Early embryo development in Fucus distichus is auxin sensitive

Abstract

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

Figure 1

IAA is present in F. distichus embryos as demonstrated by a TIC of methylated, HPLC-purified samples of free IAA from F. distichus fruiting tips. The ions at m/z 130 and 189 are the quinolinium ion and molecular ion, respectively, of the methylated ester of the endogenous IAA. Additional fragments of the endogenous IAA are an unmarked peak at m/z 77 and a peak at 103. Ions at m/z 136 and 195 are the quinolinium ion and molecular ion from the [¹³C]-IAA internal standard.

Figure 2

IAA accumulation into F. distichus zygotes is not saturable. Accumulation of [³H]IAA into F. distichus zygotes was measured in the presence of a range of unlabeled IAA concentrations but with a constant 25 nm concentration of [³H]IAA. The total IAA accumulation was calculated using the tracer IAA and is plotted as a function of added cold IAA.

Figure 3

IAA and NPA led to multiple and branched rhizoids. F. distichus embryos were grown for 48 h in the dark in ASW or in the presence of 50 μm IAA or 50 μm NPA as indicated.

Figure 4

NPA and triiodobenzoic acid (TIBA) cause abnormal embryo development in the dark. The percentage of F. distichus embryos with branched and/or multiple rhizoids are reported in response to treatment with BA, NPA, and TIBA at 50 μm after 48 h. The average and se of three separate experiments are reported. Asterisk, P < 0.005 as judged by Student's t test, showing a statistical difference from BA-treated control.

Figure 5

IAA and NPA led to dose-dependent changes in F. distichus embryo development. The percentage of the total number of embryos with multiple or branched rhizoids after culture for 48 h in ASW containing either NPA or IAA is plotted as a function of concentration of added compound. Results from a representative experiment are shown that was repeated three times.

Figure 6

Zygotes are sensitive to NPA and IAA treatment early in development. A, The number of multiple or branched embryos after treatments with 50 μm IAA or NPA, respectively, for 1 h at various times during the first 15 h AF. B, The effect of IAA and NPA treatments for 30 min during the first 6 h AF on multiple and branched rhizoids, respectively. The average and se of three or four separate experiments are reported in A and B, respectively.

Figure 7

IAA and NPA alter position of division planes in F. distichus embryos. F. distichus embryos were stained with FDA and transverse (parallel to the coverslip) views of division planes in ASW are shown for untreated embryos for 12 (A), 24 (B), and 48 (C) h or with embryos treated with 50 μm IAA for 12 (D), 24 (E) or 48 (F) h, or embryos treated with 50 μm NPA for 12 (G), 24 (H), or 48 (I) h. Size bars = 50 μm.