Knockout of GH3 genes in the moss Physcomitrella patens leads to increased IAA levels at elevated temperature and in darkness.

Two proteins of the GRETCHEN HAGEN3 (GH3) family of acyl acid amido synthetases from the moss Physcomitrella patens conjugate indole-3-acetic acid (IAA) to a series of amino acids. The possible function of altered auxin levels in the moss in response to two different growth perturbations, elevated temperatures and darkness, was analyzed using a) the recently described double knockout lines in both P. patens GH3 genes (GH3-doKO) and b) a previously characterized line harboring an auxin-inducible soybean GH3 promoter::reporter fused to β-glucuronidase (G1-GUS). The GUS activity as marker of the auxin response increased at higher temperatures and after cultivation in the darkness for a period of up to four weeks. Generally, the double knockout plants grew more slowly than the wild type (WT). The altered growth conditions influenced the phenotypes of the double knockout lines differently from that of WT moss. Higher temperatures negatively affected GH3-doKO plants compared to WT which was shown by stronger loss of chlorophyll. On the other hand, a positive effect was found on the concentrations of free IAA which increased at 28 °C in the GH3-doKO lines compared to WT plants. A different factor, namely darkness vs. a light/dark cycle caused the adverse phenotype concerning chlorophyll concentrations. Mutant moss plants showed higher chlorophyll concentrations than WT and these correlated with higher free IAA in the plant population that was classified as green. Our data show that growth perturbations result in higher free IAA levels in the GH3-doKO mutants, but in one case - growth in darkness - the mutants could cope better with the condition, whereas at elevated temperatures the mutants were more sensitive than WT. Thus, GH3 function in P. patens WT could lie in the regulation of IAA concentrations under unfavorable environmental conditions.