Pi M4: An additional Pi M subtype

Summary The authors studied Pi polymorphism using the Separator isofocusing method with slight modification. A new Pi allele was observed. Family pedigrees confirmed co-dominant inheritance with other Pi alleles. According to the electrophoretic mobility of its isoprotein bands, and to its frequency (0.04) this new allele is considered as a fourth Pi ^M subtype: Pi ^M4.     

Rice ACID PHOSPHATASE 1 regulates Pi stress adaptation by maintaining intracellular Pi homeostasis

The concentration and homeostasis of intracellular phosphate (Pi) are crucial for sustaining cell metabolism and growth. During short-term Pi starvation, intracellular Pi is maintained relatively constant at the expense of vacuolar Pi. After the vacuolar stored Pi is exhausted, the plant cells induce the synthesis of intracellular acid phosphatase (APase) to recycle Pi from expendable organic phosphate (Po). In this study, the expression, enzymatic activity and subcellular localization of ACID PHOSPHATASE 1 (OsACP1) were determined. OsACP1 expression is specifically induced in almost all cell types of leaves and roots under Pi stress conditions. OsACP1 encodes an acid phosphatase with broad Po substrates and localizes in the endoplasmic reticulum (ER) and Golgi apparatus (GA). The phylogenic analysis demonstrates that OsACP1 has a similar structure with human acid phosphatase PHOSPHO1. Overexpression or mutation of OsACP1 affected Po degradation and utilization, which further influenced plant growth and productivity under both Pi-sufficient and Pi-deficient conditions. Moreover, overexpression of OsACP1 significantly affected intracellular Pi homeostasis and Pi starvation signalling. We concluded that OsACP1 is an active acid phosphatase that regulates rice growth under Pi stress conditions by recycling Pi from Po in the ER and GA.     

The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family

The deployment of broad-spectrum resistance genes is the most effective and economic means of controlling blast in rice. The cultivar Er-Ba-Zhan (EBZ) is a widely used donor of blast resistance in South China, with many cultivars derived from it displaying broad-spectrum resistance against blast. Mapping in a set of recombinant inbred lines bred from the cross between EBZ and the highly blast-susceptible cultivar Liangjiangxintuanheigu (LTH) identified in EBZ a blast resistance gene on each of chromosomes 1 (Pish), 6 (Pi2/Pi9) and 12 (Pita/Pita-2). The resistance spectrum and race specificity of the allele at Pi2/Pi9 were both different from those present in other known Pi2/Pi9 carriers. Fine-scale mapping based on a large number of susceptible EBZ?×?LTH F(2) and EBZ?×?LTH BC(1)F(2) segregants placed the gene within a 53-kb segment, which includes Pi2/Pi9. Sequence comparisons of the LRR motifs of the four functional NBS-LRR genes within Pi2/Pi9 revealed that the EBZ allele is distinct from other known Pi2/Pi9 alleles. As a result, the gene has been given the designation Pi50(t).     

Phosphate (Pi) Starvation Effect on the Cytosolic Pi Concentration and Pi Exchanges across the Tonoplast in Plant Cells: An in Vivo 31P-Nuclear Magnetic Resonance Study Using Methylphosphonate as a Pi Analog

In vivo ³¹P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60–80 μm instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.Phosphorus, a key constituent of nucleic acids and membrane phospholipids, is an essential element for energy-mediated metabolic processes in all living organisms. In plants, the acquisition of phosphate (Pi), the size of endogenous Pi pools, and the exchange of Pi between different cell compartments have been the major focus of a great number of studies (for review, see Bieleski, 1973; Schachtman et al., 1998; Raghothama, 1999; Drobny et al., 2003; Raghothama and Karthikeyan, 2005). In particular, the concentration and homeostasis of cytoplasmic Pi (cyt-Pi) are considered as crucial for signal transduction pathways and for the regulation of many enzymes (Mimura, 1999; Poirier and Bucher, 2002). For example, Pi concentration in plastids regulates starch synthesis, phosphorylated carbohydrate metabolism, and photosynthesis (Plaxton and Carswell, 1999).Phosphorous acquisition by plants often fluctuates because this essential nutrient is one of the least available in the soil (Barber et al., 1963). After a prolonged Pi deprivation, cyt-Pi decreases strongly (Gout et al., 2001). In this case, photosynthesis and carbon fixation are severely affected (for review, see Natr, 1992), but not respiration due to the high affinity of mitochondria for Pi (Rébeillé et al., 1984) and to the induction of alternative pathways of glycolysis and mitochondrial electron transport (Theodorou and Plaxton, 1993). On the other hand, cyt-Pi homeostasis is expected to be tightly regulated (Raghothama, 1999). Indeed, during short-term Pi starvation, cyt-Pi level is assumed to be maintained relatively constant at the expense of vacuolar Pi (vac-Pi; Bieleski, 1973; Rébeillé et al., 1983; Tu et al., 1990; Mimura et al., 1996), leaving nucleoside triphosphate content unaffected in maize (Zea mays) roots (Lee and Ratcliffe, 1993). However, early changes are observed in the phospholipids of Pi-starved Arabidopsis (Arabidopsis thaliana) cells (Jouhet et al., 2003), suggesting the existence of an early Pi deprivation signal. In addition, the supply of tissues with substrates efficiently phosphorylated in the cytoplasm, like d-Man (Loughman et al., 1989), glycerol (Aubert et al., 1994), and choline (Bligny et al., 1989), triggers a decrease of hexose phosphates. These data suggested the existence of a cyt-Pi pool able to rapidly fluctuate according to the Pi supply from external and vacuolar stores and to the Pi demand for metabolism. As the Pi concentration of mitochondria and plastids is tightly regulated (Scarpa, 1979; Plaxton and Carswell, 1999), we hypothesized that the appearance of early Pi deprivation effects correlated with low Pi supply may originate from rapid Pi changes in cytosol.In this paper, we term cytosol (cyt_sol) as the cell compartment exterior to the vacuole and organelles bounded by a double membrane (mitochondria and plastids, here called org_mp). Accordingly, the cyt_sol-Pi regulation is achieved by a combination of Pi transport across plasma membrane, org_mp membranes, and vacuolar membrane (tonoplast) and Pi-related metabolism. To date, only a few publications investigating in vivo Pi transport across the tonoplast have been published, and in none of them was the cyt_sol-Pi directly measured. This is addressed in this study using in vivo ³¹P-NMR.For many years, ³¹P-NMR has permitted the noninvasive measurement of cyt- and vac-Pi pools (Rébeillé et al., 1983). However, until now, it was not possible to discriminate between cyt_sol- and org_mp-Pi signals, despite the more alkaline pHs of chloroplasts (ΔpH approximately 0.1–0.2 in the dark; Heldt, 1979) and mitochondria (ΔpH approximately 0.2–0.3; Neuburger and Douce, 1980), because the natural heterogeneity of plant tissues broadens Pi signals, thus favoring overlaps. In spinach (Spinacia oleracea) leaves, for example, we previously hypothesized that the cyt_sol-Pi signal was swamped by the chloroplast Pi signal (Bligny et al., 1990). In this study, we worked on culture cells first to improve sample homogeneity and second to finely adjust endogenous Pi level. Heterotrophic sycamore (Acer pseudoplatanus) cells were most frequently used because they contain small amyloplastids instead of big chloroplasts, thus limiting the org_mp-Pi signal intensity. Finally, perfusion parameters were optimized to narrow the Pi signals. Together, these experimental conditions permitted us to discriminate, to our knowledge for the first time, between cyt_sol-Pi and org_mp-Pi pools.To measure the fluctuations of endogenous Pi pools and analyze the movements of Pi across the tonoplast, we used Pi-starved cells containing less endogenous Pi stores and performed successive short sequences of Pi supply and starvation. We also used a Pi analog, methylphosphonate (MeP), which has several advantages: (1) it mimics Pi perception by plant cells but, unlike Pi, is not metabolized and is nontoxic in the short term (Couldwell et al., 2009); (2) its ³¹P-NMR signal displays a broad amplitude of pH-related shift with pKa at 7.5 (DeFronzo and Gillies, 1987), thus facilitating the discrimination between cyt_sol-MeP and org_mp-MeP pools (Lebrun-Garcia et al., 2002); and (3) its signals do not overlap with those of Pi and phosphorylated metabolites (Gout et al., 2001).     

Pi Mheerlen,a Pi M allele resulting in very low a 1-antitrypsin serum levels

Summary A patient with pulmonary emphysema is described, who had a very low ₁-antitrypsin serum concentration (2% of normal). After isoelectric focusing and staining, the patient's serum revealed no visible ₁-antitrypsin bands. Immunofixation, following isoelectric focusing, gave a banding pattern identical to that of a normal M type. The existence of this deficient M-allele was confirmed by family studies. Low ₁-antitrypsin concentrations, due to the presence of the deficient allele, were coupled with low serum antitrypsin activities.     

Renal Endosomal Phosphate (Pi) Transport in Normal and Diabetic Rats and Response to Chronic Pi Deprivation

Chronic renal adaptation to dietary deprivation of Pi is accompanied by increased Na⁺/Pi co-transport across the brush border membrane of the renal proximal tubule. The increased activity of this co-transport system depends on de novo protein synthesis and insulin. The present study used normal and diabetic rats to determine if the endosomal pool of Na⁺/Pi co-transporters was altered by Pi deprivation and the possible role of insulin. In response to 5 days of dietary Pi deprivation there was a significant increase in endosomal Na⁺/Pi co-transport in control rats but there was no change in diabetic rats. The increase in endosomal Pi uptake was restored in diabetic rats treated with exogenous insulin. Na⁺/Pi-independent Pi uptake and proline uptake remained unchanged in all groups. The changes in endosomal Na⁺/Pi co-transport correlated with the abundance of the specific Na⁺/Pi co-transporter protein, as determined by Western blots. The pattern of endosomal changes paralleled that observed in brush border membranes. One possibility consistent with these findings is that the endosomal fraction contains newly synthesized Na⁺/Pi co-transporters targeted for delivery to the apical brush border membrane. Increased synthesis and delivery is required to maintain the adaptation to chronic Pi deprivation. © 1997 John Wiley & Sons, Ltd.     

Skeletal muscle Pi transport and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles.

In the rat skeletal myoblast line L6 and in a rabbit skeletal muscle sarcolemma/t-tubule vesicle preparation, [32P]Pi uptake was largely dependent on the transmembrane Na gradient. Na-dependent [32P]Pi uptake had a hyperbolic relationship to [Pi] and [Na], being half-maximal at 0.2-0.3 mM [Pi] and at 25-40 mM [Na]. In vesicles the Na-dependence suggests that approx. two Na are transported with each Pi, but the inhibition of [32P]Pi uptake at high pH suggests that the Pi monoanion is the transported form. Together these imply electrogenic transport and this is confirmed by the results of manipulating the vesicle membrane potential. Thus, electrogenic Na-Pi co-transport exploits both the sodium gradient and the cell membrane potential to maintain muscle cellular [Pi] against an unfavourable electrochemical gradient. The low [Pi] for half-maximal flux may partly explain the small effect of altered extracellular [Pi] on cellular [Pi]. In L6 myoblasts most 32P was first detectable in an organic phosphate pool rather than cellular Pi, while the specific activity of cell Pi rapidly reached 40% of that of extracellular Pi and was stable for at least 3 h. These results are discussed in terms of the organisation of cellular phosphate metabolism.     

4Pi microscopy of quantum dot-labeled cellular structures

The most prominent restrictions of fluorescence microscopy are the limited resolution and the finite signal. Established conventional, confocal, and multiphoton microscopes resolve at best approximately 200nm in the focal plane and only 500nm in depth. Additionally, organic fluorophores and fluorescent proteins are bleached after 10(4)-10(5) excitation cycles. To overcome these restrictions, we synergistically combine the 3- to 7-fold improved axial resolution of 4Pi microscopy with the greatly enhanced photostability of semiconductor quantum dots. Co-localization studies of immunolabeled microtubules and mitochondria demonstrate the feasibility of this approach for routine biological measurements. In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail.     

On the genetics of the Pi serum proteins

Summary The authors report family studies (51 families with 134 children) on the inheritance of the Pi phenotypes. Combining these data with a Norwegian family material (77 families with 323 children) published by Fagerhol and Gedde-Dahl (1969) a total of 128 families with 457 children is now available, which allows the following conclusion: The Pi phenotypes are inherited by a simple codominant mode of heredity and they are determined by a set of (at least nine) alleles. As up to now no exception to the role of inheritance has been observed, the application of the Pi system in cases of disputed paternity seems to be discussible. Some methodological problems in connection with this are shown.Supported by the Deutsche Forschungsgemeinschaft.