Simon D. X. Chuong

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.

The Cytoskeleton Maintains Organelle Partitioning Required for Single-Cell C4 Photosynthesis in Chenopodiaceae Species

Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C4 photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein–tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm.

The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells

BackgroundThe plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol.ResultsWe demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations.ConclusionPlant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes.

Developing prolamine protein bodies are associated with the cortical cytoskeleton in rice endosperm cells.

Abstract. The mRNAs that encode the prolamine storage proteins in rice (Oryza sativa L.) endosperm cells are enriched on the surface of the prolamine protein bodies (PBs), a subcellular structure consisting of a prolamine intracisternal granule surrounded by rough endoplasmic reticulum membrane. Previous biochemical studies (D.G. Muench et al., 1998, Plant Physiol. 116: 559–569) have shown that prolamine mRNAs may be anchored to the PB surface via the cytoskeleton. To better understand the mechanism and role of mRNA localization in rice endosperm cells, we studied the subcellular development of prolamine PBs and their relationship with the cytoskeleton in rice endosperm cells. Confocal microscopy of endosperm cells showed that, unlike the glutelin PBs, the developing prolamine PBs are not randomly distributed within the cell, but instead are often enriched in the cortical region of the cell only a few micrometers beneath the plasma membrane. In addition, the peripheral prolamine PBs are closely associated with the cortical microtubule and actin filament networks. The cortical enrichment of rice prolamine protein bodies represents a unique example of endoplasmic reticulum subdomain localization in plant cells. The interaction of this endoplasmic reticulum subdomain with the cytoskeleton provides new insights on the possible mechanism and role of mRNA localization in plants.

Species Having C4 Single-Cell-Type Photosynthesis in the Chenopodiaceae Family Evolved a Photosynthetic Phosphoenolpyruvate Carboxylase Like That of Kranz-Type C4 Species

Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C4 photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C4 photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C4 photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO2 via PEPC from C4 acid decarboxylation and CO2 donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C4 species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C3 (Suaeda linifolia) chenopods. It was found that PEPC from both types of C4 chenopods displays higher specific activity than that of the C3 species and shows kinetic and regulatory characteristics similar to those of C4 species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C4 species possesses a Kranz-type C4 isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C4 species are in a clade with the C3 and Kranz C4 Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C4-type PEPC similar to that in Kranz C4 plants, which likely is required for effective function of C4 photosynthesis.

Flowers of Bienertia cycloptera and Suaeda aralocaspica (Chenopodiaceae) complete the life cycle performing single-cell C4 photosynthesis

Leaves and cotyledons of the terrestrial C4 plants, Bienertia cycloptera Bunge ex Boiss. and Suaeda aralocaspica (Bunge) Freitag & Schütze (Chenopodiaceae), accomplish C4 photosynthesis within individual chlorenchyma cells: each species having a unique means of intracellular spatial partitioning of biochemistry and organelles. In this study the chlorenchyma tissue in flowers and stems of these species was investigated. Flowers have an outer whorl of green tepals with a layer of chlorenchyma cells, which are located on the abaxial side, exposed to the atmosphere. Anatomical, immunocytochemical, western blots and starch analyses show that the chlorenchyma cells in tepals are specialised for performance of single-cell C4 photosynthesis like that in leaves. In the tepals of B. cycloptera, chlorenchyma cells have a distinctive central cytoplasmic compartment, with chloroplasts which contain Rubisco, separated by cytoplasmic channels from a peripheral chloroplast-containing compartment, with phosphoenolpyruvate carboxylase (PEPC) distributed throughout the cytoplasm. In the tepals of S. aralocaspica, chlorenchyma cells have chloroplasts polarised towards opposite ends of the cells. Rubisco is found in chloroplasts towards the proximal end of the cell and PEPC is found throughout the cytoplasm. Also, green stems of B. cycloptera have a single layer of the specialised C4 type chlorenchyma cells beneath the epidermis, and in stems of S. aralocaspica, chlorenchyma cells are scattered throughout the cortical tissue with chloroplasts around their periphery, typical of C3 type chlorenchyma. During reproductive development, green flowers become very conspicuous, and their photosynthesis is suggested to be important in completion of the life cycle of these single-cell C4 functioning species.

Functional compartmentation of C4 photosynthesis in the triple layered chlorenchyma of Aristida (Poaceae)

The genus Aristida (Poaceae), is composed of species that have Kranz anatomy and C4 photosynthesis. Kranz anatomy typically consists of two photosynthetic cell types: a layer of mesophyll cells where atmospheric CO2 is fixed into C4 acids, and an internal, chlorenchymatous vascular bundle sheath to which C4 acids are transferred and then decarboxylated to donate CO2 to the C3 cycle. The anatomy of Aristida species is unusual as it has three distinct layers of chlorenchyma cells surrounding the vascular tissue: an inner bundle sheath, an outer bundle sheath and the mesophyll cells. In this study of Aristida purpurea Nutt. var. longiseta, the functions of the three layers of chlorenchyma cells relative to the C4 photosynthetic mechanism were determined using ultrastructural analysis, western blots, immunolocalisation of photosynthetic enzymes and starch histochemistry. The results indicate that mesophyll cells contain high levels of phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK), and function to capture CO2 in the C4 cycle. The inner bundle sheath, which is high in Rubisco and contains NADP-malic enzyme and glycine decarboxylase, functions to transfer CO2 to the C3 cycle through decarboxylation of C4 acids and by decarboxylation of glycine in the glycolate pathway. The outer chlorenchymatous sheath is where ADPG pyrophosphorylase is mainly located, and this cell layer functions as the primary site of starch storage. The outer sheath, which has low levels of Rubisco and PEPC, may also have a role in refixation of any CO2 that leaks from the inner bundle sheath cells.

Development of structural and biochemical characteristics of C4 photosynthesis in two types of Kranz anatomy in genus Suaeda (family Chenopodiaceae)

Genus Suaeda (family Chenopodiaceae, subfamily Suaedoideae) has two structural types of Kranz anatomy consisting of a single compound Kranz unit enclosing vascular tissue. One, represented by Suaeda taxifolia, has mesophyll (M) and bundle sheath (BS) cells distributed around the leaf periphery. The second, represented by Suaeda eltonica, has M and BS surrounding vascular bundles in the central plane. In both, structural and biochemical development of C(4) occurs basipetally, as observed by analysis of the maturation gradient on longitudinal leaf sections. This progression in development was also observed in mid-sections of young, intermediate, and mature leaves in both species, with three clear stages: (i) monomorphic chloroplasts in the two cell types in younger tissue with immunolocalization and in situ hybridization showing ribulose bisphosphate carboxylase oxygenase (Rubisco) preferentially localized in BS chloroplasts, and increasing in parallel with the establishment of Kranz anatomy; (ii) vacuolization and selective organelle positioning in BS cells, with occurrence of phosphoenolpyruvate carboxylase (PEPC) and immunolocalization showing that it is preferentially in M cells; (iii) establishment of chloroplast dimorphism and mitochondrial differentiation in mature tissue and full expression of C(4) biochemistry including pyruvate, Pi dikinase (PPDK) and NAD-malic enzyme (NAD-ME). Accumulation of rbcL mRNA preceded its peptide expression, occurring prior to organelle positioning and differentiation. During development there was sequential expression and increase in levels of Rubisco and PEPC followed by NAD-ME and PPDK, and an increase in the (13)C/(12)C isotope composition of leaves to values characteristic of C(4) photosynthesis. The findings indicate that these two forms of NAD-ME type C(4) photosynthesis evolved in parallel within the subfamily with similar ontogenetic programmes.