The potential for floral evolution in response to competing selection pressures following the loss of hawkmoth pollination in Ruellia humilis.

PREMISE: In the absence of hawkmoth pollinators, chasmogamous (CH) flowers of Ruellia humilis self-pollinate by two secondary mechanisms. Other floral visitors might exert selection on CH floral traits to restore outcrossing, but at the same time preferential predation of CH seeds generates selection to increase the allocation of resources to cleistogamous (CL) flowers. METHODS: To assess the potential for an evolutionary response to these competing selection pressures, we estimated additive genetic variances ( σ A 2 ${\sigma }_{{\rm{A}}}^{2}$ ) and covariances for 14 reproductive traits and three fitness components in a Missouri population lacking hawkmoth pollinators. RESULTS: We found significant σ A 2 ${\sigma }_{{\rm{A}}}^{2}$ for all 11 floral traits and two measures of resource allocation to CL flowers, indicating the potential for a short-term response to selection on most reproductive traits. Selection generated by seed predators is predicted to increase the percentage of CL flowers by 0.24% per generation, and mean stigma-anther separation is predicted to decrease as a correlated response, increasing the fraction of plants that engage in prior selfing. However, the initial response to this selection is opposed by strong directional dominance. CONCLUSIONS: The predicted evolutionary decrease in the number of CH flowers available for potential outcrossing, combined with the apparent preclusion of potential diurnal pollinators by the pollen-harvesting activities of sweat bees, suggest that 100% cleistogamy is the likely outcome of evolution in the absence of hawkmoths. However, rare mutations with large effects, such as delaying budbreak until after sunrise, could provide pathways for the restoration of outcrossing that are not reachable by gradual quantitative-genetic evolution.

Tbx3-Mediated Regulation of Cardiac Conduction System Development and Function: Potential Contributions of Alternative RNA Processing.

In this article, we provide a brief summary of work by us and others to discover the molecular underpinnings of early conduction system development and function. We focus on how the multifunctional protein Tbx3 contributes to acquisition and homeostasis of the tissue-specific properties of the sinoatrial and atrioventricular nodes. We also provide unpublished, preliminary findings supporting the role of Tbx3-regulated alternative RNA processing in the developing conduction system.

Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2.

KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K+ current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular retention.

Functional Invalidation of Putative Sudden Infant Death Syndrome-Associated Variants in the KCNH2-Encoded Kv11.1 Channel.

BACKGROUND: Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome. METHODS: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential. RESULTS: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration. CONCLUSIONS: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants.

Genetic and environmental integration of the hawkmoth pollination syndrome in Ruellia humilis (Acanthaceae).

Background and Aims: The serial homology of floral structures has made it difficult to assess the relative contributions of selection and constraint to floral integration. The interpretation of floral integration may also be clouded by the tacit, but largely untested, assumption that genetic and environmental perturbations affect trait correlations in similar ways. In this study, estimates of both the genetic and environmental correlations between components of the hawkmoth pollination syndrome are presented for chasmogamous flowers of Ruellia humilis , including two levels of control for serial homology. Methods: A greenhouse population for quantitative genetic analysis was generated by a partial diallel cross between field-collected plants. An average of 634 chasmogamous flowers were measured for each of eight floral traits that contribute to the hawkmoth syndrome. Genetic correlations (across parents) and environmental correlations (across replicate flowers) were estimated by restricted maximum likelihood. Key Results: Stigma height, anther height and floral tube length were very tightly integrated in their responses to both genetic and environmental perturbations. The inclusion of floral disc width as a control for serial homology suggests this integration is an adaptive response to correlational selection imposed by pollinators. In contrast, integration of non-homologous traits was low. Furthermore, when comparisons between the dimensions of serially homologous structures were excluded, the genetic and environmental correlation matrices showed little congruence. Conclusions: The results suggest that hawkmoths have imposed strong correlational selection on floral traits involved in the deposition and removal of pollen, and that this is a consequence of stabilizing selection on the relative positions of stigmas and anthers in the face of substantial flower size variation. Low integration of other floral traits, and conflicting patterns of genetic and environmental correlations among these traits, suggest weak or no correlational selection within the range of variability expressed within a population.