Compartment‐specific Ca²⁺ imaging in the green alga Chlamydomonas reinhardtii reveals high light‐induced chloroplast Ca²⁺ signatures

Abstract

Summary To investigate the role of intracellular Ca2+ signaling in the perception and response mechanisms to light in unicellular microalgae, the genetically encoded ratiometric Ca2+ indicator Yellow Cameleon (YC3.6) was expressed in the model organism for green algae Chlamydomonas reinhardtii, targeted to cytosol, chloroplast, and mitochondria. Through in vivo single‐cell confocal microscopy imaging, light‐induced Ca2+ signaling was investigated in different conditions and different genotypes, including the photoreceptors mutants phot and acry. A genetically encoded H2O2 sensor was also adopted to investigate the possible role of H2O2 formation in light‐dependent Ca2+ signaling. Light‐dependent Ca2+ response was observed in Chlamydomonas reinhardtii cells only in the chloroplast as an organelle‐autonomous response, influenced by light intensity and photosynthetic electron transport. The absence of blue and red‐light photoreceptor aCRY strongly reduced the light‐dependent chloroplast Ca2+ response, while the absence of the blue photoreceptor PHOT had no significant effects. A correlation between high light‐induced chloroplast H2O2 gradients and Ca2+ transients was drawn, supported by H2O2‐induced chloroplast Ca2+ transients in the dark. In conclusion, different triggers are involved in the light‐induced chloroplast Ca2+ signaling as saturation of the photosynthetic electron transport, H2O2 formation, and aCRY‐dependent light perception.

Abiotic stress-induced chloroplast and cytosolic Ca2+ dynamics in the green alga Chlamydomonas reinhardtii

“…To investigate the role of Ca 2+ signalling in nutrient sensing in C. reinhardtii , the previously obtained transgenic C. reinhardtii lines, encoding the ratiometric genetically encoded fluorescent Ca 2+ indicator, YC3.6, at the level of the cytosol and chloroplast stroma (M. Pivato et al, 2023) were exploited. Similar experimental approach to investigate Ca 2+ -dependent nutrient sensing mechanisms previously reported in diatoms (Helliwell et al, 2021) was here applied in C. reinhardtii.…”

“…The mechanisms of perception of extracellular stresses and their subsequent transduction into physiological responses are required by nearly all eukaryotic cells to adapt to the environment. The requirement for Ca 2+ -dependent signalling pathways in many of these environmental responses has been shown to be conserved among algae, plants, or even animal cells, displaying common elements in the underlying cellular mechanisms (M. Pivato et al, 2023; Stael et al, 2012). Here, intracellular Ca 2+ signalling is demonstrated to be likely involved in the responses of green algae to several different environmental stressors, including osmotic stimuli, temperature fluctuations and Ci availability (Table 1).…”

“…The C. reinhardtii strains used in this study were UVM4 (UV-mediated mutant 4) (Neupert, Karcher, & Bock, 2009) and the previously obtained transgenic UVM4 C. reinhardtii lines expressing YC3.6 (Nagai, Yamada, Tominaga, Ichikawa, & Miyawaki, 2004) at the level of the cytosol or the chloroplast stroma (M. Pivato et al, 2023). Algal cells were cultivated in mixotrophic or photoautotrophic conditions, in Tris-acetate-phosphate (TAP) or Tris-phosphate (TP) minimal medium respectively (E. H. Harris, 2008; Kropat et al, 2011).…”

“…Calcium (Ca 2+ )-dependent signalling mechanisms has a role in stress responses to environmental abiotic stimuli of C. reinhardtii , but also in many of its motile responses and in the regulation of photosynthesis (Bickerton, Sello, Brownlee, Pittman, & Wheeler, 2016; Verret, Wheeler, Taylor, Farnham, & Brownlee, 2010; Glen L. Wheeler & Brownlee, 2008). In a previous work in vivo single-cell imaging techniques were applied to study C. reinhardtii light-dependent Ca 2+ signalling at subcellular resolution, reporting high light and/or H 2 O 2 dependent chloroplast [Ca 2+ ] transients (M. Pivato, Grenzi, Costa, & Ballottari, 2023). No cytosolic or mitochondrial Ca 2+ responses to light stimuli were detected, leading to the hypothesis of a chloroplast-autonomous light-dependent Ca 2+ response.…”

“…Exogenous supply of different Ci sources and heat stress primarily affects chloroplast [Ca 2+ ], suggesting a major role of the chloroplast in the perception of these environmental stimuli. As both light and Ci sources preferentially influence chloroplast bioenergetic processes, it is reasonable to expect that this subcellular compartment could primarily and autonomously respond to high light stress, H 2 O 2 , photo-oxidative stress (M Pivato et al, 2023). and/or Ci availability.…”