Investigations on the Mechanism of the Brassinosteroid Response: VI. Effect of Brassinolide on Gravitropism of Bean Hypocotyls

Brassinosteroids are steroidal lactones of plant origin that promote growth of a number of plant systems, and particularly the growth induced by auxins. Biologically active brassinosteroids (BR) also promote the growth of gravisensitive hypocotyls of 7-day-old light grown Phaseolus vulgaris when gravistimulated. Brassinolide-mediated promotion of curvature of gravistimulated internodes occurs in the absence of exogenously supplied indole-3-acetic acid (IAA). This is in contrast to the BR-promoted bending of vertically positioned bean hypocotyls, which is dependent upon exogenous IAA. Brassinosteroid treatment increased the graviperception of young internode tissues and the bending of the gravistimulated sections as well as the subsequent reversal of bending after the sections were placed vertically. These results indicate that BR sensitizes bean hypocotyls to gravistimulation and potentiates the action of a growth factor that induces gravitropic growth.     

Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots

We have found that chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea (Pisum sativum L. cv Alaska), specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Application of 60 microM CSI disrupts the vertically oriented elongation of wild-type roots grown on agar plates but orients the elongation of agravitropic mutant aux1-7 roots toward the gravity. The CSI-induced restoration of gravitropic response in aux1-7 roots was not observed in other agravitropic mutants, axr2 and eir1-1. Because the aux1-7 mutant is reduced in sensitivity to auxin and ethylene, we examined the effects of CSI on another auxin-resistant mutant, axr1-3, and ethylene-insensitive mutant ein2-1. In aux1-7 roots, CSI stimulated the uptake of [(3)H]indole-3-acetic acid (IAA) and induced gravitropic bending. In contrast, in wild-type, axr1-3, and ein2-1 roots, CSI slowed down the rates of gravitropic bending and inhibited IAA uptake. In the null allele of aux1, aux1-22, the agravitropic nature of the roots and IAA uptake were not affected by CSI. This close correlation between auxin uptake and gravitropic bending suggests that CSI may regulate gravitropic response by inhibiting or stimulating the uptake of endogenous auxin in root cells. CSI exhibits selective influence toward IAA versus 1-naphthaleneacetic acid as to auxin-induced inhibition in root growth and auxin uptake. The selective action of CSI toward IAA along with the complete insensitivity of the null mutant aux1-22 toward CSI strongly suggest that CSI specifically interacts with AUX1 protein.     

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

Background and Aims

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process.

Methods

A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated.

Key Results

Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones.

Conclusions

No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.     

Abscisic acid is involved in brassinosteroids-induced chilling tolerance in the suspension cultured cells from Chorispora bungeana

The objective of this study was to investigate whether abscisic acid (ABA), a second messenger in chilling stress responses, is involved in brassinosteroids (BRs)-induced chilling tolerance in suspension cultured cells from Chorispora bungeana. The suspension cells were treated with 24-epibrassinolide (EBR), ABA, ABA biosynthesis inhibitor fluridone (Flu) and EBR in combination with Flu. Their effects on chilling tolerance, reactive oxygen species (ROS) levels and antioxidant defense system were analyzed. The results showed that EBR treatment markedly alleviated the decrease of cell viability and the increases of ion leakage and lipid peroxidation induced by chilling stress, suggesting that application of EBR could improve the chilling tolerance of C. bungeana suspension cultures. In addition, similar results were observed when exogenous ABA was applied. Treatment with Flu alone and in combination with EBR significantly suppressed cell viability and increased ion leakage and lipid peroxidation under low temperature conditions, indicating that the inhibition of ABA biosynthesis could decrease the chilling tolerance of C. bungeana suspension cultures and the EBR-enhanced chilling tolerance. Further analyses showed that EBR and ABA enhanced antioxidant defense and slowed down the accumulation of ROS caused by chilling. However, Flu application differentially blocked these protective effects of EBR. Moreover, EBR was able to mimic the effect of ABA by markedly increasing ABA content in the suspension cells under chilling conditions, whereas the EBR-induced ABA accumulation was inhibited by the addition of Flu. Taken together, these results demonstrate that EBR may confer chilling tolerance to C. bungeana suspension cultured cells by enhancing the antioxidant defense system, which is partially mediated by ABA, resulting in preventing the overproduction of ROS to alleviate oxidative injury induced by chilling.     

A reappraisal of the role of abscisic acid and its interaction with auxin in apical dominance

BACKGROUND AND AIMS: Evidence from pea rms1, Arabidopsis max4 and petunia dad1 mutant studies suggest an unidentified carotenoid-derived/plastid-produced branching inhibitor which moves acropetally from the roots to the shoots and interacts with auxin in the control of apical dominance. Since the plant hormone, abscisic acid (ABA), known to inhibit some growth processes, is also carotenoid derived/plastid produced, and because there has been indirect evidence for its involvement with branching, a re-examination of the role of ABA in apical dominance is timely. Even though it has been determined that ABA probably is not the second messenger for auxin in apical dominance and is not the above-mentioned unidentified branching inhibitor, the similarity of their derivation suggests possible relationships and/or interactions. METHODS: The classic Thimann-Skoog auxin replacement test for apical dominance with auxin [0.5 % naphthalene acetic acid (NAA)] applied both apically and basally was combined in similar treatments with 1 % ABA in Ipomoea nil (Japanese Morning Glory), Solanum lycopersicum (Better Boy tomato) and Helianthus annuus (Mammoth Grey-striped Sunflower). KEY RESULTS: Auxin, apically applied to the cut stem surface of decapitated shoots, strongly restored apical dominance in all three species, whereas the similar treatment with ABA did not. However, when ABA was applied basally, i.e. below the lateral bud of interest, there was a significant moderate repression of its outgrowth in Ipomoea and Solanum. There was also some additive repression when apical auxin and basal ABA treatments were combined in Ipomoea. CONCLUSION: The finding that basally applied ABA is able partially to restore apical dominance via acropetal transport up the shoot suggests possible interactions between ABA, auxin and the unidentified carotenoid-derived branching inhibitor that justify further investigation.     

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legué et al. 1994, Physiol Plant 91: 559–566). After stimulation at 90°C or 135°, the transformed root tips curve, but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.Abbreviations DEZ distal elongation zone - ELISA enzymelinked immunosorbent assay - T-DNA DNA transferred from Agrobacterium rhizogenes to the plant genome This work was supported by the Centre National d'Etudes Spatiales.     

Effect of Lysophosphatidylethanolamine and Brassinosteroids on Development of <Emphasis Type="Italic">Arabidopsis</Emphasis> Roots

Exogenously applied lysophosphatidylethanolamine (LPE) increased the growth of primary roots and the formation of lateral roots in Arabidopsis thaliana. In the presence of brassinolide, lateral root formation induced by LPE was enhanced, implying that both LPE and brassinosteroids (BR) interact positively in the development of Arabidopsis roots. Co-treatment with LPE and BRs increased the bending activity in the rice lamina inclination assay compared to that when BRs were applied alone, suggesting that LPE seems to exert its activity via BRs activity. RT-PCR revealed that LPE did not alter the expressions of genes involved in the biosynthesis of BRs but did activate the expressions of BR signaling genes in A. thaliana. In a BR-insensitive mutant, bri1, enhanced gravitropic response by LPE in wild-type A. thaliana was diminished. In conclusion, LPE is a positive regulator for the growth and development of Arabidopsis roots, and this process seems to be enhanced by BR signaling rather than by increase in endogenous levels of BRs in A. thaliana.     

Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza

Transgenic tomato plants with reduced expression of the sucrose transporter SlSUT2 showed higher efficiency of mycorrhization suggesting a sucrose retrieval function of SlSUT2 from the peri-arbuscular space back into the cell cytoplasm plant cytoplasm thereby limiting mycorrhiza fungal development. Sucrose uptake in colonized root cells requires efficient plasma membrane-targeting of SlSUT2 which is often retained intracellularly in vacuolar vesicles. Protein-protein interaction studies suggested a link between SISUT2 function and components of brassinosteroid biosynthesis and signaling. Indeed, the tomato DWARF mutant d^x defective in BR synthesis¹ showed significantly reduced mycorrhization parameters.² The question has been raised whether the impact of brassinosteroids on mycorrhization is a general phenomenon. Here, we include a rice mutant defective in DIM1/DWARF1 involved in BR biosynthesis to investigate the effects on mycorrhization. A model is presented where brassinolides are able to impact mycorrhization by activating SUT2 internalization and inhibiting its role in sucrose retrieval.     

Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis

Plant hormone brassinosteroids (BRs) and auxin exert some similar physiological effects likely through their functional interaction, but the mechanism for this interaction is unknown. In this study, we show that BRs are required for lateral root development in Arabidopsis and that BRs act synergistically with auxin to promte lateral root formation. BR perception is required for the transgenic expression of the beta-glucuronidase gene fused to a synthetic auxin-inducible promoter (DR5::GUS) in root tips, while exogenous BR promotes DR5::GUS expression in the root tips and the stele region proximal to the root tip. BR induction of both lateral root formation and DR5::GUS expression is suppressed by the auxin transport inhibitor N-(1-naphthyl) phthalamic acid. Importantly, BRs promote acropetal auxin transport (from the base to the tip) in the root. Our observations indicate that BRs regulate auxin transport, providing a novel mechanism for hormonal interactions in plants and supporting the hypothesis that BRs promote lateral root development by increasing acropetal auxin transport.     

Involvement of brassinosteroids in the gravitropic response of primary root of maize

Exogenously applied brassinolide (BL, 10(-9)-10(-5) M) increased gravitropic curvature in maize (Zea mays) primary roots. The BL-enhanced gravitropic curvature was clearly promoted in the presence of indole-3-acetic acid (IAA, 10(-10)-10(-8) M), indicating that BL is interactive with IAA during the gravitropic response. The interactive effect between BL and IAA was completely diminished by treatment of p-chlorophenoxy isobutric acid, an auxin action antagonist. The activation of the gravitropic response by BL in the absence and in the presence of IAA was nullified by application of 2, 3,5-triiodobenzoic acid, a polar auxin transport inhibitor. The data indicate that brassinosteroids (BRs) might be involved in auxin-mediated processes for the gravitropic response. Gas chromotography-selected ion-monitoring analysis revealed that maize primary roots contained approximately 0.3 ng g(-1) fresh weight castasterone as an endogenous BR. Exogenously applied castasterone also increased the gravitropic response of maize roots in an IAA-dependent manner. This study provides the first evidence, to our knowledge, for occurrence and gravitropic activity of BRs in plant roots.     

A proteomics study of brassinosteroid response in Arabidopsis

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.     

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.     

Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.     

Phytohormones signaling and crosstalk regulating leaf angle in rice

Leaf angle is an important agronomic trait in rice (Oryza sativa L.). It affects both the efficiency of sunlight capture and nitrogen reservoirs. The erect leaf phenotype is suited for high-density planting and thus increasing crop yields. Many genes regulate leaf angle by affecting leaf structure, such as the lamina joint, mechanical tissues, and the midrib. Signaling of brassinosteroids (BR), auxin (IAA), and gibberellins (GA) plays important roles in the regulation of lamina joint bending in rice. In addition, the biosynthesis and signaling of BR are known to have dominant effects on leaf angle development. In this review, we summarize the factors and genes associated with the development of leaf angle in rice, outline the regulatory mechanisms based on the signaling of BR, IAA, and GA, and discuss the contribution of crosstalk between BR and IAA or GA in the formation of leaf angle. Promising lines of research in the transgenic engineering of rice leaf angle to increase grain yield are proposed.     

Sites and regulation of auxin biosynthesis in Arabidopsis roots

Auxin has been shown to be important for many aspects of root development, including initiation and emergence of lateral roots, patterning of the root apical meristem, gravitropism, and root elongation. Auxin biosynthesis occurs in both aerial portions of the plant and in roots; thus, the auxin required for root development could come from either source, or both. To monitor putative internal sites of auxin synthesis in the root, a method for measuring indole-3-acetic acid (IAA) biosynthesis with tissue resolution was developed. We monitored IAA synthesis in 0.5- to 2-mm sections of Arabidopsis thaliana roots and were able to identify an important auxin source in the meristematic region of the primary root tip as well as in the tips of emerged lateral roots. Lower but significant synthesis capacity was observed in tissues upward from the tip, showing that the root contains multiple auxin sources. Root-localized IAA synthesis was diminished in a cyp79B2 cyp79B3 double knockout, suggesting an important role for Trp-dependent IAA synthesis pathways in the root. We present a model for how the primary root is supplied with auxin during early seedling development.