Nonuniform distribution of glucosinolates in Arabidopsis thaliana leaves has important consequences for plant defense

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   Fig. 1.

   Structures of A. thaliana glucosinolates identified in this study and scheme for myrosinase-catalyzed hydrolysis of glucosinolates to isothiocyanates and nitriles.

   
   
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   Fig. 2.

   Feeding pattern of H. armigera larvae on mature rosette leaf of A. thaliana. Ten second-instar larvae were allowed to feed for 5 h. This photograph is representative of five trials. Photograph by Danny Kessler.

   
   
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   Fig. 3.

   Feeding preference of second-instar H. armigera larvae for regions of A. thaliana leaves. (a) Bioassay arena in a 10-mm Petri dish equipped with leaf disks from outer lamina (OL) and inner lamina (IL). Leaf disks and caterpillar are not shown to scale. (b) Bioassay results on A. thaliana wild type (n = 30) and tgg1/tgg2 mutant line (n = 25). For wild-type leaves, larvae preferred to feed on disks removed from the inner over the outer lamina (binomial test, P = 0.001). There was no significant preference for the corresponding leaf discs from the tgg1/tgg2 mutant (binomial test, P = 0.764), which has the two principal myrosinase genes knocked out and is thus unable to activate the hydrolysis of glucosinolates.

   
   
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   Fig. 4.

   Mass spectrometric imaging of A. thaliana leaves. (a) Section (m/z 400–500) of a MALDI-TOF/MS spectrum averaged from 100 consecutive pixels on a leaf sprayed on its abaxial side with 9-aminoacridine as a matrix. The molecular peaks at m/z 436.0, 447.0, and 492.0 correspond to M⁻ ions of 4-methylsulfinylbutylglucosinolate (4MSOB), indol-3-ylmethylglucosinolate (I3M), and 8-methylsulfinyloctylglucosinolate (8MSOO), respectively. The spectra were collected in a negative reflectron mode on a MALDI micro MX (Waters). (b) Three-dimensional ion intensity map of the matrix ion [m/z 193.0 ± 0.25 (M − H)⁻] from measurement of a leaf sample mounted on the MALDI target plate. The image demonstrates that the matrix was deposited homogeneously over the leaf. The spraying area was defined with a paper mask to prevent matrix dispersal during application. (c) Ion intensity map of 4MSOB (m/z 436 ± 0.25) created in ImageJ software from ≈100,000 MALDI-TOF/MS spectra (420 × 252 pixels (w × h), pixel size 200 μm). The ion intensities are displayed in a pseudocolor on an intensity scale of 0–255 shades (see Inset for the color scale). (d and e) To demonstrate that analytes on the leaf are not delocalized during matrix application and the other steps of sample processing, a permanent marker pen with methanol-soluble ink was used to write “m/z” on the leaf. The intensity image of a characteristic ion from the marker ink (m/z 663.8) obtained after coating with the 9-aminoacridine matrix corresponds well to the original ink pattern. The matrix coating and the instrumental acquisition parameters were identical to those used in the other imaging experiments. (Scale bars in c and e: 0.5 cm.)

   
   
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   Fig. 5.

   Imaging reveals nonuniform glucosinolate distribution in A. thaliana leaves. (a) Five different leaves measured and displayed as in Fig. 2 showing observed 4MSOB ion patterns. (Scale bar: 1 cm.) (b and c) Ion intensity maps of I3M (477 ± 0.25) and 8MSOO (492 ± 0.25), respectively, obtained from mass data measured on the next to last leaf in a. (d) A scatter plot of 4MSOB vs. I3M obtained with the ImageJ applet[36]. The correspondence with a line of slope y = x shows the extent to which the two compounds cooccur. Scales are in false color intensity (0–255) for both x and y axes. (e) A scatter plot of 4MSOB vs. 8MSOO.

   
   
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   Fig. 6.

   Comparison of the relative amounts of the three measured glucosinolates in three different leaf regions of A. thaliana leaves defined as in a. Normalized ion intensities were calculated for the midvein, the inner lamina, and the outer lamina areas from mass spectrometric imaging (b) and glucosinolate concentrations were measured by HPLC analysis (c). Error bars represent SEM from five mature leaves from different plants. Bars marked by different letters show significant difference at P < 0.05.