Quantitative trait locus mapping of loci influencing elongation factor 1alpha content in maize endosperm

The nutritional value of maize (Zea mays) seed is most limited by its protein quality because its storage proteins are devoid of the essential amino acid lysine (Lys). The Lys content of the kernel can be significantly increased by the opaque-2 mutation, which reduces zein synthesis and increases accumulation of proteins that contain Lys. Elongation factor 1alpha (eEF1A) is one of these proteins, and its concentration is highly correlated with the Lys content of the endosperm. We investigated the genetic regulation of eEF1A and the basis for its relationship with other Lys-containing proteins by analyzing the progeny of a cross between a high (Oh51Ao2) and a low (Oh545o2) eEF1A maize inbred. We identified 83 simple sequence repeat loci that are polymorphic between these inbreds; the markers are broadly distributed over the genome (1,402 cM) with an average interval of 17 cM. Genotypic analysis of the F(2) progeny revealed two significant quantitative trait loci that account for 25% of the variance for eEF1A content. One of these is on the short arm of chromosome 4 and is linked with a cluster of 22-kD alpha-zein coding sequences; the other quantitative trait locus is on the long arm of chromosome 7. The content of alpha-zein and gamma-zein was measured in pools of high- and low-eEF1A individuals obtained from this cross, and a higher level of alpha-zein was found to cosegregate with high eEF1A content. Allelic variation at the 22-kD alpha-zein locus may contribute to the difference of eEF1A content between Oh51Ao2 and Oh545o2 by increasing the surface area of protein bodies in the endosperm and creating a more extensive network of cytoskeletal 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.     

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.     

Aspartate kinase 2. A candidate gene of a quantitative trait locus influencing free amino acid content in maize endosperm

The maize (Zea mays) Oh545o2 inbred accumulates an exceptionally high level of free amino acids, especially lysine (Lys), threonine (Thr), methionine, and iso-leucine. In a cross between Oh545o2 and Oh51Ao2, we identified several quantitative trait loci linked with this phenotype. One of these is on the long arm of chromosome 2 and is linked with loci encoding aspartate (Asp) kinase 2 and Asp kinase (AK)-homoserine dehydrogenase (HSDH) 2. To investigate whether these enzymes can contribute to the high levels of Asp family amino acids, we measured their specific activity and feedback inhibition properties, as well as activities of several other key enzymes involved in Lys metabolism. We did not find a significant difference in total activity of dihydrodipicolinate synthase, HSDH, and Lys ketoglutarate reductase between these inbreds, and the feedback inhibition properties of HSDH and dihyrodipicolinate synthase by Lys and/or Thr were similar. The most significant difference we found between Oh545o2 and Oh51Ao2 is feedback inhibition of AK by Lys but not Thr. AK activity in Oh545o2 is less sensitive to Lys inhibition than that in Oh51Ao2, with a Lys I50 twice that of Oh51Ao2. AK activity in Oh545o2 endosperm is also higher than in Oh51Ao2 at 15 d after pollination, but not 20 d after pollination. The results indicate that the Lys-sensitive Asp kinase 2, rather than the Thr-sensitive AK-HSDH2, is the best candidate gene for the quantitative trait locus affecting free amino acid content in Oh545o2.     

Effect of the opaque and floury mutations on the accumulation of dry matter and protein fractions in maize endosperm.

Grains of nine opaque (o) and floury (fl) mutants of maize (Oh43o1, Oh43o2, B79o5, B37o7, W22o10, W22o11, W22o13, Oh43fl1 and Oh43fl2) were examined for the weight proportions of their component tissues and the content of eight nitrogen fractions in their endosperms. A linear regression was found connecting the amounts (mg per endosperm) of zeins and true proteins (crude proteins minus non-protein nitrogen) for the non-opaque2 mutants. The data points connecting zeins to true proteins present in the mature endosperms of six wild-type (+) inbred lines and their o2 versions were located outside (+) or within (o2) the 95% confidence range of the regression line. The data obtained from the developing and mature endosperms of the W22o7 inbred line (Di Fonzo et al., Plant Sci. Lett., 1979, 77) and the floury portion of mature endosperms of three other wild-type inbred lines fell practically on the regression line. The effects of genotype and environmental factors upon the relative accumulation rate of zeins were assessed from the present results and the data taken from the literature concerning the quantitative interdependence between zeins and true proteins in immature and mature endosperms.     

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.     

Changes in the levels of seven proteins involved in polypeptide folding and transport during endosperm development of two barley genotypes differing in storage protein localisation

The Russian barley cultivar Nevsky lacks ₃ hordein and accumulates most of its hordein in the lumen of the endoplasmic reticulum and only a minor portion in the vacuole. In wild type barley and all other temperate cereals, storage proteins are deposited in the vacuole. F1 crosses revealed that the Nevsky phenotype is recessive; but the extent of hordein accumulation in the endoplasmic reticulum in F2 endosperm lacking ₃ hordein was very much less than in the Nevsky parent. In order to study the Nevsky endosperm phenotype we have measured the levels of seven proteins and two mRNAs involved in protein folding in the ER lumen or ER to Golgi transport during endosperm development. The protein levels were unaltered in Nevsky as compared to the wild-type variety Bomi. When the levels of these seven proteins were correlated with the rate of hordein accumulation, four of these (HSP70, PDI, Sar1p and Sec18p) were consistently up-regulated with hordein synthesis. Accumulation of hordein in the endoplasmic reticulum appears to be determined by the absence of ₃ hordein, or the product of a gene closely linked to it, plus one or more other recessive genes.     

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.     

A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress

Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.     

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.

     

Plant Nucleases: VI. GENETIC AND DEVELOPMENTAL VARIABILITY IN RIBONUCLEASE ACTIVITY IN INBRED AND HYBRID CORN ENDOSPERMS

The nuclease activity of developing corn endosperms was found to consist mainly of plant RNase I during the period of major deposition of dry weight. The RNase concentrations in most inbred lines and hybrids increased throughout development, but there were large differences among genotypes in the enzyme levels at all stages. Crosses were made among inbreds classified as containing high or low RNase levels. In most cases, the general patterns of enzyme levels during development of the hybrid endosperms were not changed greatly, or showed intermediate levels of activity compared to the inbred parents. When Oh43 was used as a maternal parent, two contrasting developmental patterns were produced by using two low RNase inbreds as pollen parents. There appear to be genetic controls not only on the gross RNase levels, but also on the timing of RNase synthesis and on its stability after the cells mature. Environmental influences on RNase levels in the endosperm were noted one year.At 18 days after pollination, the RNase levels in the endosperm crown were as much as 10 times higher than in the base. By 35 days after pollination, the enzyme levels were generally uniform; at 50 days, the basal tissue usually contained the highest levels. In some genotypes, however, the enzyme levels fell in the crown while they rose in the base. These changes suggest that RNase may be associated with developmental controls that operate as the different portions of the endosperm cease cell division and begin synthesis of starch and zein.     

Increased lysine content in rice grains by over-accumulation of BiP in the endosperm

Over-accumulation of lysine-rich binding protein (BiP) in the rice endosperm caused strong endoplasmic reticulum (ER) stress and reduced seed storage proteins, resulting in a relative increase in nutritionally balanced non-seed storage proteins. We show that transgenic rice with over-accumulated BiP was a high-lysine rice germplasm and that the over-accumulation of BiP in the endosperm offered a unique strategy to improve the lysine content of cereal grains.     

Kernel lysine content does not increase in some maize opaque2 mutants

The recessive mutant allele of the opaque2 gene (o2) alters the endosperm protein pattern and increases the kernel lysine content of maize (Zea mays L.). In this study, sequencing results showed that the o2 mutant was successfully introgressed into 12 elite normal maize inbred lines by marker assisted selection (MAS). The average genetic similarity between these normal inbred lines and their o2 near-isogenic lines (NILs) was more than 95%. Kernel lysine content increased significantly in most of o2 NILs lines relative to normal elite inbreds, but remained unchanged in the genetic backgrounds Dan598o2 and Liao2345o2. Moreover, the kernel characteristics of these two o2 NILs did not differ from the other inbred lines. The results of lysine content analysis in the F1 hybrids between Liao2345o2 and Dan598o2 and other o2 NILs demonstrated that gene(s) other than opaque2 may control kernel lysine content in these two o2 NILs. The results of zein analysis showed that 22-kD α-zein synthesis was reduced or absent, and the 19-kD α-zein synthesis was greatly reduced compared with the recurrent parents in most o2 NILs except for Dan598o2 and Liao2345o2. Our results indicate that gene(s) other than opaque2 may play more important roles in zein synthesis and kernel lysine content in some maize genetic backgrounds.     

Characterization of monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the endosperm

A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional aspartate kinase, the nature of the monofunctional aspartate kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast aspartate kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2-Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.     

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

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