Retracted: Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

Abstract

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 a-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting a-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.T HE texture and protein quality of maize (Zea mays L.) endosperm are important factors affecting grain shipping, insect and fungal pathogen resistance, and nutritional quality. Much evidence indicates that the reduction in the amount of zeins in the endosperm leads to a decrease in the endosperm hardness and an increase in lysine content (Mertz et al. 1964;Misra et al. 1972;Schmidt et al. 1987;Dombrink-Kurtzman and Bietz 1993;Holding and Larkins 2006;. Maize have a number of opaque or floury endosperm mutants that affect the texture and protein quality of endosperm by altering zeins accumulation. Our understanding of the underlying mechanisms determining zeins accumulation comes from the study of seed mutants.There are .18 mutants that can exhibit an opaque or floury endosperm (Thompson and Larkins 1994;Hunter et al. 2002). Among them are the recessive opaque mutants (o1, o2, o5, o7, o9-o11, and o13-o17), the semidominant floury mutants (fl1, fl2, and fl3), and the dominant mutants Mucronate (Mc) and Defective endosperm B30 (De-B30) (Motto et al. 1996;Gibbon and Larkins 2005). The cloning and characterization of some of the opaque mutants has revealed important regulatory mechanisms for zeins accumulation in maize endosperm. The O2 gene, which encodes a defective basic-domain-leucine-zipper transcription factor, regulates several endosperm-expressed genes, in particular the 22-kDa a-zeins (Schmidt et al. 1987(Schmidt et al. , 1990Damerval and De Vienne

Opaque1 Encodes a Myosin XI Motor Protein That Is Required for Endoplasmic Reticulum Motility and Protein Body Formation in Maize Endosperm

“…It is believed that zein protein bodies play an important role in the vitreous endosperm phenotype at kernel maturity. Several maize opaque mutants, such as o2, o7, fl2, De-B30, and Mc (Schmidt et al, 1990;Gillikin et al, 1997;Kim et al 2004Kim et al , 2006Wang et al, 2011b), result in quantitative or qualitative alternations in zein accumulation. Consistent with a previous report (Hunter et al, 2002), we found that all the o1 alleles result in no significant changes in zein or nonzein proteins at maturity (Table 1; see Supplemental Figure 1 online).…”

“…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, 2004Kim et al, , 2006Holding 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.…”

“…O2 encodes a bZIP transcription factor that regulates expression of 22-kD a-zeins (Schmidt et al, 1990). O7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis, particularly the 19-and 22-kD a-zeins (Wang et al, 2011b). Other mutations directly affect zein genes themselves: fl2 encodes a defective 22-kD a-zein ; Mc encodes a defective 16-kD g-zein (Kim et al, 2006), and De-B30 encodes a defective 19-kD a-zein (Kim et al, 2004).…”

Genetic Enhancement of Grain Quality-Related Traits in Maize

“…The O7 gene was recently cloned by two different groups, using a combination of mapbased cloning and transposon tagging and confirmed by transgenic functional complementation Wang et al, 2011). Moreover, these last workers via sequence analysis indicated that the O7 gene showed similarities with members of the larger family of acyl-CoA synthetase-like genes (ACS), although its exact enzymatic activity is uncertain.…”

The Maize High-Lysine Mutantopaque7Is Defective in an Acyl-CoA Synthetase-Like Protein

“…Note added in proof: See Wang et al 2011Wang et al (pp. 1281Wang et al -1295) in this issue, for a related work.…”