Wheat meiosis and the Ph1 locus

The Ph1 chromosome pairing locus in wheat - April 2012 (Powerpoint)

The polyploid wheat genome

Wheat is an allopolyploid.  It possesses multiple complete sets of related chromosomes resulting from interbreeding between closely related species.  Bread wheat is a hexaploid, having three diploid genomes, A, B and D.  These genomes contain both homologous and homoeologous chromosomes.  Homologous chromosomes are of a similar size and shape, contain the same genes in the same order, but may have different alleles.  Homoeologous (related) chromosomes can have a similar gene content and order but diverge in repetitive DNA content.

Ph1 controls the ‘diploidizing’ mechanism in wheat

Despite its genome complexity, hexaploid wheat behaves as a diploid at meiosis.  Chromosome 1A only pairs with 1A (its homologue), and not with either 1B or 1D (its homoeologues), and this holds true for all seven chromosome groups.  The ability of wheat to distinguish between homologous and homoeologous chromosomes essentially diploidizes the wheat genome.  The accuracy and efficiency of the mechanisms used to achieve this diploidization have a profound influence on the fertility of the wheat plant, which is of major importance for success in breeding. The Ph1 (Pairing homoeologous 1) locus on wheat chromosome 5B controls this diploidizing mechanism in wheat.

The role of Ph1 in wheat breeding

Many wild species related to wheat carry interesting characteristics, such as disease resistance genes, that would be useful to exploit in breeding programmes.  Sexual hybridization between polyploid wheat and a wild species generally produces an interspecific hybrid containing a haploid set of polyploid and wild-relative chromosomes.  Breeders make use of these crosses to introduce desirable genes into elite wheats from exotic germplasms.  However, in many such hybrids there is only a low level of pairing and recombination between wheat and wild relative chromosomes because there are no homologous chromosomes.  The major factor preventing pairing between wheat and wild species chromosomes is the Ph1 locus.

The importance of understanding the Ph1 locus

Wheat lines carrying deletions encompassing the Ph1 locus have been used for the last 25 years in breeding.  Pairing can occur between related chromosomes in these lines, but without the Ph1 locus, the chromosomes become heavily rearranged, making it difficult for recombination to take place between the wheat and related chromosomes, and leading to infertility.  What is needed is the ability to temporarily switch off the Ph1 locus to enable related chromosomes to pair.  An understanding of the biology of this phenotype will help to achieve this goal.   

Homology of Ph1 genes with human Cdk2

We have defined the Ph1 locus on wheat chromosome 5B to a region containing a gene complex of seven Cdk (Cyclin dependent kinase)-like genes disrupted by a piece of sub-telomeric heterochromatin (Griffiths et al. 2006; Al-Kaff et al. 2008).  The homoeologous regions on chromosomes 5A and 5D contain five and two Cdk-like genes respectively.  Of the 14 Cdk-like genes in this region, at least one copy (5B2, 5A3 and 5D2) on each genome has a full open reading frame.  However, ongoing transformation experiments have revealed that whereas over-expression of both 5A3 and 5D2 have dramatic effects on plant viability and fertility, over-expression of 5B2 had no such effect, suggesting that it might be defective compared with 5A3 and 5D2. The protein sequences of 5B2, 5A3 and 5D2 are similar for the first 314 amino acids, but the final 31 amino acids of 5B2 and its stop codon are different, possibly providing an explanation for the phenotype observations.

Protein modeling studies have revealed that the Ph1 Cdk-like genes have homology to Cdk2, a mammalian gene regulating replication by controlling chromatin structure (Yousafzai et al. 2010a and b).  The human CDK2 protein interacts with cyclin-A during the cell-cycle, and detailed modeling predicts that the wheat Cdk-like protein Ta5B2 has the ability to interact with cyclin-A of Arabidopsis.

3D model of a wheat Cdk-Arabidopsis cyclin protein complex (Ta5B2-AtcyclinA2:3) (gold colour) superimposed onto the crystal structure of the human CDK2-cyclin-A complex (red).  A deletion in the wheat Cdk sequence results in a decrease in the size of a coil/turn which can be seen in the middle of the figure.

Induction of chromosome pairing using Okadaic acid

If all the 5B Cdk copies are indeed defective, then the Ph1 phenotype could be the result of a dominant negative response, in which overall CDK activity is reduced, while CDK activity is increased in the absence of the locus. Okadaic acid is a protein phosphatase inhibitor known to increase the activity of CDKs.  If pairing between related chromosomes is due to increased CDK activity in the absence of the Ph1 locus, then Okadaic acid treatment should phenocopy the effect of deleting the Ph1 locus. We have used Okadaic acid to phenocopy the Ph1 effect (See chemical modification of chromosome pairing).  We have found that Okadaic acid can induce pairing between the related chromosomes of a wheat-rye interspecific hybrid even in the presence of Ph1, thus mimicking the pairing observed in the absence of Ph1 (Knight et al., 2010).

The Ph1 locus suppresses Cdk2-type activity

The above observations imply that Cdk-type phosphorylation can regulate chromosome pairing, and that the Ph1 Cdk pseudogenes may suppress this CDK-type activity. This raises the question of whether it is possible to demonstrate that Ph1 actually suppresses CDK activity, and whether this suppression is specifically of Cdk2-type activity, as indicated by the homology of the Ph1 Cdk-like genes to Cdk2.  Mammalian Cdk2 phosphorylates many substrates, including histone H1, at specific Cdk2 consensus sites. We have used phosphoproteomics to show that wheat histone H1 is phosphorylated at similar Cdk2 consensus sites to those of mammalian histone H1 (Greer et al., 2012). Moreover, either Okadaic acid treatment, or the deletion of the Ph1 locus lead to a two-fold increase in phosphorylation of the same Cdk2-type consensus sites on histone H1 (Greer et al., 2012). Thus, the Ph1 locus does indeed suppress Cdk2-type activity.  If this Cdk2-type activity regulated by Ph1 is similar to that of mammalian Cdk2, then it should also alter the replication process. We have shown that replication progression is indeed altered by Ph1 during premeiotic S phase (Greer et al., 2012).

All our current data, both published and unpublished, leads us to the proposal that Cdk2-type activity regulates whether the chromosomes pair as homologues, homoeologues or not at all.

A model for Ph1 regulation of chromosome pairing and recombination

Prior to meiosis, each chromosome is replicated, and the respective products, sister chromatids, are held together via specific cohesion proteins. At the onset of meiosis, each chromosome derived from parent 1 must recognize its homologue (the corresponding chromosome from parent 2).  These homologues must then pair and intimately align over their entire lengths so that recombination can reassort the genetic information from the two parents.  Chromosomes pair via their telomeres, which cluster to form a bouquet at one pole of the nucleus.   Intimate chromosome pairing or synapsis is then initiated from the telomeric regions. When the telomere bouquet forms, the sister chromatids condense onto a meiosis-specific protein core, which generates the axial elements of the synaptonemal complex.  The synaptonemal complex is a zipper-like protein structure that assembles between homologous chromosomes during meiotic prophase.

During meiotic prophase I there are two stages in which the chromosomes decondense. The first stage occurs prior to the chromosomes pairing and appears to be important for Ph1’s effectin wheat hybrids. The second stage happens between pachytene and diplotene and appears to be important for Ph1’s effect in wheat itself. Altered histone H1 phosphorylation as a result of Ph1 will affect both these decondensation stages.

The effect of Ph1 on meiotic chromosome pairing in wheat itself needs to be considered separately from its effect on pairing in wheat-relative hybrids used to introduce key traits.  This is because in wheat itself there are both homologues and homoeologues present, while in wheat hybrids, just homoeologues and no homologues.  This is important because the presence of homologues triggers a conformation or condensation change in both chromosomes early in meiosis before they pair, which enables them to pair.  If this change does not happen, the chromosomes can’t pair. The phenomenon was initially reported in wheat, but has recently been reported in C elegans, suggesting that it is conserved across kingdoms.  Thus, in wheat, which possesses homologues, this condensation change happens irrespective of whether Ph1 is present or absent. However, in wheat-relative hybrids, for example wheat-rye hybrids, the homoeologues in the presence of Ph1 do not treat each other as “homologues” at meiosis, and so the conformation change is delayed and little pairing occurs. In contrast, in the absence of Ph1, the homoeologues treat each other as homologues and undergo the condensation change, and therefore there is a level of pairing.  Given that this conformation change can happen in the presence or absence of Ph1, it seems unlikely that Ph1 is directly involved in the process.

However, wheat synapsis studies reveal that the chromosomes initially synapse as multivalents in both the presence and absence of Ph1, but in the absence of Ph1, the multivalent associations fail to resolve into homologous bivalents at pachytene in contrast to the situation when Ph1 is present (Holm, 1986; Holm and Wang, 1988). This resolution normally occurs at the diffuse or decondensation stage at the end of pachytene.  The simplest explanation for these observations is that the altered chromatin structure affects how recombination sites are processed or resolved at this diffuse /decondensation stage.

In the wheat hybrids, where the condensation change is triggered in the absence of Ph1 and homoeologues are then processed as homologues, Cdk2-like activity is increased, associated with increased histone H1 phosphorylation (Greer et al., 2012). This will lead to a more open chromatin, which may explain the reduction in the stringency of homology required to trigger the meiotic condensation change. 

So how does Ph1 affect chromosome pairing and recombination in the hybrids between wheat and its relatives??  Recombination involves the formation and repair of double strand breaks (DSBs).  DSB repair exploiting homologues or homoeologues can lead to chromosome exchange, while repair using sister chromatids (sisters) leads to non-exchange. Chromosomes intimately align or synapse during early meiosis, and recombination can occur within this framework. In wheat hybrids where no homologues are present, the chromosomes can synapse in both the presence and absence of Ph1, but in the presence of Ph1 this synapsis does not lead to recombination. After dissolution of the synaptonemal complex, the associated chromosomes resolve as univalents which do not fragment, implying that DSBs have been repaired using the sister chromatids. On the other hand, in the absence of Ph1, DSBs can be repaired with homoeologues, and chromosome exchange can occur.

Previous studies have revealed that Ph1 affects the structure of centromeric heterochromatin as well as sister chromatid separation during anaphase (Aragon-Alcaide et al., 1997).  Moreover, in wheat, the addition of B chromosomes can compensate for the effect of deleting Ph1 (Dover and Riley, 1972).  Heterochromatin on B chromosomes also affects sister chromatid separation of both A and B chromosomes, and alterations in sister chromatid cohesion would be expected to affect sister chromatid separation. We therefore propose that Ph1 affects sister chromatid cohesion through altering heterochromatin decondensation, and that whether DSBs are repaired with sister chromatids or homoeologues will be changed by alterations in sister chromatid cohesion. At the onset of meiosis, heterochromatin decondenses just prior to the homologues pairing, leading to chromosome elongation and sister chromatid separation (Martinez-Perez et al., 1999) before condensing later on in meiosis. It can be argued that the separation of sisters helps in the formation of a barrier to DSB repair with sisters. The extent of this heterochromatin decondensation depends on the presence of Ph1 (Prieto et al., 2004; Colas et al., 2008).  Ph1 delays heterochromatin decondensation during the onset of meiosis in the wheat-rye hybrid, which may prevent the barrier to sister repair from being formed.  In the absence of Ph1, decondensation is not delayed and so the barrier to sister repair can be established. We propose that Ph1 regulates this process by altering the structural stability of heterochromatin. Recent work has shed some light on what provides the barrier to sister chromatid DSB repair. Asy1, an axial element protein, suppresses DSB repair via sister chromatids (Sanchez-Moran et al., 2007).  Interestingly reducing Asy1 in wheat mimics the phenotype of deleting Ph1 (Boden et al., 2009).

Future studies

We are currently engaged in assessing Ph1’s effect on sister chromatid cohesion in wheat hybrids, and are exploiting model systems to understand the effect of the Ph1 Cdks (and their homologues) on homologous chromosome pairing.

Collaborations

The cell biological studies developed in wheat to understand the effect of the Ph1 locus were undertaken in collaboration with Peter Shaw and his group. This collaboration has now been expanded to include proteomics approaches in wheat for studying the Ph1 effect for which we acknowledge Alex Jones, The Sainsbury Laboratory

Presentation

The Ph1 chromosome pairing locus in wheat - April 2012 (Powerpoint)

References

Al-Kaff N, Knight E, Bertin I, Foote T, Hart N, Griffiths S, Moore G (2008).  Detailed dissection of the chromosomal region containing the Ph1 locus in wheat Triticum aestivum: With deletion mutants and expression profiling.  Ann. Bot. 101: 863-872 doi:10.1093/aob/mcm252

Aragón-Alcaide L,  Reader S, Miller T, Moore G (1997) Centromeric behaviour in wheat with high and low homoeologous chromosome pairing Chromosoma 106: 327-333

Boden SA, Langridge P, Spangenberg G, Able J (2009) TaASY1 promotes homologous chromosome interactions and is affected by deletion of Ph1. The Plant Journal 57: 487-497 DOI: 10.1111/j.1365-313X.2008.03701.x

Colas I, Shaw P, Prieto P, Wanous M, Spielmeyer W, Mago R, Moore G (2008) Effective chromosome pairing requires chromatin remodeling at the onset of meiosis
Proceedings of the National Academy of Sciences USA 105 (16): 6075-6080
DOI:doi/10.1073/pnas.0801521105

Dover GA, Riley R (1972) Prevention of pairing of homoeologous meiotic chromosomes of wheat by an activity of supernumerary chromosomes of Aegilops. Nature 240: 159–161.

Greer E, Martin AC, Pendle A, Colas I, Jones AM, Moore G, Shaw P (2012) The Ph1 locus suppresses Cdk2-type activity during premeiosis and meiosis in wheat. Plant Cell DOI/10.1105/tpc.111.094777.

Griffiths S, Sharp R, Foote TN, Bertin I, Wanous M, Reader S, Colas I, Moore G (2006) Molecular characterization of Ph1 as a major chromosome pairing locus in polyploid wheat. Nature 439: 749-752.  Figure legends to accompany supplementary information  (Cell Research Highlight- Leading edge- Cell (2006) 124: 869)

Holm, P.B. (1986) Chromosome pairing and chiasma formation in allohexaploid wheat, Triticum aestivum, analysed by spreading of meiotic nuclei. Carlsberg Res. Commun. 51, 239-294.

Holm, P.B. and Wang X. (1988) The effect of chromosome 5B on synapsis and chiasma formation in wheat, Triticum aestivum cv Chinese Spring. Carlsberg Res.Commun. 53, 191-208.

Knight E, Greer E, Draeger T, Thole V, Reader S, Shaw P, Moore G (2010) Inducing chromosome pairing through premature condensation: analysis of wheat interspecific hybrids Funct Integr Genomics 10: 603-608 DOI: 10.1007/s10142-010-0185-0

Martinez-Perez E, Shaw P, Reader S, Aragon-Alcaide L, Miller T, Moore G (1999) Homologous chromosome pairing in wheat. J. Cell Sci. 112: 1761–1769.

Prieto P, Shaw P, Moore G (2004) Homologue recognition during meiosis is associated with a change in chromatin conformation.  Nature Cell. Biol. 6: 906-908.

Sanchez-Moran E, Santos J-L, Jones GH, Franklin FCH (2007) ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis. Genes Dev. 21: 2220–2233.

Yousafzai F, Al-Kaff N, Moore G (2010a) The molecular features of chromosome pairing at meiosis:the polyploid challenge using wheat as a reference Funct Integr Genomics 10: 147-156 DOI: 10.1007/s10142-010-0171-6

Yousafzai F, Al-Kaff N, Moore G (2010b) Structural and functional relationship between the Ph1 locus protein 5B2 in wheat and CDK2 in mammals Funct Integr Genomics 10: 157-166 DOI: 10.1007/s10142-010-0170-7

John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Top of Page