Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Abstract

Guard cells (GC)1 are highly specialized cells that form tiny pores called stomata on the leaf surface. When environmental conditions change, guard cells can rapidly change shape so that the pores open or close to control leaf gas exchange and water transpiration. Mesophyll cells (MC) are mainly parenchyma cells between the upper and lower epidermis specialized for photosynthesis. Previous studies that focused on guard cell metabolism and response to environmental signals have revealed important features of functional differentiation of GC (1, 2). Compared with MC, GC contain few chloroplasts with very limited structures and thus possess very low photosynthetic capability. The Calvin cycle in GC only assimilates 2-4% of CO 2 fixed in MC (3). In contrast, GC contain abundant mitochondria and display a high respiratory rate, suggesting that oxidative phosphorylation is an important source of ATP to fuel the guard cell machinery (4). Using microarrays covering just one-third of the Arabidopsis genome, Leonhardt et al. (5) observed a differential abscisic acid (ABA) modulation of many guard cell ABA signaling components as well as key enzymes involved in carbon metabolism in GC and MC. This only available large scale genomics study identified 1309 guard cell expressed genes of which 64 transcripts mainly involved in transcription, signaling, and cytoskeleton were preferentially expressed in GC compared with MC. However, functional grouping of the genes revealed only a 1.9% higher representation of photosynthesis genes in MC than in GC. The percentages of genes in all other categories such as protein turnover, defense, signaling, channels and transporters, and metabolism are similar between the two distinct cell types (5). These proteins are known to play specific roles in guard cell functions (6). This highlights the necessity of studying guard cell functions at the protein level.To date, there have been very few analyses of single celltype proteomes in plants. Proteome analyses of trichomes from Arabidopsis (7) and tobacco (8) and root hairs from soybean (9) exist but have identified fewer than 100 proteins per proteome. The proteomes of pollen from different species have been relatively well studied, but the pollen grains are not single cells because they contain two/three-cell gametophytes (10). A critical factor for large scale proteomics analFrom the ‡Department

Protein phosphorylation in stomatal movement

“…51 MPK4 is also strongly expressed in guard cells. [52][53][54] The role of MPK4 in Nicotiana tabacum was studied using NtMPK4-silenced tobacco plants. 55 Under normal growth conditions, stomatal density was similar in wild type and MPK4-silenced plants.…”

Abscisic Acid–Responsive Guard Cell Metabolomes of Arabidopsis Wild-Type and gpa1 G-Protein Mutants

“…Guard cells have evolved exquisite mechanisms to sense and respond to endogenous and environmental stimuli such as plant hormones, including ABA, as well as to plant water status, light, carbon dioxide, temperature, and humidity. Guard cells have become a premier model system for studies of cellular signaling (Assmann, 2010) in large part because the effects of a stimulus on guard cell ion channels, cytosolic ion concentrations, and stomatal apertures can be assayed in real time, and it is also feasible, albeit challenging, to isolate large populations of highly pure guard cells for transcriptomic (Leonhardt et al, 2004;Pandey et al, 2010;Wang et al, 2011) and proteomic (Zhao et al, 2008Zhu et al, 2009Zhu et al, , 2010 analyses. Dozens of individual cellular compounds and processes, including osmolytes, lipid metabolites, cyclic nucleotides, elevation of cytosolic free calcium concentration ([Ca 2+ ] cyt ), and production of reactive oxygen species (ROS) and nitric oxide (NO), have been implicated in ABA-responsive guard cell signaling pathways (MacRobbie, 1998;Lamattina et al, 2003;Hetherington and Brownlee, 2004;Li et al, 2006;Wang and Song, 2008;Kim et al, 2010;Roelfsema and Hedrich, 2010).…”

Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics

“…These techniques are especially powerful in studies of enriched cell types. Two-dimensional gel electrophoresis and shotgun proteomics approaches were utilized for tomato pollen (Sheoran et al, 2007), elicitor-treated opium poppy (Papaver somniferum) cell culture (Zulak et al, 2009), guard cells of Arabidopsis (Arabidopsis thaliana) and Brassica napus (Zhao et al, 2008;Zhu et al, 2009), and soybean (Glycine max) root hairs (Brechenmacher et al, 2009). Laser capture microdissection combined with proteomics was used to analyze vascular bundles from Arabidopsis stems (Schad et al, 2005) and pericycle cells from maize (Zea mays) primary roots (Dembinsky et al, 2007).…”