SO Signaling In Plants
Because of SO’s extremely high reactivity in aqueous and organic
solutions, and the abundance of ROS scavengers in cells, SO has been
estimated to have half-life of 200 ns in biological environments (Gorman
& Rodgers, 1992). Therefore, responses to intracellular sources of SO
are likely due to interactions with biomolecules close to the site of
production that initiate a chloroplast-to-nucleus retrograde signal.
These may be β-carotene near PSII reaction centers, lipids of
chloroplast membranes, or proteins embedded in the thylakoid membrane
(Wagner et al., 2004; Przybyla et al., 2008; Ramel et al., 2012b). InA. thaliana , there appears to be more than one distinct pathway
for SO retrograde signaling (Figure 2), but detection of SO by the
EXECUTOR1 protein in the grana margins or carotenoid signaling in the
grana core are of particular importance.
SO Sensing by EXECUTER1. EXECUTER1 (EX1) and its homolog
EXECUTER2 (EX2) were originally discovered as a result of work with the
conditional flu mutant (Meskauskiene et al., 2001), and they
modulate most of the phenotypes generated by SO induction in flu .
The amount of SO that accumulates in L:D:L-exposed flu mutants
varies depending upon the duration of the dark period and the light
intensity of re-exposure (Lee et al. 2007; Hou et al. 2019). As a
result, this mutant can be utilized to study both programmed cell death
(PCD) in response to high SO dosages, or stress acclimation programs
activated by sub-lethal SO doses. Experiments with the flu mutant
demonstrate that EX1 plays a role in both of these processes, and
reduces or blocks the majority of phenotypes caused by flu (e.g.
Lee et al., 2007; Zhang et al., 2014). Exposing flu to 8h of
darkness followed by reillumination halts plant growth, induces the
formation of lesions on foliage, modulates expression of a large set of
SO-responsive genes (SORGs), and activates multiple hormone signaling
pathways (Ochsenbein et al., 2006; Lee et al., 2007; Przybyla et al.,
2008). Introducing the ex1 loss-of function mutation into theflu background inhibited lesion formation, restored growth, and
blocked the induction of ~80% of SORGs, whereas EX2 was
not required for induction of most SORGs (Lee et al., 2007). EX1 also
contributes to the effects of SO on hormone signaling. Przybyla and
coworkers (2006) demonstrated that exposing flu to L:D:L shift
induced enzymatic lipid peroxidation and accumulation of the oxylipin
hormones 12-oxo phytodienoic acid (OPDA) and jasmonic acid, whereas
these responses were inhibited in the flu/ex1 double mutant
(Przybyla et al., 2008). The L:D:L shift also has been shown to cause a
rapid upregulation of ENHANCED DISEASE SUSCEPTIBILITY1(EDS1 ) and salicylic acid (SA) accumulation, which consequently
activated expression of genes encoding pathogenesis-related (PR)
proteins PR1 and PR5 (Ochsenbein et al., 2006). In
protoplasts, SA contributed to the cell death phenotype observed influ (Danon et al., 2005). Zheng and coworkers (2014) subsequently
showed that PR1 induction was compromised in theflu /ex1/ex2 triple mutant, suggesting that induction of SA
signaling by SO is dependent upon EX1.
The complex molecular processes through which EX1 and EX2 mediate plant
responses to SO are not yet fully resolved, but recent studies have made
major advances in deciphering them. The EX1 and EX2 proteins are
localized to the non-appressed region of the thylakoid membrane called
the grana margin (Wang et al., 2016), where, prior to stimulation by SO,
they complex with several other proteins, including GENOMES UNCOUPLED4
(GUN4) and GUN5, proteins involved in tetrapyrrole synthesis (Li et al.
2023). In response to SO accumulation, EX1 undergoes oxidative
modification, disassociates from the complex, and accumulates in the
nucleus, where it interacts directly with WRKY transcription factors and
gene promoters to activate expression of SORGs (Li et al. 2023). In
parallel, exposure to SO also causes a dose-dependent decline in EX1
abundance that requires a functional copy of the thylakoid
membrane-bound metalloprotease FtsH2 (Wang et al., 2016; Dogra et al.,
2017). Inactivation of FtsH2 repressed induction of
~85% of EX1-dependent SORGs in flu , implying
that proteolysis of EX1 by FtsH2 is important to its function in
SO-responsive signaling (Dogra et al., 2017). Like EX1, EX2 can also
undergo oxidative modification by SO and proteolysis by FtsH2, and the
presence of a functional copy of EX2 slows down proteolysis of EX1 and
decreases expression of EX1-dependent SORGs (Dogra et al., 2022). These
results suggest that EX2 acts as a negative modulator of EX1 signaling,
tapping the brakes on this system by competing with EX1 to interact with
SO or FtsH2.
Further work is needed to determine how and why proteolysis of EX1
promotes EX1-dependent regulation. The EX1 proteins found in the nucleus
after SO induction are full-length (Li et al. 2023), and so it appears
that there are two separate pools of EX1 in the cell—one that
moves to the nucleus to act as a transcriptional activator, and one that
remains in the chloroplast to be degraded. Somehow these pools act
synergistically to promote SORG expression. Another important question
that remains to be resolved is the source of ROS that oxidizes EX1 and
EX2 after SO induction. The typical site of SO production occurs from
active PSII in the appressed thylakoid (grana core) during
photosynthesis. However, the reactive nature and short half-life of SO
(Gorman and Rodgers, 1992) severely reduce the likelihood of this ROS
traveling from the grana core to the grana margin. It is possible that
SO in the grana core triggers production of other more stable ROS that
move to the grana margins to modify EX1 and EX2. Alternatively, it has
been proposed that there is an additional mechanism to generate SO in
the grana margin via chlorophyll precursors or damaged PSII subunits
sent to the grana margins for repair (Wang et al., 2016; Dogra and Kim,
2020). EX1 and EX2 coprecipitate with multiple proteins including the
PSII D1 and D2 proteins and proteins involved in chlorophyll synthesis,
including GUN4, GUN5, and Pchlide oxidoreductases (Dogra et al., 2022;
Li et al. 2023). GUN4 and GUN5 are upstream of Pchlide synthesis in the
chlorophyll synthesis pathway, whereas Pchlide oxidoreductases convert
Pchlide to chlorophyllide, and so the balance of activities among these
enzymes could regulate Pchlide accumulation. GUN4 has been implicated in
SO generation (Tabrizi et al., 2016), and together with GUN5, it may
promote Pchlide synthesis and SO generation in the grana margins,
causing oxidation of EX1 and activation of EX1 signaling. Additional
studies are needed to confirm the source of ROS at the grana margins and
the functional significance of EX1’s multiple interaction partners.
Carotenoids and Other SO Signaling Pathways. In addition
to the EXECUTOR pathway, which can mediate stress-responsive programmed
cell death or at lower SO dosages enable stress acclimation, β-carotene
derivatives also play a role in acclimation to high light stress (Ramel
et al., 2012a, 2013). The ch1 mutant, which accumulates excess SO
in the grana core, is commonly used to study the role of carotenoids in
SO signaling. The reaction of SO with carotenoid scavengers near the
reaction center of PSII yields aldehydes and endoperoxides through
oxidative modification (Ramel et al., 2012a). Specifically, oxidation of
the carotenoid β-carotene by SO gives rise to β-cyclocitral (β-CC), a
volatile, highly reactive electrophilic compound that can then diffuse
out of the chloroplast to signal for an acclimation response to high
light stress (Ramel et al., 2012a). Importantly, β-CC generation occurs
in the grana core of the thylakoid membrane where active PSII reside,
whereas EX1 and EX2 are localized in the grana margins, where damaged D1
and D2 proteins of the PSII reaction centers are sent for repair (Dogra
and Kim, 2020). Thus, the β-CC and EXECUTER pathways are not initiated
in the same area, or by the same SO-generating mechanism, and remain
relatively distinct from one another.
Pretreating A. thaliana with β-CC upregulated genes associated
with oxidative stress, hormone signaling, and detoxification, and
rendered plants more tolerant to high light exposure in a dose-dependent
manner (Ramel et al., 2012a). It has been proposed that the protein
METHYLENE BLUE SENSITIVITY (MBS1) is activated downstream of β-CC to
transduce the signal to the nucleus for regulation of plant growth and
development under high light stress (Shumbe et al., 2017). In addition,
D’Alessandro and colleagues (2018) identified Scarecrow-Like14(SLC14 ) as another downstream mediator of the SO signal
transduced by β-CC that acts independently of MBS1. SLC14, a GRAS family
transcription factor, further regulates the expression of NAC
transcription factors, and a transgenic line overexpressing SLC14was found to have enhanced resilience to high light stress, indicatingSLC14 is involved in photooxidative adaptation. However, the
authors also discovered that only 30% of gene expression changes inch1 mutants under high light stress were due to β-CC (Shumbe et
al., 2017). Therefore, it is likely that multiple pathways of SO
signaling exist for adaptation under high light stress and defense
against other abiotic and biotic stressors. Consistent with this
hypothesis, Wang and coworkers (2020) report the existence of an
EX1-dependent signaling pathway that is negatively regulated by a
protein in the chloroplast stroma, SAFEGAURD1, that is degraded in
response to SO (SAFE1). SAFE1 protects the grana margins from damage by
SO in flu plants, and loss of function of SAFE1 in aflu /ex1 background restores the cell death phenotype and
many of the transcriptional responses to SO that are seen in flubut normally suppressed by ex1 . Another EX1-independent response
factor is OXIDATIVE SIGNAL INDUCIBLE1 (OXI1), a kinase that mediates
SO-responsive cell death in the ch1 mutant, probably through a
jasmonate-dependent signaling mechanism (Shumbe et al. 2016). It is
unclear whether carotenoid signaling promotes OXI1 signaling. Further
studies are needed to characterize EX1-independent pathways, examine the
potential interconnections among the different SO signaling pathways,
and definitively establish their roles in wild-type responses to SO and
SO-generating stresses. However, markers associated with known SO
signaling pathways give us a good starting point to identify stresses
that activate SO signaling in plants.