Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

POLLINATION,FERTILIZATION AND OVULE ABORTION IN OXALIS MAGNIFICA

Pollination, fertilization and ovule abortion were studied in Oxalis magnifica (Rose) Knuth, a strongly self-incompatible herb that regularly matures only a fraction of its ovules. Examination of cleared ovules indicated that among 9 individuals the average number of ovules fertilized ranged from 48–92%. The remaining ovules either failed to produce female gametophytes, or more commonly contained unfertilized female gametophytes, despite large numbers of compatible pollen grains that were placed on stigmas. Abortion of fertlized ovules could be detected first by the flattened and enlarged appearance of the endosperm nuclei, followed by visible deterioration of the embryo. Among individuals the rate of embryo abortion varied from 3.4–47.9%. At lower levels of pollination an almost one-to-one relationship existed between the number of pollen grains placed on stigmas and the number of seeds matured in the capsule. No threshold number of pollen grains necessary for successful pollen tube growth and fertilization could be demonstrated. Reduction in seed number through embryo abortion provides an opportunity for selection among developing seeds. The potential for this form of selection varies widely among individuals of Oxalis magnifica, which showed a 14-fold variation in the average percentage of aborted ovules.     

RELATIVE SUCCESS OF SELF AND OUTCROSS POLLEN COMPARING MIXED- AND SINGLE-DONOR POLLINATIONS IN AQUILEGIA CAERULEA

Flowers frequently receive both self (S) and outcross (OC) pollen, but S pollen often sires proportionally fewer seeds. Failure of S pollen can reflect evolved mechanisms that promote outcrossing and/or inbreeding depression expressed during seed development. The relative importance of these two processes was investigated in Aquilegia caerulea, a self-compatible perennial herb. In the field I performed single-donor (S or OC) and mixed-donor (S plus OC) pollinations to compare the relative success of both pollen types at various stages from pollen germination to seed maturity. Single-donor S pollinations produced significantly fewer and lighter seeds (x decrease = 12% and 3%, respectively) than OC pollinations. Abortion rates differed by an average of 38% whereas fertilization rates differed by only 5%, indicating that most differences in seed number arose postzygotically. This suggests that inbreeding depression was responsible for most failure of S pollen. One prezygotic effect measured was that 10% fewer S than OC pollen tubes reached ovaries after 42 hr, suggesting S pollen might fertilize proportionately fewer ovules after mixed pollination. Using allozyme markers, I found mixed-donor pollinations produced significantly more and heavier outcrossed than selfed seeds. However, the proportion of selfed seed, fertilized ovules, and aborted seeds for mixed-donor fruits were each predictable from pollen performance in single-donor fruits, suggesting that differential paternity is best explained by inbreeding depression during seed development. Even given these similarities between mixed- and single-donor fruits in the relative performance of S and OC pollen, both individual seed weight and seed set were significantly higher in multiply-sired fruits.     

Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3‐BINDING F‐BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous reports have shown that the ebf1 ebf2 double homozygous mutant cannot be identified. In this study, the genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of the pEIN3::EIN3‐GFP transgenic lines showed that EIN3 signal was over‐accumulated at the micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in synergid cell led to the defect of pollen tube guidance. These results suggested that the over‐accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the expression of a sugar transporter, SENESCENCE‐ASSOCIATED GENE29 (SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube attraction, suggesting that SAG29 might play a role in ethylene signaling to repel pollen tube entry. Taken together, our study reveals that strict control of ethylene signaling is critical for the synergid cell function during plant reproduction.     

Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte clade

The fertilization process in Gnetum is critical to our understanding of the evolution of sexual reproduction within the Gnetales, a monophyletic group of nonfiowering seed plants that are the closest living relatives to flowering plants. Although much is known about the fertilization process in Ephedra, which is basal within the Gnetales, little is known about sexual reproduction in the derived sister groups Gnetum and Welwitschia. Ovules of Gnetum gnemon were collected at various stages after hand pollination and processed for light, fluorescence, and electron microscopy. Approximately 5 d after pollination, pollen tubes reach sexually mature female gametophytes, which are coenocytic. At that time, a binucleate sperm cell is found within each pollen tube. Within 7 d of pollination, double fertilization events occur when each of two sperm nuclei released from a pollen tube fuses with a separate, undifferentiated female nucleus within the free nuclear female gametophyte, which lacks differentiated egg cells. The products of double fertilization are two viable zygotes; endosperm is not formed. The lack of differentiated egg cells in Gnetum gnemon is unparalleled among land plants and the documentation of a regularly occurring process of double fertilization is congruent with the hypothesis that a rudimentary process of double fertilization evolved in a common ancestor of angiosperms and Gnetales.     

Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2β are involved in pollen development,pollen tube guidance and embryogenesis

Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2β) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2α and AtIPK2β revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2β act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock‐out mutants of atipk2α and atipk2β were indistinguishable from the wild type, whereas the atipk2α atipk2β double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2α and AtIPK2β resulted in severely reduced transmission of male gametophyte as a result of abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of the atipk2α atipk2β double mutant was also aborted. Expressing either catalytically inactive or substrate specificity‐altered variants of AtIPK2β could not rescue the male gametophyte and embryogenesis defects of the atipk2α atipk2β double mutant, implying that the kinase activity of AtIPK2 is required for pollen development, pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.     

Maternal Control of Male-Gamete Delivery in Arabidopsis Involves a Putative GPI-Anchored Protein Encoded by the LORELEI Gene

In Angiosperms, the male gametes are delivered to the female gametes through the maternal reproductive tissue by the pollen tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain poorly understood. Here, we describe lorelei, an Arabidopsis thaliana mutant impaired in sperm cell release, reminiscent of the feronia/sirène mutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signaling mechanisms at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of these proteins within a similar biological context.     

Mode of pollen tube growth in pistils of Ticodendron incognitum (Ticodendraceae,Fagales) and the evolution of chalazogamy

Ticodendron incognitum is the sole species of the Ticodendraceae, which was established as a new family in the Fagales less than 20 years ago. Considering the diverse modes of pollen tube growth observed in other Fagales, we investigated the growth of pollen tubes in the pistil of Ticodendron. At the time of pollination, T. incognitum had four immature ovules in a bilocular ovary, thus exhibiting delayed fertilization, as in other Fagales. During the period when fertilization was delayed, pollen tube growth in the pistil was intermittent, consisting of five steps associated with development of the ovules and embryo sacs. Four cessation sites occurred: in the style, in the tissue of the upper part of the ovary, inside and outside of the funicle and at the chalaza. A single pollen tube eventually reaches a mature embryo sac through the chalaza in one of the four ovules. While both delayed fertilization and intermittent pollen tube growth play a role in male and female gametophyte selection, as in other Fagales, the five‐step process of pollen tube growth through the chalaza (i.e. chalazogamy) is characteristic of lineages of the Casuarinaceae, Ticodendraceae and Betulaceae (the latter with the loss of one step). © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157 , 621–631.     

Differential self‐fertilization rates in response to variation in floral traits within inflorescences of Aquilegia buergeriana var. oxysepala (Ranunculaceae)

To assess variation in the proportion of self‐fertilized seeds among flowers within inflorescences and the relationship between floral traits and the rate of self‐fertilization, the proportion of self‐fertilized seeds among individual flowers was estimated using ten microsatellite markers in self‐compatible plants of Aquilegia buergeriana var. oxysepala. Within‐inflorescence variation in floral traits, such as the duration of the male and female phases, flower size, herkogamy and the number of pollen grains and ovules in two natural populations, were investigated. The first flower in an inflorescence produced more seeds and a higher proportion of self‐fertilized seeds than the second flower. The higher proportion of self‐fertilized seeds in the first flowers was accompanied by a higher number of pollen grains and ovules in the bud stage and the female phase. These results indicate that the high proportion of self‐fertilized seeds in the first flowers in an inflorescence may be due to the high number of remaining pollen grains in the female phase. This suggests that variation in floral traits within inflorescences affects seed quality and quantity among flowers within inflorescences.     

花粉管生长和极性引导的孢子体和配子体控制研究进展

双受精是被子植物特有的生殖方式,精细胞只有通过花粉管穿过花柱才能到达子房、胚珠受精。花粉管在母本组织中的生长和引导包括孢子体控制(sporophytic control)和配子体控制(gametophytic control)两个连续的过程,现已克隆出不同阶段花粉管生长和引导的基因,通过分析其表达调控揭示出花粉管生长和引导的分子机制。该文就近年来国内外有关花粉管生长和极性引导的调控机制研究进展进行综述,并对禾本科(Poaceae)和十字花科(Brassicaceae)植物花粉管引导的异同点进行了比较分析。     

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana

In flowering plants, cell–cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane‐bound proteins associated with the endomembrane system and the cell surface. While many synergid‐expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane‐bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co‐localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI‐anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane‐associated trafficking within a haploid cell actively involved in polar transport.     

Development and function of the synergid cell

The synergid cells are located in the female gametophyte and are essential for angiosperm reproduction. During the fertilization process, a pollen tube grows into one of the synergid cells, ceases growth, ruptures, and releases its two sperm cells into this cell. The synergid cells produce an attractant that guides the pollen tube to the female gametophyte and likely contain factors that control arrest of pollen tube growth, pollen tube discharge, and gamete fusion. The synergid cells contain an elaborated cell wall at their micropylar poles, the filiform apparatus that likely plays a role in pollen tube guidance and pollen tube reception. Recent genetic, molecular, and physiological studies in Arabidopsis, maize, and Torenia have provided insights into synergid cell development and the control of pollen tube growth by the synergid cell.     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.     

DUF784基因在花粉管导向中的功能分析

花粉管导向是高等植物完成双受精过程的重要环节,是受多重信号调控的复杂过程.最近的研究揭示,配子体阶段花粉管导向的诱导信号分子是一类具多态性的富含半胱氨酸的防卫素类似蛋白,如来自玉米的ZmEA1和蓝猪耳草中的LUREs在吸引花粉管进入珠孔起重要作用.但是拟南芥及其它植物中此类信号未知.转录组学分析表明,一组DUF784基因可能在花粉管导向中起到重要作用.通过RNAi技术降低一组DUF784基因的表达,分析发现在RNAi转基因植株中,出现胚珠败育现象,花粉管导向出现异常,一部分花粉管不能进入珠孔.另外,用MYB98基因的启动子携带1个DUF基因的编码区,然后转化ccg突变体,发现ccg转基因株系中胚胎败育率下降,即DUF基因能部分互补ccg突变体的表型;从这两方面证实了DUF784基因在花粉管定向导入过程中的作用.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

Arabidopsis COBRA‐LIKE 10, a GPI‐anchored protein,mediates directional growth of pollen tubes

Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)‐anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C‐terminal hydrophobic residues. GPI‐anchored proteins are widespread cell sensors in mammals, especially during egg‐sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell‐cell communications in plants.     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

The female gametophyte and the endosperm control cell proliferation and differentiation of the seed coat in Arabidopsis

Double fertilization of the female gametophyte produces the endosperm and the embryo enclosed in the maternal seed coat. Proper seed communication necessitates exchanges of signals between the zygotic and maternal components of the seed. However, the nature of these interactions remains largely unknown. We show that double fertilization of the Arabidopsis thaliana female gametophyte rapidly triggers sustained cell proliferation in the seed coat. Cell proliferation and differentiation of the seed coat occur in autonomous seeds produced in the absence of fertilization of the multicopy suppressor of ira1 (msi1) mutant. As msi1 autonomous seeds mostly contain autonomous endosperm, our results indicate that the developing endosperm is sufficient to enhance cell proliferation and differentiation in the seed coat. We analyze the effect of autonomous proliferation in the retinoblastoma-related1 (rbr1) female gametophyte on seed coat development. In contrast with msi1, supernumerary nuclei in rbr1 female gametophytes originate mainly from the endosperm precursor lineage but do not express an endosperm fate marker. In addition, defects of the rbr1 female gametophyte also reduce cell proliferation in the ovule integuments before fertilization and prevent further differentiation of the seed coat. Our data suggest that coordinated development of the seed components relies on interactions before fertilization between the female gametophyte and the surrounding maternal ovule integuments and after fertilization between the endosperm and the seed coat.     

Differential ovule development following self- and cross-pollination: the basis of self-sterility in Narcissus triandrus (Amaryllidaceae)

Self-pollination results in significantly lower seed set than cross-pollination in tristylous Narcissus triandrus. We investigated structural and functional aspects of pollen–pistil interactions and ovule–seed development following cross- and self-pollination to assess the timing and mechanism of self-sterility. Ovule development within an ovary was asynchronous at anthesis. There were no significant differences in pollen tube behavior following cross- vs. self-pollination during the first 6 d of growth, regardless of style morph type. Double fertilization was significantly higher following cross- vs. self-pollination. Aborted embryo development was not detected following either pollination type up to seed maturity. Prior to pollen tube entry, a significantly greater number of ovules ceased to develop following self- vs. cross-pollination. These results indicate that self-sterility in N. triandrus operates prezygotically but does not involve differential pollen tube growth typical of many self-incompatibility (SI) systems. Instead, low seed set following self-pollination is caused by a reduction in ovule availability resulting from embryo sac degeneration. We hypothesize that this is due to the absence of a required stimulus for normal ovule development. If this is correct, current concepts of SI may need to be broadened to include a wider range of pollen–pistil interactions.     

Inbreeding depression affects pollen performance in Cucurbita texana

Most studies of the effects of inbreeding focus on vegetative vigor and reproductive output through the female (fruit and seed) function. This study not only examines the effects of inbreeding on the female function but it also examines the effects of inbreeding on pollen performance both in vitro and in vivo. This study used Cucurbita texana, a wild gourd, and was performed under field conditions. In vivo performance was assessed by placing equal amounts of pollen from either the inbred or outcrossed plants onto a stigma together with pollen from a tester line. As with other studies, we found that outcrossed plants had greater reproductive output (male flowers and fruits) than plants produced from self pollinations. Unlike most studies of inbreeding depression, which mostly ignore the male function of plants, we also found that the pollen produced by outcrossed plants had faster pollen tube growth in vitro than the pollen produced on selfed plants. Moreover, pollen from selfed plants sired significantly fewer seeds than pollen from outcrossed plants under conditions of pollen competition (i.e. the number of pollen grains deposited onto the stigmas was larger than the number of available ovules). These findings indicate that inbreeding affects the performance of the resulting sporophytic generation and the microgametophytes they produce.