Zeolin. A new recombinant storage protein constructed using maize gamma-zein and bean phaseolin

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of gamma-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type gamma-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the gamma-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.     

Simple sequence repeat (SSR) analysis in relation to calcium transport and signaling genes reveals transferability among grasses and a conserved behavior within finger millet genotypes

Being an excellent source of calcium, finger millet crop has nutraceutical importance. Mineral accumulation, being a polygenic trait, becomes essential to target potential candidate genes directly or indirectly involved in the regulation of calcium transport and signaling in cereals and might have influence on grain calcium accumulation. In view of this, genic microsatellite markers were developed from the coding and non-coding sequences of calcium signaling and transport genes viz. calcium transporters (channels; ATPases and antiporters), calcium-binding proteins and calcium-regulated protein kinases available in rice and sorghum. In total, 146 genic "simple sequence repeat" (SSR) primers were designed and evaluated for cross-transferability across a panel of nine grass species including finger millet. The average transferability of genic SSR markers from sorghum to other grasses was highest (73.2 %) followed by rice (63.4 %) with an overall average of 68.3 % which establishes the importance of these major crops as a useful resource of genomic information for minor crops. The transfer rate of SSR markers was also correlated with the phylogenetic relationship (or genetic relatedness) of the species. Primers with successful amplification in finger millet were further used to screen for polymorphism across a set of high and low calcium containing genotypes. The results reveal a conserved behavior across the finger millet genotypes indicating that the mineral transport and the storage machinery largely remain conserved in plants and even SSR variations in them remain suppressed during the course of evolution. Single nucleotide polymorphism and differential expression patterns of candidate genes, therefore, might be a plausible reason to explain variations in grain calcium contents among finger millet genotypes.     

An opaque-2-like transcription factor from pearl millet

Pearl millet (Pennisetum glaucum L.), a species of the Poaceae family, is an important food crop in Africa, Asia and South America. Its nutritional value is due to storage prolamins accumulated in the seeds. In other species of the same family, the expression of the genes coding for storage prolamins is mediated by the regulatory protein opaque-2. In this paper we show that an opaque-2 -like protein is present in pearl millet too and is expressed during the early stages of seed development. The organization of the gene coding for this protein is similar to that of orthologous genes in other Poaceae species, i.e. six exons separated by five introns. A comparison of amino acid homologies with other described opaque-2 proteins is presented.     

Molecular pharming in cereal crops

There are many different agricultural expression systems that can be used for the large-scale production of recombinant proteins, but field-grown cereal crops are among the most attractive because recombinant proteins can be targeted to accumulate in the seed, and specifically in the endosperm, which has evolved naturally as a protein storage tissue. Within the developing endosperm, proteins are supplied with molecular chaperones and disulfide isomerases to facilitate folding and assembly, while the mature tissue is desiccated to prevent proteolytic degradation. Proteins expressed in cereal seeds can therefore remain stable for years in ambient conditions. Recent basic research has revealed a surprising diversity of protein targeting mechanisms in the endosperm, which can help to control post-translational modification and accumulation. Applied research and commercial development has seen several pharmaceutical proteins produced in cereals reach late stage preclinical development and the first clinical trials, with a number of companies now dedicated to developing cereal-based production platforms. In this review we discuss the basic science of molecular pharming in cereals, some of the lead product candidates, and challenges that remain to be addressed including the emerging regulatory framework for plant-made pharmaceuticals.     

Fermentation of a composite finger millet-dairy beverage

Fermented composite beverages of finger millet and milk are popular, nutritious, traditional foods in many parts of Zimbabwe. With the aim of commercial production, we determined what type of microbial cultures can be used to ferment a composite finger millet and skimmed milk powder gruel and the optimum conditions for its production. Composites containing between 0 and 100% finger millet gruel by volume were inoculated and incubated at various temperatures. The desired pH of 4.5 or less was obtained with incubation at 30 to 45 °C (but not at lower temperatures) with lower pH values being obtained as the temperature increased. YC380 (a yoghurt type bacterial starter culture) produced a pH of 4.5 or less only when skim milk was also present; V2 (another yoghurt type bacterial starter culture) did so at all levels of finger millet gruel and JC (a mixed strain culture developed to ferment cereals) only when finger millet gruel was present. A clear relationship between incubation temperature and syneresis could not be established but syneresis decreased significantly (p < 0.05) with increasing proportions of finger millet gruel. A thick product with a set consistency was obtained with YC380 at an incubation temperature of 45 °C and a storage temperature of 7 °C regardless of proportion of finger millet gruel. V2 produced a thick product with a set consistency at an incubation temperature of 45 °C, and storage temperature of 7 °C and when the proportions of finger millet gruel were between 0 and 50%. It appears that yoghurt type bacterial cultures can be successfully used to produce a composite fermented beverage from finger millet and skim milk, but cultures developed for fermentation of cereals are not suitable.     

Delivery of prolamins to the protein storage vacuole in maize aleurone cells

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.     

Two distinct jacalin-related lectins with a different specificity and subcellular location are major vegetative storage proteins in the bark of the black mulberry tree

Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research.     

Isolation and characterisation of a NBS-LRR resistance gene analogue from pearl millet

Plant resistance (R) proteins belonging to nucleotide-binding site–leucine-rich repeat (NBS–LRR) family are mainly involved in recognition of effectors secreted by pathogens. Pearl millet [Pennisetum glaucum (L.) R.Br] is one of the most drought tolerant cereals, staple food crop of the semi-arid tropics but is highly susceptible to the downy mildew disease caused by oomycetous Sclerospora graminicola (Sacc) schroet. Earlier studies have identified several resistance gene analogues (RGAs) in pearl millet which may be involved in resistance against downy mildew. Of these, a clone RGPM213 was shown to have more than 60% identity with R-proteins coding for NBS–LRR-like protein kinase. The exact nature and function of the R-protein encoded by this gene was not known. In the present study, the cDNA of RGPM213 encompassing NBS–LRR region was inserted into an expression vector pRSET-A and transformed into BL21 E.coli cells. The expressed recombinant fusion protein with a His tag was purified using nickel affinity purification and it had a molecular weight of 35 kDa on SDS-PAGE. Immunoaffinity purification using antibodies raised against this recombinant R-protein identified two proteins of molecular weights 55 kDa and 66 kDa from pearl millet seedling extracts. Peptide mass fingerprinting of these proteins followed by homology search in database revealed similarity of the 55 kDa protein with a protein kinase from Brassica oleracia containing serine/ threonine kinase domain.     

A Full-Length Dof1 Transcription Factor of Finger Millet and its Response to a Circadian Cycle

DOF1 (DNA binding with one finger) plays an important role in regulating C/N metabolism in cereals. In order to validate its role in the regulation of nitrogen use efficiency (NUE) and photosynthetic efficiency in finger millet, 5′–3′ RACE PCR was performed to obtain and characterize full-length Dof1 genes of high and low grain protein finger millet genotypes. The full-length DOF1 ORFs were both 1,284 nt long and were 98.8 % similar over 427 amino acids containing the characteristic Dof domain. Comparison of both the EcDof1 protein sequences with the Dof1 of other cereals revealed high sequence similarity to the Dof1 of rice. Southern hybridization carried out using the probe developed from the region encoding the highly variable C-terminal region of EcDof1 showed the presence of four copies of the DOF1 gene in finger millet, which might explain the high NUE and photosynthetic performance of finger millet. Since the genes involved in C/N metabolism are regulated diurnally and play crucial roles in determining grain protein content during grain filling, the diurnal expression of EcDOF1 was assessed in two finger millet genotypes (GE 3885 and GE 1437) with differing grain protein content (13.8 % and 6.15 % respectively). It was found that EcDOF1 exhibited diurnal regulation and peak differential pattern expression with early phasing in GE3885 and late phasing in GE1437. Differential expression of DOF1 might alter the regulation of genes involved in C/N metabolism affecting grain protein composition of finger millet genotypes.     

Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to lgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T. aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye), Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.     

Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T.aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye),Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.     

Foods for patients with celiac disease.

As a general rule patients with celiac disease must avoid five cereals--wheat rye, triticale, barley and oats. Very sensitive individuals must also avoid two products of these cereals--malt and hydrolyzed vegetable protein. Some less sensitive individuals may be able to tolerate barley and oats in small quantities. All other foods are acceptable, including the cereals corn, rice, buckwheat, millet and sorghum, as well as malt-flavored breakfast cereals. Wine, spirits, beer and ale are also acceptable unless otherwise contraindicated. Monosodium glutamate, other food additives and pharmaceutical preparations are also acceptable. The ingredients of prepackaged processed foods are listed on the labels. Patients with celiac disease must examine labels to ensure that they avoid the harmful cereals. With appropriate precautions they need not be concerned about eating away from home.     

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.     

Localization of a nuclear protein (QP47) in embryonicmeristems during seed maturation and germination andits distribution among crop plants

We report a desiccation-associated protein (QP47) which appearsto be localized in the nucleoplasm of embryonic meristems duringseed maturation and seed germination. The variations of nuclearlocalization shown by this protein during seed maturation andgermination might depend upon the nuclear and nucleolar rearrangementfollowing variations in moisture content in the seed tissues.The nucleolus is the compartment where the most obvious changesin chromatin organization take place. It is suggested that thisprotein has a role in the protection of the nucleus from desiccation.QP47 is present in nuclei of legumes, butnot in cereals or recalcitrantseeds. In cereals, other nuclear proteins with a putative functionsimilar to QP47 have been identified. The fact that the quantitativevariations of QP47 seem to be unique to an embryonic stage ofplant development suggests that this protein may act as an atypical desiccation protein with an additional function in storage.Such an additional storage function would also explain the restrictedoccurrence of QP47 to members of the Leguminosae. Key words: Nuclear protein, meristems, seed maturation, seed germination     

Glutathione transferase, but not agglutinin, is a dormancy-related protein in Castanea crenata trees.

The annual changes in Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) were investigated by both protein and RNA blotting analyses, to clarify whether CCA has a function as storage protein. In the woody part of shoots and leaves, CCA expression was only detected at both the protein and RNA levels in May and June. In buds, the CCA protein and mRNA expressions were both restricted to April. However, the amount of accumulated CCA was too low to act as a nitrogen reserve. No expression was observed in the bark at any time point, suggesting that bark does not contain either CCA or CCA-like proteins. These results suggest that CCA may be required in young organs as a defense protein, rather than as a storage protein. In addition, CCA was not related to dormancy, unlike some other woody plant bark lectins. In contrast to CCA, a 28kDa polypeptide was observed to accumulate during dormancy. Sequence analysis indicated that this polypeptide was a glutathione transferase. After cDNA cloning, RNA blot analyses indicated that this glutathione transferase was strongly expressed in woody parts during mid-winter. In shoots, this protein represented approximately 10% of the total soluble protein content. Therefore, in Japanese chestnut trees, glutathione transferase may play a nitrogen storage role in addition to its intrinsic defensive role against stresses during dormancy.     

Unusual features of cereal seed protein structure and evolution

The alcohol-soluble (prolamin) storage proteins of barley, wheat and rye vary in their structures, but all have two features in common: the presence of distinct structural domains differing in amino acid compositions, and of repeats within one of these domains. Detailed comparisons of amino acid sequences show that all appear to have evolved from a single ancestral gene consisting of three short related regions (called A, B and C). Regions related to A, B and C are also present in the minor prolamins of maize and in three other groups of seed proteins: inhibitors of alpha-amylase and/or trypsin from cereals. 25 storage globulins from several dicotyledonous species and a 2S albumin from sunflower. It is suggested that these proteins together constitute a protein superfamily with limited sequence homology.     

Deposition of storage proteins

Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.     

Protein storage vacuole acidification as a control of storage protein mobilization in soybeans

Soybean protease C1 (EC 3.4.21.25), the subtilisin-like serine protease that initiates the proteolysis of seed storage proteins in germinating soybean [Glycine max (L.) Merrill], was localized to the protein storage vacuoles of parenchyma cells in the cotyledons by immunoelectron microscopy. This was demonstrated not only in germination and early seedling growth as expected, but also in two stages of protein storage vacuole development during seed maturation. Thus, the plant places the proteolytic enzyme in the same compartment as the storage proteins, but is still able to accumulate those protein reserves. Since soybean protease C1 activity requires acidic conditions for activity, the hypothesis that the pH condition in the protein storage vacuole would support protease C1 activity in germination, but not in seed maturation, was tested. As hypothesized, acridine orange accumulation in the protein storage vacuole of storage parenchyma cells was detected by fluorescence confocal microscopy in seedlings before the onset of mobilization of reserve proteins as noted by SDS-PAGE. Accumulation of the dye was reversed by inclusion of the weak base methylamine to dissipate the pH gradient across the vacuolar membrane. Also as hypothesized, acridine orange did not accumulate in the protein storage vacuole of those parenchyma cells during seed maturation. These results were obtained using cells separated by pectolyase treatment and also using cotyledon slices.     

The role of intercropping different cereal species in controlling lepidopteran stemborers on maize in Kenya

Abstract:  The effects of mixed cropping systems containing maize, sorghum, millet and beans on infestations of cereals by lepidopteran stemborers and on associated parasitoids, as well as on yields and land equivalent ratios (LER) were assessed during four consecutive rainy seasons at two sites in the semi-arid eastern region of Kenya. Systems containing the non-host bean were more efficient in reducing pest densities than those with millet or sorghum only. Higher parasitism in diversified systems compared to monocrops was due to density-dependent effects rather than superior suitability of such systems to parasitoids. The maize–bean system, which had the highest proportion of bean plants, had LERs >1.65 while most other systems had LERs <1. It is concluded that mixed cropping with several cereal species has little advantages in terms of yield loss abatement due to stemborers and land use efficiency. However, including the drought-tolerant crops such as sorghum and millet in the system stabilizes food security in drought-prone areas such as eastern Kenya.