Abstract
LONELY GUY has been previously characterized in flowering plants to be involved in the direct activation of cytokinins. In this study, the function of the only LONELY GUY gene (CvarLOG1) from unicellular green microalga Chlorella variabilis NC64A has been investigated. CvarLOG1 expressed mainly in the lag and log phases of growth and was confirmed to be a cytokinin-activating enzyme. Overexpression of CvarLOG1 in Chlorella led to extended life in culture by almost 10–20 days, creating a “stay-green” phenotype. In the transformed alga, the cell cycle was lengthened due to delayed entry into the G2/M phase contrary to the known role of cytokinins in stimulating G2/M transition possibly due to excessive levels of this hormone. However, due to the sustained growth and delayed senescence, there was an increase in cell number by 11% and in biomass by 46% at the stationary phase, indicating a potential application for the biofuel industry. The total carbohydrate and lipid yield increased by approximately 30 and 20%, respectively. RNA-Seq-based transcriptomic analysis revealed that the genes associated with light and dark reactions of photosynthesis were upregulated, which may be the reason for the increased biomass. These data show that LOG plays an essential role during the cell cycle and in the functioning of the chloroplast and that the pathway leading to direct activation of cytokinins via LOG is functional in algae.
Highlights
The presence and role of active hormones had been long speculated in algae, but the first proof of the bioactive forms of auxin, cytokinin, Gibberellic Acid (GA), brassinosteroid, Abscisic Acid (ABA), Jasmonic Acid (JA), and polyamines came from the work of Tarakhovskaya et al (2007)
In 2010, the Chlorella variabilis NC64A genome was decoded (Blanc et al, 2010), and the presence of various classes of hormonal genes was discovered in this microalga, including those involved in cytokinin biosynthesis and signaling (Blanc et al, 2010)
A conserved domain search on NCBI revealed the presence of a lysine decarboxylase (LDC) domain in CvarLOG1 with a conserved PGGxGTxxE motif like the LONELY GUY (LOG) counterparts from seed plants (Supplementary Figure 2)
Summary
The presence and role of active hormones had been long speculated in algae, but the first proof of the bioactive forms of auxin, cytokinin, Gibberellic Acid (GA), brassinosteroid, Abscisic Acid (ABA), Jasmonic Acid (JA), and polyamines came from the work of Tarakhovskaya et al (2007). More data describing the exact levels for auxin, cytokinin, GA, brassinosteroid, and ABA were added for the microalgae (Tarakhovskaya et al, 2007; Stirk et al, 2013a,b, 2014); their exact function remained unknown. Cytokinin Activation in Chlorella auxins (Howell et al, 2003; Ferreira and Kieber, 2005; Sakakibara, 2006) Discovery of these genes in Chlorella raises questions about the role of cytokinins in the development of these microalgae. Little is known about the role of hormone-related genes in microalgae (Lu and Xu, 2015) Taken together, these studies point toward certain equivalence between hormone biosynthetic pathways in microalgae and higher plants, whereas the evidence related to the roles these perform is largely missing. Further studies on the functional role of phytohormones in microalgal systems would be necessary to understand their full potential and put this knowledge into any biotechnological use (Lu and Xu, 2015)
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