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Abstract Pollen function is critical for successful plant reproduction and crop productivity and it is important to develop accessible methods to quantitatively analyze pollen performance to enhance reproductive resilience. Here we introduce TubeTracker as a method to quantify key parameters of pollen performance such as, time to pollen grain germination, pollen tube tip velocity and pollen tube survival. TubeTracker integrates manual and automatic image processing routines and the graphical user interface allows the user to interact with the software to make manual corrections of automated steps. TubeTracker does not depend on training data sets required to implement machine learning approaches and thus can be immediately implemented using readily available imaging systems. Furthermore, TubeTracker is an excellent tool to produce the pollen performance data sets necessary to take advantage of emerging AI-based methods to fully automate analysis. We tested TubeTracker and found it to be accurate in measuring pollen tube germination and pollen tube tip elongation across multiple cultivars of tomato. Abstract FigureGraphical AbstractGraphical user interface of TubeTracker showing all supported functionalities. 

Rising temperature extremes during critical reproductive periods threaten the yield of major grain and fruit crops. Flowering plant reproduction depends on the ability of pollen grains to generate a pollen tube, which elongates through the pistil to deliver sperm cells to female gametes for double fertilization. We used tomato as amodel fruit croptodeterminehowhigh temperature affects the pollen tube growthphase, takingadvantage of cultivars noted for fruit production in exceptionally hot growing seasons. We found that exposure to high temperature solely during the pollen tube growth phase limits fruit biomass and seed set more significantly in thermosensitive cultivars than in thermotolerant cultivars. Importantly, we found that pollen tubes from the thermotolerantTamaulipas cultivar have enhanced growth in vivo and in vitro under high temperature. Analysis of the pollen tube transcriptome’s response to high temperature allowed us to define two responsemodes (enhanced induction of stress responses and higher basal levels of growth pathways repressed by heat stress) associated with reproductive thermotolerance. Importantly, we define key components of the pollen tube stress response, identifyingenhancedreactive oxygenspecies (ROS)homeostasis andpollen tubecallose synthesis and deposition as important components of reproductive thermotolerance in Tamaulipas. Our work identifies the pollen tube growth phase as a viable target to enhance reproductive thermotolerance and delineates key pathways that are altered in crop varieties capable of fruiting under high-temperature conditions. 

Abstract Key messagePollen tubes from closely related species and mutants lacking pollen tube MYB transcription factors are able to initiate FER/LRE-dependent synergid cell calcium oscillations. AbstractReproductive isolation leads to the evolution of new species; however, the molecular mechanisms that maintain reproductive barriers between sympatric species are not well defined. In flowering plants, sperm cells are immotile and are delivered to female gametes by the pollen grain. After landing on the stigmatic surface, the pollen grain germinates a polarized extension, the pollen tube, into floral tissue. After growing via polar extension to the female gametes and shuttling its cargo of sperm cells through its cytoplasm, the pollen tube signals its arrival and identity to synergid cells that flank the egg. If signaling is successful, the pollen tube and receptive synergid cell burst, and sperm cells are released for fusion with female gametes. To better understand cell–cell recognition during reproduction and how reproductive barriers are maintained between closely related species, pollen tube-initiated synergid cell calcium ion dynamics were examined during interspecific crosses. It was observed that interspecific pollen tubes successfully trigger synergid cell calcium oscillations—a hallmark of reproductive success—but signaling fails downstream of key signaling genes and sperm are not released. This work further defines pollen tube–synergid cell signaling as a critical block to interspecific hybridization and suggests that the FERONIA/LORELEI signaling mechanism plays multiple parallel roles during pollen tube reception. 

Cryogenic infrared vibrational spectroscopy of D2-tagged cyanobenzoate (CBA) derivatives are obtained and analyzed to characterize the intrinsic spectroscopic responses of the -CO2‾ head group to its location on the ring in both the isolated anions and the cationic complexes with divalent metal ions, M2+ (M=Mg, Ca, Sr). The benzonitrile functionality establishes the different ring isomers (para, meta, ortho) according to the location of the carboxylate and provides an additional reporter on the molecular response to the proximal charge center. The aromatic carboxylates display slightly smaller shifts than those observed for a related aliphatic system upon metal ion complexation. Although the CBA anions display very similar band patterns for all three ring positions, upon complexation with metal ions, the ortho isomer yields dramatically different spectral responses in both the -CO2‾ moiety and the CN group. This behavior is traced to the emergence of a tridentate binding motif unique to the ortho isomer in which the metal ions bind to both the oxygen atoms of the carboxylate group and the N atom of the cyano group. In that configuration, the -CO2‾ moiety is oriented perpendicular to the phenyl ring, and the CN stretching fundamental is both strong and red-shifted relative to its behavior in the isolated neutral. The behaviors of the metal-bound ortho complexes occur in contrast to the usual blue shifts associated with “Lewis” type binding of metal ions end-on to -CN. The origins of these spectroscopic features are analyzed with the aid of electronic structure calculations, which also explore differences expected for complexation of monovalent cations to the ortho carboxylate. The resulting insights have implications for understanding the balance between electrostatic and steric interactions at metal binding sites in chemical and biological systems.