Abstract
The development of a plant from a single-celled zygote to a mature multicellular organism is the achievement of many coordinated cell divisions and cell growth mechanisms. Because plant cells are surrounded by a rigid cell wall, cellular growth is dependent upon cell wall remodeling and expansion. This expansion relies on orchestrated cell wall loosening, secretion of cell wall material, and cell wall proteins that play structural and signalling roles. Sensing of the cell wall is attained, in part, by receptor-like kinases that can monitor cell wall composition and transduce intracellular signals that regulate its integrity. Receptor-like kinases of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) subfamily are membrane proteins that maintain cell wall integrity through the integration of signals in the cell wall such as their RAPID ALKALINIZATION FACTOR (RALF) ligands, binding of pectins, and recruitment of other receptor kinases. Loss of CrRLK1Ls, RALFs, or their co-receptors of the LORELEI (LRE) family in the flowering plant Arabidopsis thaliana leads to aberrant rupture of tip growing cells such as root hairs and pollen tubes (PTs). During A. thaliana reproduction, control of PT rupture to allow sperm release in a coordinated process called PT reception is also mediated by CrRLK1L signalling in the synergid cells of the female gametophyte. In the synergid cells, CrRLK1L signalling regulates downstream responses such as calcium influx and levels of extracellular reactive oxygen species (ROS); however, the cytosolic proteins and signals downstream of CrRLK1Ls that trigger PT rupture are still uncharacterized. This can be, in part, attributed to PT reception occurring deep within the female reproductive tissue of the flower, and which are therefore difficult to observe. To gain a better understanding of the cellular events and downstream signals of the CrRLK1L pathway during PT reception, we took a reverse genetic approach to identifying new components of the pathway in synergid cells as described in Chapter 2. Furthermore, we optimized a technique for semi-in vitro live imaging of PT reception reported on in Chapter 3. In Chapter 4, we used this live imaging method to perform the first cytological analysis of the events surrounding PT reception using three dimensional timelapse imaging with two-photon excitation microscopy. This approach revealed a previously undescribed structure, the peritubular membrane (PRM), that envelopes the PT after penetration of the receptive synergid. Using the same semi-in vitro method and state-of-the-art genetically-encoded ROS biosensors in Chapter 5, we also report distinct cytosolic oxidative bursts that occur before and after PRM formation. FER and LRE are necessary for the pre-PRM oxidative burst and the magnitude of the post-PRM oxidative burst that is associated with PT rupture. Still, the large genetic redundancy of the CrRLK1L signaling pathway and hidden nature of the synergids in the female gametophyte hinder investigations of this pathway in A. thaliana. Therefore, in Chapter 6, we analyzed the conservation of the CrRLK1L pathway in the gametophyte of the bryophyte Marchantia polymorpha, which has less redundancy in the components of the pathway. The M. polymorpha genome encodes a single CrRLK1L homolog that has a conserved role in regulating the cell wall integrity of tip-growing rhizoids; however, the two M. polymorpha homologs of its co-receptor in A. thaliana, LRE, are uncharacterized in terms of function and complex formation. Therefore, in Chapter 6, we performed mutant and expression analysis of MpLRE1 and MpLRE2, and demonstrate that MpLRE2 has a conserved role in the regulation of tip growth and calcium signalling of rhizoids, whereas the function of MpLRE1 is still unclear.
Desnoyer, Nicholas