Rapid changes in gene expression induced by plant wilting

Abstract

The response of Pisum sativum to wilting was examined at a physiological and genetic level. Pea shoots were rapidly wilted by excision and dehydration in a 23°C air stream for 20-30 min resulting in a loss of 10-15% fresh weight. Following the dehydration period, the wilted condition was maintained by incubation in a dark, humid chamber at 23°C for 0, 0.5, 1, 2, and 4 h. Isopiestic thermocouple psychrometry was used to quantify the decrease in leaf water potential and loss of turgor, a condition which was maintained through a subsequent 4 h incubation period. An HPLC method of analysis for abscisic acid (ABA) was developed utilizing a Cis reversed-phase column for initial purification of plant tissue extracts followed by a silica column quantification step. Wilting induced the biosynthesis of ABA approximately 100-fold over the non-wilted control level by the end of the 4 h incubation period. This induction appeared to be transcriptionally controlled since inhibitors of new transcription prevented ABA accumulation following wilting. Poly (A) RNA was isolated from control and wilted shoots and translated in vitro with a wheat germ extract translation system using [35S]-methionine as a radioactive tracer. The translation products were analyzed by two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis. Following fluorography and autoradiography, a set of at least 13 translation products of poly(A) RNAs were found to be rapidly induced by wilting, several responded within 30 min. A cDNA library was constructed in λgt10 from poly (A) RNA isolated from wilted shoots. Replica filters were differentially screened with cDNA probe synthesized from control and wilted shoot poly(A) RNA. Ten unique RNAs were found to be induced within 4 h of wilting. Further characterization by cDNA probing and nuclei in vitro transcription experiments identified the time course of induction, sensitivity to heat shock, exogenous ABA, rehydration, or uptake of cycloheximide prior to wilting, and localized the site of induction to leaves, stems or roots...