1148 Nucleic Acids Research, 2007, Vol. 35, No. 4 Figure 1. AtWRKY1 is partially NPR1-dependent in SA pathway. Four weeks old wild type (Col-0), npr1-3 and 35S::NPR1 Arabidopsis were sprayed with 2 mM SA and harvested at indicated time points. PR1 transcripts were detected the same as AtWRKY1 except for the diﬀerence of probe. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading. response to exogenous SA treatment. As an important marker of SAR, PR1 was also examined to monitor the SA treatment procedure. In consistence with previous reports, PR1 began to accumulate at 4 h post-treatment and the induction of PR1 was abolished in npr1-3 while increased in NPR1-H than that in wild type (Figure 1). The induced expression of AtWRKY1 was observed after 8 h post-treatment and became obvious later than that of PR1. In npr1-3, AtWRKY1 can still be expressed but at a lower level than that in wild-type plants. The opposite trend appeared in NPR1-H with higher expression level of AtWRKY1 than in wild type (Figure 1). These data suggested that AtWRKY1 was involved in SA signaling pathway and its induced expression was partially depen- dent on NPR1. The nuclear localization of AtWRKY1-C As transcriptional regulators, WRKY proteins are shown to contain functional nuclear localization signal (NLS) and are targeted to nucleus (27,48). However, the NLS sequences of WRKY proteins are not well conserved, and may be distributed at diﬀerent locations. The analysis of protein sequences of AtWRKY1 revealed a potential NLS motif KRRKK between residues 273 and 277 near the C-terminal WRKY domain, which is a conserved feature for at least nine members of the group I WRKY proteins in Arabidopsis (Figure 2D). We have thus included this potential NLS motif in the AtWRKY1-C sequence for this study. Introducing the fusion construct of AtWRKY1-C with dimeric GFP (dGFP) into onion epidermal cells, the green ﬂuorescent signal targeted speciﬁcally to the nucleus. However, the ﬂuorescence of dGFP alone and fusion protein of AtWRKY1-C NLS with dGFP were unevenly distributed throughout the cells (data not shown), which suggested that the potential NLS motif of AtWRKY1-C is indeed responsible for the nuclear localization of AtWRKY1. Structure determination of AtWRKY1-C crystal Although in the same mother liquor and temperature, AtWRKY1-C was crystallized in a diﬀerent space group, P3221, from the one published before (34). The structure was determined to 1.9 A˚ resolution by using the Single- wavelength Anomalous Dispersion (SAD) method. The Figure 2. Structure of AtWRKY1-C and multi-sequence alignment. (A) Ribbon representation of AtWRKY1-C domain. The AtWRKY1-C is composed of ﬁve b-strands (yellow ribbons), which are numbered from the N-terminus. The zinc ion is shown as a purple sphere and the zinc-coordinating residues are represented by sticks (yellow for C, red for O, blue for N, orange for S). (B) 3D-superimposition of the structure of AtWRKY1-C and the best representative NMR structure of AtWRKY4-C (model 15), using LSQ Fit in O. Macromolecular structures are shown by cartoons, with the AtWRKY1-C colored in yellow and AtWRKY4-C in cyan. The zinc ions are represented as spheres shown by magenta in AtWRKY1-C and orange in AtWRKY4- C. (C) Charge distribution on AtWRKY1-C structure surfaces given by GRASP. Positive charges are represented by blue, negative charges are represented by red. (D) Structure-based sequence alignment of both N-terminus and C-terminus of the nine Arabidopsis WRKY proteins from group I. The zinc-coordinating residues are shown on blue background. Conserved residue elements for stabilizing the structure and recognizing DNA are drawn on red and yellow background, respectively. Residues of b1, highlighted in green but missed in AtWRKY4-C structure, are rather conserved in all C-terminal domains of group I WRKY proteins. Residues marked by stars and triangles are scaﬀolds of two stable regions. Details of the interactions of the two regions are represented in Figure 3C and D. average ratio of Bijovet pairs (5| Delta F|4/5F4) was 4.6%, in accordance with the high correlation coeﬃcients (CC/all and CC/weak) 35.94/20.70, given by SHELXD (36) in zinc substructure locating. Phasing and density modiﬁcation (DM) (39) were carried out consecutively using the software of OASIS-2004 (38) and DM (CCP4). With a ﬁgure of merit (FOM) value of 0.771 and good quality of density maps, 75 residues could be docked automatically by ARP/wARP (40). However, no density could be observed between residues 266 and 292 and between residues 369 and 371. The ﬁnal model was reﬁned manually to R-factor/R-free of 17.9 and 20.4%, respectively at 1.6 A˚ resolution (Table 1). The ﬁnal model includes 76 residues corresponding to 293–368, one zinc ion, one succinic acid and 100 water molecules. Description of the overall structure of AtWRKY1-C The AtWRKY1-C structure is mainly composed of a ﬁve- stranded antiparallel b-sheet (b1, 294–300; b2, 312–318; b3, 327–332; b4, 340–345; b5, 352–358, see Figure 2D), with the disordered N-terminal 27 (including the potential NLS motif) and C-terminal three residues missing from the structure (Figure 2). WRKY protein’s deﬁning sequence, ‘WRKYGQK’, spans the entire b2 strand. Due to the long bridging loop (Thr301–Arg311) between b1 and b2 which covers one side of the b-sheet, the