Insights into widespread disturbance in gene expression and severe growth inhibition observed in transgenic rice producing polyhydroxybutyrate

Production of polyhydroxybutyrate (PHB), a kind of biodegradable polymer, was attempted using transformant rice, in which the genes involved in PHB biosynthesis in <i>Cupriavidus necator</i> were introduced. Accumulation of PHB was observed in the transformants containing the genes for β-ketothiolase (<i>phaA</i>), acetoacetyl-CoA reductase (<i>phaB</i>) and PHB synthase (<i>phaC</i>) (<i>PhaABC</i> lines) and those containing <i>phaB</i> and <i>phaC</i> (<i>PhaBC</i> lines). However, they immediately withered after regeneration due to severe growth inhibition, whereas no growth inhibition occurred in the <i>PhaAB</i> lines containing <i>phaA</i> and <i>phaB</i>, and the <i>PhaC</i> lines containing <i>phaC</i>, which did not produce PHB. Crossing between them generated sufficient quantities of F1 seeds. Many of them germinated and produced PHB, but they died at an early stage of growth. This suggests that the accumulation of PHB in the cells caused a strong growth inhibition. Microarray analysis using the <i>PhaBC</i> and <i>PhaC</i> lines revealed very similar expression profiles, suggesting that most of the changes in gene expression were mainly caused by <i>phaC</i> gene expression. <i>PhaC</i> transformants exhibited increased expression of genes involved in the stress response, certain biological processes and cellular components. These results strongly suggest that <i>phaC</i> gene expression results in perturbation of gene expression levels in various cell functions. It was concluded that the disturbance of cell function caused by <i>phaC</i> gene expression is enhanced by the intracellular production of PHB, leading to cell death.