A Multifunctional Lipoxygenase with Fatty Acid Hydroperoxide Cleaving Activity from the Moss Physcomitrella patens
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Abstract:
A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.Keywords:
Physcomitrella patens
Premise of the Study We used the model species Physcomitrella patens to examine chirality in moss gametophores. Chirality is manifested in the direction of consecutive apical cell divisions, cell plate configurations, and deviations of leaf connecting lines from the vertical course. However, the frequencies of chiral configurations of all these processes as well as their mutual dependence—especially in the case of gametophore branching—are not known. Other moss species were checked to determine the universality of our findings. Methods The gametophore structure of Physcomitrella patens grown in the laboratory under controlled conditions was investigated using light microscopy and compared with that of other moss species collected from their natural stands. Key Results In all investigated moss species, the tetrahedral apical cell exhibits either clockwise or counterclockwise consecutive divisions, and selection of this directionality in the primary axis is random. It is, however, related to cell plate configuration. If the plate is skewed, leaf‐producing segments arising from the apical cell cleavage exhibit circumferential rotation. Three parallel lines connecting the leaves deviate from a vertical course, but always in the same direction as that of leaf initiation; thus, the angular distance between consecutive leaves increases to >120°. Lateral branches are exclusively antidromous. Conclusions Gametophore chiral configuration appears to be useful in resolving problems of moss modular growth and branching. Morphological and anatomical evidence strongly suggests that an unknown direction‐sensing mechanism controls the development of moss axial organs. We propose that leaves are responsible for a horizontal gradient of sugar signals that develops along the gametophore circumference, thus influencing branching‐unit chirality.
Physcomitrella patens
Tip growth
Apical cell
Cell plate
Primordium
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Physcomitrella patens
Allelopathy
Bryophyte
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Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18‐carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20‐and 22‐carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo 13 C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly 13 C‐enriched 16‐and 18‐carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U‐ 13 C‐polyunsaturated fatty acid mixture, 13 C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; 13 C enrichment remained high in the phospholipids and cholesterol for 15 days. 13 C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in 13 C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16‐and 18‐carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long‐chain saturated and monounsaturated fatty acids and cholesterol.
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The moss Physcomitrella patens is now often used as a model system in plant molecularbiology. Its short life cycle, easy to culture, transformant analysis and allele replacement make it anexcellent material for studying plant molecular biology. Some gene targetings have succeeded and thefunction of these genes has been proved. Tagged mutant library has been established for the furtherresearches of Physcomitrella patens. The ESTs datebase on Physcomitrella patens which containsalmost 67 000 ESTs has been established.
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Volatiles are important infochemicals acting aboveground as well as belowground between organisms. In order to understand the complex volatile network of an entire ecosystem, the habitat at the border between the atmosphere and the soil has to be considered. Mosses are the dominant colonists of this habitat. Here we tested the reaction of the moss Physcomitrella patens upon exposure to rhizobacterial volatiles. In a closed test system, when CO(2) is a dominant component of the bacterial volatile mixture, P. patens growth was promoted, while in the natural-like open test system volatiles with negative influences possess their effects resulting in growth inhibitions of the moss. Growth retardation is less pronounced when the volatiles were applied in a later stage of development of the moss.
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Bryophyte
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A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.
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857 plant miRNAs were used to search 27546 Physcomitrella patens mRNAs with PatScan for complementary hits.162 miRNA families had potential targets in P.patens.The number of targets and the position in miRNA co-regulation network simultaneously indicated that miR482 and miR1168 may play crucial roles in P.patens.52 Chlamydomonas reinhardtii specific miRNAs having targets in P.patens suggested the moss occupied a unique phylogenetic position from algae to seeding plants.
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Chlamydomonas reinhardtii
Chlamydomonas
Bryopsida
Multicellular organism
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Physcomitrella patens
Plant Physiology
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Desiccation sensitivity and tolerance in the moss Physcomitrella patens: assessing limits and damage
The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from −4 (97% RH) to −273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials <−13 MPa. Upon rehydration, moss dried to water contents >0.4 g g dm−1 maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at −13 MPa. Moss cells rehydrated after drying to −273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least −273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.
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Desiccation Tolerance
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