In darkness, Arabidopsis seedlings display an apical hook. The hook structure protects the shoot apical meristem against mechanical stress during germination and early seedling growth (Vandenbussche and Van Der Straeten 2004). Adding ethylene to etiolated seedlings causes an exaggeration of the curvature of the hook. This is also one of the traits of the triple response (Guzman and Ecker 1990). This exaggeration of curvature is accompanied by a longer arc that can be explained by extra cell divisions and differential elongation of the concave versus the convex side of the hook (Vriezen et al. 2004).
The formation of the hook results from the differential growth of cells in the apical region. Cells at the outer side of the hook are larger than those at the inner side (Vriezen et al. 2004). The phenotype of etol, eto2, eto3 and ctrl mutants proves that the formation of an exaggerated hook is at least partly dependent on ethylene, since they show an exaggerated hook even when grown in the absence of ethylene (Guzman and Ecker 1990; Kieber et al. 1993). Also, ethylene is required for normal hook formation since ethylene-insensitive mutants show a reduction in curvature (Roman et al. 1995). This difference in cell elongation can result from differential ethylene production. However, the literature does not appear fully consistent on this. While AtACOl appears to be more strongly expressed at the concave side of the hook in pea (Peck et al. 1998), AtACO2 is expressed more at the convex side of the hook in Arabidopsis (Raz and Ecker 1999). Furthermore, ACC is asymmetrically localized in cells of the apical hook in bean (Schwark and Bopp 1993). However, it was proven that seedlings are only sensitive to exogenous ethylene 60-72 hours after germination (Raz and Ecker 1999).
To exert its effects on apical hook formation, ethylene interacts with other hormones (Fig. 1). There is compelling evidence for the involvement of auxins, gibberellins, and brassinosteroids in this process. Differential auxin distribution is a prerequisite for hook formation in etiolated seedlings, since blocking auxin transport results in a hookless phenotype (Lehman et al. 1996). The auxin efflux transporter PIN3 (pin formed) is required for hook formation, since dark-grown pin3 mutants show a faster opening of the apical hook (Friml et al. 2002). When ethylene is added to pinl andpin3 an exaggerated hook is not formed, as evidenced by the smaller ratios of the length of cells in the outer to the length of cells at the inner side of the arc in both ACC and untreated seedlings (De Grauwe et al. 2005). Other auxin mutants like axrl (Lincoln et al. 1990; del Pozo et al. 1998; auxin resistant, positive regulator of auxin response, as achieved by regulating activity of TIR1 SCF ubiquitin ligase), hlsl (Lehman et al. 1996; Li et al. 2004; hookless, negative regulator of auxin response, as achieved by degrading ARF2, auxin response factor), hls3 (King et al. 1995; Gopalraj et al. 1996; Lehman et al. 1996; aminotrans-ferase with higher levels of endogenous IAA, as achieved by stimulating the synthesis thereof), and yuc (Zhao et al. 2001; yucca, flavin monooxygenase-like enzyme, with elevated IAA biosynthesis) do not display a normal apical hook.
HLS1 is a downstream target of ethylene-dependent transcriptional regulators. It is equally expressed throughout the apical hook and its expression is enhanced under the influence of ethylene. HLS1 over-expression results in an exaggerated hook. In hlsl the cells where the apical hook is supposed to arise, are elongated. Cells at the outer and inner sides of the "hook" elongate two- and tenfold, respectively, compared to those in the wild type, resulting in an equal size on both sides in the hlsl mutant, which explains why no hook is formed (Lehman et al. 1996). HLS1 in turn lowers the concentration of ARF2 (Li et al. 2004). ARF2 (auxin response factor) is a negative regulator of differential growth responses (Li et al. 2004) that binds TGTCTC sequences in the promoter of primary auxin response genes (Ulmasov et al. 1999). When ethylene is applied to wild-type plants, the concentration of HLS1 is enhanced, leading to a lower concentration of ARF2, ultimately resulting in the formation of the apical hook. Adding auxin to the medium does not alter the concentration of ARF2 (Li et al. 2004).
In dark-grown seedlings, DR5::GUS expression is localized at the inner side of the apical hook. Ethylene exposure enhances this expression. The differential localization is completely abolished in the hlsl mutant and partially restored in the double mutant hlslarf2 (Li et al. 2004). These results lead to a model where ethylene modifies auxin response genes through the modulation of HLS1 and ARF2. Taking into account the equal expression of HLSl
(Lehman et al. 1996), tissue-specific signals and different auxin concentrations (Friml et al. 2002) are supposed to modify the effect of ARF2 on the expression of auxin-dependent genes.
Ethylene also interacts with gibberellin signaling to maintain the apical hook (Fig. 1). Gibberellin biosynthesis and signaling are required to form an apical hook. ACC enhances the stimulating effect of GA on cell division in the formation of an exaggerated apical hook of etiolated seedlings, and stabilizes RGA (a negative GA regulator), leading to smaller cells. There seems to be no significant difference in the stabilization of RGA between the outer and inner sides of the hook. Despite this, a stronger up-regulation of GASA1::GUS (a GA reporter line) was noticed at the outer side of the hook. This can be explained by changes in ethylene sensitivity or gibberellin sensitivity (Vriezen et al. 2004).
Finally, brassinosteroids are also required to form the apical hook. Adding ACC to BR biosynthesis mutants cbb1/dwf1 (Kauschmann et al. 1996; cab-bage1/dwarf1) and det2 (Chory et al. 1991; de-etiolated2) does not induce an exaggeration of the hook. Moreover, the expression of CPD::GUS (Fujioka and Yokota 2003; CPD is involved in brassinolide synthesis) in the apical hook is up-regulated by the addition of ACC. This expression is stronger in the convex side of the hook than in the concave side. Auxin disrupts the differential expression, whereas NPA (N-1-napthylphthalamic acid, polar auxin transport inhibitor) limits the expression to the stele. These results indicate that ethy-lene causes a stronger CPD promoter activity at the outer side of the hook (De Grauwe et al. 2005).
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