BrassinosteroidBrassinosteroids BRs are steroidal plant hormones that play an important role in the growth and development of rbassinosteroid. The brassinosteroid biosynthesis of sterols and Brassinosteroid biosynthesis as well as the signalling cascade they induce in plants have been elucidated largely through metabolic studies and the analysis of mutants in Brassinosteroid biosynthesis and rice. Only fragmentary details about BR signalling in other plant species steroids in beef canada known. Brassinosteroid biosynthesis a forward genetics strategy was used in Petunia hybridaby which 19 families with phenotypic alterations typical for BR deficiency mutants were identified. In all mutants, the endogenous BR levels were severely reduced. Interestingly, PhBEH2 also interacted with proteins implicated in other signalling pathways.
Brassinosteroids BRs are steroidal plant hormones that play an important role in the growth and development of plants. The biosynthesis of sterols and BRs as well as the signalling cascade they induce in plants have been elucidated largely through metabolic studies and the analysis of mutants in Arabidopsis and rice. Only fragmentary details about BR signalling in other plant species are known.
Here a forward genetics strategy was used in Petunia hybrida , by which 19 families with phenotypic alterations typical for BR deficiency mutants were identified. In all mutants, the endogenous BR levels were severely reduced. Interestingly, PhBEH2 also interacted with proteins implicated in other signalling pathways.
This suggests that PhBEH2 might function as an important hub in the cross-talk between diverse signalling pathways. The importance of steroidal hormones in mammalian development has already been known for almost eight decades. The idea that plant steroids have hormonal functions arose when scientists reported the isolation of brassinolide BL , a steroid with strong plant growth-promoting activity, from bee-collected pollen of Brassica napus L. Brassinosteroids BRs are widely distributed among the plant kingdom and so far they have been found in gymnosperms, monocots, dicots, a pteridophyte, a bryophyte, and a chlorophyte Bajguz and Tretyn, The subsequent identification of several BR biosynthesis and signalling mutants further supported the importance of these compounds for growth and development, and nowadays BRs are accepted as phytohormones.
BRs are involved in various growth and developmental processes such as cell division and elongation, vascular differentiation, abiotic and biotic stresses, photomorphogenesis, germination, senescence, and fertility Clouse and Sasse, ; Bajguz and Hayat, The sterol biosynthesis pathway can be divided into three domains Fig. In the first part, the mevalonate-derived squalene is converted via multiple steps into methylene lophenol. Subsequently, the pathway diverges into two downstream branches.
One branch produces sitosterol and stigmasterol, while the other produces campesterol. These sterols are integrated into cellular membrane components. In addition, the biosynthetic precursor campesterol is utilized to synthesize BRs Lindsey et al. Plants have multiple ways to synthesize BL from campesterol.
Detailed studies on the BR biosynthesis route in Catharanthus roseus and Arabidopsis thaliana led to the identification of two parallel branched routes, better known as the early and late C-6 oxidation pathways that converge at catasterone Fig. The two pathways are linked to each other, implying that they are not totally autonomous Shimada et al.
Although the early and late C-6 oxidation pathways are found throughout the plant kingdom, it seems that in species such as tomato and tobacco the late C-6 oxidation pathway is more prevalent Nomura et al. Biosynthesis of steroids and BRs, and the Arabidopsis enzymes involved. The metabolic products are shown in boxes and the enzymes involved in the pathway are depicted in red. The predominant pathway to synthesize BL runs from the campestanol CN -independent pathway and late C-6 oxodation pathway and is indicated by the red arrows.
The CN-dependent pathway is represented by the green arrows. Compounds measured in this study are indicated in a green box. A number of sterol and BR biosynthesis mutants have been described, predominantly in Arabidopsis , and many of the genes have been cloned and studied reviewed by Clouse, BR biosynthesis mutants and some sterol mutants such as dwarf7 dwf7 , diminuto dim , and dwarf5 dwf5 can be rescued by exogenous application of BR.
Presumably, the absence of these sterols interferes with membrane integrity. BR signalling has primarily been elaborated by studies in Arabidopsis. The currently known BR signalling pathway in Arabidopsis runs from membrane to nucleus reviewed by Clouse, Here an extensive analysis of BR biosynthesis and signalling in petunia is presented.
The goal of the study is to examine to what extent the biosynthesis of steroids and BRs and the signalling cascades downstream in plants are conserved.
In three cases, the tagged genes could be identified. Furthermore, several petunia homologues of key components of the BR signalling pathway in Arabidopsis were isolated and it was determined if a similar network of interactions between these factors can occur.
These interactions reveal new mechanisms, not previously recognized, that suggest extensive cross-talk between BR signalling and other hormone signalling pathways. Petunia plantlets were freeze-dried and subjected to extraction and chromatography according to Nomura et al. Sterols in petunia were analysed according to Nomura et al. Petunia DNA was isolated by grinding one young leaf in liquid nitrogen.
Here, somatic dTph1 insertions in the target gene that may occur in some cells of petunia W plants were used. Phylogenetic trees were generated using the Neighbor—Joining method of ClustalX ftp: Bootstrap mode replications was used for estimating the level of confidence assigned to the particular nodes in the tree.
The tree was visualized with the help of Treeviewer 1. Alignments were coloured using Boxshade http: The plasmids were introduced into the yeast strain PJ69 James et al.
The library screen was performed as described previously Quattrocchio et al. For expression in protoplasts, full-length coding sequences and the N- and C-terminal halves of yellow fluorescent protein YFP were amplified with primers containing attB sites facilitating Multisite Gateway cloning Invitrogen.
In order to study BR biosynthesis and signalling in petunia, populations of the mutagenic Petunia hybrida line W were visually screened Gerats et al. All mutants exhibited the reduced stature and dark round shiny leaves typical for plants with BR deficiency Fig. Due to their appearance, these mutants were named compact disc cd. Petunia BR biosynthesis and signalling mutants. B Top view of a cd1 W mutant. Mock indicates plantlets treated with a solution without epibrassinolide.
Overview of cd alleles and results of complementation experiment by brassinolide treatment. Of 19 mutant families, 18 clearly responded to the steroid, resulting in elongation of internodes, petioles, and leaves Table 1 , Fig. Only one mutant family, cd10 P , did not respond to exogenous application of epibrassinolide, suggesting that it is mutated in a BR signalling gene.
Cross-pollination between the mutant families revealed that the 19 mutants could be assigned to five different complementation groups see Table 1. As some of these families are derived from a common ancestor see Table 1 , it is likely that they represent only three different alleles. All mutants have a short robust stature with thick, small, round, and dark-green leaves, with only some slight differences in the severity of the dwarfish phenotype between the complementation groups.
The cd10 mutants remained extremely small and, even after growing for months in the greenhouse, did not flower. The biosynthesis mutants clearly grew slowly and flowered later, and fertility seemed to be reduced in the cd2 and especially in the cd1 mutants.
Wild-type petunia, like tomato, does not accumulate BL, but rather castasterone CS , as the bioactive compound Table 2. In the cd1 mutant, the levels of CS and 6-deoxoCS were greatly reduced. The amounts of sterols were similar to those of the wild type, indicating no lesion in sterol biosynthesis.
Therefore, cd1 seems to be mutated in an unknown brassinosteroid enzyme. In the cd3 mutant, massive accumulation of 6-deoxoCS was observed, suggesting that this mutant has a defect in the conversion of 6-deoxoCS to castasterone, a reaction that in tomato is catalysed by the CYP85A1 enzyme Bishop et al.
In the cd9 mutant, sterols are abundant but the pathway end-products CS and 6-deoxoCS were severely reduced. In conclusion, it was found that cd1 , cd2 , cd3 , and cd9 are all BR deficient. FW, fresh weight; ND, not detectable; lost, internal standards not recovered. Endogenous steroid levels of wild-type petunia W , cd1, and cd9 plants g kg —1 FW.
The biochemical analysis of BR content in cd mutants in some cases provided clues on the identity of the affected gene. Using a combination of database mining and primer design based on sequences of Solanaceae species e. The obtained sequence data of petunia BR biosynthesis genes were used to determine in each cd mutant if any of these known genes is disrupted by a dTph1 insertion.
When DNA isolated from cd2 plants Fig. The other three mutants cd2 P , cd2 P , and cd2 P carry a dTph1 insertion at different sites in the second exon Fig. Plants in progeny of cd2 W occasionally gave rise to revertant shoots Fig. Furthermore, a stable cd2 mutant cd2 M was identified in which excision of dTph1 created an 8bp footprint Fig. Molecular analysis of two BR biosynthesis mutant families. A Mutant phenotype of a cd2 W plant.
B A cd2 W plant that develops a revertant shoot cd2 M In D and E, boxes represent exons and the thin lines represent introns. The triangles and line illustrate the dTph1 transposon insertions and footprint in the indicated alleles. The red sequence indicates the target site duplication.
The identity of cd3 P was revealed by mining a 3D indexed petunia insertion database generated from the ENZA population Vandenbussche et al. In progeny of cd3 P , a stable mutant was shown to carry a mutant allele containing a 7bp footprint cd3 S In plants carrying the cd3 D allele, a transposable element could not be identified in the petunia CYP85A1 homologue.
However, sequencing of the CYP85A1 coding sequence from one of the progeny family cd3-K showed an 8bp footprint in exon 1, indicating the previous existence of a transposable element at this site Fig.
It must be said that the complete sequences of these genes are currently unknown. In the 3D indexed petunia insertion database Vandenbussche et al. S1 at JXB online. Similar to the other identified BRIs, PhBRI1 has an LRR domain, a putative leucine-zipper motif, a transmembrane domain to anchor the protein in the plasma membrane, and a cytoplasmic kinase domain Fig. Characterization of the BRI1 receptor from petunia.
The triangle indicates the dTph1 transposon insertion in cd10 P D Sequence analysis of the dTph1 insertion in cd The underlined sequence indicates the target site duplication. Because cd10 P failed to respond to application of epibrassinolide, cd10 was assumed to be mutated in a BR signalling gene Fig. To explore the BR signalling cascade in petunia further, the aim was to identify signalling components downstream of PhBRI1. A PCR-based approach was performed to identify potential petunia homologues.