Molecular basis of brassinosteroid-promoted growth
Final Report Abstract
The growth-promoting effect of brassinosteroids (BRs) has been studied in hundreds of articles. A large number of BR-deficient and BR-insensitive mutants in Arabidosis thaliana and in crops such as rice, tomato, and pea were identified. These mutants are generally dwarfed and exhibit rounded, dark green leaves, delayed flowering, reduced male fertility and seed set, and delayed senescence. The most prominent direct BR-effect is the modification of gene expression patterns. The physiological mechanisms underlying BR-promoted growth appear to be manifold, and depend on the tissue and developmental stage. However, in spite of the large number of published studies the primary mode of action of BR is poorely understood, and the available data do not disclose causal relationships between physiological processes and the well-studied genomic events. Furthermore, the analysis of BR-deficient mutants is complicated by the dwarfism and multiple morphological changes. The mode of action of BR at later developmental stages cannot be faultlessly determined in these mutants. In this project, an integrated analysis of the basis of BR-promoted growth was performed in established Arabidopsis leaves by means of metabolome, transcriptome, morphology, and ultrastructure analyses. Different experimental approaches were performed in parallel. The first approach used BR-deficient mutants. CPD-antisense plants and the BR-deficient cbb1/dwf1-6 mutant were treated with 300 nM brassinolide (BL) for three weeks. Wild-type plants were grown in parallel and were simultaneously treated with a control solution. BR-supplementation fully normalized the morphology of CPD-antisense plants. The CPD-antisense plants were nearly indistinguishable from the wild type. The growth defect of cbb1 plants was largely complemented by exogenous BRs. After three weeks, BR-treatment was stopped (day 0). The CPD- antisense and cbb1 plants started to run into BR-deficiency. At this point the sampling began. Samples were taken at day 0, 1, 3, 6, 9 and 14 after stopping the respective treatment. In the second approach an inhibitor of BR-biosynthesis (brassinazole) was used. Wild-type plants were grown for three weeks without any treatment. Subsequently, plants were treated with 10 μM or 20 μM brassinazole or control solution. The wild-type plants started to develop symptoms of BR-deficiency. Additional time course experiments and isotope dilution experiments after 13CO2 labelling were initiated. The time course experiments were performed three times each. Decreasing BR-levels were indicated by increasing transcript levels of the BR-biosynthetic CPD and DWF4 genes. The time-series experiments revealed that BR-action in established Arabidopsis leaves is associated with stimulated cell expansion. Palisade and spongy parenchyma cells in cbb1, CPD-antisense, and brassinazole-treated plants were significantly smaller in comparison to the control. BR-deficiency caused a decrease in the leaf index, i.e. more roundish leaves, and a significant decrease in biomass production. Effects on cell number were less evident. Examination of plastid ultrastructure by means of transmission electron microscopy revealed intact chloroplasts in BR-deficient plants. BRZ-treated, CPD-antisense, and cbb1 chloroplasts tended to develop a thylakoid network with reduced grana stacking. Starch levels were diminished in CPD-antisense, cbb1, and BRZ-treated plants from day 3 onwards. The consequences of reduced carbon supply may include impaired energy balance, reduced provision of biosynthetic precursors, and ultimately reduced growth. Furthermore, reduced release of 14CO2 from labelled glucose in brassinazole-treated plants and elevated levels of tricarboxylic acid (TCA)-cycle intermediates in brassinazole-treated plants and the cbb1 mutant suggest reduced TCA-cycle activity in BR-deficient plants. Reduced TCA-cycle activity may compromise efficient use of carbohydrates and impair growth especially during the dark period. Furthermore, the TCA-cycle provides precursors for various biosynthetic pathways. In summary, our analyses revealed that induced BR-deficiency impairs starch accumulation, TCA-cycle activity, cell expansion, and biomass production. Further studies are needed to determine metabolic fluxes and the precise impact of BRs on catabolism and anabolism. In frame of the project, a total of 495 metabolite profiles were determined. In addition, 28 profiles from C13 pulse-labelling studies were established. The analysis of this complex data set is ongoing.
Publications
- Consequences of induced brassinosteroid-deficiency in Arabidopsis leaves. BMC Plant Biol. 2014 Nov 18;14:309
F Schröder, J Lisso, T Obata, A Erban, E Maximova, P Giavalisco, J Kopka, AR Fernie, L Willmitzer, C Müssig
(See online at https://dx.doi.org/10.1186/s12870-014-0309-0)
