Genetic control of a membrane component and zein deposition in maize endosperm

Summary An association is reported between an albuminlike protein (b-70) and the semidominant locus fluory-2 (fl2) which reduces the level of zein polypeptides in the maize endosperm. The protein b-70 is present at low level in wild-type endosperms and derppressed in fl2 endosperms. A correlation between the doses of the fl2 allele and the b-70 level has been found. Moreover a concomitant loss of the regulatory role of fl2 on zein level and on b-70 overproduction is evident when fl2 is genetically associated with o2 and o7, two recessive alleles of other zein regulatory loci. Protein b-70 is located on the membrane of the protein body where zein polypeptides accumulate. The existence of a functional relationship between this protein and the zein-secretory system is suggested or, as an alternative, that b-70 is a type of storage protein different from zeins, repressed in normal endosperms and derepressed by the fl2 allele.Abbreviations DAP days after pollination - ER endoplasmic reticulum - RER rough endoplasmic reticulum - DTT dithiothreitol - EDTA ethylene-diamintetra-acetate - NADH nicotinamide-adenine dinucleotide, reduced - PMSF phenylmethylsulfonyl-fluoride - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline (0.15 M NaCl, 0.01 M Na phosphate, pH 6.8)     

Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.     

Increased expression of the maize immunoglobulin binding protein homolog b-70 in three zein regulatory mutants.

Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein.     

Increases in binding protein (BiP) accompany changes in protein body morphology in three high-lysine mutants of maize

Summary A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.Abbreviations BiP binding protein - BSA bovine serum albumin - De*-B 30 Defective endosperm B 30 - DAP day after pollination - ER endoplasmic reticulum - fl 2 floury-2 - hsp 70 70 kDa heat shock protein - Mc Mucronate - TBST 20mM Tris-HCl, pH8.2 at 20°C, 500mM NaCl, 0.3% Tween 20 - TBST-B TBST with 1% (w/v) BSA     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: Nucleic acid (cDNA) and amino acid sequences

Summary The cDNA coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of the O2 and O6 loci, has been cloned and the complete amino acid sequence of the protein derived. A lambda gt11 cDNA library from mRNA of immature maize endosperm was screened for the expression of the b-32 protein using antibodies against the purified protein. One of the positive clones obtained was used to isolate a full-length cDNA clone. By Northern analysis, the size of the b-32 mRNA was estimated to be 1.2 kb. Hybrid-selected translation assays show that the message codes for a protein with an apparent molecular weight of 30–35 kDa. The nucleotide sequence shows that several internal repeats are present. The protein has a length of 303 amino acid residues (mol. wt. 32430 dalton) and its sequence shows the following features: no signal peptide is observable; it contains seven tryptophan residues, an amino acid absent in maize storage proteins; polar and hydrophobic residues are spread along the sequence; several pairs of basic residues are present in the N-terminal region; the secondary structure allows the prediction of two structural domains for the b-32 protein that would fold up giving rise to a globular shape. The cloning of this gene may help in understanding the role of the O2 and O6 loci in regulating the deposition of zein, the major storage protein of maize endosperm.     

The maize floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD alpha-zeins in the gamma-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD alpha-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD alpha-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD alpha-zein and that this is essential for the formation of vitreous endosperm.     

Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant

A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed -zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies.     

Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations.

The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined. The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%). For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type. In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants. Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines. In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine. The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant.     

Cloning of double stranded DNAs derived from polysomal mRNA of maize endosperm: isolation and characterisation of zein clones.

Starting from polysomal RNA of developing maize endosperm and applying the cloning procedure of integrating dsDNA into the Pst I site of plasmid pBR 322, clones containing sequences complementary to endosperm mRNAs were obtained. 25 per cent of these clones were identified as containing zein specific DNA sequences which hybridized either with the zein mRNA coding for the 22 000 Mr protein or with the zein mRNA coding for the 19 000 Mr protein. The zein-specific DNA inserts of the recombinant plasmids were further characterized by restriction enzyme analysis.     

Tissue-specific zein synthesis in maize kernel

Zein may account for as much as 10% of the total protein in the mature embryo of maize inbred W64A. This protein exhibited an electrophoretic pattern on SDS gels similar to that of the endosperm. Like the endosperm system, the synthesis of zein components in the embryo was controlled by the opaque-2 and floury-2 mutations. However, unlike zein synthesis in the endosperm, zein synthesis in the embryo could not be increased by nitrogen fertilizer. Variations in amino acid composition were observed between the zein components of the embryo and those of the endosperm.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

A defective signal peptide in a 19-kD alpha-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel alpha-zein protein in De*-B30 that migrates between the 22- and 19-kD alpha-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD alpha-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD alpha-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD alpha-zein protein.     

Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize

Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.     

Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells

Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa alpha-zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.