Molecular regulation of ethylene-induced hyponasty

Abstract

Many plant species can actively reorient their organs in response to dynamic environmental conditions. Organ movement can be an integral part of plant development or can occur in response to unfavourable external circumstances. Petiole hyponasty is an upward movement driven by a higher rate of cell expansion on the lower (abaxial) compared to the upper (adaxial) side. Hyponasty is common among rosette species facing environmental stresses such as flooding, proximity of neighbours or elevated ambient temperature. The complex regulatory mechanism of hyponasty involves activation of pathways at molecular and developmental levels with ethylene playing a crucial role. Despite the broad knowledge on the functionality and hormonal regulation of ethylene-induced hyponasty and growing insights into the developmental alterations leading to upward petiole movement, there are still many important questions to be answered. The focus of this thesis is the cellular basis and molecular regulation of ethylene-induced hyponastic growth in Arabidiopsis thaliana. A detailed analysis of the epidermal cell sizes and study of cortical microtubule (CMT) reorientation events revealed that, upon ethylene treatment, longitudinal cell expansion occurs in a localized zone of the abaxial side of the petiole. Based on the cell length data a mathematical model is provided that predicts petiole angle change which is highly similar to the observed value. The thesis also describes the use of next generation Illumina sequencing technique as a way of mapping T-DNA insertions in activation-tagging lines. Identification of T-DNA loci in such lines is occasionally problematic due to the complexity of integration events. In a screen of T-DNA activation-tagging lines carrying 35S CaMV enhancers, four candidates were selected. These lines exhibited aberrant petiole angles in control conditions, ethylene and low light treatments. The genomic DNA of those lines was pooled and subjected to Illumina sequencing which resulted in identification of three out of four insertion loci. One of the selected candidates, which exhibited decreased petiole angle in all treatments and was designated ddd1. The phenotype of ddd1 is linked to the T-DNA insertion in the intragenic region of ROTUNDIFOLIA3 (ROT3) gene which encodes an enzyme involved in the synthesis of brassinosteroids (BRs). The lack of cell expansion in ddd1 upon ethylene exposure led to the hypothesis about an interrelation of ethylene and BRs during cell elongation. This is supported by the pharmacological experiments which indicate a potential role of ethylene in sensitisation the tissue to BRs. Furthermore, the thesis aims to describe the involvement of ethylene response factors (ERFs) in regulation of hyponasty. Transcriptional regulation of ERFs during ethylene treatment is shown and possible scenarios of their involvement during the induction of hyponasty are discussed. Over-expression of a core cell cycle gene, CYCA2;1, leads to enhanced hyponasty independent of endoreduplication. It is shown that ethylene inhibits the progression of a mitotic cell cycle and mathematical modelling shows that this fine-tuning process provides a subtle balance between cell expansion and cell division which controls the degree of an upward petiole movement.