Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

Structure and function of the neck cell during fertilization in Ginkgo biloba L.

Key message

Neck cells in Ginkgo biloba contribute to archegonial opening through morphological changes and might be involved in the production of fertilization liquid to attract spermatozoids toward archegonia.

Abstract

Neck cells are an essential part of the archegonium in archegoniate gymnosperms, but their function in the sexual reproductive process remains unclear, particularly in zoidogamous gymnosperms. To clarify the structural characteristics of neck cells and their role in fertilization, we examined the neck cells of Ginkgo biloba L. by means of scanning electron microscopy and transmission electron microscopy. The two curved inner neck cells, which are covered imbricately by the two turgid outer neck cells, were pushed to two sides during fertilization, which indicated that morphological changes in these cells contribute to archegonial opening. The neck cells contained many secretory organelles with some material accumulated outside the cell wall, thus the neck cells might be involved in the production of fertilization liquid to attract spermatozoids toward the archegonium. In addition, the surrounding surface cells of the female gametophyte also cooperate to produce the liquid. Taken together, these results indicate that the neck cells provide an effective mechanism by which zoidogamous gymnosperms achieve reproductive success through altering the morphology and cellular physiology of the neck cells.     

OsMLO12, encoding seven transmembrane proteins,is involved with pollen hydration in rice

Key message

MLO mediates pollen hydration.

Abstract

Hydration is the first step in pollen germination. However, the process is not well understood. OsMLO12 is highly expressed in mature pollen grains; plants containing alleles caused by transfer DNA insertions do not produce homozygous progeny. Reciprocal crosses between wild-type and OsMLO12/osmlo12 plants showed that the mutant alleles were not transmitted through the male gametophyte. Microscopic observations revealed that, although mutant grains became mature pollen with three nuclei, they did not germinate in vitro or in vivo due to a failure in hydration. The OsMLO12 protein has seven transmembrane motifs, with an N-terminal extracellular region and a C-terminal cytosolic region. We demonstrated that the C-terminal region mediates a calcium-dependent interaction with calmodulin. Our findings suggest that pollen hydration is regulated by MLO12, possibly through an interaction with calmodulin in the cytosol.     

Embryological features of Tofieldia glutinosa and their bearing on the early diversification of monocotyledonous plants

Background and Aims

Although much is known about the vegetative traits associated with early monocot evolution, less is known about the reproductive features of early monocotyledonous lineages. A study was made of the embryology of Tofieldia glutinosa, a member of an early divergent monocot clade (Tofieldiaceae), and aspects of its development were compared with the development of other early divergent monocots in order to gain insight into defining reproductive features of early monocots.

Methods

Field-collected developing gynoecial tissues of Tofieldia glutinosa were prepared for histological examination. Over 600 ovules were sectioned and studied using brightfield, differential interference contrast, and fluorescence microscopy. High-resolution digital imaging was used to document important stages of megasporogenesis, megagametogenesis and early endosperm development.

Key Results

Development of the female gametophyte in T. glutinosa is of a modified Polygonum-type. At maturity the female gametophyte is seven-celled and 11-nucleate with a standard three-celled egg apparatus, a binucleate central cell (where ultimately, the two polar nuclei will fuse into a diploid secondary nucleus) and three binucleate antipodal cells. The antipodal nuclei persist past fertilization, and the process of double fertilization appears to yield a diploid zygote and triploid primary endosperm cell, as is characteristic of plants with Polygonum-type female gametophytes. Endosperm development is helobial, and free-nuclear growth initially proceeds at equal rates in both the micropylar and chalazal endosperm chambers.

Conclusions

The analysis suggests that the shared common ancestor of monocots possessed persistent and proliferating antipodals similar to those found in T. glutinosa and other early-divergent monocots (e.g. Acorus and members of the Araceae). Helobial endosperm among monocots evolved once in the common ancestor of all monocots excluding Acorus. Thus, the analysis further suggests that helobial endosperm in monocots is homoplasious with those helobial endosperms that are present in water lilies and eudicot angiosperms.Key words: Tofieldia, Tofieldiaceae, Alismatales, monocots, embryology, female gametophyte, antipodals, development, endosperm     

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     

Effects of nitrogen fertilization and root interaction on the agronomic traits of intercropped maize, and the quantity of microorganisms and activity of enzymes in the rhizosphere

Aims

To elucidate the mechanisms of the beneficial effects of below-ground root interactions in maize plus legume intercropping system,

Methods

A pot experiment was conducted using root separation techniques.

Results

It is shown that root interaction and nitrogen fertilization increased chlorophyll content and improved plant characteristics of maize, and the effect of root interaction was significant (p<0.05). Compared to a full root separation treatment, no root separation increased the leaf and grain nitrogen contents, and economic and biological yields per maize plant by 9.3? %, 6.0? %, 14.0? %, and 6.5? %, respectively. Root interaction and nitrogen fertilization enhanced the numbers of bacteria, fungi, actinomycetes and Azotobacteria and the activities of urease, invertase, acid-phosphatase and protease in soil. Correlation analyses revealed that the quantity of microorganisms and the activity of the aforementioned enzymes were all positively or significantly (p<0.05) positively correlated with chlorophyll content, plant height and economic and biological yields per maize plant.

Conclusions

The findings demonstrate that root interactions are important in improving the soil micro-ecological environment, increasing microbial quantity and enzyme activity in soil, and enhancing crop yield.     

Decomposition of lupine seeds and seedlings as N fertilizer in organic vegetable production

Aims

We investigated the response of the perennial grass Molinia caerulea (L.) Moench to combined effects of fertilization (N, P) and drought events. We hypothesized that N fertilization increases, and drought decreases productivity, but that N addition strengthens negative effects caused by drought.

Methods

Within a full-factorial 2-year greenhouse experiment we measured biomass productivity and allocation, tissue nutrient concentrations and nitrogen allocation patterns using ¹⁵N as a tracer.

Results

N fertilization caused a strong increase in productivity, but effects of drought were almost insignificant. However, we found strongly interrelated, non-additive effects of fertilization and drought, expressed by a strong increase of necrotic tissue. Dead aboveground biomass showed the highest values for N and ¹⁵N.

Conclusions

Accelerated productivity of aboveground tissue under N fertilization resulted in increased evaporative demands and thus higher drought susceptibility. In addition ¹⁵N allocation patterns showed that fertilization-drought treatments disenabled plants’ control of their N allocation. Molinia was unable to withdraw leaf N during the dieback of aboveground tissue. Due to the lack of an adaptive strategy to the combined effects of fertilization and drought, increasing summer drought may weaken the competitive performance of species with traits comparable to those of Molinia in N-fertilized environments.     

Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

Background

The WNT4 gene plays an important role in female sex determination and differentiation. It also contributes to maintaining of the ovaries and the survival of follicles.

Methods

We sequenced the coding region and splice sites of WNT4 in 145 Han Chinese women with premature ovarian failure (POF) and 200 healthy controls.

Results

Only one novel variation, in Exon 2 (195C > T), was detected among the women with POF. However, this synonymous variation did not result in a change in amino acid sequence (65 Asp > Asp). No further variants were found in any of the samples.

Conclusion

Although we cannot provide any evidence that it is a possible disease-causing gene, this study is the first attempt to investigate the possible role of WNT4 in Han Chinese women with POF.     

Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions

Background and aims

Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels.

Methods

Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16?days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS.

Results

Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results.

Conclusions

The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.     

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.     

Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube

During angiosperm reproduction, one of the two synergid cells within the female gametophyte undergoes cell death prior to fertilization. The pollen tube enters the female gametophyte by growing into the synergid cell that undergoes cell death and releases its two sperm cells within the degenerating synergid cytoplasm to effect double fertilization. In Arabidopsis (Arabidopsis thaliana) and many other species, synergid cell death is dependent upon pollination. However, the mechanism by which the pollen tube causes synergid cell death is not understood. As a first step toward understanding this mechanism, we defined the temporal relationship between pollen tube arrival at the female gametophyte and synergid cell death in Arabidopsis. Using confocal laser scanning microscopy, light microscopy, transmission electron microscopy, and real-time observation of these two events in vitro, we demonstrate that synergid cell death initiates after the pollen tube arrives at the female gametophyte but before pollen tube discharge. Our results support a model in which a signaling cascade triggered by pollen tube-synergid cell contact induces synergid cell death in Arabidopsis.     

Soil nitrogen balance resulting from N fixation and rhizodeposition by the symbiotic association Anthyllis vulneraria/Mesorhizobium metallidurans grown in highly polluted Zn,Pb and Cd mine tailings

Background and aims

The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca.

Methods

Plants of Festuca and of Anthyllis inoculated with M. metallidurans were grown separately during eight months in pots filled with mine contaminated soil. Estimation of the N fluxes was realized using ¹⁵?N isotopic methods.

Results

Starter N fertilization (28 kg N ha^?1) improved symbiotic N₂ fixation and the growth of both species. Belowground N balance (N rhizodeposition – soil N uptake) of the non-fertilized Anthyllis at maturity was negative (?30.6 kg N ha^?1). However, the amount of N derived from fixation, including above- and belowground parts, was 78.6 kg N ha^?1, demonstrating the ability of this symbiotic association to improve soil N content after senescence.

Conclusions

i) soil N enrichment by the N₂-fixing symbiotic association occurs after plant senescence, when decaying leaves and shoots are incorporated into the soil; ii) application of a starter fertilization is an efficient solution to improve phytostabilisation of highly contaminated sites.     

Seed physiology: From ovule to maturing seed

1. The future of the seed is partly predetermined by events (flower formation, flowering, nutrient flow from mother plant, etc.) preceding fertilization and the formation of the gametophyte.
2. The environmental conditions under which the seed matures affect its final physiological constitution. This faet has mostly been neglected by seed physiologists.
3. It is not known how far the triantic nature of the diaspore (seed coat, pulp, etc., 2n of mother plant, embryon of δ +n of Φ, endosperm 2n of Φ +n of δ) affects seed development and germination.
4. The integuments of the ovules of some species have stomata. It is not known if they are functional in gas exchange or are constitutional non-functioning relics.
5. The causes of the growth-degeneration pattern of the nucellus are unknown.
6. During the development of the megaspore mother cell into the mature embryo sac dramatic cellular ultrastructural changes take place. This probably signifies a “change of guards” during which the gametophyte is freed from part of the controls by the ultrastructural units of the mother plant, preparing the ground after fertilization for a new, genetically independent sporophyte.
7. Upon closer examination, the seemingly simple processes of fertilization and embryogenesis, as described in textbooks, turn out to be very complex and full of problems. Is the role each male nucleus plays preordained or is it left to chance which male nucleus goes where? What causes the degeneration of the synergids and of the vegetative nucleus, and what protects the other two male nuclei from a similar fate? Which ultrastructural organelles are carried by the generative nuclei into their respective receptor cells and what is their role in them? Why do zygotes in some species develop after fertilization immediately into an embryo whereas in other species the zygote remains dormant for some time? What causes the polarity of the egg cell which, after fertilization, divides into one developmentally most active apical cell (giving rise to the embryo) and into another “lazy” basal cell which develops into the suspensor of “unknown function?”
8. In the source-sink relationship between photosynthesizing organs and the maturing seed there is one point at which the photosynthates pass from symplast to apoplast to symplast. The mechanism involved is largely unknown as well as the effect which environmental conditions have on this transport.

     

In Vitro fertilization failure of normozoospermic men: search for a lack of testicular isozyme of angiotensin-converting enzyme

Background

Angiotensin converting enzyme (ACE) is a metalloprotease with two isoforms. The somatic isoform is a key component of the renin-angiotensin system; its main function is to hydrolyse angiotensin I into angiotensin II. The germinal or testicular isoform (tACE) located at the plasma membrane of the spermatozoa, plays a crucial role in the spermatozoa-oocyte interaction during in vivo fertilization, in rodents. Disruption of the tACE in mice has revealed that homozygous male tACE?/? sire few pups despite mating normally. Few spermatozoa from these tACE?/? mice are bound to the zona pellucida (ZP) despite normal semen parameters. Based on these findings in mice models, we hypothesized that some infertile men that have the same phenotype as the tACE?/? mice, ie normal semen parameters and a lack of sperm bind to the ZP in vitro, may have a tACE defect.

Methods

Twenty four men participated to this study. The case subjects (n?=?10) had normal semen parameters according to the WHO guidelines (WHO 1999) but a total in vitro fertilization failure with absence of sperm fixation to the ZP. The control subjects (n?=?14) also had normal semen parameters and a normal fertilization rate ≥65%. We investigated the tACE expression in spermatozoa by Western-Blot and performed a DNA sequencing of the tACE gene.

Results

Three case-subjects and one control-subject had no tACE expression. There were no statistic differences between the two groups. No mutation was detected in the tACE DNA sequence.

Conclusions

Our results didn’t show any involvement of tACE in human fertilization especially in ZP binding.