At4g29530 is a phosphoethanolamine phosphatase homologous to PECP1 with a role in flowering time regulation.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in membranes. The biosynthesis of phospholipids occurs mainly via the Kennedy pathway. Recent studies have shown that through this pathway, choline (Cho) moieties are synthesized through the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) by phospho-base N-methyltransferase. In Arabidopsis thaliana, the phosphoethanolamine/phosphocholine phosphatase1 (PECP1) is described as an enzyme that regulates the synthesis of PCho by decreasing the PEtn level during phosphate starvation to avoid the energy-consuming methylation step. By homology search, we identified a gene (At4g29530) encoding a putative PECP1 homolog from Arabidopsis with a currently unknown biological function in planta. We found that At4g29530 is not induced by phosphate starvation, and is mainly expressed in leaves and flowers. The analysis of null mutants and overexpression lines revealed that PEtn, rather than PCho, is the substrate in vivo, as in PECP1. Hydrophilic interaction chromatography-coupled mass spectrometry analysis of head group metabolites shows an increased PEtn level and decreased ethanolamine level in null mutants. At4g29530 null mutants have an early flowering phenotype, which is corroborated by a higher PC/PE ratio. Furthermore, we found an increased PCho level. The choline level was not changed, so the results corroborate that the PEtn-dependent pathway is the main route for the generation of Cho moieties. We assume that the PEtn-hydrolyzing enzyme participates in fine-tuning the metabolic pathway, and helps prevent the energy-consuming biosynthesis of PCho through the methylation pathway.

Pi starvation-dependent regulation of ethanolamine metabolism by phosphoethanolamine phosphatase PECP1 in Arabidopsis roots.

A universal plant response to phosphorus deprivation is the up-regulation of a diverse array of phosphatases. As reported recently, the AtPECP1 gene encodes a phosphatase with in vitro substrate specificity for phosphoethanolamine and phosphocholine. The putative substrates suggested that AtPECP1 is related to phospholipid metabolism; however, the biological function of AtPECP1 is as yet not understood. In addition, whereas lipid remodelling processes as part of the phosphorus starvation response have been extensively studied, knowledge of the polar head group metabolism and its regulation is lacking. We found that AtPECP1 is expressed in the cytosol and exerts by far its strongest activity in roots of phosphate-starved plants. We established a novel LC-MS/MS-based method for the quantitative and simultaneous measurement of the head group metabolites. The analysis of Atpecp1 null mutants and overexpression lines revealed that phosphoethanolamine, but not phosphocholine is the substrate of AtPECP1 in vivo. The impact on head group metabolite levels is greatest in roots of both loss-of-function and gain-of-function transgenic lines, indicating that the biological role of AtPECP1 is mainly restricted to roots. We suggest that phosphoethanolamine hydrolysis by AtPECP1 during Pi starvation is required to down-regulate the energy-consuming biosynthesis of phosphocholine through the methylation pathway.

Structural analysis of Gossypium hirsutum fibers grown under greenhouse and hydroponic conditions.

Cotton is the one of the world's most important crops. Like any other crop, cotton growth/development and fiber quality is highly dependent on environmental factors. Increasing global weather instability has been negatively impacting its economy. Cotton is a crop that exerts an intensive pressure over natural resources (land and water) and demands an overuse of pesticides. Thus, the search for alternative cotton culture methods that are pesticide-free (biocotton) and enable customized standard fiber quality should be encouraged. Here we describe a culture of Gossypium hirsutum ("Upland" Cotton) utilizing a greenhouse and hydroponics in which the fibers are morphological similar to conventional cultures and structurally fit into the classical two-phase cellulose I model with 4.19nm crystalline domains surrounded by amorphous regions. These fibers exhibit a single crystalline form of cellulose I-Iß, monoclinic unit cell. Fiber quality bulk analysis shows an improved length, strength, whiteness when compared with soil-based cultures. Finally, we show that our fibers can be spun, used for production of non-woven fabrics and indigo-vat stained demonstrating its potential in industrial and commercial applications.

Effect of raw humus under two adult Scots pine stands on ectomycorrhization, nutritional status, nitrogen uptake, phosphorus uptake and growth of Pinus sylvestris seedlings.

Ectomycorrhiza (EM) formation improves tree growth and nutrient acquisition, particularly that of nitrogen (N). Few studies have coupled the effects of naturally occurring EM morphotypes to the nutrition of host trees. To investigate this, pine seedlings were grown on raw humus substrates collected at two forest sites, R2 and R3. Ectomycorrhiza morphotypes were identified, and their respective N uptake rates from organic (2-(13)C, (15)N-glycine) and inorganic ((15)NH(4)Cl, Na(15)NO(3), (15)NH(4)NO(3), NH(4)(15)NO(3)) sources as well as their phosphate uptake rates were determined. Subsequently, the growth and nutritional status of the seedlings were analyzed. Two dominant EM morphotypes displayed significantly different mycorrhization rates in the two substrates. Rhizopogon luteolus Fr. (RL) was dominant in R2 and Suillus bovinus (Pers.) Kuntze (SB) was dominant in R3. (15)N uptake of RL EM was at all times higher than that of SB EM. Phosphate uptake rates by the EM morphotypes did not differ significantly. The number of RL EM correlated negatively and the number of SB EM correlated positively with pine growth rate. Increased arginine concentrations and critical P/N ratios in needles indicated nutrient imbalances of pine seedlings from humus R2, predominantly mycorrhizal with RL. We conclude that different N supply in raw humus under Scots pine stands can induce shifts in the EM frequency of pine seedlings, and this may lead to EM formation by fungal strains with different ability to support tree growth.

Arabidopsis thaliana PECP1: enzymatic characterization and structural organization of the first plant phosphoethanolamine/phosphocholine phosphatase.

Maintenance of cellular phosphate homeostasis is crucial for primary and energy metabolism. In plants, low exogenous phosphate availability activates adaptive responses that include the immediate liberation of Pi from phosphorylated metabolites by yet uncharacterized intracellular phosphatases. Based on transcriptional analyses, the Arabidopsis thaliana gene At1g17710, a member of the HAD (Haloacid Dehalogenase) superfamily, was one of the most promising candidates. Here, we show by recombinant protein production and analysis of purified protein that the gene At1g17710 encodes a phosphoethanolamine/phosphocholine phosphatase (EC 3.1.3.75). Thus, the gene product was termed AtPECP1. The present study demonstrates that the Mg(2+)-dependent enzyme exhibits pronounced specificity for both substrates. The enzyme displays a broad pH optimum ranging from pH 6 to pH 8. Comparison of K(m) values indicates a slightly higher affinity for phosphocholine (0.44 mM) than for phosphoethanolamine (1.16 mM). The catalytic efficiency, however, is markedly higher for phosphoethanolamine than for phosphocholine being 1.06 × 10(4)M(-1)s(-1) and 2.34 × 10(3)M(-1)s(-1), respectively. Size exclusion chromatography, native gel electrophoresis and SAXS experiments with recombinant protein clearly point to a rapid monomer-dimer equilibrium of protein subunits. Given its established substrate specificity the enzyme is likely to be involved in the liberation of inorganic phosphate from intracellular sources and is especially in demand under phosphate-deprived conditions.

The Solanum lycopersicum RNaseLER is a class II enzyme of the RNase T2 family and shows preferential expression in guard cells.

Ribonucleases (RNases) occur in different gene families, functioning in RNA processing and degradation. In this study, we report on cloning and characterization of RNaseLER, the first class II gene of the RNase T2 family in tomato (Solanum lycopersicum). The family also includes the class I members RNaseLE and RNaseLX, and the class III group of S-RNases acting in self incompatibility. The RNaseLER gene was cloned by polymerase chain reaction (PCR)-assisted methods. Structural analyses of RNaseLER and homologous genes revealed unique key features of class II RNase T2 genes. RNaseLER is a single copy gene in tomato and codes for a primary protein of 260 amino acids. Subcellular localization analyzed with a RNaseLER-eYFP fusion protein and co-localization experiments revealed an intracellular accumulation in the endoplasmic reticulum. Transgenic Nicotiana benthamiana plants carrying the uidA reporter gene under the control of a 900-bp RNaseLER promoter sequence express the reporter gene predominantly in guard cells and trichomes. This previously unknown spatial expression of a RNase T2 gene is consistent with ubiquitous detection of low RNaseLER transcript abundances in almost all parts of tomato plants. As revealed by quantitative real-time RT-PCR analysis treatments with abscisic acid, ethylene or other abiotic and biotic stress factors did not affect RNaseLER expression significantly. Unlike tomato class I genes, RNaseLER represents a constitutively expressed gene with a cell-specific role in stomata and trichomes and no involvement in stress responses.

The Arabidopsis thaliana phosphate starvation responsive gene AtPPsPase1 encodes a novel type of inorganic pyrophosphatase.

BACKGROUND: Low inorganic phosphate (Pi) availability triggers metabolic responses to maintain the intracellular phosphate homeostasis in plants. One crucial adaptive mechanism is the immediate cleavage of Pi from phosphorylated substrates; however, phosphohydrolases that function in the cytosol and putative substrates have not been characterized yet. One candidate gene is Arabidopsis thaliana At1g73010 encoding an uncharacterized enzyme with homology to the haloacid dehalogenase (HAD) superfamily. METHODS AND RESULTS: This work reports the molecular cloning of At1g73010, its expression in Escherichia coli, and the enzymatic characterisation of the recombinant protein (33.5 kD). The Mg²(+)-dependent enzyme named AtPPsPase1 catalyzes the specific cleavage of pyrophosphate (K(m) 38.8 µM) with an alkaline catalytic pH optimum. Gel filtration revealed a tetrameric structure of the soluble cytoplasmic protein. Modelling of the active site and assay of the recombinant protein variant D19A demonstrated that the enzyme shares the catalytic mechanism of the HAD superfamily including a phosphorylated enzyme intermediate. CONCLUSIONS: The tight control of AtPPsPase1 gene expression underlines its important role in the Pi starvation response and suggests that cleavage of pyrophosphate is an immediate metabolic adaptation reaction. GENERAL SIGNIFICANCE: The novel enzyme, the first pyrophosphatase in the HAD superfamily, differs from classical pyrophosphatases with respect to structure and catalytic mechanism. The enzyme function could be used to discover unknown aspects of pyrophosphate metabolism in general.

Transport and compartmentation of phosphite in higher plant cells--kinetic and P nuclear magnetic resonance studies.

Phosphite (Phi, H(2)PO(3)(-)), being the active part of several fungicides, has been shown to influence not only the fungal metabolism but also the development of phosphate-deficient plants. However, the mechanism of phosphite effects on plants is still widely unknown. In this paper we analysed uptake, subcellular distribution and metabolic effects of Phi in tobacco BY-2 cells using in vivo(31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy. Based on the kinetic properties of the phosphate transport system of tobacco BY-2 cells, it was demonstrated that phosphite inhibited phosphate uptake in a competitive manner. To directly follow the fate of phosphate and phosphite in cytoplasmic and vacuolar pools of tobacco cells, we took advantage of the pH-sensitive chemical shift of the Phi anion. The NMR studies showed a distinct cytoplasmic accumulation of Phi in Pi-deprived cells, whereas Pi resupply resulted in a rapid efflux of Phi. Pi-preloaded cells shifted Phi directly into vacuoles. These studies allowed for the first time to follow Phi flux processes in an in vivo setting in plants. On the other hand, the external Pi nutrition status and the metabolic state of the cells had a strong influence on the intracellular compartmentalization of xenobiotic Phi.

Wound-induced RNaseLE expression is jasmonate and systemin independent and occurs only locally in tomato (Lycopersicon esculentum cv. Lukullus).

Tomato RNaseLE is induced by phosphate deficiency and wounding and may play a role in macromolecular recycling as well as wound healing. Here, we analyzed the role of jasmonate and systemin in the wound-induced RNaseLE activation. The rapid expression of RNaseLE upon wounding of leaves leading to maximal RNase activity within 10 h, appeared only locally. Jasmonic acid (JA) or its molecular mimic ethyl indanoyl isoleucine conjugate did not induce RNaseLE expression. Correspondingly, RNaseLE was expressed upon wounding of 35S::allene oxide cyclase antisense plants known to be JA deficient. RNaseLE was not expressed upon systemin treatment, but was locally expressed in the spr1 mutant which is affected in systemin perception. In tomato plants carrying a PromLE::uidA construct, GUS activity could be detected upon wounding, but not following treatment with JA or systemin. The data indicate a locally acting wound-inducible systemin- and JA-independent signaling pathway for RNaseLE expression.

Phloem-specific expression of the wound-inducible ribonuclease LE from tomato (Lycopersicon esculentum cv. Lukullus).

Ribonuclease LE (RNaseLE) from tomato (Lycopersicon esculentum Mill. cv. Lukullus) belongs to the widespread RNase T2 family of ribonucleases. With the exception of S-RNases of the solanaceous self-incompatibility system the functions of other members of the RNase T2 family are only barely understood. Using a 2.6-kbp putative promoter sequence of RNaseLE in front of the uidA reporter gene, expression of beta-glucuronidase in developing phloem tissue and, especially, in the meristematic and elongation zones at root tips was detected. The tissue-specific expression accords with the range of cis-acting elements detected in the RNaseLE promoter. RNaseLE mRNA was localized in developing phloem cells but not in mature phloem tissue, suggesting association of RNaseLE expression with phloem development. Histochemical staining of beta-glucuronidase activity as well as detailed inspection of RNaseLE at mRNA, protein and enzyme activity levels revealed that the wound-induced expression of RNaseLE was also restricted to vascular tissue. RNaseLE transcript accumulation detected by in situ hybridization occurred preferentially in phloem and cambial cells of stem sections upon wounding. The data provide evidence for a role of RNaseLE in a tissue-specific wound response and in wound healing of tomato.

Differential expression of the LePS2 phosphatase gene family in response to phosphate availability, pathogen infection and during development.

In this study, we report the cloning of the three-member LePS2 gene family of acid phosphatases via subtractive screening of a cDNA library of Pi-starved cultivated tomato cells (Lycopersicon esculentum Mill. cv. Lukullus). As members of the plant Pi-starvation response, LePS2 genes were tightly regulated in cultivated cells and tomato seedlings by Pi availability. The LePS2 enzymes which are most likely expressed in the cytoplasma could be involved in processes that are accompanied by degradation of phosphorylated organic substrates. Independently from exogenous phosphate supply LePS2 expression was detected in tomato endosperm during germination. LePS2 genes were differentially induced after infection with the bacterial pathogen Pseudomonas syringae and in the early stages of flower development. Using RT-PCR it was found that the gene LePS2B was the most abundant transcript in phosphate-depleted cells, but a reduced expression was determined in floral buds and it was not found during pathogen interaction. In this respect, it is interesting that the promoter sequences of the LePS2 genes are also divergent. LePS2 gene products may have functions in developmental processes which are restricted to distinct plant tissues or cell types.