Breaking Barriers: Leveraging LORELEI-Like Proteins to Enhance Hybridization and Reproductive Heat Tolerance in Brassicaceae

Abstract

Plant sexual reproduction is essential for fruit and seed production, and the rapid fertilization process in angiosperms has been central to the domestication of crops for human consumption. As global temperatures rise and plants increasingly face environmental stresses such as heat, soil salinity, and pathogens, breeding and engineering plants that are resilient to these challenges have become critical for sustaining crop productivity. Recent studies have focused on elucidating the molecular mechanisms underlying reproductive thermotolerance. This PhD dissertation advances our knowledge of a key signaling complex found in female and male reproductive tissues and demonstrates how it can be leveraged to overcome barriers to plant sexual reproduction through hybridization and engineer transgenic plants with enhanced tolerance to heat stress. The ability of flowering plants to reproduce successfully underlies fruit and seed production and is therefore central to global agriculture and food security. In angiosperms, pollen tube growth is the critical step that enables fertilization—delivering immotile sperm cells to the ovules deep within the pistil—and thus directly determines reproductive success and yield. However, realizing the full potential of plant breeding and engineering is often constrained by pre-fertilization incompatibility barriers that prevent hybridization between related species. During interspecific crosses, pollen tubes may germinate but frequently fail to navigate or communicate properly within the pistil, arresting before fertilization can occur. A second major limitation to advancing agriculture arises from environmental stress, particularly elevated temperatures, which disrupt pollen tube elongation and integrity, leading to failed fertilization and reduced seed set. Together, these biological and environmental barriers restrict both the diversification and resilience of crops. This dissertation advances our understanding of a key signaling complex that operates in both female and male reproductive tissues and demonstrates how manipulating this pathway can overcome one of the barriers to fertilization by enabling compatibility in previously incompatible interspecific crosses or by engineering heat-resilient reproductive function through targeted transgenic approaches. LORELEI-LIKE GPI-ANCHORED PROTEIN (LLGs) provide a unique opportunity to overcome barriers to sexual reproduction. As GPI-anchored proteins, they are tethered to the outer leaflet of the plasma membrane and can move along it, enabling them to sense environmental cues and complex with receptors to regulate downstream responses in the cell. Across both vegetative and reproductive tissues, LLGs form complexes with members of the Catharanthus roseus RECEPTOR-LIKE-KINASES (CrRLKs) family, which span the transmembrane and possess a cytoplasmic kinase domain that transmits extracellular signals upon binding of RAPID ALKALINIZATION FACTORS (RALF) peptide ligands. These interactions enable the transduction of extracellular signals into coordinated intracellular responses that regulate cell wall integrity and growth. In female reproductive tissues, LRE, an LLG member, interacts with CrRLK FERONIA (FER) to mediate pollen tube reception. Loss of function lre ovules results in pollen tube overgrowth and failed fertilization, phenotypes resembling those seen in interspecific crosses and leading to significant reductions in seed set. In chapter 1, we show that LRE has evolved an additional role in recognizing interspecific pollen tubes during interspecific crosses. By introducing cognate species LRE or changing critical residues to match the pollen tube species, we were able to enhance successful pollen tube reception and reduce pollen tube overgrowth, thereby improving cross-compatibility. These results demonstrate that precise LRE-mediated signaling is required to trigger pollen tube cell wall loosening and rupture at the synergid cells, thereby enabling pollen tube reception in crosses that would otherwise be blocked. In Arabidopsis male tissues, LLG2 and LLG3 function redundantly to regulate pollen tube growth. They complex with the CrRLKs ANXUR 1 and ANXUR 2 (ANX1 and ANX2) and BUDDHA’S PAPER SEAL 1 and 2 (BUPS1 and BUPS2), together with the peptide ligands RALF 4 and RALF19, to control downstream targets such as ROS homeostasis and callose synthesis and deposition in the pollen tube cell wall. Under heat stress, the LLG-CrRLK-RALF signaling complex is disrupted, resulting in reduced growth and premature rupture of the pollen tube. Supporting this, recent evidence in tomatoes demonstrated that loss of SlRALF1 loss of function in tomato causes leads to cultivar-specific sensitivity to heat stress. These findings implicate the LLG signaling pathway in pollen tube thermotolerance and highlight the LLG pathway’s role in maintaining cell wall integrity under stress. To test this hypothesis, I developed quantitative in vivo heat stress assays to monitor pollen tube growth under heat stress in Arabidopsis, a tool that previously did not exist (Chapter 2). This assay enabled precise quantification of pollen tube length and fertilization efficiency across temperature regimes and served as the foundation for assessing thermotolerance in transgenic lines in Chapter 3. I found that enhanced expression of tomato LLG2/3 in Arabidopsis pollen tubes significantly improved pollen tube growth under heat stress. Remarkably, transgenic pollen tubes expressing tomato LLG2/3 can outcompete non-transgenic wild-type pollen tubes under heat stress, resulting in increased siring of transgenic seeds in the next generation. Taken together, these results demonstrate that we can leverage LLGs in both female and male tissues to enhance reproductive success under adverse conditions. Hybridization barriers (Chapter 1) often arise when pollen tubes fail to burst upon reaching an ovule of a different species because they cannot perceive the female signal required to loosen the cell wall. In heat stress conditions (Chapters 2 and 3), there is reduced growth and a premature burst because the pollen tube cannot maintain its cell wall integrity. In both cases, we were able to overcome these barriers by ectopically expressing LLGs that modulate the cell wall integrity to restore normal reproductive function: either promoting controlled pollen tube rupture in synergid cells or maintaining cell wall integrity of the pollen tube during growth through the transmitting tract. Overall, our findings advance the understanding of LLG-mediated signaling in plant reproduction and highlight its potential to engineer cross-compatible or thermotolerant plants.