Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.     

Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl‐prolyl cis‐trans isomerase of the FK506‐binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over‐expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H⁺‐ATPase) is indirect: over‐expression of ROF2 activates K⁺ uptake, causing depolarization of the plasma membrane, which activates the electrogenic H⁺ pump. The depolarization of ROF2 over‐expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.     

Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H(+)-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.     

Single-stranded DNA binding factor AtWHY1 modulates telomere length homeostasis in Arabidopsis

Telomere homeostasis, a process that is essential for the maintenance of chromosome integrity, is regulated by telomerase and a collection of associated proteins. By mass spectrometry we have identified a new telomeric protein encoded by the AtWHY1 (Arabidopsis thaliana Whirly 1) gene in Arabidopsis. AtWHY1 specifically binds the single-stranded plant telomeric DNA sequences, but not double-stranded telomeric DNA. To gain insights into the function of AtWHY1 in telomere biogenesis, we have identified two Arabidopsis lines harboring T-DNA insertions in AtWHY1. These lines exhibit neither growth nor developmental defects. However, AtWHY1-deficient plants show a steady increase in the length of telomere tracts over generations. This telomere elongation is correlated with a significant increase in telomerase activity. On the contrary, transgenic plants expressing AtWHY1 show a decreased telomerase activity and shortened telomeres. The evidence presented here indicates that AtWHY1 is a new family of telomere end-binding proteins that plays a role in regulating telomere-length homeostasis in Arabidopsis.     

Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis

Emerging studies have suggested that there is a close link between inositol phosphate (InsP) metabolism and cellular phosphate (P_i) homeostasis in eukaryotes; however, whether a common InsP species is deployed as an evolutionarily conserved metabolic messenger to mediate P_i signaling remains unknown. Here, using genetics and InsP profiling combined with P_i‐starvation response (PSR) analysis in Arabidopsis thaliana, we showed that the kinase activity of inositol pentakisphosphate 2‐kinase (IPK1), an enzyme required for phytate (inositol hexakisphosphate; InsP₆) synthesis, is indispensable for maintaining P_i homeostasis under P_i‐replete conditions, and inositol 1,3,4‐trisphosphate 5/6‐kinase 1 (ITPK1) plays an equivalent role. Although both ipk1‐1 and itpk1 mutants exhibited decreased levels of InsP₆ and diphosphoinositol pentakisphosphate (PP‐InsP₅; InsP₇), disruption of another ITPK family enzyme, ITPK4, which correspondingly caused depletion of InsP₆ and InsP₇, did not display similar P_i‐related phenotypes, which precludes these InsP species from being effectors. Notably, the level of d /l ‐Ins(3,4,5,6)P₄ was concurrently elevated in both ipk1‐1 and itpk1 mutants, which showed a specific correlation with the misregulated P_i phenotypes. However, the level of d /l ‐Ins(3,4,5,6)P₄ is not responsive to P_i starvation that instead manifests a shoot‐specific increase in the InsP₇ level. This study demonstrates a more nuanced picture of the intersection of InsP metabolism and P_i homeostasis and PSRs than has previously been elaborated, and additionally establishes intermediate steps to phytate biosynthesis in plant vegetative tissues.     

Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis

The mobilization of inorganic phosphate (Pi) in planta is a complex process regulated by a number of developmental and environmental cues. Plants possess many Pi transporters that acquire Pi from the rhizosphere and translocate it throughout the plant. A few members of the high-affinity Pht1 family of Pi transporters have been functionally characterized and, for the most part, have been shown to be involved in Pi acquisition. We recently demonstrated that the Arabidopsis Pi transporter, Pht1;5, plays a key role in translocating Pi between tissues. Loss-of-function pht1;5 mutant seedlings accumulated more P in shoots relative to wild type but less in roots. In contrast, overexpression of Pht1;5 resulted in a lower P shoot:root ratio compared with wild type. Also, the rosette leaves of Pht1;5-overexpression plants senesced early and contained less P, whereas reproductive organs accumulated more P than those of wild type. Herein we report the molecular response of disrupting Pht1;5 expression on other factors known to modulate P distribution. The results reveal reciprocal mis-regulation of PHO1, miR399d, and At4 in the pht1;5 mutant and Pht1;5-overexpressor, consistent with the corresponding changes in P distribution in these lines. Together our studies reveal a complex role for Pht1;5 in regulating Pi homeostasis.     

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi‐deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi‐sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi‐deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.     

The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis

In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.     

PID1 in adipocytes modulates whole-body glucose homeostasis

The novel obesity-associated protein Phosphotyrosine Interaction Domain containing 1 (PID1) inhibits insulin-PI3K/Akt signaling pathway and insulin-stimulated glucose uptake in vitro. In this study, we generated fat tissue-specific aP2-PID1 transgenic (aP2-PID1^tg) mice and PID1 knockout (PID1^?/?) mice to explore how PID1 affects glucose metabolism in vivo. We observed insulin resistance and impaired insulin-PI3K/Akt signaling in aP2-PID1^tg mice. Consistent with these data, the PID1^?/? mice displayed improved glucose tolerance and insulin sensitivity under chow diet, with increased Akt phosphorylation in white adipose tissue (WAT). We further demonstrated that PID1 could interact with low density lipoprotein receptor-related protein 1 (LRP1) but not the insulin receptor (IR) in adipocytes, and its overexpression could lead to decreased GLUT4 level. Our results thus indentify PID1 as a critical regulator of glucose metabolism in adipocytes.     

Amino acid homeostasis modulates salicylic acid-associated redox status and defense responses in Arabidopsis

The tight association between nitrogen status and pathogenesis has been broadly documented in plant-pathogen interactions. However, the interface between primary metabolism and disease responses remains largely unclear. Here, we show that knockout of a single amino acid transporter, LYSINE HISTIDINE TRANSPORTER1 (LHT1), is sufficient for Arabidopsis thaliana plants to confer a broad spectrum of disease resistance in a salicylic acid-dependent manner. We found that redox fine-tuning in photosynthetic cells was causally linked to the lht1 mutant-associated phenotypes. Furthermore, the enhanced resistance in lht1 could be attributed to a specific deficiency of its main physiological substrate, Gln, and not to a general nitrogen deficiency. Thus, by enabling nitrogen metabolism to moderate the cellular redox status, a plant primary metabolite, Gln, plays a crucial role in plant disease resistance.     

Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter: functional characterization in Escherichia coli

In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na+-dependent inorganic phosphate (Pi) transporters. The function of ANTR1 as a Na+-dependent Pi transporter was demonstrated by heterologous expression and uptake of radioactive Pi into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated Pi uptake in a pH- and Na+-dependent manner as compared with the control cells. Among various tested effectors, Pi was the preferred substrate. Although it competed with the uptake of Pi, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a Pi transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of Pi produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H+ electrochemical gradient storage.