Photo-bleaching of Chlamydomonas reinhardtii after dark-anoxic incubation

Abstract

In aerobes, anoxia impairs mitochondrial energy generation as well as biosynthesis and degradation of essential cell components. In a recent analysis we have shown that the unicellular green alga Chlamydomonas reinhardtii responds to anaerobiosis in the dark by significant changes of the transcriptome, which, in summary, were directed at saving and economizing energy. Several of the transcriptional changes were related to photosynthesis and were accompanied by reduced amounts of chlorophylls and plastid lipids as well as lowered photosystem 2 quantum yields. A further noticeable pattern was a transcriptional upregulation of various genes encoding O₂ dependent enzymes of central biosynthetic pathways. However, cells do not divide in dark-anoxia, indicating that C. reinhardtii cannot compensate for the lack of O₂ and light. Upon return to aeration and light, cultures show severe photo-bleaching, which might be a stress reaction, but also part of an acclimation process or its disturbance.

Keywords: :

• anoxia
• chlorophyll
• darkness
• oxygen-dependent
• oxidative stress
• photo-bleaching
• re-aeration

Although they generate molecular oxygen (O₂) during oxygenic photosynthesis, many green organisms can find themselves confronted with hypoxic or anoxic conditions in shaded aquatic environments or in waterlogged soil. O₂ deficiency has a profound influence on the energy metabolism, and strategies of hypoxia-tolerant species like rice to cope with low O₂ concentrations involve quiescence strategies and the economic utilization of carbohydrates,¹ showing the importance of the energy status for surviving longer periods of anaerobiosis.

In a recent study, we analyzed the responses of the unicellular green alga Chlamydomonas reinhardtii to dark-anoxia using an RNA-Seq approach.² The alga has an elaborate and flexible ?bacterial-type? fermentation metabolism,^3,4 and the wild type grows without any difficulty in an anaerobic atmosphere in the light.^2,5-7 However, what could be deduced from transcript patterns and accompanying physiological analyses of cells subjected to anoxia in darkness was in accordance with a transition to a ?power save mode?: The cells stopped dividing and exhibited an overall decrease of the amounts of transcripts associated with growth and gene expression. A significant amount of upregulated transcripts related to starch, amino acid, and fatty acid catabolism furthermore indicated that the microalgae sought to make all internal energy sources available.

However, not only the energy metabolism is impaired when O₂ is limiting. The biosynthesis and degradation of various cell compounds such as fatty acids or tetrapyrroles requires O₂ as a substrate or reagent. Though under debate, it has been discussed that upon O₂ limitation, plants might actively downregulate the rate of respiratory O₂ uptake in order to maintain a certain O₂ threshold for essential metabolic reactions.⁸ For the transcriptome study, C. reinhardtii was simply transferred to gas-tight flasks in the dark, allowing the cells to ?anaerobize themselves? by respiratory activity. We analyzed if C. reinhardtii would consume dissolved O₂ completely when placed into a sealed container (Fig. 1). Indeed, the cells kept the respiratory rate unchanged until O₂ became undetectable (Fig. 1), indicating that no or only minute amounts of O₂ are maintained within the cells.

Figure 1. Three liquid cultures of C. reinhardtii strain CC-124 were grown in Tris Acetate Phosphate (TAP) medium on a horizontal shaker at 20 °C and with bottom-up illumination of 80 µmol photons ? m^?2 ? s^?1 up to a cell density of 2.5 (light gray symbols), 2.7 (dark gray symbols), or 3.5 (black symbols) ? 10⁶ cells ml^?1. 2-ml aliquots of the cell suspensions were transferred to the sealed measuring cell of a Hansatech O₂ electrode according to Philipps et al., 2011.¹⁹ O₂ uptake was monitored in darkness until O₂ became undetectable (A). (B) O₂ uptake rates were calculated each minute and plotted against the respective O₂ concentration.

The generation of complete anoxia notwithstanding, the RNA-Seq data of dark-anoxic C. reinhardtii cells revealed an accumulation of various transcripts encoding O₂ dependent enzymes. Among those were several fatty acid desaturases, enzymes of tetrapyrrole and sterol biosynthesis as well as prolyl-4-hydroxylases. The desaturation status of the fatty acids from dark-anoxic algal cells, however, was not enhanced,² and we concluded that the upregulation of transcripts encoding O₂-requiring enzymes is probably a?futile?compensating response. Spinning this thought further, O₂-dependent enzymatic reactions or their products might well be used as O₂ sensors in C. reinhardtii. To date, the COPPER RESPONSE REGULATOR1 (CRR1) is the only specific transcription factor that is known to regulate a subset of genes in response to anoxia in Chlamydomonas.^7,9-11 Comparing transcriptome profiles of O₂ deprived crr1 mutants with those of the rescued strain (crr1:CRR1) confirmed the well-known connection between copper and O₂ deficiency mediated by CRR1. However, the differential expression of only about 40 transcripts depended on CRR1,² showing that further O₂ sensing and -signaling strategies must exist to account for the major part of the transcriptional changes. In yeast, products of O₂-requiring biosynthetic pathways, heme and sterols, are involved in O₂ sensing, while in mammals, O₂-dependent hydroxylation by prolyl-4-hydroxylases of the central transcription factor HIF1? controls its proteasomal turnover.^12,13 Arabidopsis group VII Ethylene Response Factors (ERFs), transcription factors that regulate low oxygen responses, are destabilized in normoxia by the N-end rule: The N-terminal Cys residue becomes oxidized under normoxic conditions, possibly by an O₂-requiring enzymatic reaction.¹⁴ In parallel to these examples, and in view of the upregulation of genes coding for O₂-dependent enzymes, one might speculate that C. reinhardtii makes a virtue out of necessity and utilizes the inability to carry out these reactions as a means of O₂ sensing.

The dependence of (metabolic and/or regulative) enzymes on the O₂ molecule can also result in the inability to catalyze O₂-dependent degradation steps, and this might be a further challenge. Tetrapyrroles are good representatives, because these molecules as well as some pre-cursors and degradation intermediates are photosensitizers.^15,16 For example, an impairment of the O₂-requiring pheophorbide a oxygenase, involved in chlorophyll (Chl) breakdown, might result in the accumulation of pheophorbide a, which is a photosensitizer, but also involved in programmed cell death.¹⁷ The dark-anoxic C. reinhardtii strains analyzed by RNA-Seq had lower Chl contents and exhibited decreased abundances of transcripts encoding tetrapyrrole biosynthetic enzymes and Chl-binding proteins.² This was accompanied by a moderate upregulation of genes encoding a NON YELLOW COLORING1 (NYC1) homolog and a putative chlorophyllase (CLH1). NYC1 is a short-chain dehydrogenase likely to be a Chl b reductase and, together with NYC1-LIKE (NOL), important for Chl breakdown during senescence,^15,17 while chlorophyllase removes the phytol tail from Chl and, besides pheophytinase, might also be involved in Chl degradation.¹⁵

Because the transcriptome study indicated that dark-anoxic incubation had a significant influence on the Chl metabolism of C. reinhardtii,² possibly resulting in an accumulation of Chl degradation intermediates, a photo-bleaching phenotype after oxic or anoxic incubation in the dark for increasing periods of time (up to 72 h) was analyzed. Cells incubated in sealed flasks, i.e., anoxically, for 6 h did not show any chlorosis when transferred back to aerated and illuminated conditions (Fig. 2). However, after 24 to 72 h of incubation, severe photo-bleaching was observed, and the recovery back to green cultures took more time the longer the cells had been subjected to dark-anoxia (Fig. 2). The fact that the bleaching of the cells was dependent on light and normal aeration became apparent by re-oxygenating cells in darkness or re-illuminating them in sealed flasks. Cells such treated did not show signs of chlorosis (Fig. 2).

Figure 2.C. reinhardtii strain CC-124 cultures were grown as described in the legend of Figure 1 up to a cell density of about 3 ? 10⁶ cells ml^?1. Then they were transferred to darkness either aerated in beakers (O₂ +) or in sealed flasks (O₂ ?). Cells were incubated under these conditions for 6, 24, 48, or 72 h (indicated in the top row) and then transferred back to standard growth conditions (aeration and illumination at 80 µmol photons ? m^?2 ? s^?1; Oxic, light). Aliquots of cells incubated dark-anoxically for 72 h were also kept in sealed flasks, but re-illuminated (Anoxic, light) or re-oxygenated in the dark (Oxic, dark). Photographs were taken immediately after transfer of the cells to Erlenmeyer flasks (0h at the left) and then after 8, 24, 48, 72, and 96 h (indicated at the left).

Whether the observed photo-bleaching of C. reinhardtii is specific for a dark-anoxic influence on Chl (or tetrapyrrole) biosynthesis, -binding and -degradation remains to be examined. A potentially stressful consequence of anoxia and re-aeration is a (photosensitizer-independent) generation of reactive oxygen and nitrogen species (ROS, RNS), which are produced and employed as second messengers by plants under anoxia.¹⁸ Various transcripts related to ROS and RNS generation and -signaling indicated that C. reinhardtii shares the ubiquitously observed anoxic generation and utilization of ROS and RNS,² and we could show that NO is involved in the anoxic response of C. reinhardtii.⁵ We also noted transcript patterns speaking for an activation of ROS/RNS stress defense systems. Sudden re-aeration in combination with light might overstrain the antioxidative defense systems of C. reinhardtii cells. However, the photo-bleaching phenotype might also be part of a long-term acclimatory process to anoxia in darkness. The RNA-Seq profiles revealed many genes involved in photosynthesis and Chl binding to be downregulated, and though this transcriptional response was probably due to darkness rather than O₂ deficiency, only dark-anoxic cells exhibited strongly decreased photosystem 2 quantum yields and lowered amounts of the plastid lipids monogalactosyl- and digalactosyldiacylglycerol (MGDG and DGDG).² Furthermore, the decreased amounts of MGDG and DGDG were accompanied by the appearance of typical MGDG- and DGDG-associated unsaturated fatty acids in triacylglycerols. Though speculative for the time being, it might be proposed that dark-anoxic C. reinhardtii cells specifically dismantle their photosynthetic apparatus, either to reallocate resources or to be prepared for sudden and potentially harmful re-oxygenation and -illumination. As we additionally observed significant effects of darkness and of inhibiting photosystem 2 with 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) on transcript abundances of ?anaerobic genes?², the metabolic and regulatory interactions of the photosynthetic machinery and (dark-) anoxic responses appear to be an important theme in C. reinhardtii.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG), (He 5790/1?1 and -2, and HE 5790/3?1).