Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium.

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae.

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistronic genes, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for polycistronic expression in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed 1) a preference for weak Kozak-like sequences for ORF 1 and 2) an underrepresentation of potential initiation codons before ORF 2, which are suitable conditions for leaky scanning to allow ORF 2 translation. We used mutational analysis in Auxenochlorella protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky ribosome scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames found across eukaryotic genomes and for transgene expression in synthetic biology applications.

Mutation of negative regulatory gene CEHC1 encoding an FBXO3 protein results in normoxic expression of HYDA genes in Chlamydomonas reinhardtii.

Oxygen is known to prevent hydrogen production in Chlamydomonas, both by inhibiting the hydrogenase enzyme and by preventing the accumulation of HYDA-encoding transcripts. We developed a screen for mutants showing constitutive accumulation of HYDA1 transcripts in the presence of oxygen. A reporter gene required for ciliary motility, placed under the control of the HYDA1 promoter, conferred motility only in hypoxic conditions. By selecting for mutants able to swim even in the presence of oxygen we obtained strains that express the reporter gene constitutively. One mutant identified a gene encoding an F-box only protein 3 (FBXO3), known to participate in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1 , leads to constitutive expression of HYDA1 and other genes regulated by hypoxia, and of many genes known to be targets of CRR1, a transcription factor in the nutritional copper signaling pathway. CRR1 was required for the constitutive expression of the HYDA1 reporter gene in cehc1-1 mutants. The CRR1 protein, which is normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 acts to facilitate the degradation of CRR1. Our results reveal a novel negative regulator in the CRR1 pathway and possibly other pathways leading to complex metabolic changes associated with response to hypoxia.

Iron rescues glucose-mediated photosynthesis repression during lipid accumulation in the green alga Chromochloris zofingiensis.

Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (-Fe). Cells grown in -Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.