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

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.


RESULTS
Silencing genes that affect PD transport alters the cellular redox state ise1 and ise2 mutants and virus-induced gene silencing (VIGS) of ISE1 and ISE2 increase intercellular transport and induce the production of secondary PD (Stonebloom et al., 2009;Burch-Smith and Zambryski, 2010). ISE1 localizes to mitochondria, and both ise1 mutants and ISE1 silenced tissue exhibit increased production of ROS (Stonebloom et al., 2009). The specific effects of loss of ISE1 function on redox state in sub-cellular compartments, however, are unknown. ISE2 localizes to plastids, as has been recently demonstrated using bioinformatics, proteomics, and microscopy (Ichikawa et al., 2006;Olinares et al., 2010;Burch-Smith et al., in press), and its effect on cellular redox state has not been tested. To monitor specific redox states in subcellular compartments we utilized redox sensitive GFPs (roGFPs) that target to the cytoplasm, mitochondria or plastids.
RoGFPs are a set of GFP variants in which two cysteine residues have been introduced near the fluorophore. Formation of a disulfide bridge between the two introduced cysteines induces a structural rearrangement of amino acid side chains contacting the chromophore, favoring a neutral rather than an anionic chromophore (Cannon and Remington, 2009). The neutral and anionic forms of the chromophore possess distinct peak fluorescence excitation wavelengths at 400 nm and 495 nm, respectively. Measurement of roGFP fluorescence intensity following excitation at each excitation maximum permits an evaluation of the relative proportion of roGFP in a reduced or oxidized state. In vitro and in vivo studies have shown that roGFPs are in equilibrium with the redox potential of the glutathione (GSH) pool (Schwarzländer et al., 2008). Therefore changes in the proportion of oxidized roGFP reflect changes in the ratio of GSH to GSSG in the subcellular compartment where roGFP is targeted. roGFPs have been used in Arabidopsis to examine the cellular redox state during root development (Jiang et al., 2006), drought stress (Jubany-Marí et al., 2010), dark treatment (Rosenwasser et al., 2011) and response to treatment with metabolic inhibitors (Lehmann et al., 2009;Schwarzländer et al., 2009 We first silenced ISE1 and ISE2 via VIGS in N. benthamiana plants (as in Burch-Smith and Zambryski, 2010). Then, cytoplasmic (cyto)-roGFP1, mitochondrial (mito)-roGFP1, or plastid-roGFP2 were transiently expressed in silenced leaves and the oxidation states of six regions from at least eight leaves were measured using ratiometric fluorescence microscopy. Silencing ISE1 induced oxidation of mito-roGFP1, reduction of plastid-roGFP2, and did not significantly alter the redox state of cyto-roGFP1 ( Fig. 1). Silencing ISE2 induced reduction of plastid-roGFP2 and cyto-roGFP1, but did not affect the oxidation state of mito-roGFP1 (Fig. 1). These results indicate that the net increases in ROS accumulation seen in ise1 mutants and ISE1 silenced plants, which had been observed using techniques that cannot resolve the subcellular location of ROS accumulation (Stonebloom et al, 2009), are actually due to increases in ROS production specifically within the mitochondria. Unexpectedly, silencing either ISE1 or ISE2 induces reductive shifts in plastids, although ISE1 localizes exclusively to mitochondria. The data together reveal that oxidative shifts in mitochondria and reductive shifts in plastids lead to increased cell-to-cell transport. The data further suggest inter-organelle signaling between mitochondria and plastids in the absence of ISE1 function.
The localization of mito-roGFP1 and cyto-roGFP1 have been previously documented (Jiang et al, 2006). Figure 2 documents the plastid specific localization of the plastid-roGFP2 constructed herein.

Effects of metabolic inhibitor treatment on intercellular transport via PD
To determine if changes in individual organelle redox state are capable of regulating PD function, we selected metabolic inhibitors predicted to specifically alter the redox state of chloroplasts or mitochondria and then determined their impact on PDmediated intercellular transport. Paraquat (also called methyl viologen dichloride) is an herbicide that primarily affects plants by acting as a terminal oxidant for photo-system I and by direct oxidation of plastid reductants, including NADP + and ferredoxin (Farrington et al., 1973;Babbs et al., 1989;Foyer and Noctor, 2009). Reduced paraquat interacts with molecular oxygen to yield superoxide, and then paraquat serves as an oxidant again, creating a redox cycle that rapidly increases ROS levels and oxidizes the plastids. Salicylhydroxamic acid inhibits the mitochondrial alternative oxidase, an enzyme that allows electrons to bypass the mitochondrial electron transport chain by transferring electrons from ubiquinol to oxygen, thus limiting the translocation of protons and decreasing the efficiency of ATP generation (Schwarzländer et al., 2009). The alternative oxidase pathway is induced in response to cold and other stresses, and serves to stabilize cellular redox state (Armstrong et al., 2008). Inhibition of the alternative oxidase pathway leads to production of ROS specifically in the mitochondria (Maxwell et al., 1999).
To evaluate the effects of metabolic inhibitors on PD transport we developed a particle bombardment based cell-to-cell movement assay for Arabidopsis thaliana leaves following metabolic inhibitor treatment. Expanded true leaves of 11-day old Arabidopsis seedlings were removed and placed on agar media with varied concentrations of paraquat or salicylhydroxamic acid. We initially applied relatively high Our assay for the effects of metabolic inhibitors on cell-to-cell transport via PD involves four steps. First, true leaves from 11-day old Arabidopsis thaliana plants were isolated and placed on growth medium plates. Second, low pressure particle bombardment was used to introduce plasmid DNA containing a 35S::sGFP expression cassette into isolated epidermal cells. To ensure efficient and random transfection of DNA, the abaxial epidermis was bombarded twice in rapid succession. Third, leaves were transferred to 1/2x MS plates supplemented with 1% sucrose and metabolic inhibitors or a comparable volume of the relevant solvent. Treated leaves were incubated for 24 hours under constant light. Fourth, GFP expressing foci were imaged using confocal laser scanning microscopy. Images were captured as stacks of confocal sections through the epidermal layer in the region of each GFP expressing cell.
Examples of reconstructed projections of stacked images for typical GFP foci after treatment with each metabolic inhibitor are presented in Figure 3. Control leaves (water treatment for paraquat experiments, DMSO treatment for salicylhydroxamic acid experiments) show visible movement of GFP away from the initial transfected cell.
Leaves treated with salicylhydroxamic acid show significantly more intercellular transport of GFP than control leaves (Fig. 3A), while leaves treated with paraquat show significantly less intercellular transport of GFP than untreated leaves (Fig. 3C). Figure 3 and Tables I and II present a quantitative study of cell-to-cell movement in ~50 independent foci for each treatment, where the number of contiguous rings of cells with detectable levels of GFP away from each primary transfected cell was counted. Free GFP (27 kDa) is below the size limit of the nuclear pore, so GFP movement into cells surrounding the initial transfected cell is visible by GFP fluorescence in nuclei, where GFP can accumulate. Nuclear localization is an easily scorable marker for quantifying cell-to-cell movement in the leaf epidermis, as shown previously (Stonebloom et al., 2009;Burch-Smith and Zambryski, 2010). In control leaves, GFP was confined to 2 or fewer rings of cells in 55% of expression foci, and could travel as far as 4 cells away from the initial transfected cell. Treatment with 200 μ M salicylhydroxamic acid treatment dramatically induced cell-to-cell movement of GFP in leaves: only 28% of foci exhibited movement of GFP to 2 or fewer rings of cells, and 72 % exhibited movement to 3 or more rings of cells (Fig. 3A,B). In contrast, following treatment with 1 μ M paraquat, GFP was restricted to 2 or fewer rings of cells in 83% of foci, and was never observed in cells more than 3 rings away from the initial transfected cell (Fig. 3C,D). Thus, 1 μ M paraquat treatment markedly decreased the cell-to-cell movement of GFP. Treatment with salicylhydroxamic acid or paraquat significantly affected the measured intercellular transport in this assay (n>43, p<0.001).
To determine if a threshold exists for the oxidative shifts necessary to alter the transport abilities of leaf epidermal PD, we serially reduced the concentrations of metabolic inhibitors in our movement assay. The cell-to-cell movement assay was  (Table II). In contrast, in leaves treated with 1 μ M paraquat GFP was restricted to 1 or fewer rings of cells in 43% of foci (Table II). In summary, treatment with lowered concentrations of paraquat and salicylhydroxamic acid did not notably affect cell-to-cell movement of GFP. Thus, the higher concentrations of each compound used for the results presented in Figures 3-4 approach the minimum concentrations necessary to induce statistically significant changes in cell-to-cell movement.

Effects of metabolic inhibitor treatment on cellular redox state
To evaluate redox state in different cellular compartments following metabolic inhibitor treatment, we measured the oxidation state of roGFP in transgenic Arabidopsis seedlings expressing cyto-roGFP1, mito-roGFP1, or plastid-roGFP2. As in the cell-tocell movement assay, expanded true leaves were removed from 11-day old seedlings and placed on agar media containing inhibitors. Leaves treated for 24 hours with 200 2001), showed strong induction of ROS production in plastids, mitochondria, and cytoplasm. These data imply that an oxidative shift in the plastid leads to secondary effects in the cytoplasm and mitochondria, further demonstrating crosstalk between the organelles, as shown above when VIGS of mitochondria-localized ISE1 led to reductive shifts in plastids (Fig. 1).
Since treatment with reduced concentrations of paraquat or salicylhydroxamic acid did not significantly affect transport via PD, we tested if these lower concentrations have a significant effect on sub-cellular redox states. Leaves expressing transgenic cyto-roGFP1, mito-roGFP1, or plastid-roGFP2 were treated with the lowered concentrations of each metabolic inhibitor and examined using ratiometric fluorescence microscopy. Treatment with lowered concentrations of salicylhydroxamic acid did not significantly affect the subcellular redox states as reported by roGFP (Fig. 5, A,C,E). As a slight exception, treatment with 20 μ M salicylhydroxamic acid led to minor oxidation of mito-roGFP1, but this small change was insufficient to induce detectable changes in intercellular transport using the cell-to-cell movement assays. Treatment with 0.1 μ M or 0.01 μ M paraquat did not induce significant changes in the redox state of cyto-roGFP1, mito-roGFP1, or plastid-roGFP2 (Fig. 5, B, D, F). We conclude that the changes in intercellular transport with 1 µM paraquat or 200 µM salicylhydroxamic acid (Fig. 3) reflect the lowest concentration capable of also inducing changes in redox state. We recently proposed a model of organelle-nucleus-plasmodesmata signaling (ONPS) that connects plastid development, especially the induction of photosynthesisrelated nuclear genes during the mid-torpedo stage of embryogenesis, to PD structure, function, and biogenesis (Burch-Smith et al., in press). Besides plastid-targeted gene products, Burch-Smith et al. (in press) further demonstrate that genes for products that localize to plasmodesmata or to plant cell walls are also altered during mitochondrial (ise1 mutant) or plastid (ise2 mutant) dysfunction. Potentially, alterations in mitochondrial or plastid redox states also utilize ONPS to signal alterations in expression of genes that encode products that alter PD formation or function.
Independent studies demonstrate that a mutant for the plastid thioredoxin Similarly, loss of GAT1/TRX-m3 function likely results in a local oxidative shift in plastids, which in turn leads to oxidation of other subcellular compartments.
Beyond organelle-nucleus signaling initiated by changes in organelle redox state due to aberrant ROS production within the organelles, there are likely two distinct types of pathways where ROS may function directly or as upstream signals to modify PD.
First, ROS may act directly as oxidants at or near PD to alter their formation or function. controlled release of hydroxyl radicals (Cosgrove, 2005;Müller et al., 2009;Tsukagoshi et al., 2010). Cytological and proteomic studies further indicate that several class III peroxidases may localize to PD, where they could produce H 2 O 2 to rapidly induce changes to cell wall polysaccharides around PD to dynamically regulate PD structure (458/488 nm) and helium/neon (543 nm) lasers. GFP was excited with the 488-nm laser band at 49% laser power and emitted light was collected between 505 and 530 nm. Foci were imaged with a 25x oil immersion objective. Z-stacks were collected through the epidermis in the region of each primary expression focus.

Ratiometric measurement of roGFP
roGFP imaging and analysis of roGFP excitation ratios was conducted as in (Jiang et al., 2006). Leaves were imaged on a Nikon Diaphot Nikon