Northern blot of total RNA. A, RNA was extracted from leaf tissues of 3- to 4-week-old lima bean JWAR and F1072. Three- to 4-week-old seedlings were incubated at 40°C for 0, 30, 60, 120, or 300 min. RNA was extracted from immature leaves, separated by formaldehyde, agarose gel electrophoresis, and blotted onto Hybond N+ (Amersham, Arlington Heights, IL) membrane. The northern blot was hybridized with antisense riboprobe derived from lima bean cyt HSP100/ClpB gene fragment and an actin gene fragment simultaneously. B, Quantification of hybridizing signals. An autoradiograph of the hybridized northern blot in A was converted to a digital image and the density of the bands measured using the NIH Image program. Relative densities were adjusted based on the density of the actin signal at that time point. The least actin signal was set at a value of 1.0, and all of the other actin signals adjusted to this signal. The actin ratio was then used to normalize the relative density of the signal from the HSP100 probe for the same sample.