Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (Flmage) of freshly excised cucumber (Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-ban d (300-400 nm) excitation source centered at 360 nm. Means of relative fluo rescence intensities (RFI) from these spectral fluorescence images were com pared with spectral fluorescence emission data obtained from excitation wav elengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of di methyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (Flmage, 280EX, 380 EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2- oxo-1-imidazolidinyl) ethyl]-N'-phenylurea (EDU or ethylenediurea) as a che mical protectant against UV-B damage. Plants exhibited well known foliar gr owth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower ch lorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone h ad no effect on foliar variables, there was no evidence that EDU afforded p rotection against UV-B. Instead, EDU augmented some UV-B effects when provi ded in conjunction with UV-B irradiation (e.g., reductions in the chlorophy ll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b conte nt). Relative fluorescence intensities (RFI) in the longer visible wavelengths ( green, red, and far-red) were uncorrelated for comparisons between the Flma ge and 380EX data sets. However, blue and green RFI were significantly corr elated (0.8>r>0.6; P<0.002) for comparisons between Flmage and 280EX data s ets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both ima ges and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and Flmage stimulates processes that produce excess blue fluores cence The molecules that produce the excess blue fluorescence in both the 2 80EX and the Flmage data are different electron transfer agents that operat e in parallel. For Flmage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissue s las occurs for the extracts of leaf discs),whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer UV-B irradiated leaves showed much greater heteorgeneity of RFI in both th e green (F550(Flmag)) and the red (F680(Flmag)) bands than unirradiated lea ves; this was true irrespective of EDU treatment. Although qualitative responses in individual bands differed between Flmage and 380EX data, similar results were obtained in the detection of UV-B indu ced effects when the red/green and blue/far-red fluorescence ratios of thes e data were compared. The red/green ratio (either F680/F550(Flmage) or F675 /F525(380EX)) was lower for UV-B exposed plants in both images and 380EX da ta. UV-B exposure also significantly enhanced the blue/far-red ratio of ima ges (F450/F740(Flmage)) and the comparable 380EX ratio (F450/F730(380EX)) f or the combined UV-B/EDU group, The far-red/red ratios were not useful in s eparating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420(280EX)) was substantially reduced by UV-B exposure and was inversely related to total p hotosynthetic pigment content. These findings suggest that the red/green ra tio (Flmage, 380EX) and the UV/blue ratio (280EX) may be as useful as the b lue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-8 stress damage in plants .