The Maize High-Lysine Mutant opaque7 Is Defective in an Acyl-CoA Synthetase-Like Protein
Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA. Genetics
(Impact Factor: 5.96).
09/2011; 189(4):1271-80. DOI: 10.1534/genetics.111.133918
Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ∼1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.
Available from: David R Holding
- "Factors unrelated to prolamins are also essential for vitreous endosperm formation as demonstrated by opaque mutants such as mto140, o7, and o5 (Holding et al., 2010; Miclaus et al., 2011a; Myers et al., 2011). MTO140 encodes a member of the maize arogenate dehydrogenase family that are involved in tyrosine biosynthesis (Holding et al., 2010), while O7 encodes an acyl-CoA synthetase-like protein (Miclaus et al., 2011a). O5 encodes the major biosynthetic enzyme for synthesis of chloroplast membrane lipids, monogalactosyldiacylglycerol synthase and the mutant is specifically defective in galactolipids necessary for amyloplast and chloroplast function (Myers et al., 2011). "
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ABSTRACT: Prolamin storage proteins are the main repository for nitrogen in the endosperm of cereal seeds. These stable proteins accumulate at massive levels due to the high level expression from extensively duplicated genes in endoreduplicated cells. Such abundant accumulation is achieved through efficient packaging in endoplasmic reticulum localized protein bodies in a process that is not completely understood. Prolamins are also a key determinant of hard kernel texture in the mature seed; an essential characteristic of cereal grains like maize. However, deficiencies of key essential amino acids in prolamins result in relatively poor grain protein quality. The inverse relationship between prolamin accumulation and protein quality has fueled an interest in understanding the role of prolamins and other proteins in endosperm maturation. This article reviews recent technological advances that have enabled dissection of overlapping and non-redundant roles of prolamins, particularly the maize zeins. This has come through molecular characterization of mutants first identified many decades ago, selective down-regulation of specific zein genes or entire zein gene families, and most recently through combining deletion mutagenesis with current methods in genome and transcriptome profiling. Works aimed at understanding prolamin deposition and function as well as creating novel variants with improved nutritional and digestibility characteristics, are reported.
Available from: Rentao Song
- "A number of maize mutants with an opaque or floury kernel phenotype have been identified, including three classes: recessive mutants, semidominant mutants, and dominant mutants (Gibbon and Larkins, 2005). Four genes corresponding to recessive mutants, opaque1 (o1), o2, o5, and o7, have been cloned (Schmidt et al., 1990; Miclaus et al., 2011; Myers et al., 2011; Wang et al., 2011, 2012), the majority of which result from defects in storage protein synthesis, which affects endosperm texture. For example, O2 encodes a transcriptional factor that positively regulates the expression level of 22-kD a-zeins (Damerval and Devienne, 1993). "
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ABSTRACT: Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semi-dominant opaque mutant, floury4 (fl4). fl4 is caused by a mutated z1A 19kD α-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein (BIP) content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including endoplasmic reticulum associated degradation (ERAD), the unfolded protein response (UPR), and translational suppression by the phosphorylation of eIF2α. Programmed cell death (PCD) is also elevated, corroborating the intensity of ER stress in fl4. These results provided new insights into cellular responses caused by storage proteins with defective signal peptides.
Available from: Guifeng Wang
- "At least 18 of these have been positioned on the genetic map (Gibbon and Larkins, 2005). So far, only seven genes corresponding to these mutants have been cloned, including opaque2 (o2), o5, o7, floury1 (fl1), fl2, Mucronate (Mc), and Defective endosperm B30 (De-B30) (Schmidt et al., 1990; Coleman et al., 1997; Kim et al., 2004, 2006; Holding et al., 2007; Miclaus et al., 2011; Myers et al., 2011; Wang et al., 2011b). The majority of these mutants result from defects in zein protein synthesis . "
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ABSTRACT: Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70-interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture.
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