CHARACTERIZATION OF IRON-REGULATED PROTEINS AND THEIR TRANSCRIPTIONAL RESPONSE TO TRANSIENT IRON LEVELS IN THE OPEN OCEAN DIATOM THALASSIOSIRA OCEANICA

Abstract

Iron-requiring proteins are essential for numerous cellular processes, but low iron concentrations limit algal growth in over 30% of the ocean’s surface waters. Iron-fertilization experiments induced diatom-dominated blooms, and transcriptional analysis of iron-deprived cultures revealed novel proteins, coined iron starvation-induced proteins (ISIP). The main objectives of this thesis were to study the transcriptional and physiological response of the open ocean diatom Thalassasiosira oceanica to iron addition and to characterize the ISIP proteins. The first chapter introduces the background, explains the methods, and states the objectives of each chapter. In the second chapter, the functional characterization of ISIP proteins is explored by reviewing and comparing the iron acquisition strategies in different algal species. Through in-silico analysis and cellular localization experiments, the study reveals differences in iron-uptake proteins, iron-acquisition strategies, and ISIP proteins. Furthermore, this study hypothesizes that ISIP3 is an iron storage protein. The analysis of the high-resolution temporal response of iron-responsive genes to the addition of iron was the objective of the third chapter. Here, employing targeted transcriptomics, the dynamics of iron-regulated transcripts were followed after the addition of iron to iron-deprived cultures. Iron-uptake related transcripts were downregulated first, followed by an upregulation of transcripts for iron-containing proteins, and photosynthetic parameters recovered within a period of 6 h. In the fourth chapter, N-linked glycosylated peptides were analyzed under high- and low-iron growth conditions. N-linked glycosylated peptides were captured using the solid-phase extraction of N-linked glycopeptides (SPEG) method and identified via LC-MS/MS, revealing 118 glycosylated peptides with mostly NXT-type motifs. Peptides recovered from low-iron cells confirmed the predicted topology of ISIP1a. Chapter 5 reports on the development of a transformation system in T. oceanica to identify the cellular localization of YFP-tagged proteins. The transformation vector was designed using the Gibson® cloning technology, and cells were transformed via microparticle bombardment. T. oceanica was genetically transformable, confirmed by the expression of the resistance gene and growth in antibiotic-selective media. Overall, the findings presented here greatly expand the understanding of the response of diatoms to changes in iron concentrations. The transcription kinetics will be useful for modeling efforts at the cellular and biogeochemical level.