Pi Accumulation Research Articles

Arabidopsis Pht1;5 Mobilizes Phosphate between Source and Sink Organs and Influences the Interaction between Phosphate Homeostasis and Ethylene Signaling

Phosphorus (P) remobilization in plants is required for continuous growth and development. The Arabidopsis (Arabidopsis thaliana) inorganic phosphate (Pi) transporter Pht1;5 has been implicated in mobilizing stored Pi out of older leaves. In this study, we used a reverse genetics approach to study the role of Pht1;5 in Pi homeostasis. Under low-Pi conditions, Pht1;5 loss of function (pht1;5-1) resulted in reduced P allocation to shoots and elevated transcript levels for several Pi starvation-response genes. Under Pi-replete conditions, pht1;5-1 had higher shoot P content compared with the wild type but had reduced P content in roots. Constitutive overexpression of Pht1;5 had the opposite effect on P distribution: namely, lower P levels in shoots compared with the wild type but higher P content in roots. Pht1;5 overexpression also resulted in altered Pi remobilization, as evidenced by a greater than 2-fold increase in the accumulation of Pi in siliques, premature senescence, and an increase in transcript levels of genes involved in Pi scavenging. Furthermore, Pht1;5 overexpressors exhibited increased root hair formation and reduced primary root growth that could be rescued by the application of silver nitrate (ethylene perception inhibitor) or aminoethoxyvinylglycine (ethylene biosynthesis inhibitor), respectively. Together, these data indicate that Pht1;5 plays a critical role in mobilizing Pi from P source to sink organs in accordance with developmental cues and P status. The study also provides evidence for a link between Pi and ethylene signaling pathways.

Vacuolar Ca2+/H+ Transport Activity Is Required for Systemic Phosphate Homeostasis Involving Shoot-to-Root Signaling in Arabidopsis

Calcium ions (Ca(2+)) and Ca(2+)-related proteins mediate a wide array of downstream processes involved in plant responses to abiotic stresses. In Arabidopsis (Arabidopsis thaliana), disruption of the vacuolar Ca(2+)/H(+) transporters CAX1 and CAX3 causes notable alterations in the shoot ionome, including phosphate (P(i)) content. In this study, we showed that the cax1/cax3 double mutant displays an elevated P(i) level in shoots as a result of increased P(i) uptake in a miR399/PHO2-independent signaling pathway. Microarray analysis of the cax1/cax3 mutant suggests the regulatory function of CAX1 and CAX3 in suppressing the expression of a subset of shoot P(i) starvation-responsive genes, including genes encoding the PHT1;4 P(i) transporter and two SPX domain-containing proteins, SPX1 and SPX3. Moreover, although the expression of several PHT1 genes and PHT1;1/2/3 proteins is not up-regulated in the root of cax1/cax3, results from reciprocal grafting experiments indicate that the cax1/cax3 scion is responsible for high P(i) accumulation in grafted plants and that the pht1;1 rootstock is sufficient to moderately repress such P(i) accumulation. Based on these findings, we propose that CAX1 and CAX3 mediate a shoot-derived signal that modulates the activity of the root P(i) transporter system, likely in part via posttranslational regulation of PHT1;1 P(i) transporters.

The Phosphate Transporter Gene OsPht1;8 Is Involved in Phosphate Homeostasis in Rice

Plant phosphate transporters (PTs) are active in the uptake of inorganic phosphate (Pi) from the soil and its translocation within the plant. Here, we report on the biological properties and physiological roles of OsPht1;8 (OsPT8), one of the PTs belonging to the Pht1 family in rice (Oryza sativa). Expression of a β-glucuronidase and green fluorescent protein reporter gene driven by the OsPT8 promoter showed that OsPT8 is expressed in various tissue organs from roots to seeds independent of Pi supply. OsPT8 was able to complement a yeast Pi-uptake mutant and increase Pi accumulation of Xenopus laevis oocytes when supplied with micromolar (33)Pi concentrations at their external solution, indicating that it has a high affinity for Pi transport. Overexpression of OsPT8 resulted in excessive Pi in both roots and shoots and Pi toxic symptoms under the high-Pi supply condition. In contrast, knockdown of OsPT8 by RNA interference decreased Pi uptake and plant growth under both high- and low-Pi conditions. Moreover, OsPT8 suppression resulted in an increase of phosphorus content in the panicle axis and in a decrease of phosphorus content in unfilled grain hulls, accompanied by lower seed-setting rate. Altogether, our data suggest that OsPT8 is involved in Pi homeostasis in rice and is critical for plant growth and development.

Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of "comet tails" that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P(2) and PI(3,4,5)P(3) on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P(2) and PI(3,4,5)P(3) and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P(2) and PI(3,4,5)P(3) formation and signaling in endomembranes.

Signaling of extracellular inorganic phosphate up-regulates cyclin D1 expression in proliferating chondrocytes via the Na +/Pi cotransporter Pit-1 and Raf/MEK/ERK pathway

As chondrocytes mature, the concentration of inorganic phosphate (Pi) increases in the extracellular milieu. It was demonstrated that the progressive accumulation of Pi started from the proliferative zone and peaked in the hypertrophic zone of growth plate. Although extracellular Pi is reported to be involved in the apoptosis and mineralization of mature chondrocytes, its role in proliferating chondrocytes remains unclear. Here we investigated this role utilizing ATDC5, an established cell model of chondrocytic differentiation. In proliferating ATDC5 cells, we found that the expression of cyclin D1 was up-regulated, and that of alkaline phosphatase (ALP) was down-regulated in response to an increase in extracellular Pi within 24 h. Moreover, an increase in extracellular Pi-induced activation of the Raf/MEK/ERK pathway, and treatment with a MEK inhibitor PD98059 abolished the effects on the expression of cyclin D1 and ALP, indicating that extracellular Pi regulates the expression of these genes through the Raf/MEK/ERK pathway. Consistent with its up-regulation of cyclin D1 expression, the extracellular Pi facilitated the proliferation of ATDC5 cells. Treatment with phosphonoformic acid (PFA), an inhibitor of sodium/phosphate (Na +/Pi) cotransporters, abrogated the activation of the Raf/MEK/ERK pathway and gene expression induced by the increase in extracellular Pi. Knocking down of the type III Na +/Pi cotransporter Pit-1 diminished the responsiveness of ATDC5 cells to the increase in extracellular Pi. Interestingly, the increased extracellular Pi induced the phosphorylation of fibroblast growth factor receptor substrate 2α (FRS2α), which was also cancelled by knocking down of the expression of Pit-1. In primary chondrocytes isolated from mouse rib cages as well, increased extracellular Pi induced the phosphorylation of ERK1/2 and alterations in the expression of cyclin D1 and ALP, both of which were abolished by treatment with PFA. These results suggest that signaling by extracellular Pi is mediated by Pit-1 and FRS2α, and leads to activation of the Raf/MEK/ERK pathway and increased expression of cyclin D1, which facilitates the proliferation of immature chondrocytes.

Does OsPHR2, central Pi-signaling regulator, regulate some unknown factors crucial for plant growth?

OsPHR2, the homolog of AtPHR1, is a central Pi-signaling regulator. The Pi-signaling pathway downstream of AtPHR1, similarly of OsPHR2,1,2 involves a noncoding RNA which targets mimicry of miR399. miRNA399 mediates cleavage of PHO2.3,4 The regulating pathway downstream of OsPHR2 is negatively regulated by the Pi-signaling responsive gene OsSPX1.5,6 Overexpression of AtPHR1 and OsPHR2 leads to an increased concentration of Pi in the shoot tissues with leaf toxic symptom and growth retardation similar as the phenotype of pho2 mutant, especially under Pi abundant conditions.7,2,6 It has been known that the low affinity Pi transporter OsPT2 mainly contributes to the shoot Pi accumulation mediated by OsPHR2, and overexpression of OsPT2 results in shoot Pi accumulation and leaf toxic symptom and growth retardation under Pi abundant conditions.6 Two curious questions are emerging from the reported results: How Os SPX1 functions on the negative regulation of the pathway and what mechanism of the growth retardation mediated by OsPHR2. For the second question, our favored hypothesis is that the growth inhibition mediated by overexpression of OsPHR2 is caused by toxic physiological effects due to excessive Pi accumulation in shoots (Pi toxicity). In fact, the toxic symptoms become diminished with decreased Pi levels in growth medium. However, the plant growth retardation mediated by overexpression of OsPHR2 may be caused by some unknown genetic factor(s) regulated by OsPHR2.

OsSPX1 suppresses the function of OsPHR2 in the regulation of expression of OsPT2 and phosphate homeostasis in shoots of rice

Phosphate (Pi) homeostasis in plants is required for plant growth and development, and is achieved by the coordination of Pi acquisition, translocation from roots to shoots, and remobilization within plants. Previous reports have demonstrated that over-expression of OsPHR2 (the homolog of AtPHR1) and knockdown of OsSPX1 result in accumulation of excessive shoot Pi in rice. Here we report that OsPHR2 positively regulates the low-affinity Pi transporter gene OsPT2 by physical interaction and upstream regulation of OsPHO2 in roots. OsPT2 is responsible for most of the OsPHR2-mediated accumulation of excess shoot Pi. OsSPX1 suppresses the regulation on expression of OsPT2 by OsPHR2 and the accumulation of excess shoot Pi, but it does not suppress induction of OsPT2 or the accumulation of excessive shoot Pi in the Ospho2 mutant. Our data also show that OsSPX1 is a negative regulator of OsPHR2 and is involved in the feedback of Pi-signaling network in roots that is defined by OsPHR2 and OsPHO2. This finding provides new insight into the regulatory mechanism of Pi uptake, translocation, allocation and homeostasis in plants.

Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana

Low temperature is a major environmental stress for plants. Many important cultivated crops have limited capacity to survive below freezing/subfreezing temperatures. Low inorganic phosphate (Pi) is reportedly important in triggering cold acclimatization. SPX (SYG1/Pho81/XPR1: SYG1, suppressor of yeast gpal; Pho81, CDK inhibitor in yeast PHO pathway; XPR1, xenotropic and polytropic retrovirus receptor) domain proteins have been shown to be involved in the phosphate-related signal transduction and regulation pathways. Recently, Arabidopsis AtSPX family genes have been found to possess diverse functions in plant tolerance to phosphorus starvation, and OsSPX1 is involved in phosphate homeostasis in rice and optimizes growth under phosphate-limited conditions through a negative feedback loop. In this study, our phylogenetic and gene expression profiling approaches identified six rice OsSPX genes up-regulated during cold stress. Transgenic tobacco plants with constitutive expression of OsSPX1 were more tolerant to cold stress than were wild-type plants, and showed better seedling survival and reduced cellular electrolyte leakage. In addition, there was decreased total leaf Pi content and accumulation of free proline and sucrose in transgenic tobacco plants during cold stress. To further establish a cause-and-effect relationship between intracellular Pi level and cold acclimatization in transgenic plants, we generated transgenic Arabidopsis plants with constitutive expression of OsSPX1. Cold stress resulted in reduced leaf Pi levels in Arabidopsis transgenic relative to wild-type plants. From real-time reverse transcriptase-polymerase chain reaction analysis, several Pi starvation-related genes, such as AtSPX1 (orthologue of OsSPX1), PHO2, PLDZ2 and ATSIZ1, showed differential expression between wild-type and transgenic plants during cold stress. Our results indicate that OsSPX1 may play an important role in linking cold stress and Pi starvation signal transduction pathways.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation

Plant phosphate (Pi) transporters mediate the uptake and translocation of this nutrient within plants. A total of 13 sequences in the rice (Oryza sativa) genome can be identified as belonging to the Pi transporter (Pht1) family. Here, we report on the expression patterns, biological properties and the physiological roles of two members of the family: OsPht1;2 (OsPT2) and OsPht1;6 (OsPT6). Expression of both genes increased significantly under Pi deprivation in roots and shoots. By using transgenic rice plants expressing the GUS reporter gene, driven by their promoters, we detected that OsPT2 was localized exclusively in the stele of primary and lateral roots, whereas OsPT6 was expressed in both epidermal and cortical cells of the younger primary and lateral roots. OsPT6, but not OsPT2, was able to complement a yeast Pi uptake mutant in the high-affinity concentration range. Xenopus oocytes injected with OsPT2 mRNA showed increased Pi accumulation and a Pi-elicited depolarization of the cell membrane electrical potential, when supplied with mM external concentrations. Both results show that OsPT2 mediated the uptake of Pi in oocytes. In transgenic rice, the knock-down of either OsPT2 or OsPT6 expression by RNA interference significantly decreased both the uptake and the long-distance transport of Pi from roots to shoots. Taken together, these data suggest OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, whereas OsPT2 is a low-affinity Pi transporter, and functions in translocation of the stored Pi in the plant.

Skeletal Muscle Fatigue

The article by Jane Kent-Braun, Ph.D., FACSM, (6) combines a muscular component of aging with a neural component to hypothesize a silver lining - as people age, their muscles get weaker, but also less fatigable. The muscular component combines consistent evidence of a shift in fiber-type distribution with age and her own data showing reduced accumulation of H+ and Pi in aged compared with younger subjects' muscles during successive maximum voluntary contractions to make a compelling case for increased metabolic economy of aged muscle. To support the neural component, she reviews a small but consistent literature reporting reduced maximal motor unit discharge rates in aged subjects and hypothesizes a consequent reduced metabolic demand. Together, these mechanisms are hypothesized to increase metabolic economy and decrease accumulation of metabolic products, an adaptation that decreases fatigability in aged muscle. The literature reviewed in (6) comparing fatigability of younger with that of older people's muscles, shows mixed results attributed in this article to dynamic versus static fatiguing protocols. It is known that fatigue can be caused by many different mechanisms. Kent-Braun's technique identifies fatigue that is associated with less acidosis and lower accumulation of Pi and H2PO4-. Cairns et al. (2) pointed out that various protocols might selectively stress different mechanisms among the manifold possible causes of fatigue, the protocol effectively selecting the mechanism of fatigue. In this context, it is important to ask whether protocols that select for fatigue caused by accumulation of metabolites are the most appropriate to study fatigue mechanisms relevant to problems associated with aging. We also suggest that the reduction in maximal motor unit discharge frequency among aged subjects may not be a neural adaptation to aging, but rather a corollary to the selective cell death of larger motor neurons that occurs with aging. Motor neurons serving Type I muscle units have lower discharge rates, increased metabolic economy because of both less calcium pumping and lower myosin ATPase rates (1,8), less production of metabolic products, less sensitivity to metabolic products (4), and lower susceptibility to deficits in excitation-contraction coupling (7). We therefore suggest an alternative hypothesis: a larger proportion of Type I motor unit cross-sectional area confers resistance to some important types of fatigue. Our hypothesis is consistent with that of Kent-Braun (6), but may make the age advantage also a gender advantage, and a general advantage to all of us with slower fiber-type distributions - likely a large population, as the variance in fiber-type distribution in human populations is very large (5). Type I fibers have long been recognized as less fatigable than Type II under a restricted definition of fatigue. Future studies might relax this restriction. There are emerging experimental approaches that focus on identifying mechanisms of fatigue that are task specific (3). Selected comparisons of tasks, groups of individuals, and interventions with a task-failure approach have potential to delve into the mechanisms that limit performance of many different activities of daily living, providing a translational approach. Steven L. Lehman Department of Integrative Biology University of California-Berkeley Berkeley, CA Ladora V. Thompson Department of Physical Medicine and Rehabilitations University of Minnesota Minneapolis, MN

Molecular cloning and characterization of phosphate (Pi) responsive genes in Gulf ryegrass (Lolium multiflorum L.): a Pi hyperaccumulator

Gulf annual ryegrass has been identified as potential Pi hyperaccumulator, however the molecular mechanism remains largely unknown. A suppression subtractive hybridization (SSH) analysis was used to evaluate the phosphate (Pi) responsive genome expression pattern changes in Gulf annual ryegrass (Lolium multiflorum L.). Differential screening identified 384 putative Pi-starvation induced cDNAs. Bioinformatic analysis revealed that 116 cDNAs are nonredundant unigenes of which 108 exhibited high similarities with Genbank entries. The differential expression patterns of 13 cDNAs, representing diverse functional categories, were confirmed by RNA gel blot analysis. Further, detailed molecular analysis of three genes (LmPAP1, LmIPS1 and LmIDS1) was carried out by cloning and characterization of full-length cDNAs. LmPAP1 is 1,414 bp in length with an open reading frame (ORF) of 1,188 bp capable of encoding an N-terminal signal peptide of 26 amino acids. LmIPS1 gene is a member of TPSI1/Mt4 family that contains 3 short ORFs. The cDNA of LmIDS1 is 346 bp in size including a single ORF of 222 nucleotides that encodes 74 amino acid proteins, exhibiting homology with IDS1 with similarity to type 2 metallothionein like protein. In our preliminary screening of different genotypes of annual ryegrass for hyperaccumulation of Pi in their shoots, Gulf and Urugrary showed significant differences with values of 1.0% and 0.7%, respectively. Since it is logical to assume a plausible correlation that may exist between Pi-accumulation in the shoots and the expression of Pi-responsive genes, the expression of LmPAP1, LmIPS1 and LmIDS1 was evaluated in these two genotypes grown under different Pi regimes. Although there was a significant induction of these genes in both the genotypes grown under Pi-deprived condition, the abundance of LmPAP1 transcripts was relatively higher in the Gulf genotype as compared to that in the Urugrary genotype. A similar trend was observed in qRT-PCR data of other tested genotypes of annual ryegrasses. This suggests the potential role of LmPAP1 in accumulation of Pi in Gulf grass. In addition, Gulf grass genotype revealed higher levels of total P, (33)Pi uptake, and APase activity as compared to Urugrary. Together, these results suggest that the Gulf ryegrass has evolved mechanisms to acquire and hyperaccumulate more Pi under different Pi regimes by activating multiple Pi acquisition and mobilization mechanisms.

Role of OsPHR2 on phosphorus homoestasis and root hairs development in rice (Oryza sativa L.)

AtPHR1 plays a central role in Pi-starvation signaling in Arabidopsis (Arabidopsis thaliana). Two PHR genes were isolated from rice and designated as OsPHR1 and OsPHR2 based on amino acid sequence homology to AtPHR1. Transgenic plants with overexpression and repression of OsPHR1 and OsPHR2, respectively, were used for investigation of roles of the genes in Pi-signaling pathway and Pi homeostasis under Pi-sufficient and deficient conditions. The results showed that both of the genes are involved in the Pi-signaling pathway, while overexpression of OsPHR2 mimics Pi-starvation stress with enhanced root elongation and proliferated root hairs, and results in the excessive accumulation of Pi in shoots under Pi-sufficient conditions. OsPHR2 regulated proliferation of root hair growth and root elongation suggests that OsPHR2 is involved in both systematic and local Pi-signaling pathways.

Regulatory network of microRNA399 and PHO2 by systemic signaling.

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.

ROOT HAIR DEFECTIVE4Encodes a Phosphatidylinositol-4-Phosphate Phosphatase Required for Proper Root Hair Development inArabidopsis thaliana

Polarized expansion of root hair cells in Arabidopsis thaliana is improperly controlled in root hair-defective rhd4-1 mutant plants, resulting in root hairs that are shorter and randomly form bulges along their length. Using time-lapse fluorescence microscopy in rhd4-1 root hairs, we analyzed membrane dynamics after labeling with RabA4b, a marker for polarized membrane trafficking in root hairs. This revealed stochastic loss and recovery of the RabA4b compartment in the tips of growing root hairs, consistent with a role for the RHD4 protein in regulation of polarized membrane trafficking in these cells. The wild-type RHD4 gene was identified by map-based cloning and was found to encode a Sac1p-like phosphoinositide phosphatase. RHD4 displayed a preference for phosphatidylinositol-4-phosphate [PI(4)P] in vitro, and rhd4-1 roots accumulated higher levels of PI(4)P in vivo. In wild-type root hairs, PI(4)P accumulated primarily in a tip-localized plasma membrane domain, but in rhd4-1 mutants, significant levels of PI(4)P were detected associated with internal membranes. A fluorescent RHD4 fusion protein localized to membranes at the tips of growing root hairs. We propose that RHD4 is selectively recruited to RabA4b-labeled membranes that are involved in polarized expansion of root hair cells and that, in conjunction with the phosphoinositide kinase PI-4Kbeta1, RHD4 regulates the accumulation of PI(4)P on membrane compartments at the tips of growing root hairs.

OsPHR2 is involved in phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants.

Previous research has demonstrated that AtPHR1 plays a central role in phosphate (Pi)-starvation signaling in Arabidopsis thaliana. In this work, two OsPHR genes from rice (Oryza sativa) were isolated and designated as OsPHR1 and OsPHR2 based on amino acid sequence homology to AtPHR1. Their functions in Pi signaling in rice were investigated using transgenic plants. Our results showed that both OsPHR1 and OsPHR2 are involved in Pi-starvation signaling pathway by regulation of the expression of Pi-starvation-induced genes, whereas only OsPHR2 overexpression results in the excessive accumulation of Pi in shoots under Pi-sufficient conditions. Under Pi-sufficient conditions, overexpression of OsPHR2 mimics Pi-starvation stress in rice with enhanced root elongation and proliferated root hair growth, suggesting the involvement of OsPHR2 in Pi-dependent root architecture alteration by both systematic and local pathways. In OsPHR2-overexpression plants, some Pi transporters were up-regulated under Pi-sufficient conditions, which correlates with the strongly increased content of Pi. The mechanism behind the OsPHR2 regulated Pi accumulation will provide useful approaches to develop smart plants with high Pi efficiency.

Place Spindle Here

Nonpolarized cultured cells that adhere to the substrate on which they are growing orient their spindles parallel to the substratum, thereby allowing both daughter cells to maintain contact with the substrate. Interactions between the astral microtubules and various microtubule-organizing proteins, including the minus end-directed motor complex and the dynein-dynactin complex, are involved in connecting the spindle to the actin cytoskeleton and the cell cortex. Now Toyoshima et al . find that, in HeLa cells, integrin-mediated stimulation of phosphoinositide 3-kinase (PI3K) leading to the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] at the midsection of the cell cortex in metaphase cells was required for proper spindle orientation. PI(3,4,5)P 3 concentration was monitored with a green fluorescent protein fused to the pleckstrin homology domain of Akt (Akt-PH-GFP), and the midsection cortical localization, as well as proper spindle orientation, was lost if PI3K activity was inhibited pharmacologically or if β1 integrin was knocked down with RNAi. In contrast, polarized MDCK cells did not require PI3K signaling to achieve proper spindle orientation and instead appeared to rely on an interaction of the spindle with the adherens junction (see Gachet and Tournier). The dynein-dynactin complex associates with the astral microtubules, and inhibition of PI3K prevented the localization of the dynein-dynactin complex to the midsection of the cell. Furthermore, in cells in which PI3K was inhibited, the chromosomes rotated in the z axis, and this rotation was eliminated in cells in which dynein activity was inhibited using RNAi techniques. An open question is how integrin signaling, which occurs at the bottom of the cell at the sites of cell attachment to the substrate, leads to the accumulation of PI(3,4,5)P 3 at the midsection of the cell cortex. F. Toyoshima, S. Matsumura, H. Morimoto, M. Mitsushima, E. Nishida, PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells. Dev. Cell 13 , 796-811 (2007). [PubMed] Y. Gachet, S. Tournier, Lost your orientation? Find your way with PtdIns(3,4,5)P3! Dev. Cell 13 , 753-754 (2007). [PubMed]

MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis.

The presence of microRNA species in plant phloem sap suggests potential signaling roles by long-distance regulation of gene expression. Proof for such a role for a phloem-mobile microRNA is lacking. Here we show that phosphate (Pi) starvation-induced microRNA399 (miR399) is present in the phloem sap of two diverse plant species, rapeseed and pumpkin, and levels are strongly and specifically increased in phloem sap during Pi deprivation. By performing micro-grafting experiments using Arabidopsis, we further show that chimeric plants constitutively over-expressing miR399 in the shoot accumulate mature miR399 species to very high levels in their wild-type roots, while corresponding primary transcripts are virtually absent in roots, demonstrating shoot-to-root transport. The chimeric plants exhibit (i) down-regulation of the miR399 target transcript (PHO2), which encodes a critical component for maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi accumulation in the shoot, which is the phenotype of pho2 mutants, miR399 over-expressers or chimeric plants with a genetic knock-out of PHO2 in the root. Hence the transported miR399 molecules retain biological activity. This is a demonstration of systemic control of a biological process, i.e. maintenance of plant Pi homeostasis, by a phloem-mobile microRNA.

G protein–independent Ras/PI3K/F-actin circuit regulates basic cell motility

Phosphoinositide 3-kinase (PI3K)γ and Dictyostelium PI3K are activated via G protein–coupled receptors through binding to the Gβγ subunit and Ras. However, the mechanistic role(s) of Gβγ and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gβ null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P3. Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras–guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.

Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling

During embryonic development, cell movement is orchestrated by a multitude of attractants and repellents. Chemoattractants applied as a gradient, such as cAMP with Dictyostelium discoideum or fMLP with neutrophils, induce the activation of phospholipase C (PLC) and phosphoinositide 3 (PI3)-kinase at the front of the cell, leading to the localized depletion of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and the accumulation of phosphatidylinositol-3,4,5-trisphosphate (PI[3,4,5]P3). Using D. discoideum, we show that chemorepellent cAMP analogues induce localized inhibition of PLC, thereby reversing the polarity of PI(4,5)P2. This leads to the accumulation of PI(3,4,5)P3 at the rear of the cell, and chemotaxis occurs away from the source. We conclude that a PLC polarity switch controls the response to attractants and repellents.