Microalgae usually accumulate triacylglycerols (TAGS) under stress conditions, especially during nitrogen (N) starvation. However, little information is available regarding the underlying mechanism. In the present study, a green algae Micractinium pusillum accumulated significant amounts of TAGs after 6 d of N starvation. Aiming at fundamental elucidation of the molecular mechanisms involved in N starvation-induced TAG accumulation, the global gene expression changes were identified by using suppression subtractive hybridization (SSH). In total, 290 expressed sequence tags (ESTs) showed significant differential expression between the treated and control samples. The expression profiles of 19 ESTs have been confirmed by quantitative real-time PCR (qPCR). Homology analysis showed that the 174 N deficiency-induced ESTs were mainly involved in carbohydrate metabolism, pyruvate and acetyl-CoA synthesis, isoprenoids biosynthesis, and TAG degradation, as well as a severe down-regulation in the expression of 116 genes related to photosynthesis, cell growth, amino acid synthesis and cell cycle regulation. A significant decrease in carbohydrate content was also observed in the N-deficient algal cells. These results suggested that the carbon sources for TAG biosynthesis were largely derived from carbohydrate metabolism rather than from photosynthesis. The results of the qPCR showed that, compared to the control, the transcript levels of genes related to pyruvate and acetyl-CoA synthesis were dramatically increased over 100 times after N starvation, which suggests that pyruvate and acetyl-CoA play important roles in driving carbon flow into TAG biosynthesis. (C) 2012 Elsevier Ltd. All rights reserved.