The Plasmodesmata

Plasmodesmata [30] are channels in the plant cell wall that in conjunction with associated phloem form an intercellular communication network that supports the cell-to-cell and long-distance trafficking of small molecules as well as of a wide spectrum of endogenous proteins and ribonucleoprotein complexes [14,15,27]. The trafficking of such macromolecules is of importance in the orchestration of non-cell-autonomous developmental and physiological processes. Plant viruses encode movement proteins (MPs) that subvert this communication network to facilitate the spread of infection. Therefore, these viral proteins represent excellent experimental keys for exploring the mechanisms involved in intercellular trafficking and communication via plasmodesmata.

Plasmodesmata interconnect the cytoplasm of contiguous cells. The interconnection of cytoplasm of groups of cells creates supracellular domains [28]. Plants can regulate the cell–to-cell communication between cells in such supracellular domains by a number of means, for example by developmental changes in plasmodesmata structure, by changes in their biochemical composition or by changing the frequency of plasmodesmata between cells. Up-regulation or down-regulation of plasmodesmatal conductivity can modify the size of a communication domain and also redefine domain boundaries, resulting in modulation of extent and quality of communication with resultant changes in intercellular and interdomain interactions (reviewed in [15]). The conductivity of plasmodesmata is regulated in response to developmental and physiological cues. Thus, plasmodesmata can exist in different states, closed, open or dilated, and therefore either restrict or allow the trafficking of molecules based on their molecular size [6,21]. However, some specialized non-cell-autonomous proteins have the ability to actively target plasmodesmata and modify their size exclusion limit [21]. This group of proteins comprises transcription factors that are known to have important non-cell-autonomous roles during plant development as well as viral MPs that mediate the cell-to-cell spread of their cognate viral genomes. Especially the MPs are used as convenient keys to unravel the cellular pathways by which macromolecules target plasmodesmata [15,31]. The MP of Tobacco mosaic virus (TMV) was the first MP identified [8] and also the first plant protein expressed as a functional GFP fusion protein [16]. The MP:GFP fusion protein illuminates the localization of plasmodesmata and also cellular components such as microtubules and the endomembrane network that may be involved in the targeting of the protein to the channel [17,32]. GFP fusions with MPs of other viruses are also being intensely investigated and illustrate a wide spectrum of potential mechanisms by which MPs target plasmodesmata (e.g. [4,13,24,36,40,42,43].

The ultrastructure of plasmodesmata has been defined by numerous electron microscopy studies (for example [3,9]). The plasma membrane delineates the plasmodesmal pore, which is traversed in its axial center by the appressed membrane of the endoplasmic reticulum (ER) termed desmotubule. The plasma membrane and the desmotubule are densely covered with globular particles which segment the region between plasma membrane and desmotubule, the cytoplasmic sleeve, into 8-10 channels [9]. These channels are considered to function as conduits for diffusion of molecules between cells. The cell wall or neck region surrounding the plasmodesmal orifices is speculated to participate in the control of molecular traffic through the channel [29,34,44].

Unlike the ultrastructure, the molecular composition of plasmodesmata is poorly defined. Three main strategies to elucidate plasmodesmal composition have been employed:

(A) Direct biochemical approaches aimed to extract plasmodesmal proteins (for example [10,22,41] led to identification of a 41 kDa protein within mesocotyl cell wall fractions of Zea mays [11] and in plasmodesmal protein-enriched fractions from Arabidopsis [39]. Recently, this protein was shown to represent a member of the class1 reversibly glycosylated polypeptides (C1RGP) protein family [39]. RGPs localize to the Golgi and plasmodesmata as shown by fusion to GFP, suggesting that these proteins are secretory proteins that are delivered to plasmodesmata via the Golgi apparatus. A beta-1,3-glucanase that has been isolated from a plasmodesmata-enriched cell wall fraction of Arabidopsis may represent another potential plasmodesma-associated protein. When fused to GFP and expressed in tobacco or Nicotiana benthamiana, this protein labels cell wall puncta, which may represent plasmodesmata, as well as membranes of the endoplasmic reticulum [26]. Biochemical enrichment of a plasmodesmal protein fraction led also to purification of a casein kinase I (CKI) activity from N. tabacum suspension culture cells that was able to phosphorylate the TMV MP, which is consistent with previous findings that show TMV MP phosphorylation in planta. In transient expression assays in tobacco leaves, the kinase (CKL6) fused to GFP co-localized with TMV MP in cell wall associated puncta, suggesting its plasmodesmal localization [25].

(B) The use of antibodies against known proteins suspected to reside at plasmodesmata indicated cytoskeletal components [35, 38, 44] and calcium-binding proteins such as centrin and calreticulin as plasmodesmal proteins [1,2].

(C) The viral expression of cDNA libraries to express random proteins fused to GFP and subsequent selection for punctuate localization of fusion proteins to the cell wall, a pattern indicating plasmodesmal localization, revealed twelve GFP fusion proteins (PD01-12) potentially localizing to plasmodesmata. Plasmodesmal localization was confirmed for one of these proteins, PD01, by immuno gold labeling with antiserum against GFP [12].

In addition to these three strategies, also the isolation of MP-interacting factors can lead to the identification of novel proteins that are targeted to plasmodesmata. One example is Arabidopsis calreticulin, which shows affinity to TMV MP in vitro and colocalizes with MP in plasmodesmata [5]. Given the similarities to nuclear pore complexes with respect to flexibility in structure and permeability, plasmodesmata likely consist of many more structural proteins that need to be identified.

The currently used and perhaps most reliable markers for the labeling of plasmodesmata by the expression in fusion to fluorescent proteins are still mostly represented by viral MPs. As shown for the MP of TMV, during infection [17] or upon transient expression by microparticle bombardment [23] or agroinfiltration (Heinlein, unpublished observations), this protein accumulates to high levels and associates with various cellular components, including plasmodesmata, whereas a more selective labeling of plasmodesmata has been observed when this protein is expressed in transgenic plants [37]. Thus transgenic plants expressing viral MPs [37, 43] provide useful tools for those who would like to test the localization of other proteins to plasmodesmata. However, given the advances in protein purification and identification technologies, it can be expected that proteins other than viral MPs will soon become available as reliable markers for plasmodesmata.

GFP and GFP fusion proteins are also used to test the cell-and tissue-specific conductivity of plasmodesmata. Here, the cells expressing the proteins are tested for their ability to support the trafficking of fluorescence into adjacent cells. For example, Arabidopsis or tobacco plants that express GFP under the control of the companion cell-specific AtSUC2 promoter [19,33] were used to monitor the spreading of GFP from the companion cells in source leaves into a diversity of physiological sinks, including young leaves, root tips and ovules [19,33]. Likewise, GFP and GFP fusions, when introduced into cells either by microinjection or by ectopic expression following biolistic bombardment, were shown to passively diffuse between cells in sink leaves whereas the cell-to-cell spread in source leaves was reduced [6,7,33,45]. GFP and 2x GFP (GFP fused to GFP) were used to study the ability of plasmodesmata to traffic proteins during embryogenesis and early seedling development in Arabidopsis. These studies again supported the concept that plasmodesmata in younger tissues are more dilated and less restrictive than plasmodesmata in older tissues [20].


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