Identification and Characterization of Maizefloury4as a Novel Semidominant Opaque Mutant That Disrupts Protein Body Assembly    

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, semidominant opaque mutant, floury4 ( fl4). fl4 is caused by a mutated z1A 19-kD a-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 content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ERassociated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 a-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

Comprehensive proteomic analysis of developing protein bodies in maize (Zea mays) endosperm provides novel insights into its biogenesis

“…This result indicates that α-zeins also could be retained in the ER independently of the interaction with γ-zeins (Kim et al , 2002), probably with the aid of BiPs (Zhang and Boston, 1992). Even in some mutants with elevated ER stress, the defective zeins could still accumulate in the misshapen PBs and the ER membrane, rather than entering the protein quality control system and being degraded by the ER-associated degradation pathway (Kim et al , 2006; Wang et al , 2014). Therefore, ER chaperones play an essential role in zein folding, aggregation, and assembly into PBs, avoiding entry into the ER export pathway and degradation via the protein quality control system.…”

“…Immunoblotting was carried out as described previously (Wang et al , 2012 a ), using antibodies against ER BiP (at-95, Santa Cruz Biotechnology, 1/1000), peroxisome PEX14P (Agrisera AS08 37, 1/10000), mitochondria IDH (Agrisera AS06203A, 1/5000), ribosome L13 (Agrisera AS132650, 1/2500), Golgi COPI (Agrisera AS08 327, 1/1000), and 15-kDa zein PBs (1/500) (Wang et al , 2012 a ). Immunolocalization was performed as previously described (Kim et al , 2006; Wang et al , 2014), using an antibody against a randomly selected protein (GRMZM2G346263_P03, 1/200) for localization validation, which was produced by Shanghai ImmunoGen Biological Technology in rabbits according to standard protocols.…”

“…Simultaneous reductions in both 19- and 22-kDa α-zeins severely decreased PB size but did not induce other morphological abnormalities (Guo et al , 2013). Interestingly, substantial suppression of only 22-kDa α-zeins or accumulation of either defective 19- or 22-kDa α-zeins resulted in small and misshapen PBs (Coleman et al , 1997; Kim et al , 2006; Wu and Messing, 2010; Wang et al , 2014). Immunolocalization evidence indicates that 19-kDa α-zeins fill in the PB core while 22-kDa α-zeins preferentially deposit as an intermediary layer between the central 19-kDa α-zeins and the γ-zeins at the periphery, most likely controlled by FL1 (Holding et al , 2007).…”

Dek42 encodes an RNA‐binding protein that affects alternative pre‐mRNA splicing and maize kernel development

“…The gold‐coated samples were fixed on the electric objective table and then observed with SEM (Hitachi S‐3400N). The detailed process is described in a previously reported method (Wang et al ).…”

The Maize Imprinted Gene Floury3 Encodes a PLATZ Protein Required for tRNA and 5S rRNA Transcription through Interaction with RNA Polymerase III

“…Thus far, 11 naturally occurring opaque and floury mutants have been cloned; all but o5 are related to zein synthesis or organization in protein bodies (Myers et al, 2011). The o2 mutation affects the transcription of most zein genes (Schmidt et al, 1992;Zhang et al, 2015;Yang et al, 2016); fl2, fl4, De-B30*, and Mucronate1 affect the zein protein structure (Coleman et al, 1997;Kim et al, 2004Kim et al, , 2006Wang et al, 2014a); fl1, o1, and o10 affect zein protein deposition and protein body formation (Holding et al, 2007;Wang et al, 2012;Yao et al, 2016); and o6 (also called pro1) and o7 are related to metabolic processes that affect zein protein accumulation (Miclaus et al, 2011;Wang et al, 2011Wang et al, , 2014b. The o5 mutation is not involved in the above processes; it results from a defective monogalactosyl diacylglyerol synthase and affects the phospholipid composition of amyloplast membranes (Myers et al, 2011).…”