opaque2 and o2-defective proteins in functional complexes

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Running head: Opaque2 and o2-defective proteins in functional complexes

*Corresponding author: Angelo Viotti, Istituto Biologia e Biotecnologia Agraria, CNR, Via Bassini

15, I-20133 Milano, Italy; phone 39-2-23699437; fax 39-2-23699411; e-mail [email protected]

Plant Physiology Preview. Published on September 7, 2007, as DOI:10.1104/pp.107.103606

Copyright 2007 by the American Society of Plant Biologists

www.plantphysiol.orgon March 15, 2018 - Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved.

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Wild-type Opaque2 and Defective o2 Polypeptides Form Complexes in Maize

Endosperm Cells and Bind the O2-Zein Target Site1

Floriana Gavazzi, Barbara Lazzari2, Pietro Ciceri3, Elisabetta Gianazza4 and Angelo Viotti*

Istituto Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via E. Bassini 15, I-

20133 Milano, Italy

2 Parco Tecnologico Padano, Via Einstein – Località Cascina Codazza, 26900 Lodi, Italy,

4 Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Dipartimento di Scienze

Farmacologiche, Università degli Studi di Milano, Via G. Balzaretti 9, I - 20133 Milano, Italy



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1 This work was partially supported supported by grants from MIUR, FIRB project

3 Present address: Ambit Biosciences, 4215 Sorrento Valley Blvd, San Diego, CA 92121, USA



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ABSTRACT

The Opaque2 bZIP transcriptional activator controls the expression of several genes in maize. We

investigated the phosphorylation extent of wild-type Opaque2 and mutant-defective or mutant-

truncated o2 polypeptides in endosperm cells, their subcellular localization, participation in complex

formation, and involvement in functional activity. Beside the wild-type, four mutant alleles (o2T,

o2-52, o2It, o2-676) producing o2 polypeptides and a null transcript allele (o2R) were considered.

Observing the effects of these mutations, multi-phosphorylation events in O2 or o2 proteins were

confirmed and further investigated, and the involvement of both the NLS-B and leucine-zipper

domains in proper targeting to the nucleus was ascertained. The absence of these domains in the

o2T, o2It-S mutant-truncated forms holds them within the cytoplasm, where they are partially

phosphorylated, while the presence of NLS-B and a partial leucine-zipper domain in o2-52

distributes this mutant-truncated form in both cytoplasm and nucleus. Although mutated in the

NLS-B domain, the o2It-L and o2-676 mutant-defective forms are, respectively, partially or

completely distributed into the nucleus. Only wild-type O2 and mutant-defective o2 polypeptides

bearing the leucine-zipper are able to form complexes whose components were proven to bind the

O2-zein target site by in vitro analyses. The transcription of a subset of H-zein genes as well as H-

zein polypeptide accumulation in several o2-mutant-defective genotypes indicate the in vivo

involvement of o2-mutant-defective proteins in O2-zein target site recognition. The gathered

information broadens our knowledge on Opaque2 functional activity and our view on possible QPM

trait manipulation or plant transformation via the utilization of cisgenic elements.

Genus: Zea

Species: mays

Subspecies: mays



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INTRODUCTION

The Opaque2 transcriptional activator is specifically expressed in the subaleurone layers of maize

endosperm cells, during seed development. Opaque2 is a basic-leucine-zipper (bZIP) protein that

contains both the dimerization and DNA binding domains in the second half of the amino acidic

sequence (Schmidt et al.1990), as well as two nuclear localization signals (NLSs): the NLS-A,

located in the first half of the protein, and the bipartite NLS-B, intermingled within the basic

domain (Varagona et al., 1992).

The Opaque2 transcriptional activator regulates the transcription of several structural genes coding

for a subset of the zein multigene family, for the ribosome-inactivating protein (RIP) b-32 , and for

a number of enzyme activities involved in different metabolic pathways (Rossi et al., 2001). Both b-

32 and zein polypeptides are recovered in mature seeds, where they display their functional role

against pathogen attack and as source of storage products, respectively. Zein genes can be divided in

sequence subfamilies: SF1, SF2 and SF3, coding mainly for light class polypeptides, and SF4,

coding mainly for heavy class polypeptides (Viotti et al. 1985; Rubenstein and Geraghty 1986).

Sequences of the different subfamilies do not cross-hybridize, and are regulated by different loci

(Soave and Salamini, 1984). In particular, O2 controls the expression of the SF4 genes coding for

various size zein polypeptides of the 22 kD type, belonging to the heavy zH1 and zH2 classes

(Viotti et al., 1985; Ciceri et al., 2000). In vitro experiments showed that O2 homodimers are able to

bind the already described O2 target site (5’-TCCACGTAGA-3’) (Schmidt et al., 1990). This

sequence is an imperfect A-box type palindrome located in the P2 promoter regions of most of the

heavy zein genes at about – 300 bp from the start codon (Sturaro and Viotti, 2001). However, a

further more distal promoter, P1, is also able to drive zein genes transcription. P1 and P2 share their

consensus sequences, are able to give rise to long and short transcripts, respectively, and they both

promote reporter gene expression in homologous or heterologous cell/tissue systems (Langridge and

Feix, 1983; Quattrrocchio et al., 1990; Giovinazzo et al., 1992).

Several O2 wild-type alleles are known to code for polypeptides of different length, with two

hypervariable regions occurring in the first sixty amino acids of the amino terminal region. In

addition, several o2 mutant alleles were characterized. These contain specific nucleotide insertions

or deletions, causing frame shifts that result in the translation of mutant-truncated or mutant-

defective polypeptides (Lazzari et al., 2002). More in detail, as schematically reported in Fig. 1, the

o2T allele produces a truncated polypeptide (o2T) missing the second half of the protein and, as a

consequence, the NLS-B and the leucine-zipper motifs; the o2-52 allele codes for a differently

truncated polypeptide that lacks the carboxy-terminal region downstream the second leucine of the



6

leucine-zipper motif; the o2It allele produces two polypeptides: the former, referred to as “Long”

(o2It-L or mutant-defective), migrating in SDS-PAGE as the wild-type protein (67-68 kD) and the

latter, referred to as “Short” (o2It-S or mutant-truncated), with an SDS-PAGE relative mobility of

about 47 kD; the o2-676 allele produces a polypeptide that contains an amino acidic substitution

(R249K) in the basic region of the bZIP domain; and finally, the o2R allele is defective in

transcription and was defined as a null transcript allele (Schmidt et al., 1987).

Most of the o2 mutant alleles were shown to severely reduce the expression of the heavy class zeins,

and to only slightly affect the expression of those of the light class. The o2-676 mutation, however,

surprisingly reduces the expression of a group of zeins belonging to the light class, only slightly

affecting some components of the heavy class (Aukerman et al., 1991). In homozygous lines for any

of the o2 mutants, the observed seed phenotype is opaque and, even crossing different o2 alleles

(Ciceri et al., 2000), complementation was never observed, the translucent and vitreous phenotype

typical of the wild-type alleles never being restored (Schmidt et al., 1993). Mutant o2 alleles were

recovered in different genetic backgrounds and found in different maize collections (Schmidt et al.,

1987; Bernard et al., 1994). Our previous results clearly show that the genetic background can

differently influence the transcription of the zH genes in the absence of the O2 protein - as in o2R

genotypes - or even in the presence of defective o2 polypeptides, as in those genotypes carrying the

o2It or o2-676 alleles (Dolfini et al. 1992; Ciceri et al. 2000). We suggested that these differences

could be attributed to the presence of putative O2-vicarious protein factor(s) able to partially

complement o2 function, or to other helper factors interacting with the o2 defective proteins (Ciceri

et al., 2000; Locatelli et al., 2001).

The regulation of the expression of the O2 gene occurs at different levels. Opaque2 transcription is

tissue-specific and developmental stage-specific: O2 transcription mainly occurs in the subaleurone

layers of the endosperm (Dolfini et al., 1992), starting from about 10 days after pollination (DAP)

(Gallusci et al., 1994). Moreover, the steady-state level of the O2 transcript is also subject to diurnal

changes and appears to be regulated by a circadian clock. The highest quantity of O2 transcript is

observed at midday and the lowest at midnight (Ciceri et al., 1999). A translational control of O2

expression is exerted by three short upstream open reading frames (uORFs) located in the leader

sequence of the mRNA (Lohmer et al., 1993). At the post-translational level, phosphorylation of the

O2 protein modulates its DNA-binding affinity (Ciceri et al., 1997). The O2 polypeptide, in fact,

exists in the endosperm cells as a pool of differentially phosphorylated forms that oscillate in their

relative abundance as well as in the extent of phosphorylation. The non-phosphorylated and hypo-

phosphorylated forms bind the target DNA sequence with high affinity, whereas the hyper-

phosphorylated forms bind the target with low affinity, or acquire the capacity to bind it only after



7

in vitro enzymatic dephosphorylation (Ciceri et al., 1997). In addition, the O2 phosphorylation

pattern changes diurnally: non-phosphorylated and hypo-phosphorylated forms accumulate during

the day, while hyper-phosphorylated forms are present mainly at night. This suggests that O2

activity is down regulated at night both by a reduction in the transcript level and by hyper-

phosphorylation of the O2 protein (Ciceri et al., 1997). It was shown that O2 binding activity is also

influenced by the methylation state of the O2 binding region located in the promoter of the 22 kD

zein genes. This region is less methylated in the endosperm than in sporophytic tissues and

methylation severely reduces the O2 affinity for its binding site (Sturaro and Viotti, 2001).

Furthermore, it was suggested that the activity of the O2 protein depends upon regulatory

mechanisms involving protein-protein interaction. In fact, in vitro experiments demonstrated that

O2 interacts with another O2-like bZIP protein (OHP1) that is able to bind the O2 binding site, both

as homodimer or as heterodimer with O2 (Pysh et al., 1993). In addition, O2 interacts with another

trans-acting factor, PBF, that binds to the P-box motif located in the promoter region of the 22 kD

zeins about 30 bp upstream the O2 binding site (Vicente-Carbajosa et al., 1997; Wang et al., 1998;

Wang and Messing, 1998). However, the in vivo occurrence of these or other heterodimers was

never demonstrated. In support of the protein-protein interaction hypothesis, co-operative binding of

O2 to multiple target sites in the promoters of the regulated genes was demonstrated (Yunes et al.,

1998). Moreover, it is known that O2 binding to the DNA is, in some way, promiscuous, since O2

binds in vitro to different target sequences (Lohmer et al., 1991; Schmidt et al., 1992; Izawa et al.,

1993; Yunes et al., 1994). Finally, O2 is able to complement in vivo the bZIP function of the

General-Control-Nonderepressible-4 gene product (GCN4) in gcn4 yeast strains (Mauri et al.,

1993), as well as to activate in the endosperm different genes bearing variable O2-core binding

sequences in their promoter region (Maddaloni et al., 1996; Kemper et al., 1999).

In this report, we analyzed and compared the characteristics of the O2 wild-type and mutant

polypeptides with respect to their cellular localization, differences in binding activity and

phosphorylation state, and to their influence on zein gene expression. We also recovered protein

complexes containing the O2 wild-type at different seed developmental stages that were proven to

be able to bind the O2 target site. Similarly, we showed that defective o2 polypeptides containing

the leucine-zipper domain form complexes and their isoforms bind the O2 target site upon

dephosphorylation. The overall recruited information allowed us to extend the knowledge of the O2

protein function in the maize endosperm.

RESULTS



8

Analysis of O2 Wild-Type and o2 Mutant Proteins in Different Maize Lines

Nucleotide sequence analysis of Opaque2 genomic fragments and cDNA clones reveals a high

degree of heterogeneity among the wild-type and mutant alleles (Schmidt et al., 1990; Henry et al.,

2005). We characterized three wild-type sequences, having slightly different coding capacities, due to the

insertion or deletion of a few amino acids in the amino terminal region. They were classified as O2w1, O2w2

and O2w3 and identified to occur, respectively, in the W64A, W22 and A69Y lines (Ciceri et al. 2000;

Lazzari et al., 2002). A number of mutated o2 alleles was also collected and characterized, bearing either few

changes or severe alterations in the deriving polypeptides (Bernard et al. 1994; Lazzari et al., 2002).

These O2 and o2 polypeptides were analyzed by western blot with two different polyclonal

antibodies: O2-NT, specific for the first 24 amino acids of the N-terminal part of the O2 protein, and

O2-CT, specific for the last 170 amino acids of the carboxy-terminal part of the protein. The

immunoblot analysis, carried out on O2 and o2 enriched protein extracts from immature

endosperms from various genetic backgrounds (GBs), showed a specific polypeptide pattern for

each O2 or o2 allele (Fig. 2A). The o2R allele, an o2 null transcript mutant introgressed in the A69Y

GB, produced no immune-reacting band with both O2 antisera, with the exception of a faint

unspecific band with a relative mobility of about 53 kD, that was revealed in the various extracts

with variable intensities. The O2wt allele produced a band migrating with a relative molecular mass

of about 68 kD that was detected by both the O2 antisera. Actually, this band was composed of a

closely spaced doublet that becames evident under particular electrophoretic conditions (Bernard et

al., 1994; Ciceri et al., 1997). In A69Y and in Bianchi o2 GBs, the o2It allele produced two bands,

both detectable with O2-NT antiserum, the former migrating slightly faster than the wild-type and

the latter with a relative molecular mass of about 47 kD; this second band was not revealed by the

O2-CT antiserum. The product of the o2T allele, in the A69Y and W64A GBs, was detected only by

the O2-NT antiserum, showing a closely spaced doublet with relative molecular mass of about 43

kD. When A69Yo2It was crossed with W64Ao2T, a pattern consisting of all the three expected

bands (two specific of the o2It allele plus the lowest migrating one, specific of the o2T allele) was

detected with the O2-NT antiserum; in this cross, only the uppermost band, with relative molecular

mass of about 67 kD, was detected by the O2-CT antiserum. Also the 52 kD band produced by the

A69Yo2-52 line was detected only by the O2-NT antiserum. Even in this case, in the cross between

A69Yo2It and A69Yo2-52, the expected three-band pattern was recovered with the O2-NT

antiserum. As usual, only the uppermost band of the o2It allele was detected by the O2-CT

antiserum. Finally, the A69Yo2-676 allele produced a polypeptide that was detected by both O2



9

antisera and migrated slightly slower than the wild-type, according to its coding capacity

(Aukerman et al., 1991; Lazzari et al., 2002).

The above-mentioned closely spaced doublet is a result of the co-existence in the endosperm cells of

differentially phosphorylated O2 or o2 forms (Ciceri et al., 1997 and 1999). To better investigate the

phosphorylation extent in the O2 and o2 lines, we analyzed the charge heterogeneity of the O2 wild-

type and o2 mutant proteins by isoelectric focusing in a narrow immobilized pH gradient (IPG-IEF,

pH 4.0-5.0) (Righetti, 1990; Bjellqvist et al., 1993; Ciceri et al., 1997). Figure 2B shows the

isoelectric pattern of the O2 and o2 alleles, whose migration in SDS-PAGE is displayed in the upper

panels (Fig. 2A). After IPG-IEF electrophoresis the proteins were blotted onto nitrocellulose

membrane and O2 polypeptides were revealed by immunoreaction with the O2-NT and O2-CT

antisera. The o2R null transcript allele produced no immunoreacting band, with the exception of few

faint unspecific bands in the most basic or acid regions of the gradient. The O2wt, o2It and o2-676

alleles, which produce polypeptides in the 67-69 kD range, showed isoelectric patterns composed of

7-8 isoforms distributed along the 4.2-4.7 pH range and detectable by both the O2 antisera. The o2T

and o2-52 alleles, which produce truncated proteins, showed a lower number of bands, revealed

only with O2-NT antiserum and occurring, respectively, in the most acidic or most basic region of

the gradient. The o2It and the o2T alleles showed the same behavior in two different genetic

backgrounds: A69Y or Bianchi o2, and A69Y or W64A, respectively. The crosses between

A69Yo2It and W64Ao2T or between A69Yo2It and A69Yo2-52 showed additive patterns with

bands of higher intensity in the most acidic part of the gradient in the first cross with respect to the

second. To support this evidence, a blotted filter from a 3.5 to 5.0 pH gradient containing the wild-

type O2 protein and the o2T and o2It mutants was at first immunodecorated with O2-CT, then

stripped and re-probed with O2-NT (Fig. 2C). Clearly, in the most acidic region of the gradient,

more clean-cut bands were detected for the o2T and o2It with the O2-NT as compared to the O2-

CT. This indicates that the truncated o2T and o2It-S polypeptides undergo two or three

phosphorylation events.

Variations in the Primary Structure of the O2 Protein Affect its Cellular Localization

Opaque2 localizes within the cell nucleus and contains two nuclear localization signals, the NLS-A

and the bipartite NLS-B, located, respectively, in the first and second half of the protein (Varagona

et al., 1991, 1992).

In this report we analyzed and compared the localization patterns of the O2 and o2 polypeptides

presented in Fig. 1 in the maize endosperm cells. Opaque2 polypeptides were immunodetected by



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O2-NT and O2-CT antisera on sections from O2 wild-type and o2 mutant maize seeds at 17-20

DAP (Fig. 3). In the O2 line the immunoreacting polypeptides were mainly localized into the nuclei

of the subaleurone layers and were revealed by both O2 antisera. The same result was obtained in

the o2-676 line, whose o2 polypeptide preserves the two NLSs in its primary sequence (Lazzari et

al., 2002). The null transcript o2R allele, as expected, did not show any immunofluorescent signal

and was considered as negative control. The two o2 polypeptides from the o2-52 and the o2T lines

were not revealed by the O2-CT antiserum, as they lack the carboxy-terminal region, while they

were detected by the O2-NT antiserum. In particular, the o2-52 polypeptide was detected both

within the nuclei and in the cytoplasm, while the o2T that lacks the bipartite NLS-B and the leucine-

zipper was detected only in the cytoplasm. The o2It allele produces two polypeptides, the o2It-L,

that can be revealed with both the O2 antisera, and the o2It-S, that is revealed only with the O2-NT

antiserum (Fig. 2B and Lazzari et al., 2002). Polypeptides immunoreacting with both the O2

antisera were detected both in the cytoplasm and in the nucleus, with comparable quantitative

distribution. As o2It-S is not revealed by the O2-CT antiserum (Fig. 2A), which also does not reveal

the most acidic bands in o2It extracts (Fig. 2C), the fluorescent signal recovered in the cytoplasm

and in the nucleus with the O2-CT should be a result of the presence of the o2It-L polypeptide in

both the cellular compartments.

Opaque2 Protein Expression Occurs at Early Developmental Stages during Seed Development

and Remains until Maturity

Opaque2 gene expression starts at about 10 DAP (Gallusci et al., 1994). O2 regulation occurs at

translational level, but phosphorylation of the O2 proteins also contributes to regulate O2 activity at

post-translational level. In this work, the presence and the phosphorylation state of the O2 protein in

the time course of seed development were investigated.

Protein extracts from wild-type seeds or endosperms at various developmental stages were

fractionated according to their molecular weight and charge and revealed by O2-NT antiserum (Fig.

4). As shown in Fig. 4A, the O2 protein is present in seeds from about 8-9 DAP to the later stages

of development, and can be detected also in fully dry seeds (data not shown). The relative

abundance of all the O2 isoforms (whose presence is evident at 14 DAP) remains fairly constant

during seed development (Fig. 4B). Actually, with longer exposures faint bands of the O2 isoforms

are visible from 8-10 DAP on (data not shown).



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Opaque2 is Recovered as a Complex in Cross-linked Endosperm Extracts at Early and Late

Stages of Development

The activity of the O2 protein was suggested to depend upon regulatory mechanisms involving

protein-protein interaction. In fact, in vitro experiments demonstrated that O2 can interact with

another O2-like bZIP protein (Pysh et al., 1993) and with a trans-acting factor, PBF, that binds to

the P-box motif that lies 30 bp upstream the O2 target site within the P2 promoter region of the 22

kD zeins (Vicente-Carbajosa et al., 1997; Wang et al., 1998; Wang and Messing, 1998). However,

the occurrence in vivo of these or other heterodimers was never demonstrated.

Here we report evidence of the existence in vivo of one or more protein complex(es) containing the

O2 transcriptional activator. Protein extracts from O2 wild-type and o2R endosperms were treated

with two different cross-linking reagents, DSP and BMDB, fractionated by SDS-PAGE and blotted

onto a nitrocellulose membrane. Immunoreaction with the O2-NT antiserum revealed, only in the

wild-type genotype, one or more diffused band(s) with relative mobilities around 180 and 280 kD

and an additional faint band at about 68 kD, corresponding to the O2 monomer (Fig. 5A).

Since the DSP molecule contains a disulfide bond and is cleaved by thiol-reducing agents, the O2

cross-linking extracts were analyzed by gel electrophoresis either before or after treatment with β-

mercaptoethanol. In these analyses (Fig. 5B), we observed that the faint band corresponding to the

O2 monomer in the non-reduced extract became more intense after reduction treatment, while the

intensity of the O2 complex(es) severely decreased.

To confirm the presence of the O2 protein in the immunodetected complex, the gel lane carrying the

fractionated DSP protein complex was sliced from the gel and then treated with the reducing reagent

DTT. A second fractionation was performed by SDS-PAGE on the treated gel slice. After

immunoblotting of the resulting filter with the O2-NT antiserum, it was evident that the intensity of

the O2-containing complex had decreased, in favour of the monomeric form (Fig. 5C).

Results shown in Fig. 5D indicate that from early to late seed developmental stages most of the O2

protein is embodied in complexes that always release the O2 monomeric form by treatment with the

reducing agent.

opaque2 Mutant Polypeptides Containing the Leucine-zipper Domain Form Complexes

The o2 mutant alleles contain base changes or specific insertions/deletions that cause either amino

acid substitution or the introduction of in-frame stop codons, and consequently the formation of

defective/truncated polypeptides (Lazzari et al., 2002). The o2It-S and the o2T polypeptides lack



12

both the bipartite NLS-B and the leucine-zipper domain, while the o2-52 polypeptide is interrupted

downstream the second leucine of the leucine-zipper motif (Lazzari et al., 2002). The o2-676

polypeptide has an amino acid substitution in the NLS-B/DNA binding domain that severely

reduces its affinity for the O2-box but does not affect its nuclear localization (Aukerman et al., 1991

and Fig. 3). The o2It allele produces two polypeptides: a short one (o2It-S), consequence of a frame

shift due to a ten base pairs deletion within the first half of the NLS-B coding sequence that

introduced a stop codon, and a long one (o2It-L), with an almost identical relative mobility as the

wild-type. Experimental data (immunoreaction with the O2-CT antiserum and partial localization in

the nucleus) suggest that o2It-L contains part of the NLS-B and the downstream canonical amino

acid carboxy-terminal sequence. This can be explained assuming a read through of the frame shift in

the basic region that restores the original translation frame. This interpretation was supported by the

positive immunodetection of the o2It-L and negative immunodetection of o2It-S bands obtained

with another antiserum, O2-CT2, that recognizes the last 18 amino acids (Fig. 1) of the O2 protein

(data not shown).

To investigate whether the o2 mutant polypeptides were able to form complexes, protein extracts

from O2 wild-type and o2 mutant endosperms were treated with the DSP cross-linking reagent,

fractionated by SDS-PAGE and blotted onto a nitrocellulose membrane. The O2 and o2

polypeptides were revealed by the O2-NT antiserum (Fig. 6A). Immunodetection showed the

occurrence of complexes only for O2 or o2 polypeptides containing or supposed to contain a

leucine-zipper domain. In fact, we observed the presence of the most prominent diffuse band of

about 180 kD in the O2wt, o2-676 and o2It genotypes, and its absence in the other o2 extracts. The

faint complex band recovered in the o2It genotype was probably due to the dimerization of the o2It-

L polypeptide that should contain the leucine-zipper domain. From our data, however, it is

impossible to know whether o2It-L contains a complete leucine-zipper or only a partial structure.

Treatment of these extracts with reducing agents (Fig. 6B) eliminated the 180 kD diffuse band and

released more prominent monomeric forms (as in Fig. 5B and D). When we analyzed the diffuse

band of the cross-linking extracts after reducing agent treatment and carried out a second

fractionation, in both the o2-676 and o2It lines the monomeric form was revealed, while the

complexes almost completely disappeared (Fig. 6C and D).

Opaque2 Complex Binds the O2 Target Site in vitro



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In vitro experiments showed that O2 binds the motif 5’-TCCACGTAGA-3’, located in the promoter

of the heavy zein genes at about –300 bp from the ATG start codon (Schmidt et al., 1990, Sturaro

and Viotti, 2001).

Considering this evidence, we investigated whether the O2-complex was also able to bind in in vitro

to the O2 target site. Cross-linking extracts from two O2 wild-types (O2w1, O2w2) and o2R

(Lazzari et al., 2002) were fractionated by SDS-PAGE before and after treatment with β-

mercaptoethanol. Extracts from an E. coli strain expressing the O2w2 protein (O2be) were also

loaded as control (Ciceri et al., 1997). The filter was checked at the end of the South-western

experiments for proper protein transfer by immunoreaction with O2-NT antiserum (Fig. 7C). The

South-western experiments were performed using a double stranded oligonucleotide containing two

O2 binding sites (see Materials and Methods and Ciceri et al., 1997). In Fig. 7A we observed an

intense hybridization signal for the O2be extract corresponding to the O2 monomer. In treated or

untreated endosperm extracts, no signal was observed in the o2R sample, while faint hybridization

signals were revealed in correspondence of the O2 complexes and monomers, with some

hybridizing band in the lower region of the filter.

It is known that phosphorylation of the O2 protein modulates its DNA-binding affinity and that the

hyper-phosphorylated forms acquire a higher binding activity after Potato Acid Phosphatase (PAP)

treatment (Ciceri et al., 1997). Therefore, the probe was stripped from the filter, which was then

treated with PAP and re-hybridized with the same probe (Fig. 7B). The O2 wild-types from

endosperm extracts showed an increase of hybridization after treatment, both at the complex and at

the monomer expected relative mobilities, whereas the signal of the O2be remained almost constant.

From these data we can infer that the O2 ability to bind the O2 target site is far more influenced by

its phosphorylation state than by its monomeric/complex state, and is increased by in vitro

enzymatic dephosphorylation. Similar results on binding activity were obtained by analysis of the

O2 complex at later developmental stages, indicating that O2 functional activity is mainly exerted as

a complex partner (data not shown).

Heavy-type Zein Gene Transcription in O2 wild-type and o2 Mutant Maize Lines

Alcoholic extracts from endosperms of maize seeds contain two major protein fractions: the

sulphur-rich glutelins known as β-, γ- and δ-zeins, and the zein polypeptides sensu stricto, known as

α-zein (Shewry and Tatham, 1990; Shewry et al., 1995). Alpha-zeins can be resolved by SDS-

PAGE and IEF into several polypeptides with line-specific patterns (Viotti et al., 1985; Lund et al.,

1995). To verify a potential transcriptional activity of o2 defective proteins that however form



14

complexes, we analyzed the SDS-PAGE pattern of zein extracts from O2 wild-type and o2 mutant

lines in the A69Y genetic background (Fig 8A). In the wild-type line, α-zeins were resolved into

five discrete bands with different relative abundance: two of heavy type (H1 and H2) and three of

light type (L1, L2, L3). The pattern of each o2 mutant shows a characteristic spectrum of

combinations reflecting the presence/absence and the abundance of the five bands. In particular, the

L1 band was more intense in the o2T than in the O2 wild-type, and the H1 and H2 bands were not

detectable in the o2T, o2-52 and o2R mutants. In the o2It mutant the H1 and H2 bands were reduced

with respect to the O2 wild-type. In mutant lines that do not show H1 and H2 bands (o2T, o2-52 and

o2R) the γ-zein band showed a higher intensity that in the O2 wild-type. Finally, in the o2-676 we

observed the presence of both H1 and H2 bands with a noteworthy increase in their relative

abundance with respect to those of the wild-type line, as already reported for the o2-676 allele in

different GBs (Aukerman et al., 1991). This feature was evident also in the cross between o2-676

and o2It lines.

To extend a possible functional role of defective o2 proteins able to form complexes we analyzed

the heavy-type zein gene transcription in different GBs carrying the o2It allele. The original o2It

allele, recovered in the Bianchi o2It line, was introgressed in four GBs: W64A, A69Y, NYR, W22,

and 3316 (Bernard et al., 1994; Ciceri et al., 2000). In any case, H-zeins were recovered in mature

seeds, mainly H1 (Fig. 8B), indicating that in each of the GBs a subset of heavy-type zein genes is

transcribed, as previously evidenced in RNA gel blot analysis (see Dolfini et al., 1992; Ciceri et al.,

2000).

The Mutant Complex-forming o2 Polypeptides Bind the O2 Target Site in in vitro Functional

Analysis

Previous experimental data indicated that the two o2 defective polypeptides, o2-676 and o2It-L,

were unable to bind the O2 motif 5’-TCCACGTAGA-3’ (Aukerman et al., 1991; Ciceri et al.,

1997). The observed presence of H-zeins in the different BGs carrying the o2-676 (Fig. 8A and

Aukerman et al., 1991) and o2ItL alleles strongly suggests on the contrary that o2-676 and o2It-L -

that are localized internal to the nucleus and were proven to be able to form complexes - might bind,

by themselves or in concert with other protein factors, target sequences within the promoters of

some of the H-zein genes. To validate this interpretation, we performed a South-western

experiment. A protein gel blot of O2 wild-type, o2-676 and o2It extracts, resolved by IPG-IEF, was

probed with the double stranded oligonucleotide containing the O2 target site (Fig. 9A). The

membrane was then treated with PAP and reprobed with the same oligonucleotide (Fig. 9B and C).



15

In Fig. 9D, the same membrane was tested for protein loads by immunodetection with O2-NT

antiserum. As previously shown (Ciceri et al., 1997), dephosphorylation increases the radioactive

signals of the O2 wild-type most basic isoforms and lights up those of the most acidic ones. Also the

o2-676 and o2It-L isoforms acquire the ability to bind the O2-target sequence after PAP treatment,

even if with a reduced efficiency as compared to the wild type. This can explain the presence of H-

type zeins in the BGs carrying the o2-676 and o2ItL alleles.

DISCUSSION

Involvement of O2 Modifications and Domains in its Cellular Compartmentalization

The genotypes analyzed in these study derive from maize lines in which the introgression of the

various o2 alleles was obtained with at least six backcrosses, as reported in Ciceri et al., 2000. Most

of the results were obtained from the A69Y GB and are highly consistent in all the experiments.

Immunofluorescence data suggest that the localization of O2 and o2 polypeptides is correlated with

the primary protein structure. It is known that the O2 protein contains two nuclear localization

signals (NLSs) (Varagona et. al, 1992). In histochemical localization of O2/GUS fusion protein in

transformed tobacco and onion tissues, it was observed that the bipartite NLS-B is more efficient

than NLS-A in targeting O2 to the nucleus (Varagona et. al, 1992). Our data show that in maize

endosperm the o2T mutant polypeptide, which contains only the NLS-A, is retained in the

cytoplasm, while the O2 wild-type localizes for more than 90% into the nucleus. On the other hand,

even though the o2-52 polypeptide contains the NLS-B, its fluorescent signal was recovered both in

the nuclei and in the cytoplasm, showing that the lack of the polypeptide sequence downstream the

second leucine of the leucine-zipper domain influences to a certain extent the protein localization.

The ten base pairs deletion in the coding part of the basic domain of the o2It also affects the

localization of its gene products. In fact, the o2It-L polypeptide is detected both into the nuclei and

in the cytoplasm, with comparable quantitative distribution, while o2It-S remains almost completely

confined to the cytoplasm. Furthermore, we observed that the amino acid substitution in the NLS-

B/DNA binding domain of the o2-676 polypeptide, that severely reduces its affinity for the O2-box

(Aukerman et. al, 1991), does not affect its nuclear localization, being the o2-676 almost entirely

revealed into the nucleus. These data suggest that in the maize endosperm the two functions of the

bipartite basic domain (nuclear localization and DNA binding) are probably separated, as already

verified in onion tissues with GUS fusion proteins (Varagona et. al, 1994), and that the R249K

substitution partially affects the DNA binding affinity in in vitro experiments. In summary, the



16

presence of all the three NLS-A, NLS-B and leucine-zipper sub-domains is relevant for a proper

nuclear localization and any alteration in their sequence or relative position (Varagona et al., 1992)

may hamper O2 localization.

IEF data (Fig. 2B) showed that all the o2 polypeptides are present in the endosperm cells in several

phosphorylated forms. Since o2T shows at least three phosphorylated forms and is unable to enter

the nucleus (Fig. 2C and Fig. 3), we deduced that these phosphorylations are due to kinase activities

that are present in the cytoplasm. This evidence does not exclude the presence of additional kinase

activities able to phosphorylate the O2 protein in the nuclei. In fact, most of the O2 polypeptides are

nuclear, and the various isoforms are in almost equal relative amount. A good candidate for O2

phosphorylation seems to be the CKII serine-threonine kinase that is localized both in the cytoplasm

and in the nuclei of maize endosperm cells (Zhang et al., 1993; Grasser et al., 1989). By comparison

with the Prosite data base (Bairoch, 1991), putative target sites for protein kinases were found in the

deduced amino acid sequences of the O2 proteins (Lazzari et al., 2002). Eight locations for CKII

were found, four of which occur close to or within the NLS-B (Unger et al., 1993; Varagona et al.,

1991, 1992). It is not clear whether phosphorylation has an effect on the translocation of the O2

protein to the nucleus, but its involvement in the modulation of O2 activity is demonstrated (Ciceri

et al., 1997). We observed that all the phosphorylated isoforms of the O2 protein are present from

early to late stages of kernel development (Fig. 4B). Consequently, modulation of the O2 activity by

phosphorylation is exerted all along the endosperm development.

O2 and o2-defective Proteins Form Complex with Functional Activity

In the endosperm cell the O2 polypeptide mainly localizes into the nucleus (Fig. 3), where it plays

its essential role as transcriptional activator. Indeed, we were able to recover the O2 protein in a

nuclear complex. In preliminary experiments on nuclei-enriched fractions treated with DSP, we

found the O2-complex tightly bound to the chromatin, forming a super-complex that was

unfractionable by SDS-PAGE even after treatment with DNase I (data not shown). However, this

chromatinic complex releases the O2 polypeptide as monomeric form after reducing agent treatment

(data not shown). Furthermore, the complex was present from early to late stages of maize kernel

development, indicating that O2 carries out its function in constant partnership with other factors.

We do not know which is/are the O2 partner/s in the complex, but we can suppose that other

leucine-zipper protein/s is/are involved in the dimerization, as suggested from in vitro and in vivo

evidence (Pysh et al., 1993; Vicente-Carbajosa et al., 1997; Wang et al., 1998; Wang and Messing,

1998). In fact, only the o2 mutant polypeptides containing the entire leucine-zipper motif are able to



17

form the complex (Fig. 6), providing the first evidence that this motif is really involved in vivo in

the protein-protein interaction. In our experiments, two different cross-linking agents were used

with analogous results, further demonstrating the complex specificity. Moreover, no complex is

recovered in the o2R line or in those lines producing o2 polypeptides defective in the leucine-

zipper, supporting the assumption that the presence of a functional leucine-zipper motif is crucial

for the complex formation.

It is known that the O2 transcriptional activator is able to interact with other bZIP proteins; for

instance, O2 can form a heterodimer with Mybleu, a polypeptide encoded by the unspliced myb7

mRNA and composed by a Myb domain followed by the leucine-zipper motif. This interaction was

demonstrated in transiently transformed tobacco protoplasts, where Mybleu enhances the

transcription of GUS fused to the promoter of the b-32 gene, an endosperm O2-regulated gene

(Locatelli et al., 2001). Furthermore, O2 is able to recruit the maize co-activators GCN5 and ADA2,

to modulate the transactivation of the GUS gene fused to the b-32 promoter in transiently

transformed tobacco protoplasts (Bhat et al., 2004), showing that O2 is able to interact with proteins

other than bZIP type in heterologous systems. Our data provide the first evidence of the presence of

a complex involving the O2 protein in the tissue where O2 is typically expressed, the maize

endosperm.

South-western results supply other important information: the O2 complex is able to bind the zein

target sequence and this binding activity significantly increases after in vitro enzymatic

dephosphorylation (Fig. 7), indicating that the complex-bound O2 isoforms are to a great extent

hyper-phosphorylated. This suggests that phosphorylation controls O2 activity also as a complex

component.

Evidence of a functional activity of o2-676 and o2It-L defective proteins derives from the analysis

of the expression of a group of genes, among the several O2 targeted genes: the H-zeins. This

evidence is further supported by the recovery of complexes in o2-676 and o2It lines with

comparable mobility to that of the O2 line, and by the fact that this o2 defective polypeptides bind

to a certain extent the O2 target site. Heavy-type zein genes were clearly proven to be O2 regulated

by the analysis of o2T and o2R genotypes (Viotti et al. 1982; Schimdt et al., 1987). In general, the

upstream sequences of zein genes contain two promoters, the distal P1 and the proximal P2,

separated by about one thousand nucleotides (Langridge and Feix 1983; Quattrocchio et al., 1990

Giovinazzo et al., 1992). Experimental evidence indicated that in both promoter regions functional

binding sites for O2, O2 co-activators as PBF, or other transcriptional activators are present (Maier

et al., 1987; Vincente-Carbajosa et al., 1997, Schmidt et al., 1997). The P1 and P2 are responsible

for the production of long or short zein transcripts, being the P2 the strongest one (Langridge and



18

Feix 1983). On the other hand, O2 binds in vitro any type of ACGT box in perfect or slightly

imperfect surrounding palindromes showing for the G-type the higher affinity (Izawa et al., 1993).

However, an A-type with an imperfect palindrome is present in the zein gene promoter. Analyses of

genomic clusters of heavy-type zein sequences reported the occurrence of genes coding for mature-

heavy (245 aa) and -light (216 aa) class zein polypeptides, which are transcribed (Song and

Messing, 2003) and contain the canonical PBF and O2 boxes in the P2 region or even an additional

PBF box in the P1 region. The overall picture suggests that zein genes contain a variety of similar

but not identical regulatory motifs, and that O2 has a promiscuous behaviour towards target sites

and also towards eventual partners. Partner-specific O2 and even o2 defective complexes could also

exhibit different specificity towards the various target sites in vivo.

A flow chart of O2 transcription, post-translational modification, nuclear import and in nucleus

activity, as also deduced from the o2 mutants behavior, is reported in Fig. 10. This overview on O2

behavior broadens our knowledge about O2 activity and specificity towards its target genes, and

might be helpful in arranging its utilization for the manipulation of the QPM trait (Gibbon and

Larkins, 2005) and the achievement of cisgenic plants with proper regulators, modified regulatory

elements and even H-zein genes modified in their coding capacity for essential amino acids.

MATERIALS AND METHODS

Genetic Material and Protein Extract Preparation

Maize (Zea mays spp. mays) plants were grown in the field during the summer or in the greenhouse

during the February to May period of the 2000 and 2001 years. Genotypes carrying either the wild-

type O2 alleles or the various o2 mutant alleles were described in Bernard et al. (1994), in Ciceri et

al. (2000) and in Lazzari et al. (2002). Plants of the various genotypes were self-pollinated and

seeds were harvested at noon at different days after pollination (DAP), immediately frozen in liquid

nitrogen and stored at –80°C until use. Total protein extracts, zein extracts and O2-enhriched

protein extracts either from whole seed or from dissected endosperms were prepared as previously

described in Viotti et al. (1985) and Ciceri et al. (1997).



19

Gel Electrophoresis and Immunoblotting

Opaque2 protein extracts were fractionated by SDS-PAGE or by isoelectric focusing (IEF) in

immobilized pH gradients (IPG-IEF) and immunoblotting reactions with O2 antisera (O2-NT, O2-

CT) were performed as previously described (Ciceri et al., 1997, Lazzari et al. 2002). Opaque2

polyclonal antibodies specific for the amino-terminal part (O2-NT) were obtained by immunizing

rabbits with a synthetic oligopeptide containing the first 24 amino acids of the N-terminal region

deduced sequence of the O2 wild-type allele reported in Schmidt et al. (1990), and named O2w2 in

Lazzari et al. (2002). The carboxy-terminal O2 antiserum (O2-CT) was from the R.J. Schmidt

laboratory (UCSD, USA). In some experiment an affinity purified carboxy-terminal antibody, O2-

CT2, was utilized. The serum was obtained by immunizing rabbits with a synthetic oligopeptide of

19 amino acids containing the last 18 amino acids, underlined (C-PQDYELLGPNGAIHMDMY) of

the O2 protein, and the antibody was purified by chromatography on CNBr-Sepharose coupled with

the 19 amino acid oligopeptide.

After electrophoresis, proteins were transferred to nitrocellulose membranes and O2 polypeptides

were revealed by immuno-reaction as previously described (Ciceri et al., 1997; Lazzari et al., 2002).

Zein extracts were analyzed by fractionation on SDS-PAGE as reported in Viotti et al. (1985) and

Lund et al. (1995). Gels were stained with Comassie Brilliant Blue R250.

Immunofluorescence

Opaque2 wild-type and o2 mutant maize immature seeds were harvested at 17-20 DAP and fixed in

10 mM sodium phosphate buffer, 4% (w/v) paraformaldehyde, pH 7.0 (fix solution). Vacuum

infiltration was performed for 6 hours on ice, the solution was then renewed and the fixing reaction

was continued overnight at 4°C. The fix solution was replaced with cold 0,85% (w/v) NaCl and,

after 30 min at 4°C, with 70% (v/v) ethanol. The seeds were stored in 70% (v/v) ethanol at 4°C until

use. For inclusion, the seeds were dehydrated through an alcohol (ethanol/ter-butylic alcohol) series,

included in paraffin blocks and stored at 4°C until use. For the preparation of endosperm sections,

seeds included in paraffin blocks were sliced with a microtome to approximately 8 µm in thickness.

Sections were placed on glass slides and incubated overnight at 37°C to adhere. Each slide

contained two sections of O2 and two of each o2 genotype. To remove paraffin, the glass slides with

sections were dipped into 100% xylol for 8 min at room temperature. The sections were rehydrated

through an ethanol decreasing series, followed by treatments in 0,85% (w/v) NaCl, then in TBS

buffer (20 mM Tris-HCl, 0.9% (w/v) NaCl, pH 7.2) for 5 min each, and finally in TBS buffer with

20 mM glycine for 30 minutes. The samples were permeabilized three times in TSW buffer (10 mM

Tris-HCl, 0.9% (w/v) NaCl, 0.25% (w/v) gelatine, 0.02% (w/v) SDS, 0.2% (v/v) Triton X-100, pH



20

7.4) for 10 min each, then preincubated in TSW buffer in presence of 4% (v/v) goat serum (SIGMA

G9023) for 40 min at room temperature. The appropriate primary antibodies (O2-NT or O2-CT)

were diluted in TSW buffer with 0,05% (v/v) instead of 0,2% (v/v) Triton X-100, in presence of 1%

(v/v) goat serum, applied on sections, and the incubation was protracted at room temperature for at

least 1 h, into a moist box. After primary-antibody incubation the sections were washed three times

in TSW buffer for 10 min/each, at room temperature. The sections were then treated in the dark at

room temperature for 90 min with the secondary antibody goat anti-rabbit Alexa Fluor 488, diluted

in TSW buffer with 0,05% (v/v) instead of 0,1% (v/v) Triton X-100, followed by the addition of

1µg/mL of 4’,6’-diamidino-2-phenylindole (DAPI). The incubation was extended for 10 min, then

over-glass were applied to the sections. Sections were analyzed using a Leyca confocal microscope

and the images were acquired by Leyca Confocal Software.

Probing Protein Gel Blots with DNA, South-western Analysis

The double strand DNA probe was prepared as already described (Ciceri et al., 1997) by annealing

the oligonucleotides (5'-GGCCGCTCCACGTAGATAAGCTTCATTCCACGTAGAT-3' and 5'-

CTAGATCTACGTGGAATGAAGCTTATCTACGTGGAGC-3') containing two O2 binding sites

(underlined). Fill-in labeling with [32P-dCTP] was performed by Klenow. According to the

procedure outlined in Ciceri et al. 1997, IEF-filters or O2-complex filters were hybridized with a

radiolabeled O2-probe, washed as described and exposed to XR films. After appropriate exposure

time, filters were treated with Potato Acid Phosphatase (PAP) and then re-hybridized with the O2-

probe (Ciceri et al., 1997). Finally, O2 and o2 were revealed by immunodetection with O2-NT

antiserum.

O2-complex Formation and Analysis

Dissected endosperms were homogenized in presence of 0.1 M sodium phosphate buffer, pH 8.00,

with or without 3 mM thiol-cleavable Dithiobis[succinimidylpropionate] (DSP) cross-linker

(SIGMA D3669), or in presence of 10 mM HEPES-NaOH, 100 mM NaCl, 5 mM EDTA, pH 6.5

with or without 2.6 mM 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB) cross-linker (PIERCE

22332). The cross-linking treatments were performed at room temperature for 30 min. The active

group of the DSP was blocked with Tris-HCl, pH 7.5 to a final concentration of 35 mM, according

to manufacturer procedure. O2-complexes were analyzed by SDS-PAGE on 10% polyacrylamide

with or without treatment with thiol-reducing agents. For two-dimensional (2D) analysis of DSP

extracts, the gel strips were incubated in 120 mM Tris-HCl, 3% (w/v) SDS, 20% (w/v) sucrose, 60

mM dithiothreitol, 0.02% (v/v) bromophenol blue, pH 6.8, at 37°C for 40 min. Gel strips were



21

applied to an SDS-containing gel 10% in polyacrylamide, and electrophoresed at room temperature.

In both cases proteins were blotted on nitrocellulose filters and O2 or o2 was revealed by

immunodetection with O2-NT antiserum.

ACKNOWLEDGMENTS

We thank R.J. Schmidt for providing the O2-CT antiserum. We are grateful to Dr. E. Quattrini for

technical help in growing maize plants. This work was partially supported by a grant from FIRB-

MIUR, to A.V.

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Gallusci P, Salamini F, Thompson RD (1994) Differences in cell type-specific expression of the gene Opaque 2 in maize and transgenic tobacco. Mol. Gen. Genet. 244: 391-400 Gibbon BC, Larkins BA (2005) Molecular genetic approaches to developing quality protein maize. TRENDS Genet. 21: 227-233 Giovinazzo G, Manzocchi LA, Bianchi MW, Coraggio I, Viotti A (1992). Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts. Plant Mol. Biol. 19: 257-263. Grasser KD, Maier UG, Feix G (1989) A nuclear casein type II kinase from maize endosperm phosphorylating HMG proteins. Biochem. Biophys. Res. Commun. 162: 456-463 Henry AM, Manicacci D, Falque M, Damerval C (2005) Molecular evolution of the Opaque-2 gene in Zea mays L. J. Mol. Evol. 61: 551-558 Izawa T, Foster R, Chua NH (1993) Plant bZIP protein DNA binding specificity. J Mol Biol 230: 1131-1144 Kemper EL, Cord Neto G, Papes F, Martinez Moraes KC, Leite A, Arruda P (1999) The role of Opaque2 in the control of lysine-degrading activities in developing maize endosperm. Plant Cell 11: 1981-1993 Langridge P, Feix G (1983) A zein gene of maize is transcribed from two widely separated promoter regions. Cell 34: 1015-1022 Lazzari B, Cosentino C, Viotti A (2002) Gene products and structure analysis of wild-type and mutant alleles at the Opaque-2 locus of Zea mays. Maydica 47: 253-265 Locatelli F, Manzocchi LA, Viotti A, Genga A (2001) The nitrogen-induced recovery of α-zein gene expression in in vitro cultured opaque2 maize endosperms depends on the genetic background. Physiol. Plant. 112: 414-420 Lohmer S, Maddaloni M, Motto M, Di Fonzo N, Hartings H, Salamini F, Thompson RD (1991) The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J. 10: 617-624 Lohmer S, Maddaloni M, Motto M, Salamini F, Thompson RD (1993) Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence. Plant Cell 5: 65-73 Lund G, Ciceri P, Viotti A (1995) Maternal-specific demethylation and expression of specific alleles of zein genes in the endosperm of Zea mays L. Plant J. 8: 571-581 Maddaloni M, Donini G, Balconi C, Rizzi E, Gallusci P, Forlani F, Lohmer S, Thompson R, Salamini F, Motto M (1996) The transcriptional activator Opaque-2 controls the expression of a cytosolic form of pyruvate orthophosphate dikinase-1 in maize endosperms. Mol. Gen. Genet. 250: 647-654



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Maier UG, Brown JWS, Toloczyki C, Feix G (1987) Binding of a nuclear factor to a consensus sequence in the 5’ flanking region of zein genes from maize. EMBO J. 6: 17-22 Mauri I, Maddaloni M, Lohmer S, Motto M, Salamini F, Thompson R, Martegani E (1993) Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast. Mol. Gen. Genet. 241: 319-326 Pysh LD, Aukerman MJ, Schmidt RJ (1993) OHP1: a maize basic domain/leucine zipper protein that interacts with Opaque2. Plant Cell 5: 227-236 Quattrocchio F, Tolk MA, Coraggio I, Mol JNM, Viotti A, Koes RE (1990) The maize zein gene zE19 contains two distinct promoters wich are independently activated in endosperm and anthers of transgenic Petunia plants. Plant Mol. Biol. 15: 81-93 Righetti PG (1990) lmmobilized pH gradients: Theory and Methodology. (Amsterdam: Elsevier) Rossi V, Hartings H, Thompson RD, Motto M (2001) Genetic and molecular approaches for upgrading starci and protein fractions in maize kernels. Maydica 46: 147-158 Schmidt RJ, Burr FA, Burr B (1987) Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. Science 238: 960-963 Rubenstein I, Geraghty DE (1986) Genetic organization of zeins. In: Pomeranz Y, ed, In Advances in Cereal Science and Technology Vol VIII. American Association of Cereal Chemists Inc. Press, St. Paul (MN), pp 297-315 Schmidt RJ, Burr FA, Aukerman MJ, Burr B (1990) Maize regulatory gene opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA. Proc. Natl. Acad. Sci. USA 87: 46-50 Schmidt RJ, Ketudat M, Aukerman MJ, Hoschek G (1992) Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes. Plant Cell 4: 689-700 Schmidt RJ (1993). Opaque-2 and zein gene expression. In Control of Plant Gene Expression, D.P.S. Verma, ed (Boca Raton, FL: CRC Press), pp. 337-355 Soave C, Salamini F (1984) Organization and regulation of zein genes in maize endosperm. Phil. Trans. R Soc., Lond. B. 304: 341-347 Sturaro M, Viotti A (2001) Methylation of the Opaque2 box in zein genes is parent-dependent and affects O2 DNA binding activity in vitro. Plant Mol. Biol. 46: 549-560 Shewry PR, Tatham AS (1990) The prolamin storage proteins of cereal seeds: structure and evolution. Biochem. J. 267: 1-12 Shewry PR, Napier JA, Tatham AS (1995) Seed Storage Proteins: Structures and Biosynthesis. Plant Cell 7: 945-956 Song R, Messing J (2003) Gene expression of a gene family in maize based on noncollinear haplotypes. Proc. Natl. Acad. Sci. USA 100: 9055-9060



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Unger E, Parsons RL, Schmidt RJ, Bowen B, Roth BA (1993) Dominant negative mutants of Opaque2 suppress transactivation of a 22-kD zein promoter by Opaque2 in maize endosperm cells. Plant Cell 5: 831-841 Varagona MJ, Schmidt RJ, Raikhel NV (1991) Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants. Plant Cell 3: 105-113 Varagona MJ, Schmidt RJ, Raikhel NV (1992) Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque2. Plant Cell 4, 1213-1227. Varagona MJ, Raikhel NV (1994) The basic domain in the bZIP regulatory protein Opaque2 serves two independent functions: DNA binding and nuclear localization. Plant J. 5: 207-214 Vicente-Carbajosa J, Moose SP, Parsons RL, Schmidt RJ (1997) A maize zinc-finger protein binds the prolamine box in zein genes promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. Proc. Natl. Acad. Sci. USA 94: 7685-7690 Viotti A, Abildsten D, Pogna N, Sala E, Pirrotta V (1982) Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localization. EMBO J. 1: 53-58 Viotti A, Cairo G, Vitale A, Sala E (1985) Each zein gene class can produce polypeptides of different sizes. EMBO J. 4: 1103-1110 Wang Z, Ueda T, Messing J (1998) Characterization of the maize prolamin box-binding factor-1 (PBF-1) and its role in the developmental regulation of the zein multigene family. Gene 223: 321-332 Wang Z, Messing J (1998) Modulation of gene expression by DNA-protein and protein-protein interactions in the promoter region of the zein multigene family. Gene 223: 333-345 Yunes JA, Neto GC, Silva MD, Leite A, Ottoboni L, Arruda P (1994) The transcriptional activator Opaque2 recognizes two different target sequences in the 22-kD-like α-prolamin genes. Plant Cell 6: 237-249 Yunes JA, Vettore AL, Silva MD, Leite A, Arruda P (1998) Cooperative DNA binding and sequence discrimination by the Opaque-2 bZIP factor. Plant Cell 10: 1941-1955 Zhang S, Jin CD, Roux SJ (1993). Casein kinase II-type protein kinase from pea cytoplasm and its inactivation by alkaline phosphatase in vitro. Plant Physiol. 103: 955-962



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FIGURE LEGENDS Figure 1. Schematic representation of an O2 wild-type (O2wt) polypeptide and location of each

mutation.

For each mutation, an arrow points to the position of the truncated polypeptides. Open head arrow

indicates the amino acidic substitution (R249K) of the o2-676 polypeptide. Black bar indicates the

first 24 amino acids recognized by the O2-NT antiserum, open bar the last 170 amino acids

recognized by the O2-CT and gray bar the last 18 amino acids recognized by the O2-CT2. Drawing

not to scale.

Figure 2. Immunoblot analysis of O2 wild-type and o2 mutant polypeptides.

O2 and o2 polypeptides from different genetic backgrounds are indicated at the top. Genetic

backgrounds are abbreviated as: Y = A69Y; A = W64A; B = Bianchi o2 (Bernard et al. 1994).

A, About 20 µg of O2w3 and 50 µg of o2 mutant enriched protein extracts from immature

endosperms of the various genotypes were fractionated on a 12% polyacrylamide gel. Proteins were

blotted onto a nitrocellulose filter. Immunodetection was performed using O2-NT and O2-CT

polyclonal antibodies. Developing time of the chromogenic substrate was about five minutes. The

relative molecular mass of the different polypeptides is given on the left.

B and C, Opaque-2 wild-type and o2 mutant enriched protein extracts were run in an IPG-IEF gel.

On the left the pH of reference isoelectric points are marked. Developing time of the chromogenic

substrate was about fifteen minutes. B, About 50 µg of O2w3 or 100 µg of o2 enriched protein

extracts were resolved on a linear 4.0 to 5.0 pH range IPG slab gel, electroblotted onto a

nitrocellulose filter and immunodetected as described in (A). C, About 50 µg of O2w1 and O2w3,

or 100 µg of o2 enriched protein extracts were resolved on a linear 3.5 to 5.0 pH range IPG slab gel,

electroblotted onto a nitrocellulose filter and immunodetected with the O2-CT antiserum (left

panel); the same filter was stripped and successively re-probed with O2-NT antiserum (right panel).

Figure 3. Immunolocalization of O2 wild-type (O2wt) and o2 mutant polypeptides.

Sections, 8 µm thick, of endosperms from immature seeds at 17-20 DAP were analyzed for the

localization of O2 and o2 polypeptides within the endosperm cells. Green fluorescence derives from

the incubation with the primary antibody (O2-NT or O2-CT) followed by incubation with the

secondary antibody, Alexa Fluor 488. Nuclei were stained with DAPI and blue fluorescence

revealed by UV laser. The third panel of the upper block shows the merge between bright-field

images and the green and blue fluorescence images. O2wt and o2 mutant alleles are indicated



26

between the two blocks of panels. Red arrows indicate the outer side of seed. P = pericarp; A =

aleurone layer; SA = subaleurone layers; SE = starch endosperm region.

Figure 4. Opaque-2 occurrence and analysis from seed at various developmental stages.

A, About 20 µg of proteins from total seed or endosperm tissues at various developmental stages

were fractionated by SDS-PAGE and blotted as in Fig. 2A. Opaque-2 was revealed by O2-NT

antiserum. On the top, the developmental stages are reported as DAP. At 44 DAP about 40 µg of

total protein were loaded. Arrow indicates O2 migration. B, About 100 µg of protein extract from

total seed or endosperm were fractionated on an IPG-IEF gel and analyzed as in Fig.1B. The

developmental stages in DAP (Days After Pollination) are reported at the top.

Figure 5. Immunoblot detection of O2w3 in protein complex extracts.

Homogenates of endosperms at 20 DAP in sodium phosphate buffer were treated with cross-linking

reagents and fractionated on 10% SDS-PAGE. Proteins were blotted onto a nitrocellulose filter and

immunodetection of O2 was performed using O2-NT specific antibody. Opaque-2 alleles are

reported at the top. A, Analysis of the O2w3 polypeptide in protein complex obtained with different

cross-linking reagents (DSP or BMDB) from a wild-type line. The o2R line was used as negative

control. B, Samples of the DSP protein complex were treated with (+) or without (-) β-

mercaptoethanol and then fractionated by SDS-PAGE. C, A gel lane of the O2w3 DSP protein

complex was sliced from the SDS-PAGE, treated with DTT as reducing agent and then applied to a

10% SDS-PAGE for a second fractionation. D, Timing analysis of O2 complex: DSP cross-linking

protein extracts from O2w3 at 16, 20 and 33 DAP and from o2R at 17 DAP were analyzed by 10%

SDS-PAGE fractionation before (-) or after (+) treatment with β-mercaptoethanol as reducing agent.

Figure 6. Immunoblot detection of O2 and o2 polypeptides in protein complex extracts.

The experiments were performed as described in Fig. 5. A and B, Fractionation of DSP-protein

complex from two O2 wild-type (O2w1 and O2w2) and o2 mutant lines, respectively, before and

after treatment with β-mercaptoethanol as reducing agent. C and D, Gel lanes from the first

dimension of o2-676 and of o2It protein complexes were sliced from the SDS-PAGE, treated with

DTT as reducing agent and then applied to a 10% SDS-PAGE for the second fractionation.

Figure 7. DNA binding analysis of O2 complex.

A, Approximately 30 µg of protein cross-linking extracts from O2w1, O2w2 and o2R lines were

loaded before (-) and after (+) treatment with β-mercaptoethanol, fractionated by SDS-PAGE and



27

blotted onto a nitrocellulose membrane. On the same gel about 5 and 10 µg of the bacterial extract

from E.coli expressing the O2w2 protein (O2be) were loaded.

The protein blot was probed with the oligonucleotide containing the O2 target sequence, as

described in material and methods. B, The filter was washed in high-salt buffer to remove the probe,

treated with PAP and then reprobed as in (A). C, Immunodetection of O2 protein. After removal of

the probe, the filter was incubated with O2-NT antibody.

Figure 8. SDS-PAGE analysis of zein polypeptides from inbred and hybrid lines.

A, The profiles of inbred lines carrying the various o2 alleles in the A69Y GB are compared to the

A69YO2 (O2w3) and to the o2-676/o2It cross. B, The zein polypeptide profiles of different GBs

carrying either the corresponding O2 wild-type, as indicated, or the o2It allele are compared to the

Bianchi o2It, the line in which this allele was recovered (Bernard et al., 1994). Approximately 15 µg

of zein protein extracts from mature seeds were loaded in each lane. Genotypes are reported at the

top. The relative mobility of the H1 and H2 heavy chains and the L1, L2 and L3 light chains are

indicated at the left. Bracket indicates the mobility of the γ-zein polypeptides.

Figure 9. DNA binding analysis of O2 isoforms after IPG-IEF fractionation.

A, Protein gel blot analysis of O2 and o2 polypeptides resolved by IPG-IEF and probed with the

oligonucleotide containing the O2 target sequence. Approximately 50 µg of O2w1 and 100 µg of o2

enriched protein extracts were focalized on a 4.0 to 5.0 pH IPG slab gel, blotted onto a

nitrocellulose filter, and probed as described in material and methods. The isoelectric point of the

most cathodal isoform is indicated at left. B, The filter was washed in high-salt buffer to remove the

probe, treated with PAP and then reprobed as in (A). C, Represents a longer exposure than (B). D,

Immunodetection of O2 protein. After removal of the probe, the filter was incubated with O2-NT

antibody.

Figure 10. Schematic representation of an Opaque-2 wild-type (O2wt) and mutant gene

transcription products and proteins. The o2T transcript is depicted with a thinner wave line

representing the first intron and is either partially retained in the nucleus or after proper splicing

transferred to the cytoplasm where it is translated giving rise to the o2T (Dolfini et al., 1992;

Lazzari et al., 2002). Data derive from this report and from previous publications (Dolfini et al.

1992; Ciceri et al., 1997, 1999).



opaque2 and o2-defective proteins in functional complexes

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