Expression of a plant gene with sequence similarity to animal TGF-beta receptor interacting protein is regulated by brassinosteroids and required for normal plant development.

Brassinosteroids (BRs) regulate the expression of numerous genes associated with plant development, and require the activity of a Ser/Thr receptor kinase to realize their effects. In animals, the transforming growth factor-beta (TGF-beta) family of peptides acts via Ser/Thr receptor kinases to have a major impact on several pathways involved in animal development and adult homeostasis. TGF-beta receptor-interacting protein (TRIP-1) was previously shown by others to be an intracellular substrate of the TGF-beta type II receptor kinase which plays an important role in TGF-beta signaling. TRIP-1 is a WD-repeat protein that also has a dual role as an essential subunit of the eukaryotic translation initiation factor eIF3 in animals, yeast and plants, thereby revealing a putative link between a developmental signaling pathway and the control of protein translation. In yeast, expression of a TRIP-1 homolog has also been closely associated with cell proliferation and progression through the cell cycle. We report here the novel observation that transcript levels of TRIP-1 homologs in plants are regulated by BR treatment under a variety of conditions, and that transgenic plants expressing antisense TRIP-1 RNA exhibit a broad range of developmental defects, including some that resemble the phenotype of BR-deficient and -insensitive mutants. This correlative evidence suggests that a WD-domain protein with reported dual functions in vertebrates and fungi might mediate some of the molecular mechanisms underlying the regulation of plant growth and development by BRs.

The Arabidopsis PHD-finger protein SHL is required for proper development and fertility.

The SHL gene from Arabidopsis thaliana encodes a small nuclear protein that contains a BAH domain and a PHD finger. Both domains are found in numerous (putative) transcriptional regulators and chromatin-remodeling factors. Different sets of transgenic lines were established to analyze the physiological relevance of SHL. SHL expression driven by the CaMV 35S promoter results in reduced growth, early flowering, early senescence, and impaired flower and seed formation. Antisense inhibition of SHL expression gives rise to dwarfism and delayed development. In-frame N-terminal fusion of the SHL protein to beta-glucuronidase (GUS) directs GUS to the nucleus of stably transformed Arabidopsis plants. Thus, SHL encodes a novel putative regulator of gene expression, which directly or indirectly influences a broad range of developmental processes.

Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro.

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).

Plant development: A role for sterols in embryogenesis.

The Arabidopsis mutants fackel and sterol methyltransferase 1 have defects associated with body organization of the seedling. Molecular analysis of these mutants has revealed that plant sterols may be key signaling molecules influencing position-dependent cell fate during embryonic development.

A putative role for the tomato genes DUMPY and CURL-3 in brassinosteroid biosynthesis and response.

The dumpy (dpy) mutant of tomato (Lycopersicon esculentum Mill.) exhibits short stature, reduced axillary branching, and altered leaf morphology. Application of brassinolide and castasterone rescued the dpy phenotype, as did C-23-hydroxylated, 6-deoxo intermediates of brassinolide biosynthesis. The brassinolide precursors campesterol, campestanol, and 6-deoxocathasterone failed to rescue, suggesting that dpy may be affected in the conversion of 6-deoxocathasterone to 6-deoxoteasterone, similar to the Arabidopsis constitutive photomorphogenesis and dwarfism (cpd) mutant. Measurements of endogenous brassinosteroid levels by gas chromatography-mass spectrometry were consistent with this hypothesis. To examine brassinosteroid-regulated gene expression in dpy, we performed cDNA subtractive hybridization and isolated a novel xyloglucan endotransglycosylase that is regulated by brassinosteroid treatment. The curl-3 (cu-3) mutant (Lycopersicon pimpinellifolium ¿Jusl. Mill.) shows extreme dwarfism, altered leaf morphology, de-etiolation, and reduced fertility, all strikingly similar to the Arabidopsis mutant brassinosteroid insensitive 1 (bri1). Primary root elongation of wild-type L. pimpinellifolium seedlings was strongly inhibited by brassinosteroid application, while cu-3 mutant roots were able to elongate at the same brassinosteroid concentration. Moreover, cu-3 mutants retained sensitivity to indole-3-acetic acid, cytokinins, gibberellin, and abscisic acid while showing hypersensitivity to 2, 4-dichlorophenoxyacetic acid in the root elongation assay. The cu-3 root response to hormones, coupled with its bri1-like phenotype, suggests that cu-3 may also be brassinosteroid insensitive.

Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida.

Brassinosteroids are known to promote cell elongation in a wide range of plant species but their effect on cell division has not been as extensively studied. We examined the effect of brassinolide on the kinetics and final division frequencies of regenerating leaf mesophyll protoplasts of Petunia hybrida Vilm v. Comanche. Under optimal auxin and cytokinin conditions, 10-100 nM brassinolide accelerated the time of first cell division by 12 h but had little effect on the final division frequencies after 72-120 h of culture. One micromolar brassinolide showed the same acceleration of first cell division but inhibited the final division frequency by approximately 20%. Under sub-optimal auxin conditions, 10-100 nM brassinolide both accelerated the time of first cell division and dramatically increased the 72- to 120-h final division frequencies. Isolated protoplasts may provide a useful model system to investigate the molecular mechanisms of brassinosteroid action on cell proliferation.

A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development.

Brassinosteroids are widely distributed plant compounds that modulate cell elongation and division, but little is known about the mechanism of action of these plant growth regulators. To investigate brassinosteroids as signals influencing plant growth and development, we identified a brassinosteroid-insensitive mutant in Arabidopsis thaliana (L.) Henyh. ecotype Columbia. The mutant, termed bri1, did not respond to brassinosteroids in hypocotyl elongation and primary root inhibition assays, but it did retain sensitivity to auxins, cytokinins, ethylene, abscisic acid, and gibberellins. The bri1 mutant showed multiple deficiencies in developmental pathways that could not be rescued by brassinosteroid treatment including a severely dwarfed stature; dark green, thickened leaves; males sterility; reduced apical dominance; and de-etiolation of dark-grown seedlings. Genetic analysis suggests that the Bri1 phenotype is caused by a recessive mutation in a single gene with pleiotropic effects that maps 1.6 centimorgans from the cleaved, amplified, polymorphic sequence marker DHS1 on the bottom of chromosome IV. The multiple and dramatic effects of mutation of the BRI1 locus on development suggests that the BRI1 gene may play a critical role in brassinosteroid perception or signal transduction.

Plant hormones: brassinosteroids in the spotlight.

Recent studies on dwarf mutants of the model plant Arabidopsis thallana have provided convincing evidence that brassinosteroids-natural plant products similar to animal steroid hormones-are essential for normal plant growth and development.

Investigation of Gene Expression, Growth Kinetics, and Wall Extensibility during Brassinosteroid-Regulated Stem Elongation.

Brassinosteroids promote stem elongation in a variety of plants but little is known about the mechanism of action of these plant growth regulators. We investigated a number of physiological and molecular parameters associated with brassinosteroid-enhanced elongation. Continuous growth recordings of soybean (Glycine max L. cv Williams 82) epicotyls showed that there was a 45-min lag before 0.1 [mu]M brassinolide (BR) exerted a detectable effect on elongation. BR caused a marked increase in Instron-measured plastic extensibility, suggesting that BR may promote elongation in part by altering mechanical properties of the cell wall (wall loosening). Structure-function studies suggested that the dimensions of the brassinosteroid side chain were critical for promotion of elongation and expression of BRU1, a gene regulated specifically by active brassinosteroids. Auxin-BR interactions were examined by using small auxin up RNA (SAUR) gene probes and the auxin-insensitive diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.). We have shown that in wild-type tomato, which elongates in response to exogenous auxin, a transcript of identical size to the soybean SAUR 15A is strongly induced within 1 h by 50 [mu]M 2,4-dichlorophenoxyacetic acid or indoleacetic acid, whereas in the dgt mutant, which does not elongate in response to auxin, no transcript is expressed. Furthermore, BR promotes equal elongation of hypocotyls in both wild-type and dgt tomatoes but does not rapidly induce the SAUR 15A homolog in either genotype. BR does not cause rapid induction of SAUR 6B in elongating soybean epicotyls but does lead to increased expression after 18 h. This late BR activation of SAUR 6B is controlled, at least in part, at the transcriptional level and is not accompanied by an increase of free indoleacetic acid in the tissue. We conclude that although both BR and auxin affect wall relaxation processes, BR-promoted elongation in soybean and tomato stems acts via a mechanism that most likely does not proceed through the auxin signal transduction pathway.

Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean (Glycine max L.) epicotyls.

Brassinosteroids promote elongation and regulate gene expression in soybean (Glycine max L.) stems. We constructed a cDNA library from brassinosteroid-treated soybean epicotyls and used differential hybridization to isolate a cDNA (pBRU1) corresponding to a transcript whose abundance is increased by brassinosteroid treatment. Sequence analysis of pBRU1 revealed an open reading frame of 283 amino acids with a putative signal peptide of 29 amino acids. The sequence had extensive homology (77% identity, 89% similarity) over 114 contiguous amino acids to the meri-5 gene of Arabidopsis thaliana (J.I. Medford, J.S. Elmer, H.J. Klee [1991] Plant Cell 3: 359-370), and significant homology (48% identity, 62% similarity) to a xyloglucan endotransglycosylase localized in the cell walls of nasturtium (J. de Silva, C.D. Jarman, D.A. Arrowsmith, M.S. Stronach, S. Chengappa, C. Sidebottom, J.S. Reid [1993] Plant J 3: 701-711). RNase protection studies showed that BRU1 transcript levels are not increased by 1.0 microM auxins, cytokinins, abscisic acid, or gibberellic acid and that BRU1 expression is highest in stem tissue. Findings from studies with run-on transcripts from isolated soybean nuclei most likely indicate that the regulation of BRU1 by brassinosteroids is largely posttranscriptional. The elevated levels of BRU1 transcripts in elongating tissue and the homology with a xyloglucan endotransglycosylase suggest a possible role for the BRU1 protein in brassinosteroid-stimulated elongation.

Effect of brassinolide on gene expression in elongating soybean epicotyls.

We have studied the effect of brassinolide (BR), a plant steroidal lactone, on the expression of auxin-regulated genes in soybean (Glycine max L. cv Williams 82) epicotyls. BR caused up to 4-fold increases in epicotyl length during extended assays at 10(-7)m, in the absence of added auxin. Structurally related steroids failed to induce elongation or to alter the BR effect. Northern blot analysis, using sequences corresponding to auxin-regulated genes as probes, has shown that the molecular mechanism of BR-induced elongation is likely to differ from that of auxin-induced elongation in this system. BR does not rapidly induce members of the GH, SAUR, or JCW auxin-inducible gene families before the onset of elongation. BR enhances SAUR and GH1 transcripts after 18 h but has no effect on JCW1 or GH3 transcripts at any time examined. We have shown by two-dimensional gel analysis of in vitro translated mRNA that a submicromolar concentration of BR alters the pattern of gene expression in elongating soybean epicotyls.

Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco.

Regulatory properties of a 1.4-kilobase promoter fragment of the bean chalcone synthase CHS8 gene were examined by analysis of glucuronidase (GUS) activity in transgenic tobacco containing a CHS8-GUS gene fusion. The promoter was highly active in the root apical meristem and in petals, exclusively in those cells of the inner epidermis that accumulate anthocyanins. The gene fusion was only weakly expressed in other floral organs, mature leaves, and stems. The early stages of seedling development were characterized by an apparent wound induction of the promoter in the endosperm and strong expression in the immature root, which became localized to the apical meristem and perivascular tissue at the root-hypocotyl junction. The promoter became active during lateral root formation in both the new root and damaged tissue of the main root. The gene fusion was also expressed in greening cotyledons and primary leaves but not in the shoot apical meristem. Light modulated expression in the cotyledons and root-shoot junction but had no effect on other aspects of the developmental program. Wounding or fungal elicitor treatment of mature leaves activated the promoter in a well-defined zone adjacent to the stress site. Stress induction occurred in mesophyll and vascular tissues as well as in the epidermis. We conclude that the CHS8 promoter contains cis-elements required to establish temporal and spatial control of flavonoid biosynthesis during development and in response to diverse environmental stimuli.

Glutathione-elicited changes in chromatin structure within the promoter of the defense gene chalcone synthase.

Sites hypersensitive to digestion by DNase I have been identified within the 5'-flanking and 3'-coding sequences of genes encoding the defense enzyme chalcone synthase in bean (Phaseolus vulgaris L.). Two of the 5'-flanking hypersensitive sites are markedly induced upon elicitation of cells with glutathione and delineate sequence elements that are also present in the promoters of coordinately regulated genes. In contrast, other hypersensitive sites within the 5'-flanking sequences are expressed constitutively and one maps within an element that is also present in the promoters of coordinately regulated genes. These results suggest that the transcriptional activation of chalcone synthase genes is accompanied by structural changes in the chromatin associated with the proximal region of the promoter and that these probably reflect the binding of transcription factors tocis-regulatory elements.

Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts.

To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5' flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled that of endogenous chalcone synthase genes in suspension cultured cells. Functional analysis of 5' deletions suggests that promoter activity is determined by an elicitor-regulated activator located between the "TATA box" and nucleotide position -173 and an upstream silencer between -173 and -326. These cis-acting elements function in the transduction of the elicitation signal to initiate elaboration of an inducible defense response.

Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris.

Chalcone synthase (CHS) catalyzes the first and key regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. In bean (Phaseolus vulgaris L.) and other members of the Leguminoseae, chalcone synthase is also involved in the synthesis of the isoflavonoid-derived phytoalexin antibiotics characteristic of this family. We have demonstrated that the haploid genome of bean contains a family of about six to eight CHS genes, some of which are tightly clustered. Treatment of bean cells with fungal elicitor activates several of these genes leading to the accumulation of at least five and probably as many as nine distinct CHS transcripts encoding a set of CHS isopolypeptides of Mr 42-43 kDa but with differing pI in the range pH 6-7. In elicited cells specific transcripts and encoded polypeptides are differentially induced with respect to both the extent and kinetics of accumulation. Wounding or infection of hypocotyl tissue also activates several CHS genes with marked differences in the pattern of accumulation of specific transcripts and encoded polypeptides in wounded compared to infected tissue or elicited cells, indicating operation of more than one cue for defense gene activation. Illumination induces accumulation of a different set of CHS transcripts including only one of the set hitherto demonstrated to be induced by biological stress. The organization and differential regulation of the CHS gene family in bean are discussed in relation to the functions of this enzyme in adaptative and protective responses to diverse environmental stresses.

Rapid purification of host-specific pathotoxins from Alternaria alternata f. sp. lycopersici by solid-phase adsorption on octadecylsilane.

A simplified, rapid procedure for the purification of two phytotoxic metabolites (TA and TB) from cell-free culture filtrates of Alternaria alternata f. sp. lycopersici was developed using hydrophobic chromatography on macroporous, C18 bonded silica followed by gel filtration. Baseline separation of TA from TB was achieved in the final preparation and yields of 80% were obtained with retention of full biological activity in all assay systems tested. Toxin analysis by nuclear magnetic resonance and high-performance liquid chromatography indicated TA was at least 99% pure. The efficiency of the procedure allows rapid accumulation of pure TA and TB for studies on the molecular mode of action and host plant response to this novel host-specific phytotoxin.