An Arabidopsis mutant with a T-DNA insertion in the coding region of NIC2 was obtained and characterized (Figure 3a). No NIC2 transcript was detected in the nic2-1 mutant (Figure 3b), and Southern blotting indicated only one T-DNA insertion site in the nic2-1 genome (Figure S2). We presume therefore that this allele represents complete loss of NIC2 encoded function. The nic2-1 plants appeared to grow and develop normally. As NIC2 transcript levels were relatively high in mature seed, we investigated seed phenotype and observed a paler seed coat colour compared to Ws (parental background; Figure S3). Dissection of the seed showed that the embryo was the same colour in Ws and nic2-1, and confirmed that the nic2-1 testa had altered pigmentation. To confirm that nic2-1 seed has reduced nicotinamidase activity, we assayed for activity by following the conversion of [14C]-labelled nicotinamide to nicotinic acid. An approximately twofold decrease (P < 0.05) in nicotinamidase activity was seen in dry seed extracts of nic2-1 (Figure 4a). Although nicotinamidase activity was reduced, it was still detectable in nic2-1 extracts, suggesting that there may be other proteins capable of deaminating nicotinamide in Arabidopsis seeds. Germination experiments with moist-chilled (stratified) nic2-1 seeds showed that these seeds exhibited slightly retarded germination, but there was no difference in the final percentage germination (Figure 4b). However, in the absence of a moist chilling pre-treatment (non-stratified), germination was delayed and the germination potential of nic2-1 seeds was significantly lower than that of Ws (P < 0.05), indicating a hyperdormant phenotype (Figure 4b). Complementation of nic2-1 with a 4 kb genomic DNA fragment containing NIC2 was able to rescue normal germination (Figure 4c). Dormancy is usually removed by moist chilling or by after-ripening of seeds through dry storage (Bewley, 1997). To determine whether nic2-1 seeds show delayed after-ripening, the germination potential of stored seed was tested every 7 days, without moist chilling. After-ripening of nic2-1 seed was delayed in comparison to Ws seed, although after 21–28 days storage, the germination potential of nic2-1 reached that of Ws (Figure 4d). Seed maturation and germination are both ABA-dependent processes (Bewley, 1997), and germination of stratified nic2-1 seed was found to be hypersensitive to ABA (Figure 4e,f). This was unlikely to be due to altered permeability of the nic2-1 seed coat, as no differences in 2,3,5-triphenyltetrazolium chloride staining (Debeaujon et al., 2000) were seen between Ws and nic2-1 (data not shown). To investigate whether ABA hypersensitivity could be due to enhanced ABA levels, we estimated ABA levels in seed extracts using radioimmunoassay (Quarrie et al., 1988), and found no significant difference in ABA levels between Ws and nic2-1 seed. Both Ws and nic2-1 had an average of approximately 2 fg ABA per seed. We also found that nicotinamide is an inhibitor of Arabidopsis seed germination when added to the germination medium, and that nic2-1 seeds show hypersensitivity to nicotinamide (Figure 4g,h). At 5 mm, nic2-1 seeds were significantly (P < 0.02) more sensitive to nicotinamide than Ws. This result is in line with a previous observation that nicotinamide inhibits the growth of mungbean embryos during germination (Zheng et al., 2005).
Figure 3. Characterization of the T-DNA insertion site in nic2-1. (a) nic2-1 contains a T-DNA insertion in the coding region of NIC2. The positions of primer sequences used for analysis (Lb2, F1, R1, R2 and R3) are marked with arrows. The white box represents the NIC2 coding region, hatched boxes represent non-coding regions, and lines represent intergenic regions. The triangle indicates the position of the T-DNA insertion. (b) PCR analysis of nic2-1. Amplification of genomic DNA confirmed the T-DNA insertion in nic2-1, and amplification of cDNA showed that no full-length or truncated NIC2 transcript was present in nic2-1. Primer positions are shown in (a).
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Figure 4. Characterization of nic2-1 seed germination. (a) Nicotinamidase activity in seed extracts. Conversion of [14C]-nicotinamide to [14C]-nicotinic acid was measured in extracts resolved by TLC. Mean relative spot intensities are shown for three independent extractions from seeds pooled from 6–8 plants. Activity is significantly reduced in nic2-1 seeds (P < 0.05). Bars are SE. (b) Germination of stratified (st) and non-stratified (nst) Ws and nic2-1 seeds. Each point is the mean of experiments on seed from pools of 6–8 plants (n = 3). The germination of non-stratified nic2-1 was significantly less than that of non-stratified Ws at 5–7 days after plating (P < 0.05). Bars are SE. (c) Complementation of nic2-1. Germination potential of Ws, nic2-1 and two independent lines of nic2-1 transformed with a 4 kb Arabidopsis genomic DNA fragment containing NIC2. Percentage germination of non-stratified seed after 7 days is shown. Each point is the mean of experiments on seed from pools of 6–8 plants (n = 3). Ws and complemented lines had significantly higher germination potential than nic2-1 (Ws, P < 0.04; line 1, P < 0.04; line 2, P < 0.0 006). Bars are SE. (d) Effect of after-ripening on the germination potential of Ws and nic2-1. The percentage germination of non-stratified seed was assessed 7 days after plating following a period of dry dark storage at 20°C as indicated. The results shown are representative of three independent experiments. (e) Effect of ABA on Ws and nic2-1 germination. Seeds were sown on medium supplemented with ABA or a methanol (0.02%) solvent control and stratified. Plates were then transferred to light for the time indicated. Points are the mean of three pools of 3–6 plants. Germination indices were calculated from six measurements over 3 days. Bars are SE. (f) Comparison of ABA sensitivities. Germination indices from (e) were expressed relative to indices for plants sown on medium without ABA. Germination of nic2-1 seed was significantly more sensitive to ABA at 1 μm (P < 0.03) and 2 μm (P < 0.04) compared with Ws. (g) Effect of nicotinamide on germination. Seeds from three pools of 6–8 plants were sown on medium containing nicotinamide and stratified. Germination indices were calculated from six measurements over 3 days. Bars are SE. (h) Comparison of nicotinamide sensitivities. Germination indices from (g) were expressed relative to indices for plants without nicotinamide. At 5 mm, nic2-1 seed was significantly more sensitive to nicotinamide than Ws (P < 0.02).
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