Brassinolide-Induced Elongation - ACS Symposium Series (ACS

Chapter 22 Brassinolide-Induced Elongation Jenneth M . Sasse

Downloaded by UNIV OF ARIZONA on January 10, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch022

School of Botany, University of Melbourne, Parkville, Victoria, Australia 3052

The promotive effects of brassinosteroids on the elongation of vegetative tissue have been observed in many species, but only a few have been studied in detail. The most responsive zone in stem tissue from etiolated dwarf pea is the transition from hook to elongating and laterally enlarging stem. BR affects the morphology of segments from this zone, promoting elongation and maintaining a narrow basal diameter, in contrast to the effects of other regulators. Treatment with the recognised plant hormones affects the elongation induced by brassinolide; gibberellin has an additive effect and zeatin an inhibitory one. With auxin, there is a synergism where brassinolide allows auxin to induce elongation when it is ineffective alone. Exogenous auxin affects the kinetics of the response to brass inolide, but the synergism cannot be described by a sensitivity parameter estimator program. However, the reported synergism in cucumber can be attributed to an increase in the amplitude of the response to auxin. This is also the parameter affected when brassin olide-induced elongation in pea is inhibited by abscisic acid. Ethe phon can overcome brassinolide-induced elongation, while colchicine and specific inhibitors of protein and nucleic acid synthesis are also potent inhibitors. They are not competitive inhibitors, and elonga tion continues in the presence of an inhibitor of DNA synthesis. The data suggest mRNA synthesis and/or maintenance is essential for brassinolide-induced elongation and in aged pea segments several protein bands are affected by brassinolide in the early stages of elongation. Brassinolide-induced elongation can be markedly re tarded by an inhibitor of cellulose biosynthesis, contrasting with re ported auxin effects. Also, diagnostic concentrations of the anti auxin, p-chlorophenoxybutyric acid, do not affect BR-induced elongation, so it is proposed that BR does not depend on auxin as a mediator in the promotion of elongation in younger tissue.

0097-6156/91/0474-0255$06.00/0 © 1991 American Chemical Society In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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In the first recognition of brassinosteroid-type activity (7), a marked promotion of elongation was observed in an assay then used for gibberellins, and this was confirmed with pure brassinolide (BR) (2). Promotion of elongation has now been observed in many species, and it is probable that BR's effect on the elongation of young vegetative tissue is general. The work of Sala and Sala (3) showed that BR can impose an elongated morphology on auxin-starved single cells of carrot in culture, but in a whole plant, final height and form are likely to be the result of many influences, such as hormones, nutrient and water status, and environmental effects, with complex interrelationships between them. However, it is possible to study their effects in stem tissue that is still plastic, and it may be possible in future to use such data to model extension growth in vivo. If members of the brassinosteroid family can be shown to be present at appropriate levels in responsivetissue,together with their as yet putative receptors, the potent promotive effects of these compounds on elongation will need to be included in any such models. The most brassinolide-responsive zone in greening dwarf pea tissue is in the transition from hook to laterally enlarging and elongating stem (4). BR affects the morphology of segments from this zone, promoting elongation and maintaining a narrow basal diameter, in contrast to the effects of the recognised plant hormones (5). The segment itself is unusual in its slight relief of tension, and its lack of specific responses to plant growth regulators when split longitudinally (6). The data suggest the limiting effect of the epidermal layers and the differing sensitivities to auxin in the epidermis and cortex (7) have not yet developed in this segment from the upper stem.

Effects of other plant hormones BR-induced elongation in the segment can be markedly inhibited by cytokinin (5), abscisic acid (ABA) (8) and ethephon (6). There was no evidence suggesting competitive inhibition by these regulators but analysis and comparison of data sets using the parameter estimator program (PEST) of Weyers et al. (9) showed that the ABA effect was not just subtractive. The difference between the data sets could be accounted for by a change in the difference between the minimum and maximum response rates (the parameter R ), suggesting that the response capacity of the tissue to BR was reduced. With ethephon, there was a complex interaction where only partial recovery occurred with increasing concentrations of BR, while the effect of increasing concentrations of ethephon on BR-induced elongation was antagonistic (6). The inhibition of BR-induced elongation by the cytokinin, zeatin, is accompanied by a synergistic enhancement by BR of the lateral enlargement of stem segments induced by cytokinin. Lateral enlargement induced by IAA can also be synergistically enhanced by BR (5). Thus, if BR plays an endogenous role in the control of elongation and the morphology of the intact plant, the recognised hormones could modulate the effects of BR, and it in turn could enhance their effects. Segments incorporating both G A and BR-sensitive tissue show an additive effect for these two regulators (5,5), confirming the effects observed with AMP

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In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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peatipsegments (70), and PEST analysis showed the only difference between data sets was in the values for the minimum rates of response (8). Synergism with auxin. Hook sections from etiolated dwarf peas, which do not respond to exogenous IAA alone, show a synergistic response with combinations of BR and IAA (77), and this can be confirmed with the larger stem segments that incorporate more mature stem tissue as well (5). Other auxins also show this response, and, as the auxin concentration increases, elongation can increase synergistically then decrease (6) suggesting that the hormones reach supraoptimal levels, perhaps inducing ethylene biosynthesis in the tissue. Also, if the efflux of endogenous auxin is reduced by treating the segments with phytotropins without intrinsic auxin activity, there is a significant increase in elongation (6). Thus, if BR is supplied, IAA can enhance elongation in conditions where it is ineffective alone. A synergism between IAA and BR was also described by Katsumi (72) in green cucumber hypocotyl segments, and analysis by the PEST program showed the difference between data sets for IAA alone and IAA with fixed concentrations of BR can be accounted for by a change in the parameter R , suggesting that the response capacity of the tissue to IAA is enhanced by BR (8). Possible explanations for this effect could be increased numbers of receptors for IAA, amplification of the IAA-induced signal, or its transmission, increased transcription or translation rates for IAA-induced protein synthesis, decreased turnover of mRNA or proteins, increased rates of delivery of cell wall components, etc. Much more research is needed to examine these possibilities. Changes in the other parameters calculated by PEST might also account for a synergistic response, e.g., a steeper dose/response curve (increasing p values) or a shift of the concentration for 50% relative response ([HJ^) to lower values. In the pea stem segments, very slight alterations in the concentration of the exogenous auxin can affect the kinetics of the BR-induced elongation response markedly. With very low concentrations of auxin (0.05uM 4-chloroindole-3-acetic acid or O.OljiM IAA) and a concentration range of BR, a significant change in the parameter [H]^ was observed, but it was not in the direction of a positive synergism, as die value of [H]^ increased (Figure 1). If physico-chemical meaning can be ascribed to the parameters calculated by PEST (9), very low levels of auxin might affect the binding of BR and its dissociation to and from a receptor investigation of such a possibility must wait on the isolation and characterization of such BR-receptors. High concentrations of auxin gave data sets whose dose/response curves were not suitable for analysis by PEST, and the clear synergisms seen in the pea stem segment with conventional experimental protocols could not be detected by the PEST program. It has been proposed using data from bending responses (13,14,15) that responses induced or accelerated by BR require the presence of some auxin, either exogenously or endogenously; if this applied to extension growth, the characteristics of sustained auxin-induced growth should be observed after BR treatment, e.g., antiauxins should reduce the BR-induced elongation. Diagnostic AMP



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concentrations (where the compound is not toxic to thetissue(16,17)) of the auxin antagonist, p-chloroisobutyric acid (PCIB), do not affect BR-induced elongation in the pea stem segment (6). This is in contrast with the data of Katsumi (72), but there, more mature, auxin-sensitive greentissuewas used, and BR may have been accelerating the auxin response. The results of Katsumi's study of the order and timing of application of the hormones is consistent with this idea. Also, sustained auxin-induced elongation is remarkably insensitive to inhibition of cellulose biosynthesis by treatment with 2,4-dichlorobenzonitrile (18), but BR-induced elongation is quickly reduced by very low concentrations of this inhibitor, suggesting cellulose supply rapidly becomesrate-limitingfor extension (6). So, taken together with the work of Sala and Sala (5), data from studies with the upper stem segment from pea suggest BR has its own role in elongation, but the mechanism of its effect is as yet unknown.

Extension of responsive cells Essential to BR-induced elongation is the maintenance of axial growth, rather than isodiametric expansion. Elongation in stem cells correlates with the orientation of arrays of cellulose microfibrils which are in turn correlated with microtubules within the cytoplasm, and hormones such as gibberellin and ethylene are known to affect the orientation of such arrays (19,20). That the correlation between the microfibrils and microtubules may not be causal has been discussed recently, and is receiving some experimental support (21,22), but it is clear that treatment of plant cells with antimitotic drugs that disrupt microtubule polymerization causes isodiametric expansion instead of continuing elongation. BR-induced elongation is no exception (75), and with the lowest effective concentrations of colchicine in the pea stem section there is no evidence for competitive inhibition (Figure 2), suggesting no direct effect of BR on microtubule polymerization. However, the orientation of the microtubule array could still be affected by BR in a manner similar to gibberellin (25). Cell wall synthesis requires, as well as cellulose, hemicellulose, pectin, lignin precursors and proteins. Lignification and crosslinking of cell wall components limit extension growth, and retardation of these processes (e.g., if peroxidase levels were lower, or lignin precursor concentrations were reduced) could permit continued elongation. Some evidence exists for peroxidase reduction after BR-treatment (24), but detailed studies on the effects of BR on lignification or the crosslinking of extensin have not yet been done. Results with the inhibitors /ro/tfcinnamic acid and 2,2'-bipyridyl need very careful interpretation, as these compounds are not really specific inhibitors. It is known that protein synthesis is necessary for BR-induced effects in root tissue (25), and BR-treatment increases nucleic acid and protein synthesis in bean stem (26). In the pea stem segment, kinetic studies with selected protein and nucleic acid synthesis inhibitors showed no evidence for competitive inhibition in polypeptide chain elongation, post-transcriptional polyA addition to heterogeneous RNA, DNA-dependent RNA synthesis, or of the DNA-dependent RNA polymerase


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Concentration (M) Figure 1. Effect of 0.01 uM IAA on the BR-induced elongation of pea stem segments. Lines are curves of best fit as calculated by PEST, and comparison gave a model where a change in lnfH]^ accounted for the difference between the data sets.

• = BR alone

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Concentration (M) Figure 2. Effect of 1 and 10 jiM colchicine on BR-induced elongation of pea stem segments, 14 segments per treatment, 29.5h growth. BR and colchicine effects significant at p

Brassinolide-Induced Elongation - ACS Symposium Series (ACS

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