Reactive oxygen species are involved in regulation of pollen wall cytomechanics

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H₂O₂ and the hydroxyl radical (^?OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H₂O₂, ^?OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn‐5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)21H, 23H‐porphin, or a spin‐trap α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone, which eliminates ^?OH. The inhibiting concentrations of exogenous H₂O₂ and ^?OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo‐osmotic shock. ^?OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H₂O₂, as well as partial removal of endogenous ^?OH, over‐tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H₂O₂‐inducible inter‐polymer cross‐linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross‐links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. ^?OH is involved in local loosening of the intine in the germination pore region, while H₂O₂ is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.     

Generation of reactive oxygen species during cryopreservation may improve Lilium × siberia pollen viability

During biotic and abiotic stress in plants, reactive oxygen species (ROS) may play two very different roles: high ROS concentrations can exacerbate damage, whereas low concentrations can activate defense responses. The aim of this study was to investigate the relationship between ROS generation and pollen viability after cryopreservation. ROS generation was detected from ‘Siberia’ (Lilium?×?siberia) pollen using flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. Pollen viability was determined by 2,3,5-triphenyltetrazolium chloride staining. ROS generation was slightly increased by rapid cooling (26.13?±?4.74 vs. 15.80?±?2.30 for fresh pollen) and significantly increased by vitrification (49.74?±?1.43; P?<?0.01). Pollen viabilities after rapid cooling and vitrification were significantly increased (58.88?±?3.76% and 70.35?±?2.90%, respectively) over that of fresh pollen (46.65?±?1.61%; P?<?0.01). No significant differences in ROS generation were associated with cold acclimation at different temperatures before rapid cooling. However, sharp decreases in viability were observed with cold acclimation at 4°C and ?20°C relative to rapid cooling without acclimation (P?<?0.01). We observed nonsignificant decreases in ROS generation among vitrification treatments that omitted different steps and a significant decrease when the unloading step was omitted (P?<?0.05). Pollen viabilities were significantly reduced when the loading or dehydration steps were omitted (P?<?0.01). No significant differences were observed in ROS generation or pollen viability among the treatments when 200 U/ml catalase was added to different solutions used in the vitrification process. Comprehensive analysis of all data indicated a positive correlation between ROS generation and pollen viability (r?=?0.651, P?<?0.001). Therefore, increasing ROS generation during cryopreservation may improve the viability of ‘Siberia’ pollen.     

The effects of time,temperature and plant variety on pollen viability and its implications for gene flow risk

• Pollen viability affects the probability that a pollen grain deposited on a plant's stigma will produce a viable seed. Because a mature seed is needed before a gene flow event can occur, pollen viability will influence the risk of escape for genetically engineered (GE) crops.
• Pollen viability was measured at intervals for up to 2 h following removal of the pollen from the anthers. It was quantified at three temperatures and for different alfalfa varieties, including both conventional and Roundup Ready (RR) varieties. Pollen viability was assessed using in vitro germination.
• Time since removal from the anthers was the most prevalent factor affecting pollen viability in alfalfa. Pollen viability declined with increasing time at all three temperatures and for all varieties tested. Pollen viability was not affected by temperatures ranging between 25 and 37 °C and did not vary among plant varieties, including conventional and RR varieties.
• Bee foraging behaviour suggested pollen viability within the first 10 min following pollen removal from a flower to most affect seed production. Pollen longevity was predicted to have little impact on seed set and gene flow. Linking pollinator behaviour to pollen viability improved our understanding of its impact on gene flow risk.

     

Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature,polyamines and polyamine synthesis inhibitor

Prunus dulcis L. ‘Mamaei’ is grown widely in souhtwest of Iran. It blooms in early spring when temperatures are still low. Based on our knowledge there are no reports in the literature regarding pollen behavior of this cultivar under specified condition. Thus, the possible factors for low germination percentage in this cultivar have not been reported. The effect of three different temperatures (10, 25, or 35 °C), polyamines (putrescine, spermidine, and spermine) and polyamine synthesis inhibitor, methylglyoxals-bis (guanyl-hydrazone) (MGBG) on in vitro pollen germination and pollen tube growth were investigated in P. dulcis L. ‘Mamaei’. All temperatures and chemicals significantly affected both pollen germination percentage and pollen tube growth. In general, different polyamines stimulated the pollen germination percentage compared to the control at all temperatures, but increasing the temperature, particularly to 35 °C, had demonstrated inhibitory effects on pollen germination. At a concentration of 0.05 mM putrescine and spermidine and 0.005 and 0.025 mM spermine revealed longer pollen tube growth than that of the control at 10 °C, while higher concentrations tended to inhibit pollen tube growth. At 25 °C, most of the treatments had an inhibitory effect on pollen tube growth except for 0.25 mM putrescine and 0.005 mM spermine, which slightly stimulated pollen tube growth. Pollen germination and pollen tube growth were inhibited by MGBG at all temperatures and in all concentrations.     

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model – protoplasts obtained from lily pollen grains at the early germination stage – to reveal the effect of H₂O₂ on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS‐sensitive currents on the pollen grain plasma membrane: the hyperpolarisation‐activated calcium current, which is strongly enhanced by H₂O₂, and the outward potassium current, which is modestly enhanced by H₂O₂. We used low concentrations of H₂O₂ that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.     

Using genetic evaluation to guide conservation of remnant Juniperus communis (Cupressaceae) populations

• Many critically endangered plant species exist in small, genetically depauperate or inbred populations, making assisted gene flow interventions necessary for long‐term population viability. However, before such interventions are implemented, conservation practitioners must consider the genetic and demographic status of extant populations, which are strongly affected by species’ life‐history traits. In northwestern Europe, Juniperus communis, a dioecious, wind‐pollinated and bird‐dispersed gymnosperm, has been declining for the past century and largely exists in small, isolated and senescent populations.
• To provide useful recommendations for a recovery plan involving translocation of plants, we investigated genetic diversity and structure of populations in Belgium using four microsatellite and five plastid single‐nucleotide polymorphism (SNP) markers.
• We detected no clonality in the populations, suggesting predominantly sexual reproduction. Populations exhibited high genetic diversity (H_e = 0.367–0.563) and low to moderate genetic differentiation (F_ST ≤ 0.133), with no clear geographic structure. Highly positive inbreeding coefficients (F_IS = 0.221–0.507) were explained by null alleles, population substructuring and biparental inbreeding. No isolation by distance was observed among distant populations, but isolation at close geographic proximity was found. Patterns were consistent with high historical gene flow through pollen and seed dispersal at both short and long distances. We also tested four pre‐germination treatments among populations to improve germination rates; however, germination rates remained low and only cold‐stratification treatments induced germination in some populations.
• To bolster population regeneration, introductions of cuttings from several source populations are recommended, in combination with in situ management practices that improve seedling survival and with ex situ propagation.

     

Fruit misshapen in strawberry cultivars (Fragaria×ananassa) is related to achenes functionality

Fruit deformation is a phenomenon commonly observed in commercial strawberry cultivars (Fragaria×ananassa) with a negative impact on the economic benefit of crop production. To better understand this physiopathy, we evaluated fruit size and the relative amount of ‘small’ (<0.4 mm) and ‘big’ achenes (>0.6 mm) in misshapen (MF) and well‐formed (WF) fruits from ‘Camarosa’, ‘Ventana’ and ‘Medina’ strawberry cultivars growing in the field. In ‘Camarosa’, size‐dependent achenes functionality was assessed by analysing achenes germinability and differences in time to ripening between MF and WF. We found that the occurrence of fruit deformation was not only strongly dependent on the cultivar (i.e. genetic factor) but also was promoted by low temperatures (i.e. environmental factor). Regardless of the cultivar, MF showed higher percentage of ‘small’ non‐functional achenes than WF, indicating a failure in achene development, which could be related to the functional integrity of reproductive structures. To test this hypothesis, we performed an experiment by pollinating strawberry flowers with pollen developed under low and mild temperatures. Low temperatures reduced pollen viability, and flowers pollinated with low quality pollen led to a higher amount of MF. However, fruit deformation was not completely explained by differences in pollen quality, neither by differences in pistil maturity and receptivity nor ovule viability, suggesting that non‐functional achenes might result from a disruption in postfertilisation processes such as embryo abortion as a consequence of low temperatures.     

Brassinosteroid‐mediated reactive oxygen species are essential for tapetum degradation and pollen fertility in tomato

Phytohormone brassinosteroids (BRs) are essential for plant growth and development, but the mechanisms of BR‐mediated pollen development remain largely unknown. In this study, we show that pollen viability, pollen germination and seed number decreased in the BR‐deficient mutant d^{^im}, which has a lesion in the BR biosynthetic gene DWARF (DWF), and in the bzr1 mutant, which is deficient in BR signaling regulator BRASSINAZOLE RESISTANT 1 (BZR1), compared with those in wild‐type plants, whereas plants overexpressing DWF or BZR1 exhibited the opposite effects. Loss or gain of function in the DWF or BZR1 genes altered the timing of reactive oxygen species (ROS) production and programmed cell death (PCD) in tapetal cells, resulting in delayed or premature tapetal degeneration, respectively. Further analysis revealed that BZR1 could directly bind to the promoter of RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1), and that RBOH1‐mediated ROS promote pollen and seed development by triggering PCD and tapetal cell degradation. In contrast, the suppression of RBOH1 compromised BR signaling‐mediated ROS production and pollen development. These findings provide strong evidence that BZR1‐dependent ROS production plays a critical role in the BR‐mediated regulation of tapetal cell degeneration and pollen development in Solanum lycopersicum (tomato) plants.     

Impact of temperature on olive (Olea europaea L.) pollen performance in relation to relative humidity and genotype

Olive varieties ‘Koroneiki’, ‘Kalamata’, ‘Mastoidis’ and ‘Amigdalolia’ were employed in two experiments for 3 years to assess the effect of temperature on olive pollen germination and tube growth in relation to relative humidity and genotype. Pollen samples were subjected to pre-incubation at 10, 20, 30 or 40 °C in combination with decreased air relative humidity – 80, 40, 30 or 20%, respectively – for 24 h to simulate temperature stress that is observed during pollen dispersal; and subsequently in vitro cultured. In the second experiment, pollen was exposed at 15, 20, 25 and 30 °C for 24 h in vitro to evaluate pollen response in conditions of water and nutrients availability and to determine the optimum pollen germination and tube growth temperatures for each cultivar. The highest pre-incubation temperature treatment (40 °C) prevented pollen germination in ‘Koroneiki’ and ‘Mastoidis’, with the less affected varieties (‘Amigdalolia’ and ‘Kalamata’) having average germination percentages of only 7.6 and 2%, respectively. Pre-incubation at 30 °C had a negative impact on pollen germination in ‘Koroneiki’ (?65%), ‘Kalamata’ (?20%) and ‘Amigdalolia’ (?72%) compared to the control (20 °C). Pollen pre-incubation at 40 °C decreased significantly the pollen tube length in ‘Kalamata’ (?50%) and ‘Amigdalolia’ (?52%). In the second experiment, in vitro pollen germination increased after incubation at 25 °C for ‘Koroneiki’ (+6%), ‘Mastoidis’ (+52%), ‘Kalamata’ (+10%) and ‘Amigdalolia’ (+10%) compared to the control (20 °C). At 30 °C germination percentages for ‘Mastoidis’, ‘Kalamata’ and ‘Amigdalolia’ were 8, 6 and 14% higher, respectively, compared to the control (20 °C). Pollen tube length also increased with incubation temperature for all of the studied cultivars. Based on the cumulative stress response index (CSRI) that was calculated for high temperature stress the varieties were classified: ‘Mastoidis’ and ‘Kalamata’ as tolerant and ‘Koroneiki’ and ‘Amigdalolia’ as intermediate at 30 °C while all studied cultivars were sensitive at 40 °C. The observed strong genotype-differentiated response in high and low temperature stress could be exploited by plant breeders towards producing new tolerant olive varieties.     

ROS-induced oxidative stress is closely related to pollen deterioration following cryopreservation

Oxidative stress induced by excessive accumulation of reactive oxidative species (ROS) during cryopreservation is thought to be one factor contributing to cryodamage of biological materials. To explore the role of oxidative stress in the cryopreservation of plant pollen, germination, ROS, and malondialdehyde (MDA) levels of pollen from 20 ornamental plant species were compared before and after cryopreservation. The results showed that the germinability of cryopreserved pollen from 13 out of the 20 species was not significantly different from that of fresh pollen (group 1), only one increased significantly, while the other six declined significantly (group 2). The MDA content in cryopreserved pollen from nine species in group 1 showed no significant difference from that of fresh pollen, while four species in group 2 rose significantly. This suggested that pollen viability and MDA levels were negatively correlated. ROS generation in cryopreserved pollen from nine species in group 1 was unchanged compared to fresh pollen, while five species in group 2 increased significantly. This suggested that pollen viability was negatively correlated with ROS generation. Additionally, both ROS and MDA levels in pollen from four species in group 2 increased significantly. In conclusion, pollen from most species possesses some cryostorage tolerance, but some species are severely damaged by cryostorage. Oxidative stress induced by the cryostorage in liquid nitrogen (LN) may be a key factor for the decreased viability in pollen following cryopreservation.     

Patterns and sources of variation in pollen deposition and pollen tube formation in flowers of the endemic monoecious shrub Cnidoscolus souzae (Euphorbiaceae)

Pollen deposition and pollen tube formation are key components of angiosperm reproduction but intraspecific variation in these has rarely been quantified. Documenting and partitioning (populations, plants and flowers) natural variation in these two aspects of plant reproduction can help uncover spatial mosaics of reproductive success and underlying causes. In this study, we assess variation in pollen deposition and pollen tube formation for the endemic monoecious shrub Cnidoscolus souzae throughout its distribution range in Mexico, and determine how this variation is structured among populations, plants and flowers. We also infer the relative importance of pollen quantity and quality in determining pollination success in this species. While we found no evidence suggesting that pollen receipt limits C. souzae reproduction across 19 populations, we did find extensive variation in pollen load size and pollen tube number per flower. Total variation in pollen receipt and pollen tube number was mostly explained by intra‐individual and among‐population variance. Furthermore, pollen load size had a stronger effect on the number of pollen tubes at the base of the style than pollen germination rate, suggesting that pollen quantity may be more important than quality for pollen tube success in C. souzae. Our results suggest that both small within‐plant flower differences and broad‐scale differences in community attributes can play an important role in determining pollination success. We emphasise the need to evaluate patterns and sources of variation in pollen deposition and pollen tube formation as a first step in understanding the causes of variation in pollination success over broad spatial scales.     

Pollenkitt of some monocotyledons: lipid composition and implications for pollen germination

• The composition of pollenkitt and its role in the progamic phase of reproduction are poorly understood. With the aim of extending knowledge on these topics, we chose to study two monocotyledons rich in pollenkitt, with bi‐celled and long‐lived pollen and dry‐type stigma: Crocus vernus Hill subsp. vernus and Narcissus poeticus L.
• Fatty acids of pollenkitt were assayed with gas chromatography. Germination tests were performed in vivo by pollinating the stigmas with a beard hair under a stereomicroscope, and in vitro in liquid culture medium using pollen, either treated or not, with carbon disulphide to remove pollenkitt. The pollen tube percentages were evaluated using fluorescence microscopy techniques. Scanning electron microscopy was used to examine pollen and to follow the early post‐pollination stages.
• Pollenkitt forms bridges between pollen grains but not between grains and stigma papillae. It consists of a mixture of 25 fatty acids, most with long and unsaturated chains, among which are some omega acids. The same acids with different percentages persist on the peritapetal membrane. After its removal, the pollen loses adhesiveness and dries quickly, but retains full capacity for germination on the papillae and can even trigger germination in contiguous pollen grains that do not touch the papillae.
• The results, while confirming the key role of pollenkitt in protecting pollen and favouring pollination, suggest secondary roles in the progamic phase, and highlight the interactive ability of the pollen regardless of lipid shell. The predominance of fatty acids with 18:3 and 16:0, as already noted in Brassica napus pollenkitt, suggests their hierarchy independent of plant species.

     

Screening of natural products in the laboratory and the field for control of pollen beetles

Pollen beetles, Meligethes spp. (Coleoptera: Nitidulidae), are among the most damaging pests of oilseed rape (Brassica napus L.). Increasing populations of pyrethroid‐resistant pollen beetles coupled with the prohibition of synthetic pest control products in organic farming means that other direct control methods are needed. A laboratory study was therefore conducted to evaluate the effect of natural products, combinations of natural products and additives as well as natural and synthetic insecticides on mortality of pollen beetles. In addition, field trials under both integrated and organic production were conducted to evaluate the efficacy of six natural products, nine combinations of natural products, two synthetic insecticides (integrated‐production trials only), two natural insecticides and two additives to reduce pollen beetles. The laboratory trial, using both a curative and preventive approach, showed that two of the eight natural products (lavender oil and stone meal) caused a mortality of over 98% within the first day of application. The other natural products were only partially effective compared with the untreated control. In the field trials, the natural products reduced the number of pollen beetles 1 day after application compared with the untreated control in both trial years and production systems. Promising substances were stone meal, Silico‐Sec and liquid manure. Nevertheless, the efficacy was not consistent, and no effect on oilseed rape yield was observed. In spite of this, these products have the potential to control pollen beetles under field conditions with comparable effects to synthetic insecticides. Further research should therefore focus on timing and frequency of applications as well as on the formulation of the natural products to increase the persistency of the products under field conditions.     

Pollen competition between two sympatric Orchis species (Orchidaceae): the overtaking of conspecific of heterospecific pollen as a reproductive barrier

The frequency of hybrid formation in angiosperms depends on how and when heterospecific pollen is transferred to the stigma, and on the success of that heterospecific pollen at fertilising ovules. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Mediterranean deceptive orchid. Plants of Orchis italica and O. anthropophora were pollinated with conspecific and heterospecific pollen (first conspecific pollen then heterospecific pollen and vice versa) and molecular analysis was used to check the paternity of the seeds produced. In this pair of Mediterranean orchids, competition between conspecific and heterospecific pollen functions as a post‐pollination pre‐zygotic barrier limiting the frequency of the formation of hybrids in nature. Flowers pollinated with heterospecific pollen can remain receptive for the arrival of conspecific pollen for a long time. There is always an advantage of conspecific pollen for fruit formation, whether it comes before or after heterospecific pollen, because it overtakes the heterospecific pollen. The conspecific pollen advantage exhibited in O. italica and O. anthropophora is likely to result from the reduced germination of heterospecific pollen or retarded growth of heterospecific pollen tubes in the stigma and ovary. Overall, the results indicate that our hybrid zone represents a phenomenon of little evolutionary consequence, and the conspecific pollen advantage maintains the genetic integrity of the parental species.     

The exogenous application of AtPGLR,an endo‐polygalacturonase,triggers pollen tube burst and repair

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine‐tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark‐grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo‐PG that preferentially releases non‐methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non‐methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β‐glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.     

Pollen viability and pollen vigor

Summary Investigations were carried out to correlate pollen viability, assessed on the basis of a fluorochromatic reaction (FCR) test, with pollen vigor, assessed on the basis of the time taken for in vitro germination in pollen grains subjected to high humidity (>95% RH) and temperature (38 °C) or storage stress of Nicotiana tabacum, Agave sp., Tradescantia virginiana, and Iris sp. Both high RH and temperature, as well as storage stresses, affected pollen vigor before affecting pollen viability. The results are discussed in the light of available data on the viability and vigor of stressed pollen and of aged seeds. The need for consideration of pollen vigor, particularly in stored pollen, the inadequacy of the methods presently used, and some of the methods suitable to assess pollen vigor are elaborated.     

Heteranthery as a solution to the demand for pollen as food and for pollination – Legitimate flower visitors reject flowers without feeding anthers

• Heteranthery, the presence of feeding and pollinating anthers in the same flower, seems to mediate the evolutionary dilemma for plants to protect their gametes and yet provide food for pollinators. This study aims to elucidate the role of heteranthery in the buzz‐pollinated Senna reniformis.
• The fecundity of pollen from long‐, medium‐ and short‐sized anthers was determined by hand cross‐pollination experiments, and the quantity, size, ornamentation and viability of pollen of different anthers were compared. Rates of flower rejection by bees were measured in anther removal experiments to assess the preferences of flower visitors for feeding or pollinating anthers.
• Large bees, which were the effective pollinators of self‐incompatible S. reniformis, avoided flowers without short feeding anthers, but not those without medium or long anthers. Illegitimate small and medium‐sized bees were unresponsive to anther exclusion experiments. Long anthers deposited pollen on the back and short anthers on the venter of large bees. Pollen from long anthers had higher in vitro viability and higher fruit and seed set after cross‐pollination than pollen from other sized anthers.
• Short anthers produce feeding pollen to effective pollinators and long anthers are related to pollination of S. reniformis. Bee behaviour and size was found to directly influence the role of anthers in the ‘division of labour’. Only large bee pollinators that carry the pollinating pollen from long anthers in ‘safe sites’ associated short anthers with the presence of food. In the absence of these larger bee pollinators, the role of heteranthery in S. reniformis would be strongly compromised and its function would be lost.

     

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S‐RNase‐independent unilateral incompatibility

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self‐incompatible (SI) and the male parent self‐compatible (SC) (the ‘SI × SC rule’). Additional, as yet unknown, UI mechanisms are independent of self‐incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen‐side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S‐RNase, the pistil determinant of S‐specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18‐fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F₁ hybrid, leading to transmission ratio distortion in the F₂ progeny. CRISPR/Cas9‐generated knockout mutants (fps2) in S. pennelliiLA0716 are self‐sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F₂ progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2‐based pollen selectivity does not involve S‐RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.