Non-zein Proteins Research Articles

Accumulation of 22 kDa α-zein-mediated nonzein protein in protein body of maize endosperm.

Protein bodies (PBs), the major protein storage organelle in maize (Zea mays) endosperm, comprise zeins and numerous nonzein proteins (NZPs). Unlike zeins, how NZPs accumulate in PBs remains unclear. We characterized a maize miniature kernel mutant, mn*, that produces small kernels and is embryo-lethal. After cloning the Mn* locus, we determined that it encodes the mitochondrial 50S ribosomal protein L10 (mRPL10). MN* localized to mitochondria and PBs as an NZP; therefore, we renamed MN* Non-zein Protein 1 (NZP1). Like other mutations affecting mitochondrial proteins, mn* impaired mitochondrial function and morphology. To investigate its accumulation mechanism to PBs, we performed protein interaction assays between major zein proteins and NZP1, and found that NZP1 interacts with 22 kDa α-zein. Levels of NZP1 and 22 kDa α-zein in various opaque mutants were correlated. Furthermore, NZP1 accumulation in induced PBs depended on its interaction with 22 kDa α-zein. Comparative proteomic analysis of PBs between wild-type and opaque2 revealed additional NZPs. A new NZP with plastidial localization was also found to accumulate in induced PBs via interaction with 22 kDa α-zein. This study thus reveals a mechanism for accumulation of NZPs in PBs and suggests a potential application for the accumulation of foreign proteins in maize PBs.

Deletion of maize RDM4 suggests a role in endosperm maturation as well as vegetative and stress-responsive growth.

Opaque kernels in maize may result from mutations in many genes, such as OPAQUE-2. In this study, a maize null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4) showed an opaque kernel phenotype, as well as plant developmental delay, male sterility, and altered response to cold stress. We found that in opaque kernels, all zein proteins were reduced and amino acid content was changed, including increased lysine. Transcriptomic and proteomic analysis confirmed the zein reduction and proteomic rebalancing of non-zein proteins, which was quantitatively and qualitatively different from opaque-2. Global transcriptional changes were found in endosperm and leaf, including many transcription factors and tissue-specific expressed genes. Furthermore, of the more than 8000 significantly differentially expressed genes in wild type in response to cold, a significant proportion (25.9% in moderate cold stress and 40.8% in near freezing stress) were not differentially expressed in response to cold in rdm4, suggesting RDM4 may participate in regulation of abiotic stress tolerance. This initial characterization of maize RDM4 provides a basis for further investigating its function in endosperm and leaf, and as a regulator of normal and stress-responsive development.

Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins.

Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm.

Microtubule-Associated Protein SBgLR Facilitates Storage Protein Deposition and Its Expression Leads to Lysine Content Increase in Transgenic Maize Endosperm.

Maize (Zea mays) seed is deficient in protein and lysine content. Many studies have been made to improve the nutritional quality of maize seeds. Previously, we reported the role of a natural lysine-rich protein gene SBgLR in increasing protein and lysine content. However, how the SBgLR improves lysine and protein content remains unclear. Here, the reasons and possible mechanism for SBgLR in protein and lysine improvement have been analyzed and discussed. Through seed-specific expression of SBgLR, we obtained transgenic maize with the simultaneously increased lysine and protein contents. High-protein and high-lysine characters were stably inherited across generations. The expression of SBgLR in maize kernels increased the accumulation of both zeins and non-zein proteins. Transmission electron microscopy showed that the number of protein bodies (PBs) was increased obviously in SBgLR transgenic immature endosperms with the morphology and structure of PBs unchanged. The proteinaceous matrix was more abundant in transgenic mature endosperms under scanning electron microscopy. The stabilities of zein and lysine-rich non-zein genes were also increased in transgenic endosperms. Finally, the potential application of SBgLR in maize nutrient improvement was evaluated. This study shows that a cytoskeleton-associated protein has potential applicable value in crop nutrient improving, and provided a feasible strategy for improvement of maize grain quality.

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize.

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.

Proteome balancing of the maize seed for higher nutritional value.

Most flowering plant seeds are composed of the embryo and endosperm, which are surrounded by maternal tissue, in particular the seed coat. Whereas the embryo is the dormant progeny, the endosperm is a terminal organ for storage of sugars and amino acids in proteins and carbohydrates, respectively. Produced in maternal leaves during photosynthesis, sugars, and amino acids are transported to developing seeds after flowering, and during germination they nourish early seedlings growth. Maize endosperm usually contains around 10% protein and 70% starch, and their composition ratio is rather stable, because it is strictly regulated through a pre-set genetic program that is woven by networks of many interacting or counteracting genes and pathways. Endosperm protein, however, is of low nutritional value due mainly to the high expression of the α-zein gene family, which encodes lysine-free proteins. Reduced levels of these proteins in the opaque 2 (o2) mutant and α-zein RNAi (RNA interference) transgenic seed is compensated by an increase of non-zein proteins, leading to the rebalancing of the nitrogen sink and producing more or less constant levels of total proteins in the seed. The same rebalancing of zeins and non-zeins has been observed for maize seeds bred for 30% protein. In contrast to the nitrogen sink, storage of sulfur is controlled through the accumulation of specialized sulfur-rich proteins in maize endosperm. Silencing the synthesis of α-zeins through RNAi fails to raise sulfur-rich proteins. Although overexpression of the methionine-rich δ-zein can increase the methionine level in seeds, it occurs at least in part at the expense of the cysteine-rich β- and γ-zeins, demonstrating a balance between cysteine and methionine in sulfur storage. Therefore, we propose that the throttle for the flow of sulfur is placed before the synthesis of sulfur amino acids when sulfur is taken up and reduced during photosynthesis.

Maize Endosperm Protein Extraction and Analysis

Alcohol-solubility is the most characteristic feature of the zein proteins, the major storage protein in maize. Using sodium borate buffer system with added reducing agent, total proteins are isolated, and zein proteins are separated from non-zein proteins. The extraction effect is intuitive on a SDS-PAGE isolation system. In addition, a simple and rapid approach to extract zeins is introduced, taking full advantage of alcohol-solubility of zeins directly.

Technical note: Quantification of zeins from corn, high-moisture corn, and corn silage using a turbidimetric method: Comparative efficiencies of isopropyl and tert-butyl alcohols

Zeins are corn endosperm storage proteins that encapsulate starch granules into a protein matrix, which can act as a barrier to starch accessibility and digestion. Laboratory methods to quantify zein are seldom used because they are considered arduous and time-consuming. A recently published rapid turbidimetric method (mTM) was reinvestigated by changing the solution originally used for the zein solubilization step. In particular, the aim was to explore whether, and to what extent, the use of tert-butyl alcohol (t-BuOH-mTM) in lieu of isopropyl alcohol (i-PrOH-mTM) was able to improve the quantification of zeins from dry corn, high-moisture corn, and corn silage samples. The nature of the alcohol influenced the zein extraction values, and t-BuOH-mTM gave higher zein values in corn (3.6 vs. 3.3g/100g of dry matter) and corn silage samples (1.2 vs. 0.9g/100g of dry matter) compared with i-PrOH-mTM. In contrast, similar zein extraction values were obtained for high-moisture corn (2.1 vs. 1.9g/100g of dry matter, respectively). Sodium dodecyl sulfate-PAGE analysis revealed no contamination by nonzein proteins with the use of tert-butyl alcohol. Overall, these findings indicated that tert-butyl alcohol has a greater ability to solubilize zein compared with isopropyl alcohol and thus the t-BuOH-mTM allowed greater extraction of zeins. Considering the growing interest of animal nutritionists in zein proteins, such results should provide useful information for routine laboratory analysis.

RNA Interference Can Rebalance the Nitrogen Sink of Maize Seeds without Losing Hard Endosperm

BackgroundOne of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels.MethodsThe most predominant zeins are the 22- and 19-kDa α-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both α-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed.ConclusionsThe α-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.

γ-Zeins are essential for endosperm modification in quality protein maize

Essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of zein storage proteins that lack these amino acids. A natural mutant, opaque 2 (o2) causes reduction of zeins, an increase of nonzein proteins, and as a consequence, a doubling of lysine levels. However, o2's soft inferior kernels precluded its commercial use. Breeders subsequently overcame kernel softness, selecting several quantitative loci (QTLs), called o2 modifiers, without losing the high-lysine trait. These maize lines are known as "quality protein maize" (QPM). One of the QTLs is linked to the 27-kDa gamma-zein locus on chromosome 7S. Moreover, QPM lines have 2- to 3-fold higher levels of the 27-kDa gamma-zein, but the physiological significance of this increase is not known. Because the 27- and 16-kDa gamma-zein genes are highly conserved in DNA sequence, we introduced a dominant RNAi transgene into a QPM line (CM105Mo2) to eliminate expression of them both. Elimination of gamma-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to gamma-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2/+; o2/o2; gammaRNAi/+ genotype suggests that gamma-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2, the 22-kDa alpha-zein, gamma-zein, and beta-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that gamma-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding.

Compositional and transcriptional analyses of reduced zein kernels derived from the opaque2 mutation and RNAi suppression

Corn protein is largely made up of a group of nutritionally limited storage proteins known as zein. The reduction of zein can be achieved by a transcriptional mutation, opaque2 (o2), or a transgene targeting zein through RNA interference (RNAi). Zein reduction results in an increase of more nutritionally balanced non-zein proteins, and therefore enhance the overall quality of corn protein. In this study, the composition of mature kernels and the transcriptional profile of developing kernels of these two types of zein reduced kernels were compared. Both zein reduced kernels contained higher levels of lysine and tryptophan and free amino acids were 10-20-folds more abundant than the wild-type counterpart. We also found that free lysine contributed partially to the increased lysine in o2 kernels while protein-bound lysine was mainly responsible for the increased lysine in transgenic zein reduction (TZR) kernels. Although they had relatively similar gene expression patterns in developing endosperm, o2 kernels had greater transcriptional changes than TZR kernels in general. A number of transcripts that were specifically down-regulated in o2 were identified. Many promoter sequences of these transcripts contain putative O2 binding motifs, suggesting that their expression is directly regulated by O2.

Identification of non‐zein proteins in BR473 maize protein bodies by LC‐nanoESI‐MS/MS

The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.

High Lysine and High Tryptophan Transgenic Maize Resulting from the Reduction of Both 19- and 22-kD α-zeins

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD alpha-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, aspartate and glutamate, was observed in the zein reduction kernels.

Maize Endosperm Proteins That Contribute to Endosperm Lysine Content

ABSTRACTNonzein proteins were analyzed using SDS‐PAGE to study their relationship to lysine level in mature endosperm of three isogenic lines and 29 inbred lines. The majority of nonzein proteins are glutelins that contain a relatively high lysine content. Trinitrobenzene‐sulfonic acid was used to visualize high‐lysine proteins on nitrocellulose membrane. Seven intense yellow bands (high‐lysine proteins) were found in the glutelin fraction: 35, 43, 48, 52, 84, 92, and 100 kDa. These seven glutelin proteins were also significantly correlated to each other. Lysine content of the triads correlated with three glutelin proteins (35, 43, and 52 kDa), while lysine content of 29 inbred lines correlated with six glutelin proteins (all except 52‐kDa protein). Immunoassays based on these higher lysine glutelin proteins provide an effective screening method in the presence of variable zein levels. Retention or improvement of lysine content is possible using this information in a maize improvement program.

The origin of lysine-containing proteins in opaque-2 maize endosperm

The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 micrograms of lysine compared with 350 micrograms in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.

New Methods for Extraction and Quantitation of Zeins Reveal a High Content of γ-Zein in Modified opaque-2 Maize

We have developed methods for quantitative extraction and analysis of zeins from maize (Zea mays L.) flour. Extraction involved solubilization of total endosperm proteins in an alkaline buffer containing SDS and 2-mercaptoethanol with subsequent precipitation of nonzein proteins by the addition of ethanol to 70%. Analysis of these proteins by SDS-PAGE with Coomassie blue staining and by Western blotting and ELISA assay with zein antibodies revealed that this extraction method is more quantitative than the traditional Landry-Moureaux procedure, especially for the beta- and gamma-zeins. This method was used to extract and analyze the zein content of several ;Quality Protein Maize' (QPM) varieties developed by the International Maize and Wheat Improvement Center. QPM varieties contain ;modifier genes' that confer a vitreous phenotype on opaque-2 genotypes, while maintaining the elevated levels of lysine and tryptophan characteristic of this mutant. This analysis revealed that the QPM types contain 2 to 4 times the amount of the gamma-zein than unmodified opaque-2 or normal maize varieties. Possible relationships between the high expression of the gamma-zein and the modified opaque phenotype are discussed.