Loss of INCREASED SIZE EXCLUSION LIMIT (ISE)1 or ISE2 increases the formation of secondary plasmodesmata

Abstract

Plasmodesmata (PD) transport developmentally important nucleic acids and proteins between plant cells. Primary PD form during cell division and are simple, linear channels. Secondary PD form in existing cell walls after cell division and are simple, twinned, or branched. PD function undergoes a marked reduction at the mid-torpedo stage of Arabidopsis embryogenesis. Two mutants, increased size exclusion limit (ise)1 and ise2, fail to undergo this transition, and their null mutations are embryonically lethal. We investigated the ultrastructure of PD in early-, mid-, and late-torpedo-stage embryos and in young leaves. Wild-type (WT) embryos contain twinned and branched (T/B) PD at all stages, but ise1 and ise2 embryos contain significantly higher proportions of T/B PD than WT embryos. WT T/B PD formation occurs in a stage- and tissue-specific pattern that is reversed in ise1 embryos. Silencing ISE1 in Nicotiana benthamiana leaves increases the frequency of secondary PD in existing cell walls. Silencing ISE2 increases the proportion of T/B secondary PD formed. Silenced tissues exhibit increased PD-mediated movement of green fluorescent protein tracers. Thus, silencing of ISE1 and ISE2 phenocopies ise1 and ise2 mutant embryos: when wild-type ISE1 and ISE2 functions are lost, de novo production of PD occurs, leading to increased intercellular transport.

Figure 1. Analysis of PD structure in embryonic cotyledons and hypocotyls

(A) Simple PD. (B) Twinned PD. (C-D) Branched PD may be Y or H-shaped. Scale bar represents 200 nm in (A) and 100 nm in (B, C and D). (E) The fraction of twinned and branched (T/B) PD were counted in cotyledons and hypocotyls from early-, mid-, and late-torpedo embyos. *p<0.05 compared to wild type tissues. Refer to Figure S1.

Figure 2. Frequency of PD per um² and T/B PD formed in N. benthamiana cell walls

(A) Representative TEM image of an epidermal cell from the youngest leaf. Left, epidermal-epidermal cell wall (arrowhead). Bottom, epidermal-mesophyll cell wall (arrow). (B) Mean PD frequency um^-2 in epidermal-epidermal (black bars) or epidermal-mesophyll (grey bars) cell walls. *p<0.05 compared to non-silenced control leaves. (C) Proportions of T/B PD in epidermal-epidermal (black bars) and epidermal-mesophyll (grey bars) cell walls. *p<0.05 compared to non-silenced control leaves.

Figure 3. P30-2XGFP and 2XGFP cell-to-cell transport via PD

(A-B) Z-series projections from non-silenced N. benthamiana leaf tissue 48 hpi (A) or 72 hpi (B) with Agrobacterium carrying the P30-2XGFP expression cassette. The brightest cell is the primary transformed cell and the surrounding cells are cells into which the P30-2XGFP has moved via PD. P30-2XGFP marks PD as bright punctae (inset). (C) Mesophyll cells under the focus in (B) contain P30-2XGFP. Arrows, mesophyll cell walls containing P30-2XGFP. (D) Cartoon of side view of P30-2XGFP foci. The primary transformed cell is bright green. P30-2XGFP moves cell-to-cell and localizes to PD (green punctae) of neighbouring epidermal cells and mesophyll cells below. Dotted lines represent cross-section planes for cells shown in top-down view in (E). (E) Cartoon of top-down view of cross-section of mesophyll cells in (D). P30-2XGFP punctae in PD (green punctae), as observed in (C). (F) Z-stack projection through a 2XGFP focus in a non-silenced leaf at 72 hpi. Bright cell is the primary, transformed cell where GFP localizes throughout the cell. Low 2XGFP fluorescence occurs in the cytoplasm and nuclei of the surrounding ring of cells. (G) Z-stack projection through a 2XGFP focus in an ISE1-silenced N. benthamiana leaf at 72 hpi. 2XGFP moves extensively into the neighboring 2 rings of cells. There is a gradient of GFP fluorescence from the primary cell to the second ring of cells. Asterisks mark the nuclei of the second ring of cells.

Figure 4. Cell-to-cell transport is altered in ISE1 and/or ISE2-silenced leaves

(A) Size of foci measured as numbers of rings of cells containing P30-2XGFP punctae around a transformed cell 48 hpi in control or silenced leaves. (B) Size of P30-2XGFP foci at 72 hpi in control or silenced leaves. (C) Number of foci with P30-2XGFP in the mesophyll PD at 48 hpi scored as a percent of the total number of foci observed. *p <0.05 compared to non-silenced controls. (D) Fraction of foci with P30-2XGFP in the mesophyll PD at 72 hpi.