Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata

Abstract

Recent studies suggest that intercellular transport via plasmodesmata (PD) is regulated by cellular redox state. Until now, this relationship has been unclear, as increased production of reactive oxygen species (ROS) has been associated with both increased and decreased intercellular transport via PD. Here, we show that silencing two genes that both increase transport via PD, INCREASED SIZE EXCLUSION LIMIT1 (ISE1) and ISE2, alters organelle redox state. Using redox-sensitive green fluorescent proteins targeted to the mitochondria or plastids, we show that, relative to wild-type leaves, plastids are more reduced in both ISE1- and ISE2-silenced leaves, whereas mitochondria are more oxidized in ISE1-silenced leaves. We further show that PD transport is positively regulated by ROS production in mitochondria following treatment with salicylhydroxamic acid but negatively regulated by an oxidative shift in both chloroplasts and mitochondria following treatment with paraquat. Thus, oxidative shifts in the mitochondrial redox state positively regulate intercellular transport in leaves, but oxidative shifts in the plastid redox state counteract this effect and negatively regulate intercellular transport. This proposed model reconciles previous contradictory evidence relating ROS production to PD transport and supports accumulating evidence that mitochondria and plastids are crucial regulators of PD function.

Figure 1.

VIGS of ISE1 and ISE2 in N. benthamiana alters organelle redox state. The proportion of oxidized mito-roGFP1 (A), plastid roGFP2 (B), or cyto-roGFP1 (C) following tobacco rattle VIGS of ISE1, ISE2, or a control construct. *P < 0.001.

Figure 2.

Plastid-roGFP2 localizes to plastids. Chloroplast autofluorescence, shown in green (A), and plastid-roGFP2 fluorescence, shown in magenta (B), colocalize, shown in white in the merged image (C). Scale bars, 20 μm.

Figure 3.

Effects of salicylhydroxamic acid and paraquat on cell-to-cell movement of GFP. GFP often moves to at least three rings of cells in leaves treated with 200 μm salicylhydroxamic acid (A) or is restricted to one or fewer rings in leaves treated with 1 μm paraquat (C), as shown in these representative images from the intercellular movement assay. Intercellular movement was determined by size of fluorescent foci, measured as numbers of rings of cells containing GFP in the nuclei of cells surrounding a single transfected cell 24 h after particle bombardment in leaves treated with several concentrations of salicylhydroxamic acid in DMSO (B) or paraquat in water (D). *P < 0.001.

Figure 4.

Salicylhydroxamic acid (SH) and paraquat treatment affect the organelle redox state. Proportion of oxidized mito-roGFP1 (A), plastid roGFP2 (B), or cyto-roGFP1 in Arabidopsis leaves following treatment with 200 μm SH in DMSO or 1 μm paraquat (PQ) in water. *P < 0.001.

Figure 5.

The effects of reduced concentrations of metabolic inhibitors on organelle redox state. The proportion of oxidized mito-roGFP1 (A and B), plastid roGFP2 (C and D), or cyto-roGFP1 (E and F) after treatment with varying concentrations of salicylhydroxamic acid in DMSO (A, C, and E) or paraquat in water (B, D, and E). *P < 0.001.

Figure 6.

Organelle redox states differentially regulate PD. VIGS of ISE1 or ISE2 or treatment with salicylhydroxamic acid or paraquat (PQ) alter (increase, blue arrow; decrease, orange bar) PD-mediated transport and induce shifts in the redox states of plastids (green) and/or mitochondria (pink). See text for details. Purple, Endoplasmic reticulum; yellow, cell wall.