Fermentation or anoxic metabolism allows unicellular organisms to colonize environments that become anoxic. Free-living unicellular algae capable of a photoautotrophic lifestyle can also use a range of metabolic circuitry associated with different branches of fermentation metabolism. While algae that perform mixed-acid fermentation are widespread, the use of anaerobic respiration is more typical of eukaryotic heterotrophs. The occurrence of a core set of fermentation pathways among the algae provides insights into the evolutionary origins of these pathways, which were likely derived from a common ancestral eukaryote. Based on genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism has been examined in more detail in Chlamydomonas reinhardtii (Chlamydomonas) than in any other photosynthetic protist. This green alga is metabolically flexible and can sustain energy generation and maintain cellular redox balance under a variety of different environmental conditions. Fermentation metabolism in Chlamydomonas appears to be highly controlled, and the flexible use of the different branches of fermentation metabolism has been demonstrated in studies of various metabolic mutants. Additionally, when Chlamydomonas ferments polysaccharides, it has the ability to eliminate part of the reductant (to sustain glycolysis) through the production of H₂, a molecule that can be developed as a source of renewable energy. To date, little is known about the specific role(s) of the different branches of fermentation metabolism, how photosynthetic eukaryotes sense changes in environmental O₂ levels, and the mechanisms involved in controlling these responses, at both the transcriptional and post-transcriptional levels. In this review, we focus on fermentation metabolism in Chlamydomonas and other protists, with only a brief discussion of plant fermentation when relevant, since it is thoroughly discussed in other articles in this volume.

Chloroplasts are the site of numerous biochemical reactions including photosynthesis, but they also produce reactive oxygen species (ROS) that negatively affect chloroplast integrity. The chaperone-like CDC48 complex plays critical roles in ubiquitin-dependent protein degradation in yeast and mammals, but its function in plants is largely unknown. Here, we show that defects in CDC48A and its cofactors UFD1 and NPL4 lead to the accumulation of ubiquitinated chloroplast proteins in Arabidopsis thaliana. We reveal that two plastid genome-encoded proteins, RbcL and AtpB, associate with the CDC48 complex. Strikingly, RbcL and AtpB are ubiquitinated and degraded by the 26S proteasome pathway upon ROS stress, and these processes are impaired by defects of the CDC48 complex. Functional analysis demonstrates that the CDC48 complex is required for plant tolerance to ROS. This study reveals a role for the plant CDC48 complex in modulating ubiquitin-dependent degradation of intra-chloroplast proteins in response to oxidative stress.

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.