Differential gene expression in proteoid root clusters of white lupin (Lupinus albus).

Proteoid root clusters are induced by P deficiency in white lupin. In their mature stage, these roots excrete organic acids (mainly citrate), thus allowing this species to acquire P from sparingly soluble sources. To screen for P-regulated genes expressed during the period of citrate efflux, an experimental model based on proteoid root clusters contrasting in citrate efflux was developed. The feasibility of this model in identifying differential gene expression was assessed over a population of mRNAs from P-starved and P-starved rescued proteoid root clusters, sampled 24 and 72 h after P addition to 24 days P-starved white lupin. Approximately 1500 bands of cDNA were displayed by differential display of 21-primer pair's combination; 52 differentially expressed bands, either up- or down-regulated after P addition, were observed. Sequence analysis of 17 of them revealed that they represent distinct cDNAs. A subsample of seven cDNAs was analysed by northern-blot, showing that six were truly differential products. Transcripts coding for enzymes involved in carbon flux (glyceraldehyde 3-phosphate dehydrogenase), glycolytic bypass (phosphoenolpyruvate carboxylase), Pi recycling (sulpholipid synthase), and two unknown cDNAs were shown to be down-regulated by P supply. Besides, an up-regulated transcript coding for a putative auxin-induced protein was identified, whereas P addition did not significantly affect expression of a transcript for cyclophilins. These results show the feasibility of using P-starved and P-starved rescued proteoid root clusters as an experimental model to detect and examine the molecular changes occurring in root clusters during the period of citrate efflux in white lupin.

Phosphate deficiency regulates phosphoenolpyruvate carboxylase expression in proteoid root clusters of white lupin.

Proteoid roots play a major role in enabling white lupin (Lupinus albus L.) to adapt to phosphate (Pi) deficiency. Such roots release citrate from proteoid rootlets, which allows this species to mobilize Pi from sparingly soluble Pi sources. Release of citrate is preceded by a significant accumulation of organic acids, in which a Pi deficiency-inducible phosphoenolpyruvate carboxylase (PEPC) activity has been involved. To gain an insight into this adaptive mechanism, the expression of three different transcripts coding for PEPC was examined in proteoid rootlets of Pi-starved and Pi-starved-and-rescued white lupin. Semi-quantitative reverse transcriptase (RT)-PCR experiments performed with gene-specific primers targeted to the 3'-end region of the corresponding cDNAs revealed that the transcripts for these three PEPCs differentially accumulate in both Pi-starved and Pi-starved-and-rescued proteoid rootlets. Semi-quantitative RT-PCR analysis in Pi-starved proteoid rootlets sampled at different times after being rescued from Pi deficiency showed that Pi levels differentially down-regulated the three PEPC transcripts. RT-PCR experiments were further extended to Pi-starved and Pi-fed whole roots, cotyledons, and leaves on which a tissue-specific, Pi-dependent PEPC expression was observed. These results indicate that there exists at least three different transcripts coding for PEPC in proteoid root clusters of white lupin, whose expression are differentially regulated by Pi.