Characterization of novel genes in brassinosteroid biosynthesis and signaling pathways in monocotyledonous plants

Abstract

Brassinosteroids (BRs) are growth-promoting hormones in plants that play important roles in plant architecture, and as a consequence plant yield, a subject of great agronomic interest. Components and mechanisms of BR responses have been elucidated mainly using the model dicotyledonous plant Arabidopsis thaliana, but interesting differences with monocotyledonous plants have been revealed after the increasing use of Oryza sativa (rice) for the study of BRs in grasses. These differences prompt the study of BRs directly in monocots and thus the development of genomic resources and search for new tools. I joined the quest to find new ways to elucidate BR responses by studying plants with agronomic interest such as rice or model plants more closely related to cereal grasses. I first characterize the use of Propiconazole (Pcz) as a BR inhibitor for the use in field or large-scale experiments needed in monocots. Pcz treatments succeed to mimic BR-deficient mutant phenotypes on various monocot plants (i.e. maize, rice and Brachypodium), proving that its use was feasible. Using Pcz and available genomic and genetic tools I characterized the general response to BRs of the model grass Brachypodium distachyon and found several homologs genes from the BR signal (i.e. BIN2) and BR biosynthetic (i.e. DWF4, CPD and BR6OX2) pathways. Likewise, I used Pcz to screen a T-DNA mutant population in rice aimed to find new components of the BR response. From the screened lines, I further analyzed the denominate propiconazole-resistant 1 (pzr1-D) which presented various BR-related phenotypes such as increase sensitivity to BR and grain yield when compared with wild-type plants. The gene affected by the mutation is likely to be a rice ortholog of Arabidopsis DPb, a transcription factor which dimerizes with E2F and together control transcription of proteins involved in cell division. The increment of PZR1 transcript levels by the T-DNA activation-tagging in pzr1-D mutants and PZR1 overexpressing lines produce more number of tiller, panicles, and panicle branches, all resulting in increased grain number compared with wild type. Plant phenotypes along with whole-transcriptome analysis present evidence that cell cycle and brassinosteroid signal may be linked through PZR1. Finally, as another tool for the study of BRs-related processes I developed and demonstrated the use of a specific antibody anti BZR1, a key transcription factor and positive regulator of the BR signal. Anti-BZR1 antibody is able to detect active dephosphorylated and inactive phosphorylated forms of BZR1 in Arabidopsis and orthologs in rice, Setaria and Sorghum. In future, this antibody may be used to elucidate mechanisms in which the BZR1 is involved, specifically regarding its interaction with other proteins and the cross-talk between different plant signaling pathways.