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

Wheat NAC transcription factor <i>NAC5‐1</i> is a positive regulator of senescence

Wheat (Triticum aestivum L.) is an important source of both calories and protein in global diets, but there is a trade‐off between grain yield and protein content. The timing of leaf senescence could mediate this trade‐off as it is associated with both declines in photosynthesis and nitrogen remobilization from leaves to grain. NAC transcription factors play key roles in regulating senescence timing. In rice, OsNAC5 expression is correlated with increased protein content and upregulated in senescing leaves, but the role of the wheat ortholog in senescence had not been characterized. We verified that NAC5‐1 is the ortholog of OsNAC5 and that it is expressed in senescing flag leaves in wheat. To characterize NAC5‐1, we combined missense mutations in NAC5‐A1 and NAC5‐B1 from a TILLING mutant population and overexpressed NAC5‐A1 in wheat. Mutation in NAC5‐1 was associated with delayed onset of flag leaf senescence, while overexpression of NAC5‐A1 was associated with slightly earlier onset of leaf senescence. DAP‐seq was performed to locate transcription factor binding sites of NAC5‐1. Analysis of DAP‐seq and comparison with other studies identified putative downstream target genes of NAC5‐1 which could be associated with senescence. This work showed that NAC5‐1 is a positive transcriptional regulator of leaf senescence in wheat. Further research is needed to test the effect of NAC5‐1 on yield and protein content in field trials, to assess the potential to exploit this senescence regulator to develop high‐yielding wheat while maintaining grain protein content

Wheat NAC transcription factor <i>NAC5‐1</i> is a positive regulator of senescence

Wheat (Triticum aestivum L.) is an important source of both calories and protein in global diets, but there is a trade‐off between grain yield and protein content. The timing of leaf senescence could mediate this trade‐off as it is associated with both declines in photosynthesis and nitrogen remobilization from leaves to grain. NAC transcription factors play key roles in regulating senescence timing. In rice, OsNAC5 expression is correlated with increased protein content and upregulated in senescing leaves, but the role of the wheat ortholog in senescence had not been characterized. We verified that NAC5‐1 is the ortholog of OsNAC5 and that it is expressed in senescing flag leaves in wheat. To characterize NAC5‐1, we combined missense mutations in NAC5‐A1 and NAC5‐B1 from a TILLING mutant population and overexpressed NAC5‐A1 in wheat. Mutation in NAC5‐1 was associated with delayed onset of flag leaf senescence, while overexpression of NAC5‐A1 was associated with slightly earlier onset of leaf senescence. DAP‐seq was performed to locate transcription factor binding sites of NAC5‐1. Analysis of DAP‐seq and comparison with other studies identified putative downstream target genes of NAC5‐1 which could be associated with senescence. This work showed that NAC5‐1 is a positive transcriptional regulator of leaf senescence in wheat. Further research is needed to test the effect of NAC5‐1 on yield and protein content in field trials, to assess the potential to exploit this senescence regulator to develop high‐yielding wheat while maintaining grain protein content