Abstract
Triacylglycerols are produced in abundance through chloroplast and endoplasmic reticulum pathways in some microalgae exposed to stress, though the relative contribution of either pathway remains elusive. Characterization of these pathways requires isolation of the organelles. In this study, an efficient and reproducible approach, including homogenous batch cultures of nitrogen-deprived algal cells in photobioreactors, gentle cell disruption using a simple custom-made disruptor with mechanical shear force, optimized differential centrifugation and Percoll density gradient centrifugation, was developed to isolate chloroplasts from Chlamydomonas reinhardtii subjected to nitrogen stress. Using this approach, the maximum limited stress duration was 4 h and the stressed cells exhibited 19 and 32% decreases in intracellular chlorophyll and nitrogen content, respectively. Chloroplasts with 48 – 300 μg chlorophyll were successfully isolated from stressed cells containing 10 mg chlorophyll. These stressed chloroplasts appeared intact, as monitored by ultrastructure observation and a novel quality control method involving the fatty acid biomarkers. This approach can provide sufficient quantities of intact stressed chloroplasts for subcellular biochemical studies in microalgae.
Highlights
The chloroplast is a specialized subcellular compartment of plant and algal cells that is derived from cyanobacteria through endosymbiosis (Moreira et al, 2000; Okamoto and Inouye, 2005)
Multiple other processes occur in chloroplast, including biosyntheses of chlorophyll (Chl), amino acids, and fatty acids as well as membrane lipid assembly and trafficking (Baginsky and Gruissem, 2004; Block and Jouhet, 2015)
Chlamydomonas reinhardtii has emerged as a model green microalga for investigations of many aspects of lipid metabolism (Li-Beisson et al, 2015), especially biosynthesis of the promising biofuel feedstock triacylglycerol (TAG) (Ruiz et al, 2016; Zienkiewicz et al, 2016)
Summary
The chloroplast is a specialized subcellular compartment of plant and algal cells that is derived from cyanobacteria through endosymbiosis (Moreira et al, 2000; Okamoto and Inouye, 2005). Increasing evidences reveal that parallel TAG biosynthesis pathways in the chloroplast and endoplasmic reticulum (ER) concomitantly exist in microalgae, especially the plastid pathways including de novo TAG biosynthesis and turnover of chloroplast membrane lipids into TAG (Goodson et al, 2011; Legeret et al, 2015; Goold et al, 2016; Xu et al, 2016; Balamurugan et al, 2017), which are distinct from the relatively known pathways present in higher plants and yeast (Xu et al, 2016). As the chloroplastidic lipid remodeling that occurs in C. reinhardtii following stress conditions, nitrogen deprivation in particular, is proposed to be closely correlated to TAG synthesis (Li-Beisson et al, 2015; Du and Benning, 2016; Xu et al, 2016), it is essential to obtain stressed chloroplasts from this alga. Despite a brief mention by Fan et al (2011), without any data presented, chloroplast isolation from stressed C. reinhardtii has yet to be achieved
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