Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states

Abstract

Plant cells rely on plasmodesmata for intercellular transport of small signaling molecules as well as larger informational macromolecules such as proteins. A green fluorescent protein (GFP) reporter and low-pressure microprojectile bombardment were used to quantify the degree of symplastic continuity between cells of the leaf at different developmental stages and under different growth conditions. Plasmodesmata were observed to be closed to the transport of GFP or dilated to allow the traffic of GFP. In sink leaves, between 34% and 67% of the cells transport GFP (27 kD), and between 30% and 46% of the cells transport double GFP (54 kD). In leaves in transition transport was reduced; between 21% and 46% and between 2% and 9% of cells transport single and double GFP, respectively. Thus, leaf age dramatically affects the ability of cells to exchange proteins nonselectively. Further, the number of cells allowing GFP or double GFP movement was sensitive to growth conditions because greenhouse-grown plants exhibited higher diffusion rates than culture-grown plants. These studies reveal that leaf cell plasmodesmata are dynamic and do not have a set size exclusion limit. We also examined targeted movement of the movement protein of tobacco mosaic virus fused to GFP, P30::GFP. This 58-kD fusion protein localizes to plasmodesmata, consistently transits from up to 78% of transfected cells, and was not sensitive to developmental age or growth conditions. The relative number of cells containing dilated plasmodesmata varies between different species of tobacco, with Nicotiana clevelandii exhibiting greater diffusion of proteins than Nicotiana tabacum.

Figure 1

Schematic representation of regions A and B of plants analyzed for protein movement

Figure 2

CF loading. CF was loaded into the phloem through a cut that severs the root system. B through E, Images of leaves after a 30-min loading period; A and G, after a 10-min loading. The leaves analyzed in this study and subsequent figures are represented by A, B, and D through G. A is a low magnification view of loading in a sink leaf; the densely spaced trichome hairs illustrate that the leaf is unexpanded. B shows symplastic coupling between the cells in this sink leaf. For comparison, C shows minimal loading in a source leaf (leaves below region B, Fig. 1). D and F show the tip and mid-blade regions of a leaf in transition (from sink to source). E and G show high (30 min) and low (10 min) symplastic unloading in transition leaves. A, Scale bar = 1 mm; B through G, scale bar = 200 μm.

Figure 3

Non-targeted diffusion through plasmodesmata. All images were captured 16 to 20 h post-bombardment using a CCD camera and epifluorescent microscope equipped with a fluorescein isothiocyanate filter set. Scale bars = 10 μm. A, GFP expression in sink tissue (region A) of N. tabacum. B, GFP expression in transition leaf (region B) of N. tabacum. C, Localization and spread of 2×GFP is similar to GFP in sink leaf. D, 2×GFP is often restricted to the transfected cell as seen here in a transition leaf. E, 3×GFP does not leave the transfected cell in sink tissues. F, 3×GFP movement is limited also in transition leaves.

Figure 4

P30::GFP is targeted to punctae in the cell wall in region A and region B leaves. All images were captured using a CCD camera and epifluorescent microscope equipped with a fluorescein isothiocyanate filter set. Scale bars are 10 μm A, P30::GFP moves efficiently and localizes to cell wall punctae in sink tissue of cultured N. tabacum. B, P30::GFP moves frequently to adjacent cells in transition leaves. Fluorescent punctae of P30::GFP are seen here in underlying mesophyll cells of cultured N. tabacum (focused through overlying epidermal cells). Mesophyll cells are smaller and more oval-shaped than puzzle-shaped cells of epidermis seen in A, C, and D. C, P30::GFP punctae in the cell wall of a sink leaf of a soil-grown N. tabacum. D, P30::GFP localizes to punctae in transitioning leaves of soil-grown N. tabacum.

Figure 5

Different plasmodesmata states within the leaf. We detect different frequencies of movement with the various probes used to assess plasmodesmata aperture. Some transfected cells do not exhibit GFP movement; thus, their plasmodesmata are completely closed or only open to small molecules. Closed plasmodesmata would not allow any transport, and could be permanently (as in stomata) or temporarily sealed (as has been reported in the shoot apical meristem; Rinne and Van Der Schoot, 1998; Gisel et al., 1999; van der Schoot and Rinne, 1999). Open plasmodesmata would allow for the exchange of nutrients and small dyes (i.e. sugars, CF, and 8-hydroxypyrene 1,3,6 trisulfonic acid). Other cells of the leaf have dilated plasmodesmata of varying apertures that allow for macromolecular trafficking through plasmodesmata. One population of cells allows GFP diffusion, implying their plasmodesmata are dilated to a sufficient degree to allow this 27-kD molecule to transit. A smaller population of transfected cells have plasmodesmata that are dilated to a higher degree, as they allow 2×GFP (54 kD) transit. These results suggest that the leaf is a mosaic where cells exist with plasmodesmata in varying states of distention and that dilated plasmodesmata do not have a single-set aperture.