The two major research projects ongoing in our laboratory examine the molecular regulation and evolution of nitrogen assimilating enzymes in diverse lineages of marine and freshwater algae (described below) and the ecophysiology of salt marsh plants.
MOLECULAR REGULATION NITROGEN ASSIMILATING ENZYMES IN ALGAE
The assimilation of inorganic nitrogen (N) into organic compounds is a key process regulating the growth and productivity of photosynthetic eukaryotes. Diatoms are unicellular photoautotrophs that contribute significantly to global biochemical cycles. They exhibit rapid growth in response to increases in N availability, which in marine ecosystems, varies over several spatial (meters to kms) and temporal scales (hours to months). Thus, the ecological success of diatoms can, in part, be attributed to their ability to rapidly sense and respond to fluctuations in N source and supply.
In all living cells, the regulation of gene expression is a multifaceted and dynamic process. Cells integrate intrinsic and environmental signals into multiple regulatory pathways allowing for coordinated gene expression and cellular function. While there has been much focus on patterns of coordinated gene transcription, there are now examples from bacteria, kinetoplastids, plants, fungi, and animals of coordinated post-transcriptional regulation of mRNAs encoding functionally related proteins. This project explores the general hypothesis that post-transcriptional regulation of genes involved in N transport and assimilation in marine diatoms allows for rapid metabolic response to perturbations in nutrient source or supply and is mediated by changes in mRNA stability.
EVOLUTION OF NITROGEN ASSIMILATING ENZYMES IN ALGAE
The assimilation of nitrogen into organic molecules is a highly regulated process involving enzymes in the cytosol, chloroplast, and mitochondria. Nitrate and ammonium are the the principle forms of inorganic nitrogen assimilated by photosynthetic organisms in both aquatic and terrestrial ecosystems. While many of the enzymes in nitrate and ammonium assimilation are well-conserved among the distinct lineages of photosynthetic eukaryotes, our work has shown there are many striking differences in the evolutionary history of these enzymes in chromalveolates (diatoms, dinoflagellates, haptophytes, and cryptophytes) and green algae and vascular plants.
Much of our earlier work has focused on the molecular evolution of the glutamine synthetase (GS) gene family. Glutamine synthetase catalyzes the ATP-dependant condensation of ammonium and glutamate producing glutamine. The nitrogen incorporated into glutamine by GS is transferred to 2-oxoglutarate by the activity of glutamine 2-oxoglutarate amidotransferase (GOGAT) yielding two molecules of glutamate. The glutamine and glutamate produced by the GS:GOGAT cycle are used in a variety of essential biosynthetic reactions in all cells.
The GS gene family has three phylogenetically distinct classes that encode the subunits of the holoenzymes (GSI, GSII, and GSIII). Each holoenzyme is comprised of the same class of subunts but the number and size of the subunits differ among the classes. In most photosynethic eukaryotes, multiple GS enzymes are expressed and function in the chloroplast and cytosol, or in some species, the mitochondrion. Within the vascular plants, the GS isoenzyems are members of the GSII fmaily and appear to have evolved by a recent gene duplication event, with an expansion of the cytosolic gene family in many lineages. GSII isoenzymes are also observed in green algae and early diverging plants however, our phylogenetic studies have shown that in these groups, the chloroplast-targeted enzyme likely evolved via a horizontal gene transfer from the eubacteria.
Multiple GS isoenzymes are also expressed in chromalveolates. However, in constrast to vascular plants and green algae, the chromalveolates express GSII and GSIII isoenzymes. Our recent phylogentic studies have provided evidence that the chloroplast-targeted GSII enzyme likely evolved by endosymbiotic gene transfer while the GSIII enzyme may have been present the nucleus early in the evolution of eukaryotic cells. Although we have provided only a glimpse into the molecular evolution of the GS gene family here, overall,our work, along with others, has uncovered a complex evolutionary history for these gene families in the algae. This history includes the presence of orthologous and paralogous genes, ancient and recent gene duplications, gene losses and replacements, and the potential of both endosymbiotic and horizontal gene transfers. There are still many other secrets to be uncovered are continuing to explore the evoluaiton of this essential gene family, taking advantage of emerging genomic and transcriptome data.
In addition to our work with GS, we are also exploring the evolutionary history of other nitrogen assimilating enzymes focuing on chromalveolates. The chromalveolates evolved via secondary endosymbiosis, a symbiotic association between a heterotrophic eukaryotic host cell and a photosynthetic eukaryotic symbiont. During evolution, as the endosymbiont evolved into an organelle (the chloroplast), there was a great deal of gene loss, gene duplication, and gene transfer to the nucleus. We have already shown that the GSII and GSIII gene in chromalveloates to have different evolutionary histories (endosymbiotic gene transfer and present in the host cell nucleus, respectively) and are exploring the evolution of the other enzymes to better understand how genes are recruited and retained in the genomes of these chimeric organisms.