Plant KASH proteins SINE1 and SINE2 have synergistic and antagonistic interactions with actin-branching and actin-bundling factors.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of outer nuclear membrane KASH proteins, interacting in the nuclear envelope lumen with inner nuclear membrane SUN proteins and connecting the nucleus and cytoskeleton. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light-dark transitions and abscisic acid (ABA), which requires F-actin reorganization. SINE2 influences actin depolymerization and SINE1 actin repolymerization. The actin-related protein 2/3 (ARP2/3) complex, an actin nucleator, and the plant actin-bundling and -stabilizing factor SCAB1 are involved in stomatal aperture control. Here, we have tested the genetic interaction of SINE1 and SINE2 with SCAB1 and the ARP2/3 complex. We show 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 opposing roles. The actin repolymerization defect observed in sine1-1 is partially rescued in scab1-2 sine1-1, while SINE2 is epistatic to SCAB1. In addition, SINE1 and ARP2/3 act synergistically in lateral root development. The absence of SINE2 renders trichome development independent of the ARP2/3 complex. Together, these data reveal complex and differential interactions of the two KASH proteins with the actin-remodeling apparatus and add evidence to the proposed differential role of SINE1 and SINE2 in actin dynamics.

A nuclear localization signal targets tail-anchored membrane proteins to the inner nuclear envelope in plants.

Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment, and both monopartite and bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery. This article has an associated First Person interview with the first author of the paper.

A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development.

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3',5'-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca(2+)-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response.

The Arabidopsis KASH protein SINE3 is involved in male and female gametogenesis.

KEY MESSAGE: The Arabidopsis KASH protein SINE3 is involved in male and female gametophyte development, likely affecting the first post-meiotic mitosis in both cases, and is required for full seed set. Linker of nucleoskeleton and cytoskeleton (LINC) complexes are protein complexes spanning the inner and outer membranes of the nuclear envelope (NE) and are key players in nuclear movement and positioning. Through their roles in nuclear movement and cytoskeletal reorganization, plant LINC complexes affect processes as diverse as pollen tube rupture and stomatal development and function. KASH proteins are the outer nuclear membrane component of the LINC complex, with conserved C-termini but divergent N-terminal cytoplasmic domains. Of the known Arabidopsis KASH proteins, SUN-INTERACTING NUCLEAR ENVELOPE PROTEIN 3 (SINE3) has not been functionally characterized. Here, we show that SINE3 is expressed at all stages of male and female gametophyte development. It is located at the NE in male and female gametophytes. Loss of SINE3 results in a female-derived seed set defect, with sine3 mutant ovules arresting at stage FG1. Pollen viability is also significantly reduced, with microspores arresting prior to pollen mitosis I. In addition, sine3 mutants have a minor male meiosis defect, with some tetrads containing more than four spores. Together, these results demonstrate that the KASH protein SINE3 plays a crucial role in male and female gametophyte development, likely affecting the first post-meiotic nuclear division in both cases.

Distinct Roles for KASH Proteins SINE1 and SINE2 in Guard Cell Actin Reorganization, Calcium Oscillations, and Vacuolar Remodeling.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a protein complex spanning the inner and outer membranes of the nuclear envelope. Outer nuclear membrane KASH proteins interact in the nuclear envelope lumen with inner nuclear membrane SUN proteins. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light-dark transitions and ABA. Previous studies have shown F-actin organization, cytoplasmic calcium (Ca2+) oscillations, and vacuolar morphology changes are involved in ABA-induced stomatal closure. Here, we show that SINE1 and SINE2 are both required for actin pattern changes during ABA-induced stomatal closure, but influence different, temporally distinguishable steps. External Ca2+ partially overrides the mutant defects. 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. We show first evidence for nuclear Ca2+ oscillations during ABA-induced stomatal closure, which are disrupted in sine2-1. Vacuolar fragmentation is impaired in both mutants and is partially rescued by F-actin depolymerization. Together, these data indicate distinct roles for SINE1 and SINE2 upstream of this network of players involved in ABA-based stomatal closure, suggesting a role for the nuclear surface in guard cell ABA signaling.

Regulation of nuclear shape and size in plants.

Nuclear shape and size changes have long been used by cytopathologists to diagnose, stage, and prognose cancer. However, the underlying causalities and molecular mechanisms are largely unknown. The current eukaryotic tree of life groups eukaryotes into five supergroups, with all organisms between humans and yeast falling into the supergroup Opisthokonta. The emergence of model organisms with strong molecular genetic methodology in the other supergroups has recently facilitated a broader evolutionary approach to pressing biological questions. Here, we review what is known about the control of nuclear shape and size in the Archaeplastidae, the supergroup containing the higher plants. We discuss common themes as well as differences toward a more generalized model of how eukaryotic organisms regulate nuclear morphology.

Nuclei in motion: movement and positioning of plant nuclei in development, signaling, symbiosis, and disease.

While textbook figures imply nuclei as resting spheres at the center of idealized cells, this picture fits few real situations. Plant nuclei come in many shapes and sizes, and can be actively transported within the cell. In several contexts, this nuclear movement is tightly coupled to a developmental program, the response to an abiotic signal, or a cellular reprogramming during either mutualistic or parasitic plant-microbe interactions. While many such phenomena have been observed and carefully described, the underlying molecular mechanism and the functional significance of the nuclear movement are typically unknown. Here, we survey recent as well as older literature to provide a concise starting point for applying contemporary molecular, genetic and biochemical approaches to this fascinating, yet poorly understood phenomenon.