Abstract
Many plant tissues fluoresce due to the natural fluorophores present in cell walls or within the cell protoplast or lumen. While lignin and chlorophyll are well-known fluorophores, other components are less well characterized. Confocal fluorescence microscopy of fresh or fixed vibratome-cut sections of radiata pine needles revealed the presence of suberin, lignin, ferulate, and flavonoids associated with cell walls as well as several different extractive components and chlorophyll within tissues. Comparison of needles in different physiological states demonstrated the loss of chlorophyll in both chlorotic and necrotic needles. Necrotic needles showed a dramatic change in the fluorescence of extractives within mesophyll cells from ultraviolet (UV) excited weak blue fluorescence to blue excited strong green fluorescence associated with tissue browning. Comparisons were made among fluorophores in terms of optimal excitation, relative brightness compared to lignin, and the effect of pH of mounting medium. Fluorophores in cell walls and extractives in lumens were associated with blue or green emission, compared to the red emission of chlorophyll. Autofluorescence is, therefore, a useful method for comparing the histology of healthy and diseased needles without the need for multiple staining techniques, potentially aiding visual screening of host resistance and disease progression in needle tissue.
Highlights
Plant tissues contain many fluorescent compounds [1]
Where fluorophores exist in spatial isolation, it is possible to identify the fluorophore by histochemistry, by comparison with purified standards, or potentially by using other forms of spectroscopy such as FTIR or Raman microspectroscopy [1,2,3,4]
We provide a characterization of cell wall components including lignin, suberin, ferulate, and flavonoids, extractives such as oleoresin and terpenes, and chlorophyll, including a comparison between histochemistry and autofluorescence where possible
Summary
Plant tissues contain many fluorescent compounds [1]. This natural fluorescence is called autofluorescence and can be used for imaging cells and tissues. Autofluorescence can be used to image tissues even when the fluorophore has not been identified with the advantage of high contrast and spatial resolution [4,5]. Autofluorescence may either complement or interfere with staining or labeling protocols, especially when the fluorescence is relatively bright as is the case with chlorophyll [6,7]. As more plant fluorophores are characterized, autofluorescence will become an increasingly important imaging tool in plant science
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