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Essay 24.1

Brassinosteroids and the Apical Hook—An Ongoing Story in Plant Architecture

Jenneth Sasse, University of Melbourne, Melbourne, Australia

August, 2006

As discussed in the text (Chapter 22), the hook in etiolated dicot seedlings protects the apical meristem during seedling emergence, and its structure is maintained over time (Chapter 16). This asymmetric growth is dramatically altered upon exposure to light; photomorphogenesis begins and the hook straightens. Perception of the light signal by the phytochrome system in the hook can be transduced rapidly but is followed by slower responses, leading to developmental changes (Chapter 17).

Plant hormone signalling also contributes, and the development, maintenance, and opening of the apical hook is an example of crosstalk between the hormones, and with the light signals. Early work was often done with legumes or mustard, and treatments with exogenous auxin or ethylene were shown to enhance curvature of the hook, with a "window" of ethylene sensitivity when exaggeration can occur. Now the model plant of choice is Arabidopsis, and the effect of ethylene on it is illustrated in the text (textbook Figure 22.5).

Work by Kang and Ray in 1969 (cited in Yopp et al. 1981) suggested ethylene acted as a mediator for auxin- and light-induced responses, and the effect of exogenous auxin on hook angle (measured as hook opening) in light and dark can be seen in Figure 1. Kang and Ray also tested the effects of carbon dioxide, gibberellin, cytokinin, abscisic acid, and selected metal ions, and their data buttressed the case that the auxin effect was probably mediated through ethylene. Yopp et al. (1981) showed that exogenous brassinosteroid (BR) was also effective in Kang and Ray′s system, and that there was a marked synergism between BR and auxin leading to enhanced closure of the hook in both the light and the dark (see Figure 1). Subsequent discoveries of further properties of brassinolide (and the benefits of hindsight) suggest that ethylene biosynthesis could be induced in the isolated hooks by these treatments, and that BR, or its precursors, were contributing to the synergism observed.

Figure 1   Effect of brassinolide (BR) and indole-3-acetic acid (IAA), alone and in combination, on apical hook angle, measured as hook opening, of bean segments in continuous light or darkness for 36 hours. Least significant difference between means (LSD, P = 0.01). (Redrawn from Yopp et al. 1981, with permission for reproduction from Blackwell, Oxford.) (Click image to enlarge.)

With the availability of selected mutants of Arabidopsis and pea, the techniques of molecular biology, and our increasing knowledge of signalling cascades, it is now possible to learn much more about the control of plant architecture, particularly the effects of light and endogenous hormones, and the crosstalk between them. Ross et al. (2005) and Vandenbussche and Van Der Straeten (2004) have recently reviewed the control of shoot architecture, and highlighted the role of endogenous hormones and light signals for the formation and opening of the apical hook.

Studies with mutants suggest the distinctive structure of the hook depends not only on endogenous ethylene and auxin but also on asymmetric distribution of auxin. This implies control of its transport, and treatment with an auxin efflux inhibitor does disrupt hook formation. Location and recycling of PIN proteins (see Chapter 19), particularly PIN3, is important for the lateral transport of auxin; indeed, the mutant pin3 lacks the hook structure. Relocalization of PIN3, which is frequently detected in vesicles (Chapter 1), depends on actin-dependent cycling between the plasma membrane and an endosomal compartment. Also, enhanced expression of an auxin reporter gene construct, DR5::GUS, can be demonstrated on the inner side of the hook (Friml et al. 2002, cited in De Grauwe et al. 2005). The latter authors confirmed this, both for control and after treatment with the biosynthetic ethylene precursor 1-amino-cyclopropane-1-carboxylic acid (ACC), using a green fluorescent protein construct (Figure 2A). Auxin effects in the hook are mediated by DELLA proteins, which are regulators of gibberellin signalling as well (Chapter 20), and exaggeration of the hook by ethylene treatment depends on DELLA protein stability (Vriezen et al. 2004, cited in Vandenbussche and Van Der Straeten 2004).

Figure 2   Responses in apical hook tissue of Arabidopsis. A) Fluorescence of the auxin reporter gene DR5::GFP in control (low nutrient medium, LNM) and after treatment with the ethylene precursor, ACC. B) Differential staining after ACC treatment of the construct carrying the BR biosynthesis reporter, CPD::GUS, and its quantification. C) Effects of ACC and BR, alone and in combination, on hook tissue of wild type (Col-0), the mutant pin3-3, and the construct 35S::PIN1 in the dark, and quantification of the hook angle. (Reproduced from De Grauwe et al. 2005, with permission from Professor Van Der Straeten and Oxford University Press.) (Click image to enlarge.)

The presence of gibberellin itself is also essential for hook maintenance in the dark. In pea, while moderately GA-deficient mutants display an etiolated phenotype, the severely deficient mutant na does not develop an apical hook, and has expanded leaflets (illustrated in Ross et al. 2005). In normal plants, the transient drop in gibberellin levels after exposure to light may contribute to the disappearance of the hook structure, so that gibberellin may be part of the transduction chain from the environmental signal (Ross et al. 2005). In Arabidopsis, no hook is formed in the dark in the absence of gibberellin, and gibberellin-induced elongation is modulated by treatment with ACC. In the model proposed by Vandenbusche and Van Der Straeten (2004) for auxin and gibberellin effects, ethylene induces the gibberellin biosynthesis gene GA1, which responds all over the hook, but extension of the inner side of the hook is reduced by higher sensitivity to ethylene there, together with the asymmetric distribution of auxin. DELLA proteins are again considered crucial, with gibberellin promoting their degradation and thus relieving growth restraint (Vriezen et al. 2004, cited in Vandenbussche and Van Der Straeten 2004).

Mutants have also proved invaluable for studying the effects of BR in the development of the apical hook. In pea, a double-mutant impaired in both biosynthesis and perception of BR lacks the apical hook, but, unlike the na mutant, does not display leaflets. In Arabidopsis, plants defective in BR biosynthesis or perception lack, or have a more open, apical hook ( det2 and cbb1 are illustrated in De Grauwe et al. 2005). Many display their cotyledons in darkness, and at least one has documented acceleration of hook-opening in the dark, and that mutant can go on to anthesis (Laxmi et al. 2004).

The role of BR endogenously has been studied in detail by De Grauwe et al (2005), especially interactions with ethylene and auxin, using not only mutants but also appropriate promoter-reporter constructs. A reporter line for BR biosynthesis (CPD::GUS), when treated with ACC, showed quantitatively more CPD expression on the outer side (Figure 2B). This effect could be inhibited when an ethylene response inhibitor was added, and addition of an auxin transport inhibitor (NPA, see textbook Figure 19.17) abolished the differential staining. With two biosynthetic mutants (det 2 and cbb1), ACC could not induce hook exaggeration in the dark. Also, hook formation in the dark in a wild-type Arabidopsis could be partially inhibited by exogenous treatment with an inhibitor of BR biosynthesis, and those seedlings were less responsive than controls to exogenous ACC. So, one can conclude that BR, as well as auxin and gibberellin, are essential for hook formation, and that the response to ethylene is dependent on BR downstream from the ethylene signal.

But how are BR and ethylene effects related to those of auxin? Are there effects of BR on the transport of auxin and thus on its asymmetric location? De Grauwe et al (2005) approached these questions with the use of the pin3-3 mutant and an overexpressor line, 35S::PIN1. Both the mutant and the construct showed less curvature of the hook when compared to wild type, and their responses to exogenous ACC were not as exaggerated. BR treatment enhanced elongation of the hook zone on both sides when compared with control, with the construct responding the most. Hook angle was significantly less acute than control in pin3-3, but equivalent to control in 35S::PIN1. When compared to ACC alone, treatment of BR-treated plants with ACC slightly restored hook curvature in pin3-3, but fully restored it in 35S::PIN1 (Figure 2C). So, how might these results be interpreted? To quote De Grauwe et al. (2005) "...normal auxin transport through active PIN proteins is necessary for BR- and ethylene-related phenotypes in darkness...." It may be that BR can modulate auxin transport by differentially affecting the production and translocation of PIN proteins; such effects may well vary in the light and in the dark.

These authors have put forward a model for the interactions between ethylene, auxin, and BR in the hook of etiolated seedlings in the dark, based on their own and others′ results (De Grauwe et al. 2005, and references cited therein). A simplified, more general scheme, including gibberellin effects (see also Vandenbussche and Van Der Straeten 2004) is shown in Figure 3.

Figure 3   A simplified scheme for the interactions between ethylene, auxin, and BR in the hook of etiolated seedlings in the dark. Biosynthetic pathways for the regulators are indicated by dashed lines. (Click image to enlarge.)

What else might be involved in the formation of the apical hook? Clearly, the control of biosynthesis and catabolism of those plant hormones discussed above, and the details and fine controls of the signal transduction pathways involved, are important. Any roles of other plant growth regulators, such as cytokinins, polyamines, abscisic acid, and sugars also need further work. As flagged by Vandenbussche and Van Der Straeten (2004), cytokinins are of particular interest, as at least one hookless mutant, amp1, is a cytokinin overproducer. So morphological changes in the hook, and the dramatic changes in architecture after a plant′s initial exposure to light, as well as its continuing response to light and shade, will continue to provide many challenges for plant biologists.

Acknowledgements

I wish to thank Professor Van Der Straeten for helpful discussion, suggestions, and permission to reproduce figures. I also wish to thank Oxford University Press and Blackwell, Oxford, for permissions.

References

De Grauwe, L., Vandenbussche, F., Tietz, O., Palme, K., and Van Der Straeten, D. (2005) Auxin, ethylene and brassinosteroids: Tripartite control of growth in the Arabidopsis hypocotyl. Plant Cell Physiol. 46: 827–836.

Laxmi, A., Paul, L. K., Peters, J. L., and Khurana, J. P. (2004) Arabidopsis constitutive photomorphogenic mutant, bls1, displays altered brassinosteroid response and sugar sensitivity. Plant Molec. Biol. 56: 185–201.

Ross, J. J., Reid, J. B., Weller, J. L. and Symons, G. M. (2005) Shoot architecture I: Regulation of stem length. In C. G. N. Turnbull, (Series Ed.) Annual Plant Reviews: Vol. 17. Plant Architecture and its Manipulation, Blackwell, Oxford.

Vandenbussche, F., and Van Der Straeten, D. (2004) Shaping the shoot: A circuitry that integrates multiple signals. Trends Plant Sci. 9: 499–506.

Yopp, J. H., Mandava, N. B., and Sasse, J. M. (1981) Brassinolide, a growth-promoting steroidal lactone I: Activity in selected auxin bioassays. Physiol. Plant. 53: 445–452.