Gene-sequence-tag expression analyses of 1,800 genes related to chloroplast functions

Abstract.

Quantification of the expression levels of nuclear genes encoding plastid proteins under different genetic or environmental conditions can contribute to the genetic dissection of plastid functions. To facilitate such measurements, a set of 1,827 Arabidopsis thaliana genes coding for plastid proteins was PCR-amplified from genomic DNA and spotted on nylon membranes to generate an array of chloroplast-specific gene-sequence-tags (GSTs). The sensitivity and reliability of the experimental system was evaluated and a procedure was developed for detecting differential gene expression. The GST array was found to serve as a reliable monitor of changes in gene expression induced by environmental and genetic alteration of chloroplast functions. Based on comparisons of dark- versus light-grown seedlings, and wild-type versus prpl11-1 plants, lists of differentially expressed genes are provided which include 193/7 and 25/42 up/down-regulated genes, respectively. The cut-off values for differential expression were 2.5-times (up) and 0.40 (down). Additional up-regulated genes with relatively low expression ratios (from 1.5- to 2.5-times) or down-regulated with relatively high ratios (0.4–0.67) can be accessed at the website: http://www.mpiz-koeln.mpg.de/~richly/GST-array.html. A sample of genes analysed by quantitative reverse transcription PCR confirmed the expression profiles monitored by the GST array. Differential hybridisation experiments with the prpl11-1 mutant revealed the existence of regulatory networks sensing the protein state of the chloroplast and transmitting the signal to the nucleus.