<i>Arabidopsis</i> AtPARP1 and AtPARP2 are functional poly(ADP-ribose) polymerases.

<p>(<b>A</b>) AtPARP1 and AtPARP2 <i>in vitro</i> activity detected by α-PAR antibody. MBP, MBP-AtPARP1 or MBP-AtPARP2 proteins were incubated with activated DNA with or without NAD⁺. 3-AB is a competitive inhibitor of PARP and could block PAR reactions. The PARylated proteins were detected in a Western blot using α-PAR antibody (top Panel) and the protein inputs were indicated by Coomassie Brilliant Blue (CBB) staining (bottom panel). The blot exposure time for AtPARP1 and AtPARP2 was same. (<b>B</b>) AtPARP1 and AtPARP2 <i>in vitro</i> activity detected by Biotin-labelled NAD⁺. (<b>C</b>) AtPARP1 and AtPARP2 <i>in vitro</i> activity detected by autoradiograph with ³²P-NAD⁺. (<b>D</b>) AtPARP2 PARylates <i>Arabidopsis</i> Histone H1.1 and H1.3 <i>in vitro</i>. The PARylated proteins were detected by autoradiography with ³²P-NAD⁺. (<b>E</b>) flg22 treatment enhances AtPARP2 activity <i>in vivo</i>. Protoplasts were transfected with <i>AtPARP2-HA</i>, treated with 100 nM flg22 for 30 min and fed with ³²P-NAD⁺. The AtPARP2-HA proteins were immunoprecipitated with an α-HA antibody and detected by autoradiography (Top panel). The input of AtPARP2 proteins is shown with an α-HA Western blot (middle panel), and the protein loading control is shown by Ponceau S staining for RBC (bottom panel). (<b>F</b>) AtPARP2 localizes in nucleus. <i>AtPARP2-GFP</i> was transiently expressed in <i>N. benthamiana</i> and the images were taken with a confocal microscope 2 days after inoculation. For nuclear staining, Hoechst 33342 (1 µg/ml) was infiltrated into the <i>N. benthamiana</i> leaf one hour before imaging. Scale bar is 20 µm. The above experiments were repeated at least 3 times with similar results.