Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress

Abstract

SummaryWhite lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to )P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA i )-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under )P stress. Here, we show that LaMATE had high expression in proteoid roots not only under )P, but also under )Fe, )N, )Mn and þAl stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein.LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA i -based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.

Plant adaptations to severely phosphorus-impoverished soils

“…Increased P-transport activity and expression of genes encoding P transporters have been reported [60]. Furthermore, a L. albus MATE homologue to the citrate exporter FRD3 (ferric reductase defective 3) in Arabidopsis is highly induced in cluster roots [61]. Plasma-membrane H + -ATPases also show increased activity [62], and proton and citrate exudation are correlated, although protons are not the only counterions during carboxylate release [48].…”

Genomic and Genetic Control of Phosphate Stress in Legumes

“…Transcript accumulation of white lupine LaPT1, MATE, and LaSAP1 genes was increased in stems and in normal and cluster roots of P-stressed plants, although there was virtually no transcript accumulation of these genes in P-sufficient plants Uhde-Stone et al, 2005). The direct involvement of LaPT1, MATE, and LaSAP1 genes in P starvation has been verified by studies using transgenic lupine and alfalfa plants that contained the promoters and 5#-untranslated intron fused to a reporter GUS gene Uhde-Stone et al, 2005). Roots of transgenic alfalfa plants grown under P limitation showed strong GUS staining and enzyme activity.…”

The multidrug and toxic compound extrusion (MATE) family in plants

“…In P deficient conditions, white lupin (Lupinus albus) was found to develop cluster roots, in which a MATE transporter, LaMATE, was highly expressed (Uhde-Stone et al 2005). Findings showing that Lupinus releases high amounts of malate and citrate under P stress (Cheng et al 2011), and that LaMATE shows high sequence similarity with citrate-transporting MATEs (Figure 1), suggested that LaMATE functions as a citrate efflux carrier in cluster roots.…”

“…Findings showing that Lupinus releases high amounts of malate and citrate under P stress (Cheng et al 2011), and that LaMATE shows high sequence similarity with citrate-transporting MATEs (Figure 1), suggested that LaMATE functions as a citrate efflux carrier in cluster roots. However, LaMATE could not complement an atfrd3 mutant with a native LaMATE promoter (Uhde-Stone et al 2005). …”