Iron (Fe) is an essential cofactor of multiple enzymes, but to avoid its hazardous
effects on oxidative stress, signalling of the Fe status, especially in plant cells
organelles is essential, although hardly known. Since 80–90% of Fe in the mesophyll
cells is targeted to the chloroplasts to operate the photosynthetic apparatus, the
control over plastidial Fe status has prime importance. Sensing the cytoplasmic Fe
is based on hemerythrin motif-containing E3 ubiquitin ligases that are negative regulators
of Fe deficiency responses. Indeed, no mechanisms are described that monitor the Fe
status of the organelles. In vitro chloroplast Fe uptake (Solti et al., 2012; Müller
et al. 2019) and ferric chelate reductase enzyme studies (Solti et al. 2014; Sági-Kazár
et al. 2021) suggests that Fe in the chloroplasts has a feedback regulatory effect
on the affinity of the plastidial Fe acquisition machinery. Nitric oxide (NO) is long
known to regulate Fe uptake of roots, but also generated in chloroplasts under increasing
Fe availability. It is involved in post translation modification of proteins that
directly affect the function of those. Since the synthesis and accumulation of NO
in chloroplasts has not been confirmed so far, we aimed here to detect and quantify
NO in response to altered Fe nutrition chloroplasts.
Sugar beet (Beta vulgaris cv. Orbis) model was grown on Fe(III)-EDTA source. Fe deficiency
(Fe-def) was achieved with a complete depletion of Fe in the four-leaf stage. Mesophyll
cell protoplasts and chloroplasts were isolated. Abundance of the NO signal in the
protoplast population and the subcellular NO locations in the protoplasts were analysed
applying DAF-FM diacetate in fluorescence activated cell sorting and confocal microscopy,
respectively. The proportion of DAF-FM positive protoplasts significantly increased
under iron deficiency, together with a higher apparent DAF-FM signal intensity in
the protoplasts. Applying BioXol membrane staining of the chloroplast envelopes, chloroplast
stroma origin of the DAF-FM signal was approved. DAF-FM signal of isolated chloroplasts
was analysed by epifluorescent microscope, also indicating an increased ratio and
pixel density of DAF-FM positive chloroplasts under Fe-def compared to the control.
Electron paramagnetic resonance (EPR) spectroscopy based semiquantitative NO determination,
applying N-methyl-D-glucamine dithiocarbamate spin trap and 150 K environment indicated
a concentration of NO in the Fe-def chloroplasts around the lower detection limit,
that of attomol NO chloroplast-1 dimension. NO in the control chloroplasts remained
below the detection limit. Tyr nitration pattern detected by anti-nitro-Tyr immunoblotting
of total chloroplast protein showed no significant changes in the Tyr nitration status
indicating that nitrosative stress level has not increased significantly.
This work was supported by the grant financed by the National Research, Development
and Innovation Office, Hungary (NKFIH K-135607). Á.S. was also supported by the Bolyai
János Research Scholarship of the Hungarian Academy of Sciences (BO/00113/23/8).
References
Müller et al. (2019) Planta 249:751.
Sági-Kazár et al. (2021) Front Plant Sci 12:748.
Solti et al. (2014) New Phytol 202:920.
Solti et al. (2012) Plant Physiol Biochem 52:91
