ISOLATION OF CDNA CLONES OF GENES WITH ALTERED EXPRESSION LEVELS IN PHOSPHATE-STARVED BRASSICA-NIGRA SUSPENSION CELLS

Differential gene expression at the transcriptional level was examined as an initial step in the investigation of the P-i starvation response of Brassica nigra suspension cells. Total RNA was extracted from 7-day old cells grown in media containing either no P-i, 1.25 mM or 10 mM P-i. In vitro translation was carried out using their respective poly(A)(+) RNA isolates and the resultant polypeptides were separated on a high-resolution SDS-PAGE gel. Scanning densitometry identified four polypeptides (ca. 31.7, 32.3, 52.5 and 64.8 kDa) present only in the P-i-starved samples. Screening by differential hybridization was performed on a cDNA library constructed from mRNA isolated from P-i-starved cells. Probes prepared from mRNA. from P-i-defficient and P-i-sufficient cells identified a number of clones representing mRNA species that were preferentially transcribed under P-i deficiency. These phosphate starvation-responsive (psr) clones were placed into eleven groups as determined by cross-hybridization. Northern blots showed that the corresponding genes are inducible in both mild and severe P-i starvation conditions. Preliminary sequencing identified one of the clones as being homologous to beta-glucosidases from several plant species. The possible role of beta-glucosidase during P-i starvation and the identities of the other psr genes are discussed.