Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression

Materials and Reagents

1. 1.5 ml tube
   
2. Leaf discs
   Note: Ensure that the leaf discs are fully submerged in the DMF solution.
   
3. N, N-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: D4551-250 ml )
   

Equipment

1. Spectrophotometer (Spectronic Biomate3) (Thermo Fisher Scientific, catalog number: 335904P )
   
2. Glass spectrophotometer cuvettes (Sigma-Aldrich, catalog number: Z276898 )
   Note: Product Z276898 has been discontinued.
   

Procedure

1. Place three leaf discs (e.g., 4 mm radius, from Eppendorf lid) into a 1.5 ml tube containing 1 ml of dimethylformamide (DMF). Use a cork borer to excise leaf disk. Include five replicates for each sample type. Ensure that the leaf discs are fully submerged in the DMF solution. Allow the chlorophyll to dissolve into the DMF solution by still incubation overnight at 4 °C.
   
2. Mix 300 μl of sample solution with 600 μl of DMF in a fresh Eppendorf tube (2 volumes of DMF per volume of sample). Read the absorbance (A) in spectrophotometer at 647 nm and 664.5 nm wavelengths using a glass cuvette.
   Chlorophyll a content (µg/ml):
   = (12 x A_664.5)-(2.79 x A₆₄₇)
   Chlorophyll b content (µg/ml):
   = (20.78 x A₆₄₇)-(4.88 x A_664.5)
   Total chlorophyll content (µg/ml) = Chla + Chlb
   Sample area (for 3 leaf discs at 4 mm radius) (mm²) = 3 x πr²
   Total chlorophyll content (µg/mm²) = (Chla + Chlb)/Sample area
   

Notes

This protocol is most suitable for comparing treatments within an experiment, as the absolute chlorophyll content will vary between different sets of plants and over time. If measuring chlorophyll content after leaf infiltration assays, it is preferable to infiltrate all the treatment types to be compared on a single leaf. This can be repeated, in different configurations, on multiple leaves. To minimize the effects of inter-leaf variability, all the leaf discs from the same treatment type can then be pooled. A minimum of 15 leaf discs (3 per sample, five replicates) for each treatment type is required. While our experience with this protocol has been using leaves of Nicotiana benthamiana and N. tobacum, it has also been applied to (at least) Arabidopsis thaliana (Pruzinska et al., 2005) - as a proxy for leaf senescence - and could in principle be applied to many other plant species where chlorophyll content is of physiological relevance.

Acknowledgments

We would like to thank Sylvain Aubry for his inspiration in the establishing this protocol. Research in the Baulcombe laboratory is supported by the ERC Advanced Investigator grant ERC-2013-AdG 340642 TRIBE. C. J. H was supported by a BBSRC Ph.D. Studentship. D. C. B. is the Royal Society Edward Penley Abraham Research Professor.

References

1. Harris, C. J., Slootweg, E. J., Goverse, A. and Baulcombe, D. C. (2013). Stepwise artificial evolution of a plant disease resistance gene. Proc Natl Acad Sci U S A 110(52): 21189-21194.
   
2. Pruzinska, A., Tanner, G., Aubry, S., Anders, I., Moser, S., Muller, T., Ongania, K. H., Krautler, B., Youn, J. Y., Liljegren, S. J. and Hortensteiner, S. (2005). Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol 139(1): 52-63.
   
3. Strain, H. H., Cope, B. T. and Svec, W. A. (1971). Analytical procedures for the isolation, identification, estimation and investigation of the chlorophylls. Methods Enzymol 23: 452-476.