Systematic Identification, Evolution and Expression Analysis of the Zea mays PHT1 Gene Family Reveals Several New Members Involved in Root Colonization by Arbuscular Mycorrhizal Fungi

Abstract

The Phosphate Transporter1 (PHT1) family of genes plays pivotal roles in the uptake of inorganic phosphate from soils. However, there is no comprehensive report on the PHT1 family in Zea mays based on the whole genome. In the present study, a total of 13 putative PHT1 genes (ZmPHT1;1 to 13) were identified in the inbred line B73 genome by bioinformatics methods. Then, their function was investigated by a yeast PHO84 mutant complementary experiment and qRT-PCR. Thirteen ZmPHT1 genes distributed on six chromosomes (1, 2, 5, 7, 8 and 10) were divided into two paralogues (Class A and Class B). ZmPHT1;1/ZmPHT1;9 and ZmPHT1;9/ZmPHT1;13 are produced from recent segmental duplication events. ZmPHT1;1/ZmPHT1;13 and ZmPHT1;8/ZmPHT1;10 are produced from early segmental duplication events. All 13 putative ZmPHT1s can completely or partly complement the yeast Pi-uptake mutant, and they were obviously induced in maize under low Pi conditions, except for ZmPHT1;1 (p < 0.01), indicating that the overwhelming majority of ZmPHT1 genes can respond to a low Pi condition. ZmPHT1;2, ZmPHT1;4, ZmPHT1;6, ZmPHT1;7, ZmPHT1;9 and ZmPHT1;11 were up-regulated by arbuscular mycorrhizal fungi (AMF), implying that these genes might participate in mediating Pi absorption and/or transport. Analysis of the promoters revealed that the MYCS and P1BS element are widely distributed on the region of different AMF-inducible ZmPHT1 promoters. In light of the above results, five of 13 ZmPHT1 genes were newly-identified AMF-inducible high-affinity phosphate transporters in the maize genome. Our results will lay a foundation for better understanding the PHT1 family evolution and the molecular mechanisms of inorganic phosphate transport under AMF inoculation.

Keywords: AMF-inducible; Zea mays; evolution; expression; phosphate transporters; promoter.

Figure 1

Phylogenetic analysis of PHT1 transporters between maize and other plants. Roman numerals (I–IV) indicate the four PHT1 subfamilies. Transporters from maize (ZmPHT1;1 to ZmPHT1;13), rice (OsPHT1;1 to OsPHT1;13), sorghum (SbPHT1;1 to SbPHT1;11), Brachypodium (BdPHT1;1 to BdPHT1;13) and some recently-identified AM-inducible PHT1 transporters [20]. ZmPHT1s are indicated by filled red circles, and AMF-inducible PHT1s in other plants are indicated by filled blue triangles.

Figure 2

Phylogenetic tree, motif and gene structure of ZmPHT1 genes. (a) The phylogenetic tree contains only ZmPHT1s, which were divided into two groups (Class A and Class B); (b) exons are indicated by grey boxes, and introns are indicated by single lines; black lines represent untranslated regions (UTR); (c) motifs’ analysis; different colors represent different motifs.

Figure 3

Distribution and synteny of ZmPHT1 genes. (a) Distribution of ZmPHT1 genes on the maize chromosomes. On the top of each bar is the chromosome number; (b) synteny of ZmPHT1 genes. Zm1–10 represented maize chromosome 1–10, indicated by colored boxes. Chromosome box numbers represent sequence lengths in megabases. All of the syntenic genes were located in the map and linked by red lines.

Figure 3

Distribution and synteny of ZmPHT1 genes. (a) Distribution of ZmPHT1 genes on the maize chromosomes. On the top of each bar is the chromosome number; (b) synteny of ZmPHT1 genes. Zm1–10 represented maize chromosome 1–10, indicated by colored boxes. Chromosome box numbers represent sequence lengths in megabases. All of the syntenic genes were located in the map and linked by red lines.

Figure 4

Heat map of maize ZmPHT1 genes. The expression level of each ZmPHT1 genes can be estimated based on the scale to the right. Red indicates high expression level; yellow indicates medium expression level; and blue indicates low expression level. H, hours; DAS, days after sowing; GH, greenhouse; V, vegetative; DAP, days after pollination; VT, vegetative tasseling; R, reproductive.

Figure 5

Functional characterizations of ZmPHT1s in a yeast inorganic phosphate (Pi) transport mutant. Under two Pi conditions three repeat experiments were set respectively. Repeat experiments were represented by a1, a2, a3 under 20 µM and b1, b2, b3 under 100 µM Pi conditions.

Figure 6

Expression pattern analyses of ZmPHT1 genes in root related to Pi availability. Maize was grown in nutrient solution containing 50 µM Pi and 5 mM Pi and sampled 40 days after treatment initiation. Data points are the means ± SD (n = 3 biological replicates of three plants each).

Figure 7

qRT-PCR analysis of ZmPHT1 genes in root samples in response to AMF inoculation under low Pi supply. Maize were grown in a low Pi (50 µM) condition and an inoculation with autoclaved and active Glomus etunicatum, respectively. Data points are the means ± SD (n = 3 biological replicates of three plants each).

Figure 8

Analysis of regulatory elements in the promoters of ZmPHT1 genes. Three previously reported Pi-responsive motifs (P1BS, PHO and W-box) and two AM-activated motif (MYCS and OSEROOTNODULE) were searched by DNA-pattern matching arithmetic. PHO (CACGTG), P1BS (GNATATNC), W-BOX (TTGACY), MYCS (TTTCTTGTTCT, or with one nucleotide variation), OSEROOTNODULE (AAAGAT).