High Resolution Analysis of Meiotic Chromosome Structure and Behaviour in Barley (Hordeum vulgare L.)

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM)

The role of the Arabidopsis small GTPase Arac7 (Rop9) in hormone signaling

Small GTP-binding proteins of the Rac family are important signaling switches that regulate plant growth and development due to their ability to shuttle between the inactive GDP-bound and the active GTP-bound form. The ratio between the two forms is tightly regulated in the cell. The Arabidopsis genome encodes eleven Rac proteins designated Arac1-11. Based on the C-terminus, Aracs can be divided into typeI and typeII. TypeI Aracs associate with the membrane by prenylation while typeII Aracs associate with the membrane by palmitoylation. Differences in the effector-binding region and the C-terminal hypervariable region place Arac7 in a separate phylogenetic group than the other typeII Aracs. The work described in this dissertation examines the expression patterns, functions and regulation of the Arac7 protein and provides evidence that supports a distinct role for Arac7 within type II Aracs. Arac7 expression is shown to repress auxin-induced gene expression and to enhance ABA-stimulated gene expression in Arabidopsis. Plants overexpressing Arac7 are less responsive to auxin but show increased responses to ABA while the opposite is observed in plants with decreased levels of Arac7 mRNA. Arac7 expression is high in the lateral root primordia and can be traced back to the first pericicle divisions that give rise to these primordia. This, together with the increased number of lateral roots of plants overexpressing Arac7, is consistent with a role in lateral root formation for this small GTPase. Interestingly, transcription from the Arac7 promoter is stimulated by auxin but repressed by ABA. Together with the observed functions on auxin and ABA signaling, the regulated expression of Arac7 provides a feedback mechanism to ensure that cells maintain sensitivity to signals. This work also shows that Arac7 is constitutively associated with the plasma membrane where it partitions into detergent-resistant membrane domains (DRMs). Moreover, at the plasma membrane, the majority of Arac7 partitions into high molecular weight complexes. These properties contrast with those observed for the typeI Arac5 which is distributed between the cytosol and non-DRMs regions of the plasma membrane and exists predominantly in low molecular weight forms. Together, these observations suggest different functions and regulatory mechanisms for typeI and typeII Aracs

Quantitative high resolution mapping of HvMLH3 foci in barley pachytene nuclei reveals a strong distal bias and weak interference

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species

FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase

Plant growth and development are controlled by a delicate balance of hormonal cues. Growth-promoting hormones and growth-inhibiting counterparts often antagonize each other in their action, but the molecular mechanisms underlying these events remain largely unknown. Here, we report a cross-talk mechanism that enables a receptor-like kinase, FERONIA (FER), a positive regulator of auxin-promoted growth, to suppress the abscisic acid (ABA) response through activation of ABI2, a negative regulator of ABA signaling. The FER pathway consists of a FER kinase interacting with guanine exchange factors GEF1, GEF4, and GEF10 that, in turn, activate GTPase ROP11/ARAC10. Arabidopsis mutants disrupted in any step of the FER pathway, including fer, gef1gef4gef10, or rop11/arac10, all displayed an ABA-hypersensitive response, implicating the FER pathway in the suppression mechanism. In search of the target for the FER pathway, we found that the ROP11/ARAC10 protein physically interacted with the ABI2 phosphatase and enhanced its activity, thereby linking the FER pathway with the inhibition of ABA signaling