Jason A. Able

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

BackgroundIn 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.ResultsTranscriptome 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.ConclusionsNot 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.

Microarray expression analysis of meiosis and microsporogenesis in hexaploid bread wheat.

BackgroundOur 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.ResultsWe 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.ConclusionThis resource is now available to support research into the molecular basis of pairing and recombination control in the complex polyploid, wheat.

The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum

SummaryThis report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding β-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (rs=0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actinl and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actinl and CaMV 35S constructs. Stable callus sectors (on 2 mg 1−1 bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.

TaASY1 promotes homologous chromosome interactions and is affected by deletion of Ph1.

During meiosis, chromosomes are sorted into homologous pairs as a preface to their intimate association via recombination and synapsis. However, little is known about the mechanism used to distinguish homologous chromosomes from other chromosomes present in the nucleus. Studies in wheat (Triticum aestivum) have shown that the Pairing homoeologous 1 (Ph1) locus is required to suppress interactions between genetically similar homoeologous chromosomes. Here we show that absence of Ph1 causes increased transcription of Asynapsis 1 (ASY1), a gene that encodes an axial-element-associated protein that is essential for synapsis and cross-over formation in Arabidopsis and rice. Localisation of ASY1 during meiosis is also affected by deletion of Ph1. In addition, transgenic wheat mutants with decreased activity of TaASY1 display reduced synapsis during prophase I and exhibit pairing between homoeologous chromosomes at metaphase I. These results suggest that ASY1 is required to promote interactions between homologous chromosomes in bread wheat, and that Ph1 has a gene regulatory role, which is consistent with its suggested genetic identity as a Cdk-like gene. Broader implications of this research suggest that we could use the Taasy1 mutants to assess their efficacy in alien chromatin introgression studies, as seen with the ph1b mutant.

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

BackgroundChromosome 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.ResultsSequencing 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.ConclusionResults 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.

Genome-Wide Identification of MicroRNAs in Leaves and the Developing Head of Four Durum Genotypes during Water Deficit Stress.

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in plant development and abiotic stress responses. The miRNA transcriptome (miRNAome) under water deficit stress has been investigated in many plant species, but is poorly characterised in durum wheat (Triticum turgidum L. ssp. durum). Water stress during early reproductive stages can result in significant yield loss in durum wheat and this study describes genotypic differences in the miRNAome between water deficit tolerant and sensitive durum genotypes. Small RNA libraries (96 in total) were constructed from flag leaf and developing head tissues of four durum genotypes, with or without water stress to identify differentially abundant miRNAs. Illumina sequencing detected 110 conserved miRNAs and 159 novel candidate miRNA hairpins with 66 conserved miRNAs and five novel miRNA hairpins differentially abundant under water deficit stress. Ten miRNAs (seven conserved, three novel) were validated through qPCR. Several conserved and novel miRNAs showed unambiguous inverted regulatory profiles between the durum genotypes. Several miRNAs also showed differential abundance between two tissue types regardless of treatment. Predicted mRNA targets (130) of four novel durum miRNAs were characterised using Gene Ontology (GO) which revealed functions common to stress responses and plant development. Negative correlation was observed between several target genes and the corresponding miRNA under water stress. For the first time, we present a comprehensive study of the durum miRNAome under water deficit stress. The identification of differentially abundant miRNAs provides molecular evidence that miRNAs are potential determinants of water stress tolerance in durum wheat. GO analysis of predicted targets contributes to the understanding of genotypic physiological responses leading to stress tolerance capacity. Further functional analysis of specific stress responsive miRNAs and their interaction with targets is ongoing and will assist in developing future durum wheat varieties with enhanced water deficit stress tolerance.

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

BackgroundPairing 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.ResultsThe 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 Ta ASY1 interacts with chromatin that is associated with both axial elements before SC formation as well as lateral elements of formed SCs.ConclusionTaASY1 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.

Cotton bunchy top: an aphid and graft transmitted cotton disease

A new disease, termed cotton bunchy top (CBT), has been observed in Australian cotton fields since the 1998–99 cotton-growing season. Symptoms included short petioles and internodes, pale, light-green, angular patterns on the leaf margins, and a leathery texture of mature leaves. Affected plants had a reduced photosynthetic rate, leaf area, plant height, number of bolls, dry weight of bolls, roots and stem and ultimately yield. CBT was demonstrated to be graft-transmissible in glasshouse experiments. In the field, CBThotspots appeared to correlate with cotton aphid (Aphis gossypii) density and this species was identified as a CBT vector in controlled transmission tests. CBT symptoms and plant responses recorded in graft and aphid-inoculated plants were similar to those seen in the field. Seed transmission of CBT appears unlikely as none of 3930 plants grown from seed of CBT-affected plants developed symptoms.

Translating knowledge about abiotic stress tolerance to breeding programmes

Plant breeding and improvements in agronomic practice are making a consistent contribution to increasing global crop production year upon year. However, the rate of yield improvement currently lags behind the targets set to produce enough food to meet the demands of the predicted global population in 2050. Furthermore, crops that are exposed to harmful abiotic environmental factors (abiotic stresses, e.g. water limitation, salinity, extreme temperature) are prone to reduced yields. Here, we briefly describe the processes undertaken in conventional breeding programmes, which are usually designed to improve yields in near-optimal conditions rather than specifically breeding for improved crop yield stability under stressed conditions. While there is extensive fundamental research activity that examines mechanisms of plant stress tolerance, there are few examples that apply this research to improving commercial crop yields. There are notable exceptions, and we highlight some of these to demonstrate the magnitude of yield gains that could be made by translating agronomic, phenological and genetic solutions focused on improving or mitigating the effect of abiotic stress in the field; in particular, we focus on improvements in crop water-use efficiency and salinity tolerance. We speculate upon the reasons for the disconnect between research and research translation. We conclude that to realise untapped rapid gains towards food security targets new funding structures need to be embraced. Such funding needs to serve both the core and collaborative activities of the fundamental, pre-breeding and breeding research communities in order to expedite the translation of innovative research into the fields of primary producers.

Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5′ RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.