A circadian transcriptional sub-network and EARLY FLOWERING 3 control timing of senescence and grain nutrition in bread wheat

Bread wheat (Triticum aestivum cv. Mace) mature and senescent flag leaves were collected over a 48 h time course in continuous conditions to investigate changes in circadian clock regulation that occur during leaf senescence. Overall design: Wheat seeds of the Australian cultivar Mace were sown in a single growth room in two staggered batches, 28 days apart. Leaves were sampled concurrently when 'mature' plants were at ear emergence (Zadok's stage 51-59) and 'senescent' plants had evident chlorophyll degradation in flag leaves. At dawn (Zeitgeber Time 0; ZT0), the growth room was set to continuous light and temperature (20ºC) and whole flag leaves from the primary tiller of four plants were collected every 2 h from ZT45 to ZT91 (48 h). Samples were immediately flash frozen in liquid nitrogen and stored at -80°C. RNA from two individual leaf samples was equally pooled to provide two replicates for sequencing.