Download tissue-specificity of storage protein genes has evolved

Maydica 54 (2009): 409-415
TISSUE-SPECIFICITY OF STORAGE PROTEIN GENES HAS EVOLVED
WITH YOUNGER GENE COPIES
Y. Wu, J. Messing*
Waksman Institute of Microbiology, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA
Received September 23, 2009
ABSTRACT - Seed storage proteins are critical for nitrogen storage in seeds and the nutrition of livestock and
humans. In maize synthesis begin 10-12 days after pollination (DAP) and peaks about 18-24 DAP. The major fraction of storage proteins in maize is classified as prolamins, which are rich in the amino acids proline and glutamine. Although storage proteins can accumulate in embryo tissue, the maize prolamins, also called zeins, accumulate in the triploid endosperm. Localization in either
embryo or endosperm, however, requires the onset of the
synthesis of tissue-specific trans-acting factors in a temporal fashion. It was therefore unexpected that one of the
zein genes is strongly expressed in tissue culture. When a
chimeric gene controlling expression of the green fluorescence protein (GFP) was introduced into immature cultured embryos by Agrobacterium-mediated transformation, the GFP gave rise to green callus, indicating that the
27-kDa promoter of the gamma zein gene was active.
Subsequent analysis of the endogenous 27-kDa gammazein gene showed that 27-kDa gamma-zein protein was
made as well, indicating that expression of this gene was
not due to the transformation procedure itself. This could
be further demonstrated by the fact that the general transacting factor of prolamin genes, the prolamin-box-binding
factor (PBF), was also expressed in the green callus.
However, the O2 transcription factor that is required for
the expression of a subset of alpha zein genes was not
expressed, suggesting that tissue-specificity evolved into
the combinatorial action of different trans-acting factors
along with younger target genes arising from gene duplications.
KEY WORDS: Storage proteins; Gamma zein; Maize; Promoter specificity; Developmental gene expression.
Dedicated to Ronald L. Phillips upon his retirement from the
faculty of the University of Minnesota and in recognition of his
pioneering contribution to maize tissue culture.
* For correspondence (fax: +1 732-445-0072; e.mail: messing@
waksman.rutgers.edu).
INTRODUCTION
An interesting question in cell differentiation and
morphogenesis is the nature of the specificity of
transcription as the first step in gene expression. In
general, a promoter has to switch to an active state
of transcription so that upon the synthesis of the
necessary trans-acting factors the transcription machinery can act on the promoter of a gene. Therefore, gene expression is also a measure of the activity of trans-acting factors as well. In this study, we
found unexpectedly activity of a storage protein
gene in cultured cells as opposed to endosperm
cells. Storage proteins are needed for the plant as a
mechanism to channel photosynthates from leaves
to seeds during senescence and convert them into a
format that can be stably stored until germination
(SABELLI and LARKINS, 2009). During germination,
when there is no photosynthesis, the plant can
draw from the stored nutrients to grow and develop, closing the life cycle. Failure to convert amino
acids into seed storage proteins would disrupt the
plant life cycle and deprive us from the most important source of nutrition. In Zea mays, the majority of
storage proteins accumulate in the triploid endosperm and not in the embryo. Therefore, expression of the corresponding genes must be tightly
controlled in time and place (HUNTER et al., 2002).
Maize storage proteins seem to have evolved
from water-soluble (globulins) to water-insoluble
ones (prolamins) during the evolution of the grasses
(XU and MESSING, 2009). The prolamins are rich in
proline and glutamine and can be divided into 4
groups, alpha, beta, gamma, and delta prolamins
(ESEN, 1987). Interestingly, within the grass family
the gamma prolamin genes seem to be the oldest
and most diverged and present in all species analyzed so far, while the alpha and delta zein genes
are the youngest ones; they are also specific for the
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Panicoideae, the subfamily of the Poaceae that includes maize, sorghum, sugarcane, and the millets
(XU and MESSING, 2008b). This grouping is not only
based on amino acid sequence homology but also
on different roles during compartmentalization in the
endosperm cell. Zeins are synthesized by polyribosomes of the rough endoplasmatic reticulum (RER)
and translocated into the lumen of the RER, where
they assemble into protein bodies (WOLF et al., 1967;
LARKINS and DALBY, 1975; BURR and BURR, 1976; LENDING and LARKINS, 1992). Translocation involves a signal peptide that is cleaved off. Although this step is
common to all zeins, they seem to play a different
role in the organization of protein bodies. While the
cysteine-rich gamma and beta zeins are located at
the periphery of the protein bodies, the alpha and
delta zeins accumulate in the center (LUDEVID et al.,
1984; LENDING and LARKINS, 1989). Being associated
with the protein membrane and having a rather bipolar structure, gamma zeins have been proposed to
act as osmoregulators in respect to the water content
in protein bodies and their surrounding environment
(WU and MESSING, ms in prep).
Given this role gamma-zein genes also commence expression before alpha- and delta-zein
genes and one can envision how they initiate the
formation of protein bodies and how the alpha
zeins, representing the bulk of the maize prolamins,
expand the size of protein bodies during endosperm development (LENDING and LARKINS, 1989).
Although little is known about the transcriptional
regulation of storage protein genes, their promoters
all share a common upstream sequence element,
called the prolamin box or P-Box (KREIS et al., 1985;
BORONAT et al., 1986; UEDA et al., 1994). Biochemical
studies suggest that this element is recognized by
the prolamin-box-binding factor (PBF), which is also expressed during endosperm development (UEDA
et al., 1994; VICENTE-CARBAJOSA et al., 1997; WANG et
al., 1998). In addition, most storage protein gene
promoters contain another sequence motif that is
recognized by bzip transcription factors, related to
the O2 protein (DE FREITAS et al., 1994). This protein
is conserved throughout the evolution of the grasses
and also expressed during endosperm development
(XU and MESSING, 2008a). The gene encoding this
transcription factor was duplicated in an ancient
whole genome duplication event preceding the progenitors of rice, maize, and sorghum. Both copies
underwent further duplication in tandem and
through a whole genome duplication event of
maize, resulting in a high degree of redundancy.
Still, only one copy represents the o2 locus that
controls a subset of alpha zein genes (PYSH et al.,
1993). The role of the other gene copies has not
been validated yet, but one can envision that zein
genes are controlled by a number of transcription
factors to coordinate their timely and tissue-specific
expression.
Indeed, the tissue-specificity of zein promoters
has been used in the construction of chimeric genes
to achieve expression of transgenic proteins in
maize endosperm (LAI and MESSING, 2002). Here we
have constructed a chimeric reporter gene with the
green fluorescence protein (GFP). To reduce the
complexity of the integration event of the transgene, we used Agrobacterium-mediated transformation rather than the biolistic approach (FRAME et al.,
2002). After growth in the presence of the selectable
marker and phosphinothricin in the media, callus
tissue surprisingly turned green, indicating the expression of GPF under the 27-kDa gamma-zein promoter. Therefore, the aim of this study was to investigate as to why this gene was expressed in non-differentiated tissue.
MATERIALS AND METHODS
Plasmid construction and Plant transformation
A GFP expressing construct (PTF102-p27GFPTdzs10) for
Agrobacterium-mediated transformation has been assembled.
The GFP gene is driven by the 27-kDa gamma-zein promoter
amplified from maize inbred line B73 with the primer pair, P27EcoR1 and p27-Nco1 (5’-CCAGAATTCCTTTATAATCAACCCGCACTC-3’ and 5’- AATACCATGGTGTCGATCGGGTTCTTC-3’); the
GFP gene was amplified from the plasmid pEGFP (Clontech)
with the primer pair GFP-Nco11 and GFP-BspE1 (5’- TATTCCATGGTGAGCAAGGGCGAGG-3’ and 5’- TAATGGTACCTTACTTGTACAGCTCGTCC-3’); the termination and polyadenylation signals are conferred by a 961-bp sequence downstream
from the stop codon of the 10-kDa delta-zein gene, amplified
with the primer pair Tdzs10-BspE1 and Tdzs10-HindIII (5’AAAGCTGTACAAGTAAATAGAAATATTTGTGTTGTATCG-3’ and
5’-ATCAAGCTTCTTTATGCTGATGGGGTTAC-3’).
Hi-II F1 (B x A) immature embryos (1.5-2.0 mm) were dissected from the ears growing in an environmental chamber
(Waksman Institute, Rutgers) 10 to 11 days after pollination. The
construct was delivered into the embryos by Agrobacterium-mediated transformation according to the protocol released by Iowa
State University Plant Transformation Facility (http://www.agron.
iastate.edu/ptf/) (FRAME et al., 2002). After the second selection, a
portion of callus for each event was saved to extract DNA by
CTAB. Positive events could be screened by PCR with the primer
pair p27F and GFPR (5’-ATGCTTACAGCTCACAAGAC-3’ and 5’TTACTTGTACAGCTCGTCC-3’) (Fig. 1). To propagate the positive
event, a portion of callus was transferred to the fresh Selection II
medium every other week. A negative event was also saved to
extract RNA as a control for further research.
PROMOTER SPECIFICITY IN MAIZE
FIGURE 1 - Schematic illustration of the GFP expression construct driven by the 27-kDa gamma-zein gene promoter. The termination and polyadenylation signals are provided by a 961-bp
sequence downstream from the stop codon of the 10-kDa deltazein gene. Two primer pairs, p27F and GFPR, and GFPF and GFPR, indicated in the figure are used to screen positive events and
detect GFP gene expression, respectively.
RT-PCR
Total RNAs from 18-DAP endosperms and calli were extracted by using TRIzol reagent (Invitrogen). 5 µg RNA was digested
with DNase I (Invitrogen) and then reverse-transcribed. 25 ng of
each cDNA was applied for RT-PCR. The primer pairs for PCR
amplification are: 50-kDaFR, (5’-ATGAAGCTGGTGCTTGTGGTTC-3’ and 5’-TAATGTCATTGCTGCTGCATGG-3’); 27-kDaFR,
(5’-ATGAGGGTGTTGCTCGTTGC-3’
and
5’-ACTCAACTAGCTAGCTAGCC-3’); 22-kDaFR, (5’-ACACCATATGTTCATTATTCCACAATGCTCA-3’ and 5’-TTAAGGATCCTATATAATCTAAAAGATGGCA-3’); 16-kDaFR, (5’-TCGACACCATGAAGGTGCTG-3’ and 5’TGGTGATGGGTGACACTACG-3’); 15-kDaFR, (5’-AGGATCGTCGAACAGAACAGC-3’ and 5’-AGATGGATAGAGGAGATTTCCC-3’);
10-kDaFR, (5’-ATACTCTAGGAAGCAAGGAC-3’ and 5’-TAAGAACATGGGTGGAATCG-3’); pbfFR, (5’-ATGGACATGATCTCCGGCAG-3’ and 5’-ACTAACCTTATTGTCCCTTG-3’); opaque2FR, (5’ATGGAGCACGTCATCTCAATG-3’ and 5’-CCTTATTCAGCGACGCCTG-3’); GFPF and GFPFR (5’-ATGGTGAGCAAGGGCGAGG-3’
and 5’-TTACTTGTACAGCTCGTCC-3’).
Western analysis
100 mg powder of finely ground callus or 18-DAP endosperm was mixed and vortexed with 400 µl of 70% ethanol/2%
2-mercaptoethanol (vol/vol), then kept on the bench at room
temperature for more than two hours; the mixture was centrifuged at 1,300 rpm for 10 min, then 100 µl of the supernatant
liquid was transferred to a new tube and added to 10 µl of 10%
SDS; the mixture was dried by vacuum and resuspended in 50 µl
(callus) or 100 µl (endosperm) of distilled water.
0.2 µl of the endosperm total zeins and 4 µl of the zeins extracted from callus were separated on 15% SDS-PAGE gel, and
then transferred to an Immun-Blot PVDF membrane (Bio-Rad).
The membrane was hybridized with a 1:5000 diluted antibody
against the 27-kDa gamma zein.
RESULTS
Construction of chimeric gene
Besides the regulation of gene expression by
transcription, we also could show that zein genes
were regulated at the post-transcriptional level
through mRNA stability (CRUZ-ALVAREZ et al., 1991;
SCHICKLER, 1993). We could show that the stability of
delta-zein mRNAs was regulated by sequence ele-
411
ments in their non-translated region, i.e. 5’ or 3’
UTR (LAI and MESSING, 2002). Although the study of
this regulation was not the subject here, the construct designed for the test of this regulation led to
our unexpected results of gene expression in tissue
culture. In one of the test constructs, we combined
a 961-bp sequence immediately downstream from
the stop codon of the 10-kDa delta-zein gene with
the promoter of the 27-kDa gamma-zein gene to investigate the role of the 3’UTR of the delta zein
gene. With this construct, we wanted to test in a
gain-of-function experiment the post-transcriptional
control of the delta-zein gene using a visible marker. To develop such a system, we took advantage of
the green fluorescent protein (GFP) as reporter system and inserted its coding sequence behind the
27-kDa gamma zein promoter as shown in Fig. 1.
The chimeric gene was inserted into the Agrobacterium vector pTF102 and used to transform Hi-II
immature embryos following a standard protocol as
described previously (FRAME et al., 2002).
Expression of a chimeric gene in callus
Phosphinothricin resistant callus was grown on
suitable medium (FRAME et al., 2002). After prolonged growth on selective medium, two independent events growing vigorously were observed both
to turn green (Fig. 2A). One of them was further
characterized and confirmed by PCR with a primer
pair specific for the chimeric gene (Fig. 2B). The
unique properties of GFP indicated that the
chimeric gene was expressed. To confirm expression, RNA was isolated from the green callus and
subjected to RT-PCR. Indeed, RT-PCR produced a
transcript consistent with the fluorescent color seen
in the callus (Fig. 2B).
Expression of endogenous zein genes
in callus culture
Expression of the chimeric gene could be explained by a position effect because of an enhancer
close to the integration event of the transgene. It
has been shown in so-called enhancer-trap experiments that enhancer-free chimeric reporter genes
can be used in searching the genome for enhancers
and promoter elements and their tissue-specificity
(SUNDARESAN et al., 1995). To distinguish between
such an event and the function of the 27-kDa gamma-zein promoter we further examined the extracted mRNA whether it also indicated expression of
the endogenous 27-kDa gamma-zein gene. Using
appropriate primer pairs, a strong band was ob-
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FIGURE 2 - Activation of GFP expression driven by the 27-kDa
gamma-zein gene promoter in the transgenic callus. Panel A:
GFP-expression callus showing strong green color. Upper row:
Left, a negative callus as a control; right, the transgenic callus.
Picture was taken under natural light with a digital camera. Bottom row: The same samples, but picture was taken with a fluorescence microscope. Panel B: Transgenic event confirmation
and RT-PCR analysis of GFP expression. Genomic DNA and cDNA of the green callus were amplified with primer pair p27F and
GFPR, respectively (lane 1 and 2). Because the promoter region
is not transcribed, only the genomic DNA gave a positive band.
Primer pair GFPF and GFPR designed from the GFP gene-coding
region (see Fig. 1) was applied to detect its expression and gave
a strong band with the appropriate size (lane 3). Size markers of
2 kb, 1.4 kb, 1.3 kb, 1 kb, 750 bp, 500 bp, 400 bp, 300 bp and
200 bp (from top to bottom) are shown in the left lane.
FIGURE 3 - RT-PCR analysis of zein-, PBF- and O2-gene expression. All zein-gene members and the Pbf and O2 genes were
highly expressed in the Hi-II hybrid 18-DAP old endosperm
(lower panel). Comparatively, high accumulation of the 27-kDa
gamma-zein transcripts was detected in the GFP expressing (upper panel) and the negative calli (middle panel), while the other
zeins are silent or expressed at very low levels. The Pbf gene is
also activated in the GFP expressing and the negative calli, although at much lower levels than in the endosperm. However,
the transcript of O2 gene was not detected. Size markers of 1.4
kb, 1.3 kb, 1 kb, 750 bp, 500 bp and 400 bp (from top to bottom) are shown in the left lane of all panels.
served for the 27-kDa gamma zein mRNA, whereas
only very weak amplification was noted for the 16kDa gamma-zein mRNA (Fig. 3). However there
was no indication of the expression of alpha zein
genes, consistent with a recent analysis of alpha
zein promoters showing a closed chromatin structure in non-endosperm tissue (LOCATELLI et al.,
2009). To rule out the possibility that the transactivation of the 27-kDa gamma-zein promoter was
caused by transformation, a slow-growing negative
callus on selection medium was used as a control to
screen all zein-gene expression. Like the positive
green callus, the endogenous 27-kDa gamma-zein
gene was also expressed (Fig. 3). In addition, weak
expression of the 16-kDa gamma-zein and 18-kDa
delta-zein was detected (Fig. 3).
To ensure that transcripts were intact and translated properly, protein was extracted from green
callus and immature endosperm as a reference following a standard zein fractionation protocol. Western blot analysis indeed indicated that gamma zein
accumulates in callus tissue although at a reduced
level compared to endosperm (Fig. 4). Therefore, it
appeared that not only the transgene but also the
endogenous gene was expressed in callus tissue.
Expression of trans-acting factors
in callus culture
Expression of the 27-kDa zein gene in non-differentiated cells poses the question whether one of
the endosperm-specific trans-acting factors is also
expressed in callus tissue. As already described
PROMOTER SPECIFICITY IN MAIZE
FIGURE 4 - Immunoblot analysis of the 27-kDa gamma-zein protein. Material was prepared and analyzed as described under Materials and Methods. Hi-II hybrid 18-DAP endosperm (lane 1-3);
GFP expressing callus (lane 4-6).
above the most common promoter element of prolamin genes is the P-Box, which has been shown to
interact with the zinc-finger protein PBF (UEDA et
al., 1994; VICENTE-CARBAJOSA et al., 1997). Therefore,
the green callus cDNAs were also used to investigate PBF-gene expression with a specific primer
pair flanking the 2-kb intron, which could be easily
detected by size if the PCR product was amplified
from any contaminated genomic DNA. As shown in
Fig. 3, transcript from the PBF-gene was indeed
present explaining the transcription of the 27-kDa
zein gene. Because the P-Box is common to all zein
genes, what would distinguish between the expression of the 27-kDa zein gene and the other zein
genes, in particular the alpha zein genes, which are
not expressed at all? Therefore, RT-PCR was also
performed with primers that amplify the O2 gene.
However, in contrast to PBF gene, no transcript was
observed (Fig. 3), which is consistent with the absence of the expression of the 22-kDa alpha-zein
genes in green tissue callus.
DISCUSSION
We have shown in this study that the transcriptional activator PBF is unexpectedly expressed in
non-differentiated cells. Previously, PBF has been
proposed to be a general activator of endospermspecifically expressed genes through the interaction
of the P-Box, located about 300 bp upstream of the
translation start codon (KREIS et al., 1985; BORONAT et
al., 1986; UEDA et al., 1994). This cis-acting element
(TGTAAAG) is very much conserved in most prolamin genes and because of the tissue-specific expression of prolamin genes in endosperm is also re-
413
ferred to as the endosperm motif. Based on these
observations, it was assumed that PBF is expressed
only in the endosperm.
However, there are two other features of transcription that have to be taken into consideration:
one, additional cis-acting elements that interact with
other trans-acting factors dominating transcriptional
activation of gene expression, and two, the chromatin structure of the target gene. Indeed, comparison of the promoter regions of prolamin genes yield
a second conserved sequence motif that is present
in many but not all prolamin genes. This motif is
called the GCN4-like motif (GLM) (A/G)TGAGTCAT
and usually is located close to the endosperm motif
(KREIS et al., 1985; BORONAT et al., 1986; UEDA et al.,
1994). Such a close distance would argue for additional protein-protein interaction between two transcriptional activators. Indeed, it has been shown
that PBF interacts with the O2 DNA-binding protein
(WANG and MESSING, 1998). O2 specifically interacts
with a subset of alpha zein promoters, but belongs
to a gene family where orthologous and parologous
copies could play the same role at other prolamin
genes (XU and MESSING, 2008a). Interestingly, there
are 22-kDa alpha zein genes that are expressed in
o2 homozygous seeds, indicating that other transacting factors than O2 can function in endosperm
(SONG et al., 2001). Recent studies have shown that
O2 target genes need to undergo tissue-specific
chromatin modification at the DNA binding site for
O2 to function, suggesting that the tissue specificity
of prolamin genes is also regulated at the chromatin
level (LOCATELLI et al., 2009), which would also suggest that chromatin remodeling has to occur before
transcriptional activation can begin.
Given these consideration one could argue that
selection could act on multiple levels to achieve organ morphogenesis and identity. Previous studies of
an epiallele of the P-locus has shown that some sort
of presetting occurs at the promoter site because
the epiallele had a chromatin structure that was resistant to tissue-specific modification occurring with
the normal allele during seed development (LUND et
al., 1995). Such gene silencing would prevent gene
expression even if trans-acting factors were produced as it becomes clear from variegated tissue,
where the epigenetic state is unstable and can revert (DAS and MESSING, 1994).
Obviously, DNA binding depends on the interaction between protein and DNA. Here, alleles of
trans-acting factors could have different affinities to
DNA binding sites (AUKERMAN et al., 1991). In addi-
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Y. WU, J. MESSING
tion, dimerization or heterodimerization of transacting factors could be important for transcriptional
activation (PYSH et al., 1993; GROTEWOLD et al.,
2000). Because DNA can bend physical interaction
between two DNA binding proteins they could also
be important in exposing the moiety that interacts
with the RNA polymerase II complex. Furthermore,
activation could come from a protein that does not
even bind to DNA but interacts with the DNA-binding protein through protein-protein interaction
(WANG and MESSING, 1998).
How these various models apply here, is not yet
clear. However, it seems that from all prolamin
genes PBF expression preferentially turns on the expression of the 27-kDa gamma-zein gene. In contrast, all other zein genes are weakly or not expressed at all outside the endosperm, providing a
unique feature to only one of the zein genes. Interestingly, this coincides with the observation that this
might be the oldest and longest conserved prolamin
gene in maize (XU and MESSING, 2009). Therefore,
one could argue that its regulation might have predated the differential role required for optimal seed
development and is one of those functions co-opted
early for seed function. In this respect it is worthy
to note that PBF has been suggested to be one of
the few loci domesticated from teosinte to maize
(JAENICKE-DESPRES et al., 2003).
ACKNOWLEDGEMENTS - We thank Brian A. Larkins for his generous provision of the 27-kDa gamma-zein antibody. The research described in this manuscript was supported by the Selman
A. Waksman Chair in Molecular Genetics and a grant from the
DOE (# DE-FG05-95ER20194) to JM.
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