Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis

Abstract

Plant hormone brassinosteroids (BRs) and auxin exert some similar physiological effects likely through their functional interaction, but the mechanism for this interaction is unknown. In this study, we show that BRs are required for lateral root development in Arabidopsis and that BRs act synergistically with auxin to promte lateral root formation. BR perception is required for the transgenic expression of the beta-glucuronidase gene fused to a synthetic auxin-inducible promoter (DR5::GUS) in root tips, while exogenous BR promotes DR5::GUS expression in the root tips and the stele region proximal to the root tip. BR induction of both lateral root formation and DR5::GUS expression is suppressed by the auxin transport inhibitor N-(1-naphthyl) phthalamic acid. Importantly, BRs promote acropetal auxin transport (from the base to the tip) in the root. Our observations indicate that BRs regulate auxin transport, providing a novel mechanism for hormonal interactions in plants and supporting the hypothesis that BRs promote lateral root development by increasing acropetal auxin transport.

Figure 1.

Brassinosteroids promote lateral root formation A, Low concentration of BL promotes lateral root formation. Col-0 seedlings were grown vertically for 8 d on 1/2 MS plates containing 0, 1, 2, 5, 10, 50, and 100 nm BL, respectively. The number of lateral roots and visible lateral root primodia per centimeter of primary root were counted. Values in the figure represent the means ± se of four replicates. B, BL promotion of lateral root primodium development is suppressed by NPA. Five-day-old DR5::GUS seedlings grown vertically on 1/2 MS plates were transferred to plates containing 50 nm BL and/or 2 μm NPA for another 2 d. After histochemical GUS staining, the number of LRPs was counted under microscope. CK, control; B, BL; N, NPA; B + N, BL plus NPA. Values in the figures represent the means ± se of three replicates. C, BL promotes the development of the early stages of lateral root primordia. Different stages of lateral root primordia (stages 1–7) were determined according to Malamy and Benfey (1997).

Figure 2.

BL and IAA act synergistically to promote lateral root development. Col-0 seedlings were grown vertically on 1/2 MS plates containing 0, 1, 5, 20, and 50 nm IAA with or without 1 nm BL, respectively, for 8 d. The number of lateral roots and visible lateral root primodia per centimeter of the primary root was counted. Data are represented as the ratio of the number of lateral root within unit length in each treatment relative to the number of lateral root in 1 nm BL treatment. Values in the figures represent the means ± se of three replicates.

Figure 3.

Brassinosteroids induce DR5::GUS expression. A, DR5::GUS expression in WT seedlings treated with BL or Brz or in the bri1-119 mutant. Four-day-old DR5::GUS seedlings grown in liquid medium were treated for 48 h with 50 nm BL or 1 μm Brz, respectively. DR5::GUS/ bri1 seedlings were grown in liquid medium for 6 d before histochemical GUS staining was performed. Images show different parts of seedlings: cotyledons (Cotyl), first true leaves, shoots, root tips, LRP and mature lateral root. Scale bar = 100 μm. B, BL dose response of BL-induced DR5::GUS expression. Four-day old liquid-cultured DR5::GUS seedlings were treated with 1 μm Brz, or 0, 10, 50, 100, or 1,000 nm BL, respectively for 24 h. Whole seedlings were used for the assay. Values in the figure represent the means ± se of three replicates. C, Time-course analysis of BL effect on DR5::GUS expression. Four-day-old DR5::GUS seedlings were treated with 50 nm BL or dimethyl sulfoxide mock treatment for 0, 12, 24, and 48 h, respectively. Whole seedlings were used for the assay. Data are represented as a ratio of GUS activity per microgram protein from the BL treated samples relative to the mock-treated samples. Values in the figure represent the means ± se of three replicates.

Figure 4.

Effects of BRs on free IAA levels. The bri1-119 and wild-type Col-0 seedlings were grown in liquid medium for 7 d. The wild type seedlings were treated with 50 nm BL or 1 μm Brz, respectively, for 24 h before IAA extraction. Ten milligrams of whole seedlings were used for the extraction. Values in the figure represent the means ± se of three replicates.

Figure 5.

Effects of NPA on BR induction of DR5::GUS expression. DR5::GUS seedlings were grown in liquid medium for 4 d and were treated with dimethyl sulfoxide (mock treatment), 50 nm BL, 2 μm NPA, or 50 nm BL plus 2 μm NPA for 48 h before histochemical GUS staining as shown in Figure 3. Scale bar =100 μm.

Figure 6.

BR promotes auxin acropetal transport in the root. Wild-type Col-0 seedlings were grown vertically on 1/2 MS soild medium containing 0, 1, 5, or 25 nm BL for 7 d and the roots were used for transport assay as described in text. The unit in the y axis Avg pm indicates average pmoles of IAA moved as described in text.