- American Society of Plant Biologists
Synergid cells are two specialized cells that lie adjacent to the egg cell in the female gametophyte of angiosperms and play an essential role in pollen tube guidance and function. The term synergid comes from the Greek “synergos,” which means “working together” and was reportedly coined by Eduard Strasburger, a famous 19th century botanist who was one of the first to note that these cells somehow assist fertilization of the egg (Vesque, 1878; reviewed in Higashiyama, 2002).
After a pollen grain germinates on the surface of the stigma, it forms a pollen tube that grows through the style and is guided through the micropyle (a minute opening at one end of the ovule). The pollen tube grows toward and into one of the synergid cells, which begins to degenerate as the pollen tube enters it (or shortly before). The pollen tube then ceases growth, ruptures, and releases its two sperm cells, which migrate to the egg and central cell, fertilization of which gives rise to the embryo and endosperm, respectively. The synergid cells are necessary for pollen tube guidance in the ovule, as shown by Higashiyama et al. (2001) using a laser ablation technique in Tourenia to selectively remove different cells within the female gametophyte. The synergids are also essential for the cessation of pollen tube growth and release of the sperm cells.
The synergid cell wall forms a highly thickened structure called the filiform apparatus at the micropylar end, consisting of numerous finger-like projections into the synergid cytoplasm. This structure greatly increases the surface area of the plasma membrane in this region, which is also associated with an elaborated endoplasmic reticulum. It is thought that the filiform apparatus mediates the transport of molecules into and out of the synergid cells (Willemse and van Went, 1984; Huang and Russell, 1992).
In this issue of The Plant Cell, Punwani et al. (pages 2557–2568) show that the R2R3 Myb protein MYB98 functions as a transcriptional regulator of genes that are expressed in synergid cells and are required for formation of the filiform apparatus and pollen tube guidance. Kasahara et al. (2005) previously showed that, within the female gametophyte, MYB98 is expressed exclusively in the synergid cells and that myb98 mutants show defects in pollen tube guidance and development of the filiform apparatus. Punwani et al. extend these observations by showing that MYB98 is localized to the nucleus and binds DNA and that it is required for the expression of at least 16 genes previously identified (Steffen et al., 2007) as being expressed predominantly in synergid cells. Interestingly, many of these genes (11 of 16) encode small, Cys-rich proteins, and further investigation of five of these showed that the encoded proteins are secreted into the filiform apparatus. These data show that MYB98 is a key regulator of transcriptional events in the synergid cells, and it points to small Cys-rich proteins as playing a role in pollen tube guidance and successful fertilization.
Punwani et al. found that a fusion construct of MYB98:green fluorescent protein (MYB98:GFP) driven by the MYB98 promoter localized to the nuclei of synergid cells (see figure), and in vitro DNA mobility shift assays showed that MYB98 binds a consensus binding sequence for mammalian c-Myb, which has an R2R3 Myb domain highly similar to that of MYB98. The authors then used real-time RT-PCR to investigate the expression of 18 genes shown by Steffen et al. (2007) to be expressed predominantly in synergid cells, which showed that 17 of these genes are strongly downregulated in the female gametophyte of myb98 mutant plants. The expression of native promoter:GFP constructs in myb98 mutant plants confirmed that MYB98 is required for the expression of 16 of these genes.
Localization of GFP Fusion Proteins in the Female Gametophyte.
GFP:MYB98 localizes to the nucleus of synergid cells (top panel). DD4:GFP localizes to the filiform apparatus of synergid cells (bottom panel). DD4 encodes a Cys-rich protein and is one of numerous synergid-expressed genes whose expression is severely reduced in myb98 mutant gametophytes. scn, synergid cell nucleus; sc, synergid cell; fa, filiform apparatus. Bars = 20 μg. Figure reproduced from Punwani et al. (2007).
These experiments strongly suggest that MYB98 functions as a positive regulator of transcription in synergid cells. Additional experiments are necessary to elucidate this function more precisely, for example, to identify the specific binding site and discover which genes are direct rather than indirect targets of MYB98. Punwani et al. show that MYB98 binds specifically to a core TAAC sequence present in the c-Myb consensus sequence. However, TAAC forms the core of many of the binding sites known for R2R3 binding proteins and, as noted by the authors, is found one or more times in virtually every promoter in the Arabidopsis genome. The use of a random binding site selection assay (e.g., Huang et al., 1993; Tang and Perry, 2003) would be an important step to establish the specific binding site of MYB98. In addition, although Punwani et al. show that MYB98 is required for the expression of 16 genes expressed in synergid cells, it is not clear if all of these genes are direct targets. Activation assays (e.g., Pasquali et al., 1994) testing the potential of MYB98 to activate transcription from the various target promoters would be one way to distinguish direct from indirect targets.
In another set of experiments, the authors chose six of the synergid-expressed genes downregulated in myb98 mutant gametophyte tissue and investigated their subcellular localization patterns by transforming wild-type plants with GFP fusion constructs containing complete coding sequences and upstream regions. One of these genes, which encodes a Cys-rich protein lacking a putative signal peptide, was found to be expressed throughout the cytoplasm of synergid cells. Expression of the remaining five, all of which contained N-terminal signal peptides, was strongly associated with the filiform apparatus and not detected in the cytoplasm. Expression of a GFP construct containing only the putative signal peptide from one of these genes (driven by the MYB98 promoter) was also localized to the filiform apparatus. This suggests that an N-terminal signal peptide is sufficient to localize a synergid-expressed protein to the filiform apparatus.
The authors hypothesize that the synergid-expressed genes regulated by MYB98 (whether directly or indirectly) play roles in the formation and function of the filiform apparatus. Several of the Cys-rich proteins share similarity to other proteins known to play specialized roles in signaling. These include defensins, which are small Cys-rich proteins that have antimicrobial properties and function in signaling (Thomma et al., 2002), and the S proteins from Papaver, which are incompatibility factors that induce a signaling pathway resulting in death of incompatible pollen tubes (Thomas and Franklin-Tong, 2004). Two of the genes encode predicted cell wall–modifying proteins (a polygalacturonase and a galacosyltransferase) that may have a function in the specialized cell wall of the filiform apparatus. Further investigation of the functions of these synergid-expressed genes will be critical in determining how the filiform apparatus functions in pollen tube guidance and fertilization in angiosperms. This work represents an important step toward understanding the various functions of the synergids, the associated signaling events, and underlying gene regulatory network.
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