What Pairing in Euploid Wheat Itself Tells Us?
Is the effect on chromosome exchange the whole explanation of Ph1's action? As stated previously from analysis of hybrids, Ph1 can't prevent related chromosomes synapsing (Martin et al. 2014), however Ph1 does prevent related chromosomes from synapsing in wheat itself. Wheat synapsis studies reveal that the chromosomes are essentially synapsed as bivalents at pachytene when Ph1 is the present, but synapse as multivalents when Ph1 is absent (Holm 1986; Holm and Wang 1988). This implies that in wheat itself where there are both homologues and homoeologues, the overall effect of Ph1 on chromosome pairing as distinct from recombination must be the promotion of homologue pairing rather than specifically suppressing pairing between the related chromosomes. How does this work? Euploid wheat homologues synchronously elongate prior to pairing at the onset of meiosis (Prieto et al. 2004; Colas et al. 2008). The degree of homologue elongation reflects the level of homology between the two parental chromosomes (Colas et al. 2008). This promotes homologue pairing which is disrupted in the absence of Ph1 leading to incorrect pairing. Thus the level of chromosome homology in the presence of Ph1 influences conformational changes required for initial pairing, which determines the extent of chromosome pairing in wheat. Thus if two segments within homologous chromosomes are too distinct with respect to each other, they can't associate or synapse at all and if they are related, they can partially pair or synapse. Recent studies have reported a similar effect in C. elegans suggesting that this is a general meiotic phenomenon (Nabeshinia et al. 2011). They are identified some of the proteins involved, whose activity will depend on phosphorylation levels. The data suggests that these proteins bind along the chromosome triggering the conformational changes at a regional level. The increased Cdk2 activity through deleting Ph1 will also affect the phosphorylation of the protein SUN1 early in meiosis. This may well affect the functioning of the telomere bouquet, and therefore pairing of chromosomes via the telomeres early in meiosis. Reduced shaking of chromosomes by the telomere bouquet will enable homoeologous associations to be maintained, while more rigorous shaking will rip such associations apart leaving just homologous associations between the chromosomes. We are currently studying the effect of Ph1 on telomere bouquet function, and its subsequent effect on chromosome pairing early in meiosis (Richards et al. 2012).
Independent Centromere Pairing
Finally proper segregation of chromosomes to daughter cells requires that the paired chromosomes correctly orientate themselves so that the spindle fibres attach to the centromeres and pull the chromosomes in opposite directions. To achieve balanced gametes, the homologous centromeres must be correctly paired. We isolated an element Hi-10, which is found at the centromeres of all cereals (Aragon-Alcaide et al. 1996) and exploited it as an in situ marker for studying centromere pairing behaviour during meiosis. We reported that wheat centromeres pair independently from the rest of the chromosome, which associate and synapse from the telomeres during the telomere bouquet stage at the onset of meiosis. Our studies were controversial at the time, however it has been since reported that centromeres pair independently from the rest of the chromosome for meiosis in Arabidopsis, rice, Brachypodium and maize. It has been elegantly demonstrated in some of these systems that the centromeres synapse homologously, and independently of the rest of the chromosome (Da Ines et al. 2012). Tetraploid and hexaploid wheat possess 28 and 42 chromosomes respectively, or two and three copies of seven sets of chromosomes. During anther development in wheat-wild relative hybrids, tetraploid and hexaploid wheat, the centromeres associate as pairs (Aragon-Alcaide et al. 1997; MartinezPerez et al. 1999; Martinez-Perez et al. 2003). During premeiotic replication, the pairs engage in a sorting process reducing to seven centromere sites at the onset of meiosis, again as the telomeres cluster to form a telomere bouquet (Martinez-Perez et al. 2003; Greer et al. 2012). Ph1 increases the stringency of this independent centromere pairing process (Martinez-Perez et al. 2001), and therefore will affect the correct segregation of chromosomes, and the production of balanced gametes.
We have shown that Ph1 has two distinct effects on chromosome pairing and recombination. It promotes homologous pairing through: influencing conformational changes required for initial pairing, increasing the stringency of independent centromere pairing, and finally altering telomere bouquet formation (and possibly function, we are currently studying this). Ph1 also stalls Double Holliday Junctions from resolving as crossovers on paired homoeologues. The Ph1 locus has been delineated to a region containing a cluster of Cdk2-like genes containing a large segment of heterochromatin. Interestingly recent mouse studies also reveal that Cdk2 has two distinct effects on this system, one on telomere bouquet function, and one at the stage when Double Holliday Junctions are being resolved.
Acknowledgments I thank Peter Shaw, who has been a collaborator and great colleague at JIC for the last 15 years on the cell biological, proteomics and model systems aspects of the Ph1 story. I also thank the following people who have also contributed in my group or as collaborators: Tracie (nee Foote) Draeger, Simon Griffiths, Masahiro Yano, Michael Roberts, Lijia Qu, Terry Miller, Steve Reader, Sebastien Allouis, Rebecca Sharp, Kath Mortimer, Emilie Knight, Nadia Al-Kaff, Vera Thole, Azahara Martin, Ali Pendle, Alex Jones, Isabelle Colas, Faridoon Yousafzai, Shahl Abbo; Luis Aragon, Enrique (Fadri) Martinez-Perez, Pilar Prieto, Mike Wanous, Emma Greer, Danielle Monk, Ruoyu Wen, Mike Gale, Dick Flavell, Nori Kurata, Tim Helenjaris, Helene Lucas, Boulos Chalhoub, Shahryar Kianian, Phillippe Vain, John Doonan, Dupont-Pioneer and finally funding from BBSRC. My sincere apologies to anybody I have forgotten…