To further confirm the possibility that H2S might be involved in NAA-induced lateral root formation, an H2S scavenger, hypotaurine (HT), was applied. First, we observed that the addition of HT significantly decreased lateral root density and length; meanwhile, slight but not significant decreases in lateral root number and the number of lateral root primordia were observed (Fig. 5A–C). These results could be consistent with the decreasing effects of HT on endogenous H2S production, supported by the down-regulation of SlDES1 transcript and decreased DES activity ( Fig. 5D and E). Subsequent results showed that the above HT responses were significantly reversed by the cotreatment with NaHS. By contrast, NAA failed to reverse above responses elicited by HT pretreatment.