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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.

Exploring the Role of a Cytokinin-Activating Enzyme LONELY GUY in Unicellular Microalga Chlorella variabilis