky20, Arabidopsis WRKY transcription factor 20; H. v. SUSIBA2, barleySUSIBA2; I. b. SPF1, sweet potato SPF1; L. e. hyp, tomato hypothetical proteins; N. t. wrky, tobacco WRKY protein; R. r. d-i, white broom drought-induced protein.

consistent with the results reported by Sørensen et al. (1989).To study the correlation between sucrose and susiba2 expres-sion, we monitored the endogenous sucrose concentrationsduring endosperm development using an enzymatic assay andthin layer chromatography. The sucrose concentrations weredetermined in seeds from the same batches used for transcriptanalysis. A comparison between the temporal expression pat-terns for susiba2 and iso1 transcript accumulation and sucroselevels showed a good correlation (Figures 8B and 8C).

No susiba2 transcripts were detected in untreated barleyleaves. However, ectopic susiba2 expression in leaves wasachieved after supplying exogenous sugar (Figure 9A). The samewas true for iso1, in agreement with previous results (Sun et al.,1999). As a comparison, the photosynthesis genes rbcS andrbcL, which encode the small and large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, were ex-pressed abundantly in control leaves (0 mM sucrose). The nuclearrbcS was downregulated dramatically by sucrose, whereas theplastidic rbcL was not affected notably. The sugar-induced ex-pression levels for susiba2 and iso1 in leaves were quite low com-pared with those observed in endosperm and were detected onlyafter prolonged exposure. The expression studies were extendedto the protein level, using both EMSA with nuclear extracts (Fig-ure 9B) and protein gel blot analyses with the SUSIBA2 anti-body (Figure 9C). Consistent with the transcript assessments,nuclear extracts from barley leaves gave rise to a complex withthe SURE-a probe after but not before sugar treatment. The im-munological examination showed that SUSIBA2 was detectedin endosperm but not in leaves. We failed to obtain satisfactoryprotein gel blot results with extracts from sugar-treated leaves.

In Vivo Analysis of Transformed Barley Endosperm Demonstrates the Relevance of the SURE Element for iso1 Promoter Activity

To test the function of the SURE element in the regulation of iso1expression, we performed transient assays in which the tran-scription of a chimeric iso1 reporter construct was studied intransformed barley endosperm. In this experiment, the entire(1.5 kb) iso1 promoter was fused to the green fluorescent protein(gfp) reporter gene. The exploitation of the GFP reporter systemfor the analysis of promoter activity has been used successfullyin transformed barley embryos (Ahlandsberg et al., 1999, 2002b).Because the iso1 gene is not expressed in embryos (Sun et al.,1999), we needed first to establish a protocol for the transfor-mation of barley endosperm. This was accomplished recently(S. Palmqvist, S. Ahlandsberg, C. Sun, and C. Jansson, unpub-lished results), allowing us to perform transient assays with gfpreporter constructs also in transformed barley endosperm.

The activity of the iso1 promoter, manifested as GFP fluores-cence, was followed in transformed barley endosperm in thepresence or absence of a transcription factor oligodeoxynucle-otide (ODN) decoy containing the SURE-a sequence (Figure10A). The ODN decoy approach involves flooding the cells withenough double-stranded decoy to compete for binding of tran-scription factors with their consensus sequences in target genes.If present in high enough concentrations, these decoys can ne-gate the ability of the transcription factor to regulate gene expres-sion. The transcription factor ODN technology is used widely inmedical investigations (Mann and Dzau, 2000). Here, we chose toadopt the strategy for our studies on barley endosperm. As anegative control, we constructed a decoy containing a sequencefrom the rbcL gene. As demonstrated in Figure 10B, the presenceof the SURE-a decoy in the endosperm cells blocked most, al-though not all, iso1 promoter activity, whereas the rbcL decoyhad no obvious effect. The results of nine independent experi-ments are summarized in Figure 10C. To affirm that the SURE-adecoy did not interfere nonspecifically with the transcriptionalmachinery, we fused the gfp gene downstream of the constitu-tive intronic actin promoter (Act-1) from rice (McElroy et al.,1990, 1991; Ahlandsberg et al., 1999) and monitored its activityin transformed barley endosperm (Figure 10D). No inhibitory ef-fect on GFP fluorescence was detected in the presence of theSURE-a decoy.

Figure 3. Purification of SUSIBA2.

His-tagged SUSIBA2 was subjected to fast protein liquid chromatogra-phy (FPLC) purification on a Ni affinity column after overexpression ofthe susiba2 cDNA construct in E. coli. Amino acid sequences obtainedby microsequencing of tryptic fragments are shown at right. Molecularmasses (in kilodaltons) are indicated at left. fr., fraction.

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Nuclear Localization of SUSIBA2

The subcellular localization of SUSIBA2 was determined in atransient assay system using transformed barley endospermcells (Figure 11). For this investigation, the gfp reporter gene wasfused in frame C terminal to the susiba2 open reading frame. Aconstruct that encodes GFP alone served as a control. Endospermcells transformed with the control construct showed GFP fluores-cence throughout the entire cytosol and the nucleus. By contrast,cells transformed with the SUSIBA2:GFP fusion protein showedGFP fluorescence exclusively in the nucleus.

Figure 5. Titration of SUSIBA2 Oligomerization.

The SURE-a probe was used for EMSA in the absence (�) or presenceof different concentrations (0.035, 0.07, 0.14, and 0.28 �g, in 50-�L re-actions, corresponding to 0.01, 0.02, 0.04 and 0.08 �M proteins, re-spectively) of overproduced SUSIBA2. The position of the free probe isindicated.

Figure 4. Binding of SUSIBA2 to DNA Sequences Containing the SP8a,SURE, and W-Box Elements.

(A) A portion of the iso1 promoter and restriction fragments spanningthe SP8a element and flanking sequences. The positions of the threeSURE elements (a, b, and c) and the W-box (W) are indicated.(B) EMSA using the different SP8a-containing restriction fragments in-cubated in the presence (�) or absence (�) of SUSIBA2. The positionsof the DNA-SUSIBA2 complexes and free probes are indicated.

(C) The sequences of five oligonucleotides containing the SP8a, SURE-a,SURE-b, SURE-c, and W-box sequences. The SP8a and W-box se-quences are shown in boldface.(D) EMSA using the different oligonucleotides incubated in the presence(�) or absence (�) of overproduced SUSIBA2 or barley nuclear extract.The position of the free probe is indicated.

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Isolation of cDNA for SUSABA2 Orthologs in Riceand Wheat

The involvement of the SUSIBA2 transcription factor in the reg-ulation of iso1 expression in barley endosperm suggested thatSUSIBA2 orthologs also should be found in sink tissues in otherplants. The same primers used for cloning of the susiba2 cDNAwere used for RT-PCR amplification of total RNA isolated fromrice and wheat endosperm. For both species, only one majorRT-PCR product was obtained (data not shown). The PCR prod-ucts were subcloned and sequenced. The deduced amino acidsequences from the amplified regions in the rice and wheatclones share a high degree of identity with the correspondingsegment in SUSIBA2 (see supplemental data online). By contrast,in this region between the conserved WRKY domains, SUSIBA2and its rice and wheat orthologs all are significantly different fromthe SP8a binding SPF1 protein. The entire sequence for the riceortholog could be retrieved from GenBank with the temporaryname WRKY20. SUSIBA2 and the rice ortholog share a highdegree of amino acid identity (80%) along their entire lengths.

DISCUSSION

We have identified a class of transcription factors, the SUSIBAs,and are studying the influence of the SUSIBA isomers on theregulation of starch synthesis in barley. To date, we have iso-lated cDNA clones for three members of the SUSIBA family:SUSIBA1, SUSIBA2, and SUSIBA3. The PCR product used inthe barley library screening that resulted in the isolation of thesusiba family was generated based on sequence similaritiesbetween cDNAs for SPF1 and SPF1-like proteins. Thus, the weakinteraction between SUSIBA2 and the SP8a element might seemsurprising. However, as is evident in Figure 2, the PCR primersused correspond to regions in the conserved WRKY domains,and, apart from these two domains, the overall degree of identitybetween SUSIBA2 and SPF1 is rather low (28%; see supplemen-tal data online). Furthermore, as has been suggested (Eulgem et al.,2000), it has not been shown conclusively that SPF1 binds to thereported SP8a element, because the oligonucleotide used in thebinding assay also contained a W-box. Sequence analysis ofSUSIBA1, SUSIBA2, and SUSIBA3 shows that they are closelyrelated and belong to the WRKY superfamily of plant transcrip-tion factors. The major difference between SUSIBA1 andSUSIBA2 relates to the number of WRKY domains (see supple-mental data online). SUSIBA2 contains two WRKY domains andbelongs to group 1 of the WRKY proteins, whereas SUSIBA1contains one WRKY domain and belongs to group 2. SUSIBA3has not been characterized in detail. In the work described here,we focus on SUSIBA2 and its communication with the iso1 pro-moter.

Figure 6. EMSA with Engineered SURE-a Oligonucleotides and Over-produced SUSIBA2.

(A) Sequences of SURE-a, the SURE-a 5� probe (SURE-a 5�), theSURE-a 3� probe (SURE-a 3�), and the engineered SURE-a 3� probes(SURE-a 3� m1 to m4). Engineered nucleotides are shown in boldface.(B) EMSA with the different SURE-a probes in the presence (�) or ab-

sence (�) of overproduced SUSIBA2. The position of the free probe isindicated.(C) EMSA with the different SURE-a probes in the presence (�) or ab-sence (�) of nuclear extracts from barley endosperm. The position ofthe free probe is indicated.

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trehalose and trehalose-6-phosphate in plant sugar signalinghas been proposed (Goddijn and Smeekens, 1998).

As has been demonstrated here, purified SUSIBA2 bindsstrongly to the SURE element and the W-box but not to theSP8a motif (Figures 4 to 6). Binding to the W-box is consistentwith SUSIBA2 being a WRKY protein, whereas binding to theSURE element displays a novel feature for a WRKY protein. TheWRKY proteins seem to be specific to the plant kingdom andare best known for their participation in various stress responsesand senescence (Eulgem et al., 2000). Our results demonstratethat their sphere of activities has to be expanded to include car-bohydrate anabolism. We show that SUSIBA2 is a regulatorytranscription factor in starch synthesis and that it binds to theSURE element. Although the binding of nuclear protein frac-tions to the SURE element in the potato patatin (Grierson et al.,1994) and maize sbeI (Kim and Guiltinan, 1999) promoters, tothe SP8a sequence in the barley iso1 promoter (Sun et al.,1999), and to the Bbl element in the second intron of the barleysbeIIb gene (Ahlandsberg et al., 2002b) has been documentedpreviously, the interacting trans factors have not been isolatedor identified.

Analysis of the SUSIBA2 amino acid sequence shows that itconforms well to the general features of group 1 of the WRKYtranscription family (Figure 2). Our understanding of structure-function relationships in WRKY proteins is poor. It is believedgenerally that the C-terminal WRKY domain mediates sequence-specific binding to the cognate DNA elements, whereas theN-terminal WRKY domain facilitates DNA binding or engages inprotein–protein interactions (Eulgem et al., 2000). The zinc fin-gers in each WRKY domain might be involved in binding toeither DNA or proteins. What aspects of SUSIBA2 that areresponsible for binding to the SURE element are not known.Site-directed mutagenesis in combination with binding assaysis under way in our laboratory to address this question. It isprobable that the unique properties of SUSIBA2, comparedwith other, previously known WRKY proteins, are attributable toregions outside of the two WRKY domains, where the aminoacid sequences are relatively divergent (Figure 2). Another re-gion that merits attention is the insertion in SUSIBA2 right afterthe C-terminal WRKY domain (Figure 2).

Using RT-PCR, we found orthologs to SUSIBA2 in rice andwheat (see supplemental data online). The deduced amino acidsequences from the PCR products include the region betweenthe two WRKY domains, and analysis of these segments fromthe rice and wheat sequences shows that they share a highdegree of identity between themselves and with SUSIBA2. Be-cause the open reading frame for the rice clone could beobtained in its entirety, we were able to compare the completeprimary sequence between SUSIBA2 and the rice ortholog (seesupplemental data online). The two proteins are very similar inthe extreme C and N termini. Notably, the SUSIBA2-specificC-terminal insertion (Figure 2) is present in the rice sequence.Preliminary data suggest that we have identified SUSIBA2orthologs in potato and Arabidopsis (data not shown).

Given the large number (�70) of WRKY genes in Arabidopsisand the conservation of the WRKY domains, it is an apparentlyunexpected outcome that the initial RT-PCR amplification of

Figure 7. Protein Analysis of the Complex Formed between the SUREElement and Nuclear Extracts.

EMSA with the SURE-a 5� probe and a nuclear extract from barley en-dosperm was performed, and the complex produced was subjected toSDS-PAGE and protein gel blot analysis with antibodies againstSUSIBA2 (anti-SUSIBA2) serum before immunization with SUSIBA2(preimmune serum) or antibodies against a 6-His tag (anti-His tag). Mo-lecular masses (in kilodaltons) are indicated.

The SUSIBA2 Transcription Factor

Purified SUSIBA2 appeared in two forms, SUSIBA2-70 andSUSIBA2-68, of �70 and 68 kD, respectively (Figure 3). It ispossible that SUSIBA2-68 is a truncated version of SUSIBA2-70. This notion is supported by preliminary results, which indi-cate that the level of SUSIBA2-70 increases at the expense ofSUSIBA2-68 with increasing concentrations of protease inhibi-tors. Whether the truncation is at the N or C terminus, and if it isof biological significance in vivo, remain to be elucidated. Be-cause the purified SUSIBA2 still contains the N-terminal Histag, it seems less likely that the truncation should be at the Nterminus, because that would leave too few His residues onSUSIBA2-68 to bind to the Ni column. Alternatively, the sizedifference between SUSIBA2-70 and SUSIBA2-68 is an effectof post-translational modification.

The SUSIBA2 preparation also contained the E. coli enzymetrehalose 6-phosphate synthase (Figure 3). This enzyme alsocopurified with the SUSIBA2 polypeptides during repeated runson the Ni column. There do not seem to be enough His resi-dues in trehalose 6-phosphate synthase to generate high affin-ity to the Ni resin. Instead, it is conceivable that the copurifica-tion was attributable to the association between SUSIBA2 andtrehalose 6-phosphate synthase. Although such an associationis likely to be nonspecific, we must consider the possibility thatit reflects an interaction of biological significance betweenSUSIBA2 and barley trehalose 6-phosphate synthase. A role for

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Figure 8. Temporal Expression Profiles for susiba2 and iso1.

(A) RNA gel blot analysis of susiba2 and iso1 gene activity during barleyendosperm development. Total RNA was isolated from barley en-dosperm on different days after pollination (d.a.p.) and used for RNA gelblot analysis. The blots were hybridized with probes specific forsusiba2, iso1, or paz1. The sizes for the hybridizing fragments andethidium bromide (EtBr)–stained rRNA are shown.(B) Thin layer chromatography analysis of endogenous sucrose levels indeveloping barley endosperm from different days after pollination. Ex-tracts from 0.2 mg dry weight of developing endosperm from each timepoint were applied to thin layer chromatography analysis.(C) Correlation between endogenous sucrose content and relativesusiba2 and iso1 mRNA levels in developing endosperm. Sucrose con-

tent was determined by an enzymatic assay and is presented in milli-grams per gram dry weight of tissue (DW). Relative mRNA levels (in per-centage of the levels at 12 days after pollination) were determined fromthe RNA gel blots in (A) using NIH Image software.

barley, rice, and wheat RNA yielded only one major product.One likely explanation for this finding is that the RNA was iso-lated from developing endosperms, in which only a subset ofWRKY genes is expressed.

As demonstrated using a SUSIBA2:GFP chimeric protein,SUSIBA2 is targeted to the nucleus. Nuclear import can occurby diffusion or carrier-mediated active transport (Görlich andMattaj, 1996). As a result of its predicted molecular mass of 62kD, translocation of SUSIBA2 across the nuclear pore is likelyto require active transport via NLS-dependent receptor bind-ing. Several NLSs were found in SUSIBA2, one of which in-volved a WRKY motif (Figure 2). Whether one or more ofthese NLSs is responsible for the nuclear import of SUSIBA2remains to be established. As shown by Robatzek andSomssich (2001), a classic NLS is not always a prerequisite fornuclear targeting.

Transcriptional Analyses of the susiba2 Gene

The expression pattern of the susiba2 gene mimicked that ofbarley iso1. The temporal expression of both genes followedchanges in the endogenous sucrose levels and peaked at 12days after pollination (Figure 8). Both genes are expressed inthe endosperm but not in leaves. However, they can be ex-pressed ectopically in leaves after sugar treatment (Figure 9A).Again, the expression levels followed the sucrose concentra-tion. The spatial expression of susiba2 was confirmed at the pro-tein level; SUSIBA2 was present in the endosperm but not inleaves (Figure 9C). That SUSIBA2 was produced in sugar-treated leaves is suggested by the sugar-induced SURE bind-ing activity in nuclear extracts from barley leaves (Figure 9B).We did not get good immunological signals from sugar-treatedbarley leaves using our SUSIBA2 antibody. A probable reasonis that the SUSIBA2 protein was present at concentrations toolow to be detected by protein gel blot analysis, which is lesssensitive than EMSA. The data from the expression analysesare consistent with a role for SUSIBA2 as a positive regulatorytranscription factor for iso1 activity. Experimental evidence forthis view was obtained recently in antisense inhibition studies(data not shown). They also suggest that the expression of thesusiba2 gene itself is controlled via sugar signaling.

The coding region of susiba2 contains an 18-nucleotide se-quence that is exactly complementary to the human IGF1 re-ceptor gene (Figure 1). We present this observation without anyattempt at explanation. The sequence is not conserved in anyof the susiba2 orthologs except for that of wheat. It also is notpresent in the recently deposited partial EST sequences forbarley WRKY proteins implicated in drought tolerance (Ozturket al., 2002). However, the 18-mer sequence is conserved in

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susiba1, susiba2, and susiba3, and it might serve as a signaturesequence for susiba genes.

We noted that the expression of iso1 in transformed barleyendosperm was confined mainly to the periphery of the en-dosperm, toward the aleurone layer (Figure 10B). The significanceof this finding is unclear. The endosperms used for transformationwere from seeds 20 days after pollination, which is near maturity.Because programmed cell death of the starchy endospermtissue in barley progresses from the center, the uneven distri-bution of iso1 activity might indicate that at 20 days after polli-nation, only endosperm cells along the aleurone layer are meta-bolically viable (for a recent review of endosperm development,see Olsen, 2001).

The Nature and Relevance of the SURE Element

The importance of the SURE cis element for the sucrose induc-tion of gene activity in plants was first demonstrated by Griersonet al. (1994) from work on the potato patatin promoter and hasbeen corroborated by a large number of studies in different plants(Kim et al., 1991; Rhee and Staswick, 1992; Fu et al., 1995; Mitaet al., 1995; Kim and Guiltinan, 1999; Li et al., 2002). In some ofthese studies, the assignment of the SURE sequence as a regu-latory element was inferred from experimental data (i.e., bindingassays), whereas in other cases, it was based primarily on com-putational analyses. Here, we show that three SURE elements,SURE-a, SURE-b, and SURE-c, also are located in the barley iso1promoter and that they provide binding sites for the SUSIBA2transcription factor (Figures 4 to 7).

Most of our attention in this study was directed toward SURE-a,because it appeared to have the strongest SUSIBA2 binding ac-tivity (Figure 4). The fact that SURE-a produced a similar bindingpattern with purified SUSIBA2 and nuclear extracts (Figure 4) in-dicates that the in vivo assembly of the SUSIBA2 complex on theiso1 promoter does not involve regulatory proteins other thanSUSIBA2 itself. The binding assays also suggest that SUSIBA2can oligomerize in a concentration-dependent manner (Figures4 and 5). The structural features of the SURE elements in thebarley iso1 promoter need to be assessed. As judged by muta-tion analyses of the SURE-a sequence (Figure 6), a large num-ber of A nucleotides does not in itself constitute a SUSIBA2binding element. In fact, the presence of long poly(A) stretchesin the upstream region (SURE-a 3� m1), the middle region(SURE-a 5� and SURE-a 3� m2), or the entirety (SURE-a 3� m4)of SURE-a diminished or abolished its SUSIBA2 binding activ-ity (Figure 6).

Figure 9. Tissue-Specific and Ectopic Expression of susiba2 and iso1in Barley Leaves.

(A) Barley leaves were treated with exogenous sucrose at the concen-trations indicated. Total RNA was isolated and hybridized to probesspecific for susiba2, iso1, rbcS, and rbcL. The sizes for the hybridizingmolecules and ethidium bromide (EtBr)–stained rRNA are shown.(B) EMSA was performed with the SURE-a 3� probe in the absence (�)or presence of barley nuclear extracts isolated from endosperm orleaves. The leaves were treated with sucrose and harvested at the timesindicated. The position of the free probe is indicated.(C) Protein gel blot analysis of susiba2 expression in barley endospermand leaves.

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Figure 10. Effects of the SURE-a ODN Decoy on iso1 Expression in Transformed Barley Endosperm.

(A) Scheme of the iso1:gfp construct and sequences of the SURE-a and rbcL ODN decoys.(B) Transient expression assays of GFP fluorescence in transformed barley endosperm cells. Barley endosperm cells were transformed by micro-projectile bombardment with the iso1:gfp construct in the absence (top) or presence of the rbcL (middle) or SURE-a (bottom) ODN decoy. Forcotransformation, the iso1:gfp construct and the ODN decoys were mixed at a molar ratio of 1:100.(C) Average number (means � SE) of fluorescent foci per plate from nine independent transformation events. An F test indicated significant differencebetween means for the absence and presence of the SURE-a decoy (P 0.0017) and no significant difference between means for the absence andpresence of the rbcL decoy (P 0.62).(D) Transient expression assays of GFP fluorescence in transformed barley endosperm cells. Barley endosperm cells were transformed by micro-projectile bombardment with the Act-1:gfp construct in the absence (left) or presence (right) of the SURE-a ODN decoy. For cotransformation, theAct-1:gfp construct and the ODN decoy were mixed at a molar ratio of 1:100.

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Given the large number of DNA binding proteins in nuclei, itis plausible that the complex formations observed for SURE-a5� and SURE-a 3� m4 with nuclear extracts are attributable toproteins other than SUSIBA2 (Figures 6A and 6C). Notably, ex-tending the poly(A) stretch in the downstream half of SURE-a(SURE-a 3� m3) significantly augmented the affinity for the puri-fied SUSIBA2 protein, although not for nuclear extracts, andpromoted the assembly of high Mr complexes (Figure 6). Theapparent lower SURE-a 3� m3 binding capacity for nuclear ex-tracts compared with that for purified SUSIBA2 is not under-stood. It might reflect the fact that the amount of purified SUSIBA2used in the binding assays exceeded that present in the addednuclear extracts.

The complex formed between the SURE-a 5� element andthe nuclear extract harbored two polypeptides of 68 and 66 kDthat reacted with the SUSIBA2 antibodies (Figure 7). Thesedata demonstrate that the SURE binding activity in the nuclearextracts was attributable to SUSIBA2. Whether the SUSIBA2duplex observed in Figure 7 corresponds to the overproducedSUSIBA2-70 and SUSIBA2-68 is not known. Occasionally, twoadditional faint bands in the 56- to 54-kD region were dis-cerned on the protein gel blot (data not shown), possibly as aresult of degradation during the extraction procedure. The na-

ture of the 96-kD polypeptide recognized by the His tag anti-bodies is unknown.

Its presence in the barley iso1 and maize sbe1 promoterssuggests that the SURE element also should be found in otherstarch synthesis genes subject to sugar induction. Indeed, adatabase search of the proximal and distal promoter regions ofsequences revealed that SURE-like sequences are present inpromoters of several genes that encode enzymes involved instarch synthesis. Among those are barley sbeIIb and the barleygenes for starch synthase I (ssI) (Gubler et al., 2000) and thesmall subunit of ADP–glucose pyrophosphorylase (agpaseS)(Thorbjørnsen et al., 2000). Although all three SURE sequencesin the barley iso1 promoter, as well as those identified in othergenes, bear a good resemblance to the patatin SURE sequencein pair-wise comparisons, it is difficult to arrive at a consensussequence for the SURE element. An alignment of a subset of re-ported SURE sequences is shown in the supplemental data on-line. The compilation also includes the SURE sequences frombarley ssI and agpaseS.

As can be seen, the SURE element is best described as anA-rich sequence with the consensus core AA/TAA. Other A-richor A/T-rich promoter elements with demonstrated or impliedprotein binding activity are known from both plants and otherorganisms, such as the A-box in gbssI from wheat (Kluth et al.,2002) and developmentally regulated genes from Drosophila(Gasser and Laemmli, 1986), the A-stretch in psbD from tobacco(Allison and Maliga, 1995), the A-rich sequence in the Npg1gene from tobacco (Tebbutt et al., 1994), the A/T-rich regionin URA3 from yeast (Roy et al., 1990), the A-rich site in the -MHCgene from rat (Molkentin and Markham, 1994), the A-rich regionin the Ig� gene from mouse (Costa and Atchison, 1996), andthe A/T-rich AT hook binding (Dragan et al., 2003) and SPKKbinding (Churchill and Suzuki, 1989) regions in eukaryotic DNA.A/T-rich regions also might be expected in some promoters,because they facilitate DNA unwinding.

We noted that, in contrast to the barley sbeIIb promoter,which contains three SURE elements, no SURE-like sequencewas found in the promoter of the barley sbeIIa gene. BothsbeIIb and sbeIIa contain a W-box (data not shown). We re-ported previously (Sun et al., 1998) that the expression of bar-ley sbeIIb is endosperm specific, whereas that of sbeIIa is not.We suggest that the W-box in the iso1, sbeIIb, and sbeIIa pro-moters serves as a general activating element, whereas thesugar responsiveness conferred to iso1 and sbeIIb by the SUREelements contributes to their endosperm specificity (by thehigher sugar levels in sink tissues compared with embryos andvegetative tissues). Further control of endosperm-specific ex-pression probably is exerted via the binding of repressor pro-teins in nonexpressing tissues. From earlier work, we concludedthat repressors are recruited to the SP8a element in the barleyiso1 promoter (Sun et al., 1999) and to the Bbl element in thesecond intron of barley sbeIIb (Ahlandsberg et al., 2002b).

The importance of the SURE element for iso1 expression isdemonstrated by the decoy experiment (Figure 10). The activityremaining in the presence of the SURE-a decoy could indicatethat the decoy did not efficiently trap the SURE binding activity.More likely, it demonstrates that elements other than SURE aresufficient to maintain a basic level of iso1 activity. The involve-

Figure 11. Nuclear Localization of the SUSIBA2:GFP Fusion Protein.

Transient expression assays of GFP fluorescence in transformed barleyendosperm cells. Barley endosperm cells were transformed by micro-projectile bombardment with a construct encoding GFP only (top) orwith a construct encoding the SUSIBA2:GFP fusion protein (bottom).

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ment of the SURE element in the regulation of iso1 expressionis in agreement with the ectopic expression of iso1 in sugar-treated barley leaves (Sun et al., 1999). The emerging transcrip-tion factor ODN technology offers a powerful and convenientmeans to assess transcription factor function and the involve-ment of cis elements both in vitro and in vivo. The strategy hasgained rapid popularity in animal sciences in studies of genetherapy (Morishita et al., 1998; Mann and Dzau, 2000). Thepresent report demonstrates the applicability of the transcrip-tion factor ODN technology also in plant biology.

The enrichment of W-boxes in the promoters of defense-relatedwrky genes suggests that these genes are subject to autoregula-tion and/or controlled by other members of the WRKY superfamily(Dong et al., 2003). It is worth considering whether the barleysusiba2 gene participates in such a self-regulatory network. Wehave not completed the sequencing of the susiba2 genomic cloneand do not know if the susiba2 promoter contains W-boxes orSURE elements. However, thanks to the rice genome project, wewere able to analyze the entire locus for the orthologous gene,WRKY20, in rice. Inspection of the 1.2-kb region upstream of thepredicted transcription start site revealed that the WRKY20 geneharbors one W-box sequence (TGACT; nucleotides �796 to �792)and one SURE sequence (see supplemental data online). Whetherthese putative elements are present in barley susiba2, and to whatextent they would contribute to the regulation of susiba2 activityand starch synthesis, are questions that remain to be addressed.

METHODS

Plant Material

Barley (Hordeum vulgare cv Pongo), rice (Oryza sativa), and wheat (Triti-cum aestivum) were grown in soil in a climate chamber under a 16-h-light/8-h-dark photoperiod as described by Sun et al. (1998, 1999).

Oligonucleotides

The following oligonucleotides were used: 1, 5�-CCAAGAAGTTATTAC-AAGTG-3�; 2, 5�-TGGTTATGTTTTCCTTCGTA-3�; 3, 5�-GGAATTCCA-TATGTCCCCCGCGCGGCTGCC-3�; 4, 5�-CGGATCCGGCTGAACTGA-CTTGTAAC-3�; 5, 5�-CCCTCGTGGAAGCAAAACTGTGTTTCTCGC-3�;6, 5�-GCGAGAAACACAGTTTTGCTTCCACGAGGG-3�; 7, 5�-TTCCGA-AAAAAACTAAGAAAGACCGATGGAAAACCGAAAT-3�; 8, 5�-ATTTCGGTT-TTCCATCGGTCTTTCTTAGTTTTTTTCGGAA-3�; 9, 5�-GGAAAACCGAAA-TACCAAAAAATAATAATAAAATAATAAT-3�; 10, 5�-ATTATTATTTTATTA-TTATTTTTTGGTATTTCGGTTTTCC-3�; 11, 5�-CCGCGTGCGAAAAAATAA-AGAAAATGAAATCTGAAGGAAG-3�; 12, 5�-CTTCCTTCAGATTTCATT-TTCTTTATTTTTTCGCACGCGG-3�; 13, 5�-TCGCTAACCAGTGACTTCCAC-GTTTCATCATTTATT-3�; 14, 5�-AATAAATGATGAAACGTGGAAGTCACT-GGTTAGCGA-3�; 15, 5�-TTCCGAAAAAAACTAAGAAA-3�; 16, 5�-TTTCTT-AGTTTTTTTCGGAA-3�; 17, 5�-GACCGATGGAAAACCGAAAT-3�; 18, 5�-ATTTCGGTTTTCCATCGGTC-3�; 19, 5�-GAAAAATGGAAAACCGAAAT-3�;20, 5�-ATTTCGGTTTTCCATTTTTC-3�; 21, 5�-GACCGAAAAAAAACC-GAAAT-3�; 22, 5�-ATTTCGGTTTTTTTTCGGTC-3�; 23, 5�-GAAAAAAAA-AAAAAAAAAAT-3�; 24, 5�-ATTTTTTTTTTTTTTTTTTC-3�; 25, 5�-ATG-ACTCGAGCAGATTTTGGATTGCTAATGA-3�; 26, 5�-ATGACCATGGGC-CACCTCGTGTTGGTTCTTCGT-3�; 27, 5�-ATTACGGTAGAGCGTGTT-ATGAGTGTCTACGCGGTGGACT-3�; and 28, 5�-AGTCCACCGCGT-AGACACTCATAACACGCTCTACCGTAAT-3�.

Computational Analyses

Searches of the NCBI databases were performed with the Basic LocalAlignment Search Tool (BLAST) service (http://www.ncbi.nlm.nih.gov/BLAST/). Alignments of nucleotide and amino acid sequences were per-formed using the CLUSTAL W service at the European Bioinformatics In-stitute (http://www.ebi.ac.uk/clustalw), except for the alignment of sweetpotato, cucumber, and parsley SPF1 cDNA sequences, which was per-formed with the MacVector program (Accelrys, Paris, France), and align-ment of the SURE element sequences, which were run on the T-Coffeeserver (http:// www.ch.embnet.org/software/TCoffee.html). For presenta-tion purposes, the ALN output from the CLUSTAL W program was fedinto the BOXSHADE server (http://www.ch.embnet.org/software/BOX_form.html).

Isolation of cDNA Clones

Oligonucleotides 1 and 2 were constructed according to the consensussequences for the SPF1 and SPF1-like cDNA sequences. Total RNA wasisolated from developing barley, rice, and wheat endosperm accordingto Sun et al. (1999). First-strand cDNAs were produced as described(Frohman et al., 1988) using oligonucleotide 2. Reverse transcriptase–mediated (RT) PCR was performed according to standard protocols(Sambrook et al., 1989) using oligonucleotides 1 and 2. The PCR prod-ucts were subcloned and sequenced. The RT-PCR product from barleywas used as a probe to screen a barley cDNA library from developingendosperm 10 days after pollination. Library construction and screening,subcloning, DNA gel blot analysis, and sequence analysis were as de-scribed (Sun et al., 1998, 1999).

Overproduction of SUSIBA2 in Escherichia coli, Microsequencing, Antibody Production, and Protein Gel Blot Analysis

Two primers were designed: oligonucleotide 3, with an NdeI restrictionsite, and oligonucleotide 4, with a BamHI restriction site. These primerswere used for PCR amplification of the susiba2 cDNA from the isolatedfull-length cDNA clone. The amplified cDNA was inserted into the ex-pression vector pET-15b (Novagen, Madison, WI) between the NdeI andBamHI sites. The constructed plasmid was transformed into E. coli strainBL21(DE3). Overexpression was performed according to the manualprovided by the manufacturer, except that the isopropylthio-�-galacto-side induction was performed at 37�C for 2 h. The overproduced proteinwas purified by fast protein liquid chromatography on a column with aNi� chelating resin. SDS-PAGE was run as described previously (Sun etal., 1997).

Trypsin digestion and microsequencing were performed in collabora-tion with Amersham Pharmacia Biotech. Antisera were produced byAgriSera (Vännäs, Sweden) after immunizing rabbits with the CoomassieBrilliant Blue R 250–stained SUSIBA2-70 and SUSIBA2-68 polypeptidebands excised from the SDS gel. Protein gel blot analysis was performedas described (Sun et al., 1997). Polyclonal His tag antibodies were pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA).

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays (EMSAs) were performed essen-tially as described by Sun et al. (1999). For protein gel blot analysis of theEMSA complex formed between the nuclear protein extract and theSURE element, a polyacrylamide gel with a SURE-a 5� protein complexwas positioned over the x-ray film and a 1-cm-tall section of the la-beled complex was sliced out from the gel. The gel pieces were homog-enized in loading buffer and applied directly to an SDS gel.

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RNA Gel Blot Analysis

RNA isolation and RNA gel blot analysis were performed as describedpreviously (Sun et al., 1998, 1999).

Sucrose Isolation and Analysis, Exogenous Sucrose Induction, and Nuclear Protein Isolation

Sucrose isolation, enzymatic sucrose analysis, thin layer chromatogra-phy, and sucrose induction of ectopic gene expression in barley leaveswere performed as described (Sun et al., 1998, 1999). Nuclear proteinswere isolated from leaves and endosperm as described by Sun et al.(1999).

Transformation and GFP Assay of Barley Endosperm

The 1504-bp promoter region of the iso1 gene was amplified by PCR us-ing oligonucleotide primers 25 and 26 and fused to the gfp plasmidpN1473GFP as described by Ahlandsberg et al. (1999, 2002a, 2002b),yielding the construct p48. The SURE-a and rbcL oligodeoxynucleotidedecoys were generated by annealing oligonucleotides 7 and 8, and 27and 28, respectively, in 14 mM Tris-HCl, pH 8.0, and 7 mM MgCl2. TheAct-1:gfp construct was as described (Ahlandsberg et al., 2002b). TheSUSIBA2:GFP fusion protein was constructed by ligating the NcoI-SacIfragment of the susiba2 cDNA into NcoI-SacI–digested pN1473GFP.Transformation of barley endosperm by microprojectile bombardmentusing the DuPont PDS 1000 He Biolistic Delivery System (Bio-Rad Lab-oratories, Hercules, CA) and transient assay of GFP fluorescence wereperformed as described by Ahlandsberg et al. (2002a) with the followingexceptions. Barley caryopses at 19 to 22 days after pollination were bi-sected longitudinally and placed endosperm side up on DG3B mediumcontaining Murashige and Skoog (1962) medium supplemented with 100g/L sucrose, 1.0 mg/L thiamine-HCl, 0.25 g/L myo-inositol, 1.0 g/Lcasein hydrolase, and 0.69 g/L Pro solidified by 5.0 g/L Gelrite (Duchefa,Haarlem, The Netherlands) for 2 h before bombardment. Six bisectedcaryopses were placed in the center of a 20- � 90-mm Petri dish withDG3B medium, 13 cm below the stopping screen, and bombarded onceusing a 7.6-MPa rupture disc. After bombardment, the caryopses werekept for 48 h in darkness on plates before GFP fluorescence assays.

Upon request, materials integral to the findings presented in this pub-lication will be made available in a timely manner to all investigators onsimilar terms for noncommercial research purposes. To obtain materials,please contact Christer Jansson, [email protected].

Accession Numbers

The susiba2 cDNA sequence has been deposited in GenBank with the ac-cession number AY323206. The cDNA sequences for the rice and wheatsusiba2 orthologs have been deposited with accession numbers AY324392and AY324393, respectively. The GenBank accession numbers for the othersequences referred to in this work are as follows: Arabidopsis gene for�-amylase, S77076; Arabidopsis hypothetical protein, T08930; ArabidopsisWRKY transcription factor 20, Q93WV0; Arabidopsis putative protein,NP567752; Arabidopsis unknown protein, AAK76566; barley agpaseS,AJ239130; barley iso1, AF142589; barley sbeIIb, AF064563; barley ssI,AF234163; barley WRKY proteins implicated in drought tolerance,BM816210 and BM816211; E. coli trehalose-6-phosphate synthase,S33584; human IGF1 receptor, NM_000875; maize sbeI, AF072724; potatopatatin class-I gene, M18880; potato gene for proteinase inhibitor II,X04118; potato gene for sucrose synthase, U24087; soybean gene for veg-etative storage protein, M76980; sweet potato SPF1, S51529; tobaccoWRKY protein, BAB61056; tomato hypothetical proteins, CAC36397 and

CAC36402; white broom drought-induced protein, AAL32033; and rice pu-tative WRKY transcription factor 20, BAC55609.

ACKNOWLEDGMENTS

We thank the reviewers for thorough work and constructive criticism.This work was supported by grants from the Swedish Research Councilfor Environment, Agricultural Sciences, and Spatial Planning, the NordicJoint Committee for Agricultural Research, the Foundation for StrategicResearch, and Helge Ax:son Johnsons Stiftelse.

Received June 11, 2003; accepted June 19, 2003.

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a novel wrky transcription factor, susiba2, participates in sugar

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