Regulatory network of microRNA399 and PHO2 by systemic signaling

Abstract

Recently, we showed that microRNA399s (miR399s) control inorganic phosphate (Pi) homeostasis by regulating the expression of PHO2 encoding a ubiquitin-conjugating E2 enzyme 24. Arabidopsis (Arabidopsis thaliana) plants overexpressing miR399 or the pho2 mutant overaccumulate Pi in shoots. The association of Pi translocation and coexpression of miR399s and PHO2 in vascular tissues suggests their involvement in long-distance signaling. In this study, we used reciprocal grafting between wild-type and miR399-overexpressing transgenic plants to dissect the systemic roles of miR399 and PHO2. Arabidopsis rootstocks overexpressing miR399 showed high accumulation of Pi in the wild-type scions because of reduced PHO2 expression in the rootstocks. Although miR399 precursors or expression was not detected, we found a small but substantial amount of mature miR399 in the wild-type rootstocks grafted with transgenic scions, which indicates the movement of miR399 from shoots to roots. Suppression of PHO2 with miR399b or c was less efficient than that with miR399f. Of note, findings in grafted Arabidopsis were also discovered in grafted tobacco (Nicotiana benthamiana) plants. The analysis of the pho1 mutant provides additional support for systemic suppression of PHO2 by the movement of miR399 from Pi-depleted shoots to Pi-sufficient roots. We propose that the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Moreover, PHO2 small interfering RNAs mediated by the cleavage of miR399s may function to refine the suppression of PHO2. The regulation of miR399 and PHO2 via long-distance communication in response to Pi deficiency is discussed.

Figure 1.

Reciprocal grafting between wild-type and miR399f-overexpressing transgenic Arabidopsis plants. A, Pi concentration in the scions. Each spot indicates one data point from an independent grafted plant. B, Relative levels of PHO2 mRNA in the scions (black bars) and rootstocks (gray bars) of grafted plants. C, Relation between the Pi concentration in scions and the expression of PHO2 in rootstocks. D and E, Measurement of pri-miR399f (D) and mature miR399f (E) in scions (black bars) and rootstocks (gray bars), respectively. Amount of mature miR399f in wild-type self-grafting plants subjected to 1- or 2-d Pi starvation treatment were included in E. F, Relative expression of GFP:GUS, pri-miR399f, and mature miR399f in the rootstocks carrying miR399f promoter∷GFP:GUS transgene grafted with miR399f-overexpressing scions (399f/P_399f) or self-grafted controls (P_399f/P_399f). Grafting combinations are shown as scion/rootstock. RNA was analyzed by quantitative RT-PCR and the levels could be compared with those shown in Figure 2. The error bars represent sd (n = 4–5 independent grafted plants). ND, Nondetectable. The difference between two dashed lines in E indicates the potential amount of moved miR399f.

Figure 2.

Reciprocal grafting between wild-type and miR399b-overexpressing transgenic Arabidopsis plants. A, Pi concentration in the scions. Each spot indicates one data point from an independent grafted plant. B, Relative levels of PHO2 mRNA in the scions (black bars) and rootstocks (gray bars) of grafted plants. C and D, Measurement of pri-miR399b (C) and mature miR399b/c (D) in scions (black bars) and rootstocks (gray bars), respectively. The level of mature miR399b/c in wild-type self-grafted plants subjected to 1- or 2-d Pi starvation treatment were included in D. Because of the identical sequences, the level of mature miR399b and c cannot be distinguished. The error bars represent sd (n = 4–5 independent grafted plants). ND, Nondetectable. The difference between two dashed lines in D indicates the potential amount of moved miR399b.

Figure 3.

Sequence alignments among different miR399 species and five potential targets on PHO2 mRNA (A) and the mimicked target sequence on At4/IPS1 noncoding RNAs (B). Mismatched nucleotides are indicated in bold and GU base-pairings are marked in gray. The position of nucleotide 13 is highlighted. Solid triangles indicate the cleavage sites of target sequences by miR399s.

Figure 4.

Reciprocal grafting between wild-type and miR399-overexpressing transgenic tobacco plants. Pi concentration in the scions (A and B) and the relative level of PHO2 mRNA (C and D), pri-miR399 (E and F), and mature miR399 (G and H) in scions (black bars) and rootstocks (gray bars) of grafted plants were measured. A, C, E, and G represent data from grafts between wild-type and miR399f-overexpressing plants. Results from B, D, F, and H are from grafts between wild-type and miR399b-overexpressing plants. The error bars represent the sd (n = 4–5 independent grafted plants). ND, Nondetectable.

Figure 5.

A and B, Time-course analyses of different pri-miR399s (a, b, c, d, and f; A) and mature miR399 species (a, b+c, d, and f; B) during the initiation of Pi deprivation. The dashed and solid lines indicate the expression levels in shoots and roots, respectively. The level of PHO2 mRNA was included in B as a gray solid line. All miR399 species are shown except miR399e because of its low expression. The level of mature miR399b and c cannot be distinguished because of identical sequences. C and D, Relative expression levels of pri-miR399s (C) and mature miR399s (D) in shoots (black bars) and roots (gray bars) after 5 d of Pi starvation treatment. Please note that the mature miR399a and b/c could have been overestimated in B and D (see Supplemental Fig. S5). One of two biological replicates is presented, and the error bars indicate the sd of two technical replicates.

Figure 6.

Quantitative RT-PCR analyses of different pri-miR399s (a–f; A) and mature miR399 species (a, b+c, d, e, and f; B) and PHO2 mRNA (C) in the shoots (black bars) and roots (gray bars) of wild-type plants and pho1 mutant. Please note that the mature miR399a and b/c could have been overestimated in B (see Supplemental Fig. S5). The error bars represent the sd of two technical replicates. ND, Nondetectable.

Figure 7.

Detection of PHO2 siRNA. The location of probe (double-pointed black arrow) and sequenced small RNAs (single-pointed gray arrows indicating the direction of 5′ to 3′) corresponding to the PHO2 transcript are marked in A. The vertical bars within the second exon indicate the five miR399 target sites. B and C, RNA gel-blot analysis of PHO2 siRNAs in Pi-sufficient (+Pi) and 5-d Pi-starved (−Pi) shoots (B) and roots (C) of wild-type (wt), pho2, and miR399-overexpressing (399b and 399f) plants. The siRNA could be detected by both SP and ASP. D, Detection of PHO2 siRNA (ASP) and miR399 in the roots of small RNA biogenesis mutants subjected to 5-d Pi-starvation treatment. E, RNA gel-blot analysis of PHO2 mRNA in the roots of different mutants under Pi-sufficient or -deficient conditions. Staining of 5S ribosomal RNA and tRNA (B–D) and 25S and 18S ribosomal RNAs (E) is shown as the loading control.

Figure 8.

A proposed model for the regulation of miR399 and PHO2 through signal communication between roots and shoots in response to Pi deficiency. SRS, Systemic root-born signal; SSS, systemic shoot-born signal; LRS, local root-born signal. Movement of PHO2 siRNAs is not known and is indicated as a dashed line. Allocation and recycling of Pi between shoots and roots may be involved in this long-distance signaling network and are indicated as circular arrows. See text for a detailed description.