The cell death negative regulator, CaRLK1 gene, causes metabolic alterations of pyruvate and alanine via the glyoxylate cycle under hypoxia. Nicotiana tabacum

A custom-made microarray was used for comparative transcriptome analysis of transgenic suspension cell line of Nicotiana tabacum that overexpresses the CaRLK1 gene. Ectopic expression of the CaRLK1 preferentially upregulated many amino acid biosynthetic genes under hypoxia: a 1.7-fold higher steady-state concentration of total free amino acids. Free Ala level was predominantly increased by 3-4 times and reached more than half of total free amino acid content. A significantly and markedly increased pyruvate content was also observed. These accumulations are associated with the glyoxylate cycle positively regulated by the CaRLK1. Overall design: A total of 12 chips were used for microarray. Total RNAs were extracted from BY-2 suspension cells (control), RLK1ox suspension cells (RLK), auxin-free RLK1ox suspension cells (RA), and 10 uM DPI-treated RLK1ox suspension cells (RD) with triple biological replicates.