Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis

Abstract

Members of the Arabidopsis thaliana phosphate transporter1 (PHT1) family are key players in acquisition of Pi from the rhizosphere, and their regulation is indispensable for the maintenance of cellular Pi homeostasis. Here, we reveal posttranslational regulation of Pi transport through modulation of degradation of PHT1 proteins by the RING-type ubiquitin E3 ligase, nitrogen limitation adaptation (NLA). Loss of function of NLA caused high Pi accumulation resulting from increases in the levels of several PHT1s at the protein rather than the transcript level. Evidence of decreased endocytosis and ubiquitination of PHT1s in nla mutants and interaction between NLA and PHT1s in the plasma membranes suggests that NLA directs the ubiquitination of plasma membrane-localized PHT1s, which triggers clathrin-dependent endocytosis followed by endosomal sorting to vacuoles. Furthermore, different subcellular localization of NLA and phosphate2 (pho2; a ubiquitin E2 conjugase) and the synergistic effect of the accumulation of PHT1s and Pi in nla pho2 mutants suggest that they function independently but cooperatively to regulate PHT1 protein amounts. Intriguingly, NLA and PHO2 are the targets of two Pi starvation-induced microRNAs, miR827 and miR399, respectively. Therefore, our findings uncover modulation of Pi transport activity in response to Pi availability through the integration of a microRNA-mediated posttranscriptional pathway and a ubiquitin-mediated posttranslational regulatory pathway.

Figure 1.

NLA Is Involved in the Regulation of Protein Abundance of PHT1s. (A) Gene structure and mutation sites of NLA. Black bars, untranslated region; gray bars, coding region. Col, Columbia; Ws, Wassilewskija. (B) Pi content of the wild type and nla mutants under Pi-sufficient conditions. n = 10; error bar indicates se. Student’s t test, mutants versus the wild type, **P < 0.01 and ***P < 0.005. FW, fresh weight. (C) qRT-PCR analysis of PHT1;1, PHT1;2, PHT1;3, and PHT1;4 mRNA in the roots of the wild type and nla mutants. n = 3; error bar indicates se. Student’s t test, mutants versus the wild type, *P < 0.05. CT, cycle threshold. (D) Immunoblot analysis of NLA, PHT1;1/2/3, and PHT1;4 proteins in the 19-d-old wild type and nla mutants under Pi-sufficient conditions.

Figure 2.

NLA-Mediated Regulation Is Important to Avoid Pi Overaccumulation. (A) Shoot Pi content after Pi replenishment in the wild type and nla-2 mutants. n = 8; error bar indicates se. Student’s t test, mutants versus the wild type, *P < 0.05 and ***P < 0.005. FW, fresh weight. (B) Immunoblot analysis of PHT1;1/2/3, PHT1;4, and NLA proteins in roots of the wild type and nla-2 mutants after Pi replenishment. The detection of ARF1 was used as a loading control.

Figure 3.

NLA Is Predominantly Localized in the PM. (A) Subcellular localization of CFP-NLA and NLA-CFP in tobacco leaves. The “Merge” panel is an overlaid image of the CFP and bright field. Bars = 20 μm. (B) Subcellular localization of truncated NLA proteins, NLA^SPX and NLA^RING, in tobacco leaves. Bars = 20 μm. (C) and (D) Immunoblot analyses revealed predominant localization of NLA in the PM protein fraction rather than the EM, nuclear (N), or soluble protein (S) fractions of Arabidopsis roots. TM, total microsomal proteins. Col, Columbia. (E) Immunoblot analysis of PHT1;1/2/3 and PHT1;4 in different membrane fractions of the wild type and nla-2 mutants. Specific protein markers for different subcellular compartments were as follows: PIP1 and H⁺-ATPase, PM protein; PHF1, ER protein; cFBPase, cytosolic protein; and HIS3, nuclear protein.

Figure 4.

The RING Domain of NLA Is Required for the Degradation of PHT1;1 and PHT1;4. Coexpression of At-NLA, At-NLA^C279A, or Os-NLA1 with At-PHT1;1 (A) or At-PHT1;4 (B) in tobacco leaves. GFP was used as the control for the infiltration event. Note that “+++” and “++” denotes fivefold and 2.5-fold increases in volume of infiltration relative to “+,” respectively.

Figure 5.

PHT1;1 and PHT1;4 Are the Ubiquitination Substrates of NLA. (A) and (B) In vivo ubiquitination of PHT1;1/2/3 (A) and PHT1;4 (B) in the roots of the wild type and nla-2 mutants under Pi-sufficient conditions and after 1 d of Pi recovery, respectively. The corresponding antibodies were used for immunoprecipitation (IP) (bound). Immunoprecipitation without adding antibodies (unbound) was used as negative control. PHT1;1/2/3 and PHT1;4 protein levels of the immunoprecipitation products are shown in the bottom panels. Col, Columbia. (C) and (D) BiFC analysis of the interaction between NLA^SPX and PHT1;1 or PHT1;4 (C) and NLA^SPX and CHL1 or IRT1 (D). Reconstituted fluorescence signals were detected in the PM of tobacco leaf cells when NLA^SPX-nYFP was coexpressed with PHT1;1- or PHT1;4-cYFP but not with CHL1-cYFP or IRT1-cYFP. Coexpression of nYFP or cYFP with the corresponding PHT1;1-cYFP or PHT1;4-cYFP or NLA^SPX-nYFP constructs was used as an additional control. The top panels are YFP images, and the bottom panels are overlaid images of YFP and bright field. Bars = 20 μm. (E) The interaction between NLA^SPX and PHT1;4 by split-ubiquitin yeast two-hybrid analysis. NubI and NubG, the wild type and mutated N-terminal fragment of ubiquitin with isoleucine and glycine at position 13, respectively; Cub, C-terminal ubiquitin. Dolichyl-phosphate beta-glucosyltransferase (Alg)-Cub is the control that could interact with NubI but not with NubG and NubG-NLA^SPX.

Figure 6.

NLA-Mediated Ubiquitination Triggers Clathrin-Dependent Endocytosis of PHT1;1 and PHT1;4 from the PM and Sorting into the Endosomal Trafficking Pathway. (A) The punctate signals of YFP (arrowheads) were observed when PHT1;1-YFP or PHT1;4-YFP was coexpressed with NLA but not with NLA^C279A in tobacco leaves. Bars = 10 μm. (B) The punctate structure of PHT1;1-YFP caused by NLA was diminished by applying Tyr A23, an inhibitor of clathrin-dependent endocytosis, but not by its analog, Tyr A51. The signal was observed 24 h after the chemicals were applied. Bars = 10 μm. (C) and (D) The effect of E-64d on the localization of PHT1;1-YFP in the wild type (C) and nla-2 mutant plants (D). The samples were observed 3 h after treatment with E-64d and FM4-64. DMSO was used as the negative control. The aggregation of PHT1;1-YFP in the wild type (C) is indicated by arrowheads. Bars = 20 μm.

Figure 7.

NLA and PHO2 Function Cooperatively to Regulate the Degradation of PHT1 Proteins. (A) and (B) The Pi content (A) and protein expression of PHT1;1/2/3, PHT1;4, and PHF1 (B) in nla-2 pho2 double and single mutants. n = 3; error bar indicates se; Student’s t test, single mutants versus the wild type, *P < 0.05, **P < 0.01, and ***P < 0.005; nla-2 pho2 versus pho2, ⁺P < 0.05 and ⁺⁺⁺P < 0.005. Col, Columbia; FW, fresh weight. The detection of ARF1 was used as a loading control. (C) and (D) qRT-PCR analysis of PHO2 mRNA in the root (C) and the shoot Pi content (D) of the wild type and nla mutants during plant growth. n = 3; error bar indicates se; Student’s t test, mutants versus the wild type, *P < 0.05, **P < 0.01, and ***P < 0.005. CT, cycle threshold.

Figure 8.

A Working Model of the Regulation of Pi Uptake via MiRNA-Mediated Posttranscriptional and Ubiquitin-Mediated Posttranslational Regulation. NLA and PHO2 function in the PM and EM system, respectively, to direct the degradation of PHT1s through protein ubiquitination machinery. Degradation of PHT1s is relieved by miR827- and miR399-mediated posttranscriptional cleavages on the transcripts NLA and PHO2, respectively, upon Pi deficiency. UBC designates a ubiquitin E2 conjugase that works together with NLA to ubiquitinate PHT1s in the PM.