Celery Leaf Research Articles

Effects of aqueous extract of celery (Apium graveolens L.) leaves on spermatogenesis in healthy male rats.

Nowadays, a lot of attention has been paid to the therapeutic properties of herbs, including evaluation of the effects of these plants on fertility in laboratory animals. Apium graveolens L. (celery) has been widely used in traditional medicine for treatment of various disorders including impotency. Therefore, this study was designed to investigate the effects of aqueous extract of A. graveolens on testicular tissue and spermatogenesis in healthy male rats. In this research, 24 apparently healthy male rats were divided into three groups, including eight rats in each. The first group as control received only distilled water 1 ml/animal/day. The second and third groups orally received 100 and 200 mg/kg b.w. of the extract, respectively, for 30 days. The day after the last administration of the extract, the rats were sacrificed, the testes were removed entirely, and the morphometric studies were carried out. Epididymal sperm count and histological studies of testicular tissue were conducted. The comparison between the treated and control groups revealed a remarkable increase in the seminiferous tubules diameter, testes volume (p≤0.001), and the number of spermatogonia, primary spermatocytes and spermatozoa. Furthermore, the increase in the number of spermatids and epididymal weight were only significant at high doses of the extract (p≤ 0.05). The results from this study indicated that administration of celery leaf extract may improve spermatogenesis process and also be useful for some sperm fertility parameters.

Impact of Foliar Application of Tytanit on Zn, Li, Ni, Cr, Pb, and Cd Contents in Celery Leaves

Impact of Foliar Application of Tytanit on Zn, Li, Ni, Cr, Pb, and Cd Contents in Celery Leaves

Effect of foliar fertilization with tytanit on the content of selected macroelements and sodium in celery

AbstractFoliar application of growth regulators or fertilizers containing biostimulators can influen-ce the uptake and accumulation of mineral elements by plants. A pot experiment carried out in two annual series (year of experiment I and II) examined the effect of various concentrations of Tytanit applied once or twice against mineral fertilization (NPK), in comparison with the control object (without fertilization) and the object with mineral fertilization (NPK), on the total content of potassium, calcium, magnesium and sodium in petioles and leaf blades of celery. The experi-ment involved the application of various concentrations of Tytanit, ranging from 0.001% to 3.6%. The content of selected elements in the plant material was determined after dry mineralization, using the ICP – AES method. The total content of potassium in petioles of celery fertilized with NPK + 3.6% was higher or the same as in the control and in plants fertilized with NPK (I and II series). The results were similar in the case of leaf blades (except for series I). The highest concentration of Tytanit (3.6%) resulted in a reduction of the total calcium content in both of the tested parts of celery and magnesium in petioles, compared with the control. On the other hand, the total content of sodium increased along with the growth of the fertilizer concentration. The highest bioaccumulation of calcium was observed in celery cultivated in the object without ferti-lization (control). A significantly higher potassium content (mean from both series of research) and over two-fold higher content of calcium were found in celery leaf blades than in petioles, while a higher content of magnesium and sodium appeared in petioles than in leaf blades. The values of (K+Na) : (Ca+Mg) ratios in leaf blades were found to be over two-fold higher than in petioles of celery fertilized with Tytanit.Key words: petioles and leaf blades of celery, Tytanit concentration, potassium, calcium, mag-nesium, sodium.prof. dr hab. Stanislaw Kalembasa, Chair of Soil Science and Agricultural Chemistry, Siedlce University of Natural Sciences and Humanities, B. Prusa 14 Street, 08-114 Siedlce, Poland; e-mail: kalembasa@uph.edu.pl

Separation and identification of celery proteins

Background Allergy to Apium graveolens is one of the more severe food allergies. Six celery allergens have been characterised, Api g 1-6, while additional allergens have characterised from other members of the Magnoliospida class, including kiwi, peanut and ragweed, suggesting homologous allergens may also be present in celery. The participants of the Omics Allergy School 2013 were provided with celery bulb and leaf extracts for two dimensional separation and immunoblotting.

In vitro and in vivo antioxidant activities of a flavonoid isolated from celery (Apium graveolens L. var. dulce)

In the present article, a flavonoid was separated and purified from celery leaf through ethanol extraction, column chromatography and crystallization. The product was identified as apiin by LC/ESI-MS, and its antioxidant activities were evaluated in vitro, including by 1,1-diphenl-2-picrylhyrazyl free radical (DPPH˙), superoxide radical (O2(-)˙) and hydroxyl radical (OH˙) scavenging assays. IC50 values were 68.0 μg ml(-1) in the DPPH assay, 0.39 mg ml(-1) in the O2(-)˙ assay and 48.0 μg ml(-1) in the OH˙ assay. The antioxidant activities were investigated in vivo with the use of mice models. All data were measured including the contents of maleic dialdehyde (MDA) and lipofuscin (LPF), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (TAOC), in the serum, brain, heart, liver and kidney. Results showed that apiin had a remarkable scavenging activity on MDA and LPF, promoted TAOC and significantly enhanced the activities of SOD, GSH-Px and CAT.

Hepatoprotective action of celery (Apium graveolens) leaves in acetaminophen-fed freshwater fish (Pangasius sutchi)

Acetaminophen (APAP)-induced liver damage is one of the most common problems among the population. Therefore, the study was aimed to investigate the hepatoprotective effect of celery leaves on APAP-induced toxicity in a freshwater fish, Pangasius sutchi. Fish were divided into four experimental groups of 6 fish each. Group 1 served as control. Group 2 fish were exposed to APAP (500 mg/kg) for 24 h. Groups 3 and 4 fish were exposed to APAP + celery leaf powder (CE) (500 mg/kg) and CE for 24 h, respectively. The severity of liver damage, hepatic lipid, glycogen, ions status and histological alterations was examined. The characterization of CE extract was also performed. APAP-exposed fish showed elevated levels of both circulating and tissue hepatotoxic markers (AST, ALT and ALP), reduced hepatic glycogen and lipid contents (TG and cholesterol), increased tissue lipid peroxidation markers (TBARS, LHP and PCO), altered tissue levels of enzymatic (SOD, CAT, GPx and GST) and non-enzymatic (GSH) antioxidants and cellular thiol levels (T-SH, P-SH and NP-SH), and reduced hepatic ions (Na(+), K(+) and Ca(2+)) and abnormal liver histology. The abnormalities associated with APAP exposure were reversed on treatment with CE. The TLC separation and HPLC quantification of petroleum ether/acetone extract of CE showed the peaks for highly efficient flavonoids such as rutein, quercetin and luteolin. The observed hepatoprotective effect of CE might be due to its rich flavonoids.

First Report of Leaf Curl on Celery Caused by Colletotrichum acutatum in the United States.

In June 2010 and July 2011, celery (Apium graveolens) samples cv. Tango were submitted to the Penn State Plant Disease Clinic from Franklin and Dauphin Counties, PA, respectively. Plants exhibited curling and twisting of leaves and petioles and dark, brownish-black necrotic lesions at the base of the plant, extending up the petioles. A fungal organism with morphology consistent to Colletotrichum acutatum J.H. Simmonds was isolated from plant lesion tissue excised from the Dauphin Co. sample. Grown on half strength potato dextrose agar (PDA), the colony had gray aerial mycelium and a pink reverse. Conidia were 5.1 to 14.5 × 2.6 to 5.1 μm, aseptate, hyaline, elliptical, with one or both ends slightly pointed, and formed from the mycelium or in dense orange masses of acervuli on the aerial surface of the culture. Setae were not present. To test pathogenicity, five 23-week-old plants of the cv. Sonora and five 11-week-old plants each of the cvs. Tango and Tall Utah were sprayed until runoff with a conidial suspension (1.3 × 106 conidia/ml and 1.4 × 106 conidia/ml, respectively) and 0.025% Tween. One plant of each cv. was sprayed with milliQ water and 0.025% Tween as a control. Plastic bags were sprayed with the conidial suspension (milliQ water for the control), and secured over the individual plants for 24 h to create a humidity chamber. Plants were incubated in a growth chamber with a 16-h photoperiod, 25°C day/18°C night temperatures, and 70% humidity. Post-inoculation, all of the cv. Tango plants exhibited leaf cupping and curling after 7 days and most plants had dark stem lesions after 3 weeks, consistent with celery leaf curl symptoms. Plants of cvs. Tall Utah and Sonora developed malformed leaves and leaf curl symptoms 16 days and 10 days post-inoculation, respectively. None of the control plants developed symptoms. Infected tissue was excised from diseased plants, surface disinfested in 0.5% sodium hypochlorite for 45 s and plated on half strength PDA. Fungal colonies consistent with C. acutatum were recovered from all inoculated celery tissues (except two of the five inoculated cv. Tall Utah plants and the negative controls). To verify morphological identification, the internal transcribed spacer (ITS) rDNA region was amplified and sequenced for our original isolate and those recovered from the inoculated plants using ITS1 and ITS4 primers (2) (GenBank Accession No. JQ794875). Sequence homology revealed 99 to 100% similarity to accessioned isolates of C. acutatum, which included the holotype and a paratype of C. acutatum (Accession Nos. AF411700 and AF411701, respectively). Celery leaf curl has been reported to have caused devastating crop losses on celery in Australia (1, 3) and to our knowledge, C. acutatum causing leaf curl of celery has not been officially reported in the United States. Infected celery plants are unmarketable because of the leaf malformation and eventual plant necrosis caused by C. acutatum. As such, this disease could have serious negative implications for celery growers in the United States.

First Report of Colletotrichum acutatum sensu lato Causing Leaf Curling and Petiole Anthracnose on Celery (Apium graveolens) in Michigan.

In September 2010, celery plants with leaf cupping and petiole twisting were observed in commercial production fields located in Barry, Kent, Newago, and Van Buren Counties in Michigan. Long, elliptical lesions were observed on petioles but signs (mycelia, conidia, or acervuli) were not readily observed. Celery petioles were incubated in humid chambers (acrylic boxes with wet paper towels). After 24 h, conidia corresponding to the genus Colletotrichum were observed. Isolations were performed by excising pieces of celery tissue from the lesion margin and placing them on potato dextrose agar (PDA) amended with 30 ppm of rifampicin and 100 ppm of ampicillin. Plates were incubated at 21 ± 2°C under fluorescent light for 5 days. Fungal colony morphology was gray with salmon-colored masses of spores when viewed from above, and carmine when viewed from below. Isolates were single-spored and placed on 30% glycerol in -20°C, and cryoconservation media (20% glycerol, 0.04% yeast extract, 0.1% malt extract, 0.04% glucose, 0.02% K2HPO4) at -80°C. Conidia were 8.5 to 12.0 × 2.8 to 4.0 μm and straight fusiform in shape. Three isolates were confirmed as C. acutatum sensu lato based on sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA and the 1-kb intron of the glutamine synthase gene (3), both with 100% similarity with Glomerella acutata sequences. Sequences were submitted to GenBank (Accession Nos. JQ951599 and JQ951600 for ITS and GS, respectively). Additionally, C. acutatum specific primer CaIntg was used in combination with the primer ITS4 on 54 isolates from symptomatic celery plants, obtaining the expected 490-pb fragment (1). Koch's postulates were completed by inoculating 4-week-old celery seedlings of cultivars Sabroso, Green Bay, and Dutchess using three plants per cultivar. Prior to inoculation, seedlings were incubated for 16 h in high relative humidity (≥95%) by enclosing the plants in humid chambers. Seven-day-old C. acutatum s. l. colonies were used to prepare the inoculum. Seedlings were spray-inoculated with a C. acutatum s. l. conidial suspension of 1 × 106 conidia/ml in double-distilled water plus Tween 0.01%. Two control seedlings per cultivar were sprayed with sterile, double-distilled water plus 0.01% Tween. Plants were enclosed in bags for 96 h post inoculation and incubated in a greenhouse at 27°C by day/20°C by night with a 16-h photoperiod. Leaf curling was observed on all inoculated plants of the three cultivars 4 days after inoculation (DAI). Petiole lesions were observed 14 to 21 DAI. Conidia were observed in lesions after incubation in high humidity at 21 ± 2°C for 24 to 72 h. Symptomatic tissue was excised and cultured onto PDA and resulted in C. acutatum colonies. Control plants remained symptomless. C. acutatum (4) and C. orbiculare (2) were reported to cause celery leaf curl in Australia in 1966 (2,4). To our knowledge, this is the first report of C. acutatum s. l. infecting celery in Michigan.

In vitro antioxidant activity, total phenolics and flavonoids from celery (Apium graveolens) leaves

Apium graveolens has several beneficial properties including antioxidant activity. The leaves of A. graveolens was extracted with methanol and partitioned with water, ethyl acetate and butanol. The phenolic content of the extracts was determined by Folin-Coicalteu method and antioxidant activity was assayed through some in vitro models such as antioxidant capacity by radical scavenging activity using α, α-diphenyl-β-picrylhydrazyl (DPPH), β-carotene-linoleate, reducing power, metal chelating effects and phosphomolybdenum method. The phenolic content of the extracts were expressed as gallic acid equivalents and was found to be highest in methanol (51.09 mg/g) followed by water extract (46.40 mg/g), ethyl acetate (22.70 mg/g) and butanol extract (19.43 mg/g). At 250 µg/ml concentration, the antioxidant and free radical scavenging activities of the extracts assayed through DPPH and reducing power were found to be highest with methanol extract followed by water, ethyl acetate and butanol extract. The antioxidant activity of butylated hydroxyl toluene (BHT) was higher than the extracts at each concentration points. The results indicated that the extent of antioxidant activity of the extract is in accordance with the amount of phenolics present in that extract and the celery leaf vegetable being rich in phenolics may provide a good source of antioxidants. Key words: Apium graveolens, antioxidant activity, α-diphenyl-β-picrylhydrazyl (DPPH), flavonoid content, reducing power, total phenolic content.

Development and validation of a duplex real-time PCR method for the simultaneous detection of celery and white mustard in food

Celery (celery root: Apium graveolens var. Rapaceum; leaf celery: A. g. var. Secalinum; celery stalks: A. g. var. Dulce) and mustard (white or yellow mustard: Sinapis alba; black mustard: Brassica nigra; brown or oriental mustard: Brassica juncea) are frequently used as ingredients in sauces, spices, sausages and other meat-products as well as in convenience products. Within the European Union, the presence of potentially allergenic celery and mustard in foodstuffs has to be declared according to the EU legislative 2007/68/EC. The aim of the present study was to develop and validate a duplex real-time PCR method allowing the simultaneous detection of traces of celery and white mustard in food. Primers and TaqMan probes were designed for the Apium graveolens NADPH-dependent mannose-6-phosphate reductase mRNA as well as the Sinapis alba mRNA for MADS D protein. PCR was performed on the RotorGene RG-3000 from Corbett Life Sciences. With the optimized duplex assay DNA extracted from celery root, leaf celery and celery stalks as well as DNA from white mustard was amplified. The assay did not show any cross-reactivity with more than 60 food matrices, among them important members of the plant families Apiaceae and Brassicaceae, spices and different meat species. The LOD in serially diluted DNA extracts from celery root, leaf celery, celery stalks and white mustard was found to be 2 pg/μL (10 pg absolute). The PCR efficiency was 99.4% for celery root, 108.3% for celery stalks, 96.5% for leaf celery and 99.0% for white mustard. The LOD in DNA extracts obtained by extraction of model sausages was 0.005% (50 mg/kg) for celery and 0.001% (10 mg/kg) for white mustard, in both raw and brewed model sausages. The PCR efficiency was 90.0% (celery) and 101.1% (white mustard) in raw model sausages and 85.8% (celery) and 91.2% (white mustard) in brewed model sausages.

Content of Selected Flavonoids in 100 Edible Vegetables and Fruits

The objective of this work was to quantify the potential anticancer flavonoids (quercetin, kaempferol, isorhamnetin, apigenin and luteolin) by a rapid, reliable and accurate reverse phase high performance liquid chromatography (RP-HPLC) among 100 edible vegetables and fruits commonly consumed in Harbin city, China. The predominant flavonoids found in the largest abundance in all of the analyzed foods were kaempferol, followed by luteolin and quercetin. The maximum content of quercetin, kaempferol, isorhamnetin, apigenin, luteolin and total flavonoids in foods amounted to 379.7±19.7mg kg-1, 482.0±21.0mg kg-1, 333.6±14.4mg kg-1, 139.3±5.2mg kg-1, 179.5±10.6mg kg-1 and 853.0mg kg-1 fresh weight (FW), respectively. Potherb mustard, lettuce (red), toona leaf, celery leaf, garlic, garlic stalk, apple, date, ginger (with peel) and radish leaf (red root, small) were major food sources of flavonols and flavones. This study contribute to the establishment of China's flavonoid database and provide a basis for the assessment of the daily flavonoid intake.

The relationship between fasting plasma concentrations of selected flavonoids and their ordinary dietary intake

Epidemiological studies suggest that a diet high in flavonoids protects against chronic diseases such as CVD and cancer. The objective of the present study was to evaluate the relationship between the intake of quercetin, kaempferol, isorhamnetin, apigenin and luteolin and their corresponding plasma concentrations, and further to explore whether these flavonoids can serve as biomarkers of their intake. Flavonoid intake and their plasma concentrations were analysed in ninety-two subjects consuming their habitual diet. Flavonoid intake was estimated with 7-d dietary records using available data on the flavonoid content of food. Plasma flavonoid concentrations were quantified by HPLC. In addition, we undertook a dietary intervention study to investigate plasma apigenin concentration after the consumption of celery leaf. The mean intake estimates of quercetin, kaempferol, isorhamnetin, apigenin and luteolin amounted to 13.58, 14.97, 12.31, 4.23 and 8.08 mg/d, respectively. The corresponding mean plasma concentrations were 80.23, 57.86, 39.94, 10.62 and 99.90 nmol/l. The mean 7 d intake of five flavonoids was positively correlated to their corresponding plasma concentrations, with correlation coefficients ranging from 0.33 to 0.51 (P < 0.05). In the dietary intervention study, the plasma apigenin concentration rose after celery leaf ingestion, and fell within 28 h to below the limit of detection (2.32 nmol/l). The present results suggest that quercetin, kaempferol, isorhamnetin, apigenin and luteolin are bioavailable from the diet. The levels of fasting plasma flavonoids seem to be suitable biomarkers of short-term intake. The combination of plasma flavonoids with their intake may prove useful when the possible health-protective effects of flavonoids are studied.

AKTIVITAS ANTIDIARE EKSTRAK ETANOL DAUN SELEDRI (Apium graveolens L) PADA MENCIT JANTAN

Celery (Apium graveolens L.) is one of plants that initially only used for cooking. Based on the research by Wolski, et al., 2002, celery contains chemical compound such as tannins and polyphenols. Tannins may be useful as an anti-diarrheal because of the adstringent effect. This study aims to determine the antidiarrheals activity of celery leaf in male mice strain Balb-C that was induced by castor oil. The ethanolic extract of celery leaves divided into three dose group (5,10 and 20 mg/kgBW). This research was done by two methods, protection of castor oil induced diarrhea and intestinal transit. After five hours treatment, frequency of defecation, number of soft/liquid feses and weight of stools were calculated. The gastrointestinal transit rate was expressed as the percentage of longest distance traversed by the charcoal divided by the total length of the small intestine.From the protection of castor oil induced diarrhea method, ethanol extract of celery 20 mg/20gBW doses decrease frequency of defecation, quantity of the soft / liquid stool and weight of stool after 5 hours treatment in male mice that had been induced by castor oil. The effectiveness of ethanol extract 20mg/20gBW dose equivalent to loperamid 0.06mg/20gBW (one way anova test at the α = 95%). Besides, the ethanol extract of celery can prolong intestinal transit of mice, but lower than loperamid 0.06mg/20g (one-way anova test at the α = 95%). Keywords: antidiarrheal, castor oil, celery, intestinal transit, taninns

Effect of Fumitory, Celery and Lemon on Blood Glucose and Histopathology of Pancreas of Alloxan Diabetic Rats

Jelodar, G., Maleki, M. and Sirus, S. 2007. Effect of fumitory, celery and lemon on blood glucose and histopathology of pancreas of alloxan diabetic rats. J. Appl. Anim. Res., 31: 101–104. This study was performed to investigate hypoglycemic effect of fumitory leaf, celery leaf and lemon recommended in Persian folklore medicine as beneficial in treatment of diabetes and their possible effect on pancreatic tissue. Diabetes mellitus was induced in 20 adult male Sprague Dawley rats, by intraperitoneal injection of alloxan (185 mg/kg). The diabetic rats were divided into four groups; three of them fed a diet supplemented with 15 grams of the mentioned plants for 15 days. Fourth diabetic untreated group (positive control) and a non-diabetic group (negative control) received standard diet. Volume density of β cells and islets, percent of β cells, number of islets per square centimeter and average area of islets was recorded. Fumitory and celery reduced blood glucose significantly compared with diabetic untreated group (10.71 and 11.17 vs. 14.36 mmol/1) (P<0.05). In control positive and groups treated with plants all mentioned histomorphometric factors were significantly changed in comparison with control negative except number of islets per square centimeter, which increased in fumitory group. In conclusion fumitory leaf and celery have shown potential hypoglycemic effect and the results support the traditional use of these plants for controlling hyperglycemia.

(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.

Antipyretic Effect of Celery (Apium graveolens.) Extracts in Mice

Extracts of celery (Apium graveolens. L.; Apiaceae) leaves prepared in solvents of different polarity, viz., ether, chloroform, ethyl acetate, n.-butanol, and water, were used to study their antipyretic effect in mice. Experiments were conducted on white laboratory mice divided into five groups. On the first day of the experiment, their rectal temperatures were measured every 30 min during 5 h (basal temperature). On the second day, the animals were given 12% yeast suspension to induce a pyrogenic effect and on the third day the same dose of yeast and the appropriate celery extract, and the rectal temperatures were measured in the same way as basal temperature. By plotting the measured temperatures versus time, the corresponding areas-under-the-curve (AUC) were obtained for each group of animals. The AUC values were used to determine statistically significant differences between them. The results showed that extracts of celery leaf decreased (annuled) the pyrogenic effect of 12% yeast suspension.

Opinion of the Scientific Panel on Dietetic products, nutrition and allergies [NDA] to a notification from EFFA on celery leaf oil, celery seed oil and celery oleoresin pursuant to Article 6 paragraph 11 of Directive 2000/13/EC

Opinion of the Scientific Panel on Dietetic products, nutrition and allergies [NDA] to a notification from EFFA on celery leaf oil, celery seed oil and celery oleoresin pursuant to Article 6 paragraph 11 of Directive 2000/13/EC

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.

NADPH Supply and Mannitol Biosynthesis. Characterization, Cloning, and Regulation of the Non-Reversible Glyceraldehyde-3-Phosphate Dehydrogenase in Celery Leaves

Mannitol, a sugar alcohol, is a major primary photosynthetic product in celery (Apium graveolens L. cv Giant Pascal). We report here on purification, characterization, and cDNA cloning of cytosolic non-reversible glyceraldehyde-3-P dehydrogenase (nr-G3PDH, EC 1.2.1. 9), the apparent key contributor of the NADPH required for mannitol biosynthesis in celery leaves. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified nr-G3PDH showed a molecular mass of 53 kD. A 1,734-bp full-length cDNA clone (accession no. AF196292) encoding nr-G3PDH was identified using polymerase chain reaction and rapid amplification of cDNA ends techniques. The cDNA clone has an open reading frame of 1,491 bp encoding 496 amino acid residues with a calculated molecular weight of 53,172. K(m) values for the celery nr-G3PDH were low (6.8 microM for NADP(+) and 29 microM for D-glyceraldehyde-3-P). NADPH, 3-phosphoglycerate, and ATP were competitive inhibitors, and cytosolic levels of these three metabolites (as determined by nonaqueous fractionation) were all above the concentrations necessary to inhibit activity in vitro, suggesting that nr-G3PDH may be regulated through feedback inhibition by one or more metabolites. We also determined a tight association between activities of nr-G3PDH and mannose-6-P reductase and mRNA expression levels in response to both leaf development and salt treatment. Collectively, our data clearly show metabolic, developmental, and environmental regulation of nr-G3PDH, and also suggest that the supply of NADPH necessary for mannitol biosynthesis is under tight metabolic control.

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.