Allelopathy of red pine: isolation and identification of an allelopathic substance in red pine needles

Since red pine (Pinus densiflora Sieb. et Zucc.) often forms sparse forest floors where herbaceous plants do not grow well, allelopathy of red pine was investigated. A growth inhibitory substance was isolated from an aqueous methanol extract of red pine needles and determined by spectral data as abscisic acid-β-d-glucopyranosyl ester (ABA-GE). This substance inhibited root and shoot growth of cress and E. crus-galli seedlings at concentrations greater than 0.1 μM. The concentrations required for 50% growth inhibition on roots and shoots of cress were 0.23 and 0.61 μM, respectively, and those of E. crus-galli were 1.1 and 2.8 μM, respectively. The activity of ABA-β-d-glucosidase, which liberates free ABA from ABA-GE, in cress and E. crus-galli seedlings was 13–29 nmol mg⁻¹ protein min⁻¹. Endogenous concentration of ABA-GE in the pine needles was 4.1–21.5 μmol kg⁻¹ and the concentration in soil water of the pine forest was 2.5 μM. The effectiveness of ABA-GE on growth inhibition and the occurrence of ABA-GE in pine needles and soil water suggest ABA-GE may play an important role in the allelopathy of red pine resulting in the formation of sparse forest floors.     

Phytotoxic activity of Cachrys pungens Jan,a mediterranean species: separation,identification and quantification of potential allelochemicals

In continuous research for bioactive compounds obtained from plants to use for weed control in sustainable agriculture, the aerial parts of Cachrys pungens Jan (Umbelliferae) were extracted with methanol and then fractionated using hexane, chloroform (CHCl₃) and ethyl acetate (AcOEt). The potential phytotoxicity of total methanolic extract and each fraction was assayed in vitro on seed germination and root elongation of lettuce (Lactuca sativa L.) and the most active fractions were assayed on three of the most common weeds (Lolium perenne, Amaranthus retroflexus, Echinochloa crus-galli). Non linear regression that allowed to obtain the ED₅₀ index for both physiological processes was applied. The fraction bioassays indicated the following hierarchy of phytotoxicity for both processes: CHCl₃ ≥ AcOEt > hexane. Moreover, in the present work was chemically characterized for the first time (through HPTLC) the polar fraction of this species pointing out the high presence of flavonoids and phenolic acids. In particular six of them have been chemically characterized and quantified (naringin, quercetin, catechin, caffeic acid, ferulic acid and gallic acid). These results make C. pungens Jan a potential source of natural compounds employable for an eco-friendly agriculture.     

Phytotoxicity of extracts and fractions of Ouratea spectabilis (Mart. ex Engl.) Engl. (Ochnaceae)

Among the numerous plant species occurring in the Cerrado, Ouratea spectabilis stands out because of the lack of species that grow beneath its canopy. Therefore, this study aimed to evaluate the phytotoxic potential of different extracts and fractions of the hydroethanolic extract from leaves of O. spectabilis through laboratory bioassays of the pre-and post-emergence of seeds of Lactuca sativa L., determination of the mitotic index in root cells of Allium cepa L., antioxidant activity and phytochemical screening of different classes present in extracts and ethyl acetate fractions. It was possible to verify that different extracts and ethyl acetate fractions of O. spectabilis interfered with germination rates, as reduced germination was observed when compared with the control. Similarly, growth and development was affected in lettuce seedlings, as shown by the reduced length of primary roots and hypocotyls compared with the control. In addition, the mitotic index was reduced in treated groups compared with the negative control. HPLC-PAD analysis for both the hydroethanolic extract and its ethyl acetate fraction, showed a predominance of flavonoid compounds belonging to the groups of isoflavones and catechins in ethyl acetate fractions of hydroethanolic extracts. Thus, it was concluded that this species synthesizes phytotoxic compounds capable of interfering in the stabilization and development of other species.     

The Multiple Forms of alpha-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz : A Comparative Study

α-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of α-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific α-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five α-amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of α-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins.     

Organ-specific-active allelopathic substance in red pine needles

An organ-specific-growth inhibitory substance was isolated from an aqueous methanol extract of red pine needles and determined by spectral data as 1-mono(16-hydroxyhexadecanoyl)glycerol. This substance inhibited root growth of cress (Lepidium sativum L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv) seedlings at concentrations greater than 0.01 and 0.03???M, respectively. The concentrations required for 50?% growth inhibition on roots of cress and barnyard grass were 0.16 and 3.4???M, respectively. However, the inhibitory activity of the substance on shoots of cress and barnyard grass was very weak. The endogenous concentration of 1-mono(16-hydroxyhexadecanoyl)glycerol in the pine needles was 4.6???mol?kg^?1. Two related compounds, 1-monohexadecanoylglycerol and 16-hydroxyhexadecanlic acid had no activity up to 1,000???M on cress roots and shoots. The effectiveness of 1-mono(16-hydroxyhexadecanoyl)glycerol on root growth inhibition and the occurrence of 1-mono(16-hydroxyhexadecanoyl)glycerol in pine needles suggest the substance may play an important role in the allelopathy of red pine. Root-specific-growth-inhibition by the substance may be one of the strategies for red pine to compete with neighboring plants for nutrients and space because root growth of competitive plants may be very important for their whole plant development.     

Isolation and identification of a plant growth inhibitor from Tinospora tuberculata Beumee

Tinospora tuberculata Beumee has been used widely as a folk medicine and several bioactive compounds have been isolated. However, no herbicidal compound has been reported in this species. Therefore, we investigated the presence of phytotoxins in T. tuberculata. The aqueous methanol extracts of T. tuberculata inhibited the growth of roots and shoots of cress (Lepidum sativum L.), lettuce (Lactuca sativa L.), timothy (Phleum pratense L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.). The extract was then purified by several chromatographic runs with monitoring the inhibitory activity and the main phytotoxic substance was isolated. The chemical structure of the compound was determined by spectral data as syringin (4-[(1E)-3-Hydroxyprop-1-en-1-yl]-2,6-dimethoxyphenyl β-d-glucopyranoside). It inhibited the root and shoot growth of all test plant species at concentrations >10 µM. The concentrations required for 50 % inhibition of root and shoot growth of cress and lettuce ranged from 78.2 to 412 μM, and that of timothy and barnyard grass renged from 9.8 to 73.2 µM. Effectiveness of syringin on monocotyledonous (timothy and barnyard grass) plants was greater than that on dicotyledonous (cress and lettuce) plants. These results suggest that syringin may contribute to the allelopathic effect caused by the T. tuberculata extract.     

Arabidopsis nudix hydrolase 7 plays a role in seed germination

Arabidopsis nudix hydrolase 7 (Atnudt7) mutants exhibit reduced seed germination phenotype following after-ripening. The role of AtNUDT7 in seeds and during early stages of imbibition was examined. Seeds of Atnudt7-1 and Col-0 following 3 days of imbibition were used to profile changes in NADH- and ADP-ribose pyrophosphohydrolase enzyme activities, expression of nudix family genes closely related to AtNudt7, and AtNUDT7 protein levels. Changes in pyridine nucleotides, phytohormones, reactive oxygen species and poly(ADP-ribose) levels in after-ripened seeds and 1 day after imbibition were also analyzed. Changes in AtNUDT7 gene expression, protein levels and enzyme activities in WT seeds and during early stages of imbibition were correlated. Atnudt7-1 seeds lacked NADH pyrophosphohydrolase activity that led to very high catabolic redox charge. Abscisic acid (ABA) levels were higher in Atnudt7-1 mutant while salicylic acid, gibberellic acid, and reactive oxygen species (ROS) levels were higher in WT seeds. In Atnudt7-1, there was excess ROS accumulation 1 day after imbibition. PAR levels were significantly higher in Atnudt7-1 mutant when compared to WT during imbibition. Based on these observations, we conclude NADH pyrophosphohydrolase activity conferred by AtNUDT7 is important for NAD:NADH homeostasis in seeds. Perturbations to this key redox couple alter ABA and ROS levels in the seeds that in turn lowers germination.     

A Potent Phytotoxic Substance in Aglaia odorata Lour.

Aglaia odorata Lour . (Meliaceae) was found to have very strong allelopathic activity and a bioherbicide PORGANIC^? was developed from its leaf extracts. However, the phytotoxic substances causing the strong allelopathic activity of the plants have not yet been determined. Therefore, we investigated allelopathic properties and phytotoxic substances in A. odorata. Aqueous EtOH extracts of A. odorata leaves inhibited root and shoot growth of garden cress (Lepidum sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), timothy (Phleum pratense), ryegrass (Lolium multiflorum), and Echinochloa crus‐galli with the extract concentration‐dependent manner. The extracts were then purified and a major phytotoxic substance with allelopathic activity was isolated and identified by spectral data as rocaglaol. Rocaglaol inhibited the growth of garden cress and E. crus‐galli at concentrations > 0.3 and 0.03 μm , respectively. The concentrations required for 50% inhibition ranged from 0.09 to 2.5 μm . The inhibitory activity of rocaglaol on the weed species, E. crus‐galli, was much greater than that of abscisic acid. These results suggest that rocaglaol may be a major contributor to the allelopathic effect of A. odorata and bioherbicide PORGANIC^?.     

Nephroprotective,cytotoxic and antioxidant activities of Euphorbia paralias

ObjectiveTo investigate the effect of the ethyl acetate fraction of the aerial parts of E. paralias L. F. Euphorbiaceae on nephroprotective, cytotoxic, and antioxidant.Methodsdifferent spectroscopic and spectrophotometric methods were applied to identify phytoconstituents. The nephroprotective potential of E. paralias ethyl acetate fraction (Ep EtOAc) was evaluated in male rats with thioacetamide-induced kidney injury, as wll as cytotoxic activity was evaluated using a viability assay, and the antioxidant activity was evaluated using the DPPH method. Results: quantitative estimation of total phenolics and flavonoids of E. paralias was performed using unique spectrophotometric methods. The polyphenolic compounds gallic acid (1), ellagic acid (2), kaempferol-3-O-(6″-O-galloyl-β-D-glucopyranoside) (3), quercetin-3-O-β-D-glucopyranoside (4) and quercetin-3-O-β-D-arabinoside (5) were isolated from the ethyl acetate fraction of the aerial parts of E. paralias. The thioacetamide administration resulted in marked nephrotoxicity, but pretreatment with Ep EtOAc significantly attenuated the nephrotoxicity through alteration of kidney biomarkers, thereby improving the redox status of the tissue and restoring serum biochemical parameters nearly to normal levels. This study revealed a significant cytotoxic and strong antioxidant effect. Conclusion: we conclude that the Ep EtOAc may be used in the future as nephroprotective, cytotoxic, and antioxidant agent derived from a natural source.     

Growth and chlorophyll a fluorescence in Erythrina crista-galli L. plants under flooding conditions

The present study aimed to analyse the alterations in the growth and chlorophyll content of Erythrina crista-galli plants in response to flooding and to evaluate possible photosystem II (PSII) damage through chlorophyll a fluorescence transient analysis using the JIP test. Plants cultured from seeds were grown in 0.5-L pots in a greenhouse and transferred to 5-L pots. Two treatments were employed: plants were either flooded at the root and maintained with a sheet of water above the soil or not flooded (control). Evaluations were performed at 10, 20, 30 and 40 days after the treatment was initiated. The experiment was set up in a randomised block design, and the results were subjected to analysis of variance (ANOVA). The means were compared by Tukey’s test at a 5 % significance level. The flooding of the plants induced the formation of lenticels, adventitious roots and larger intercellular spaces in the root cortex and inhibited the growth of shoots at early time points compared to control plants. However, growth resumed by the end of the experimental period. The Erythrina crista-galli plants manifested a reduction in chlorophyll content as a function of the duration of exposure to flooding. However, there were changes in the chlorophyll fluorescence parameters, indicating impairment of the structure and function of PSII, and the performance indices were the most representative parameters to describe the effects of flooding. The results showed that the Erythrina crista-galli tolerated flooding by initially investing energy into the formation of morpho-anatomical structures to maintain oxygen diffusion and ATP production. The effect of flooding on the leaves was reflected in increased energy dissipation through chlorophyll a fluorescence, likely as a mechanism to protect the photosynthetic apparatus.     

Phytotoxic activity of Cleome arabica L. and its principal discovered active compounds

The aim of this study was to assess the phytotoxic potential of Cleome arabica L, as well as to isolate the main bioactive compounds. Phytotoxicity was evaluated on germination and seedling growth of Lactuca sativa, Raphanus sativus, Peganum harmala and Silybum marianum, through testing aqueous and organic extracts of different C. arabica organs (roots, shoots, siliquae and seeds). Results showed that siliquae methanol extract caused the greatest negative effect on lettuce germination and growth. For the bioactive subfractions (petroleum ether, ethyl acetate and methanol–water), the ethyl acetate induced highly significant reduction, showing 100% inhibition of lettuce growth at 6 g/L. The bioactive ethyl acetate subfraction was chromatographed and subjected to NMR techniques. Based on bio-guided chromatographic fractionation, five bioactive allelochemical compounds were isolated from ethyl acetate extract of siliquae of C. arabica. The most inhibitory compound on lettuce seedling growth was elucidated as 11-α-acetylbrachy-carpone-22(23)-ene.     

Biological activity of Xanthium strumarium seed extracts on different cancer cell lines and Aedes caspius,Culex pipiens (Diptera: Culicidae)

Effects of methanol extracts of Xanthium strumarium on different cancer cell lines and on the mortality rates of Aedes caspius, Culex pipiens (Diptera: Culicidae) were investigated. Among the cell lines tested, the Jurkat cell line was the most sensitive to the methanol extract and ethyl acetate fraction, with reported LC₅₀ values of 50.18 and 48.73 μg/ml respectively. Conversely, methanol extracts were not that toxic to the A549 cell line though the toxicity increased on further purification. The percentage of growth inhibition was dose dependent for the methanol extract and ethyl acetate fraction. The ethyl acetate fraction showed higher toxicity to all cell lines tested when compared to the methanol extract. The results showed that methanol extracts of plant seeds caused 100% mortality of mosquito larvae at a concentration of 1000 μg/ml after 24 h of treatment. The LC₅₀ and LC₉₀ values of X. strumarium were found to be 531.07 and 905.95 μg/ml against Ae. caspius and 502.32 and 867.63 μg/ml against Cx. Pipiens, respectively. From the investigations, it was concluded that the crude extract of X. strumarium showed a weak potential for controlling the larval instars of Ae. caspius and Cx. pipiens. However, on further purification the extract lost the larvicidal activity. The ethyl acetate fraction showed higher toxicity to all cell lines tested when compared to the methanol extract. The ethyl acetate fraction investigated in this study appears to have a weak larvicidal activity but a promising cytotoxic activity. Future studies will include purification and investigation in further detail of the action of X. strumarium on Cancer Cell Lines and mosquitoes.     

A novel allelopathic substance, 13-epi-orthosiphol N,in Orthosiphon stamineus

Orthosiphon stamineus (Java tea) has been widely used as traditional herb and several bioactive compounds against animal cells have been isolated. However, no bioactive compound against plants has been reported. Therefore, we investigated possible allelopathic properties and substances in O. stamineus. Aqueous methanol extracts of O. stamineus inhibited root and hypocotyl growth of cress (Lepidium sativum) and lettuce (Lactuca sativa) seedlings. Increasing the extract concentration increased the inhibition, which suggests that O. stamineus may have allelopathic properties. When the extract was divided into an ethyl acetate and an aqueous fraction, the ethyl acetate fraction showed the stronger inhibitory effect. Thus, the ethyl acetate phase was further purified, and the main allelopathic substance was isolated and identified as 13-epi-orthosiphol N, a novel compound, by spectral data. 13-epi-Orthosiphol N inhibited root and hypocotyl growth of cress and lettuce at concentrations greater than 10 μmol/L. The concentrations required for 50% inhibition ranged from 41 to 102 μmol/L. These results suggest that 13-epi-orthosiphol N may be an allelochemical and main contributor to the growth inhibitory effect of O. stamineus and may have potential as a template for the development of new plant control substances.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

Effects of Power Frequency Electromagnetic Fields on Growth of Germinating Vicia faba L., the Broad Bean

Experimental investigations were carried out to evaluate the effect of continuous and delayed exposure of power frequency electromagnetic fields at 5, 50 and 100 μT on germinating Vicia faba seedlings as a model system. These studies included physical parameters (length and girth of primary roots, number as well as length of lateral roots and imbibition), major biochemical constituents (total sugar, protein, and fat) and activities of important housekeeping enzymes (amylases, proteases, and lipase) at 2, 4, and 8 days of growth. Also, mitotic index and rate of DNA synthesis were studied at day 8 of growth. There was no significant change in physical parameters and major biochemical constituents between control and experimental groups. Also, the comparison between the control and experimental group of seeds showed that α-amylase activity significantly decreased at 5, 50 and 100 μT on day 2 and 4 of growth. β-amylase and protease (37^○C & 50^○C) showed a significant decrease in activity on day 2 and 4 of growth at 100 μT, whereas activity of lipase significantly decreased only on day 2 of growth at 100 μT. At day 8 of growth, all enzyme activities reverted back to the same as control. Also, there was a significant increase in mitotic index as well as ³H-thymidine uptake at 100 μT delayed exposure on day 8. The present study suggests that exposure to power frequency electromagnetic fields up to 100 μT on germinating seedlings does not cause any permanent damage since the initial alteration under the magnetic fields in some important housekeeping enzymes involved in the onset of seed germination were returened to control values on day 8 of growth. Also, the growth of the germinated seedlings was found to be enhanced by the application of power frequency magnetic fields (100 μT) as evidenced by mitotic index and ³H-thymidine uptake.     

The first bioactivity studies of Acantholimon lycopodioides from high altitude Karakoram-Himalayan desert

Couple of ethnopharmacological surveys in the Indian Ladakh and Pakistani Shigar valleys has reported the medicinal use of Acantholimon lycopodioides against cardiac and gastric disorders that however, remains without scientific rationale or experimental validations. Here, we assess the in vitro bio/therapeutic activities of A. lycopodioides extracts as well as chloroform, ethyl acetate, n-butanol and aqueous fractions. The in vitro β-carotene-linoleic acid bleaching and DPPH radical scavenging methods demonstrated a very high anti-oxidative property of chloroform and ethyl acetate fractions compared to others. Cell viability assay (MTT) on human cervical (HeLa), breast (MDA-MB321) and liver (HepG2) cancer cells revealed their differential cytotoxicity, except the chloroform fraction. Of these, the precipitate exerted highest cytotoxicity on HepG2 cells followed by aqueous fraction on MDA-MB321 cells. Notably, the non-cytotoxicity of chloroform fraction coincided with its highest anti-oxidative activity. Further, the chloroform fraction showed marked hepatoprotection (up to 84%) against 3′7′dichlorofluorescin triggered free radicals induced oxidative damage. Also, the hepatoprotective chloroform fraction mildly activated CYP3A4 in HepG2 cells (dual-luciferase assay). Moreover, the A. lycopodioides extracts and fractions showed differential anti-bacterial and anti-fungal activities. Of these, while S. aureus was more sensitive to the water-insoluble extract, ethyl acetate fraction showed moderate activity against E. coli and C. albicans. On the other hand, the chloroform fraction showed promising activity against S. Aureus, C. albicans, P. vulgaris and E. faecalis. In conclusion, our data for the first time, demonstrated promising anti-oxidative, hepatoprotective, anti-cancer, anti-microbial and CYP3A4 activating salutations of A. lycopodioides. This warrants further studies towards isolation and identification of its therapeutically active principles.     

Possible involvement of aquaporins in water uptake by pea seeds differing in quality

Using the method of room temperature phosphorescence (RTP), we divided air-dry pea (Pisum sativum L.) seeds subjected to accelerated ageing (40°C, 85% relative humidity) into three fractions: (I) high-quality seeds, (II) weakened seeds, and (III) dead seeds. In the process of ageing, seed germinability firstly decreased and then increased due to so-called “improved” seeds of fraction II, which returned to fraction I as judged from the RTP level; the germinability of these seeds became equal to that of fraction I seeds. Seeds capable of germination (fractions I and II) differed in the rates of imbibition, which depended on plasma membrane permeability (opened or closed water channels) but not on the presence of the seed coat. A low activation energy of seed imbibition in fraction II (less than 5 kcal/mol) indicates that water channels are open. A mercury-containing compound (5 μM p-chloromercuribenzoate (PCMB) reduced the rate of water uptake by these seeds, and dithiothreitol restored it. A high activation energy of fraction I seed imbibition (more than 12 kcal/mol) corresponded to the water uptake mainly across the lipid bilayer when water channels are closed. PCMB did not affect the rate of fraction I seed imbibition. We supposed that mature air-dry pea seeds had open water channels. During the first stages of fraction I seed imbibition, these channels were closed, limiting water uptake. NaF (100 μM), an inhibitor of phosphatase, prevented channel closing and accelerated the imbibition of fraction I seeds. It did not affect the imbibition rate of fraction II seeds, indicating their water channels to be opened. However, NaF did not affect the water uptake of “improved” fraction II seeds as well. It seems likely that their channels were closed during accelerated ageing but otherwise than via dephosphorylation. The results obtained indicate the possibility of water inflow regulation in the weakened seeds via the state of aquaporins, which form water channels in the membranes.     

Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

Increased ��-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis ��-tocopherol methyltransferase

Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The ??-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. ??-tocopherol methyltransferase (??-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of ??- and ??-isomers to ??- and ??-isomers. In present study, the Arabidopsis ??-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the ??-/??-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the ??-isomers were converted to ??-isomers, especially the ??- and ??-tocotrienol levels were dramatically decreased. As a result, the ??-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the ??-tocotrienol content. The results showed that the ??-/??-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on ??-tocopherol level, which may reflect the fact that ??-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign ??-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world??s most important food crops.     

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA(1)

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A₁ solutions for 24 hours at 20°. Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA₁, and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA₁ brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA₁. Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.