The THO/TREX Complex Active in miRNA Biogenesis Negatively Regulates Root-Associated Acid Phosphatase Activity Induced by Phosphate Starvation

Abstract

Induction and secretion of acid phosphatases (APases) is an adaptive response that plants use to cope with P (Pi) deficiency in their environment. The molecular mechanism that regulates this response, however, is poorly understood. In this work, we identified an Arabidopsis (Arabidopsis thaliana) mutant, hps8, which exhibits enhanced APase activity on its root surface (also called root-associated APase activity). Our molecular and genetic analyses indicate that this altered Pi response results from a mutation in the AtTHO1 gene that encodes a subunit of the THO/TREX protein complex. The mutation in another subunit of this complex, AtTHO3, also enhances root-associated APase activity under Pi starvation. In Arabidopsis, the THO/TREX complex functions in mRNA export and miRNA biogenesis. When treated with Ag(+), an inhibitor of ethylene perception, the enhanced root-associated APase activity in hps8 is largely reversed. hpr1-5 is another mutant allele of AtTHO1 and shows similar phenotypes as hps8 ein2 is completely insensitive to ethylene. In the hpr1-5ein2 double mutant, the enhanced root-associated APase activity is also greatly suppressed. These results indicate that the THO/TREX complex in Arabidopsis negatively regulates root-associated APase activity induced by Pi starvation by inhibiting ethylene signaling. In addition, we found that the miRNA399-PHO2 pathway is also involved in the regulation of root-associated APase activity induced by Pi starvation. These results provide insight into the molecular mechanism underlying the adaptive response of plants to Pi starvation.

Figure 1.

Analysis of Pi starvation-induced root APase activity in 8-d-old Arabidopsis seedlings of the wild type and hps8. A, Histochemical staining of APase activity on the root surface of the seedlings using BCIP as a substrate. B, Root surface-associated APase activity as determined with BCIP as the substrate. C, Root intracellular APase activity as determined with BCIP as the substrate. Values in B and C represent means with se of three replicates. Asterisks indicate significant difference (P < 0.05) from the wild type according to Student’s t test.

Figure 2.

Root morphological phenotypes of 8-d-old Arabidopsis seedlings of the wild type and hps8 grown under Pi sufficiency (P⁺) and deficiency (P⁻) conditions. A, The whole seedlings showing root growth characteristics. B, Patterns of root hair formation.

Figure 3.

Molecular and genetic analyses of hps8. A, A diagram showing the structure of the AtTHO1 gene and the positions of the point mutation in the hps8 and hpr1-5 mutants and the insertions in the attho1 and hpr1-4 mutants. The black box and the horizontal line represent the exons and introns, respectively. The positions of start (ATG) and stop (TGA) codons are shown. B, Histochemical staining of APase activity on the root surface of 8-d-old seedlings of the wild type, hps8, attho1, hpr1-4, hpr1-5, and 35S:AtTHO1 grown on P⁺ medium. C, Root hair patterns of the seedlings corresponding to those shown in B. D, Morphologies of 3-week-old plants of the wild type, hps8, and 35S:AtTHO1 grown in soil.

Figure 4.

Expression pattern and subcellular localization of AtTHO1. A, Relative expression levels of AtTHO1 mRNA in roots, stems, leaves, flowers, and siliques. B, Relative expression levels of AtTHO1 mRNA in shoots and roots of 8-d-old wild-type seedlings grown under P⁺ and P⁻ conditions. Asterisks indicate values that are significantly different from that of the wild type under the same growing conditions (Student’s t test, P < 0.05). C, Subcellular localization of GFP-AtTHO1 in roots of transgenic plants. Top, Root meristem and elongation zones (left) and maturation zone (right). Bottom, a magnified view of the meristematic region at the root tip (left) and near the elongation zone (right).

Figure 5.

Root surface-associated APase activity and root hair patterns in various mutants. A, BCIP staining of APase activities on the root surface of 8-d-old seedlings of the wild type, hps8, attho3, and attho6 grown on P⁺ and P⁻ media. B, Patterns of root hairs in the seedlings corresponding to those in A.

Figure 6.

Effects of the ethylene perception inhibitor Ag⁺ on the root-associated APase activity and root hair production in the wild type and hps8. A, BCIP staining of root-associated APase activity of 8-d-old seedlings of the wild type and hps8 grown on P⁺ and P⁻ medium with or without addition of 10 μM Ag⁺. B, Root hair patterns of the seedlings corresponding to those shown in A.

Figure 7.

The root-associated APase activity and root hair production of the wild type and various mutants grown under P⁺ and P⁻ conditions. A, Histochemical staining using BCIP as a substrate of APase activity on the root surface of 8-d-old seedlings. B, Root hair patterns of 8-d-old seedlings.

Figure 8.

qPCR analysis of relative levels of three mature miRNA399s in the roots of 8-d-old seedlings of the wild type and hps8 grown under P⁺ and P⁻ conditions. Values are means ± se with three replicates. Asterisks indicate that the mean is significantly different from that of the wild type (t-test, P < 0.05).

Figure 9.

Analysis of Pi starvation-induced root APase activity in 8-d-old Arabidopsis seedlings of the wild type, miRNA399 OX, and pho2 grown under P⁺ and P⁻ conditions. A, Histochemical staining of APase activity on the root surface of the seedlings using BCIP as a substrate. B, Root surface-associated APase activity as determined with BCIP as the substrate. C, Root intracellular APase activity as determined with BCIP as the substrate. Values in B and C represent means with se of three replicates. Asterisks indicate a significant difference (P < 0.05) from the wild type according to Student’s t-test.