The down-regulation of Mt4-like genes by phosphate fertilization occurs systemically and involves phosphate translocation to the shoots

Abstract

Mt4 is a cDNA representing a phosphate-starvation-inducible gene from Medicago truncatula that is down-regulated in roots in response to inorganic phosphate (Pi) fertilization and colonization by arbuscular mycorrhizal fungi. Split-root experiments revealed that the expression of the Mt4 gene in M. truncatula roots is down-regulated systemically by both Pi fertilization and colonization by arbuscular mycorrhizal fungi. A comparison of Pi levels in these tissues suggested that this systemic down-regulation is not caused by Pi accumulation. Using a 30-bp region of the Mt4 gene as a probe, Pi-starvation-inducible Mt4-like genes were detected in Arabidopsis and soybean (Glycine max L.), but not in corn (Zea mays L.). Analysis of the expression of the Mt4-like Arabidopsis gene, At4, in wild-type Arabidopsis and pho1, a mutant unable to load Pi into the xylem, suggests that Pi must first be translocated to the shoot for down-regulation to occur. The data from the pho1 and split-root studies are consistent with the presence of a translocatable shoot factor responsible for mediating the systemic down-regulation of Mt4-like genes in roots.

Figure 1

The systemic down-regulation of Mt4 by Pi fertilization. A, Illustration of a split-root plant. B, Northern blot of total RNA isolated from the roots of M. truncatula grown in a split-root design with one-half of the root system receiving high-Pi (+Pi half of split-root) and one-half receiving low-Pi (−Pi half of split-root) fertilizer. In split-root controls both halves received high-Pi (+Pi control) or low-Pi (−Pi control) fertilizer. The blot was probed with a ³²P-labeled Mt4 cDNA (top panel) and a ³²P-labeled pSR1-2B3 (18S rRNA, bottom panel).

Figure 4

The nucleotide and translated amino acid sequences of At4. Bases are numbered at right. Predicted ORFs are shaded. The region of nucleotide identity with Mt4 from M. truncatula and TPSI1 from tomato is in bold italics. The primer pair used in PCR experiments is underlined.

Figure 2

The systemic down-regulation of Mt4 by mycorrhizal colonization. Northern blots of total RNA isolated from the roots of M. truncatula grown in a split-root design with one-half of the root system grown with (+F half of split-root) and one-half grown without (−F half of split-root) G. versiforme colonization for two plants (A and B). In split-root controls one-half of the root system was mock inoculated or both halves were uncolonized (−F control). The blot was probed with a ³²P-labeled Mt4 cDNA (top panel) and a ³²P-labeled pSR1-2B3 (18S rRNA, bottom panel).

Figure 3

Detection of Mt4-like genes in Arabidopsis, tomato, and soybean. A, Northern blot of total RNA isolated from plants receiving either low-Pi (−Pi) or high-Pi (+Pi) fertilizer. The blot was probed with the conserved sequence (Oligo), the Mt4 cDNA, the At4 cDNA, or a ³²P-labeled pSR1-2B3 (rRNA). B, Expression of At4 in Arabidopsis roots. RT-PCR products from RNA extracted from leaves and roots of Pi-deprived Arabidopsis plants (wild type and pho1 mutant) using At4-specific primers. The blot was hybridized with ³²P-labeled At4 cDNA.

Figure 5

Comparison of the conserved nucleotide sequences of Mt4 from M. truncatula, TPSI1 from tomato, and At4 from Arabidopsis. Perfectly conserved nucleotides are represented by asterisks and well-conserved nucleotides are represented by dots.

Figure 6

Southern blot of Arabidopsis genomic DNA. Hybridization of a ³²P-labeled At4 cDNA to a gel blot of DNA digested with five different restriction enzymes. Size markers are on the right.

Figure 7

Southern blots of RT-PCR products derived from wild-type Arabidopsis and the Pi-accumulation mutant pho1. Total RNA was extracted from plants grown with (+Pi) or without (−Pi) phosphate fertilization, reverse transcribed, standardized, amplified by PCR using a primer pair based on the At4 sequence, blotted, and probed with either a ³²P-labeled At4 cDNA (At4) or an end-labeled oligonucleotide identical to the conserved sequence (Oligo) for two independent experiments (A and B).