A modified 10 kD zein protein produces two morphologically
distinct protein bodies in transgenic tobacco
Jennifer Randall
a, Suman Bagga
a,b, Henry Adams
c, John D. Kemp
a,*
aDepartment of Entomology,Plant Pathology and Weed Science,Gene Lab,New Mexico State Uni6ersity,Box3GL,Las Cruces,
NM88003,USA
bDepartment of Agronomy and Horticulture,New Mexico State Uni6ersity,Box3GL,Las Cruces,NM88003,USA cDepartment of Cell Biology,Baylor College of Medicine,1Baylor Plaza,Houston,TX77025,USA
Received 10 March 1999; received in revised form 14 July 1999; accepted 15 July 1999
Abstract
The 10 kD zein protein contains an N-terminal signal peptide that directs the protein into the endoplasmic reticulum (ER) of developing corn seeds. Subsequent to signal peptide removal, the mature protein is folded into its tertiary conformation and deposited into protein bodies. In transgenic tobacco leaves, the 10 kD zein protein accumulates and forms novel ER derived protein bodies (S. Bagga, H. Adams, F. Rodriquez, J.D. Kemp, C. Sengupta-Gopalan, Coexpression of the maized-zein and
b-zein genes results in stable accumulation ofd-zein in endoplasmic reticulum-derived protein bodies formed byb-zein, The Plant Cell 9 (1997) 1683 – 1696). In this study, the amino acid sequence of the 10 kD zein signal peptide was modified to determine the effect on cleavage and accumulation patterns. The modified zein gene was constitutively expressed in tobacco where its protein accumulates in novel protein bodies in leaves. Amino acid sequencing of the accumulated protein indicates that the cleavage site for the signal peptide was altered so that the mature protein includes three additional amino acids on the N-terminus. Electron microscopy (EM) analysis of leaves from transgenic plants containing the modified gene indicates the presence of two morphologically distinct protein bodies. Furthermore, immunolocalization analysis shows that the modified protein is localized in both types of protein bodies, which are described as spherical and aggregate in this report. This is in contrast to the accumulation of unmodified 10 kD zein protein in transgenic leaves where only spherical protein bodies are observed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Zein; Signal peptide; Targeting; Transgenic plants; Protein bodies
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1. Introduction
Zeins are alcohol soluble seed storage proteins found in the endosperm of maize. They are
differ-entiated into four classes: a, b, d, and g based
upon their solubility and their mobility on a SDS-PAGE gel [2]. All zein proteins contain an N-ter-minal signal peptide that directs them into the endoplasmic reticulum (ER). Once translocation of the protein into the ER is complete, a signal peptidase cleaves the signal peptide from the ma-ture protein. The mama-ture protein is then folded
into its tertiary configuration and subsequently deposited into ER derived protein bodies.
In the endosperm of maize seeds the production of zeins is developmentally regulated and each protein class is deposited in a specific manner
within protein bodies [3]. The a and d zeins
com-prise the core of the protein body with the beta and gamma zeins surrounding the periphery [4]. It is not clear how the zeins are retained in the ER. They do not contain a known ER retention signal such as the KDEL or the HDEL motif found in other ER resident proteins [5]. However, high levels of binding protein (BiP), an ER resident protein that acts as a chaperone, are found associ-ated with the protein bodies [6 – 8]. This associa-* Corresponding author: Tel.: +1-505-6465453; fax: +
1-505-6461302.
E-mail address:[email protected] (J.D. Kemp)
tion may be responsible for the retention of the zein proteins in the ER [7]. Protein bodies are also found associated with the cytoskeleton.
Elonga-tion factor 1a(EF-1a) is also localized around the
periphery of zein protein bodies. Since EF-1a is
known to associate with actin in the cytoskeleton, it is possible that the cytoskeleton directs or an-chors zein mRNAs to the site in which protein body formation will occur [9].
Signal peptides are essential for zein proteins to be directed into the ER where protein bodies and
stable proteins accumulate (Randall et al.,
manuscript in progress). Modifications between the signal peptide cleavage point and the mature protein can dramatically alter protein structure or function. A single amino acid substitution of an
alanine for a valine at the −1 position of an
alpha zein protein is the cause of abnormal protein
body formation in the floury-2 mutant [10]. The
signal peptide for this alpha zein is not cleaved and therefore anchors the protein to the ER mem-brane [11]. Von Heijne’s rules predict that disrup-tion of signal peptide cleavage would occur due to this substitution [12]. Predictions of signal peptide cleavage by Von Heijne’s rules are not absolute. The gene that codes for alcohol dehydrogenase was fused with a signal peptide from a zein gene (preZad) [13]. Although, the signal peptide junc-tion was maintained and Von Heijne’s rules would predict that signal peptide cleavage would occur, cleavage of the signal peptide was prevented. The attached signal peptide thus anchored alcohol de-hydrogenase to the ER membrane [13].
The delta zein class consists of the 10 kD and 18 kD polypeptides which are each encoded by only one gene in the haploid genome [14]. The 10 kD zein gene was expressed in transgenic tobacco under the constitutive control of the cauliflower mosaic virus (CaMV) 35 S promoter [1]. Further-more, the 10 kD zein protein is localized within novel spherical protein bodies in the leaves of the transgenic tobacco cells [1].
In this study, the amino acid sequence of the 10 kD zein signal peptide was modified to determine its effect on cleavage and protein accumulation. Three amino acids glycine, serine, and methionine have been inserted between the signal peptide cleavage site and the mature 10 kD zein protein. Our data shows that the modified signal peptide was cleaved, and furthermore, a novel
morpholog-ically different protein body is observed in the leaves of transgenic tobacco accumulating this modified 10 kD zein protein.
2. Materials and methods
2.1. Plasmid construction
The plasmid pDspz was constructed using the polymerase chain reaction. The regions that code for the signal peptide and the mature coding re-gion of the protein were independently amplified. The primers utilized for the amplification of the
signal peptide are: 5%
CTACAAGATCTGATAT-CATCGATG 3% and 5%
CCGGATCCACTTAG-TGGCGCTTGC 3%. The primers utilized for the
amplification of the mature coding region are:
5%
CCAGATCTATGGCGACCCATATTCCAGG-GC 3% and 5%
GAACGATCGGGAATTCTCG-AGG 3%. The cycle conditions were as follows:
initial denaturation at 95° for 2 min, thirty cycles of 95°C for 45 s, annealing at 49°C for 1 min, extension of 72°C for 2 min, with a final extension of 72°C for 5 min. Both products were cloned into Promega’s pGem T vector and completely se-quenced. Two sites were created for the facilitation
of cloning. A Bam HI site was created at the 3%
end of the signal peptide and a Bgl II site created
at the 5% end of the mature coding region. These
sites were used to insert three amino acids (glycine,
serine, and methionine) between the −1 and −3
position at the junction between the signal peptide and the mature coding sequence. The amplifica-tion products were ligated together at the created
Bam HI/Bgl II sites and then ligated into the
multiple cloning region of pMon 316 which con-tains the cauliflower mosaic virus (CaMV) 35 S constitutive promoter and the nopaline synthase
(NOS) 3% terminator [15].
2.2. Tobacco transformation
Transfer of pDspz into Agrobacterium tumefa
-ciens (strain pTiT37ASE) was accomplished
through tri-parental matings [15]. Nicotiana
tabacum plants were transformed using the leaf
disc transformation system [16]. Transformants
were selected on MS media containing 100 mg/ml
2.3. RNA Analysis
RNA was isolated from mesophyll tissue of transformed plants via the lithium chloride proce-dure [17]. Twenty micrograms of total RNA was separated on a 1% agarose formaldehyde gel. The fractionated RNA was then transferred and fixed to a nylon membrane. The membrane was hy-bridized to digoxigenin-labeled 10 kD zein DNA probes generated by PCR amplification. Detection of hybridization was accomplished using a chemi-luminescent assay [18].
2.4. Protein analysis
Protein was isolated from leaf tissue of trans-formed plants as described by Bagga et al. [16].
Zein proteins were detected after separating 2.5mg
of ethanol soluble protein on a 16% SDS-PAGE gel [19] and challenging the blot with anti-10 kD zein polyclonal rabbit antisera [20] diluted 1:1000 in RIPA with 1% BSA. BiP proteins were detected
by separating 50 mg of PBS soluble protein on an
8% SDS-PAGE gel [19] and challenging the blot with anti-BiP polyclonal rabbit antisera [21] di-luted 1:1000 in RIPA with 1% BSA.
2.5. Protein sequencing
The ethanol soluble protein fraction from leaves
of transformed plants was isolated and 500mg was
separated on a 16% SDS-PAGE gel. The protein was then transferred to PVDF membrane and coomassie stained. The coomassie stained bands were then N-terminally sequenced on Applied Biosystems 473A Protein Sequencer using stan-dard Edman degradation.
2.6. Electron microscopy analysis
Leaves from transformed plants were sectioned and fixed as described by Bagga et al. [16]. The grids were observed using a Hitachi H700 trans-mission electron microscope. Immunolocalization was performed as described by Bagga et al. [16]. The sections were then labeled with the polyclonal 10 kD antibody, followed by anti-rabbit IgG con-jugated to 10 nm gold particles.
3. Results
3.1. Expression of altered 10 kD zein
3.1.1. Transcript analysis
The 10 kD modified zein gene in which three amino acids (glycine, serine, and methionine) were inserted, was positioned behind the CaMV 35 S constitutive promoter and introduced into to-bacco. The presence of transcript was detected by northern analysis in five independently trans-formed plants containing the modified 10 kD zein (pDspz) construct. The quantity of transcript ob-served in each independently transformed plant varied (Fig. 1). The high levels observed in some plants (lanes A and B) indicate that the ability of the gene to be transcribed was not compromised by altering the gene. The range of mRNA accumu-lation is approximately tenfold when comparing transformed plants A or B to plant D (Fig. 1). The varied amounts of transcript observed between the independently transformed plants is more than likely due to the position of where the gene was inserted within the plant genome.
3.1.2. Protein analysis
Five independently transformed plants were an-alyzed for the presence of the 10 kD zein protein
Fig. 2. Western analysis of independently transformed plants accumulating the modified 10 kD zein protein. (A) PBS (phosphate buffer saline) equivalent ethanol soluble protein (2.5mg) was separated on a 20% SDS-PAGE gel prior to transfer to nitrocellulose and immunodetected using the 10 kD zein antiserum. Lane A is the modified 10 kD zein protein from a transgenic tobacco plant expressing the pDSPz construct. Lane P is the unmodified 10 kD zein protein positive control from a transgenic tobacco plant. (B) PBS equivalent ethanol soluble protein (2.5mg) was separated on a 16% SDS-PAGE gel prior to transfer to nitrocellulose and immunodetected using the 10 kD antiserum. Lanes A – E are independently transformed plants which contain the pDSPz construct. Lane P is the unmodified 10 kD zein positive control from a transgenic tobacco plant. Lane N is the non-transformed tobacco negative control. Lane M is the molecular weight marker. The arrow indicates the protein band of interest.
by western analysis (Fig. 2). Accumulation of the altered 10 kD zein protein varied between the
independently transformed plants and
corre-sponded to the amount of transcript observed in each plant. Separation of proteins on a 20% SDS-PAGE gel indicates that the modified 10 kD zein protein migrates faster on an SDS-PAGE gel than the original 10 kD zein protein.
3.1.3. Protein sequencing
The modified 10 kD protein was N-terminally sequenced to determine where processing oc-curred. The N-terminal sequence of the mature modified 10 kD zein protein is serine, methionine, alanine, and threonine indicating that the N-termi-nal sigN-termi-nal peptide was processed. However, the location for processing was altered. The threonine
continues to be the −3 position whereas the
added glycine is now the −1 position (see Fig. 3).
This new −1 position with the −3 position still
conform to Von Heijne’s ‘−1, −3 rule’. The
modified 10 kD mature zein protein has an addi-tion of three amino acids at the N-terminus. The native 10 kD zein has a molecular weight of 14.5 kD when calculated from the amino acid sequence whereas, the altered mature 10 kD zein protein has a calculated molecular weight of 14.8 kD. How-ever, on a SDS-PAGE gel the native zein runs at a mobility equivalent to 10 kD [20] and the altered 10 kD zein at approximately 9 kD.
3.1.4. EM Analysis
The 10 kD zein protein has been localized in ER derived protein bodies in the leaves of transgenic tobacco plants [1]. EM analysis was performed on leaves of transgenic plants containing the modified 10 kD zein gene to determine if protein body morphology would be altered due to the extra three amino acids in the mature coding region. Two morphologically different protein bodies were observed in the leaves of these plants (Figs. 4 and 5). The modified 10 kD zein protein was localized inside both types of protein bodies (Fig. 4b, and 5d). The spherical type of protein body observed is the same classical type of protein body observed in leaves containing the unmodified 10 kD zein protein [1] (Fig. 4). This protein body has a dis-tinct spherical shape with a densely stained mem-brane surrounding it. The aggregate type of protein body does not have a distinct shape as the spherical protein body and has not been previ-ously observed in transgenic plants containing other zein constructs (Fig. 5). The aggregate type
Fig. 3. The amino acid differences of the original 10 kD zein signal peptide and the modified signal peptide. The arrows indicate where signal peptide cleavage occurs. The −1 and
Fig. 4. Spherical protein bodies observed during EM analysis of leaf tissue from transgenic plants accumulating the modified 10 kD zein protein. (A) Conventionally fixed and stained section of a leaf from a tobacco plant accumulating the modified 10 kD zein protein. The spherical protein bodies are indicated by arrows. The larger spherical body has the dimensions of approximately 1.5mm in width and 1.2 mm in length. The marker denotes one micron. (B) Immunolocaliza-tion of the 10 kD zein protein within the spherical protein bodies using the rabbit anti-10 kD zein antibody diluted 1:50 followed by 10 nm diameter gold conjugated goat anti-rabbit IgG. The arrows indicate gold particle labeling within the spherical protein body.
mately 1.5mm in width and 1.2mm in length. The
aggregate protein body observed in Fig. 5c is
approximately 15 mm in width and 10 mm in
length.
3.1.5. Protein analysis of BiP
BiP is an ER resident chaperone protein which aids in folding of proteins upon their translocation into the ER. BiP is also associated with zein protein bodies in maize and implicated in protein body formation [6 – 8]. Induction of BiP protein occurs in transgenic tobacco plants accumulating zein protein [1]. To determine if the transgenic tobacco plants accumulating the modified 10 kD zein protein also have a higher accumulation of BiP, western analysis was performed with the use of a BiP antisera (Fig. 6). BiP is induced in the leaves of transgenic tobacco plants accumulating the modified zein protein. The plants that accumu-lated higher levels of the modified 10 kD zein protein accumulated higher levels of BiP and the plants that accumulated lower levels of the modified 10 kD zein protein accumulated lower levels of BiP. However, although a trend is ob-served between the zein and BiP protein levels in these transgenic plants a quantative relationship can not be made.
4. Discussion
In this study, the amino acid sequence of the 10 kD zein signal peptide was modified to determine its effect on cleavage and accumulation patterns. Modification of the protein could have prevented cleavage of the N-terminal signal peptide which would anchor the protein to the membrane of the
ER as has been observed with thefloury-2 mutant
[10] and the alcohol dehydrogenase pre-zad fusion [13]. Instead, the signal peptide of the modified 10 kD zein protein was cleaved upon entry into the ER and further, the cleavage point between the signal peptide and mature protein was altered. This altered cleavage point creates a protein that has three extra amino acids (serine, methionine, and alanine) at the N-terminal end of the mature protein. Protein analysis of transgenic tobacco leaves expressing this altered gene indicate that this modified protein accumulates to levels equal to or greater than accumulation of the unmodified 10 kD zein protein in transgenic plants [1]. This protein bodies were only observed in the leaves of
approxi-suggests that the stability of the protein was not compromised with the modifications. However, the modified 10 kD zein protein does migrate faster on an SDS-PAGE gel than the original 10 kD zein protein. Perhaps the addition of the three amino acids which are hydrophobic in nature in-creases the hydrophobic character of the protein in such a way that it actually migrates faster on an SDS-PAGE gel, thus explaining the observed smaller size. Induction of BiP, which has been shown in transgenic plants accumulating zein protein [1], is also observed in plants expressing the modified 10 kD zein gene.
EM Analysis of leaves from transgenic tobacco plants accumulating this altered protein indicates that two morphologically distinct protein bodies are produced, spherical and aggregate. Spherical
protein bodies were originally observed in leaves of transgenic plants accumulating 10 kD zein protein. We also observe them in the leaves accu-mulating the modified 10 kD zein protein. Aggre-gate protein bodies, on the other hand, have not been previously observed in transgenic plants ac-cumulating other zein proteins. The modified 10 kD zein protein has been immunolocalized within both types of protein bodies. It is possible that the tertiary structure of the 10 kD zein protein has been slightly altered with the addition of the three extra amino acids on the N-terminus. This change in configuration could account for the change in protein body morphology, perhaps by changing zein deposition of the protein bodies. However, since two different protein bodies are observed and only one protein population of the modified
Fig. 6. BiP accumulation in independently transformed to-bacco plants accumulating the modified 10 kD zein protein. PBS soluble protein (50mg) from leaves was separated on an 8% SDS-PAGE gel prior to immunoblotting with the use of a maize BiP antiserum. Lanes A – E are independently trans-formed tobacco plants accumulating the modified 10 kD zein protein. Lane N is the non-transformed tobacco negative control. Lane M is the molecular weight marker. The arrow indicates the protein band of interest.
modified 10 kD zein protein reported in this paper may be altered in such a way that a large concen-tration of the protein bodies induces aggregation only in plants accumulating the modified 10 kD zein protein.
Acknowledgements
The authors would like to thank Dr Joachim Messing for the use of the 10 kD zein antibody, and Dr Rebecca Boston for the use of the maize BiP antibody. We would also like to thank Ed Towers of Macromolecular Resources, Depart-ment of Biochemistry, Colorado State University, for protein sequencing and Dr Paul Jackson of Los Alamos National Laboratory for the primers. We would especially like to thank Daniel Kim and Dr Jose Ortega for their technical assistance and Dr Carol Potenza for her technical assistance and critical review of this manuscript. This work was
supported by USDA/CSREES grant c
95-34250-1413, and the Agricultural Experiment Station at New Mexico State University.
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