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Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F 2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The short-day plant pharbitis (also called Japanese morning glory), Ipomoea nil (formerly Pharbitis nil), was induced to flower by poor-nutrition stress. This stress-induced flowering was inhibited by aminooxyacetic acid (AOA), which is a known inhibitor of phenylalanine ammonia-lyase (PAL) and the synthesis of indole-3-acetic acid (IAA) and 1-aminocycropropane-1-carboxylic acid (ACC) and thus regulates endogenous levels of salicylic acid (SA), IAA and polyamine (PA). Stress treatment increased PAL activity in cotyledons, and AOA suppressed this increase. The observed PAL activity and flowering response correlate positively, indicating that AOA functions as a PAL inhibitor. The inhibition of stress-induced flowering by AOA was also overcome by IAA. An antiauxin, 4-chlorophenoxy isobutyric acid, inhibited stress-induced flowering. Both SA and IAA promoted flowering induced by stress. PA also promoted flowering, and the effective PA was found to be putrescine (Put). These results suggest that all of the pathways leading to the synthesis of SA, IAA and Put are responsive to the flowering inhibition by AOA and that these endogenous factors may be involved in the regulation of stress-induced flowering. However, as none of them induced flowering under non-stress conditions, they may function cooperatively to promote flowering., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Glycosylation is an important post-translational modification involved in the modulation of a wide variety of cellular processes. Because glycosydases are central, the aim of this study was to investigate the glycosyl activity present in the cotyledons of the seeds of an important crop legume, Lupinus albus, as well as potential natural substrates of the detected enzymes. The glycosyl activity detected in the cotyledons beginning at seed imbibition and continuing until 9 days after, was due to a β-N-acetylhexosaminidase (β-NAHase), which was molecularly and biochemically characterized after purification. Two isoenzymes with molecular masses of 64 and 61 kDa were detected, each having five isoenzymes with pIs 5.3-5.6. The 64 and 61 kDa isoenzymes had the same protein core showing different degrees of glycosylation. The N-terminal sequence of the enzyme protein core was determined [VDSEDLI(EN)AFKIYVEDDNEHLQGSVD] and to our knowledge, is the first reported protein sequence from a plant β-NAHase. L. albus β-NAHase had Km values of 2.59 mM and 2.94 mM and V values of 18.40 μM min(-1) and 2.73 μM min(-1), for pNP-GlcNAc and pNP-GalNAc, an optimum pH of 5.0 and 4.0 and temperature of 50 °C and 60 °C were detected toward pNP-GlcNAc and pNP-GalNAc. In the presence of AgNO3, CoCl2, CuSO4, FeCl3, CdCl2 and ZnCl2 the enzymatic activity decreased more than 50%, and when in the presence of sugars, an activity reduction of no more than 25% was observed. A physiological role for β-NAHase in L. albus storage protein mobilization was investigated. β-NAHase has already been implicated in several biological processes, namely in glycoprotein processing during seed germination and seedling growth. However, the natural substrates used by this enzyme are not yet completely clarified. By gathering in vivo and in vitro data for β-NAHase activity together with globulin degradation, we suggest that L. albus β-NAHase is involved in the mobilization of storage protein degradation, with α-conglutin being a potential natural substrate for this enzyme., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

We previously reported that native tropical zone plants showed high acetylcholinesterase (AChE) activity during heat stress, and that AChE activity in endodermal cells of maize seedlings was increased by heat treatment. However, the physiological role of AChE in heat stressed plants is still unclear. Here we report (1) tissue-specific expression and subcellular localization of maize AChE, (2) elevation of AChE activity and possible post-translational modifications of this enzyme under heat stress, and (3) involvement of AChE in plant heat stress tolerance. Maize AChE was mainly expressed in coleoptile nodes and seeds. Maize AChE fused with green fluorescent protein (GFP) was localized in extracellular spaces of transgenic rice plants. Therefore, in maize coleoptile nodes and seeds AChE mainly functions in the cell wall matrix. After heat treatment, enhanced maize AChE activity was observed by in vitro activity measurement and by in situ cytochemical staining; transcript and protein levels, however, were not changed. Protein gel blot analysis revealed two AChE isoforms (upper and lower); the upper-form gradually disappeared after heat treatment. Thus, maize AChE activity might be enhanced through a post-translational modification response to heat stress. Finally, we found that overexpression of maize AChE in transgenic tobacco plants enhanced heat tolerance relative to that of non-transgenic plants, suggesting AChE plays a positive role in maize heat tolerance., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP(+)-dependent (EC 1.1.1.42) and NAD(+)-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Sphingolipids are components of plant cell membranes that participate in the regulation of important physiological processes. Unlike their animal counterparts, plant sphingolipids are characterized by high levels of base C4-hydroxylation. Moreover, desaturation at the Δ8 position predominates over the Δ4 desaturation typically found in animal sphingolipids. These modifications are due to the action of C4-hydroxylases and Δ8-long chain base desaturases, and they are important for complex sphingolipids finally becoming functional. The long chain bases of sunflower sphingolipids have high levels of hydroxylated and unsaturated moieties. Here, a C4-long chain base hydroxylase was functionally characterized in sunflower plant, an enzyme that could complement the sur2Δ mutation when heterologously expressed in this yeast mutant deficient in hydroxylation. This hydroxylase was ubiquitously expressed in sunflower, with the highest levels found in the developing cotyledons. In addition, we identified a new Δ8-long base chain desaturase gene that displays strong homology to a previously reported desaturase gene. This desaturase was also expressed in yeast and was able to change the long chain base composition of the transformed host. We studied the expression of this desaturase and compared it with that of the other isoform described in sunflower. The desaturase form studied in this paper displayed higher expression levels in developing seeds., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The ability of cells to control the degradation of their own DNA is a common feature of most living organisms. In plants, extensive hydrolysis of nuclear DNA occurs during different forms of programmed cell death (PCD). In addition to the removal of unwanted cells, the PCD process allows for the remobilization of cellular constituents, including the products of DNA hydrolysis. Although programmed cell death occurs widely during normal development and plant defense responses to pathogens, only one class of deoxyribonucleases, the S1 type, involved in these processes, has been well characterized. Using DNA-SDS-PAGE, we identified the activities of 14 deoxyribonucleases expressed in different organs of cauliflower seeds, seedlings and the flower head. These enzymes represent several classes based on their substrate specificity and ion dependency. In addition to four Zn(2+)-dependent enzymes, we identified five Ca(2+)-dependent, two Mg(2+)-dependent, three Ca(2+)/Mg(2+)-dependent and one nuclease whose activities seem to be independent of any divalent cations. We also identified a set of DNases whose expression seems to be common for different organs and different stages of development, as well as a few highly tissue-specific nucleases. Expression of three nucleases was inducible by drought stress and hydrogen peroxide., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Levels of sucrose and raffinose family oligosaccharides (RFOs) (raffinose and stachyose) were determined in beech (Fagus sylvatica L.) seeds during development, maturation, desiccation and storage. An increase in RFOs and a marked decrease in the S:(R+St) ratio (i.e. mass ratio of sucrose to the sum of RFOs) were observed at the time of desiccation tolerance (DT) acquisition by seeds. In seeds stored at -10 degrees C through 1, 4, 7, and 12 years, changes in sucrose, raffinose and stachyose levels and in alpha-galactosidase activity were noted. The S/R+St ratio and alpha-galactosidase activity significantly increased in seeds after 7 and 12 years of storage, when a marked decrease in viability, measured as germination capacity, was recorded. Germination capacity was found to be strongly correlated with sucrose content, the S:(R+St) ratio, and alpha-galactosidase activity. A strong positive correlation was found between germination capacity and stachyose content. The results clearly indicated that the composition of RFOs in beech seeds is closely related to DT acquisition and seed viability during storage.

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

The light- and indole-3-acetic acid (IAA)-regulated 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (PnACS) from Pharbitis nil was isolated. Here, it was shown that the gene was expressed in cotyledons, petioles, hypocotyls, root and shoot apexes both in light- and dark-grown seedlings. The highest expression level of PnACS was found in the roots. IAA applied to the cotyledons of P. nil seedlings caused a clear increase of PnACS messenger accumulation in all the organs examined. In this case, the most IAA-responsive were the hypocotyls. Our studies revealed that the PnACS transcript level in the cotyledons exhibited diurnal oscillations under both long-day (LD) and short-day (SD) conditions. IAA applied at the beginning of inductive darkness caused a dramatic increase in the expression of PnACS, suggesting that the inhibitory effect of IAA on P. nil flowering may result from its stimulatory effect on ethylene production.

Beneficial effects of low-concentration chemical stressors have been investigated previously in different model systems. The symptoms of stimulation are known from earlier studies, but information about the mechanism is at an initial stage. In the present work, the mechanism of stimulation of low-concentration Cd (5 x 10(-8)M) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 10(-7)M) was investigated in barley seedlings. In treated plants, the amount of cytokinins increased in roots and, after being transported to the leaves, they caused stimulation there. To identify the signal transduction pathway(s) involved in the primary stimulation of cytokinin synthesis (and/or activation) in roots, specific phosphatidylinositol-4,5-bisphosphate-inositol-1,4,5-triphosphate/diacylglycerol (PIP(2)-IP(3)/DAG) and mitogen activated protein kinase (MAPK) signaling pathway inhibitors were added to the nutrient solution, and all proved to be effective, eliminating the stimulation by the stressors. Measurements of superoxide dismutase (SOD, EC 1.15.1.1) activity and the amount of malonyldialdehyde (MDA) showed that the increased amount of Cd did not cause oxidative stress in the roots, and no oxidative stress was found in the leaves, where Cd did not even accumulate. DCMU slightly increased the activity of SOD after 1 week in roots, but did not cause lipid peroxidation. In leaves, there was no oxidative stress upon treatment with DCMU. Thus, oxidative stress cannot be responsible for the stimulation with low-concentration stressors, as they changed the activity of SOD differently, while being equally stimulative for the plants.

The seeds of Azadirachta indica were successfully cryopreserved for 12 months with 45% survival following drying to 0.16 g H(2)O g(-1) dry mass (DM). Highest survival (94-96%) was recorded during the first month of cryostorage. Subsequent cryopreservation up to 12 months resulted in decreasing germination. Post-thawing pre-heat treatment enhanced the recovery marginally in seeds cryopreserved from 3 to 12 months. Viability of cryostored seeds was negatively correlated with leachate conductivity and accumulation of thiobarbituric acid reactive substances (TBRS) estimated in cotyledons and axes. Leachate conductivity of imbibed seeds was low during the first month of cryostorage but increased gradually with the duration of cryostorage to a maximum after 12 months. TBRS accumulation was gradual throughout cryostorage. Relatively low amounts of active oxygen species (AOS) detected during the first month of cryostorage were closely associated with very high activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in seeds (cotyledons and axes). Marked accumulation of AOS from 3 to 12 months was associated with decrease in antioxidant enzyme activity.

The relationship between coleoptile elongation and ethanolic fermentation was investigated in rice (Oryza sativa L.) coleoptiles of four cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress; however, the elongation of cvs Yukihikari and Nipponbare was much greater than that of cvs Leulikelash and Asahimochi. The stress did not significantly increase lactate dehydrogenase (LDH) activity or lactate concentration, but increased alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activities, as well as ethanol concentration in the coleoptiles of all cultivars. The elevated ADH and PDC activities and ethanol concentration in cvs Yukihikari and Nipponbare were much greater than those of cvs Leulikelash and Asahimochi, suggesting that ethanolic fermentation is likely more active in cvs Yukihikari and Nipponbare than in cvs Leulikelash and Asahimochi. ATP concentration in cvs Yukihikari and Nipponbare in anoxia was also greater than that in cvs Leulikelash and Asahimochi in anoxia. The ethanol concentration in the coleoptiles was correlated with anoxia tolerance with respect to the ATP concentration and coleoptile elongation. These results suggest that the ability to increase ethanolic fermentation may be one of the determinants in anoxia tolerance of rice coleoptiles.

The role of beta-glucosidase during the phototropic response in maize (Zea mays) coleoptiles was investigated. Unilateral blue light illumination abruptly up-regulated the activity of beta-glucosidase in the illuminated halves, 10 min after the onset of illumination, peaking after 30 min and decreasing thereafter. The level of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which is released from DIMBOA glucoside (DIMBOA-Glc) by beta-glucosidase, and its degradation compound 6-methoxy-benzoxazolinone (MBOA) were elevated within 30 min in the illuminated halves as compare to the shaded halves, prior to the phototropic curvature. Furthermore, beta-glucosidase inhibitor treatment significantly decreased the phototropic curvature and decreased growth suppression in the illuminated sides. These results suggest that blue light induces the activity of beta-glucosidase in the illuminated halves of coleoptiles causing an increase in DIMBOA biosynthesis and the growth inhibition that leads to a phototropic curvature.

Narciclasine (NCS), isolated from mucilage of Narcissus bulb, showed inhibitory effects on growth and plastid development of excised radish cotyledons. NCS (0.1 mumol/L) started to show inhibitory effects on isocitrate lyase and hydroxypyruvate reductase activities after 24 h incubation in light. When NCS concentration was increased to 10 mumol/L, the activities of both enzymes are completely inhibited. From ultrastructural studies, NCS markedly prevented the degradation of protein bodies and lipid bodies, as well as chloroplast formation of excised radish cotyledons. There was only little degradation of protein and lipid bodies, and almost no chloroplast formation in the excised radish cotyledon treated with 1 mumol/L NCS. Therefore, our results provide clear evidence that NCS inhibited the transition of glyoxysomes and peroxisomes, and chloroplast development.

Plants have a variety of glycosidic conjugates of hormones, defense compounds, and other molecules that are hydrolyzed by beta-glucosidases (beta-D-glucoside glucohydrolases, E.C. 3.2.1.21). Workers have reported several beta-glucosidases from maize (Zea mays L.; Poaceae), but have localized them mostly by indirect means. We have purified and partly characterized a 58-Ku beta-glucosidase from maize, which we conclude from a partial sequence analysis, from kinetic data, and from its localization is not identical to any of those already reported. A monoclonal antibody, mWP 19, binds this enzyme, and localizes it in the cell walls of maize coleoptiles. An earlier report showed that mWP19 inhibits peroxidase activity in crude cell wall extracts and can immunoprecipitate peroxidase activity from these extracts, yet purified preparations of the 58 Ku protein had little or no peroxidase activity. The level of sequence similarity between beta-glucosidases and peroxidases makes it unlikely that these enzymes share epitopes in common. Contrary to a previous conclusion, these results suggest that the enzyme recognized by mWP19 is not a peroxidase, but there is a wall peroxidase closely associated with the 58 Ku beta-glucosidase in crude preparations. Other workers also have co-purified distinct proteins with beta-glucosidases. We found no significant charge in the level of immunodetectable beta-glucosidase in mesocotyls or coleoptiles that precedes the red light-induced changes in the growth rate of these tissues.