Thomas Dresselhaus

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1

In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K(+)-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K(+) channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K(+) influx. We further suggest that K(+) influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.

Male-Female Crosstalk during Pollen Germination, Tube Growth and Guidance, and Double Fertilization

Sperm cells of flowering plants are non-motile and thus require transportation to the egg apparatus via the pollen tube to execute double fertilization. During its journey, the pollen tube interacts with various sporophytic cell types that support its growth and guide it towards the surface of the ovule. The final steps of tube guidance and sperm delivery are controlled by the cells of the female gametophyte. During fertilization, cell-cell communication events take place to achieve and maximize reproductive success. Additional layers of crosstalk exist, including self-recognition and specialized processes to prevent self-fertilization and consequent inbreeding. In this review, we focus on intercellular communication between the pollen grain/pollen tube including the sperm cells with the various sporophytic maternal tissues and the cells of the female gametophyte. Polymorphic-secreted peptides and small proteins, especially those belonging to various subclasses of small cysteine-rich proteins (CRPs), reactive oxygen species (ROS)/NO signaling, and the second messenger Ca(2+), play center stage in most of these processes.

Egg Cell–Secreted EC1 Triggers Sperm Cell Activation During Double Fertilization

Double Delivery During Plant Fertilization Double fertilization is a defining feature of flowering plants and involves two nonmotile male gametes (sperm cells) and two female gametes (egg cell and central cell). Both fertilization events are necessary for reproductive success. It is not clear how flowering plants ensure the reliable and on-time fusion of the two pairs of gametes, while preventing polyspermy. Sprunck et al. (p. 1093; see the Perspective by Snell) now show that gamete interactions in Arabidopsis depend on small cysteine-rich EGG CELL 1 (EC1) proteins that accumulate in storage vesicles of the egg cell and that are released during sperm-egg interaction. EC1 peptides trigger the delivery of a fusogen to the sperm cell surface. An intercellular link connects the two sperm cells throughout the gamete fusion process and could play a role in preventing the spontaneous fusion of activated sperm cells. The cysteine-rich proteins of Arabidopsis egg and central cells enable fusion with just one sperm each. Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple–sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.

Tackling Drought Stress: RECEPTOR-LIKE KINASES Present New Approaches

Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops.

Micropylar pollen tube guidance and burst: adapted from defense mechanisms?

After the first description of fertilization in flowering plants some 125 years ago (Strasburger E: Neue-Untersuchungen über den Befruchtungsvorgang bei den Phanerogamen als Grundlage für eine Theorie der Zeugung. Gustav Fischer; 1884), we are finally beginning to understand the various molecular mechanisms leading to sperm delivery and discharge inside the hidden micropylar region of the female gametophyte (embryo sac). The last phase of pollen tube guidance culminating in tube burst and explosive release of tube contents requires extensive crosstalk between both male and female gametophytes. The first molecules identified that play key roles in these processes represent highly polymorphic proteins, similar to major components of the plant innate immune system. Here we summarize recent advances and briefly discuss the underlying molecular mechanisms also in respect to prezygotic barriers of reproductive isolation.

Activation of the imprinted Polycomb Group Fie1 gene in maize endosperm requires demethylation of the maternal allele

Imprinting refers to the epigenetic regulation of gene expression that is dependent upon gene inheritance from the maternal or paternal parent. Previously, we have identified two maize homologs of the single Arabidopsis Polycomb Group gene FIE. Here, we report on the expression pattern of these genes in individual gametes before and after fertilization, and on the role of DNA methylation in determining the maternal expression of the Fie1 gene. We found that Fie1 is neither expressed in the sperm, egg cell nor central cell before fertilization. Activation of the Fie1 maternal allele occurs around two days after pollination (DAP) in the primary endosperm and peaks at 10–11 DAP coinciding with endosperm transition from mitotic division to endoreduplication. In contrast, Fie2 is expressed in the egg cell and more intensively in the central cell similar to Arabidopsis FIE, which strongly supports the hypothesis that it functions as a repressor of endosperm development before fertilization. Using MSRE-PCR and bisulfite sequencing, we could show that the methylated inactive state is the default status of Fie1 in most tissues. In the endosperm the paternal Fie1 allele remains methylated and silent, but the maternal allele appears hypomethylated and active, explaining mono-allelic expression of Fie1 in the endosperm. Taking together, these data demonstrate that the regulation of Fie1 imprinting in maize is different from Arabidopsis and that Fie1 is likely to have acquired important novel functions for endosperm development.

Male-female communication triggers calcium signatures during fertilization in Arabidopsis

Cell–cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+]cyto) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+]cyto signals during the entire double fertilization process in Arabidopsis. [Ca2+]cyto oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+]cyto transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+]cyto transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+]cyto signature differing by timing and behaviour from [Ca2+]cyto waves reported in mammals.

Overcoming Hybridization Barriers by the Secretion of the Maize Pollen Tube Attractant ZmEA1 from Arabidopsis Ovules

A major goal of plant reproduction research is to understand and overcome hybridization barriers so that the gene pool of crop plants can be increased and improved upon. After successful pollen germination on a receptive stigma, the nonmotile sperm cells of flowering plants are transported via the pollen tube (PT) to the egg apparatus for the achievement of double fertilization. The PT path is controlled by various hybridization mechanisms probably involving a larger number of species-specific molecular interactions. The egg-apparatus-secreted polymorphic peptides ZmEA1 in maize and LURE1 and LURE2 in Torenia fournieri as well as TcCRP1 in T. concolor were shown to be required for micropylar PT guidance, the last step of the PT journey. We report here that ZmEA1 attracts maize PTs in vitro and arrests their growth at higher concentrations. Furthermore, it binds to the subapical region of maize PT tips in a species-preferential manner. To overcome hybridization barriers at the level of gametophytic PT guidance, we expressed ZmEA1 in Arabidopsis synergid cells. Secreted ZmEA1 enabled Arabidopsis ovules to guide maize PT in vitro in a species-preferential manner to the micropylar opening of the ovule. These results demonstrate that the egg-apparatus-controlled reproductive-isolation barrier of PT guidance can be overcome even between unrelated plant families.

The two male gametes share equal ability to fertilize the egg cell in Arabidopsis thaliana.

Summary The seed of a flowering plant develops from an ovule containing two distinct female gametes — the egg cell and the central cell — that are fertilized by a pair of non-motile sperm cells conveyed by the pollen tube. With a few exceptions [1], the two sperm cells, derived from a symmetrical mitosis, are isomorphic and seem to express a similar gene repertoire [2]. Since the discovery of double fertilization in flowering plants at the end of the 19th century, it has been a long standing question whether the two sperm cells are functionally equivalent, that is, whether they are capable of fertilizing the egg cell and the central cell in equal measure.