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.

Plant hormones have been shown to regulate key processes during embryogenesis in the model plant Arabidopsis thaliana, but the mechanisms that determine the peculiar embryo pattern formation of monocots are largely unknown. Using the auxin and cytokinin response markers DR5 and TCSv2 (two-component system, cytokinin-responsive promoter version #2), as well as the auxin efflux carrier protein PIN1a (PINFORMED1a), we have studied the hormonal response during early embryogenesis (zygote towards transition stage) in the model and crop plant maize. Compared with the hormonal response in Arabidopsis, we found that detectable hormone activities inside the developing maize embryo appeared much later. Our observations indicate further an important role of auxin, PIN1a and cytokinin in endosperm formation shortly after fertilization. Apparent auxin signals within adaxial endosperm cells and cytokinin responses in the basal endosperm transfer layer as well as chalazal endosperm are characteristic for early seed development in maize. Moreover, auxin signalling in endosperm cells is likely to be involved in exogenous embryo patterning as auxin responses in the endosperm located around the embryo proper correlate with adaxial embryo differentiation and outgrowth. Overall, the comparison between Arabidopsis and maize hormone response and flux suggests intriguing mechanisms in monocots that are used to direct their embryo patterning, which is significantly different from that of eudicots.

Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter- and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50–100 μm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.

We propose to develop maize (Zea mays) as a model to investigate the above-described processes to understand the associated intra- and interspecific crossing barriers in grasses. Many genetic, cellular and biotechnological tools including the completion of a reference genome (inbred line B73) have been established in the last decade and many more maize inbred genomes are expected to be available soon. Moreover, a cellular marker line database as well as large transposon insertion collections and improved Agrobacterium transformation protocols are now available. Additionally, the processes described above are well studied at the morphological level and a number of mutants have been described already, awaiting disclosure of the relevant genes. The identification of the first key players in pollen tube growth, guidance and burst show maize to be an excellent grass model to investigate these processes in more detail. Here we provide an overview of our current understanding of these processes in Poaceae with a focus on maize, and also include relevant discoveries in eudicot model species.

Unlike in animals, female gametes of flowering plants are not the direct products of meiosis but develop from a functional megaspore after three rounds of free mitotic divisions. After nuclei migration and positioning, the eight-nucleate syncytium differentiates into the embryo sac, which contains two female gametes as well as accessory cells at the micropylar and chalazal pole, respectively. We report that an egg-cell-specific gene, ZmEAL1, is activated at the micropylar pole of the eight-nucleate syncytium. ZmEAL1 translation is restricted to the egg cell, resulting in the generation of peptide-containing vesicles directed toward its chalazal pole. RNAi knockdown studies show that ZmEAL1 is required for robust expression of the proliferation-regulatory gene IG1 at the chalazal pole of the embryo sac in antipodal cells. We further show that ZmEAL1 is required to prevent antipodal cells from adopting central cell fate. These findings show how egg cells orchestrate differentiation of the embryo sac.

Pollen tube growth and guidance in the female tissues of flowering plants is a long-studied and anatomically well-described process. A large number of gene products and chemical compounds involved have been identified in the last 20 years, and some underlying molecular mechanisms including self-incompatibility in the Brassicaceae, Solanaceae and Papaveraceae are now well understood. However, the largest part of the pollen tube pathway inside the transmitting tract towards the ovule harbouring the female gametophyte still requires intensive investigations. Especially in the economically most import plant family, the Poaceae or grasses, progamic pollen tube development is barely understood. Using maize as a model, we propose to divide pollen tube germination, growth and guidance towards the female gametophyte into five distinct phases. The model is adapted from Arabidopsis thaliana, taking anatomical differences and novel genetic and cellular studies into consideration. With the exception of Phase V, all phases seem to be under sporophytic control in grasses.