UC Berkeley

The ability to adapt to a changing climate while sustaining the nutritional needs of a growing human population are major challenges of modern human history. Basic plant research and plant biotechnology are key solutions to meeting both challenges. However, iron (Fe)-deficiency both in the oceans and on arable land undermines those efforts. Iron is an essential nutrient for growth and energy production in all forms of life as a cofactor in a myriad of enzymatic reactions. When environmental Fe availability can no longer support intracellular Fe demands, photosynthetic organisms degrade Fe-dependent proteins, especially proteins involved in photosynthesis, to reduce the intracellular Fe content. Photosystem I (PSI) is a primary target for this degradation owing to its high Fe demands within three Fe4S4 clusters. Therefore, the mechanism by which photosynthesis is maintained with limited photosynthetic components remains an area of great research interest. Nevertheless, certain essential Fe-dependent processes are maintained for “housekeeping functions” to allow for cell maintenance. The metabolic pathway that is preserved or degraded in Fe limitation suggests a hierarchy of Fe-dependent processes which I systematically investigated in the following chapters of this dissertation. To better understand the hierarchy of Fe-dependent processes in the face of Fe-limitation, I examined the transcriptome, proteome, protein structure, and physiology of a well-studied, freshwater alga, Chlamydomonas reinhardtii (Chlamydomonas), and two extremophile, marine algae, Dunaliella salina Bardawil and Dunaliella tertiolecta, as they transitioned into Fe-starvation. I studied this with three specific aims: 1) Capturing the temporal view of Chlamydomonas acclimating from an Fe-replete condition to Fe limitation using transcriptomics; 2) Cataloging Fe-binding proteins from Chlamydomonas, D. salina Bardawil, and D. tertiolecta and exploring their responses to both trophic and Fe nutrition stages through proteomics; and 3) Discovering an Fe deficiency induced structural modification to PSI in D. salina Bardawil and D. tertiolecta. In Aim 1, Chlamydomonas was transferred from an Fe-replete to an Fe-limited medium where samples were taken for RNA-seq analysis to capture both short- and long-term transcriptome changes over 48 h. The analysis revealed a hierarchy among the pathways responding to Fe starvation. Among the Fe transporters, FRE1 transcripts were the most highly upregulated (10,000-fold increase) within 30 min in Fe-starved medium, and this was a result of transcriptional regulation shown with a promoter-reporter analysis. Of the bioenergetic pathways, transcripts for chlorophyll biosynthesis and photosynthetic components were the first to respond to low Fe medium while transcripts for respiratory components responded last and were comparatively maintained. I supplemented the transcriptomic observations with measurements for protein abundance and physiology such as chlorophyll content, intracellular metal content, and growth. The physiology measurements showed Chlamydomonas experienced the distinct Fe nutrition stages of Fe-replete, Fe deficiency, and Fe limitation over the 48 h experiment. In Aim 2, I cataloged Fe-binding proteins from Chlamydomonas, D. salina Bardawil, and D. tertiolecta using both computational predictions and orthology to known Fe-binding proteins with manual curation. Proteomics of Chlamydomonas grown in two different trophic conditions (mixotrophic and phototrophic) and four different Fe nutritional stages (Fe-limited, Fe-deficient, Fe-replete, and Fe-excess) were used to probe the different roles of Fe-containing proteins in the cell. D. salina Bardawil and D. tertiolecta Fe-binding proteins were also investigated using proteomics from cells grown in Fe-replete and Fe-starved conditions. Through this analysis, we identified 388, 292, and 277 Fe-containing proteins in C. reinhardtii, D. salina Bardawil, and D. tertiolecta, respectively. Finally, in Aim 3, I used single-particle cryogenic electron microscopy to unveil the Fe-starved PSI structure from D. salina Bardawil and D. tertiolecta. This Fe-starved PSI-light harvesting complex (LHC) I supercomplexes included a second LHCI belt with a novel chlorophyll-binding protein called TIDI1 (thylakoid iron deficiency induced protein) compared to the Fe-replete PSI-LHCI supercomplexes. Altogether, my studies provide a comparative view of both dynamic and static acclimation strategies to low Fe nutrition in both a well-studied alga, Chlamydomonas, and two extremophile, marine algae, D. salina Bardawil and D. tertiolecta, and these chapters showcase the diversity of the responses to Fe limitation among green algae.