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IN THIS ISSUE

Insights into Plant Cellular Mechanisms: Of Phosphate Transporters and Arbuscular Mycorrhizal Infection

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The high demand for phosphorus as an essential macronutrient coupled with limitations in phosphate availability or accessibility in soils has led to the evolution in plants of various mechanisms for facilitating and enhancing the uptake of inorganic phosphate. Uptake of phosphate into plant cells occurs against a steep concentration gradient, as the concentration within plant cells (1 to 10 mM) is 10,000-fold higher than that of the soil solution (Rausch and Bucher, 2002). Two important mechanisms for phosphate uptake in higher plant roots are the activity of high affinity phosphate transporters and the symbiotic association with arbuscular mycorrhizal (AM) fungi. Both of these phenomena involve orchestration of a complex series of events within the cell. On the one hand, traffic through the secretory pathway is required to direct localization of phosphate transporters to the plasma membrane, and on the other, the plant cell allows for penetration and elaboration of a complex network of fungal arbuscular hyphae that facilitates exchange of minerals and nutrients between the plant cell and fungal symbiont. Two articles in this issue of The Plant Cell present significant new information on cellular mechanisms associated with these processes of fundamental importance to higher plants. In one article, González et al. (pages 3500–3512) identify an accessory protein associated with targeting of phosphate transporters to the plasma membrane in Arabidopsis. In a separate article, Genre et al. (pages 3489–3499) present a detailed intracellular view of the early stages of root colonization by AM fungi in Medicago truncatula and show that the host plant plays a key role in orchestrating the AM infection process.

	ACCESSORY PROTEIN GUIDES ENDOPLASMIC RETICULUM EXIT OF A PHOSPHATE TRANSPORTER

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Plants contain a large number of phosphate transporters (reviewed in Rausch and Bucher, 2002). The PHT1 family includes high affinity phosphate transporters that have 12 membrane spanning domains and are presumed to be targeted to the plasma membrane. PHT1 is a relatively large family in Arabidopsis (nine members) and rice (at least 13), and patterns of gene expression suggest functional specialization within the family (Mudge et al., 2002). Thus, some PHT1 proteins may function primarily in phosphate uptake at the soil–root interface, whereas others may participate predominantly in translocation to other parts of the plant and/or transport within certain tissues or cell types. Plant organelles appear to contain separate families of phosphate transporters; for example, PHT2 proteins and another type called phosphate translocators may be found in chloroplasts, whereas PHT3 proteins are targeted to mitochondria.

González et al. isolated a mutant of Arabidopsis that displays constitutive expression of a phosphate starvation responsive reporter gene, suggesting mutation in a gene associated with phosphate perception or transport (i.e., perception and transport of phosphate normally inhibits the expression of phosphate starvation responsive genes, which will only be induced when the inorganic phosphate concentration falls below a certain threshold). Analysis of the mutant plants suggested that phosphate accumulation was reduced due to a specific effect on phosphate uptake (uptake of other nutrients was unaffected). The corresponding gene, named PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1), was found to encode a Sec12-related plant-specific protein required for localization of PHT1 to the plasma membrane. PHT1 was shown to be localized to the plasma membrane in wild-type cells but was retained in the endoplasmic reticulum (ER) in the phf mutant (Figure 1).

View larger version (68K): [in this window] [in a new window]   Figure 1. Subcellular Localization of PHT1 in Wild-Type and phf1 Mutant Plants. Micrographs of leaf epidermal cells from transgenic wild type (left) and phf1 mutant (right) harboring a PHT1:GFP construct driven by the cauliflower mosaic virus 35S promoter. PHT1:GFP is localized to the plasma membrane in wild-type cells, but is retained in the ER in the phf1 mutant. (Figure courtesy of Esperanza González and Javier Paz-Ares.)

The ER in yeast and animals also contains a number of substrate- or cargo-specific accessory proteins that assist in various ways with the recruitment, packaging, and transport of cargo in COPII vesicles (Herrmann et al., 1999). For example, mammalian RanB2 and Drosophila NinaA, related to cyclophilins known to be involved in protein folding, are involved in the ER–Golgi transport of specific seven-transmembrane photoreceptors. Various other plasma membrane–bound proteins require the presence of accessory proteins for transport through the secretory pathway. Indeed, the yeast PHT1-related phosphate transporter PHO84 requires the accessory protein PHO86 for exit from the ER (Lau et al., 2000).

Although PHF1 shares homology with Sec12 and no structural or sequence similarity to PHO86, González et al. show that it is functionally similar to PHO86, acting as a substrate-specific accessory protein for the transport of PHT1 out of the ER, rather than a general activator of Sar1 GTPase similar to yeast Sec12. First, although PHF1 shows a high degree of structural similarity to Sec12 and is localized to the ER, it lacks a number of the most highly conserved residues present in Sec12 proteins from a number of organisms. Arabidopsis contains a close homolog of Sec12, STL2, and the authors showed that heterologously expressed STL2 was able to complement the sec12 yeast mutant, whereas PHF1 was not. Secondly, the phf1 mutant did not exhibit generalized impairment of protein trafficking out of the ER, as would be expected by disruption of Sec12 activation of Sar1 GTPase. The authors confirmed this observation by visualization of a green fluorescent reporter fused to the plasma membrane marker protein PIP2A, which was correctly targeted to the plasma membrane in phf1 mutant plants.

Phylogenetic analysis conducted by González et al., together with the functional characterization, suggests that PHF1 proteins originated from a plant-specific Sec12 ancestral protein and evolved functional specialization as accessory proteins guiding ER exit of phosphate transporters. This work provides new information about the secretory pathway in plants, as it reveals the likely existence of plant-specific accessory proteins that participate in targeting of proteins to the plasma membrane.

	A DETAILED VIEW OF THE AM INFECTION PROCESS

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Most terrestrial plants participate in a symbiotic relationship with AM fungi that facilitates uptake of nutrients, particularly phosphorus, by plant roots and provides the fungus with essential carbohydrates (reviewed in Hause and Fester, 2005). AM fungi are obligate biotrophs that depend entirely on the host plant as a carbon source. All AM fungi belong to the phylum Glomeromycota, which is divided into four orders and 150 known species (Harrison, 2005). Glomus and Gigaspora species are the best characterized and most studied. Colonization of roots by AM fungi and development of the arbuscular hyphae within root cortical cells is a highly complex process. Plants must recognize AM fungi as beneficial nonpathogenic intruders and facilitate both the initial entry of the fungus and the subsequent development of the arbuscular network (reviewed in Harrison, 2005). In legumes such as Medicago and Lotus, a subset of genes is required for establishing both the AM association and root nodulation associated with nitrogen-fixing bacteria, indicating that common pathways are likely to be involved in initiating both types of symbioses (Parniske, 2004; Kistner et al., 2005).

Enhanced uptake of inorganic phosphate is one of the major benefits that plants derive from AM fungi, and indeed plants appear to contain phosphate transporters specialized for this purpose. In M. truncatula, transcript and protein levels of the high affinity plasma membrane phosphate transporters PT1 and PT2 are induced in roots in response to phosphate starvation but are not expressed in roots colonized by AM fungi (Chiou et al., 2001). Instead, the phosphate transporter MtPT4 is expressed specifically in mycorrhizal roots, where it is likely localized to the periarbuscular membrane and functions specifically in uptake of phosphate released by the AM fungus (Harrison et al., 2002). Karandashov et al. (2004) found that AM-specific phosphate transporter genes related to MtPT4 are conserved across phylogenetically distant plant species.

Despite knowledge of plant genes that are required for or whose expression is altered by the association with AM fungi, relatively little is known about the mechanisms that control AM development. Genre et al. studied in vivo cellular dynamics within M. truncatula root epidermal cells during the early stages of colonization by Gigaspora AM fungi, making use of green fluorescent protein (GFP) reporters to visualize the plant cytoskeleton and ER. Three different GFP constructs were used to visualize the microtubular cytoskeleton, actin filaments, and the ER. The authors modified a targeted AM inoculation technique developed by Chabaud et al. (2002) to allow continuous microscopic observation during AM colonization of roots. The resulting images provide a detailed picture of intracellular events associated with AM infection.

AM root infection initiates with the formation of surface appressoria at points of contact between fungal hyphae and the root epidermal cell. Notably, the results of Genre et al. have shown that the epidermal cell nucleus undergoes two distinct phases of movement, both preceding fungal penetration. After appressoria formation, the epidermal cell nucleus rapidly moves toward and positions itself directly below the appressorium contact site. During a second trans-cellular phase of nuclear movement, a hollow structure comprising ER and cytoskeletal components is progressively assembled between the appressorium contact site and the nucleus. This process takes 4 to 5 h following appressorium formation, and fungal penetration occurs only after completion of this structure, termed the prepenetration apparatus (PPA; Figure 2). Furthermore, since hyphal growth occurs strictly along and within the trans-cellular path created by the PPA, the authors propose that the role of this apparatus is to synthesize the apoplastic compartment, which always surrounds and isolates the infection hypha (Bonfante et al., 2000). After fungal penetration, the epidermal cell nucleus repositions at the cell periphery. The authors provide a supplemental animated video depicting the entire AM infection process and the roles of the plant cell nucleus, cytoskeleton, and ER in formation of the PPA.

View larger version (77K): [in this window] [in a new window]   Figure 2. Intracellular Dynamics in the M. truncatula Root Epidermis Preceding AM Infection. The illustration shows the transient PPA, comprising cytoskeletal/ER components, which is assembled in response to surface hyphal contact and appressorium formation. The PPA is assembled within the cytoplasmic column created during transcellular nuclear migration and likely plays a central role in the synthesis of the apoplastic compartment through which the AM infection hypha subsequently crosses the epidermal barrier. Color coding: cell nucleus, dark brown; plasma membrane, light brown; microtubules, green; actin bundles, red; ER, white. (Figure courtesy of Andrea Genre.)

The work of Genre et al. provides significant new insights into the early stages of AM infection and in particular the active role of the host plant in regulating this process. These results also provide direct evidence for the pivotal role of epidermal cells in recognition and accommodation of the AM symbiont and raise the likelihood of direct analogies with the cellular mechanism involved in the entry of rhizobia via the root hair apoplastic invagination known as the infection thread.

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This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Eckardt, N. A. Search for Related Content PubMed Articles by Eckardt, N. A. Agricola Articles by Eckardt, N. A.