The isolation and characterisation of the wheat molecular ZIPper I homologue, TaZYP1

Extent: 13p.Background: 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. Results: 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. TaZYP1 has a putative DNA-binding S/TPXX motif in its C-terminal region and we provide evidence that TaZYP1 interacts non-preferentially with both single- and double-stranded DNA in vitro. 3-dimensional dual immunofluorescence localisation assays conducted with an antibody raised against TaZYP1 show that TaZYP1 interacts with chromatin during meiosis but does not co-localise to regions of chromatin where TaASY1 is present. The TaZYP1 signal lengthens into regions of chromatin where TaASY1 has been removed in wild-type but this appears delayed in the ph1b mutant. The localisation profile of TaZYP1 in four Taasy1 knock-down mutants is similar to wild-type but TaZYP1 signal intensity appears weaker and more diffused. Conclusions: 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 TaZYP1 signal only lengthens into regions of chromatin after TaASY1 signal is being unloaded. However, the observation that TaZYP1 loading appears delayed in both the ph1b and Taasy1 mutants suggests that TaASY1 may still be essential for TaZYP1 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.Kelvin HP Khoo, Amanda J Able and Jason A Abl

Poor Homologous Synapsis 1 Interacts with Chromatin but Does Not Colocalise with ASYnapsis 1 during Early Meiosis in Bread Wheat

Chromosome pairing, synapsis, and DNA recombination are three key processes that occur during early meiosis. A previous study of Poor Homologous Synapsis 1 (PHS1) in maize suggested that PHS1 has a role in coordinating these three processes. Here we report the isolation of wheat (Triticum aestivum) PHS1 (TaPHS1), and its expression profile during and after meiosis. While the TaPHS1 protein has sequence similarity to other plant PHS1/PHS1-like proteins, it also possesses a unique region of oligopeptide repeat units. We show that TaPHS1 interacts with both single- and double-stranded DNA in vitro and provide evidence of the protein region that imparts the DNA-binding ability. Immunolocalisation data from assays conducted using antisera raised against TaPHS1 show that TaPHS1 associates with chromatin during early meiosis, with the signal persisting beyond chromosome synapsis. Furthermore, TaPHS1 does not appear to colocalise with the asynapsis protein (TaASY1) suggesting that these proteins are probably independently coordinated. Significantly, the data from the DNA-binding assays and 3-dimensional immunolocalisation of TaPHS1 during early meiosis indicates that TaPHS1 interacts with DNA, a function not previously observed in either the Arabidopsis or maize PHS1 homologues. As such, these results provide new insight into the function of PHS1 during early meiosis in bread wheat

Comparative Transcriptomics Reveals 129 Transcripts That Are Temporally Regulated during Anther Development and Meiotic Progression in Both Bread Wheat (Triticum aestivum) and Rice (Oryza sativa)

Meiosis is a specialised type of cell division in sexually reproducing organisms that generates genetic diversity and prevents chromosome doubling in successive generations. The last decade has seen forward and reverse genetic approaches identifying many genes in the plant kingdom which highlight similarities and differences in the mechanics of meiosis between taxonomic kingdoms. We present here a high throughput in silico analysis, using bread wheat and rice, which has generated a list of 129 transcripts containing genes with meiotic roles and some which are currently unknown

TaMSH7: A cereal mismatch repair gene that affects fertility in transgenic barley (Hordeum vulgare L.)

Background: Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family. Results: Sequencing of the three MSH7 genes, located on the short arms of wheat chromosomes 3A, 3B and 3D, has revealed no significant sequence divergence at the amino acid level suggesting conservation of function across the homoeogroups. Functional analysis of MSH7 through the use of RNAi loss-of-function transgenics was undertaken in diploid barley (Hordeum vulgare L.). Quantitative real-time PCR revealed several T0 lines with reduced MSH7 expression. Positive segregants from two T1 lines studied in detail showed reduced MSH7 expression when compared to transformed controls and null segregants. Expression of MSH6, another member of the mismatch repair family which is most closely related to the MSH7 gene, was not significantly reduced in these lines. In both T1 lines, reduced seed set in positive segregants was observed. Conclusion: Results presented here indicate, for the first time, a distinct functional role for MSH7 in vivo and show that expression of this gene is necessary for wild-type levels of fertility. These observations suggest that MSH7 has an important function during meiosis and as such remains a candidate for Ph2.Andrew H Lloyd, Andrew S Milligan, Peter Langridge, and Jason A Abl

Expression and functional analysis of TaASY1 during meiosis of bread wheat (Triticum aestivum)

Background: Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. Results: The full length wheat cDNA and genomic clone, TaASY1, has been isolated, sequenced and characterised. TaASY1 is located on chromosome Group 5 and the open reading frame displays significant nucleotide sequence identity to OsPAIR2 (84%) and AtASY1 (63%). Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occur. Immunolocalisation using transmission electron microscopy showed TaASY1 interacts with chromatin that is associated with both axial elements before SC formation as well as lateral elements of formed SCs. Conclusion: TaASY1 is a homologue of ScHOP1, AtASY1 and OsPAIR2 and is the first gene to be isolated from bread wheat that is involved in pairing and synapsis of homologous chromosomes.Scott A Boden, Nadim Shadiac, Elise J Tucker, Peter Langridge and Jason A Abl

Analysis of anther transcriptomes to identify genes contributing to meiosis and male gametophyte development in rice

Extent: 20p.Background: In flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development. Results: Transcriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far. Conclusions: Not only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation.Priyanka Deveshwar, William D Bovill, Rita Sharma, Jason A Able and Sanjay Kapoo

Microarray expression analysis of meiosis and microsporogenesis in hexaploid bread wheat

BACKGROUND: Our understanding of the mechanisms that govern the cellular process of meiosis is limited in higher plants with polyploid genomes. Bread wheat is an allohexaploid that behaves as a diploid during meiosis. Chromosome pairing is restricted to homologous chromosomes despite the presence of homoeologues in the nucleus. The importance of wheat as a crop and the extensive use of wild wheat relatives in breeding programs has prompted many years of cytogenetic and genetic research to develop an understanding of the control of chromosome pairing and recombination. The rapid advance of biochemical and molecular information on meiosis in model organisms such as yeast provides new opportunities to investigate the molecular basis of chromosome pairing control in wheat. However, building the link between the model and wheat requires points of data contact. RESULTS: We report here a large-scale transcriptomics study using the Affymetrix wheat GeneChip(® )aimed at providing this link between wheat and model systems and at identifying early meiotic genes. Analysis of the microarray data identified 1,350 transcripts temporally-regulated during the early stages of meiosis. Expression profiles with annotated transcript functions including chromatin condensation, synaptonemal complex formation, recombination and fertility were identified. From the 1,350 transcripts, 30 displayed at least an eight-fold expression change between and including pre-meiosis and telophase II, with more than 50% of these having no similarities to known sequences in NCBI and TIGR databases. CONCLUSION: This resource is now available to support research into the molecular basis of pairing and recombination control in the complex polyploid, wheat

A major root architecture QTL responding to water limitation in durum wheat

The optimal root system architecture (RSA) of a crop is context dependent and critical for efficient resource capture in the soil. Narrow root growth angle promoting deeper root growth is often associated with improved access to water and nutrients in deep soils during terminal drought. RSA, therefore is a drought-adaptive trait that could minimize yield losses in regions with limited rainfall. Here, GWAS for seminal root angle (SRA) identified seven marker-trait associations clustered on chromosome 6A, representing a major quantitative trait locus (qSRA-6A) which also displayed high levels of pairwise LD (r2 = 0.67). Subsequent haplotype analysis revealed significant differences between major groups. Candidate gene analysis revealed loci related to gravitropism, polar growth and hormonal signaling. No differences were observed for root biomass between lines carrying hap1 and hap2 for qSRA-6A, highlighting the opportunity to perform marker-assisted selection for the qSRA-6A locus and directly select for wide or narrow RSA, without influencing root biomass. Our study revealed that the genetic predisposition for deep rooting was best expressed under water-limitation, yet the root system displayed plasticity producing root growth in response to water availability in upper soil layers. We discuss the potential to deploy root architectural traits in cultivars to enhance yield stability in environments that experience limited rainfall

Nitrogen Starvation-Responsive MicroRNAs Are Affected by Transgenerational Stress in Durum Wheat Seedlings

Stress events have transgenerational effects on plant growth and development. In Mediterranean regions, water-deficit and heat (WH) stress is a frequent issue that negatively affects crop yield and quality. Nitrogen (N) is an essential plant macronutrient and often a yield-limiting factor for crops. Here, the response of durum wheat seedlings to N starvation under the transgenerational effects of WH stress was investigated in two genotypes. Both genotypes showed a significant reduction in seedling height, leaf number, shoot and root weight (fresh and dry), primary root length, and chlorophyll content under N starvation stress. However, in the WH stress-tolerant genotype, the percentage reduction of most traits was lower in progeny from the stressed parents than progeny from the control parents. Small RNA sequencing identified 1534 microRNAs in different treatment groups. Differentially expressed microRNAs (DEMs) were characterized subject to N starvation, parental stress and genotype factors, with their target genes identified in silico. GO and KEGG enrichment analyses revealed the biological functions, associated with DEM-target modules in stress adaptation processes, that could contribute to the phenotypic differences observed between the two genotypes. The study provides the first evidence of the transgenerational effects of WH stress on the N starvation response in durum wheat