Human health is dependent on a plentiful and nutritious supply of food, primarily derived from crop plants. Rhythmic supply of light as a result of the day and night cycle led to the evolution of circadian clocks that modulate most plant physiology, photosynthesis, metabolism, and development. To regulate crop traits and adaptation, breeders have indirectly selected for variation at circadian genes. The pervasive impact of the circadian system on crops suggests that future food production might be improved by modifying circadian rhythms, engineering the timing of transgene expression, and applying agricultural treatments at the most effective time of day. We describe the applied research required to take advantage of circadian biology in agriculture to increase production and reduce inputs.

Plant breeders have indirectly selected for variation at circadian-associated loci in many of the world’s major crops when breeding to increase yield and improve crop performance. Using an eight-parent Multiparent Advanced Generation Inter-Cross (MAGIC) population, we investigated how variation in circadian-clock-associated genes contributes to the regulation of heading date in UK and European winter wheat (Triticum aestivum) varieties. We identified homoeologues of EARLY FLOWERING 3 (ELF3) as candidates for the Earliness per se (Eps) D1 and B1 loci under field conditions. We then confirmed a single-nucleotide polymorphism within the coding region of TaELF3-B1 as a candidate polymorphism underlying the Eps-B1 locus. We found that a reported deletion at the Eps-D1 locus encompassing TaELF3-D1 is instead an allele that lies within an introgression region containing an inversion relative to the Chinese Spring D genome. Using Triticum turgidum cv. Kronos carrying loss-of-function alleles of TtELF3 we showed that ELF3 regulates heading, with loss of a single ELF3 homoeologue sufficient to alter heading date. These studies demonstrated that ELF3 forms part of the circadian oscillator; however, loss of all homoeologues was required to affect circadian rhythms. Similarly, loss of functional LUX ARRHYTHMO (LUX) in T. aestivum, an orthologue of a protein partner of Arabidopsis (Arabidopsis thaliana) ELF3, severely disrupted circadian rhythms. ELF3 and LUX transcripts are not co-expressed at dusk, suggesting the structure of the wheat circadian oscillator might differ from that of Arabidopsis. Our demonstration that alterations to ELF3 homoeologues can affect heading date separately from effects on the circadian oscillator suggests a role for ELF3 in cereal photoperiodic responses that could be selected for without pleiotropic deleterious alterations to circadian rhythms.

Optimising the seasonal control of flowering in the major crops is an important component of breeding to match crop adaptation to the target environment. Using an eight parent Multiparent Advanced Generation Inter-Cross (MAGIC) population we investigated the contribution of variation at circadian clock-associated genes to the regulation of heading date (flowering) in UK and European winter wheat varieties. We identified homoeologues of EARLY FLOWERING 3 (ELF3) as candidate genes for the Earliness per se (Eps) D1 and B1 loci in field conditions. We confirmed that a SNP within the coding region of TaELF3-B1 is the likely causal polymorphism underlying the Eps-B1 locus. We also identified that a reported deletion at the Eps-D1 locus encompassing TaELF3-D1, is in fact a novel allele that lies within an introgression region that contains an inversion relative to the Chinese Spring D genome. Our findings that ELF3 might be associated with the regulation of heading date prompted us to investigate whether ELF3 is a circadian oscillator gene in wheat, as it is in Arabidopsis. Using T. turgidum cv. Kronos carrying loss of function alleles for both copies of TtELF3 we found that circadian rhythms were severely disrupted. Furthermore, in T. aestivum, we found that loss of functional LUX ARRHYTHMO (LUX), an orthologue of the protein partner of ELF3 in Arabidopsis, also severely disrupted circadian rhythms. Whilst these data suggest a function for both ELF3 and LUX in the wheat circadian oscillator, that oscillator might be structured differently to that of Arabidopsis because wheat ELF3 and LUX transcripts are maximal at the end of the night and day respectively, rather than co-expressed at dusk as they are in Arabidopsis. We conclude that there is sufficient allelic diversity within the three wheat ELF3 homoeologues for selection to delay or advance heading, and that this can be achieved without pleiotropic deleterious alterations to circadian rhythms.

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA-sequencing of maize mesophyll and bundle sheath strands over a 24-h time-course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene co-expression analyses suggested members of the DNA binding with One Finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators as well as peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.