Self-incompatibility in Papaver Pollen:Programmed Cell Death in an Acidic Environment

Self-Incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilisation and thus encourages outbreeding and genetic diversity. During pollination, most SI systems utilise cell-cell recognition to reject incompatible pollen. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy), which involves the interaction between the two S-determinants, a stigma-expressed secreted protein (PrsS) and a pollen-expressed plasma-membrane localised protein (PrpS). This interaction is the critical step in determining acceptance of compatible pollen or rejection of incompatible pollen. Cognate PrpS-PrsS interaction triggers a signalling network causing rapid growth arrest and eventually programmed cell death (PCD) in incompatible pollen. In this review, we provide an overview of recent advances in our understanding of the major components involved in the SI-induced PCD (SI-PCD). In particular, we focus on the importance of SI-induced intracellular acidification and consequences for protein function, and the regulation of soluble inorganic pyrophosphatase (Pr-p26.1) activity by post-translational modification. We also discuss attempts at the identification of protease(s) involved in the SI-PCD process. Finally, we outline future opportunities made possible by the functional transfer of the P. rhoeas SI system to Arabidopsis

ATP depletion plays a pivotal role in self-incompatibility, revealing a link between cellular energy status, cytosolic acidification and actin remodelling in pollen tubes

Self-incompatibility (SI) involves specific interactions during pollination to reject incompatible ('self') pollen, preventing inbreeding in angiosperms. A key event observed in pollen undergoing the Papaver rhoeas SI response is the formation of punctate F-actin foci. Pollen tube growth is heavily energy-dependent, yet ATP levels in pollen tubes have not been directly measured during SI. Here we used transgenic Arabidopsis lines expressing the Papaver pollen S-determinant to investigate a possible link between ATP levels, cytosolic pH ([pH]cyt ) and alterations to the actin cytoskeleton. We identify for the first time that SI triggers a rapid and significant ATP depletion in pollen tubes. Artificial depletion of ATP triggered cytosolic acidification and formation of actin aggregates. We also identify in vivo, evidence for a threshold [pH]cyt of 5.8 for actin foci formation. Imaging revealed that SI stimulates acidic cytosolic patches adjacent to the plasma membrane. In conclusion, this study provides evidence that ATP depletion plays a pivotal role in SI upstream of programmed cell death and reveals a link between the cellular energy status, cytosolic acidification and alterations to the actin cytoskeleton in regulating Papaver SI in pollen tubes

Characterization of the metabolism of eupalinolide A and B by carboxylesterase and cytochrome P450 in human liver microsomes

Eupalinolide A (EA; Z-configuration) and eupalinolide B (EB; E-configuration) are bioactive cis-trans isomers isolated from Eupatorii Lindleyani Herba that exert anti-inflammatory and antitumor effects. Although one pharmacokinetic study found that the metabolic parameters of the isomers were different in rats, metabolic processes relevant to EA and EB remain largely unknown. Our preliminary findings revealed that EA and EB are rapidly hydrolyzed by carboxylesterase. Here, we investigated the metabolic stability and enzyme kinetics of carboxylesterase-mediated hydrolysis and cytochrome P450 (CYP)-mediated oxidation of EA and EB in human liver microsomes (HLMs). We also explored differences in the hydrolytic stability of EA and EB in human liver microsomes and rat liver microsomes (RLMs). Moreover, cytochrome P450 reaction phenotyping of the isomers was performed via in silico methods (i.e., using a quantitative structure-activity relationship model and molecular docking) and confirmed using human recombinant enzymes. The total normalized rate approach was considered to assess the relative contributions of five major cytochrome P450s to EA and EB metabolism. We found that EA and EB were eliminated rapidly, mainly by carboxylesterase-mediated hydrolysis, as compared with cytochrome P450-mediated oxidation. An inter-species difference was observed as well, with faster rates of EA and EB hydrolysis in rat liver microsomes. Furthermore, our findings confirmed EA and EB were metabolized by multiple cytochrome P450s, among which CYP3A4 played a particularly important role

Easy and green route towards nanostructured ZnO as active sensing material with unexpected H2S dosimeter-type behaviour

Nanostructured ZnO particles were prepared through a straightforward, quick and low\u2010temperature synthesis route involving coprecipitation of the metal precursor salts with oxalic acid, followed by hydrothermal treatment at 135 or 160 \ub0C. The synthesised nanostructured powders were thoroughly characterised by a wide array of analytical techniques from the morphological (Scanning Electron Microscopy \u2013SEM\u2010, Transmission Electron Microscopy \u2010TEM\u2010, Energy\u2010dispersive X\u2010ray Spectroscopy \u2010EDXS\u2010), structural (Powder X\u2010Ray Diffraction \u2010PXRD\u2010, Selected Area Electron Diffraction \u2010SAED\u2010), compositional (X\u2010ray Photoelectron Spectroscopy \u2010XPS\u2010) and physical (thermal stability) point of view. As far as functional applications are concerned, the powders were tested as gas sensor materials for H2S detection. Thereby these ZnO particles show unexpected gas dosimeter behaviour at 150 \ub0C. Based on these observations and on a comparison with literature a new model for the interaction of ZnO nanostructures with H2S is proposed

New opportunities and insights into Papaver selfincompatibility by imaging engineered Arabidopsis pollen

Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS–PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS–green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI

A hybrid backtracking algorithm for automatic test data generation

Kao osnovno pitanje u ispitivanju softvera, automatsko generiranje podataka za ispitivanje je od najveće važnosti, što je u biti problem zadovoljavanja ograničenja, a rješava se algoritmima pretraživanja. U našem prethodnom istraživanju za rješenje ograničenja predložena je metoda grananja i ograničavanja, a elaborirane su unaprijedne metode. Zasnovan na intervalnom aritmetičkom i simboličkom izvršenju, ovaj je rad usredotočen na unatražnu metodu, a to je povezivanje unaprijednog provjeravanja i unatražnog ispitivanja usmjerenog konflikta, u cilju poboljšanja učinkovitosti unatražnog praćenja u postupku traženja. Zatvaranja varijabli se koriste kako bi se olakšalo lociranje nesuglasica koje dovode do neriješenih rezultata (dead ends). Empirijski eksperimenti dokazuju učinkovitost predložene hibridne unatražne metode i njenu primjenljivost u inženjerstvu.As a fundamental issue in software testing, automatic test data generation is of crucial importance, which is essentially a constraint satisfaction problem and solved by search algorithms. In our previous research, branch and bound was proposed as our constraint solver and the look-ahead methods were elaborated. Based on interval arithmetic and symbolic execution, this paper focuses on the look-back or backtracking method, which is the hybridization of forward checking and conflict-directed backjumping, with the aim of improving the efficiency of backtracking in the search procedure. The closures of variables are used to facilitate the localization of the conflicts which cause dead ends. Empirical experiments prove the effectiveness of the proposed hybrid backtracking method and its applicability in engineering