Developmental Distribution of the Plasma Membrane-Enriched Proteome in the Maize Primary Root Growth Zone

Abstract

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Apoplastic Hydrogen Peroxide in the Growth Zone of the Maize Primary Root. Increased Levels Differentially Modulate Root Elongation Under Well-Watered and Water-Stressed Conditions

“…For example, Zhu et al (2007) provided evidence for apoplastic accumulation of ascorbate peroxidase, which oxidizes ascorbate in the presence of H 2 O 2 , in the apical region of water-stressed roots. Also, plasma membrane proteomic analyses showed an increased abundance of ascorbatedependent cytochrome b 561 protein in the apical region of well-watered (Zhang et al, 2013) and water-stressed roots (Voothuluru et al, 2016). This protein can be oxidized by monodehydroascorbate and reduced by ascorbate, and has been implicated in trans-plasma membrane electron transport (Preger et al, 2009).…”

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses

“…As an independent approach to assess the subcellular localization of FLS2, we used a simple and rapid biochemical method to analyze the PM-associated accumulation of FLS2 protein in eps1-2 null mutant and wild-type seedlings. This method uses differential centrifugation and the detergent Brij-58 to enrich for PM proteins by depleting contaminating organelles (Zhang and Peck, 2011;Zhang et al, 2013;Collins et al, 2017). Fractionation efficacy in whole seedling samples was verified by immunoblot analyses of soluble, microsomal, and enriched PM (ePM) subcellular fractions using compartment-specific antibodies (Fig.…”

“…PM proteomics was performed as described (Zhang and Peck, 2011;Zhang et al, 2013). Briefly, equal amounts of PM-enriched proteins from each genotype were separated using 8% SDS-PAGE.…”

“…Peptides were resuspended in 1% (v/v) formic acid before analysis using an LTQ-Orbitrap LC-MS/MS device (Thermoelectron) controlled by XCalibur v. 2.2.1. Mass spectrometer measurements were obtained (Zhang and Peck, 2011;Zhang et al, 2013). Mascot distiller 2.0 (Matrix Science) was used to deconvolute the mass spectra, and then Mascot (server v. 2.3; Matrix Science) and X!…”

Modification of antioxidant systems in cell walls of maize roots by different nitrogen sources

“…Since the growing zone of maize primary root is limited to a region of about 1.2 cm from the apex (Zhang et al, 2013), primary root segment, 2-7 cm from tip to base, was used as the sample with decreased growth rate. Lateral roots, with a maximal length of about 1.5 cm (two days after initiation), were taken as intensive growth zone.…”