Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus

Abstract

Phosphorus is a major nutrient acquired by roots via high-affinity inorganic phosphate (Pi) transporters. In this paper, we describe the tissue-specific regulation of tomato (Lycopersicon esculentum L.) Pi-transporter genes by Pi. The encoded peptides of the LePT1 and LePT2 genes belong to a family of 12 membrane-spanning domain proteins and show a high degree of sequence identity to known high-affinity Pi transporters. Both genes are highly expressed in roots, although there is some expression of LePT1 in leaves. Their expression is markedly induced by Pi starvation but not by starvation of nitrogen, potassium, or iron. The transcripts are primarily localized in root epidermis under Pi starvation. Accumulation of LePT1 message was also observed in palisade parenchyma cells of Pi-starved leaves. Our data suggest that the epidermally localized Pi transporters may play a significant role in acquiring the nutrient under natural conditions. Divided root-system studies support the hypothesis that signal(s) for the Pi-starvation response may arise internally because of the changes in cellular concentration of phosphorus.

Figure 1

A, Alignment of the deduced amino acid sequence of LePT1 and LePT2 with that of A. thaliana (AtPT1 and AtPT2), potato (STPT1 and STPT2), and C. roseus (PIT1) phosphate transporters. Identical amino acids are indicated by asterisks and conserved substitutions are indicated by dots. The membrane-spanning domains of LePT1 and LePT2 as predicted by TopPred (Claros and von Heijne, 1994) are underlined and their numbering is indicated by roman numerals (I–XII). The open and boxed sequences are consensus sites for phosphorylation by casein kinase II, and boxed and shaded sequences are consensus sites for phosphorylation by protein kinase C. B, Summary of the percentage of amino acid identity between tomato and other plant phosphate transporters.

Figure 2

Southern-blot analysis of tomato genomic DNA digested with PstI (P), HindIII (H), or EcoRI (E). Blots were hybridized with ³²P-labeled cDNA fragments specifically recognizing LePT1 and LePT2 genes. DNA markers are indicated in kilobases.

Figure 3

Northern-blot analysis of the expression of tomato phosphate-transporter genes. Total RNA from roots (R) or leaves (L) of tomato plants grown aeroponically and misted with a solution containing 250 μm (+) or no (−) phosphate was hybridized with a labeled probe from either LePT1 or LePT2. An ethidium bromide-stained gel picture indicating uniform loading and integrity of RNA samples is shown at the bottom.

Figure 4

Expression of LePT1 and LePT2 transcripts during Pi starvation. Northern blot of total RNA isolated from the roots of plants misted with nutrient solutions containing either 250 μm (+) or no (−) phosphate for the indicated periods. A picture of the gel showing uniform loading of RNA is shown.

Figure 5

Effect of different nutrient starvation on the expression of LePT1 and LePT2 transcripts. The roots of tomato plants grown in aeroponics were sprayed with nutrient solutions deficient in phosphate (P), potassium (K), nitrogen (N), or iron (Fe) and a control solution containing all necessary nutrients (C) for 5 d. Total RNA isolated from the roots of these plants was analyzed by northern blotting. A picture of the gel showing uniform loading of RNA is shown.

Figure 6

Effect of different concentration of Pi on the expression of LePT1 and LePT2 transcripts. Northern blot of total RNA isolated from roots of tomato plants sprayed with nutrient solution containing the indicated micromolar concentration of Pi for 5 d. A picture of the gel showing uniform loading of RNA is shown.

Figure 7

Expression of LePT1 and LePT2 genes is reversible upon the resupply of phosphate to Pi-starved plants. Tomato plants were sprayed with nutrient solution containing 250 μm (+) or no (−) Pi. After 5 d the Pi-starved plants were replenished with 250 μm Pi (R) or continued to grow in Pi-deficient medium (C) for the indicated time. Total RNA isolated from the roots of these plants was used for northern-blot analysis. A picture of the gel showing uniform loading of RNA is shown.

Figure 8

Expression of phosphate transporters and TPSI1 is regulated by the internal concentration of phosphate in plants. In this divided-root experiment roots of plants were exposed to either 250 μm Pi (C+) or 0 μm (C−) Pi. Roots of another set of plants were separated into two portions, and each portion was placed in an aerated hydroponic solution containing 250 μm (D+) or 0 μm (D−) Pi for 5 d. Total RNA isolated from roots and leaves of the plants was analyzed by northern blotting. A picture of the gel showing uniform loading of RNA is shown.

Figure 9

In situ localization of tomato phosphate-transporter transcripts in roots and leaves. Tissue localization of the message was done using digoxigenin-labeled LePT1 and LePT2 sense (A and C) and antisense (B and D) RNA probes. Sections are from roots and leaves of plants grown with (A and B) and without (C and D) phosphorus for 5 d. ep, Epidermis; cp, cortical parenchyma; cc, central cylinder; pp, palisade parenchyma; and sp, spongy parenchyma.