Advances in imaging technologies have opened new avenues for understanding the physiological basis of plant reproduction. Here, we apply synchrotron-based X-ray fluorescence (XRF) microscopy to investigate the spatial distribution of essential micronutrients, particularly copper (Cu), during reproductive development. Cu deficiency affects approximately 30% of arable land and significantly limits crop yield, yet the mechanisms by which Cu supports fertility remain poorly understood. By combining XRF imaging techniques with molecular genetics, we show that two transcription factors, Squamosa Promoter Binding Protein-Like 7 (SPL7) and Copper-Deficiency Induced Transcription Factor 1 (CITF1), are required for reproductive success in Arabidopsis thaliana. Using high-resolution XRF mapping and XRF-computed tomography, we find that the citf1 spl7 double mutant exhibits severely depleted Cu levels in flowers, particularly in pistils, anthers, and pollen grains, leading to both female and male infertility. We further demonstrate that the CITF1–SPL7 regulatory pathway is essential for stigma development, pollen viability, germination, seed Cu distribution, and embryo formation. Extending these findings to Brachypodium distachyon, we show that Cu delivery to florets is similarly critical for fertility, grain quality, and yield. This work highlights the power of synchrotron-based imaging to elucidate micronutrient function in reproductive biology and offers insights into improving crop fertility and resilience under Cu-deficient conditions.
