The maize gamma-zein sequesters alpha-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.

Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing alpha-zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of alpha-zein required simultaneous synthesis of gamma-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presence of gamma-zein, there was a turnover of alpha-zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.     

Divergent properties of prolamins in wheat and maize

Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of γ-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires γ-zeins for its proper subcellular organization in maize.     

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.     

Lysine-containing proteins in maize endosperm: a major contribution from cytoskeleton-associated carbohydrate-metabolizing enzymes

We measured fresh weight, dry weight, total protein, and the amounts of several individual proteins during endosperm development in three varieties of maize ( Zea mays L.): W64A wild-type (WT) and opaque-2 (o2), and sweet corn (SW). By 28 days after pollination (DAP), fresh weight was much higher in WT and SW than in o2, but o2 had a higher dry weight and thus a much lower water content. By 28 DAP, protein concentration [mg (g tissue(-1))] was highest in o2 and lowest in WT, while the protein content (microg seed(-1)) was lowest in o2. The storage proteins, alpha- and gamma-zeins, were low initially, but by 28 DAP they comprised over 50% of the total protein in WT and SW, but only about 30% in o2. In all varieties, the cytoskeleton proteins, actin, tubulin and eEF1alpha, sedimented with the protein bodies at 30 g to 27,000 g in tissue homogenized in cytoskeleton-stabilizing buffer. Other cytoskeleton-associated proteins increased during development, including UDP-glucose starch glucosyltransferase (UDP-GSGT, EC 2.4.1.11), sucrose synthase 1 (SuSy-1, EC 2.4.1.13) and fructose-1,6 bisphosphate aldolase (FBA, EC 4.1.2.13). At 28 DAP, these cytoskeleton-associated proteins combined make up 27% (WT), 23% (SW) and 33% (o2) of the total protein. These proteins are all rather high (5-11%) in lysine, and so they contribute about 75% (WT), 67% (o2), and 51% (SW) of the total endosperm lysine. We conclude that efforts to elevate the levels of these proteins could make a significant contribution to the nutritional value of corn.     

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.     

EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells

By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

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     

Relationship between structural and biochemical characteristics and texture of corn grains

The texture of corn grains is a fundamental characteristic for the industry as well as for grain producers and processors. To further understand the mechanisms involved in grain hardness, contrasting corn cultivars for grain hardness and protein quality were evaluated. The cultivars were Cateto L237/67 (hard endosperm and low protein value), QPM BR 451 (semi-hard endosperm and high protein value); Bolivia-2 (floury endosperm and low protein value), and Opaque-2 (floury endosperm and high protein value). Evaluations were carried out at 10, 20, 30, 40, 50, and 60 days after pollination in developing corn grains and in the mature grain. In developing grains, evaluation consisted in the determination of the area, percentage of starch granules, distribution of starch granules, and protein bodies in the endosperm. In mature corn grains, the parameters evaluated were: density, percentage of total proteins, levels of lysine and tryptophan, and banding pattern of zeins. The results indicate that the higher physical resistance of corn grains from the cultivars analyzed is influenced by the high percentage of total proteins, high synthesis of 27-kDa zeins, presence of protein bodies, and perfect organization of starch granules in the endosperm, independent of their sizes.     

The accumulation of alpha-zein in transgenic tobacco endosperm is stabilized by co-expression of beta-zein

The cysteine-poor alpha-zein is the major prolamin storage protein fraction in maize endosperm and is localized in the interior of protein bodies with delta-zein, whereas the hydrophobic cysteine-rich beta- and gamma-zein are found on the exterior of the PB. In transgenic tobacco endosperm expressing zein genes, alpha-zein was unstable unless co-expressed with gamma-zein. Here we showed that alpha-zein was also stabilized by beta-zein. Small accretions of alpha- and beta-zeins, similar in appearance to maize protein bodies, were localized to the endoplasmic reticulum within tobacco endosperm cells. The zein proteins were also localized to protein storage vacuoles in a more dispersed pattern, suggesting that they were transported there after they were post-translationally sequestered into the ER.     

Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins.     

Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants

Plant lipid transfer proteins (LTPs) are soluble proteins which are characterized by their in vitro ability to transfer phospholipids between two membranes. We have compared the functional properties of two LTPs purified from maize and wheat seeds knowing that, despite a high degree of sequence identity, the two proteins exhibit structural differences. It was found that wheat LTP had a lower transfer activity than the maize LTP, consistent with a lower kinetics of fatty acid binding. The lower affinity for the fatty acids of the wheat LTP could be explained by a narrowing occurring in the middle part of the binding site, as revealed by comparing the fluorescence spectra of various anthroyloxy-labeled fatty acids associated with the two LTPs. The affinity for some natural fatty acids was studied by competition with fluorescent fatty acids toward binding to the protein. Again, wheat LTP had a lower affinity for those molecules. All together, these observations reveal the complexity of the LTP family in plants, probably reflecting the multiple roles played by these proteins.     

Protein accumulation in aleurone cells,sub-aleurone cells and the center starch endosperm of cereals

There are mainly three endosperm storage tissues in the cereal endosperm: aleurone cells, sub-aleurone cells and the center starch endosperm. The protein accumulation is very different in the three endosperm storage tissues. The aleurone cells accumulate protein in aleurone granules. The sub-aleurone cells and the center starch endosperm accumulate protein in endoplasmic reticulum-derived protein bodies and vacuolar protein bodies. Proteins are deposited in different patterns within different endosperm storage tissues probably because of the special storage properties of these tissues. There are several special genes and other molecular factors to mediate the protein accumulation in these tissues. Different proteins have distinct functions in the protein body formation and the protein interactions determine protein body assembly. There are both cooperation and competition relationships between protein, starch and lipid in the cereal endosperm. This paper reviews the latest investigations on protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm. Useful information will be supplied for future investigations on the cereal endosperm development.     

Efficient recovery of recombinant proteins from cereal endosperm is affected by interaction with endogenous storage proteins

Cereal seeds are versatile platforms for the production of recombinant proteins because they provide a stable environment for protein accumulation. Endogenous seed storage proteins, however, include several prolamin-type polypeptides that aggregate and crosslink via intermolecular disulfide bridges, which could potentially interact with multimeric recombinant proteins such as antibodies, which assemble in the same manner. We investigated this possibility by sequentially extracting a human antibody expressed in maize endosperm, followed by precipitation in vitro with zein. We provide evidence that a significant proportion of the antibody pool interacts with zein and therefore cannot be extracted using non-reducing buffers. Immunolocalization experiments demonstrated that antibodies targeted for secretion were instead retained within zein bodies because of such covalent interactions. Our findings suggest that the production of soluble recombinant antibodies in maize could be enhanced by eliminating or minimizing interactions with endogenous storage proteins.     

Conformation of alpha zeins in solid state by Fourier transform IR

The major maize storage proteins (alpha zeins) are deposited as an insoluble mass in the protein bodies of the endosperm. Because they are insoluble in water, most structural studies are performed in alcohol solutions. To solve the question raised by several authors about denaturation of the alpha zein structure by alcohol, we analyze the secondary structure of alpha zeins prepared with and without solubilization in alcohol (corn gluten meal and protein bodies with high concentrations of alpha zeins and traces of beta zeins). The secondary structures of alpha zeins are analyzed in the solid state by Fourier transform IR spectroscopy (FTIR) in KBr pellets and solid-state 13C-NMR spectroscopy. The proportion of secondary structures obtained by FTIR of alpha zeins prepared with and without solubilization in alcohol yield almost identical proportions of alpha helices and beta sheets. The proportion of alpha helices (43%) agrees with that measured by circular dichroism in an alcohol solution. However, the proportion of beta sheets (28%) is higher than the one measured by the same technique. Gluten and protein body samples with high beta zein content showed higher beta sheet and lower alpha helix proportions than that obtained for alpha zein preparations. The solid-state 13C-NMR spectra show the carbonyl peak for the alpha zeins at delta 176 and for the sample rich in beta zeins at delta 172, which demonstrates the presence of a high content of alpha helices and beta sheets, respectively. These results indicate that alcohol solubilization does not affect the conformation of alpha zeins, validating the secondary structure measurements in solution.     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds

Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein 1. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.     

BiP and zein binding domains within the delta zein protein

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.