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

Azama, Kishu; Abe, Shunnosuke; Sugimoto, Hideki; Davies, Eric

doi:10.1007/s00425-003-1016-5

Cited by 37 publications

(32 citation statements)

References 37 publications

(76 reference statements)

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“…Aldolase also has been reported to interact with V-type ATPases, leading to the suggestion that glycolysis may be localized to regions of high ATP demand (Lu et al, 2001). In plants, phosphoglycerate kinase, glyceraldehyde-3-P dehydrogenase, and aldolase have been shown to be present in the nuclei of pea leaf cells (Anderson et al, 1995), and glyceraldehyde-3-P dehydrogenase and aldolase also were shown to associate with the cytoskeleton in developing maize endosperm (Azama et al, 2003). Very recently, glycolytic enzymes were identified in a proteomic survey of human mitochondria (Taylor et al, 2003), although the functional significance of this observation was not investigated further.…”

Section: Discussionmentioning

confidence: 99%

Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells

et al. 2003

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Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase-and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of 13 C-glucose to 13 C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

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Section: Discussionmentioning

confidence: 99%

Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells

et al. 2003

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“…4B, spot b1 was detected within 0.5 h after gravitational stimulation, but the shift in molecular weight up to the b2 position occurred at 3 h after gravitational stimulation. Spots b1 and b2 were putatively identified as fructose bisphosphate aldolase (15226185), which corresponds to an enzyme involved in carbohydrate metabolism, i.e., in glycolysis (Angeles-Castillejo et al 2004, Azama et al 2003. In the panel shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Proteomic analysis of Arabidopsis root gravitropism

2005

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In gravitropism of Arabidopsis root, the Arabidopsis root proteins induced and/or changed by gravitational stimulation were analyzed using a proteomic method. In this study, it was found that the levels of expression of some proteins related to the cytoskeleton and the calcium signal transmission system had changed during gravitational stimulation. In addition, the β subunit of the E1 component of pyruvate dehydrogenase, fructose bisphosphate aldolase, and 20S proteasome β subunit E1 protein appeared in two different molecular weight types during gravitational stimulation. The transitory changes in molecular weight in these three identified proteins were important findings although their functions in the gravity response in the roots of Arabidopsis seedlings remain uncertain. As far as we know, this is the first study involving the molecular weight shift-change in the same protein during the gravitropism response in plants using proteomic analysis.

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“…Interestingly, some of these enzymes, such as Suc synthase (Carneiro, 1998;Winter et al, 1998), glyceraldehyde-3-phosphate dehydrogenase, and Fru-1,6-bisphosphate aldolase were recently reported to be associated with the cytoskeleton (Azama et al, 2003). The Lys content of these proteins varies from about 5% to 8.5%, and their masses, together with those of structural cytoskeletal proteins (actin, tubulin, and eEF1A), were reported to contribute significantly (approximately 67%) to the total endosperm Lys content of W64Ao2 (Azama et al, 2003). Several endosperm proteins found to associate with actin in yeast two-hybrid interactions (Carneiro, 1998) included a Ser-Thr protein kinase, 60S acidic ribosomal protein, E2 ubiquitin-conjugating enzyme, and genes encoding these proteins were also upregulated in high eEF1A genotypes (Table I).…”

Section: Discussionmentioning

confidence: 99%

“…Cytoskeleton-associated proteins are not as abundant as zeins, but combined, their masses appear to make a substantial contribution to the Lys content of the grain (Azama et al, 2003). By selecting for maize genotypes, particularly Quality Protein Maize lines (Moro et al, 1996;Zarkadas et al, 2000) with high levels of eEF1A, it should be possible to create maize kernels that meet the Lys requirement for humans and other monogastric animals.…”

Section: Discussionmentioning

confidence: 99%

Cytoskeletal Proteins Are Coordinately Increased in Maize Genotypes with High Levels of eEF1A

et al. 2004

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The opaque2 (o2) mutation increases the Lys content of maize (Zea mays) endosperm by reducing the synthesis of zein storage proteins and increasing the accumulation of other types of cellular proteins. Elongation factor 1A (eEF1A) is one of these proteins, and its concentration is highly correlated with the amount of other Lys-containing proteins in the endosperm. We investigated the basis for this relationship by comparing patterns of protein accumulation and gene expression between a high (Oh51Ao2) and a low (Oh545o2) eEF1A inbred, as well as between high and low eEF1A recombinant inbred lines obtained from their cross. The content of a-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. To extend this analysis, we used an endosperm expressed sequence tag microarray to examine steady-state levels of RNA transcripts in developing endosperm of these genotypes. We identified about 120 genes coordinately regulated in association with eEF1A content. These genes encode proteins involved in several biological structures and processes, including the actin cytoskeleton, the endoplasmic reticulum, and the protein synthesis apparatus. Thus, higher levels of eEF1A in o2 mutants may be related to a more extensive cytoskeletal network surrounding the rough endoplasmic reticulum and increased synthesis of cytoskeleton-associated proteins, all of which contribute significantly to the Lys content of the endosperm.The discovery that the opaque2 (o2) mutation almost doubles the Lys content of maize (Zea mays) endosperm (Mertz et al., 1964) led to extensive efforts to develop genotypes with better nutritional quality for human and animal diets. Unfortunately, the inferior agronomic traits associated with this mutation, including reduced yield and protein content, soft kernels, and pathogen susceptibility, made it difficult to develop o2 in breeding programs (Glover and Mertz, 1987). To overcome these problems, maize breeders at International Center for Development of Maize and Wheat, Mexico (Villegas et al., 1992) and the University of Natal, South Africa (Gevers and Lake, 1992) developed modified o2 mutants called Quality Protein Maize (QPM). These genotypes combine the improved nutritional quality trait of o2 with those responsible for vitreous endosperm and normal yield. However, additional improvement of protein quality is required for Quality Protein Maize to meet the minimal Lys content (5 mg/100 mg of protein) recommended for human nutrition (Young et al., 1998).Several studies have documented a broad range of variability in Lys content among normal, o2, and modified o2 maize genotypes (Moro et al., 1996;Zarkadas et al., 2000), showing that the effect of the mutation is highly dependent on the genetic background. These studies suggested the Lys content of the kernel can be improved by appropriate selection. However, identification of more nutritional maize genotypes has been limited by knowledge of the genetic...

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

Cited by 37 publications

References 37 publications

Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells

Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells

Proteomic analysis of Arabidopsis root gravitropism

Cytoskeletal Proteins Are Coordinately Increased in Maize Genotypes with High Levels of eEF1A

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