These results, together with the coexpression profiles of NARS1 and NARS2 (see Supplemental Figure 3 online) (Obayashi et al., 2007) and a high degree of amino acid sequence similarity between NARS1 and NARS2 in the NAC domain (Figure 1A), indicate that NARS1 and NARS2 redundantly regulate seed morphogenesis. The phenotype was similar to those of transgenic plants with ProNARS1: NARS1-SRDX and ProNARS2:NARS2-SRDX (Figures 1E and 1F). nars1 nars2 produced abnormally shaped, shrunken seeds ( Figure 2E). To explore redundant regulation of seed morphology by NARS1 and NARS2, we generated the double knockout mutant nars1 nars2 by crossing nars1 and nars2. Unexpectedly, however, neither nars1 nor nars2 exhibited any abnormality in seed shapes (Figures 2C and 2D). RT- PCR revealed that nars1 and nars2 did not express transcripts of NARS1 and NARS2, respectively ( Figure 2B). To determine which gene was responsible for aberrant seed morphogenesis, we isolated nars1 knockout mutants (SM line, SM_3_28017) and nars2 knockout mutants (WiscDs-Lox line, WiscDsLox364F11) ( Figure 2A). Since NARS1 and NARS2 are closely related, this raised the possibility that NARS1 and NARS2 chimeric repressors coin- cidentally suppressed the expression of genes regulated by NARS2 and NARS1, respectively. Previously, we reported that CRES-T suppresses not only the function of the respective transcription factor of interest but also the function of closely related transcription factors (Hiratsu et al., 2003). This result indicates that expression of chimeric NARS1 and NARS2 repressors causes aberrant morphogenesis of seeds. These transgenic plants produced aberrantly shaped seeds, as did the plants with Pro35S:NARS1-SRDX and Pro35S: NARS2-SRDX (Figures 1E and 1F).
To determine whether the seed shape abnormality of the two CRES-T mutants was caused by ectopic expression of Pro35S: NARS1-SRDX and Pro35S:NARS2-SRDX, we generated transgenic plants that expressed either NARS1-SRDX or NARS2-SRDX under the control of the predicted promoter region of NARS1 ( ProNARS1:NARS1-SRDX ) or NARS2 ( ProNARS2:NARS2-SRDX ), respectively. NARS1 and NARS2 were localized in the nucleus and exhibited transactivation activity (see Supplemental Figure 2 online), indicating that NARS1 and NARS2 function as transcription factors. Pro35S:NARS2-SRDX ( Figure 1D) had rough shapes, whereas seeds of the wild type were more round (Figure 1B).