Abstract
In maize developing seeds, transfer cells are prominently located at the basal endosperm transfer layer (BETL). As the first filial cell layer, BETL is a gateway to sugars, nutrients and water from mother plant; and anchor of numerous functions such as sucrose turnover, auxin and cytokinin biosynthesis/accumulation, energy metabolism, defense response, and signaling between maternal and filial generations. Previous studies showed that basal developing endosperms of miniature1 (mn1) mutant seeds lacking the Mn1-encoded cell wall invertase II, are also deficient for hexose. Given the role of glucose as one of the key sugars in protein glycosylation and proper protein folding; we performed a comparative large scale glycoproteome profiling of total proteins of these two genotypes (mn1 mutant vs. Mn1 wild type) using 2D gel electrophoresis and glycosylation/total protein staining, followed by image analysis. Protein identification was done by LC-MS/MS. A total of 413 spots were detected; from which, 113 spots matched between the two genotypes. Of these, 45 showed >20% decrease/increase in glycosylation level and were selected for protein identification. A large number of identified proteins showed decreased glycosylation levels in mn1 developing endosperms as compared to the Mn1. Functional classification of proteins, showed mainly of post-translational modification, protein turnover, chaperone activities, carbohydrate and amino acid biosynthesis/transport, and cell wall biosynthesis. These proteins and activities were related to endoplasmic reticulum (ER) stress and unfolded protein response (UPR) as a result of the low glycolsylation levels of the mutant proteins. Overall, these results provide for the first time a global glycoproteome profile of maize BETL-enriched basal endosperm to better understand their role in seed development in maize.
Highlights
The mn1 mutation in maize is associated with a loss of 70% seed weight at maturity due to a loss-of-function mutation at the Mn1 locus that codes for the endosperm-specific cell wall invertase 2 (INCW2)
TWO DIMENSIONAL GEL ANALYSIS, PROTEIN IDENTIFICATION AND GLYCOPROTEIN (GP) DETECTION In order to determine the differences in glycosylation patterns of basal endosperm transfer cell layer (BETL) between the wild type (WT) and the mutant (M), total protein extracts were prepared from the one-third lower part of maize kernels
A total www.frontiersin.org of 413 spots were detected in the four gels of the three replicates (Supplemental Figure 1) representing the wild type and mutant endosperm extracts; of these, 254 spots with p-values smaller than 0.05 based on ANOVA test were selected for further analysis
Summary
The mn mutation in maize is associated with a loss of 70% seed weight at maturity due to a loss-of-function mutation at the Mn1 locus that codes for the endosperm-specific cell wall invertase 2 (INCW2). The INCW2 protein is entirely localized in basal endosperm transfer cell layer (BETL), a major if not the sole gateway for the intake of sugars and nutrients from maternal cells into the filial tissue (Miller and Chourey, 1992; Cheng et al, 1996). The most detailed studies of GPs in maize developing endosperm have been on the storage protein zein, stored in ERderived protein bodies (Vitale and Boston, 2008; Arcalis et al, 2010). Comparative GP profiles of the BETL enriched proteins of the mn relative to the Mn1 revealed potential changes due to both the hexose deficiency
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