Examining the role of plant LINC complexes in stomatal dynamics, fertility, and development

The nucleus is a dynamic organelle whose nuclear envelope (NE) is composed of a double membrane with a distinct but continuous outer nuclear membrane (ONM) and inner nuclear membrane (INM). Linker of the nucleoskeleton and cytoskeleton (LINC) complexes are located at the nuclear envelope and are composed of Klarsicht/ANC-1/Syne Homology (KASH) ONM proteins and Sad1/UNC-84 (SUN) INM proteins. SUN and KASH proteins interact in the NE lumen, forming a bridge between the nucleoplasm and cytoplasm. While plants do not encode homologs of animal KASH proteins, functional analogs have been identified. The Arabidopsis thaliana WPP domain-interacting proteins (WIPs) were the first plant KASH proteins identified. These proteins interact with WPP domain-interacting tail-anchored (WIT) proteins at the ONM, and with Arabidopsis SUN proteins in the INM. Together, a LINC complex comprised of SUN, WIP, and WIT is necessary for proper vegetative nuclear movement and morphology in pollen tubes.In Arabidopsis pollen tubes, the vegetative nucleus (VN) precedes the sperm cells (SCs) and maintains a fixed distance from the pollen tube tip during growth. In pollen tubes of wip and wit mutants, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Pollen tubes frequently fail to burst upon reception at the ovule, leading to a significant fertility defect. In vitro-induction of reactive oxygen species (ROS) has been shown previously to lead to pollen tube growth arrest and rupture in a Ca2+-dependent manner. I hypothesized that the WIT-WIP-SUN LINC complex is required for ROS-induced pollen tube rupture. I report a decrease in Ca2+-dependent ROS-induced pollen tube rupture in both wip and wit mutants. The ROS hyposensitivity correlates with an increased distance of the VN from the pollen tube tip. In addition, I report the first evidence of nuclear Ca2+ fluctuations in the VN and show that the patterns during growth and ROS-mediated pollen tube burst are altered in the mutants. The proximity of the VN to the pollen tube tip is required for WT-like response to exogenous ROS and nuclear Ca2+ fluctuations.In addition to WIPs, four additional KASH proteins were bioinformatically identified and named SUN domain-interacting nuclear envelope (SINE) proteins. SINE1 and SINE2 are paralogous proteins that have been shown to function during stomatal dynamics induced by light-dark transitions and ABA. ABA is a plant hormone that induces stomatal closure in response to stress, involving the activation of guard cell anion channels, reactive oxygen species (ROS) production, cytoplasmic calcium (Ca2+) oscillations, and filamentous actin (F-actin) reorganization. I show that ABA-induced ROS production is not perturbed in sine1-1 and sine2-1 mutants. However, ABA-induced cytoplasmic Ca2+ oscillations are diminished in sine2-1 but not sine1-1, and this defect can be rescued by both exogenous Ca2+ and F-actin depolymerization. ABA-induced cytoplasmic Ca2+ oscillations are also diminished in sine1-1 sine2-1 and this defect can be rescued by exogenous Ca2+, but not F-actin depolymerization. I show the first evidence for nuclear Ca2+ oscillations during ABA-induced stomatal closure, which are disrupted in sine2-1.Loss of SINE1 and SINE2 has been shown to impair F-actin reorganization during ABA-induced stomatal closure, in which SINE1 is involved in actin polymerization and/or stabilization and SINE2 is involved in actin depolymerization. To further elucidate the role SINE1 and SINE2 play during actin reorganization during ABA-induced stomatal closure, I generated double mutants with SINE1 and SINE2 and the two known actin-remodeling factors SCAB1 and the ARP2/3 complex and observed actin reorganization during stomatal closure. I demonstrate that ABA-induced actin reorganization is impaired in sine1-1 sine2-1, similar to sine2-1. I demonstrate that SINE1 and the ARP2/3 complex function in the same pathway during ABA-induced stomatal closure, while SINE2 and the ARP2/3 complex play opposite roles. The additional loss of SCAB1 can partially rescue the actin reorganization defect observed in sine1-1, but not sine2-1. Additionally, loss of SINE1 and the ARP2/3 complex impairs shoot biomass and lateral root development while absence of SINE2 renders trichome development independent of the ARP2/3 complex.Plants have an alternating two-staged life cycle between the haploid gametophyte generation and the diploid sporophyte generation. Flowering plants generate male and female multi-cellular haploid gametophytes for sexual reproduction. In these gametophytes, haploid cells undergo two to three rounds of post-meiotic mitosis, to form gametes and the accessory cells required for reproduction. I show that SINE3, which had not been functionally characterized, plays a role in both male and female gametogenesis. Loss of SINE3 results in a female-derived fertility defect, with sine3 mutant ovules arresting at developmental stage FG1 prior to mitosis. Pollen viability is also significantly reduced in sine3 mutants, as a result of pollen microspores arresting prior to pollen mitosis I. In addition, I demonstrate that sine3 mutants have a minor male meiosis defect, with some tetrads containing more than four spores of different sizes. I show that SINE3 is expressed at all stages of male and female gametophyte development at the nuclear envelope, and that this localization is dependent on its C-terminal KASH domain.The work presented here expands our knowledge on the functions of plant KASH proteins in various plant cellular processes, such as male fertility, stomatal dynamics, and male and female gametogenesis.