Mannose-6-phosphate Reductase Research Articles

Influence of intergenotypic competition on multigenerational persistence of abiotic stress resistance transgenes in populations of Arabidopsis thaliana.

Reducing crop losses due to abiotic stresses is a major target of agricultural biotechnology that will increase with climate change and global population growth. Concerns, however, have been raised about potential ecological impacts if transgenes become established in wild populations and cause increased competitiveness of weedy or invasive species. Potential risks will be a function of transgene movement, population sizes, and fitness effects on the recipient population. While key components influencing gene flow have been extensively investigated, there have been few studies on factors subsequent to transgene movement that can influence persistence and competitiveness. Here, we performed multiyear, multigenerational, assessment to examine fitness effects and persistence of three mechanistically different abiotic stress tolerance genes: C‐repeat binding factor 3/drought responsive element binding factor 1a (CBF3/DREB1a); Salt overly sensitive 1 (SOS1); and Mannose‐6‐phosphate reductase (M6PR). Transgenic Arabidopsis thaliana overexpressing these genes were grown in pure populations and in competition with wild‐type (WT) parents for six generations spanning a range of field environment conditions. Growth, development, biomass, seed production, and transgene frequency were measured at each generation. Seed planted for each generation was obtained from the previous generation as would occur during establishment of a new genotype in the environment. The three transgenes exhibited different fitness effects and followed different establishment trajectories. In comparison with pure populations, CBF3 lines exhibited reduced dry weight, seed yield, and viable seed yield, relative to WT background. In contrast, overexpression of SOS1 and M6PR did not significantly impact productivity measures in pure populations. In competition with WT, negative fitness effects were magnified. Transgene frequencies were significantly reduced for CBF3 and SOS1 while frequencies of M6PR appeared to be subject to genetic drift. These studies demonstrate the importance of fitness effects and intergenotype competition in influencing persistence of transgenes conferring complex traits.

Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals.

Salinity is a major limiting factor affecting crops production, survival and distribution worldwide. Engineering dehydration stress tolerance in commercial crops is a trait of economic importance, especially in saline-affected areas. In this work, we are reporting the cloning of the M6PR gene homolog (encoding a key enzyme, mannose-6-phosphate reductase, for mannitol biosynthesis in celery) from Egyptian celery plants. Using RACE technique, the full-length Egyptian-M6PR gene (1333bp) was cloned into pRI-201AN plant expression vector. Analysis of the cloned gene revealed that both American and Egyptian clones had both start and stop codons in frame and was found to be 930 base long. The newly cloned EM6PR gene was found to be 126 base longer than its American counterpart at the non-coding region. Six differences at nucleotide level between the Egyptian and American sequences were observed, three of which in the coding region resulting in three polymorphic amino acids differences (tryptophan vs. leucine, glutamine vs. histidine and isoleucine vs. leucine). The newly cloned gene was introduced to tobacco via Agrobacterium and PCR analysis of T0 plants indicated the presence of the EM6PR gene into 10 out of 38 tobacco individuals. Moreover, RT-PCR analysis confirmed the presence of EM6PR transcripts in 9 out of the 10 PCR positive plants. GC/MS analysis of some RT positive individuals indicated the accumulation of mannitol in transgenics tobacco, while mannitol was absent in non-transgenic controls.

Advanced Technologies for Parasitic Weed Control

Parasitic weeds such as Phelipanche and Orobanche are obligate holoparasites that attack roots of almost all economically important crops in semiarid regions of the world. A wide variety of parasitic weed control strategies (chemical, biological, cultural, and resistant crops) has been tried. Unfortunately, most are partially effective and have significant limitations. The current mini review will discuss the needs for alternative methods and will summarize current and new biotechnology-based approaches for broomrape control. At present, we have generated transgenic tobacco plants expressing a cecropin peptide (sarcotoxin IA), under the control of the inducible HMG2 promoter. Transgenic lines enhanced host resistance to the parasitic weed; transgenes showed higher numbers of aborted parasitization events, reduced Phelipanche biomass, and increased host biomass. Sarcotoxin IA had no obvious effect on growth and development of transgenic host plants. Mannitol content in the parasite is regulated by Mannose 6-Phosphate Reductase (M6PR) gene, an essential process to broomrape species for water and nutrient uptake from the host. In our study, we used the inverted repeat technique to silence the parasite target gene, M6PR. In this study it was shown that the endogenous M6PR mRNA from P. aegyptiaca tubercles or shoots grown on transgenic tomato plants harboring the M6PR silencing construct was reduced by 60 to 80%. The number of dead tubercles was also increased significantly on transgenic plants as compared with the control plants. The strategies presented here are potentially superior to other methods in that they are effective, have a low cost of implementation for producers, and are safe for the environment.

Global gene expression analysis of transgenic, mannitol-producing, and salt-tolerant Arabidopsis thaliana indicates widespread changes in abiotic and biotic stress-related genes.

Mannitol is a putative osmoprotectant contributing to salt tolerance in several species. Arabidopsis plants transformed with the mannose-6-phosphate reductase (M6PR) gene from celery were dramatically more salt tolerant (at 100 mM NaCl) as exhibited by reduced salt injury, less inhibition of vegetative growth, and increased seed production relative to the wild type (WT). When treated with 200 mM NaCl, transformants produced no seeds, but did bolt, and exhibited less chlorosis/necrosis and greater survival and dry weights than the WT. Without salt there were no M6PR effects on growth or phenotype, but expression levels of 2272 genes were altered. Many fewer differences (1039) were observed between M6PR and WT plants in the presence of salt, suggesting that M6PR pre-conditioned the plants to stress. Previous work suggested that mannitol is an osmoprotectant, but mannitol levels are invariably quite low, perhaps inadequate for osmoprotectant effects. In this study, transcriptome analysis reveals that the M6PR transgene activated the downstream abscisic acid (ABA) pathway by up-regulation of ABA receptor genes (PYL4, PYL5, and PYL6) and down-regulation of protein phosphatase 2C genes (ABI1 and ABI2). In the M6PR transgenic lines there were also increases in transcripts related to redox and cell wall-strengthening pathways. These data indicate that mannitol-enhanced stress tolerance is due at least in part to increased expression of a variety of stress-inducible genes.

Gene silencing of mannose 6‐phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

Response of mannitol-producing Arabidopsis thaliana to abiotic stress.

In celery, mannitol is a primary photosynthetic product that is associated with celery's exceptional salt tolerance. Arabidopsis plants transformed with celery's mannose-6-phosphate reductase (M6PR) gene produce mannitol and grow normally in the absence of stress. Daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant and specific leaf weight) over a 12-day period showed less effect of salt (100 mm NaCl) on the M2 transformant than wild type (WT). Following a 12-day treatment of WT, M2 and M5 plants with 100 or 200 mm NaCl the total shoot fresh weight, leaf number, and leaf area were significantly greater in transformants than in WT plants. The efficiency of use of energy for photochemistry by PSII was measured daily under growth conditions. In WT plants treated with 100 mm NaCl, the PSII yield begin decreasing after 6 days with a 50% loss in yield after 12 days, indicating a severe loss in PSII efficiency; whereas, there was no effect on the transformants. Under atmospheric levels of CO2, growth with 200 mm NaCl caused an increase in the substomatal levels of CO2 in WT plants but not in transformants. It also caused a marked decrease in carboxylation efficiency under limiting levels of CO2 in WT compared with transformants. When stress was imposed and growth reduced by withholding water for 12 days, which resulted in a similar decrease in relative water content to salt-treated plants, there were no differences among the genotypes in PSII yields or growth. The results suggest mannitol, which is known to be a compatible solute and antioxidant, protects photosynthesis against salt-related damage to chloroplasts.

(139) A Capacity for Mannitol Biosynthesis is a Critical Component of Salt Tolerance in Celery

Mannitol, a sugar alcohol that appears to serve as an osmoprotectant/compatible solute to cope with salt stress, is synthesized in celery (Apium graveolens L.) via the action of a NADPH dependent mannose-6-phosphate reductase (M6PR). To evaluate the abiotic stress effects of mannitol biosynthesis, we transformed celery with an antisense construct of the celery leaf M6PR gene under control of the CaMV 35S promoter. Unlike wild type (WT) celery, independent antisense M6PR transformants did not accumulate significant amounts of mannitol in any tissue, with or without salt stress. In the absence of NaCl, and despite the lack of any significant accumulation of mannitol that is normally the major photosynthetic product, antisense transformants were mostly phenotypically similar to the WT celery. However, in the presence of NaCl, mature antisense transgenic plants were significantly less salt-tolerant, with reduced growth and photosynthetic rates, and some transformant lines were killed at 200 mM NaCl, a concentration that WT celery can normally withstand. Although mannitol biosynthesis is normally enhanced in salt-treated WT celery, no such increase was observed in the antisense transformants. Like our previous gain of function results showing enhanced salt tolerance in Arabidopsis plants transgenic for a sense M6PR construct, these loss of function results, using an antisense construct in celery, demonstrate a major role for mannitol biosynthesis in developing salt-tolerant plants.

Expression of a celery mannose 6‐phosphate reductase in Arabidopsis thaliana enhances salt tolerance and induces biosynthesis of both mannitol and a glucosyl‐mannitol dimer

ABSTRACTMannitol, a sugar alcohol that may serve as a compatible solute to cope with salt stress, is synthesized via the action of a mannose‐6‐phosphate reductase (M6PR) in celery (Apium graveolens L). In contrast to previous approaches that have used a bacterial gene to engineer mannitol biosynthesis in plants and other organisms, Arabidopsis thaliana, a non‐mannitol producer, was transformed with the celery leaf M6PR gene under control of the CaMV 35S promotor. In all independent Arabidopsis M6PR transformants, mannitol accumulated throughout the plants in amounts ranging from 0·5 to 6 µmol g−1 fresh weight. A novel compound, not found in either celery or Arabidopsis, 1‐O‐β‐d‐glucopyranosyl‐d‐mannitol, also accumulated in vegetative tissues of mature plants in amounts up to 4 µmol g−1 fresh weight, but not in flowers and seeds. In the absence of NaCl, all transformants were phenotypically the same as the wild type; however, in the presence of NaCl, mature transgenic plants showed a high level of salt tolerance, i.e. growing, completing normal development, flowering, and producing seeds in soil irrigated with 300 mm NaCl in the nutrient solution. These results demonstrate a major role in developing salt‐tolerant plants by means of introducing mannitol biosynthesis using M6PR.

Isolation of mannose 6-phosphate reductase cDNA, changes in enzyme activity and mannitol content in broomrape (Orobanche ramosa) parasitic on tomato roots.

We are interested in developing a control strategy efficient at the early stages of subterranean development of Orobanche in the inhibition of mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), the key enzyme of mannitol production in the parasite. We examined M6PR gene expression during pre-conditioning, germination, procaulome growth, underground shoot development and emergence of Orobanche ramosa L. attached to tomato roots, the enzyme activity at each of the above stages and the level of stored mannitol in the parasite. A 1120-pb length cDNA isolated by 3' and 5'RACE was identified as a M6PR sequence by cDNA expression in E. coli and M6PR activity measurement. Only one M6PR gene was detected in O. ramosa following southern blot analysis. M6PR expression, analysed by RT-PCR, was constant from the pre-conditioned seed to the emergence of broomrape, i.e. M6PR expression is constitutive in Orobanche. M6PR activity was also detected in pre-conditioned seeds and attachment to tomato roots resulted in a two-fold increase in enzyme activity during tubercle enlargement and crown root formation. Hexose and mannitol accumulation was strongly enhanced in the attached parasite, with accumulation primarily in the shoot. These results support the prospect of utilizing M6PR inhibitors as early applied herbicides to control this parasite in the early stages of its development.

Purification and characterization of mannose 6-phosphate reductase, a potential target for the control of Striga hermonthica and Orobanche ramosa

Mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), a potential target for the chemical control of the parasitic angiosperms that produce mannitol, was purified from the photosynthetic parasite Striga hermonthica (Del.) Benth. and the achlorophyllous parasite Orobanche ramosa L. Except for the Km values for NADPH, most of the characteristics of the enzymes are similar in these species, i.e. a monomeric structure of 38–39 kDa molecular mass, a pH optimum of 7.0–7.5 suggesting a cytosolic location, a relatively high optimum temperature (35–45˚C), a low Km value for mannose 6-phosphate of 5.9–8.6 mM and, lastly, a significant preference for NADPH as nucleotidic cofactor.

Comment. Foliar d15N is negatively correlated with rainfall along the IGBP transect in Australia. Response. Interpretation of increased foliar d15N in woody species along a rainfall gradient in northern Australia

Mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), a potential target for thechemical control of the parasitic angiosperms that produce mannitol, waspurified from the photosynthetic parasiteStriga hermonthica (Del.) Benth. and the achlorophyllousparasite Orobanche ramosa L. Except for theKm values for NADPH, most of thecharacteristics of the enzymes are similar in these species, i.e. a monomericstructure of 38–39 kDa molecular mass, a pH optimum of 7.0–7.5suggesting a cytosolic location, a relatively high optimum temperature(35–45˚C), a low Kmvalue for mannose 6-phosphate of 5.9–8.6 mM and, lastly, a significantpreference for NADPH as nucleotidic cofactor.

The Effects of Development and Salinity Stress on Mannitol Biosynthesis in Celery Leaves

In celery (Apium graveolens L.), up to 50% of newly assimilated carbon may be partitioned into mannitol in mature leaves. Mannitol biosynthesis involves three unique enzymatic steps, and mannose 6-phosphate reductase (M6PR) is the critical regulatory step in the pathway. We measured M6PR enzyme activities, M6PR protein levels (using an immunological method) and M6PR transcript levels (by Northern blotting) to assess effects of leaf development on mannitol biosynthesis. M6PR was limited to green tissues and was under tight transcriptional regulation during leaf initiation, expansion, and maturation. M6PR expression was also closely correlated with the capacity of leaves to partition newly fixed carbon into mannitol (measured by 14C pulse/chase on intact leaves). Previous studies have also shown salt stress to lead to mannitol accumulation in celery. Using the methods outlined above we also investigated the combined effects of salt stress and leaf development on M6PR expression and the capacity of leaves to partition C to mannitol. Under salt stress M6PR expression and the capacity to synthesize mannitol occurred in younger leaves than in control plants. Thus, the increase in mannitol pool size in salt-stressed celery plants is due, in part, to enhanced de novo synthesis in young leaves. The data also confirmed the relationship between development of photosynthetic capacity, mannitol synthesis and M6PR activity. Supported by USDA-NRI grant # 940-1439.

Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery.

Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.

Cloning of Mannose 6-phosphate Reductase from Celery.

In celery, photosynthetic carbon partitioning between mannitol and sucrose is highly dependent on developmental (leaf age) and environmental (salt stress) factors. Mannose 6-phosphate reductase (M6PR) mediates a key step in mannitol biosynthesis and may regulate partitioning between sucrose and mannitol. We have constructed a cDNA library and have isolated M6PR-specific clones. Before library construction, poly(A)+ RNA, extracted from newly fully expanded leaves, was translated in vitro. A single polypeptide (35.1 kD), immunoprecipitated with M6PR-specific antisera, accounted for ≈5% of the total 35S incorporated into TCA-precipitated products. Parity between the molecular masses of the immunoprecipitated product and authentic M6PR indicated minimal posttranslational modification. The unidirectional primary library, constructed in UniZap XR vector (Stratagene), consisted of 1.53 million plaque forming-units (pfus) of which <0.4% were nonrecombinant, as estimated by “blue/white”' screening. After a single amplification, ≈0.14% of the 200,000 pfus screened with M6PR-specific antisera were identified as putative M6PR clones. Following two further rounds of screening and in vivo excision of the pBluescript phagemids their identity as full length M6PR clones was confirmed as follows: 1) IPTG-induced expression of M6PR activity in crude extracts; 2) IPTG-induced expression of a polypeptide that specifically interacted with M6PR antisera and with identical mobility (on SDS gels) to authentic M6PR; 3) 100% sequence homology to an internal peptide from a tryptic digest of purified M6PR. Based on these criteria, we conclude that we successfully cloned M6PR. The sequence is similar to several reductases from both plants and animals including an aldose 6-phosphate reductase from apple. Supported by USDA-NRI grant 940-1439.

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity.

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.

Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves.

Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens L.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (Fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (M6PR), and immunocytochemical techniques, we have resolved both the inter-cellular and intracellular sites of mannitol synthesis. In leaves, M6PR was found only in cells containing ribulose-1,5-bisphosphate carboxylase/oxygenase. M6PR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by M6PR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that M6PR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of M6PR is, therefore, in the assimilation of carbon being exported from the chloroplast, and it seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.