Maize opaque10 Encodes a Cereal-Specific Protein That Is Essential for the Proper Distribution of Zeins in Endosperm Protein Bodies
Authors: Rentao Song
Huiling Ling
Gang Wang
Xia Li
Guifeng Wang
Shumei Yan
Weiwei Qi
Qing Yang
Dongsheng Yao
Journal: PLoS Genetics (2016)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Cereal storage proteins; Prolamins; Protein bodies; Maize opaque10; Endosperm
Affiliations: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai, China., Coordinated Crop Biology Research Center (CBRC), Beijing, China., National Maize Improvement Center of China, China Agricultural University, Beijing, China.

Abstract:  Cereal storage proteins are major nitrogen sources for humans and livestock. Prolamins are the most abundant storage protein in most cereals. They are deposited into protein bodies (PBs) in seed endosperm. The inner structure and the storage mechanism for prolamin PBs is poorly understood. Maize opaque10 (o10) is a classic opaque endosperm mutant with misshapen PBs. Through positional cloning, we found that O10 encodes a novel cereal-specific PB protein. Its middle domain contains a seven-repeat sequence that is responsible for its dimerization. Its C terminus contains a transmembrane motif that is required for its ER localization and PB deposition. A cellular fractionation assay indicated that O10 is initially synthesized in the cytoplasm and then anchored to the ER and eventually deposited in the PB. O10 can interact with 19-kD and 22-kD α-zeins and 16-kD and 50-kD γ-zeins through its N-terminal domain. An immunolocalization assay indicated that O10 co-localizes with 16-kD γ-zein and 22-kD α-zein in PBs, forming a ring-shaped structure at the interface between the α-zein-rich core and the γ-zein-rich peripheral region. The loss of O10 function disrupts this ring-shaped distribution of 22-kD and 16-kD zeins, resulting in misshapen PBs. These results showed that O10, as a newly evolved PB protein, is essential for the ring-shaped distribution of 22-kD and 16-kD zeins and controls PB morphology in maize endosperm. 

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PubPeer comments:
 # **Fig. 4 G and S2F Fig.** # Fig. 4 G ![file] # S2F Fig. ![file] , 
 # **Fig. 5 A** ![file] , 
 Figure 5B showed the localization of O10 protein in the PB bodies, where seven PB bodies were designated with arrows showing the gold-labeled O10 protein localization. In comparison with the diagram in Figure 6B, the O10 localization in Figure 5B appears to look very different from the red dots illustrating O10 localization in the WT diagram in Figure 6B. Although the diagram in Figure 6B is representing 10 replicated PBs for the same assay as that in Figure 5B, there are seven out of totally 13 gold-labeled O10 dots in Figure 5B localized differently from what was illustrated in Figure 6B. Blue arrows indicate the differently localized gold-labeled O10 dots in the PB bodies in the overlaid picture ![file] , 
 In  The construction of transgene vectors and their transformation paragraph in the Materials and Methods, there is no SalI site in the primers ![file] , 
 **In Figure 4 A-D, there is no nucleus marker showing what structure is representing nuclei.** # Figure 4 ![file] , 
 **There is no NedI site in the pGADT7 vector** ![file] , 
 # Fig. 4 In the Fig. 4 legend, it is quite confusing that a co-expressed  ER maker (mCherry-HDEL) co-localized with the YFP-10, so the YFP-O10 was related to the formation of endoplasmic reticulum made by the  ER maker (mCherry-HDEL) , which could be a new function of the  mCherry-HDEL . ![file] , 
 # Fig. 4E, F, and G, and the related text in the article **Fig. 4 E, F, G, H** ![file] As it is said in the article, u201cCompared to the O10 in the wild-type, the o10 in the mutant kernels had similar protein accumulation levels at 15 DAP and 18 DAP. However, o10 dramatically decreased at 21 DAP in the mutant (Fig 4E). u201d According to this, the total O10 and o10 mutant proteins accumulated to similar level while at 21 DAP, o10 mutant dropped dramatically compared to O10. It is also said that u201cThe total proteins extracted from developing kernels of wild-type and o10 at 15 DAP, 18 DAP and 21 DAP were then separated into soluble and total membrane fractions, respectively.u201d So, there were two fractions for both the O10 o10 protein, which were soluble, and membrane associated, respectively. Considering that u201cBoth O10 and o10 had significant protein levels in the soluble (cytoplasmic) fractions, and no difference was found between wild-type and mutant kernels at 15 DAP, 18 DAP and 21 DAP (Fig 4F). u201d The soluble fraction should account for the major proportion of O10 o10 proteins because it is said in the article that, 1.t u201cCompared to the O10 in the wild-type, the o10 in the mutant kernels had similar protein accumulation levels at 15 DAP and 18 DAP.u201d (Fig 4E) 2.tAt 15, 18 and 21 DAP u201cin terms of total membrane fractions, o10 dramatically decreased in the mutant kernels in compared to the wild-type (Fig 4G). u201d According to this, the total level of o10 mutant accumulated to the same as O10 at 15 and 18 DAP, even o10 mutant influenced the u201cpartitioning and accumulationu201d in the membrane fraction at all time points because the soluble protein took the major weight. However, at 21 DAP, although the soluble protein level, as the major proportion, of O10 and o10 was the same, as shown in Fig. 4F. (Also said as u201cBoth O10 and o10 had significant protein levels in the soluble (cytoplasmic) fractions, and no difference were found between wild-type and mutant kernels at 15 DAP, 18 DAP and 21 DAPu201d), the total level of o10 mutant u201cdramatically decreasedu201d. How did it come that when the major soluble portion was the same, and the influence of o10 mutant was the same throughout 15, 18 and 21 DAP, while the o10 mutant only dropped at 21 DAP? Is it possible some fractions were missed? In Fig. 4E, and most assays in this article, Tubulin was as loading control. While In Fig. 4F, no Tubulin and instead, it is said the in the figure legend, u201c(50 u03bcg of protein per lane)u201d. Is the discrepancy because of different loading weight controls were used? A piece of further evidence is that, when comparing the membrane fraction in Fig. 4G, the protein level were constant for either O10 or o10 mutant throughout 15, 18 and 21 DAP, and there were BIP used as control in Fig. 4G. BIP is an endoplasmic reticulum lumen marker, which was supposed to be constant or it could not be used as control if it was influenced by the o10 mutant. It is understandable either fraction, soluble or membrane, accounted for the majority of the total O10 o10 protein. It is not that understandable that at 15 and 18 DAP soluble fraction accounted while at 21 DAP they did not, and instead, the membrane fraction accounted. , 
 In the S2 Fig. and the figure legend , it is confusing which represents transgenic negative and which is positive ![file] , 
 **In the Extract buffer for the HPLC Assay, the Concentration of the Sodium Acetate is 0.5 (W V), that is 50g 100 ml. Actually, the maximal solubility of Sodium Acetate in 70% Ethanal is around 0.35 (W V).** ![file] , 
 # S4B Fig. # ![file] , 
 # S8 FIG. # ![file] ,