The isolation and characterisation of the wheat molecular ZIPper I homologue, Ta ZYP1
© Khoo et al; licensee BioMed Central Ltd. 2012
Received: 14 November 2011
Accepted: 18 February 2012
Published: 18 February 2012
The synaptonemal complex (SC) is a proteinaceous tripartite structure used to hold homologous chromosomes together during the early stages of meiosis. The yeast ZIP1 and its homologues in other species have previously been characterised as the transverse filament protein of the synaptonemal complex. Proper installation of ZYP1 along chromosomes has been shown to be dependent on the axial element-associated protein, ASY1 in Arabidopsis.
Here we report the isolation of the wheat (Triticum aestivum) ZYP1 (TaZYP1) and its expression profile (during and post-meiosis) in wild-type, the ph1b deletion mutant as well as in Taasy1 RNAi knock-down mutants. Ta ZYP1 has a putative DNA-binding S/TPXX motif in its C-terminal region and we provide evidence that Ta ZYP1 interacts non-preferentially with both single- and double-stranded DNA in vitro. 3-dimensional dual immunofluorescence localisation assays conducted with an antibody raised against Ta ZYP1 show that Ta ZYP1 interacts with chromatin during meiosis but does not co-localise to regions of chromatin where Ta ASY1 is present. The Ta ZYP1 signal lengthens into regions of chromatin where Ta ASY1 has been removed in wild-type but this appears delayed in the ph1b mutant. The localisation profile of Ta ZYP1 in four Taasy1 knock-down mutants is similar to wild-type but Ta ZYP1 signal intensity appears weaker and more diffused.
In contrast to previous studies performed on plant species where ZYP1 signal is sandwiched by ASY1 signal located on both axial elements of the SC, data from the 3-dimensional dual immunofluorescence localisation assays conducted in this study show that Ta ZYP1 signal only lengthens into regions of chromatin after Ta ASY1 signal is being unloaded. However, the observation that Ta ZYP1 loading appears delayed in both the ph1b and Taasy1 mutants suggests that Ta ASY1 may still be essential for Ta ZYP1 to play a role in SC formation during meiosis. These data further suggest that the temporal installation of ZYP1 onto pairing homologous chromosomes in wheat is different to that of other plant species and highlights the need to study this synaptonemal complex protein on a species to species basis.
The generation of gametes in sexually-reproducing organisms occurs through the reductional division process of meiosis that involves a single DNA replication event followed by two consecutive cell division events resulting in the formation of four haploid gametes from a single diploid progenitor cell. During the early substages of prophase I, the homologous chromosomes roughly align and pair with one another while axial element (AE) components, such as the ASYnapsis 1 (ASY1) protein, are installed along the lengths of the paired homologues. During leptotene to zygotene, transverse filament (TF) proteins of the proteinaceous ultrastructure known as the synaptonemal complex (SC) are installed to span the gap between the AE backbones. The central region of the SC consists of the polymerising ends of the TF that interact with one another to hold the homologous chromosomes together. During pachytene, the chromosomes are completely synapsed with the SC completely installed throughout the lengths of the chromosomes. Disassembly of the SC during diplotene leaves the homologous chromosomes attached only by chiasmata formed through the genetic recombination cross-over process [1, 2].
While the structure of the SC has been studied using cytology techniques extensively since the 1950s, the first plant synaptonemal complex (SC) proteins, ZYP1a and ZYP1b (collectively known as ZYP1) were only recently identified and characterised in Arabidopsis thaliana. These were named after the Saccharomyces cereviseae homologue, Molecular ZIPper 1 (ScZIP1). Characterisation of Sc ZIP1 previously revealed that its globular C-terminal domain interacts with the AE (also known as the lateral elements) while the N-terminus of the protein is able to dimerise with the N-termini of other ZIP1 molecules to form the central element of the SC . In Arabidopsis, AtZYP1a and AtZYP1b arose from a gene duplication event and encode proteins that share structural and functional similarities to Sc ZIP1 [3, 4]. Both At ZYP1a and At ZYP1b form the TF of the proteinaceous tripartite SC during early meiosis and are functionally redundant . More recently, two other plant homologues of ZYP1 have also been studied in Secale cereale (Sc ZYP1) and Oryza sativa (Os ZEP1) [5, 6]. Although the SC appears to be a well-conserved ultrastructure required for meiosis-specific chromosome pairing in various species, amino acid sequence conservation of its TF components across species is quite limited. A clear example of this can be seen in the ZIP1, SYP1, C(3)G, SCP1 and ZEP1 TF proteins of the SC that have previously been characterised in various species such as budding yeast, Drosophilia melanogaster, Caenorhabditis elegans, Mus musculus, and more recently, in some plant species such as Arabidopsis thaliana and Oryza sativa [1, 2, 4, 6–12]. Although these proteins have limited conservation at both the DNA and amino acid sequence levels, they all share the same central α-helical coiled-coil tertiary structure capped at both termini with globular domains and perform an identical function within their respective species .
In plants, the immuno-localisation profiles of the ZYP1 TF homologues also differ slightly from species to species, with ZYP1 signal first appearing as foci in leptotene stage meiocytes of Arabidopsis and rice but as short linear tracts in rye [4–6]. These ZYP1 foci are only observed after ASY1 has been loaded onto the AE backbones of the chromatin [4, 5]. Using the Arabidopsis spo11 and dmc1 mutants, Higgins and colleagues  deduced that ZYP1 was recruited to double-strand break (DSB) sites during the early stages of single-stranded DNA invasion and that ZYP1 loading was independent of recombination initiation. However, lengthening of the ZYP1 signal along the chromatin was dependent on successful recombination which in turn relied on ASY-mediated loading of the DMC1 recombinase onto chromatin . In rice and Arabidopsis, the ZYP1 signal lengthens from zygotene to pachytene and localises to the central region of the SC where it is sandwiched between ASY1 signals that are associated with the AE backbones of the homologous chromosomes on both sides [4, 6].
Given that the synaptonemal complex is a well-conserved structure present in many species and the fact that ASY1 has been shown to be necessary for synapsis and homologous chromosome pairing in bread wheat [14, 15], a ZYP1-like protein is also likely to be involved in these processes during the early stages of wheat meiosis. Indeed, knock-out of the ZYP1 protein in Arabidopsis leads to non-homologous interactions and the formation of bivalents/multivalents. Such abnormal chromosome interactions have also been observed in Taasy1 RNAi knock-down mutants as well as in the wheat pairing homologous (Ph) deletion mutant, ph1b .
The rice ZIP1 homologue (Os ZEP1) was also shown to perform similar roles to that of At ZYP1 . However, unlike other ZYP1 homologues, ZEP1 was shown to be reloaded two more times onto decondensed chromatin after the diakinesis stage of prophase I, specifically during the prophase II and telophase II stages. Based on their observations, Wang and colleagues  inferred that ZEP1 may also maintain chromatin in a decondensed state during early microsporogenesis.
The functional differences and limited sequence conservation seen in the SC TF proteins of various species highlight the need to study these proteins on a species-specific basis to determine whether these proteins may have additional roles to their already well-characterised TF role. Here we report the isolation of the TaZYP1 coding sequence, analysis of its predicted amino acid sequence and the stage-specific TaZYP1 gene expression profiles in both wild-type and the ph1b mutant. We show that Ta ZYP1 interacts with both single- and double-stranded DNA in vitro and also report on the temporal and spatial localisation profile of Ta ZYP1 within wheat meiocytes of two wild-type cultivars and the ph1b mutant using 3-dimensional dual immuno-fluorescence localisation techniques. Due to the importance of ASY1 in synapsis, maintenance of homologous chromosome interactions, and its reported role in the complete installation of ZYP1 along the lengths of Arabidopsis chromosomes, the localisation profile of Ta ZYP1 was also investigated in four Taasy1 RNAi knock-down mutants.
TaZYP1 encodes a predicted protein product with a putative S/TPXX DNA binding motif at the C-terminus
The predicted Ta ZYP1 amino acid sequence shares high levels of conservation with its homologues in close relatives, including the rice transverse element protein [GenBank: ADD69817] (Identities = 80%, Positivies = 91%, E-value = 0.00) and maize ZYP1 [GenBank: HQ116413] (Identities = 75.9%, Positives = 88%, E-value = 0.0) (Figure 1C). However, the level of sequence conservation was reduced when compared with the Arabidopsis homologues, At ZYP1b [GenBank: NP_564164.1] (Identities = 40%, Positives = 64%, E-value = 1e-156) and At ZYP1a [GenBank: NP_173645.3] (Identities = 39%; Positives = 63%, E-value = 1e-152) and even further reduced in species from other kingdoms as shown in the phylogenetic analysis (Figure 1C).
Amino acid sequence analysis revealed that both the N- and C-terminal regions of Ta ZYP1 (aa positions 1-68 and 723-863 respectively) have high pI values of 10.22 and 10.05. In addition, 18.75% of the C-terminus consisted of arginine and lysine residues. A putative DNA-binding S/TPXX motif was also found within this region (aa positions 761 to 764) (Figure 1A). Conserved domain analysis revealed that the Ta ZYP1 amino acid sequence retained modest similarities with two known Structural Maintenance of Chromosomes (SMC) conserved domains characterised in archaea, namely SMC_prok_B.and SMC_prok_A (E-values = 3.65e-09 and 3.00e-04 respectively) (data not shown). 3-dimensional protein modelling predicted that the central region of Ta ZYP1 forms a coiled-coil domain structure (from aa position 69 to 722) (data not shown).
Ta ZYP1 interacts with DNA and is up-regulated during early meiosis
Ta ZYP1 is loaded onto regions of chromatin after Ta ASY1 is removed
Having determined that TaZYP1 expression levels were slightly elevated in the ph1b mutant (Figure 3A), dual immunofluorescence localisation assays were performed to determine whether Ta ZYP1 loading was affected within the ph1b mutant. As previously reported by Boden et al. , the Ta ASY1 signal was observed to be stronger in the ph1b mutant with the nucleoplasm of some observed meiocytes completely saturated even at pre-meiotic interphase (data not shown). Furthermore, the Ta ASY1 signal appeared disordered on the condensing chromatin and persisted into diplotene. Aggregates of Ta ASY1 protein, thought to be polycomplexes, were sometimes observed as large foci in nucleolar regions of the ph1b meiocytes. Similar to wild-type, Ta ZYP1 was first observed as faint punctate foci during leptotene in the ph1b mutant (Figure 4; row 2, panel 2). However, in contrast to wild-type, lengthening of the signal from foci to form short tracts was delayed until pachytene (Figure 4; row 2, panel 4). In addition, Ta ZYP1 signal persisted at higher residual levels in diplotene compared to wild-type, possibly as a consequence of the delayed loading of Ta ZYP1 in the ph1b mutant (Figure 4; row 2, panel 5).
Ta ZYP1 signal along chromosomes in the Taasy1 knock-down mutants appear similar to wild-type albeit weaker and more diffuse
Ta ZYP1 localisation was identical both temporally and spatially in the Bob White MPB26 and Chinese Spring wild-types (compare Figure 5; row 1 with Figure 4 row 1). Similar to observations of both wild-type meiocytes (Figures 4 and 5; row 1), Ta ZYP1 and Ta ASY1 signals were sometimes seen populating regions of chromatin simultaneously but were never co-localised. Amongst the Taasy1 mutants, TaZYP1 signals were generally similar to wild-type, first appearing as faint punctate foci on regions of chromatin during leptotene and lengthening into short tracts during zygotene (Figure 5; rows 2 to 5, panels 1 to 3). The Ta ZYP1 signal then condensed and lengthened further during pachytene, populating regions of chromatin where the Ta ASY1 signal was being unloaded (Figure 5; rows 2 to 5, panel 4). Of the Taasy1 mutants analysed, ZYP1 loading in the Taasy1-1.9.2 mutant during pachytene appeared to be weaker, more diffuse and disrupted in addition to showing a delayed lengthening of the ZYP1 signal during the zygotene stage compared to wild-type (Figure 5; row 3, panel 3).Ta ZYP1 signal in the Taasy1 mutants was only present at residual levels during diplotene comparable to levels seen in the wild-type (compare Figure 5; row 1, panel 5 with Taasy1 mutant panels down column 5).
This study has characterised the ZYP1 bread wheat homologue at both the genetic and protein levels. Characterisation of the Ta ZYP1 amino acid sequence revealed that it shares similar physiochemical properties with its previous characterised homologues in other species with predictive modeling of Ta ZYP1 showing that it is a coiled-coil protein, capped with globular domains on both ends and a C-terminal region that possesses a high pI and a S/TPXX DNA-binding motif. Expression profiling of TaZYP1 revealed that TaZYP1 transcript numbers are elevated from pre-meiotic interphase to pachytene before reducing from diplotene onwards. Competitive DNA-binding assays confirm that Ta ZYP1 interacts with both ss- and ds-DNA indiscriminately in vitro with data from 3-dimensional fluorescence localisation assays conducted with an anti-Ta ZYP1 polyclonal antibody further confirming that Ta ZYP1 interacts with chromatin during the early stages of meiosis. Ta ZYP1 was localised to chromatin from leptotene to pachytene and while the Ta ZYP1 foci signal was sometimes observed to be present on the same regions of chromatin as the Ta ASY1 signal, both signals did not co-localise. In general, Ta ZYP1 signal only lengthened into regions of chromatin after the Ta ASY1 signal was unloaded. In the ph1b mutant, lengthening of the Ta ZYP1 signal appeared delayed until pachytene and persisted at higher levels in diplotene compared to wild-type. Localisation of Ta ZYP1 in four Taasy1 mutants revealed that Ta ZYP1 loading was similar to wild-type but most severely affected in the Taasy1-1.9.2 mutant (which had the lowest TaASY1 expression).
In order to characterise the wheat ZYP1 homologue, the full-length coding sequence of the wheat ZYP1 homologue was isolated using sequence information from a rice EST that showed high levels of similarity to the functionally-redundant AtZYP1a and AtZYP1b genes . However, unlike Arabidopsis , allohexaploid bread wheat appears to have only one ZYP1 gene, with copies on chromosome 2A, B and D. This was not unexpected as Higgins et al.  previously hypothesised that the two Arabidopsis ZYP1 genes were caused by a duplication event that occurred after the divergence of the Arabidopsis and brassica genera and that the AtZYP1 genes were more closely-related to each other than they were to the only ZYP1 homologue found in Brassica oleracea. More recent data supports this hypothesis, as both Oryza sativa and Zea mays have been reported to possess only a single ZYP1 homologue [6, 16]. Although TaZYP1 has been physically mapped to chromosome group 2 using the nullisomic/tetrasomic Southern blot assays, further research must be conducted to determine which orthologue of TaZYP1 has been isolated and whether its orthologues on the other genomes are transcriptionally active as well as whether their protein products are functionally redundant. The observation that the sequence of ZYP1 was not highly-conserved between other non-plant species was not unexpected as SC proteins have been previously reported to share conserved function but lack sequence conservation .
ZYP1 proteins form rod-like homodimers that are capped at the ends by globular N- and C-termini with the globular N-terminus necessary for ZYP1 homodimer formation . ZYP1 molecules attach to the lateral elements of sister chromosomes via their C-termini and extend outward allowing the N-termini to interact with one another thus forming the central element of the SC [reviewed by 10; and references therein]. Amino acid sequence analysis of Ta ZYP1 revealed that both the N and C-termini of the protein are basic with high pI values, while predictive protein modeling revealed that the central region of Ta ZYP1 has a coiled-coil central region. The high pI value of the C-terminus is particularly interesting as it may allow the lysine and arginine residues within it to bind DNA when the physiological pH of the cell is more acidic than the pI of the C-termini (which is 10.03). Positively-charged lysine and arginine residues have previously been shown to bind DNA in a non-specific manner in leucine zipper and helix-loop-helix motifs [17, 18]. In addition, the presence of a S/TPXX motif, previously characterised by Suzuki , within the Ta ZYP1 C-terminus provides further evidence that the Ta ZYP1 should also have a similar localisation pattern and arrangement within the SC as its previously characterised homologues. Furthermore, cDART database searches with Ta ZYP1 amino acid sequence returned moderate hits to two partial SMC conserved domains previously characterised in archaea (data not shown). Both these domains have previously been reported in archaea proteins that interact with DNA [20, 21], and these domains also appear to be present in higher eukaryotic species .
To determine whether Ta ZYP1 binds to DNA, purified Ta ZYP1 protein heterologously-expressed in E. coli was used in competitive DNA-binding assays. The equal levels of retardation of both the ss- and dsDNA species indicates that Ta ZYP1 is able to bind both species of DNA indiscriminately under in vitro conditions (Figure 2). The observation that Ta ZYP1 is able to interact with both ss- and dsDNA was not unexpected as studies in both mice and C. elegans zyp1 homologue deletion mutants have shown that the recruitment of the ZYP1 homologues to DNA recombination sites is essential for the unloading of the recombination machinery during meiosis within both organisms [7, 23]. Under wild-type conditions in mice and C. elegans, loading of the ZYP1 signal precedes the removal of the RAD51 foci signal (thus indicating that ZYP1 recruitment to regions of chromatin with RAD51 recombinase, and hence regions of DNA where single-stranded DNA invasion have occurred, is required before the removal of the recombination machinery). Furthermore, work conducted by Higgins and colleagues  on the Arabidopsis spo11 and dmc1 knock-out mutants show that the Arabidopsis ZYP1 homologues failed to localise to chromatin in the spo11 mutant but were localised as foci to sites of unsynapsed chromosome axes in the dmc1 mutant. Taken together, these data indicate that ZYP1 localisation to chromatin in Arabidopsis is dependent on double-stranded break (DSB) formation but independent of recombination initiation, thus inferring that ZYP1 is most likely recruited to sites of recombination during the very early stages of single-strand DNA invasion while ZYP1 polymerisation along the length of the chromosomes is dependent on recombination occurring .
Q-PCR profiling showed that TaZYP1 expression exhibits the profile of a protein typically required during early meiosis (Figure 3A). The relatively high expression level during the stages of pre-meiosis through to pachytene correlated well with observations in the immunofluorescence localisation assays where Ta ZYP1 signal was first observed in leptotene and peaked at pachytene (Figures 4 and 5; row 1). The reduction in expression post-pachytene was also reflected in the immunolocalisation loading with the Ta ZYP1 signal quickly diminishing post-pachytene (Figures 4 and 5; row 1). Comparisons of the TaZYP1 expression profiles between the wild-type and ph1b mutant showed that while the overall expression profiles were similar, transcript levels were elevated in the ph1b mutant at all stages examined (Figure 3A). Whether this increased TaZYP1 expression can be linked to the deletion of the Ph1 locus or whether it is a physiological reaction to the increased Ta ASY1 protein levels within the ph1b mutant, is yet to be determined. However, TaZYP1 expression was not affected in the Taasy1 mutants (Figure 3B).
Although Ta ZYP1 and Ta ASY1 were both present on chromatin, they did not co-localise (Figures 4 and 5). Even in the few instances where both signals were observed on the same regions of chromatin simultaneously, no co-localisation signals were detected. This observation indicates that Ta ZYP1, like its homologues in other species [4–6], appears to be a transverse filament protein and would therefore not be expected to co-localise with Ta ASY1 (which associates with the axial and lateral elements of chromatin). However, we also observed Ta ZYP1 signal being loaded onto regions of chromatin where Ta ASY1 was being unloaded. Interestingly, this aspect of the Ta ZYP1 localisation pattern is in contrast to the localisation patterns of its homologues in Arabidopsis thaliana and Secale cereale, where the ZYP1 homologues are load onto the chromatin while ASY1 signal is still present, forming a co-aligned ZYP1 signal that is sandwiched between the ASY1 signal associated to axial elements on both sides [4, 5]. The delayed lengthening of the Ta ZYP1 signal along the chromatin may suggest that Ta ZYP1 could have an additional and as yet undefined role during the process of chromosome synapsis or that chromosome synapsis in bread wheat occurs through a slightly different mechanism compared to other plant species. It is also noteworthy that the rye ZYP1 homologue signal already appears as linear tracts during leptotene , unlike the punctate foci observed during the leptotene stage of both Arabidopsis , rice  and bread wheat in this study. This clearly shows that differences are present within the temporal loading patterns of the ZYP1 protein in different species.
The presence of relatively high levels of Ta ASY1 signal in meiocytes of the ph1b and Taasy1 mutants during diplotene (Figure 4; row 2 and Figure 5; rows 2-5) is also of interest as the Arabidopsis ZYP1 signal fails to lengthen in the asy1 knock-down mutant, possibly indicating that ASY1 may directly/indirectly mediate ZYP1 loading onto chromatin . These increased levels of Ta ASY1 observed are probably the result of different mechanisms; as Ta ASY1 protein levels are significantly higher in the ph1b mutant but significantly lower in the Taasy1 knock-down mutants . Boden and colleagues  previously hypothesised that the Ta ASY1 signal seen in ph1b diplotene meiocytes could be a result of too much Ta ASY1 protein present within the cells thus requiring more time for the cellular machinery to unload the Ta ASY1 molecules which leads to persistent Ta ASY1 signal in later stages of meiosis. In contrast, the persisting Ta ASY1 signal in the Taasy1 mutants could be explained by a cellular compensation mechanism that is delaying the unloading of the Ta ASY1 protein. Previous work in Taasy1 and asy1 homologue mutants have shown that chromosomes in these mutants lack a true pachytene stage and that levels of chromosome synapsis were reduced [14, 24, 25]. Perhaps cell cycle check-point mechanisms are trying to lengthen the duration for which Ta ASY1 is loaded onto the axial elements to allow proper synapsis to occur along the lengths of the homologous chromosomes. This then raises the question of whether Ta ZYP1 loading is completed within the Taasy1 mutants due to presence of Ta ASY1 signal in the diplotene stage where Ta ZYP1 signal was no longer observed. Work conducted in the Arabidopsis asy1 mutant previously showed that the At ZYP1 homologues are loaded onto the chromatin as foci during leptotene but subsequently fail to lengthen into linear tracts . This again is at odds with the observations in this study which clearly show that the Ta ZYP1 signal still forms linear tracts in the Taasy1 mutants analysed. One possible explanation for this is the variation in severity of the asy1 knock-down effect in the mutants [14, 26]. While the ASY1 gene expression data reported in both species are incomparable due to different gene expression analysis techniques being used, inferences could be made about the severity of the ASY1 knock-down effect by analysis of the fertility levels reported. While the Arabidopsis asy1 mutant has a more severe knock-down effect with only 10% fertility, the average fertility of the Taasy1 T2 mutants was 77.74%. This possibly indicates that higher levels of ASY1 protein were present in the Taasy1 mutants compared to that in the Atasy1 mutant thus leading to a less severe effect on Ta ZYP1 loading in the Taasy1 mutant meiocytes.
In conclusion, this study has isolated and characterised the bread wheat ZYP1 homologue to further the current knowledge-base of bread wheat meiosis as well as to determine the role of ZYP1 in a complex hexaploid. Characterisation of Ta ZYP1 in vitro determined its DNA-binding capabilities while dual immunofluorescence localisation experiments with a polyclonal anti-Ta ZYP1 antibody on meiocytes of Chinese Spring and Bob White MPB26 wild-types as well as the ph1b and four Taasy1 mutants detailed its localisation profile during the early stages of meiosis both temporally and spatially. The localisation profile of Ta ZYP1 showed both similarities as well as differences with its previously characterised homologues in other species highlighting the need to further study this protein on a species-specific basis. While characterisation of Ta ZYP1 as a component of the SC has been achieved, further work is required to fully unravel the localisation profile of Ta ZYP1 and its other possible functions during the early stages of meiosis in bread wheat given the delayed lengthening of the Ta ZYP1 localisation signal with respect to Ta ASY1, in both wild-type and mutants (ph1b and Taasy1), compared to the localisation profiles of ZYP1 homologues previously reported in other species.
Hexaploid wheat (Triticum aestivum L.) cv. Chinese Spring plants, four Taasy1 RNAi knock-down mutants (Taasy1-1.9, Taasy1-1.9.2, Taasy1-2.2, and Taasy1-2.2.2) and a Chinese Spring mutant lacking the Ph1 locus (ph1b) were grown in a glasshouse with a 16/8 h photoperiod at 23°C. Harvesting and staging of meiotic anthers from both wild-type and mutant plants, for quantitative real-time PCR (Q-PCR) and fluorescence immunolocalisation, were conducted as per Boden et al. (2009). Whole meiotic spike tissue was collected for the isolation and amplification of the cDNA Triticum aestivum ZYP1 (TaZYP1) sequence.
RNA isolation and cDNA synthesis
Collected tissues for RNA isolation were initially ground in liquid nitrogen. Total RNA was extracted using Trizol reagent (Gibco-BRL, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentration was determined using a Nanodrop (ND-1000) (Nanodrop, Wilmington, DE, USA). Total cDNA libraries for the wild-type and all the mutants analysed were synthesised from 2 μg of total RNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. A Chinese Spring 3' RACE library was also synthesised from 2.5 μg of Chinese Spring total RNA per reaction using a GeneRacer Kit (Invitrogen) according to the manufacturer's instructions.
cDNA amplification and sequencing of the ZYP1 coding sequence
Primers (see Additional file 1: Table S1) for isolating the TaZYP1 ORF were designed using the OsZYP1 sequence [TIGR Rice Annotation number: LOC_Os04g37960] (that showed sequence similarity to the Arabidopsis ZYP1a and ZYPb sequences) identified through a TIGR rice expressed sequence tag (EST) BLAST search (accessed 21st October 2008).
Each PCR contained 100 ng cDNA, 0.2 mM dNTPs, 0.2 μM primers, 1U FastStart high fidelity Taq polymerase (Roche Applied Science, Mannheim, Germany) in 25 μL of 1× high fidelity buffer supplemented with 1× GC-RICH solution (Roche). PCR products were cloned into pCR8/GW/TOPO (Invitrogen) for DNA sequencing (15× coverage). Sequencing PCR was conducted using the GW1 and GW2 primers (see Additional file 1: Table S1). Secondary sets of primers were designed on the sequenced products to specifically amplify the TaZYP1 ORF. Amplification and sequencing was repeated as above. PCR cycling parameters were denaturation at 95°C for 5 min, followed by 35 cycles of 96°C for 30 s, Tm°C for 30 s, 72°C for 2 min, with a final elongation step at 72°C for 10 min (see Additional file 1: Table S1 for Tm of primer sets). The assigned NCBI accession number for TaZYP1 is JN129259.
DNA sequence alignments and comparisons were conducted with AlignX and Contig Express (Informax, VNTI Advance, Version 11, Frederick, MC, USA) software programs. VNTI software was also used to predict the molecular weight and pI of the protein. Detection for conserved domains was performed using the NCBI Conserved Domain Search Tool (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), InterPro Scan (http://www.ebi.ac.uk/Tools/InterProScan/) and Pfam 23.0 (http://pfam.janelia.org/). Amino acid alignments and comparisons of full-length ZYP1 sequences (obtained from various BLAST searches using the NCBI, TIGR, and PredictProtein [http://www.predictprotein.org/;] databases), and subsequent construction of the phylogenetic tree (neighbour-joining method)  was completed using Molecular Evolutionary Genetics Analysis (MEGA) software (version 4.0) . Default parameters were used except for the following: the pair-wise deletion option was used, the internal branch test bootstrap value was set at 10,000 re-samplings, and the model setting was amino acid: Poisson correction with predicted gamma parameters set at 2.0. Accession numbers of the sequences used were: Ta - Ta ZYP1 [GenBank: JN129259]; Bd [Phytozome: Bradi5g12010]; Os [TIGR Rice Annotation Number: LOC_Os04g0452500]; Zm [GenBank: HQ116413]; Rc [GenBank: XP_002513917]; Vv [UniProt/TrEMBL: CBI19158.1]; At-a [GenBank: NP_173645]; At-b [GenBank: NP_564164]; Bo [GenBank: ABO69625]; Mm [GenBank: NP_035646]; and Sc [GenBank: NP_013498].
Southern blot hybridisation
A 561 bp fragment of the TaZYP1 gene was used as the template for the synthesis of an α-32P dCTP labelled probe that was hybridised to a Chinese Spring nullisomic-tetrasomic membrane as per Lloyd et al. . Autoradiography films were developed using an AGFA CP1000 Developer (AGFA, Nunawading, VIC, Australia).
Q-PCR was conducted in triplicate according to Crismani et al. . Amplification of products was completed using gene specific Q-PCR primers (see Additional file 1: Table S1). The optimal acquisition temperature for TaZYP1 was 79°C. Statistical analysis to determine the mean ± SE of the three replicate experiments was conducted with GenStat software (version 11.0, Numerical Algorithms Group).
The TaZYP1 insert within the pCR8/GW/TOPO vector was cloned into a pDEST17 expression plasmid (Invitrogen) according to the manufacturer's LR clonase protocol. BL-21 A1 E. coli were transformed with the pDEST17-TaZYP1 ORF vector, and protein production was induced with 0.4% L-(+)-arabinose (w/v) (Sigma-Aldrich, St Louis, MO, USA). Protein isolation and purification was performed using nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen, Clifton, VIC, Australia) according to the manufacturer's extraction protocols. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using NuPAGE Novex 4-12% Bis-Tris 7 cm mini-gels (Invitrogen) according to the manufacturer's protocol. Staining and destaining of gels were performed as previously reported .
The identity of the recombinant Ta ZYP1 protein was confirmed by ion trap liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-MS/MS). Gel slices containing the recombinant Ta ZYP1 protein were washed with 100 mM ammonium bicarbonate, dried, rehydrated with 100 mM ammonium carbonate and subjected to in-gel tryptic digestion. LC-MS/MS of the digested peptides was then conducted as reported by March et al. .
Polyclonal antibody production
The amino acid sequence of Ta ZYP1 was assessed for hydrophobic and antigenic regions using Kite-Doolittle and Hopp plots. The peptide sequence chosen for peptide synthesis was: CLRAYHKEELQRIRS. The peptide was synthesised by Mimetopes (Mimetopes, Clayton, Victoria, Australia) and conjugated to keyhole limpet using a hemocyanin maleimidocaproyl-N-hydroxysuccinimide (MCS) linker. The Ta ZYP1 peptide antigen was delivered to Institute of Medical and Veterinary Science for immunisation of two rats (Rattus rattus). The antigen was dissolved in 1× PBS (10 μg μL-1), added with an equivalent amount of Freund's complete adjuvant (Sigma-Aldrich) and used for primary immunisation of two rats via subcutaneous injection. Three subsequent immunisations were administered in three-week intervals, with Freund's incomplete adjuvant (Sigma-Aldrich) added to the dissolved antigen in 1× PBS. All immunisation doses contained 200 μg of Ta ZYP1 antigen. Immune sera was collected 10.5 weeks after the first injection.
Competitive DNA binding assay
Recombinant full length Ta ZYP1 extracted under native conditions was quantified using the Bradford assay . Competitive DNA binding assays were conducted as described by Pezza et al.  with modifications as per Khoo et al. . The DNA binding abilities of Ta ZYP1 was tested with ΦX174 circular single-stranded DNA (ssDNA) (virion) (30 μM per nucleotide) (New England Biolabs, Beverly, MA, USA) and ΦX174 linear double-stranded DNA (dsDNA) (RFI form Pst1-digested) (15 μM per base pair) (New England Biolabs).
Fluorescence immunolocalisation of Ta ASY1 and Ta ZYP1 was performed as per Franklin et al.  and Boden et al.  with the following changes: anthers were fixed with 2% paraformaldehyde and cells permeabilised for 3 h. For detecting the localisation pattern of Ta ZYP1, a rat anti-Ta ZYP1 antibody (1:100) and an AlexaFluor® 488 conjugated donkey anti-rat antibody (1:50; Molecular Probes, Invitrogen) was used. Optical sections (90-120 per nucleus) of meiocytes were collected using a Leica TCS SP5 Spectral Scanning Confocal Microscope (Leica Microsystems, http://www.leica-microsystems.com/) equipped with an oil immersion HCX Plan Apochromat 63 ×/1.4 lens, a 405 nm pulsed laser and an Argon laser using an excitation wavelength of 468 nm. Images were processed using Leica Application Suite Advanced Fluorescence (LAS-AF; version 1.8.2, build 1465, Leica Microsystems) software to generate maximum intensity projections of each nucleus.
This research was supported in part by the Australian government under the Australia-India Strategic Research Fund (AISRF) and the School of Agriculture, Food & Wine, The University of Adelaide. KHPK was supported by an Australian Postgraduate Award (APA).
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