Genome-wide investigation and expression analysis suggest diverse roles and genetic redundancy of Pht1 family genes in response to Pi deficiency in tomato

Abstract

Background: Phosphorus (P) deficiency is one of the major nutrient stresses limiting plant growth. The uptake of P by plants is well considered to be mediated by a number of high-affinity phosphate (Pi) transporters belonging to the Pht1 family. Although the Pht1 genes have been extensively identified in several plant species, there is a lack of systematic analysis of the Pht1 gene family in any solanaceous species thus far.

Results: Here, we report the genome-wide analysis, phylogenetic evolution and expression patterns of the Pht1 genes in tomato (Solanum lycopersicum). A total of eight putative Pht1 genes (LePT1 to 8), distributed on three chromosomes (3, 6 and 9), were identified through extensive searches of the released tomato genome sequence database. Chromosomal organization and phylogenetic tree analysis suggested that the six Pht1 paralogues, LePT1/3, LePT2/6 and LePT4/5, which were assigned into three pairs with very close physical distance, were produced from recent tandem duplication events that occurred after Solanaceae splitting with other dicot families. Expression analysis of these Pht1 members revealed that except LePT8, of which the transcript was undetectable in all tissues, the other seven paralogues showed differential but partial-overlapping expression patterns. LePT1 and LePT7 were ubiquitously expressed in all tissues examined, and their transcripts were induced abundantly in response to Pi starvation; LePT2 and LePT6, the two paralogues harboring identical coding sequence, were predominantly expressed in Pi-deficient roots; LePT3, LePT4 and LePT5 were strongly activated in the roots colonized by arbuscular mycorrhizal fungi under low-P, but not high-P condition. Histochemical analysis revealed that a 1250-bp LePT3 promoter fragment and a 471-bp LePT5 promoter fragment containing the two elements, MYCS and P1BS, were sufficient to direct the GUS reporter expression in mycorrhizal roots and were limited to distinct cells harboring AM fungal structures. Additionally, the four paralogues, LePT1, LePT2, LePT6 and LePT7, were very significantly down-regulated in the mycorrhizal roots under low Pi supply condition.

Conclusions: The results obtained from this study provide new insights into the evolutionary expansion, functional divergence and genetic redundancy of the Pht1 genes in response to Pi deficiency and mycorrhizal symbiosis in tomato.

Figure 1

Predicted amino acid sequences of the eight tomato Pht1 genes LePT1 to 8. Sequence alignment analysis was carried out using multiple alignment algorithm wrapped within the DNAMAN 7.0 program (http://www.lynnon.com/). Identical amino acids are shaded and gaps are indicated by dots. The consensus sites for phosphorylation by protein kinase C and casein kinase II are shown by two arrowheads with red colour and the conserved N-glycosylation residue is shown by a green arrowhead. The characteristic Pht1 signature was underlined. The transmembrane domains (broken underline) were predicted by the Toppred algorithm (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html).

Figure 2

Distribution of Pht1 genes (LePT1 to 8 and StPT1 to 10) on the tomato (T) and potato (P) chromosomes. Chromosome numbers are shown at the top of each bar. The arrows next to the gene names indicate the direction of transcription. LePTx, StPTx1 and StPTx2 are putative Pht1 pseudogenes residual in the tomato and potato genomes. The genes from other two families, Pht3 and Pht4, encoding putative Pi transporter or carrier with no homology to the Pht1 proteins, were also labeled on the corresponding chromosomes of the two plants.

Figure 3

Phylogenetic analysis of tomato Pht1 genes and other plant Pht1 homologs. An unrooted phylogenetic tree of the plant Pht1 proteins was constructed using the neighbor-joining method with MEGA 5.0 program. Transporters and corresponding plant species are: tomato, LePT1 to 8 [30,42], this study; potato, StPT1 to 10 [30,43,44], this study; tobacco, NtPT1 to 5 [45,46]; eggplant, SmPT1 to 5 [45,46]; Arabidopsis thaliana, AtPT1 to 9 [47]; Medicago truncatula, MtPT1 to 6 [8,48,49]; Lotus japonicus, LjPT1 to 4 [31,50]; Soybean, GmPT1 to 14 [13]; Rice, OsPT1 to 13 [28]; Barley, HvPT1 to 12 [14,51]; Maize, ZmPT1 to 6 [18,29].

Figure 4

Tissue-specific expression analysis of tomato Pht1 genes. The RNA were prepared from different tissues, including roots (R), stems (S), young leaves (L), flowers (FL), as well as fruits at green (GF) and ripe (RF) stages. The relative expression levels of each of the tomato Pht1 genes were indicated as percentage of the constitutive Actin expression activity. Each bar was the mean of three biological replications with standard error.

Figure 5

Effects of mycorrhizal fungal colonization on tomato biomass (fresh weight, FW) and P concentrations under low Pi (0.05 mM) and high Pi (1 mM) supply condition. –P and + P represent supply of 0.05 mM and 1 mM Pi, respectively; –M and + M represent the inoculation with autoclaved and active inoculum containing arbuscular mycorrhizal fungi Glomus intraradices (Gi), respectively. Error bars indicate SE (n = 3).

Figure 6

Real-time RT-PCR analysis of the Pht1 genes in tomato roots and leaves in response to mycorrhizal fungi colonization under high and low Pi supply conditions. The plants were incubated for two months with a mycorrhizal inoculum containing Glomus intraradices. LP and HP represent supply with 0.05 mM and 1 mM Pi, respectively; -M and + M represent the inoculation with autoclaved and active inoculum, respectively. The relative expression levels of each tomato Pht1 gene was also shown as percentage of the constitutive Actin expression activity. Each bar was the mean of three biological replications with standard error.

Figure 7

Analysis of the tomato Pht1 gene promoters. (A) Comparative analysis of putative cis-regulatory elements responsible for the Pi- and AM-regulated expression between the eight tomato Pht1 promoters. Two previously reported Pi-responsive motifs (P1BS and W-box) and one AM-activated motif (MYCS) were searched using the DNA-pattern matching arithmetic (http://rsat.ulb.ac.be/rsat/). P1BS, GNATATNC; MYCS, TTCTTGTTC; W-box, TTGACY. (B) Histochemical analysis for the promoter activity of the two AM-induced Pht1 members, LePT3 and LePT5. (a-d) Localization of β-glucuronidase (GUS) activity (a and b, Magenta GUS; c and d, blue GUS) in mycorrhizal roots driven by the promoters of LePT3(a, c) and LePT5 (b, d), respectively. (e, f) Co-localization of GUS activity (indicated by the purple color, from the overlay of the Magenta-GUS and Trypan Blue stains) showed that the LePT3and LePT5 promoter fragments (pLePT3_-1250 and pLePT5_-471) were sufficient to direct GUS expression in mycorrhizal roots and were confined to distinct cortical cells containing AM fungal structures (arbuscules or intracellular hyphae). Green arrows indicate arbuscule or arbusculate hyphae, yellow arrows indicate intracellular hyphae and red arrows indicate noncolonized cells.