The NAM-B1 transcription factor and the control of grain composition in wheat

The NAM-B1 transcription factor increases grain protein content, alters grain micronutrient content and accelerates monocarpic senescence, often without imposing a yield penalty. The aim of this thesis was to understand the mechanisms by which NAM-B1 influences nutrient remobilisation and monocarpic senescence to cause these effects. To achieve this I have examined the expression patterns of NAM-B1 and its homologues during development. I have studied the effects of NAM-B1 on nutrient transport, photosynthetic capacity and grain filling using a range of molecular biology and physiological techniques. Finally to understand the network of genes which NAM-B1 regulates I have used chromatin-immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify downstream targets, and compared these to differentially expressed genes in plants with down-regulated expression of NAM-B1 homologues (NAM RNAi plants). I have found that NAM-B1 expression increases after anthesis in both vegetative and reproductive tissues, including the grain. In stem and leaf tissues I identified that NAM genes are highly expressed in the vascular bundles, which might be important for nutrient transport. However I did not find evidence for NAM genes altering xylem or phloem transport. I found that in NAM RNAi plants, grain development was decoupled from flag leaf senescence. In RNAi plants starch synthesis enzymes were less active during the middle of grain filling than in control plants, potentially resulting in the reallocation of photosynthate to the stems as water soluble carbohydrates. Many of the putative NAM-B1 target genes identified by ChIP-seq have functions related to photosynthesis and validation of these candidate genes is ongoing. In summary I have identified putative NAM-B1 target genes and found that NAM-B1 may act in a tissue specific manner to regulate monocarpic senescence and grain filling. Furthermore I have highlighted novel functions related to carbohydrate metabolism in stems and the grain

Genome-Wide Sequence and Expression Analysis of the NAC Transcription Factor Family in Polyploid Wheat

Many important genes in agriculture correspond to transcription factors (TFs) that regulate a wide range of pathways from flowering to responses to disease and abiotic stresses. In this study, we identified 5776 TFs in hexaploid wheat (Triticum aestivum) and classified them into gene families. We further investigated the NAC family exploring the phylogeny, C-terminal domain (CTD) conservation, and expression profiles across 308 RNA-seq samples. Phylogenetic trees of NAC domains indicated that wheat NACs divided into eight groups similar to rice (Oryza sativa) and barley (Hordeum vulgare). CTD motifs were frequently conserved between wheat, rice, and barley within phylogenetic groups; however, this conservation was not maintained across phylogenetic groups. Three homeologous copies were present for 58% of NACs, whereas evidence of single homeolog gene loss was found for 33% of NACs. We explored gene expression patterns across a wide range of developmental stages, tissues, and abiotic stresses. We found that more phylogenetically related NACs shared more similar expression patterns compared to more distant NACs. However, within each phylogenetic group there were clades with diverse expression profiles. We carried out a coexpression analysis on all wheat genes and identified 37 modules of coexpressed genes of which 23 contained NACs. Using gene ontology (GO) term enrichment, we obtained putative functions for NACs within coexpressed modules including responses to heat and abiotic stress and responses to water: these NACs may represent targets for breeding or biotechnological applications. This study provides a framework and data for hypothesis generation for future studies on NAC TFs in wheat

Genome-Wide Transcription During Early Wheat Meiosis Is Independent of Synapsis, Ploidy Level, and the Ph1 Locus

Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologs and homoeologs at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-seq data derived from six different genotypes: wheat, wheat–rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the Ph1 locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologs is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the Ph1 locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. Further studies in wheat and other polyploid species will be required to reveal whether these observations are specific to wheat meiosis