PbrRALF5/10 prevents incompatible pollen tube death by reconstructing the methyl-esterified pectin and reactive oxygen species metabolism of pear in vitro.

Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca(2+) increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.

Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system, and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca2+ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.

Pear (Pyrus pyrifolia L.) has a S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. No studies, however, have examined the extent of organelle alterations during the SI response in Pyrus pyrifolia. Consequently, this study focused on the alterations to mitochondria and nuclear DNA in incompatible pollen tubes of the pear. Methylthiazolyldiphenyl-tetrazolium bromide was used to evaluate the viability of pollen tubes under S-RNase challenge. The results showed that the viability of the control and compatible pollen tubes decreased slightly, but that of the incompatible pollen and pollen tubes began to decline at 30 min. The mitochondrial membrane potential (Delta psi(mit)) was also tested with rhodamine 123 30 min after SI challenge, and was shown to have collapsed in the incompatible pollen tubes after exposure to S-RNase. Western blotting 2 h after SI challenge confirmed that the Delta psi(mit) collapse induced leakage of cytochrome c into the cytosol. Swollen mitochondria were detected by transmission electron microscopy as early as 1 h after SI challenge and the degradation of nuclear DNA was observed by both 4,6-diamidino-2-phenylindole and transferase-mediated dUTP nick-end labeling. These diagnostic features of programmed cell death (PCD) suggested that PCD may specifically occur in incompatible pollen tubes.

Pear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca(2+) current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Rapid alkalinization factors (RALFs) are cysteine-rich peptides that play important roles in a variety of biological processes, such as cell elongation and immune signaling. Recent studies in Arabidopsis have shown that RALFs regulate pollen tube growth via plasma membrane receptor-like kinases (RLKs). However, the downstream signal transduction mechanisms of RLKs in pollen tubes are unknown. Here, we identified PbrRALF2, a pear (Pyrus bretschneideri) pollen RALF peptide that inhibits pollen tube growth. We found that PbrRALF2 interacts with a malectin-like domain-containing RLK, PbrCrRLK1L13. The relative affinity between PbrRALF2 and PbrCrRLK1L13 was at the submicromolar level, which is consistent with the values of ligand–receptor kinase pairs and the physiological concentration for PbrRALF2-mediated inhibition of pollen tube growth. After binding to its extracellular domain, PbrRALF2 activated the phosphorylation of PbrCrRLK1L13 in a dose-dependent manner. We further showed that the MAP kinase PbrMPK18 is a downstream target of PbrCrRLK1L13 that mediates PbrRALF2-elicited reactive oxygen species (ROS) production. The excessive accumulation of ROS inhibits pollen tube growth. We show that MPK acts as a mediator for CrRLK1L to stimulate ROS production, which might represent a general mechanism by which RALF and CrRLK1L function in signaling pathways.

Flowering plants have diversified and conquered the land. To do so, they have evolved strategies to avoid incest and promote compatible outcrossing. Philip Hunter explores some of the latest research in this area and its application to agriculture and horticulture.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.

Self-incompatibility (SI) is an important mechanism in higher plants that promotes outcrossing and prevents self-fertilization. ‘Banpeiyu’ ( Citrus maxima ) and ‘Hyuganatsu’ ( Citrus tamurana ), two of the Citrus cultivars distributed in Kyusyu, Japan, show gametophytic SI. In this study, we used the Citrus mature pollen culture system and stylar crude protein extracts to simulate compatible (C) and SI responses in ‘Banpeiyu’ pollen tubes. We analyzed the protein changes in pollen tubes with the C- and SI-like treatments by nano-liquid chromatography–mass spectrometry (nano-LC-MS); 14 and 27 proteins were identified in C- and SI-like treatments, respectively. We picked up some candidate genes that were particularly prevalent in SI-like treatment and analyzed their expression level changes during C- and SI-like treatments in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes. The expression levels of copper/zinc superoxide dismutase ( Cu/Zn SOD ), manganese SOD ( Mn SOD ), catalase ( CAT ), and cysteine protease ( CYP ) increased after SI-like treatment. We used a fluorescent probe to visualize reactive oxygen species (ROS) level changes in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes after C- and SI-like treatments and found that 2-hour SI-like treatment induced ROS levels to increase in the pollen tubes of both cultivars. These results suggest that an ROS increase could be one of the key phenomena in the SI response of Citrus and that gene expression changes were responses to ROS generation.

Self-incompatibility (SI) has been widely investigated at both molecular and cellular levels in pear. This trait is controlled by a single multi-allelic locus encoding at least two components from the pollen and the pistil. The stylar-S determinant is an S-glycoprotein (S-RNase) that can inhibit pollen tube growth in a self-pistil, and induces a series of changes in reactive oxygen species (ROS), calcium (Ca2+), actin cytoskeleton, and phosphatidic acid, leading to programmed cell death in incompatible pollen tubes. At present, a total of 67 S-RNase genes have been identified and have served in selecting appropriate pollinators in pear orchards. The pollen-S determinant has also been investigated in pear. Although a group of F-box genes have been identified in the S-locus, it remains unclear as to which gene(s) are involved in self-incompatibility reactions. In pear, only a few cultivars have experienced loss of self-incompatibility, due to either stylar or pollen mutations, or due to polyploidy. Except for the deletion of S4-RNase in cultivar Osa-Nijisseiki, other stylar-tissue mutations, including abnormal expression and post-transcript modification, are difficult to study, and are yet to be explained at the molecular level. Similarly, the mechanism of pollen tissue mutation and polyploidy require further investigations in future studies.

In the process of pollination, haploid pollen germinates on the stigma surface and a pollen tube grows through the diploid tissues of the pistil toward the ovary. The pistil has two basic functions: to prevent unwanted pollen from gaining access to the ovary and to support the growth of desirable pollen. Pollen-pistil signaling allows these different types of pollen to be distinguished. Self-incompatibility (SI) systems, controlled by the S locus, are the best-understood pollen-pistil signaling systems. Other SI systems have been investigated at the molecular level, but the physiology of pollen tube rejection is best understood in the field poppy, Papaver rhoeas. This species has a gametophytic SI system: Pollen is rejected when its S haplotype is the same as either of the two S haplotypes expressed in the diploid pistil. Recent advances reveal new ways that SI controls pollen tube metabolism. A soluble pyrophosphatase is down-regulated as part of the rapid SI response, and, over the long term, perturbations of the actin cytoskeleton lead to programmed cell death in incompatible pollen tubes. Manipulating incompatible pollen tube metabolism in this way may leave more resources available for supporting the growth of compatible pollen tubes, the complementary function of the pistil.

Plant reproduction requires long-distance growth of a pollen tube to fertilize the female gametophyte. Prior reports suggested that mutations altering synthesis of flavonoids, plant specialized metabolites that include flavonols and anthocyanins, impair pollen development in several species, but the mechanism by which flavonols enhanced fertility was not defined. Here, we used genetic approaches to demonstrate that flavonols enhanced pollen development by reducing the abundance of reactive oxygen species (ROS). We further showed that flavonols reduced high-temperature stress-induced ROS accumulation and inhibition of pollen tube growth. The anthocyanin reduced (are) tomato mutant had reduced flavonol accumulation in pollen grains and tubes. This mutant produced fewer pollen grains and had impaired pollen viability, germination, tube growth, and tube integrity, resulting in reduced seed set. Consistent with flavonols acting as ROS scavengers, are had elevated levels of ROS. The pollen viability, tube growth and integrity defects, and ROS accumulation in are were reversed by genetic complementation. Inhibition of ROS synthesis or scavenging of excess ROS with an exogenous antioxidant treatment also reversed the are phenotypes, indicating that flavonols function by reducing ROS levels. Heat stress resulted in increased ROS in pollen tubes and inhibited tube growth, with more pronounced effects in the are mutant that could be rescued by antioxidant treatment. These results are consistent with increased ROS inhibiting pollen tube growth and with flavonols preventing ROS from reaching damaging levels. These results reveal that flavonol metabolites regulate plant sexual reproduction at both normal and elevated temperatures by maintaining ROS homeostasis.

Background: Abnormal redox equilibrium is a major contributor to tumor malignancy and treatment resistance. Understanding reactive oxygen species (ROS) metabolism is a key to clarify the tumor redox status. However, we have limited methods to evaluate ROS in tumor tissues and little knowledge on ROS metabolism across human cancers.Methods: The Cancer Genome Atlas multi-omics data across 22 cancer types and the Genomics of Drug Sensitivity in Cancer data were analyzed in this study. Cell viability testing and xenograft model were used to validate the role of ROS modulation in regulating treatment efficacy.Results: ROS indexes reflecting ROS metabolic balance in five dimensions were developed and verified. Based on the ROS indexes, we conducted ROS metabolic landscape across 22 cancer types and found that ROS metabolism played various roles in different cancer types. Tumor samples were classified into eight ROS clusters with distinct clinical and multi-omics features, which was independent of their histological origin. We established a ROS-based drug efficacy evaluation network and experimentally validated the predicted effects, suggesting that modulating ROS metabolism improves treatment sensitivity and expands drug application scopes.Conclusion: Our study proposes a new method in evaluating ROS status and offers comprehensive understanding on ROS metabolic equilibrium in human cancers, which provide practical implications for clinical management.

Due to self-incompatibility (SI) prevents self-fertilization, natural or artificial cross-pollination has been conducted in many orchards to stabilize fruit yield. However, it is still puzzled which routes of self S-RNase arresting pollen tube growth. Herein, 17 COBRA genes were isolated from pear genome. Of these genes, the pollen-specifically expressed PbCOB.A.1 and PbCOB.A.2 positively mediates pollen tube growth. The promoters of PbCOB.A.1 and/or PbCOB.A.2 were bound and activated by PbABF.E.2 (an ABRE-binding factor) and PbC2H2.K16.2 (a C2H2-type zinc finger protein). Notably, the expressions of PbCOB.A.1, PbCOB.A.2, and PbC2H2.K16.2 were repressed by self S-RNase, suggesting that self S-RNase reduces the expression of PbCOB.A.1 and PbCOB.A.2 by decreasing the expression of their upstream factors, such as PbC2H2.K16.2, to arrest pollen tube growth. PbCOB.A.1 or PbCOB.A.2 accelerates the growth of pollen tubes treated by self S-RNase, but can hardly affect level of reactive oxygen species and deploymerization of actin cytoskeleton in pollen tubes and cannot physically interact with any reported proteins involved in SI. These results indicate that PbCOB.A.1 and PbCOB.A.2 may not relieve S-RNase toxicity in incompatible pollen tube. The information provides a new route to elucidate the arresting pollen tube growth during SI reaction.Graphical

S-RNase-mediated attenuation of FviPP2Ac-FviRbohH binding promotes ROS accumulation and arrests pollen tube growth in Fragaria viridis.