3.1 ROS signaling to regulate plasmodesmata
Callose, a β-1,3-glucan, plays a critical role in regulating PD-mediated intercellular trafficking. Accumulation of callose in the cell walls surrounding PD is associated with decreased intercellular trafficking, presumably via the physical occlusion of the PD pore. Conversely, removal or degradation of callose from around PD results in increased intercellular trafficking (Zavaliev et al., 2011). CALLOSE SYNTHASE (CALS) enzymes, (also called GLUCAN SYNTHASE LIKE, GSLs), are responsible for callose deposition at PD while β-1,3-glucanases degrade callose surrounding PD. ROS can influence callose deposition and degradation through the activation of enzymes like callose synthases and β-1,3-glucanases (Fig. 1) . Direct application of H2O2 shows dose-dependent effects on PD trafficking (Rutschow et al., 2011; Cui and Lee, 2016). One of the best-characterized mechanisms for ROS-induced callose deposition at PD involves PD LOCATED PROTEIN (PDLP)5 and CALS10/GSL8 (Saatian et al., 2023). Several other CALS/GSL proteins associate with PDLPs, and forming complexes that regulate PD trafficking during development or in response to stress. CALS3/GSL12 is involved in regulating intercellular trafficking during root development (Vaten et al., 2011). GSL8/CALS10 is required for guard cell differentiation and stomata development, and loss of GSL8 leads to increased intercellular trafficking CALS1 (Guseman et al., 2010). CALS1 and CALS8 have been implicate in stress-dependent PD regulation, with CALS8’s activity in callose deposition being induced in response the H2O2 (Cui and Lee, 2016). Further evidence linking ROS to CALS and PD regulation comes from studies with Roxy1 and Roxy2, two Glutaredoxins (GRXs) small oxidoreductases. Mutations in these genes have been shown to affect the expression of CALS5 and two β-1,3 glucanase genes, consequently impacting the accumulation of callose in the pollen cell wall (Xing and Zachgo, 2008). (Xing and Zachgo, 2008, The Plant Journal).
ROS can act as secondary messengers, triggering intracellular signaling pathways. For instance, ROS can initiate calcium ion (Ca2+) influx, leading to changes in cytosolic Ca2+ concentrations. The rise in Ca2+ levels is recognized as a universal intracellular signal that can initiate a range of cellular responses including changes in PD permeability (Yoneyama et al., 2004; Gilroy et al., 2014; Evans et al., 2016).
Calreticulin is an ER localized Ca2+ binding protein important for buffering Ca2+ levels, among other functions (Costa et al., 2018). Maize calreticulin was found to be associated with PD (Baluska et al., 1999) as was calreticulin from tobacco (N. tabacum ) BY2 (Laporte et al., 2003) and Arabidopsis (Bayer et al., 2004) cell cultures. Presumably, PD-associated calreticulin resides in the lumen of the PD-associated ER, the desmotubule. Increased calreticulin levels have been detected when callose has accumulated at PD to limit cell-to-cell trafficking, as in the case of stress due to Aluminium toxicity (Sivaguru et al., 2000). Aluminum stress also induced elevated calreticulin at PD ofMedicago truncatula roots associated with arbuscular mycorrhizal fungi (AMF) (Sujkowska-Rybkowska and Znojek, 2018). Calreticulin has also been implicated in callose accumulation at PD in response to chilling stress in maize (Zea mays ) (Bilska and Sowinski, 2010). Calreticulin levels at PD increased as early as four hours after chilling in chilling sensitive maize lines were accompanied by reduced carbon export and physical restriction of PD pores as revealed by transmission electron microscopy. Interestingly, this increase in calreticulin at PD and PD occlusion at early time points during chilling was not associated with callose accumulation, which did not occur until later times. These findings could be interpreted as indicating that calreticulin and its roles in Ca2+ regulation may have a distinct functions in regulating PD, physically modulating pore size (Demchenko et al., 2014). This hypothesis is supported by observations of the nitrogen-fixing nodules of Casuarina glauca, where PD lost calreticulin as the size of PD apertures decreased during nodule maturation (Demchenko et al., 2014). The PD in these mature cells also do not appear to be associated with callose.
Supporting a role for calreticulins in regulating PD trafficking during stress, a PD-targeted calreticulin was found to interact with the Tobacco mosaic virus (TMV) movement protein, and overexpression of this protein impaired the cell-to-cell trafficking of the movement protein itself and redirects the movement protein from the PD to microtubules (Chen et al., 2005). Concordant with these observations, intercellular viral spread was inhibited when the calreticulin was overexpressed. These findings with TMV contrast with recent findings with Bamboo mosaic virus (BaMV). N. benthamiana calreticulin 3 (NbCRT3) along with ER luminal-binding protein 4 (BiP4), both ER proteins, were found to interact with the TBGp3 movement protein of BaMV to facilitate viral cell-to-cell movement (Huang et al., 2023). Increased levels of NbCRT3 resulted in increased viral cell-to-cell spread, possibly due to increased or more efficient targeting of another movement proteinTGBp1 to PD. It remains to be determined whether the effects of NbCRT3 on trafficking of TGBp1 are simply due to increased endomembrane trafficking or if downstream signaling mediated by NbCRT3 is at play.
Besides calreticulin, calmodulins are class of Ca2+sensing proteins that associated with PD (Fernandez-Calvino et al., 2011). Calmodulin-like protein 41 (CML41) from Arabidopsis is involved in responses to bacterial infection where it triggers callose accumulation at PD, resulting in reduced intercellular trafficking of GFP probes (Xu et al., 2017). Indeed, increased PD callose levels and reduced PD trafficking were observed as early as 30 minutes after application of flg22, the elicitor derived from bacterial flagellin, and this response was dependent on Ca2+ levels as application of the chelator EGTA abolished PD inhibition in response to flg22. These rapid defenses were absent in plants where CML41expression was knocked down, and there was constitutively elevated callose levels at PD in plant overexpressing CML41 . CML41 interacts with the receptor-like kinase NOVEL CYS-RICH RECEPTOR KINASE (NCRK) as part of a module that causes ROS-induced callose accumulation at PD (Vu et al., 2022). This interaction is crucial because NRCK phosphorylates CML41, which is necessary for the accumulation of callose at the PD mediated by GLS4.
It has been well established that altering redox state in the chloroplast or the mitochondria affects PD permeability and thus cell-to-cell trafficking of biomolecules. The maize mutant sucrose export defective1 (sxd1) failed to export photosynthate from sites of photosynthesis and had reduced intercellular trafficking (Russin et al., 1996). It was proposed that was caused by accumulation of callose at PD located at the bundle-sheath and vascular parenchyma interface (Russin et al., 1996; Botha et al., 2000), although other evidence suggests that this was likely not solely due to altered PD function (Asensi-Fabado et al., 2015). SXD1 is the maize homolog of Arabidopsis VTE1 which encodes a chloroplast tocopherol cyclase required for production of the antioxidant vitamin E (Provencher et al., 2001; Porfirova et al., 2002). These observations led to the hypothesis that chloroplasts could regulate PD and intercellular trafficking. Subsequent analyses in the Arabidopsis ise1 and ise2 mutants with defects in a mitochondrial and chloroplast RNA helicase, respectively, supported this hypothesis (Burch-Smith et al., 2011). These mutants were isolated from a genetic screen of embryonically lethal mutants on the premise that defects in PD would drastically disrupt development leading to unviability (Kim et al., 2002). In ise1 and ise2Arabidopsis embryos and in N. benthamiana leaves whereISE1 or ISE2 expression was knocked down by virus-induced gene silencing (VIGS) increased intercellular trafficking was correlated with increased numbers of PD (Burch-Smith and Zambryski, 2010). Loss of ISE1 or ISE2 led to massive changes in gene expression, with chloroplast-associated genes representing the largest class of affected genes (Burch-Smith et al., 2011). These findings suggest that defects in organelles resulted in changes in nuclear gene expression, fine tuning expression of not only genes impinging on the defective organelles but also genes affecting organelles that were functionally linked. This hypothesis was described as ONPS when it described effects on PD (Burch-Smith et al., 2011; Brunkard and Burch-Smith, 2018). In particular, signals from chloroplasts may regulate PD-associated nuclear gene expression and thereby intercellular trafficking mediated by PD to control flux of chloroplast metabolites including fixed carbon (Brunkard and Burch-Smith; Ganusova et al., 2020).
A possible clue about the signaling involved in ONPS came using with redox-sensitive GFP (ro-GFP) probes. When expressed in a specific subcellular compartment, ro-GFP can report changes in the redox status of glutathione pools of that compartment (Bombarely et al., 2012) (Goodin et al., 2008). Analyses with these probes revealed that chloroplasts were more reduced in N. benthamiana leaves whereISE1 or ISE2 was silenced (Stonebloom et al., 2012). In ISE1 -silenced mitochondria were more oxidized compared to non-silenced leaves. The link between organelle redox status and intercellular trafficking capacity was supported by application of drugs that induced ROS production in specific organelles. Salicylhydroxamic acid (SHAM) induced mitochondrial ROS production and this apparently increased intercellular trafficking, consistent with observations from ISE1 -silenced leaves. In contrast, paraquat induced chloroplast ROS production and led to decreased intercellular trafficking as expected from results with ISE2-silenced plants. These redox-related findings are also consistent with those from an independent mutant with defective PD function. The Arabidopsis gfp arrested trafficking (gat)1 mutation also links PD function to chloroplast since it encodes plastid THIOREDOXIN m3, and intercellular trafficking was reduced in gat1 seedlings (Benitez-Alfonso et al., 2009). Analysis of this and related mutants revealed that plastid oxidative stress decreased PD-mediated trafficking (Benitez-Alfonso and Jackson).
Other mutants with defects in chloroplasts and PD-mediated have recently been identified. In the Arabidopsis dig8 mutant, defects in chloroplasts were associated with increased ROS levels and induction of ROS-related genes, concomitant reduced PD-mediated intercellular trafficking (Zhang et al., 2023). DIG8 encodes a protein predicted to be a chloroplast peptide release factor and therefore important for chloroplast gene expression. The chloroplast protein Kunitz peptidase inhibitor-like protein (KPILP) is a positive regulator of intercellular trafficking (Ershova et al., 2022). Overexpression led to increased intercellular trafficking of a 2xGFP proteins in N. benthamiana leaves. While ROS levels in tissues with increased KPILP levels were not reported, it is interesting that KPILP is induced by prolonged darkness and viral infection, conditions that are known to disturb chloroplast function and ROS production.
Curiously, only a few studies have explicitly examined the relationship between light and intercellular trafficking. Early work found that intercellular trafficking of GFP decreased in leaves kept in the dark (Liarzi and Epel, 2005). In contrast, a more recent study found that intercellular trafficking of GFP was strongly regulated by light, with longer light exposures leading to higher rates of trafficking (Brunkard et al., 2020). Importantly, this study made two other observations relevant to the current discussion. First, it demonstrated that ATP levels are important for intercellular trafficking as plants with reduced chloroplast ATP levels caused by silencing the chloroplastAtpC gene has reduced capacity for trafficking GFP. Second, the difference in trafficking between day and night was not dependent on PD callose levels, hinting at other mechanisms at play in regulating PD function (Brunkard et al., 2020). Further support for ATP (energy) levels as important determinant of PD trafficking capacity come from studies showing that TOR signaling is an important regulator of intercellular trafficking (Brunkard et al., 2020). Independent confirmation of the importance of ATP levels in modulating PD function was supported by work with the Arabidopsis ch1-3. Studies with this mutant under varying light regimes revealing that ATP and NADPH levels were important for regulating PD-mediated intercellular trafficking (Dmitrieva et al., 2021). Importantly, one finding of this study is that chloroplast ROS levels and redox states are likely not important regulators of intercellular trafficking. Further investigation of the roles of chloroplast redox state and chloroplast-generated ROS in ONPS is therefore warranted. In response to high light stress in Arabidopsis, the production of reactive oxygen species (ROS) resulted in an augmentation of intercellular carboxyfluorescein transport and an enlargement of plasmodesmata (PD) pores. This effect was contingent on the presence of PDLP1 and PDLP5 {Fichman, 2021}.