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.     

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.     

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.Thirty-and 35-DAP seeds when removed from the fruit tissue and imbibed on water, complete germination by 120 h after isolation. Only seeds which have developed to 35 DAP produce viable seedlings. The inability of isolated 30-DAP seed to form viable seedlings appears to be related to a lack of stored nutrients, since the germinability of excised embryos (20 DAP and onwards) placed on Murashige and Skoog (1962, Physiol. Plant. 15, 473–497) medium is high. The switch from a developmental to germinative mode in the excised 30- and 35-DAP imbibed seeds is reflected in the pattern of in-vivo protein synthesis. Developmental and germinative proteins are present in the embryo and endosperm of the 30- and 35-DAP seeds 12 h after their isolation from the fruit. The mature seed (60 DAP) exhibits germinative protein synthesis from the earliest time of imbibition. The fruit environment prevents precocious germination of developing seeds, since the switch from development to germination requires only their removal from the fruit tissue.Abbreviations DAP days after pollination - kDa kilodaltons - SP1-4 storage proteins 1–4 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - HASI hours after seed isolation - MS medium Murashige and Skoog (1962) medium This work is supported by National Science and Engineering Research Council of Canada grant A2210 to J.D.B.     

Cell wall invertase-deficient miniature1 kernels have altered phytohormone levels

The Zea mays (maize) miniature1 (Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer (BETL) of developing kernels and catalyzes conversion of sucrose into glucose and fructose. Mutations in Mn1 result in numerous changes that include a small kernel phenotype resulting from both decreased cell size and number. To explore the pleiotropic effects of this mutation, we investigated the levels of indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in basal regions, upper regions, and embryos of developing kernels in the inbred line W22. We measured phytohormones from 6 to 28 days after pollination (DAP) in wild type (WT) and two alleles of mn1, mn1-1 and mn1-89. IAA was the predominant hormone in kernels, with WT levels of free IAA accumulating over time to more than 2microg/g of fresh weight. Kernels of mn1-1 accumulated up to 10-fold less IAA than WT, and levels of IAA sugar conjugates were similarly reduced. Although less abundant, differences were also observed in levels of ABA, JA, and SA between WT and the mn1 alleles. SA levels were increased by as much as 10-fold in mn1-1, and mn1-89 displayed intermediate SA levels at most timepoints. These findings indicate that invertase-mediated sucrose cleavage directly or indirectly regulates the levels of key plant hormones during seed development.     

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.     

eEF1A isoforms change in abundance and actin-binding activity during maize endosperm development

Eukaryotic elongation factor 1A (eEF1A) appears to be a multifunctional protein because several biochemical activities have been described for this protein, in addition to its role in protein synthesis. In maize (Zea mays) endosperm, the synthesis of eEF1A is increased in o2 (opaque2) mutants, and its concentration is highly correlated with the protein-bound lysine content. To understand the basis of this relationship, we purified eEF1A isoforms from developing endosperm and investigated their accumulation and their functional and structural properties. Formation of three isoforms appears to be developmentally regulated and independent of the o2 mutation, although one isoform predominated in one high lysine o2 inbred. The purified proteins differ in their ability to bind F-actin in vitro, suggesting that they are functionally distinct. However, they share similar aminoacyl-tRNA-binding activities. Tandem mass spectrometry revealed that each isoform is composed of the four same gene products, which are modified posttranslationally by methylation and phosphorylation. The chemical differences that account for their different actin-binding activities could not be determined.     

Narrow A/T-rich zones present at the distal 5′-flanking sequences of the zein genes Zc1 and Zc2 bind a unique 30 kDa HMG-like protein

Nuclear extracts from maize endosperm were used to investigate protein-DNA interactions in the 5-upstream region of the Zc1 and Zc2 genes. These genes encode for zeins of apparent molecular mass (MW_app) 16 and 28 kDa, respectively, which accumulate in the endosperm during seed maturation. Binding assays revealed specific binding of a nuclear protein to three A/T-rich elements, 0.9–1.0 kbp upstream from the initiation codon. One of these elements (41 bp, 88% A/T), present in Zc1, contained a 13 nucleotide duplication. The other two (28 bp, 86% A/T; 42 bp alternating A-T) are consecutive elements in Zc2. Competition experiments strongly suggest that the three elements bind to the same protein. Protein-DNA interaction was detected in endosperm nuclear extracts of 8 to 21 days after pollination (DAP), as well as in 25 DAP embryos and in different tissues from plantlets. The protein factor has an MW_app of ca. 30 kDa. This factor has properties suggesting it is an HMG-like protein. These results are consistent with a growing accumulation of data for a number of genes indicating that A/T-rich elements, located at distal and proximal zones of the 5-flanking sequences, interact with HMG-like proteins.     

Characterization of maize elongation factor 1A and its relationship to protein quality in the endosperm.

The protein synthesis elongation factor 1A (eEF1A) is a multifunctional protein in eukaryotic cells. In maize (Zea mays L.) endosperm eEF1A co-localizes with actin around protein bodies, and its accumulation is highly correlated with the protein-bound lysine (Lys) content. We purified eEF1A from maize kernels by ammonium sulfate precipitation, ion-exchange, and chromatofocusing. The identify of the purified protein was confirmed by microsequencing of an endoproteinase glutamic acid-C fragment and by its ability to bundle actin. Using purified eEF1A as a standard, we found that this protein contributes 0.4% of the total protein in W64A+ endosperm and approximately 1% of the protein in W64Ao2. Because eEF1A contains 10% Lys, it accounts for 2.2% of the total Lys in W64A+ and 2.3% of the Lys in W64Ao2. However, its concentration predicts 90% of the Lys found in endosperm proteins of both genotypes, indicating that eEF1A is a key component of the group of proteins that determines the nutritional quality of the grain. This notion is further supported by the fact that in floury2, another high-Lys mutant, the content of eEF1A increases with the dosage of the floury2 gene. These data provide the biochemical basis for further investigation of the relationship between eEF1A content and the nutritional quality of cereals.     

Amino acid requirement of growing pigs depending on amino acid efficiency and level of protein deposition 1st communication: lysine

The study was conducted to evaluate the variability of efficiency of lysine utilisation in different feed proteins for growing pigs including wheat, unprocessed soybean flakes (SF), hydrothermal processed SF, corn gluten meal (CGM), two batches of soybean protein concentrate (SPC-1, SPC-2), different batches of peas (Pisum sativum) and field beans (Vicia faba). Data about efficiency of lysine are important for further conclusions related to lysine requirement in dependence on level of daily protein deposition. In N-balance studies 161 growing barrows (40-65 kg BW) of the genotype [Piétrain x (Duroc x Landrace)] were randomly allotted into 23 experimental treatments (n = 7) with diets in which lysine was the first limiting amino acid. Data from the N-balance trials were used to calculate efficiency of lysine and consequently lysine requirement based on an exponential N-utilisation model. Results from the present studies indicate that efficiency of lysine in different feed proteins varies in a very wide range and this variation greatly affected the calculated lysine requirement. Therefore, the variation in efficiency of lysine should be taken into account in requirement calculations and consequently in diet formulation for pigs. The results of model calculation for the lysine requirement depending on daily protein deposition (130, 145 and 160 g/d) and efficiency of lysine are given for different standards for comparison (g x kg(-0.67) x d(-1), g x d(-1) and percentage of lysine in the diet). The calculated lysine requirement of growing barrows (50 kg BW) corresponding to an average lysine efficiency was 15.5, 18.0 and 21.1 g/d for daily protein deposition of 130, 145 and 160 g, respectively. The results of the model calculation for 145 or 160 g daily protein deposition are in agreement with actual studies and recommendations of NRC (1998) and DLG (2002) for lysine supply.     

玉米O2/o2近等基因系胚乳中基因表达差异分析

对玉米Hz85(O2/O2)和 Hz85(o2/o2)以及S7913(O2/O2)和 S7913 (o2/o2)两种不同核背景下的近等基因系(NIL),采用cDNA芯片杂交技术研究玉米o2突变基因及赖氨酸形成和胚乳发生相关基因在RNA水平上的表达差异。在两套NILs中检测到共同的表达差异克隆87个,对其序列分析得到26个TUGs（tentative unique genes）、11个未命名蛋白和6个新序列。这些TUGs分别参与了生长发育、胁迫响应、物质运输、信号转导、电子传递链、细胞防御、代谢等细胞过程,以及作为细胞组分和贮存蛋白。基于O2/o2 NILs间胚乳发育中基因表达的差异,讨论了o2籽粒中的不透明粉质胚乳形成的分子机制。     

Molecular characterization of BET1, a gene expressed in the endosperm transfer cells of maize.

A cDNA clone, BET1 (for basal endosperm transfer layer), was isolated from a cDNA bank prepared from 10-days after pollination (DAP) maize endosperm mRNA. BET1 mRNA was shown to encode a 7-kD cell wall polypeptide. Both the mRNA and protein were restricted in their distribution to the basal endosperm transfer layer and were not expressed elsewhere in the plant. BET1 expression commenced at 9 DAP, reached a maximum between 12 and 16 DAP, and declined after 16 DAP. The initial accumulation of the BET1 polypeptide reached a plateau by 16 DAP and declined thereafter, becoming undetectable by 20 DAP. The antibody raised against the BET1 protein reacted with a number of polypeptides of higher molecular mass than the BET1 monomer. Most of these were present in cytosolic fractions and were found in nonbasal cell endosperm extracts, but three species appeared to be basal cell specific. This result and the reactivity of exhaustively extracted cell wall material with the BET1 antibody suggest that a fraction of the protein is deposited in a covalently bound form in the extracellular matrix. We propose that the BET1 protein plays a role in the structural specialization of the transfer cells. In addition, BET1 provides a new molecular marker for the development of this endosperm domain.     

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.     

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.     

Physicochemical properties of endosperm and pericarp starches during maize development

Endosperm starch and pericarp starch were isolated from maize (B73) kernels at different developmental stages. Starch granules, with small size (2–4 μm diameter), were first observed in the endosperm on 5 days after pollination (DAP). The size of endosperm-starch granules remained similar until 12DAP, but the number increased extensively. A substantial increase in granule size was observed from 14DAP (diameter 4–7 μm) to 30DAP (diameter10–23 μm). The size of starch granules on 30DAP is similar to that of the mature and dried endosperm-starch granules harvested on 45DAP. The starch content of the endosperm was little before 12DAP (less than 2%) and increased rapidly from 10.7% on 14DAP to 88.9% on 30DAP. The amylose content of the endosperm starch increased from 9.2% on 14DAP to 24.2% on 30DAP and 24.4% on 45DAP (mature and dried). The average amylopectin branch chain-length of the endosperm amylopectin increased from DP23.6 on 10DAP to DP26.9 on14DAP and then decreased to DP25.4 on 30DAP and DP24.9 on 45DAP. The onset gelatinization temperature of the endosperm starch increased from 61.3 °C on 8DAP to 69.0 °C on 14DAP and then decreased to 62.8 °C on 45DAP. The results indicated that the structure of endosperm starch was not synthesized consistently through the maturation of kernel. The pericarp starch, however, showed similar granule size, starch content, amylose content, amylopectin structure and thermal properties at different developmental stages of the kernel.     

On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.