Plant gamete ‘companion’ cells: one step forward – one step back

Like in animals, plant gametes have helper or companion cells that assist them during development (reviewed in Feng and Dickinson, 2013). Spectacularly, the main male companion cell (a.k.a. the vegetative cell) – by developing into the pollen tube – even delivers the two sperms to the egg. Over the past few years molecular analysis of pollen grains and embryo sacs (the male and female ‘gametophytes’) has pointed to a further and unexpected role for these cells. In a 2009 paper in Cell, Keith Slotin and co-authors reported that in young pollen, transposable elements (TEs; ‘parasitic’ stretches of DNA) in the vegetative cell became active through removal of silencing methylation and generate small interfering (si)RNAs. These RNAs then moved into the sperms and silenced potentially deleterious TEs by DNA methylation. This mirrors in some ways the PIWI and MIWI systems of flies and mammals, which also silence TEs, but here siRNAs are generated within the germ cells themselves. Other authors have subsequently suggested that the large numbers of siRNAs found in the endosperm (the successor to the female companion cell, a.k.a. central cell) may carry out a similar function and suppress TE activity in the egg (reviewed in Feng and Dickinson, 2013). Such a process may not of course only affect TEs, but could also silence coding genes in the germ cells.

While an attractive idea, the evidence provided was, however, of the smoking gun variety. For while Slotkin and co-authors showed that TE-derived siRNAs were being formed in the vegetative cell, evidence for the transport to and activity of these RNAs once in the sperms was a little fuzzy. In this year’s Annals of Botany Special Lecture at the Oxford University Department of Plant Sciences, Dr Xiaoqi Feng from UC Berkeley reported how data from her latest Science paper (Ibarra et al., 2012) showed that, in arabidopsis mutants where demethylation of TEs fails in pollen vegetative cells (and thus no siRNAs are generated), the same TEs in the neighbouring sperm cells were undermethylated. Dr Feng’s experiments strongly suggest that, since the sperms have all the machinery to silence these transposons through RNA-dependent DNA methylation (RdDM), companion cell (vegetative cell) siRNAs are required for this process to take place.

So far so good – but how do the siRNAs get from the vegetative cell, through two plasma membranes and a vestigial cell wall, and into the sperms? This has been a problem since Slotkin and coauthors’ idea hit the newsstands in 2009. Certainly there is some evidence from microscopic studies that the separation between the germline cells (sperms) and their companion vegetative cell may not be complete, but both cell-specific RNA sequencing data and the identification of an increasing number of vegetative cell- and sperm-specific marker proteins suggest that these two cell types are fully independent. In response, Slotkin and coauthors would point to experiments showing that a GFP-targeting artificial micro RNA (amiR) expressed in the vegetative cell can suppress the expression of a sperm-specific GFP marker gene. However, as has subsequently been pointed out (Grant-Downton et al., 2013), the vegetative cell promoter used to express the amiR (LAT52) is not truly cell-type specific and comes on late in microspore development, meaning that the TE siRNAs generated by the late microspore nucleus could be inherited by the germline through the cytoplasm they once shared. Indeed when a GFP amiR was expressed using a very ‘tight’ vegetative cell promoter (VCK1), it failed to silence a sperm-cell-expressed GFP gene (Grant-Downton et al., 2013). A problem with these experiments may lie in the use of amiRs, for while the intercellular movement of small RNAs is well documented in plants, most of the experiments have involved siRNAs and not miRNAs (amiRNAs or otherwise). It may simply be that microRNAs do not move from cell to cell with the facility of siRNAs.

So – where are we now? The sequencing data certainly show a convincing congruence between the TEs unmethylated in mutant lines and the lack of silencing in their gamete counterparts. However, key questions relating to when the siRNAs – which are certainly key pieces in this game of intercellular chess – are generated by the vegetative nucleus, and whether they are inherited by, or are transported into the germline, will have to await a better understanding of molecular events in the microspore – particularly the timing of TE demethylation in relation to cytokinesis and to the integrity of the division products.

REFERENCES

Feng, X, Zilberman, D, Dickinson, H. (2013) A Conversation across Generations: Soma-Germ Cell Crosstalk in Plants Developmental Cell. 24 (3): 215-225 doi:10.1016/j.devcel.2013.01.014
Grant-Downton, R, Kourmpetli, S, Hafidh, S, Khatab, H, Le, Trionnaire G, Dickinson, H, Twell, D. (2013). Artificial microRNAs reveal cell-specific differences in small RNA activity in pollen Current Biology. 23 (14) doi:10.1016/j.cub.2013.05.055
Ibarra, A.C., Feng, X., Schoft, V.K., and Hsieh, T.F., Uzawa, R., Rodriguez, J.A., Zemach, A., Chumak, N., Machlicova, A., Nishimura, T., Rojas, D., Fischer, R.L., Tamaru, H. and Zilberman, D. (2012). Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337: 1360-1364
Slotkin, R.K., Vaughn, M., Borges, F., Tanurdzic, M., Becker, J.D., Feijo, J.A., and Martienssen, R.A. (2009) Epigenetic reprogramming and small RNA silencing of transposable elements. Cell 136: 461-472

Hugh Dickinson
Oxford University Department of Plant Sciences.