ASU Scholarship Showcase

The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms - including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms - while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.

The unicellular green microalga Desmodesmus sp. S1 can produce more than 50% total lipid of cell dry weight under high light and nitrogen-limitation conditions. After irradiation by heavy ¹²C⁶⁺ ion beam of 10, 30, 60, 90 or 120 Gy, followed by screening of resulting mutants on 24-well microplates, more than 500 mutants were obtained. One of those, named D90G-19, exhibited lipid productivity of 0.298 g L^-1⋅d^-1, 20.6% higher than wild type, likely owing to an improved maximum quantum efficiency (Fv/Fm) of photosynthesis under stress. This work demonstrated that heavy-ion irradiation combined with high-throughput screening is an effective means for trait improvement. The resulting mutant D90G-19 may be used for enhanced lipid production.

Background: Blindness has evolved repeatedly in cave-dwelling organisms, and many hypotheses have been proposed to explain this observation, including both accumulation of neutral loss-of-function mutations and adaptation to darkness. Investigating the loss of sight in cave dwellers presents an opportunity to understand the operation of fundamental evolutionary processes, including drift, selection, mutation, and migration.

Results: Here we model the evolution of blindness in caves. This model captures the interaction of three forces: (1) selection favoring alleles causing blindness, (2) immigration of sightedness alleles from a surface population, and (3) mutations creating blindness alleles. We investigated the dynamics of this model and determined selection-strength thresholds that result in blindness evolving in caves despite immigration of sightedness alleles from the surface. We estimate that the selection coefficient for blindness would need to be at least 0.005 (and maybe as high as 0.5) for blindness to evolve in the model cave-organism, Astyanax mexicanus.

Conclusions: Our results indicate that strong selection is required for the evolution of blindness in cave-dwelling organisms, which is consistent with recent work suggesting a high metabolic cost of eye development.

The most common evolutionary events at the molecular level are single-base substitutions, as well as insertions and deletions (indels) of short DNA segments. A large body of research has been devoted to develop probabilistic substitution models and to infer their parameters using likelihood and Bayesian approaches. In contrast, relatively little has been done to model indel dynamics, probably due to the difficulty in writing explicit likelihood functions. Here, we contribute to the effort of modeling indel dynamics by presenting SpartaABC, an approximate Bayesian computation (ABC) approach to infer indel parameters from sequence data (either aligned or unaligned). SpartaABC circumvents the need to use an explicit likelihood function by extracting summary statistics from simulated sequences. First, summary statistics are extracted from the input sequence data. Second, SpartaABC samples indel parameters from a prior distribution and uses them to simulate sequences. Third, it computes summary statistics from the simulated sets of sequences. By computing a distance between the summary statistics extracted from the input and each simulation, SpartaABC can provide an approximation to the posterior distribution of indel parameters as well as point estimates. We study the performance of our methodology and show that it provides accurate estimates of indel parameters in simulations. We next demonstrate the utility of SpartaABC by studying the impact of alignment errors on the inference of positive selection. A C ++ program implementing SpartaABC is freely available in http://spartaabc.tau.ac.il.

Under models of isolation-by-distance, population structure is determined by the probability of identity-by-descent between pairs of genes according to the geographic distance between them. Well established analytical results indicate that the relationship between geographical and genetic distance depends mostly on the neighborhood size of the population which represents a standardized measure of gene flow. To test this prediction, we model local dispersal of haploid individuals on a two-dimensional landscape using seven dispersal kernels: Rayleigh, exponential, half-normal, triangular, gamma, Lomax and Pareto. When neighborhood size is held constant, the distributions produce similar patterns of isolation-by-distance, confirming predictions. Considering this, we propose that the triangular distribution is the appropriate null distribution for isolation-by-distance studies. Under the triangular distribution, dispersal is uniform over the neighborhood area which suggests that the common description of neighborhood size as a measure of an effective, local panmictic population is valid for popular families of dispersal distributions. We further show how to draw random variables from the triangular distribution efficiently and argue that it should be utilized in other studies in which computational efficiency is important.

Background: Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes.

Results: Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5 Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species.

Conclusion: This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy for intragenus phylotyping of microalgae was proposed which might be generally applicable to other microalgal genera and should serve as a valuable tool in the expanding algal biotechnology industry.

Major progress has been made in the past decade towards understanding of the biosynthesis of red carotenoid astaxanthin and its roles in stress response while exploiting microalgae-based astaxanthin as a potent antioxidant for human health and as a coloring agent for aquaculture applications. In this review, astaxanthin-producing green microalgae are briefly summarized with Haematococcus pluvialis and Chlorella zofingiensis recognized to be the most popular astaxanthin-producers. Two distinct pathways for astaxanthin synthesis along with associated cellular, physiological, and biochemical changes are elucidated using H. pluvialis and C. zofingiensis as the model systems. Interactions between astaxanthin biosynthesis and photosynthesis, fatty acid biosynthesis and enzymatic defense systems are described in the context of multiple lines of defense mechanisms working in concert against photooxidative stress. Major pros and cons of mass cultivation of H. pluvialis and C. zofingiensis in phototrophic, heterotrophic, and mixotrophic culture modes are analyzed. Recent progress in genetic engineering of plants and microalgae for astaxanthin production is presented. Future advancement in microalgal astaxanthin research will depend largely on genome sequencing of H pluvialis and C. zofingiensis and genetic toolbox development. Continuous effort along the heterotrophic-phototrophic culture mode could lead to major expansion of the micro algal astaxanthin industry.

Nitrogen availability and cell density each affects growth and cellular astaxanthin content of Haematococcus pluvialis, but possible combined effects of these two factors on the content and productivity of astaxanthin, especially under outdoor culture conditions, is less understood. In this study, the effects of the initial biomass densities IBDs of 0.1, 0.5, 0.8, 1.5, 2.7, 3.5, and 5.0 g L-1 DW and initial nitrogen concentrations of 0, 4.4, 8.8, and 17.6 mM nitrate on growth and cellular astaxanthin content of H. pluvialis Flotow K-0084 were investigated in outdoor glass column photobioreactors in a batch culture mode. A low IBD of 0.1 g L-1 DW led to photo-bleaching of the culture within 1-2 days. When the IBD was 0.5 g L-1 and above, the rate at which the increase in biomass density and the astaxanthin content on a per cell basis was higher at lower IBD. When the IBD was optimal (i.e., 0.8 g L-1), the maximum astaxanthin content of 3.8% of DW was obtained in the absence of nitrogen, whereas the maximum astaxanthin productivity of 16.0 mg L-1 d(-1) was obtained in the same IBD culture containing 4.4 mM nitrogen. The strategies for achieving maximum Haematococcus biomass productivity and for maximum cellular astaxanthin content are discussed.

Mutation is the ultimate source of all genetic variation and is, therefore, central to evolutionary change. Previous work on Paramecium tetraurelia found an unusually low germline base-substitution mutation rate in this ciliate. Here, we tested the generality of this result among ciliates using Tetrahymena thermophila. We sequenced the genomes of 10 lines of T. thermophila that had each undergone approximately 1,000 generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach that directly models the design of an MA experiment and accommodates the noise introduced by mismapped reads. Our probabilistic mutation-calling method provides a straightforward way of estimating the number of sites at which a mutation could have been called if one was present, providing the denominator for our mutation rate calculations. From these methods, we find that T. thermophila has a germline base-substitution mutation rate of 7.61 × 10 ^-12 per-site, per cell division, which is consistent with the low base-substitution mutation rate in P. tetraurelia. Over the course of the evolution experiment, genomic exclusion lines derived from the MA lines experienced a fitness decline that cannot be accounted for by germline base-substitution mutations alone, suggesting that other genetic or epigenetic factors must be involved. Because selection can only operate to reduce mutation rates based upon the "visible" mutational load, asexual reproduction with a transcriptionally silent germline may allow ciliates to evolve extremely low germline mutation rates.

Inbreeding in hermaphroditic plants can occur through two different mechanisms: biparental inbreeding, when a plant mates with a related individual, or self-fertilization, when a plant mates with itself. To avoid inbreeding, many hermaphroditic plants have evolved self-incompatibility (SI) systems which prevent or limit self-fertilization. One particular SI system—homomorphic SI—can also reduce biparental inbreeding. Homomorphic SI is found in many angiosperm species, and it is often assumed that the additional benefit of reduced biparental inbreeding may be a factor in the success of this SI system. To test this assumption, we developed a spatially-explicit, individual-based simulation of plant populations that displayed three different types of homomorphic SI. We measured the total level of inbreeding avoidance by comparing each population to a self-compatible population (NSI), and we measured biparental inbreeding avoidance by comparing to a population of self-incompatible plants that were free to mate with any other individual (PSI).

Because biparental inbreeding is more common when offspring dispersal is limited, we examined the levels of biparental inbreeding over a range of dispersal distances. We also tested whether the introduction of inbreeding depression affected the level of biparental inbreeding avoidance. We found that there was a statistically significant decrease in autozygosity in each of the homomorphic SI populations compared to the PSI population and, as expected, this was more pronounced when seed and pollen dispersal was limited. However, levels of homozygosity and inbreeding depression were not reduced. At low dispersal, homomorphic SI populations also suffered reduced female fecundity and had smaller census population sizes. Overall, our simulations showed that the homomorphic SI systems had little impact on the amount of biparental inbreeding in the population especially when compared to the overall reduction in inbreeding compared to the NSI population. With further study, this observation may have important consequences for research into the origin and evolution of homomorphic self-incompatibility systems.