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}.