Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion

In many species, environmental stress reduces plant fertility. In Arabidopsis thaliana, a significant fraction of this reduction in plant fertility results from ovule abortion and embryo senescence. In this species, environmental conditions were identified that induced 94% of the developing ovules to either undergo stress-induced ovule abortion or embryo senescence (Sun et al. Plant Physiol 135:2358–2367, 2004). Following salt stress, physiological and anatomical changes were first detected in the female gametophyte of an aborting ovule. Two to four hours after a period of salt stress that induces most ovules to abort, the mitochondrial membrane potential dissipated. Subsequently, cells in the gametophyte accumulated reactive oxygen species, which are known to be molecules that promote programmed cell death (PCD). Because mitochondria often play an important role in PCD, these organelles were closely examined for changes in structure. Although the anatomy of mitochondria varied, reproducible changes in mitochondria structure were not observed. Nonetheless, other changes in ultrastructure were found. In some aborting gametophytes, concentric rings of endoplasmic reticulum were formed. In a fraction of the aborting ovules, cytoplasmic contents and organelles were invaginated into the vacuole. Even in cryofixed sections, many of these bodies appeared indistinct, which is consistent with the degradation of their contents.     

Ovule abortion in Arabidopsis triggered by stress

Environmental stresses frequently decrease plant fertility. In Arabidopsis, the effect of salt stress on reproduction was examined using plants grown in hydroponic medium. Salt stress inhibited microsporogenesis and stamen filament elongation. Because plants grown in hydroponic media can be rapidly and transiently stressed, the minimum inductive treatment to cause ovule abortion could be determined. Nearly 90% of the ovules aborted when roots were incubated for 12 h in a hydroponic medium supplemented with 200 mm NaCl. The anatomical effects of salt stress on maternal organs were distinct from those in the gametophyte. A fraction of cells in the chalaza and integuments underwent DNA fragmentation and programmed cell death. While three-fourths of the gametophytes aborted prior to fertilization, DNA fragmentation was not detected in these cells. Those gametophytes that survived were fertilized and formed embryos. However, very few of these developing embryos formed seeds; most senesced during seed development. Thus, during seed formation, there were multiple points where stress could prematurely terminate plant reproduction. These decreases in fecundity are discussed with respect to the hypothesis of serial adjustment of maternal investment.     

Postfertilization causes of differential success of pollen donors in Erythronium grandiflorum (Liliaceae): nonrandom ovule abortion

Seed paternity in Erythronium grandiflorum does not fully reflect the proportion of pollen on the stigma. When two types of pollen are simultaneously applied to the stigma, outbred seeds are produced over inbred, and seeds from more distant donors are produced over seeds from donors nearby. I looked for postfertilization causes of these previously reported patterns of differential success of pollen donors. I simultaneously pollinated stigmas with pollen from two donors and observed ovule development through a window sliced in the ovary. Pollen donor pairs were self and cross, donors 1 and 100 m from the recipient, and two donors each 100 m from the recipient. Since one donor was always the alternate homozygote from the recipient at the malate dehydrogenase locus, I could determine the paternity of developing seeds. When it appeared that ovules were aborting, I removed them and determined their paternity using starch gel electrophoresis. Ovules fertilized by self pollen were more likely to abort than ovules fertilized by cross pollen, and ovules fertilized by nearby donors were more likely to abort than ovules fertilized by distant donors. Ovules fertilized by donors 100 m from the recipient were equally likely to abort. There was not a significant relationship between the proportion of ovules fertilized by a pollen donor and the probability of those fertilized ovules developing into seeds. There was no relationship between ovule position within a fruit and ovule abortion. I manipulated available resources by removing leaves and by permitting only one fruit to develop per plant. Decreasing the amount of resources increased the proportion of aborted ovules. Abortion of ovules of lesser quality appears to release resources that can then be used to develop other offspring.     

Pollen-tube growth pattern and chalazogamy in<Emphasis Type="Italic"> Casuarina equisetifolia</Emphasis> (Casuarinaceae)

For a better understanding of pollen-tube guidance in relation to pollen-pistil interaction, we investigated the mode of pollen-tube growth in pistils of Casuarina equisetifolia, a monoecious, wind-pollinated species that undergoes chalazogamous fertilization. The pistil is bicarpellate, but only one of the two carpels develops with two ovules. One of these ovules develops more than four embryo sacs. Pistils usually require more than 1 month to reach maturity after pollen grains have been deposited on the stigmas. During that period, pollen-tube growth proceeds discontinuously in five distinct steps that lead up to fertilization: (1) from the stigma to the upper region of the style, (2) from the upper region of the style to a septum in the ovary, (3) from the septum to the surface of the funiculus, (4) from the funiculus to chalaza in the ovule, and (5) from the chalaza to an egg apparatus. Probably because of competitive interaction between male and female gametophytes (or ovules), one pollen tube is selected from among many during the first step (just before the second step), one ovule from the two during the second and third steps, and one embryo sac from more than four during the fourth and fifth steps. On the basis of our results, erroneous drawings and explanations reported in earlier publications on chalazogamy in Casuarinaceae should be brought into question.     

Influence of drought stress on pistil physiology and reproductive success of two Gossypium hirsutum cultivars differing in drought tolerance

The causes of reproductive failure under drought stress (DS) are poorly understood. We hypothesized that reproductive failure was related to drought-induced changes in pistil biochemistry. To address this hypothesis, a water deficit-induced experiment was conducted with two cotton cultivars (Dexiamian 1, drought tolerant; Yuzaomian 9110, drought sensitive). Results showed that DS decreased the photosynthesis of subtending leaf and downregulated sucrose transporter gene (GhSUT-1) expression in pistil for both cultivars, resulting in lower pistil carbon accumulation which was reflected in the decreased starch accumulation. Lower starch, as potential energy, and adenosine triphosphate (ATP), as direct energy, in droughted pistils suggested less energy for pollen tube entrance into ovules, reducing the fertilized ovule number and fertilization efficiency. Further, although pistil peroxidase activity increased under DS, a higher hydrogen peroxide (H₂O₂) level still was measured in droughted pistils than well-watered pistils, damaging reproductive activities. Moreover, larger decreases in photosynthesis, pistil GhSUT-1 expression, carbon accumulation, starch and ATP contents caused by DS for Yuzaomian 9110 than Dexiamian 1, and different responses of superoxide dismutase and catalase activities, and ascorbic acid and H₂O₂ contents to DS between the two cultivars might be the reasons causing a greater decrease in fertilization efficiency for Yuzaomian 9110 than Dexiamian 1 under DS. Thus, we suggest that decreased ovule fertilization under DS was related to the disorganized carbohydrate metabolism and inefficient antioxidant defense in droughted pistils, and the effects of DS on pistil carbohydrate metabolism and antioxidant defense were more significant for drought-sensitive cultivars than drought-tolerant cultivars.     

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.     

Homogalacturonan deesterification during pollen–ovule interaction in Larix decidua Mill.: an immunocytochemical study

Studies on angiosperm plants have shown that homogalacturonan present in the extracellular matrix of pistils plays an important role in the interaction with the male gametophyte. However, in gymnosperms, knowledge on the participation of HG in the pollen–ovule interaction is limited, and only a few studies on male gametophytes have been reported. Thus, the aim of this study was to determine the distribution of HG in male gametophytes and ovules during their interaction in Larix decidua Mill. The distribution of HG in pollen grains and unpollinated and pollinated ovules was investigated by immunofluorescence techniques using monoclonal antibodies that recognise high methyl-esterified HG (JIM7), low methyl-esterified HG (JIM5) and calcium cross-linked HG (2F4). All studied categories of HG were detected in the ovule. Highly methyl-esterified HG was present in the cell walls of all cells throughout the interaction; however, the distribution of low methyl-esterified and calcium cross-linked HG changed during the course of interaction. Both of these categories of HG appeared only in the apoplast and the extracellular matrix of the ovule tissues, which interact with the male gametophyte. This finding suggests that in L. decidua, low methyl-esterified and calcium cross-linked HG play an important role in pollen–ovule interaction. The last category of HG is most likely involved in adhesion between the pollen and the ovule and might provide an optimal calcium environment for pollen grain germination and pollen tube growth.     

焦核与大核龙眼雌配子体发育、授粉受精及早期胚胎发育的比较研究

利用人工授粉,采用压片法对大核龙眼‘九月乌’和焦核龙眼‘闽焦64-1’、‘闽焦64-2’、‘白核’等的自交与杂交后花粉管的生长特性进行研究,同时应用常规石蜡切片技术对大核与焦核龙眼的雌配子体以及合子胚早期发育进行观察。结果表明,龙眼胚珠在单核胚囊形成前就开始败育,且焦核品种(系)的败育率显著高于大核品种。不同亲本组合的授粉率存在差异,所有授粉组合在授粉36～48 h后均能观察到1个花粉管生长并进入胚囊受精。焦核品种(系)的胚胎在谢花后10 d开始败育,且败育率明显高于大核品种。受精是龙眼子房发育的首要条件,胚珠败育的雌蕊在谢花后10 d不膨大,不能发育形成焦核果实。谢花后10～30 d的早期胚胎败育是形成焦核龙眼的主要原因。焦核品种‘白核’胚乳具有成胚能力。约有24%的‘闽焦64-1’胚珠在胚胎发育过程中,其助细胞、合点端细胞及胚乳发生异常,这可能与早期胚胎败育有关。     

In vitro gynogenesis in cotton (<Emphasis Type="Italic">Gossypium</Emphasis> sp.)

The effects of genotype, pollen or growth regulator-pretreatment of pistils, developmental stage of the ovule (embryo sac) and culture media on induction of gynogenesis, and subsequent plantlet regeneration in vitro were assessed in interspecific Gossypium barbadense × G. hirsutum cotton hybrids. Gynogenesis occurred in all genotypes used when the pistils had been pre-treated with pollen from Hibiscus cannabinus and ovaries were harvested 5 or 10 days after anthesis. The use of culture media, SH and MS, showed no significant differences in responding ovules, embryogenic ovules or embryo germination frequency. Recovered progeny were characterized cytogenetically and microscopically to help documenting their reproductive basis. Root tip chromosome counts of 17 plants established from ovule culture revealed that chromosome numbers ranged from 27 to 44. Although the reproductive mechanisms need to be characterized more extensively by cytological and molecular means, the observations suggest that gynogenesis in cotton involves some unusual reproductive events. Aneuploids could be useful for functional genomic characterization of genome shock, deletion mapping, and germplasm introgression.     

Freezing injury to ovules, pollen and seeds in winter rape

Winter rapeseed (Brassica napus, cv. Samouraï) flowersearly in spring and, under field conditions, short freezingperiods can occur. Unacclimatized plants were freeze-stressed(–3°C for 4 h) at different developmental stages ofbuds, open flowers and seeds. The dissection of pistils from stressed plants showed that freezingresults in shrivelled ovules. We assessed freezing injury onthe basis of per cent of shrivelled ovules: ovule sensitivitybegins early (8 d before anthesis) but increases up to anthesis.Crosspollination of stressed pistils with non-stressed pollenshowed that recording of freeze-injured ovules is a good methodfor early estimation of the effect of stress on seed yields. On the other hand, stress does not reduce the viability of pollen,except when it was applied at the binucleate pollen stage. Useof frozen pollen x nonstressed pistils has little effect onseed yields. Freeze injury on seeds was assessed by seed filling:seeds are very susceptible just after fertilization until 20d after fertilization (DAF). Freezing seems to inhibit seedfilling. A germination test of stressed seeds during their developmentindicated that embryo viability is not affected if the stressoccurs after 35 DAF. As the embryos develop, resistance to stressincreases. Key words: Brassica napus, rapeseed, freeze injury, pollen and ovule, seed filling     

Control of fecundity through abortion in Epilobium montanum L.

Summary Plants of the inbreeding perennial herb Epilobium montanum L. were defoliated at two stages in development and the formation and abortion of flower buds, flowers, copsules and ovules was compared with control plants. A comparison was also made of the effects of self- and cross-pollination on fecundily. Defoliation reduced the number of flower buds formed but its greatest effect was to increase the abortion of flower buds. Defoliation also caused earlier abortions which were generally lower on the racemes of the treated than on the control plants. The abortion of flowers and capsules was much less significant. The number of ovules formed per capsule decreased with height on the raceme and the abortion rate of ovules was increased by defoliation. The pattern of ovule abortion within the capsules differed between cross- and self-pollinated plants but the number of seeds ripened was not affected. The overall survivorship of ovules to mature seeds was 89.3% which compares with the average of 85% found by Wiens for inbreeding annuals. The findings suggest that such high values of ovule survivorship may be characteristics of normally inbreeding species irrespective of whether they are annuals or perennials.     

OVULE ABORTION IN ‘NONPAREIL’ ALMOND (PRUNUS DULCIS [MILL.] D. A. WEBB)

The earliest indication of ovule abortion in almond (Prunus dulcis [Mill.] D. A. Webb ‘Nonpareil‘) is the deposition of callose (as indicated by aniline blue fluorescence) 2 days after pollination which is 2 days before clear histological symptoms of ovule degeneration are evident and 6 days before fertilization of the viable ovule. Callose deposition begins in the chalazal region of the nucellus where the funicular trace enters the ovule and ramifies into the integuments. As ovule abortion progresses, callose deposition in the inner integument extends as a ring around the nucellus. Movement of the fluorescent dye disodium fluorescein (uranin) indicated that translocation from the vascular trace into abortive ovules becomes blocked at the chalazal position. The dye freely penetrates and diffuses into viable ovules but fails to penetrate abortive ovules. Lack of, or delayed and irregular, megagametophyte development was another characteristic of abortive ovules. Biochemical and histochemical analyses of abortive and viable ovules indicated that carbohydrate depletion parallels ovule abortion. These observations lead to the conclusion that ovule abortion is accompanied by blockage in metabolite supply although whether this blockage is the primary cause or a consequence of ovule abortion is uncertain.     

Self pollination and resource availability affect ovule abortion inCassia fasciculata (Caesalpiniaceae)

We examined the effects of pollen source and resource availability on ovule abortion in the annual legumeCassia fasciculata. Pollen source was controlled by hand-pollinating flowers with cross- or self-pollen. Resource availability to developing fruits was controlled by adjusting fruit loads (heavy versus light) on each plant and exposing plants to different photoperiod cycles (16 vs 12 h of light; short days favor fruit growth at the expense of vegetative growth). In mature fruits the proportion of ovules expanding (showing some development over virgin ovules) ranged from 89–95% and did not increase with resource availability, suggesting that unexpanded ovules were either unfertilized or obligately aborted shortly after fertilization. The proportion of expanded ovules maturing in mature fruits was near 97% for both self- and crosspollinations in the treatment with highest resource availability (light load, short days) and lower in the remaining treatments, where self-pollination resulted in up to 9% lower seed maturation than cross-pollination. In the latter three treatments a pollen source effect was dependent upon the maternal plant; in some plants selfing increased abortion and in others it did not. Collectively, the results suggest that (1) both pollen source and resource availability affect ovule abortion, (2) at least some abortion is facultative, and (3) when resources are limited, self-pollination increases abortion in some but not all maternal plants.     

Identification of genes expressed in the Arabidopsis female gametophyte

The angiosperm female gametophyte typically consists of one egg cell, two synergid cells, one central cell, and three antipodal cells. Each of these four cell types has unique structural features and performs unique functions that are essential for the reproductive process. The gene regulatory networks conferring these four phenotypic states are largely uncharacterized. As a first step towards dissecting the gene regulatory networks of the female gametophyte, we have identified a large collection of genes expressed in specific cells of the Arabidopsis thaliana female gametophyte. We identified these genes using a differential expression screen based on reduced expression in determinant infertile1 (dif1) ovules, which lack female gametophytes. We hybridized ovule RNA probes with Affymetrix ATH1 genome arrays and validated the identified genes using real-time RT-PCR. These assays identified 71 genes exhibiting reduced expression in dif1 ovules. We further validated 45 of these genes using promoter::GFP fusions and 43 were expressed in the female gametophyte. In the context of the ovule, 11 genes were expressed exclusively in the antipodal cells, 11 genes were expressed exclusively or predominantly in the central cell, 17 genes were expressed exclusively or predominantly in the synergid cells, one gene was expressed exclusively in the egg cell, and three genes were expressed strongly in multiple cells of the female gametophyte. These genes provide insights into the molecular processes functioning in the female gametophyte and can be used as starting points to dissect the gene regulatory networks functioning during differentiation of the four female gametophyte cell types.