Identifying the mechanistic basis to nitrogen responsiveness in two contrasting Setaria italica accessions.

Nitrogen (N) is a macronutrient limiting crop productivity with varied requirements across species and genotypes. Understanding the mechanistic basis of N responsiveness by comparing contrasting genotypes could inform the development and selection of varieties with lower N demands, or inform agronomic practices to sustain yields with lower N inputs. Given the established role of millets in ensuring climate-resilient food and nutrition security, we investigated the physiological and genetic basis of nitrogen responsiveness in foxtail millet (Setaria italica L.). We had previously identified genotypic variants linked to N responsiveness, and here, we dissect the mechanistic basis of the trait by examining the physiological and molecular behaviour of N responsive (NRp-SI58) and non-responsive (NNRp-SI114) accessions at high and low N. Under high N, NRp-SI58 allocates significantly more biomass to nodes, internodes and roots, more N to developing grains, and is more effective at remobilising flag leaf N compared to NNRp-SI114. Post anthesis flag leaf gene expression suggests that differences in N induce much higher transcript abundance in NNRp-SI114 than NRp-SI58, a large proportion of which are potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.

Prediction of near-term climate change impacts on UK wheat quality and the potential for adaptation through plant breeding.

Wheat is a major crop worldwide, mainly cultivated for human consumption and animal feed. Grain quality is paramount in determining its value and downstream use. While we know that climate change threatens global crop yields, a better understanding of impacts on wheat end-use quality is also critical. Combining quantitative genetics with climate model outputs, we investigated UK-wide trends in genotypic adaptation for wheat quality traits. In our approach, we augmented genomic prediction models with environmental characterisation of field trials to predict trait values and climate effects in historical field trial data between 2001 and 2020. Addition of environmental covariates, such as temperature and rainfall, successfully enabled prediction of genotype by environment interactions (G × E), and increased prediction accuracy of most traits for new genotypes in new year cross validation. We then extended predictions from these models to much larger numbers of simulated environments using climate scenarios projected under Representative Concentration Pathways 8.5 for 2050-2069. We found geographically varying climate change impacts on wheat quality due to contrasting associations between specific weather covariables and quality traits across the UK. Notably, negative impacts on quality traits were predicted in the East of the UK due to increased summer temperatures while the climate in the North and South-west may become more favourable with increased summer temperatures. Furthermore, by projecting 167,040 simulated future genotype-environment combinations, we found only limited potential for breeding to exploit predictable G × E to mitigate year-to-year environmental variability for most traits except Hagberg falling number. This suggests low adaptability of current UK wheat germplasm across future UK climates. More generally, approaches demonstrated here will be critical to enable adaptation of global crops to near-term climate change.

Trade-offs in the genetic control of functional and nutritional quality traits in UK winter wheat.

A complex network of trade-offs exists between wheat quality and nutritional traits. We investigated the correlated relationships among several milling and baking traits as well as mineral density in refined white and whole grain flour. Our aim was to determine their pleiotropic genetic control in a multi-parent population over two trial years with direct application to practical breeding. Co-location of major quantitative trait loci (QTL) and principal component based multi-trait QTL mapping increased the power to detect QTL and revealed pleiotropic effects explaining many complementary and antagonistic trait relationships. High molecular weight glutenin subunit genes explained much of the heritable variation in important dough rheology traits, although additional QTL were detected. Several QTL, including one linked to the TaGW2 gene, controlled grain size and increased flour extraction rate. The semi-dwarf Rht-D1b allele had a positive effect on Hagberg falling number, but reduced grain size, specific weight, grain protein content and flour water absorption. Mineral nutrient concentrations were lower in Rht-D1b lines for many elements, in wholemeal and white flour, but potassium concentration was higher in Rht-D1b lines. The presence of awns increased calcium content without decreasing extraction rate, despite the negative correlation between these traits. QTL were also found that affect the relative concentrations of key mineral nutrients compared to phosphorus which may help increase bioavailability without associated anti-nutritional effects of phytic acid. Taken together these results demonstrate the potential for marker-based selection to optimise trait trade-offs and enhance wheat nutritional value by considering pleiotropic genetic effects across multiple traits.

Defining the physiological determinants of low nitrogen requirement in wheat.

Nitrogen (N) is a major nutrient limiting productivity in many ecosystems. The large N demands associated with food crop production are met mainly through the provision of synthetic N fertiliser, leading to economic and ecological costs. Optimising the balance between N supply and demand is key to reducing N losses to the environment. Wheat (Triticum aestivum L.) production provides food for millions of people worldwide and is highly dependent on sufficient N supply. The size of the N sink, i.e. wheat grain (number, size, and protein content) is the main driver of high N requirement. Optimal functioning of temporary sinks, in particular the canopy, can also affect N requirement. N use efficiency (i.e. yield produced per unit of N available) tends to be lower under high N conditions, suggesting that wheat plants are more efficient under low N conditions and that there is an optimal functioning yet unattained under high N conditions. Understanding the determinants of low N requirement in wheat would provide the basis for the selection of genetic material suitable for sustainable cereal production. In this review, we dissect the drivers of N requirement at the plant level along with the temporal dynamics of supply and demand.

Impairment in karrikin but not strigolactone sensing enhances root skewing in Arabidopsis thaliana.

Roots form highly complex systems varying in growth direction and branching pattern to forage for nutrients efficiently. Here mutations in the KAI2 (KARRIKIN INSENSITIVE) α/ß-fold hydrolase and the MAX2 (MORE AXILLARY GROWTH 2) F-box leucine-rich protein, which together perceive karrikins (smoke-derived butenolides), caused alteration in root skewing in Arabidopsis thaliana. This phenotype was independent of endogenous strigolactones perception by the D14 α/ß-fold hydrolase and MAX2. Thus, KAI2/MAX2 effect on root growth may be through the perception of endogenous KAI2-ligands (KLs), which have yet to be identified. Upon perception of a ligand, a KAI2/MAX2 complex is formed together with additional target proteins before ubiquitination and degradation through the 26S proteasome. Using a genetic approach, we show that SMAX1 (SUPPRESSOR OF MAX2-1)/SMXL2 and SMXL6,7,8 (SUPPRESSOR OF MAX2-1-LIKE) are also likely degradation targets for the KAI2/MAX2 complex in the context of root skewing. In A. thaliana therefore, KAI2 and MAX2 act to limit root skewing, while kai2's gravitropic and mechano-sensing responses remained largely unaffected. Many proteins are involved in root skewing, and we investigated the link between MAX2 and two members of the SKS/SKU family. Though KLs are yet to be identified in plants, our data support the hypothesis that they are present and can affect root skewing.

Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots.

Phosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca2+ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca2+ ([Ca2+]cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca2+]cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca2+]cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca2+]cyt, Pi starvation strongly affects stress-induced [Ca2+]cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

Annexin 1 regulates the H2O2-induced calcium signature in Arabidopsis thaliana roots.

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca(2+) ([Ca(2+) ]cyt ) as a second messenger, with activation of plasma membrane Ca(2+) -permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca(2) (+) -permeable Stelar K(+) Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS-regulated Ca(2+) transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca(2+) ]cyt response to stress levels of extracellular hydrogen peroxide. Peroxide-stimulated [Ca(2+) ]cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide-stimulated net Ca(2+) influx and K(+) efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion-selective microelectrodes. Peroxide induction of GSTU1 (Glutathione-S-Transferase1 Tau 1), which is known to be [Ca(2+) ]cyt -dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca(2+) influx. Differential regulation of annexin expression was evident, with AtANN2 down-regulation but up-regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide-induced [Ca(2+) ]cyt signature and downstream signalling.

The hydroxyl radical in plants: from seed to seed.

The hydroxyl radical (OH(•)) is the most potent yet short-lived of the reactive oxygen species (ROS) radicals. Just as hydrogen peroxide was once considered to be simply a deleterious by-product of oxidative metabolism but is now acknowledged to have signalling roles in plant cells, so evidence is mounting for the hydroxyl radical as being more than merely an agent of destruction. Its oxidative power is harnessed to facilitate germination, growth, stomatal closure, reproduction, the immune response, and adaptation to stress. It features in plant cell death and is a key tool in microbial degradation of plant matter for recycling. Production of the hydroxyl radical in the wall, at the plasma membrane, and intracellularly is facilitated by a range of peroxidases, superoxide dismutases, NADPH oxidases, and transition metal catalysts. The spatio-temporal activity of these must be tightly regulated to target substrates precisely to the site of radical production, both to prevent damage and to accommodate the short half life and diffusive capacity of the hydroxyl radical. Whilst research has focussed mainly on the hydroxyl radical's mode of action in wall loosening, studies now extend to elucidating which proteins are targets in signalling systems. Despite the difficulties in detecting and manipulating this ROS, there is sufficient evidence now to acknowledge the hydroxyl radical as a potent regulator in plant cell biology.

Salinity-induced calcium signaling and root adaptation in Arabidopsis require the calcium regulatory protein annexin1.

Salinity (NaCl) stress impairs plant growth and inflicts severe crop losses. In roots, increasing extracellular NaCl causes Ca²âº influx to elevate cytosolic free Ca²âº ([Ca²âº](cyt)) as a second messenger for adaptive signaling. Amplification of the signal involves plasma membrane reduced nicotinamide adenine dinucleotide phosphate oxidase activation, with the resultant reactive oxygen species triggering Ca²âº influx. The genetic identities of the Ca²âº-permeable channels involved in generating the [Ca²âº](cyt) signal are unknown. Potential candidates in the model plant Arabidopsis (Arabidopsis thaliana) include annexin1 (AtANN1). Here, luminescent detection of [Ca²âº](cyt) showed that AtANN1 responds to high extracellular NaCl by mediating reactive oxygen species-activated Ca²âº influx across the plasma membrane of root epidermal protoplasts. Electrophysiological analysis revealed that root epidermal plasma membrane Ca²âº influx currents activated by NaCl are absent from the Atann1 loss-of-function mutant. Both adaptive signaling and salt-responsive production of secondary roots are impaired in the loss-of-function mutant, thus identifying AtANN1 as a key component of root cell adaptation to salinity.

Maintenance of UK bread baking quality: Trends in wheat quality traits over 50 years of breeding and potential for future application of genomic-assisted selection.

Improved selection of wheat varieties with high end-use quality contributes to sustainable food systems by ensuring productive crops are suitable for human consumption end-uses. Here, we investigated the genetic control and genomic prediction of milling and baking quality traits in a panel of 379 historic and elite, high-quality UK bread wheat (Triticum eastivum L.) varieties and breeding lines. Analysis of the panel showed that genetic diversity has not declined over recent decades of selective breeding while phenotypic analysis found a clear trend of increased loaf baking quality of modern milling wheats despite declining grain protein content. Genome-wide association analysis identified 24 quantitative trait loci (QTL) across all quality traits, many of which had pleiotropic effects. Changes in the frequency of positive alleles of QTL over recent decades reflected trends in trait variation and reveal where progress has historically been made for improved baking quality traits. It also demonstrates opportunities for marker-assisted selection for traits such as Hagberg falling number and specific weight that do not appear to have been improved by recent decades of phenotypic selection. We demonstrate that applying genomic prediction in a commercial wheat breeding program for expensive late-stage loaf baking quality traits outperforms phenotypic selection based on early-stage predictive quality traits. Finally, trait-assisted genomic prediction combining both phenotypic and genomic selection enabled slightly higher prediction accuracy, but genomic prediction alone was the most cost-effective selection strategy considering genotyping and phenotyping costs per sample.

Over-expression of TaDWF4 increases wheat productivity under low and sufficient nitrogen through enhanced carbon assimilation.

There is a strong pressure to reduce nitrogen (N) fertilizer inputs while maintaining or increasing current cereal crop yields. We show that overexpression of TaDWF4-B, the dominant shoot expressed homoeologue of OsDWF4, in wheat can increase plant productivity by up to 105% under a range of N levels on marginal soils, resulting in increased N use efficiency (NUE). We show that a two to four-fold increase in TaDWF4 transcript levels enhances the responsiveness of genes regulated by N. The productivity increases seen were primarily due to the maintenance of photosystem II operating efficiency and carbon assimilation in plants when grown under limiting N conditions and not an overall increase in photosynthesis capacity. The increased biomass production and yield per plant in TaDWF4 OE lines could be linked to modified carbon partitioning and changes in expression pattern of the growth regulator Target Of Rapamycin, offering a route towards breeding for sustained yield and lower N inputs.

GWAS identifies genetic loci underlying nitrogen responsiveness in the climate resilient C4 model Setaria italica (L.).

INTRODUCTION: N responsiveness is the capacity to perceive and induce morpho-physiological adaptation to external and internal Nitrogen (N). Crop productivity is propelled by N fertilizer and requires the breeding/selection of cultivars with intrinsically high N responsiveness. This trait has many advantages in being more meaningful in commercial/environmental context, facilitating in-season N management and not being inversely correlated with N availability over processes regulating NUE. Current lack of its understanding at the physio-genetic basis is an impediment to select for cultivars with a predictably high N response. OBJECTIVES: To dissect physio-genetic basis of N responsiveness in 142 diverse population of foxtail millet, Setaria italica (L.) by employing contrasting N fertilizer nutrition regimes. METHODS: We phenotyped S. italica accessions for major yield related traits under low (N10, N25) and optimal (N100) growth conditions and genotyped them to subsequently perform a genome-wide association study to identify genetic loci associated with nitrogen responsiveness trait. Groups of accessions showing contrasting trait performance and allelic forms of specific linked genetic loci (showing haplotypes) were further accessed for N dependent transcript abundances of their proximal genes. RESULTS: Our study show that N dependent yield rise in S. italica is driven by grain number whose responsiveness to N availability is genetically underlined. We identify 22 unique SNP loci strongly associated with this trait out of which six exhibit haplotypes and consistent allelic variation between lines with contrasting N dependent grain number response and panicle architectures. Furthermore, differential transcript abundances of specific genes proximally linked to these SNPs in same lines is indicative of their N dependence in a genotype specific manner. CONCLUSION: The study demonstrates the value/ potential of N responsiveness as a selection trait and identifies key genetic components underlying the trait in S. italica. This has major implications for improving crop N sustainability and food security.

Analysis of leaf and root transcriptomes of soil-grown Avena barbata plants.

Slender wild oat (Avena barbata) is an annual grass dominant in many grassland ecosystems in Mediterranean climate. This species has been the subject of ecological studies aimed at understanding the effect of global climate change on grassland ecosystems and the genetic basis for adaptation under varying environmental conditions. We present the sequencing and analysis of cDNA libraries constructed from leaf and root samples collected from A. barbata grown on natural soil and under varying rainfall patterns. More than 1 million expressed sequence tags (ESTs) were generated using both GS 454-FLX pyrosequencing and Sanger sequencing, and these tags were assembled into consensus sequences. We identified numerous candidate polymorphic markers in the data set, providing possibilities for linking the genomic and the existing genetic information for A. barbata. Using the digital Northern method, we showed that genes involved in photosynthesis were down-regulated under high rainfall while stress-related genes were up-regulated. We also identified a number of genes unique to the root library with unknown function. Real-time reverse transcription-PCR was used to confirm the root specificity of some of these transcripts such as two genes encoding O-methyl transferase. Also we showed differential expression of five root-specific genes under three water levels and two developmental stages. Through a combination of Sanger and 454-based sequencing technologies, we were able to generate a large set of transcribed sequences for A. barbata. This data set provides a platform for further studies of this important wild grass species.

New perspectives on glutamine synthetase in grasses.

Members of the glutamine synthetase (GS) gene family have now been characterized in many crop species such as wheat, rice, and maize. Studies have shown that cytosolic GS isoforms are involved in nitrogen remobilization during leaf senescence and emphasized a role in seed production particularly in small grain crop species. Data from the sequencing of genomes for model crops and expressed sequence tag (EST) libraries from non-model species have strengthened the idea that the cytosolic GS genes are organized in three functionally and phylogenetically conserved subfamilies. Using a bioinformatic approach, the considerable publicly available information on high throughput gene expression was mined to search for genes having patterns of expression similar to GS. Interesting new hypotheses have emerged from searching for co-expressed genes across multiple unfiltered experimental data sets in rice. This approach should inform new experimental designs and studies to explore the regulation of the GS gene family further. It is expected that understanding the regulation of GS under varied climatic conditions will emerge as an important new area considering the results from recent studies that have shown nitrogen assimilation to be critical to plant acclimation to high CO(2) concentrations.

Phytohormones Interplay: Karrikin Signalling Promotes Ethylene Synthesis to Modulate Roots.

A role for karrikin signalling in regulating root development is now well established, although the nature of the perceived signal and the complete signalling cascade remain elusive. In a recent report, Carbonnel et al. demonstrate a role for ethylene in mediating karrikin signalling effect on root development.

Variation for Nitrogen Use Efficiency Traits in Wheat Under Contrasting Nitrogen Treatments in South-Eastern Europe.

Wheat cultivars differ in their response to nitrogen (N) fertilizer, both in terms of its uptake and utilization. Characterizing this variation is an important step in improving the N use efficiency (NUE) of future cultivars while maximizing production (yield) potential. In this study, we compared the agronomic performance of 48 diverse wheat cultivars released between 1936 and 2016 at low and high N input levels in field conditions to assess the relationship between NUE and its components. Agronomic trait values were significantly lower in the low N treatment, and the cultivars tested showed a significant variation for all traits (apart from the N remobilization efficiency), indicating that response is genotype-dependent, although significant genotype × environment effects were also observed. Overall, we show a varietal improvement in NUE over time of 0.33 and 0.30% year-1 at low and high N, respectively, and propose that this is driven predominantly by varietal selection for increased yield. More complete understanding of the components of these improvements will inform future targeted breeding and selection strategies to support a reduction in fertilizer use while maintaining productivity.

Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots.

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root's mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsisthaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

A Roadmap for Lowering Crop Nitrogen Requirement.

Increasing nitrogen fertilizer applications have sustained a growing world population in the 20th century. However, to avoid any further associated environmental damage, new sustainable agronomic practices together with new cultivars must be developed. To date the concept of nitrogen use efficiency (NUE) has been useful in quantifying the processes of nitrogen uptake and utilization, but we propose a shift in focus to consider nitrogen responsiveness as a more appropriate trait to select varieties with lower nitrogen requirements. We provide a roadmap to integrate the regulation of nitrogen uptake and assimilation into varietal selection and crop breeding programs. The overall goal is to reduce nitrogen inputs by farmers growing crops in contrasting cropping systems around the world, while sustaining yields and reducing greenhouse gas (GHG) emissions.