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1. Disruption of pollen tube homogalacturonan synthesis relieves pollen tube penetration defects in the Arabidopsis O-FUCOSYLTRANSFERASE1 mutant

   https://doi.org/10.1007/s00497-023-00468-5  

   Robichaux, Kayleigh J.; Smith, Devin K.; Blea, Madison N.; Weigand, Chrystle; Harper, Jeffrey F.; Wallace, Ian S. (April 2023, Plant Reproduction)

   During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma-style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma-style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition. 

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2. Mismatch between pollen and pistil size causes asymmetric mechanical reproductive isolation across Phlox species

   https://doi.org/10.1093/evolut/qpae128  

   Feller, Anna_F; Burgin, Grace; Lewis, Nia_F; Prabhu, Rohan; Hopkins, Robin; Baucom, ed., Regina; Connallon, ed., Tim (September 2024, Evolution)

   Abstract Characterizing the mechanisms of reproductive isolation between lineages is key to determining how new species are formed and maintained. In flowering plants, interactions between the reproductive organs of the flower—the pollen and the pistil—serve as the last barrier to reproduction before fertilization. As such, these pollen–pistil interactions are both complex and important for determining a suitable mate. Here, we test whether differences in style length (a part of the pistil) generate a postmating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that the heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier, we assess pollen tube growth in vivo and in vitro. We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short-styled species also have smaller pollen and that both within- and between-species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such reproductive isolating mechanisms, combined with additional pollen–pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators. 

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3. An Extensive Study on Cross-Project Predictive Mutation Testing

   https://doi.org/10.1109/ICST.2019.00025  

   Mao, Dongyu; Chen, Lingchao; Zhang, Lingming (April 2019, IEEE Conference on Software Testing, Validation and Verification (ICST))

   Mutation testing is a powerful technique for evaluating the quality of test suite which plays a key role in ensuring software quality. The concept of mutation testing has also been widely used in other software engineering studies, e.g., test generation, fault localization, and program repair. During the process of mutation testing, large number of mutants may be generated and then executed against the test suite to examine whether they can be killed, making the process extremely computational expensive. Several techniques have been proposed to speed up this process, including selective, weakened, and predictive mutation testing. Among those techniques, Predictive Mutation Testing (PMT) tries to build a classification model based on an amount of mutant execution records to predict whether coming new mutants would be killed or alive without mutant execution, and can achieve significant mutation cost reduction. In PMT, each mutant is represented as a list of features related to the mutant itself and the test suite, transforming the mutation testing problem to a binary classification problem. In this paper, we perform an extensive study on the effectiveness and efficiency of the promising PMT technique under the cross-project setting using a total 654 real world projects with more than 4 Million mutants. Our work also complements the original PMT work by considering more features and the powerful deep learning models. The experimental results show an average of over 0.85 prediction accuracy on 654 projects using cross validation, demonstrating the effectiveness of PMT. Meanwhile, a clear speed up is also observed with an average of 28.7× compared to traditional mutation testing with 5 threads. In addition, we analyze the importance of different groups of features in classification model, which provides important implications for the future research. 

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4. Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species

   https://doi.org/10.3390/ijms221910603  

   Matzke, Courtney M.; Hamam, Hasan J.; Henning, Paige M.; Dougherty, Kyra; Shore, Joel S.; Neff, Michael M.; McCubbin, Andrew G. (October 2021, International Journal of Molecular Sciences)

   Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen. 

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5. Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

   https://doi.org/10.1111/tpj.14286  

   Luria, Gilad; Rutley, Nicholas; Lazar, Itay; Harper, Jeffery F.; Miller, Gad (March 2019, The Plant Journal)

   <bold>Summary</bold> Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROSpollen germinated with a frequency that was 35‐fold higher than the low‐ROSpollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantifyROSdynamics within a large pollen population was shown by dose‐dependent alterations inDCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increasedROSlevels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen. 

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