Active Brassinosteroids Research Articles

Structure-Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity.

A number of novel brassinosteroid analogues were synthesized and subjected to the rice leaf lamina inclination bioassay. Modified B-ring analogues included lactam, thiolactone, cyclic ether, ketone, hydroxyl, and exocyclic methylene derivatives of brassinolide. Those derivatives containing polar functional groups retained considerable bioactivity, whereas the exocyclic methylene compounds were devoid of activity. Analogues containing normal alkyl and cycloalkyl substituents at C-24 (in place of the isopropyl group of brassinolide) showed an inverse relationship between activity and chain length or ring size, respectively. The corresponding cyclopropyl and cyclobutyl derivatives were significantly more active than brassinolide and appear to be the most potent brassinosteroids reported to date. When synergized with the auxin indole-3-acetic acid (IAA), their bioactivity can be further enhanced by 1-2 orders of magnitude. The cyclopropyl derivative, when coapplied with the auxin naphthaleneacetic acid, gave a significant increase in yield of wheat in a field trial. Certain 25- and 26-hydroxy derivatives are known metabolites of brassinosteroids. All of the C-25 stereoisomers of 25-hydroxy, 26-hydroxy, and 25,26-dihydroxy derivatives of brassinolide were prepared and shown to be much less active than brassinolide. This indicates that they are likely metabolic deactivation products of the parent phytohormone. A series of methyl ethers of brassinolide was synthesized to block deactivation by glucosylation of the free hydroxyl groups. The most significant finding was that the compound where three of the four hydroxyl groups (at C-3, C-22, and C-23) had been converted to methyl ethers retained substantial bioactivity. This type of modification could, in theory, allow brassinolide or 24-epibrassinolide to resist deactivation and thus offer greater persistence in field applications. A series of nonsteroidal mimetics of brassinolide was designed and synthesized. Two of the mimetics showed significant bioactivity and one had bioactivity comparable to brassinolide, but only when formulated and coapplied with IAA. They thus represent the first nonsteroidal analogues possessing brassinosteroid activity.

BRing it on: new insights into the mechanism of brassinosteroid action.

Several recent breakthroughs have filled in key details of the brassinosteroid (BR) response. Identification of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further defined the downstream events in this pathway, confirming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered.

Physiological Roles of Brassinosteroids in Early Growth of Arabidopsis: Brassinosteroids Have a Synergistic Relationship with Gibberellin as well as Auxin in Light-Grown Hypocotyl Elongation

We examined the physiological effects of brassinosteroids (BRs) on early growth of Arabidopsis. Brassinazole (Brz), a BR biosynthesis inhibitor, was used to elucidate the significance of endogenous BRs. It inhibited growth of roots, hypocotyls, and cotyledonous leaf blades dose-dependently and independent of light conditions. This fact suggests that endogenous BRs are necessary for normal growth of individual organs of Arabidopsis in both photomorphogenetic and skotomorphogenetic programs. Exogenous brassinolide (BL) promoted hypocotyl elongation remarkably in light-grown seedlings. Cytological observation disclosed that BL-induced hypocotyl elongation was achieved through cell enlargement rather than cell division. Furthermore, a serial experiment with hormone inhibitors showed that BL induced hypocotyl elongation not through gibberellin and auxin actions. However, a synergistic relationship of BL with gibberellin A3 (GA3) and indole-3-acetic acid (IAA) was observed on elongation growth in light-grown hypocotyls, even though gibberellins have been reported to be additive to BR action in other plants. Taken together, our results show that BRs play an important role in the juvenile growth of Arabidopsis; moreover, BRs act on light-grown hypocotyl elongation independent of, but cooperatively with, gibberellins and auxin.

Brassinosteroids: modes of BR action and signal transduction

In humans and other animals, steroid hormones regulate transcription via membrane-bound receptors. These receptors have an extracellular ligand-binding domain and an intracellular domain that are responsible for transducing the signal to the next member along the signaling pathway. Brassinosteroids (BRs) are structurally similar to the animal steroid hormones found in vertebrates and insects. Plants also use steroids as signaling molecules. BRs regulate the expression of numerous genes associated with plant development, and require the activity of a serine/threonine (Ser/ Thr) receptor kinase to realize these effects. Signaling in dicotyledonous (e.g.,Arabidopsis thaliana)and monocotyle-donous (e.g.,Oryza sativa) models, is mediated by the receptor kinases BRI1 and OsBRI1, respectively. The extracellular domain of BRI1 perceives BRs, and the signal is then mediated via an intracellular kinase domain that autophosphorylates Ser and Thr residues and potentially, other substrates. BRI1 transduces steroid signals across the plasma membrane and mediates genomic effects.

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2α,3α-dihydroxy-17β-(3-methylbutyryloxy)-7-oxa-B-homo-5α-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2α,3α,17β-trihydroxy-7-oxa-B-homo-5α-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2α- and 3α-hydroxy groups it yielded the 2α,3α-isopropylidenedioxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2α,3α-dihydroxy-17β-(2-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one) in position 17β stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with β-oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.

CYP72B1-mediated inactivation of active brassinosteroids

CYP72B1-mediated inactivation of active brassinosteroids

A novel brassinolide-enhanced gene identified by cDNA microarray is involved in the growth of rice.

Brassinosteroids (BRs) are growth-promoting natural substances required for normal plant growth and development. To understand the molecular mechanism of BR action, a cDNA microarray containing 1265 rice genes was analyzed for expression differences in rice lamina joint treated with brassinolide (BL). A novel BL-enhanced gene, designated OsBLE2, was identified and cloned. The full-length cDNA is 3243 bp long, encoding a predicted polypeptide of 761 amino acid residues and nine possible transmembrane regions. OsBLE2 expression was most responsive to BL in the lamina joint and leaf sheath in rice seedlings. Besides, auxin and gibberellins also increased its expression. OsBLE2 expressed more, as revealed by in situ hybridization, in vascular bundles and root primordia, where the cells are actively undergoing division, elongation, and differentiation. Transgenic rice expressing antisense OsBLE2 exhibits various degrees of repressed growth. BL could not enhance its expression in transgenic rice expressing antisense BRI1, a BR receptor, indicating that BR signaling to the enhanced expression of OsBLE2 is through BRI1 . BL effect in the d1 mutant rice was much weaker than that in its wild-type control, indicating that heterotrimeric G protein may be a component of BRs signaling. These results suggest that OsBLE2 is involved in BL-regulated growth and development processes in rice.

Microarray analysis of brassinosteroid-regulated genes in Arabidopsis.

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. Although insights into the functions of BRs have been provided by recent studies of biosynthesis and sensitivity mutants, the mode of action of BRs is poorly understood. With the use of DNA microarray analysis, we identified BR-regulated genes in the wild type (WT; Columbia) of Arabidopsis and in the BR-deficient mutant, det2. BR-regulated genes generally responded more potently in the det2 mutant than in the WT, and they showed only limited response in a BR-insensitive mutant, bri1. A small group of genes showed stronger responses in the WT than in the det2. Exposure of plants to brassinolide and brassinazole, which is a specific inhibitor of BR biosynthesis, elicited opposite effects on gene expression of the identified genes. The list of BR-regulated genes is constituted of transcription factor genes including the phytochrome-interacting factor 3, auxin-related genes, P450 genes, and genes implicated in cell elongation and cell wall organization. The results presented here provide comprehensive view of the physiological functions of BRs using BR-regulated genes as molecular markers. The list of BR-regulated genes will be useful in the characterization of new mutants and new growth-regulating compounds that are associated with BR function.

Promotion of transcript accumulation of novel Zinnia immature xylem-specific HD-Zip III homeobox genes by brassinosteroids.

We isolated three novel homeobox genes (ZeHB-10, -11 and -12) from Zinnia elegans to elucidate the molecular mechanism underlying vascular system formation. ZeHB-10, -11 and -12 encode for HD-Zip proteins of the class III to which Arabidopsis Athb-8, -9, -14, -15 and IFL1 belong. In situ hybridization analysis demonstrated that the ZeHB-10, -11 and -12 mRNAs accumulated preferentially in procambium and immature xylem cells in 14-day-old plants. Transcripts for the three genes also accumulated in cultured Zinnia cells in a xylogenesis-specific manner. The accumulation of transcripts for all of ZeHB-10, -11 and -12 in cultured Zinnia cells was suppressed strongly by uniconazole, an inhibitor of brassinosteroid synthesis, and such suppression was reversed by the addition of brassinolide, a biologically active brassinosteroid. Thus the expression of ZeHB-10, -11 and -12 may be regulated by endogenous levels of brassinosteroids. Taken together with the fact that ZeHB-10, -11 and -12 proteins can bind to each other in yeast, the roles of HD-Zip III genes in vascular development are discussed.

Synthesis and bioactivity of C-2 and C-3 methyl ether derivatives of brassinolide

The following six novel methyl ether derivatives of brassinolide were prepared and their brassinosteroid activity was measured by means of the rice leaf lamina inclination bioassay: 2- O-methylbrassinolide, 3- O-methylbrassinolide, 2,22,23-tri- O-methylbrassinolide, 3,22,23-tri- O-methylbrassinolide, 2- O-methyl-25-methoxybrassinolide and 3- O-methyl-25-methoxybrassinolide. Brassinolide was used as a standard for comparison. All six compounds were also tested in the presence of 1000 ng of indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids. The 2- O-methyl- and 3- O-methylbrassinolide derivatives showed weak activity at high doses, which was enhanced by IAA, especially in the case of the 3- O-methyl derivative. Similarly, the 2,22,23-tri- O-methyl- and 3,22,23-tri- O-methyl derivatives displayed weak bioactivity on their own, but significantly stronger activity when applied with IAA. The 3- O-methyl and 3,22,23-tri- O-methyl analogues plus IAA were comparable in bioacivity to brassinolide alone, but were less active than brassinolide plus IAA. In each case, O-methylation at C-2 resulted in a greater loss of activity than O-methylation at C-3 under the same conditions. The relatively strong activity of 3,22,23-tri- O-methylbrassinolide in the presence of IAA is especially noteworthy as it indicates that free hydroxyl groups at C-3, C-22, and C-23 are not essential for bioactivity. Finally, 2- O-methyl- and 3- O-methyl-25-methoxybrassinolide were essentially inactive alone, and showed only a modest increase in bioactivity when coapplied with IAA.

Brassinosteroids Plant counterparts to animal steroid hormones?

Brassinosteroids are polyhydroxylated derivatives of common plant membrane sterols such as campesterol. They occur throughout the plant kingdom and have been shown by genetic and biochemical analyses to be essential for normal plant growth and development. Numerous reviews have detailed the recent progress in our understanding of the biosynthesis, physiological responses, and molecular modes of action of brassinosteroids. It is clear that like their animal steroid counterparts, brassinosteroids have a defined receptor, can regulate the expression of specific genes, and can orchestrate complex physiological responses involved in growth. This review summarizes the current status of BR research, pointing out where appropriate the similarities and differences between the mechanism of action of brassinosteroids and the more thoroughly studied animal steroid hormones.

Bioactivity of 25-hydroxy-, 26-hydroxy, 25,26-dihydroxy- and 25,26-epoxybrassinolide

The bioactivity of 25-hydroxybrassinolide, (25 S)- and (25 R)-26-hydroxybrassinolide, (25 S)- and (25 R)-25,26-dihydroxybrassinolide, and of (25 R)-25,26-epoxybrassinolide was tested in the rice leaf lamina inclination assay. The 25- and (25 S)-26-hydroxy derivatives are known metabolites of the naturally-occurring phytohormone brassinolide, whereas the other compounds are novel, but closely related, congeners. When tested alone, all showed either no activity or only weak activity at relatively high doses. When coapplied with indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids, enhanced bioactivity was observed for each compound. However, even when applied together with IAA, none of the compounds proved more bioactive than brassinolide with or without IAA. We conclude from these results that enzymatic hydroxylation of endogenous brassinolide at C-25 and/or C-26 does not enhance brassinosteroid activity, and so does not comprise an activation pathway in brassinolide biosynthesis. Instead, these hydroxylations result in modest to appreciable metabolic deactivation.

Steroid signaling in plants: from the cell surface to the nucleus.

Steroid hormones are signaling molecules important for normal growth, development and differentiation of multicellular organisms. Brassinosteroids (BRs) are a class of polyhydroxylated steroids that are necessary for plant development. Molecular genetic studies in Arabidopsis thaliana have led to the cloning and characterization of the BR receptor, BRI1, which is a transmembrane receptor serine/threonine kinase. The extracellular domain of BRI1, which is composed mainly of leucine-rich repeats, can confer BR responsivity to heterologous cells and is required for BR binding. Although downstream components of BR action are mostly unknown, multiple genes whose expression are regulated by BRs have been identified and suggest mechanisms by which BRs affect cell elongation and division.

Design, synthesis, and bioactivity of the first nonsteroidal mimetics of brassinolide.

Ten novel compounds, each consisting of two subunits and a linker, were designed with the aid of molecular modeling to resemble the natural steroidal phytohormone brassinolide. The mimetics were synthesized and subjected to the rice leaf lamina inclination bioassay to test for brassinosteroid activity. Most of the mimetics displayed very weak or no bioactivity, but two were strongly active when coapplied with the auxin indole-3-acetic acid (IAA), which synergizes the activity of brassinosteroids. Thus, 1-(4,6 alpha,7 alpha-trihydroxy-5,6,7,8-tetrahydronaphthyl)-2-(6 alpha',7 alpha'-dihydroxy-5',6',7',8'-tetrahydronaphthyl)ethyne (4) and (E)-1,2-bis[trans-(4a alpha,8a beta)-4-oxo-6 alpha,7 alpha-dihydroxy-4a,5,6,7,8,8a-hexahydro-(3H)-naphthyl]ethylene (11) showed exceptional activity at doses as low as 0.01 ng and 0.001 ng/plant, respectively. These compounds are the first biologically active nonsteroidal brassinolide mimetics.

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre, are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.

BRI1 is a critical component of a plasma-membrane receptor for plant steroids.

Most multicellular organisms use steroids as signalling molecules for physiological and developmental regulation. Two different modes of steroid action have been described in animal systems: the well-studied gene regulation response mediated by nuclear receptors, and the rapid non-genomic responses mediated by proposed membrane-bound receptors. Plant genomes do not seem to encode members of the nuclear receptor superfamily. However, a transmembrane receptor kinase, brassinosteroid-insensitive1 (BRI1), has been implicated in brassinosteroid responses. Here we show that BRI1 functions as a receptor of brassinolide, the most active brassinosteroid. The number of brassinolide-binding sites and the degree of response to brassinolide depend on the level of BRI1 protein. The brassinolide-binding activity co-immunoprecipitates with BRI1, and requires a functional BRI1 extracellular domain. Moreover, treatment of Arabidopsis seedlings with brassinolide induces autophosphorylation of BRI1, which, together with our binding studies, shows that BRI1 is a receptor kinase that transduces steroid signals across the plasma membrane.

A novel stress-inducible 12-oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between Brassinosteroid-action and Jasmonic-acid synthesis

To isolate brassinosteroid (BR) inducible genes, a subtractive cDNA-cloning strategy was applied. One of the isolated genes encodes a plant homologue to yeast old yellow enzymes (OYE) with strong sequence similarity to two cloned 12-oxo-phytodienoic acid reductases ( OPR1 and OPR2 ) from A. thaliana and was termed 12-oxophytodienoate reductase 3 ( OPR3 ; accession number: AJ238149). The expression of the OPR3 gene is induced by brassinosteroids, jasmonic acid (JA), and by a variety of stimuli like UV-light, touch, wind, wounding, and application of a detergent. Recombinant OPR3 protein converts 12-oxophytodienoate (OPDA) into 12-oxo phytoenoic acid (OPC8: 0), indicating the participation of OPR3 in the biosynthesis of JA from linolenic acid via the Vick-Zimmerman-pathway. In plants, OPC8: 0 is inevitably metabolized to JA by three cycles of β-oxidation. Both OPDA and JA are signal molecules involved in developmental processes and stress responses. Depending on environmental or developmental conditions, OPR potentially regulates the ratio between these two signal molecules. The yeast old yellow enzymes act on various enones and phenols including steroids, catalyzing reduction and disproportionation reactions. Thus, in addition to OPDA to OPC8: 0 conversion, OPR3 might be involved in further biosynthetic or degradative pathways in plants. As OPR3 expression is increased through treatment with brassinosteroids, it provides a potential link between brassinosteroid action and JA synthesis. BRs may thus influence the stress responses of plants through stimulation of JA synthesis.

Effect of chain length and ring size of alkyl and cycloalkyl side-chain substituents upon the biological activity of brassinosteroids. Preparation of novel analogues with activity exceeding that of brassinolide.

A series of brassinosteroids with different alkyl or cycloalkyl substituents in place of the isopropyl group at C-24 of brassinolide (1) were prepared by the CuCN-catalyzed addition of Grignard reagents to (threo-2R,3S,5alpha,22R,23R,24S)-23,24-epoxy-6, 6-(ethylenedioxy)-2,3-(isopropylidenedioxy)-26, 27-dinorcholestan-22-ol (9), followed by deketalization and Baeyer-Villiger oxidation. Compound 9 was employed as part of a 70:30 threo/erythro mixture of epoxides 9 and 10, from which the erythro-epoxide 10 was recovered intact after the Grignard additions. Thus, the corresponding n-dodecyl, n-hexyl, n-propyl, tert-butyl, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl analogues of brassinolide were obtained. A rearrangement byproduct was observed during the preparation of the cyclopropyl-substituted brassinosteroid when ether was used as the solvent in the Grignard reaction, but could be avoided by the use of THF. A method for recycling the undesired erythro-epoxide 10 was developed on the basis of deoxygenation with tellurium and lithium triethylborohydride. The rice leaf lamina inclination assay was then used to measure the bioactivity of the products. In general, increasing activity was observed as the length or ring size of the C-24 hydrocarbon substituent decreased. The novel cyclobutyl- and cyclopropyl-substituted analogues of brassinolide (1) were ca. 5-7 times as active as 1 and thus appear to be the most potent brassinosteroids reported to date. Further enhancement of the bioactivity of all of the above brassinosteroids, except that of the inactive n-dodecyl derivative, was observed when the brassinosteroid was applied together with an auxin, indole-3-acetic acid (IAA). The synergy between the brassinosteroids and IAA thus increased the bioactivity of the brassinosteroids, including the cyclopropyl and cyclobutyl derivatives, by ca. 1-2 orders of magnitude.

Physiology and molecular mode of action of brassinosteroids

Brassinosteroids (BRs) comprise a group of polyhydroxysteroids, which show close structural similarity to steroid hormones from arthropods and mammals. BRs are now accepted as a new class of phytohormones due to their ubiquitous occurrence in plants, their highly effective elicitation of various responses and the identification of mutants defective in BR-biosynthesis or -response. Important steps of BR-biosynthesis were elucidated with precursor-feeding experiments and by the analysis of BR-biosynthesis-deficient mutants. The altered phenotypes of these mutants, particularly in Arabidopsis, revealed the essential nature of BRs for normal growth and development. A major role of BRs is the positive regulation of cell expansion. Furthermore, BRs modulate plant responses to biotic and abiotic stresses and to other phytohormones, and influence differentiation processes of cells and tissues. BR-insensitive mutants such as bri1 hold the potential for uncovering BR-signalling pathway(s) at the molecular level. The identification of BR-regulated genes demonstrates a genetic basis for BR mode of action with reference to their multiple effects. This review focuses on the relevance of BRs to the control of various physiological processes, BR-signalling and underlying molecular mechanisms by considering known mutants.

Auxin and brassinosteroid differentially regulate the expression of three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.).

Indole-3-acetic acid (IAA) markedly increased ethylene production by inducing the expression of three 1aminocyclopropane-1-carboxylate (ACC) synthase cDNAs (pVR-ACS1, pVR-ACS6 and pVR-ACS7) in mung bean hypocotyls. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by a distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, and VR-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the beta-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tabacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR. A scheme of the multiple regulatory pathways for the expression of ACC synthase multigene family by auxin and BR is presented.