Time-resolved X-ray fluorescence analysis of element distribution and concentration in living plants: An example using manganese toxicity in cowpea leaves

The distribution and concentration of nutrients and contaminants affect almost every metabolic process in plants but analytical limitations have hindered the determination of microscopic changes over time within living plant tissues. We developed a novel method using synchrotron-based micro X-ray fluorescence (mu-XRF) that, for the first time, allows quantification of the spatial and temporal changes of multiple elements in the same area of living leaves. The utility of this approach was tested by examining changes over 48 h in unifoliate leaves of 7-d-old cowpea (Vigna unguiculata) plants simultaneously at 0.2 and 30 mu M Mn in nutrient solution, with 30 mu M Mn known to be toxic to cowpea and cause the formation of Mn-dense lesions. The fast X-ray fluorescence detector system reduced dwell on living leaf samples. This produced no evidence of tissue damage through repeated mu-XRF scanning, thereby overcoming previously noted experimental artifacts. This permitted, for the first time, visual and quantitative assessments of spatial and temporal changes in nutrient concentrations. By focusing on changes in Mn status, this study illustrated extension of two-dimensional mu-XRF scans to a three-dimensional geometry of Mn kinetics in the same area of leaves. The multi-element potential of this method was exemplified through the measurement of distributions and concentrations of K, Ca, Fe, Cu, and Zn within living plant leaves. This novel method and accompanying information on changes in Mn distribution showed the potential for microscopic, time-resolved, in vivo examination of changes in elemental distribution. We consider that this method will be of benefit for a wide range of studies, including functional characterization of molecular biology, examining changes in the distribution of nutrients, and understanding the movement and toxicity of contaminants.