Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Abstract

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants.

Attenuation of Phosphate Starvation Responses by Phosphite in Arabidopsis

“…One example of this ability is Arabidopsis, which develops normally on synthetic media containing purified nucleic acid substrates as the only source of phosphorus. When grown on ϪPi/ ϩDNA or ϪPi/ϩRNA media, Arabidopsis plants induce Pi starvation-inducible nucleolytic enzymes such as nucleases and acid phosphatase to metabolize exogenous nucleic acids (Chen et al, 2000). As a consequence, plants grown under such conditions do not show severe symptoms of Pi deficiency and are comparable in appearance and fresh weight with plants that are cultivated on ϩPi medium (see Fig.…”

“…Roots of seedlings grown on ϪPi medium developed root hairs and were more branched, whereas roots of ϩPi seedlings were less branched and appeared largely devoid of root hairs (data not shown), which is consistent with previous reports (Bates and Lynch, 1996). Because plant growth is severely inhibited in our ϪPi conditions (Chen et al, 2000), the effect of Pi limitation on root hair formation is best seen when roots of plants that were grown on ϪPi/ϩRNA and ϩPi/ϩRNA medium are compared. The external Pi concentration generated by the hydrolysis of exogenous RNA is sufficient to sustain continued plant growth but is seemingly low enough to promote pronounced development of root hairs.…”

“…These include Pi starvation-inducible acid phosphatases with broad substrate specificity (Duff et al, 1994;del Pozo et al, 1999;Baldwin et al, 2001), phosphoenolpyruvate phosphatase and pyrophosphate-dependent phosphofructokinase (Theodorou and Plaxton, 1993), ribonucleases (Green, 1994), phosphodiesterases (Abel et al, 2000), Pi transporters (Raghothama, 2000), and several genes of unknown function (Liu et al, 1997;Burleigh and Harrison, 1999). However, Pi sensory mechanisms and components of the signal transduction pathway(s) that integrate the Pi starvation response of plants have not been identified.…”

“…However, Pi sensory mechanisms and components of the signal transduction pathway(s) that integrate the Pi starvation response of plants have not been identified. To elucidate Pi starvation signaling pathway(s) in plants, we designed a conditional genetic screen in Arabidopsis that is based on the facultative ability of wild-type plants to utilize organophosphates such as nucleic acids in Pi-limiting conditions (Chen et al, 2000). We isolated several recessive mutant lines that grow on high Pi medium, but which show severe growth inhibition on medium containing nucleic acids as the only source of phosphorus.…”

“…We isolated several recessive mutant lines that grow on high Pi medium, but which show severe growth inhibition on medium containing nucleic acids as the only source of phosphorus. For a subset of these lines, the conditional growth defect is likely to be caused by reduced induction of multiple Pi-scavenging enzymes in response to low Pi, which suggests loss-of-function mutations in positive factors of plant pho gene regulation (Chen et al, 2000).…”