Cadmium translocation and accumulation in developing barley grains

Soil cadmium (Cd) contamination has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide. In order to study the transport of Cd into the developing grains, detached ears of two-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing the markers for phloem (rubidium) and xylem (strontium) transport. Cd concentration in each part of detached spikes increased with external Cd levels, and Cd concentration in various organs over the three Cd levels of 0.5, 2, 8 μM Cd on 15-day Cd exposure was in the order: awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grains with the proportion of 51.0% relative to the total Cd amount in the five parts of detached spikes. Cd accumulation in grains increased not only with external Cd levels but the time of exposure contrast to stem, awn, rachis and glume. Those four parts of detached spike showed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 and increase again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cd concentration in grains, and sucrose or zinc (Zn) addition to the medium and higher relative humidity (RH) also induced dramatic reduction in Cd transport to developing grains. The results indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as an important physiological process influencing the quality of harvested grains. Our results suggested that increasing RH to 90% and Zn addition in the medium at grain filling stage would be beneficial to decrease Cd accumulation in grains.     

Auxin-, gibberellin-like substances and cytokinins in the seed and root exudates of groundnut

Seed and root exudates were collected aseptically and analysed for auxins, gibberellins and cytokinins using paper, thin-layer chromatography, bioassay and gas-chromatographic techniques.Gas-chromatographic analysis revealed the presence of IAA and GA₃ in seed exudates only. Two cytokinins were found in seed and one in root exudates, respectively.     

In situ and in vitro ruminal starch degradation of grains from different rye,triticale and barley genotypes

In recent years, advances in plant breeding were achieved, which potentially led to modified nutritional values of cereal grains. The present study was conducted in order to obtain a broad overview of ruminal digestion kinetics of rye, triticale and barley grains, and to highlight differences between the grain species. In total, 20 genotypes of each grain species were investigated using in situ and in vitro methods. Samples were ground (2 mm), weighed into polyester bags, and incubated in situ 1 to 48 h in three ruminally cannulated lactating dairy cows. The in vitro gas production of ground samples (1 mm) was measured according to the ‘Hohenheim Gas Test’, and cumulative gas production was recorded over different time spans for up to 72 h. There were significant differences (P<0.05) between the species for most parameters used to describe the in situ degradation of starch (ST) and dry matter (DM). The in situ degradation rate (c) and effective degradability (assuming a passage rate of 8%/h; ED8) of ST differed significantly between all grains and was highest for rye (rye: 116.5%/h and 96.2%; triticale: 85.1%/h and 95.0%; barley: 36.2%/h and 90.0% for c and ED₈, respectively). With respect to DM degradation, the ranking of the species was similar, and predicted c values exhibited the highest variation within species. The in vitro gas production rate was significantly higher (P<0.05) for rye than for triticale and barley (rye: 12.5%/h; triticale: 11.5%/h; barley: 11.1%/h). A positive relationship between the potential gas production in vitro and the maximal degradable DM fraction in situ was found using all samples (r=0.84; P<0.001) as well as rye (P=0.002) and barley (P<0.001) alone, but not for triticale. Variation in ruminal in situ degradation parameters within the grain species resulted from the high c values, but was not reflected in the ED estimates. Therefore, the usage of mean values for the ED of DM and ST for each species appears reasonable. Estimated metabolisable energy concentrations (ME, MJ/kg DM) and the estimated digestibility of organic matter (dOM, %) were significantly lower (P<0.05) for barley than for rye and triticale. Rye and triticale dOM and ME values were not significantly different (P=0.386 and 0.485).     

Abscisic acid in developing grains of wheat and barley genotypes differing in grain weight

Two genetically related wheat lines growing in cabinets were given different temperatures during grain filling, and abscisic acid (ABA) was measured in whole grains by gas chromatography with an electron-capture detector. Three genetically related barley lines grown in the field were assayed for ABA content in endosperm and embryo fractions separately by radiommunoassay.Maximum grain growth rate and final weight per grain of the two wheat lines differed by 50–60% at low temperature and 30–40% at high temperature. During grain development two peaks in ABA level were observed at low temperature but only one at high temperature. At times when differences in grain growth rate between genotypes and between temperature treatments were large, the corresponding differences in ABA concentration were small. In barley, one line (Iabo 14) had 30% heavier grains than the other two (Onice and Opale). Endosperm ABA concentrations showed no clear differences between genotypes until grain filling was nearly complete. Embryo ABA levels were up to 10-times greater than those in the endosperm, with Opale having significantly less ABA in the embryo than the other two cultivars.Our experiments did not provide evidence for a causal relationship between ABA levels during grain filling and grain growth rate or final weight.Abbreviations ABA Abscisic acid - DAA days after anthesis - DW dry weight - FW fresh weight     

Enzymes of carbohydrate metabolism in developing grains of high lysine barley mutant

Activities of UDP(ADP)-sucrose synthetase, hexokinase, phosphoglucoisomerase and phosphoglucomutase have been studied in both a high lysine mutant barley, Notch-2 and its parent NP 113 during development. The Notch-2 mutant had higher average activities of UDP(ADP)-sucrose synthetase, hexokinase and phosphoglucomutase and lower activity on a grain basis of phosphoglucoisomerase than NP 113. This reflected the decreased dry matter in the mutant grain. In general, the average activities of hexokinase and phosphoglucomutase per grain did not differ significantly between Notch-2 and NP 113. It is suggested that the lower level of phosphoglucoisomerase in Notch-2 compared with NP 113 would limit the synthesis of glucose 6-phosphate, which in turn would result in reduced starch synthesis.     

Effects and interactions of gibberellic acid and cytokinins on the retention of chlorophyll and phosphate in barley leaf segments

In leaf segments of barley ( Hordeum vulgare L. cv. Mozoncillo), the cytokinin specificity for retarding the loss of phosphate is different from that of retarding the loss of chlorophyll. Some cytokinins require the simultaneous application of gibberellins to delay the loss of phosphate. Although both chlorophyll and phosphate losses occur in senescence, they are apparently controlled by cytokinins through different mechanisms.     

A proteomic analysis of 14-3-3 binding proteins from developing barley grains

14-3-3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare L.) 14-3-3A was over-expressed, immobilised and used to affinity purify 14-3-3 binding proteins from developing barley grains. Binding was shown to be phosphorylation-dependent. These proteins were fractionated by PAGE and identified by MALDI-TOF MS. In total, 54 14-3-3 binding proteins were identified, 49 of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14-3-3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co-ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14-3-3 in a dosage-dependent manner. The second-largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14-3-3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence.     

Differential callus type formation by auxins and cytokinin in in vitro cultures of pepper (Capsicum annuum L.)

ABSTRACT

Two types of callus were produced by pepper explants cultured in various media containing auxins, the cytokinin 6-benzylaminopurine (BAP) and the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA). Callus produced on media containing auxins alone was friable, grey-green or green-orange in colour and more compact, whereas when BAP was added to culture media with a low concentration of auxin or when the medium contained TIBA alone, the callus produced was white and very hard. This type of callus was also produced in cultures of older tissues and of young tissues cultured on hormonefree medium. Results are discussed in relation to the role of cytokinins in wounding, phenylpropanoid metabolism and lignin biosynthesis.     

The effect of auxins, time exposure to auxin and genotypes on somatic embryogenesis from mature embryos of wheat

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98–100%), Dicamba was more effective for the induction of embryogenic callus (21.8–38.3%). Maximum embryogenic callus formation and high number of regenerated plants were observed at 12 mg l⁻¹ of Dicamba. The time exposure to Dicamba (7, 14, 21 and 28 days) had a significant effect on efficiency of somatic embryogenesis. When contact of explants with callus induction medium was increased from 7 to 21 days the rate of somatic embryogenesis and number of regenerated plants per embryogenic callus gradually increased from 13.0 to 38.4% and 3.6 to 8.0%, respectively. Supplement of additional auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA)) to callus induction medium with Dicamba had a positive effect on the rate of embryogenic callus formation, while the average number of regenerated shoots was not affected. The best rate of somatic embryogenesis was observed at the addition of 0.5 mg l⁻¹ IAA with Dicamba (61.0%). The optimum combination of Dicamba and IAA increased the efficiency of somatic embryogenesis and plant regeneration from seven spring and winter wheat genotypes, thought overall morphogenic capacity was still genotype dependent.     

Inhibitors of chymotrypsin and microbial serine proteases in barley grains

Barley grains contain two imrnunochemically distinct inhibitors of chymotrypsin and microbial serine proteases. Both inhibitors are rich in lysine (9.5 and 11.5 g Lys/g protein). Hiproly high-lysine barley contains twenty-fold higher, high-lysine mutant 1508 five-fold higher amounts of these inhibitors than normally cultivated varieties. Inhibitors were extracted from Hiproly barley, and ammonium sulfate fractionation followed by gel filtration resulted in a neariy complete separation of the two inhibitors. No inactive protein impurities could be detected in a number of isoinhibitor preparations obtained in subsequent cation exchange chrotnatography steps. One inhibitor (CI-1) was composed of at leas# 4 molecular forms with isoelecfric points in the range 4.75–5.55 and a monomer molecular size of about 9 000. Most of this inhibitor was apparently present as dimer forms in grain extracts. The other inhibitor (CI-2) included at least 7 different molecular forms with isoelectric points in the range 6.05–7.90 and different molecular sizes in the range 6 500–9 000. Both dimer and monomer forms were present in grain extracts. In contrast to previously purified protease inhibitors of plant origin, the two barley inhibitors contain no cysteine. No interactions between the two inhibitors and trypsin were observed, but the inhibitors were immediately inactivated by pepsin at pH 2.0. Monospecific antibodies towards the two inhibitors were obtained after immunization with glutaraldehyde-polymerized inhibitor.
Inhibitor CI-1 is identical with an inhibitor of microbial alkaline proteases previously purified (Mikola and Suolinna 1971. Arch. Biochem. Biophys. 144: 566–575).     

A novel late embryogenesis abundant protein and peroxidase associated with black point in barley grains

Black point of barley grain is a disorder characterised by a brown-black discolouration at the embryo end of the grain. Black point is undesirable to the malting industry and results in significant economic loss annually. To identify proteins associated with barley black point we utilised a proteomic approach with 2-DE to compare proteins from whole grain samples of black pointed and healthy grain. From this comparison two condition-specific proteins were identified: a novel 75 kDa late embryogenesis abundant (LEA) protein and a barley grain peroxidase 1 (BP1) that were specifically more abundant in healthy grain and black pointed grain, respectively. Although LEA protein was less abundant in black pointed grain, LEA gene expression was greater suggesting protein degradation had possibly occurred in black pointed grain. Similarly, the increase in BP1 in black pointed grain could not be explained by gene expression. Western blot analysis also revealed that the identified LEA protein is biotinylated in vivo. The role that each of these proteins might have in black point development is discussed.     

Tissue-specific localization of aspartic proteinase in developing and germinating barley grains

Resting seeds of several plant species, including barley grains, have been reported to contain aspartic proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. aspartic proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.Abbreviations DAA days after anthesis - DTT dithiothreitol - HvAP Hordeum vulgare aspartic proteinase Both authors have contributed equally to this workWe thank Mart Saarma, Pia Runeberg-Roos, Alan Schulman and Yrjö Helariutta for helpful discussions during the study, Tiina Arna and Sari Makkonen for their help in proteinase activity experiments as well as Jaana Korhonen (Department of Pathology, University of Helsinki), Salla Marttila and Ilkka Porali (Department of Biology, University of Jyväskylä, Jyväskylä, Finland) for their advice on microscopical techniques. We also thank Liisa Pyhälä and Leena Liesirova for the production of the antibodies to HvAP at the National Public Health Institute, Helsinki. This study was supported by grants from the Ministry of Agriculture and Forestry and the Academy of Finland.     

Antioxidant defense system and cadmium uptake in barley genotypes differing in cadmium tolerance

Using two barley (Hordeum vulgare) cultivars (cvs. Tokak and Hamidiye) nutrient solution experiments were conducted in order to study the genotypic variation in tolerance to Cd toxicity based on (i) development of leaf symptoms, (ii) decreases in dry matter production, (iii) Cd concentration and (iv) changes in antioxidative defense system in leaves (i.e., superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, ascorbic acid and non-protein SH-groups). Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 15, 30, 60 and 120 micromol/L Cd) for different time periods. Of the barley cultivars Hamidiye was particularly sensitive to Cd as judged by the severity and earlier development of Cd toxicity symptoms on leaves. Within 48 h of Cd application Hamidiye rapidly developed severe leaf Cd toxicity symptoms whereas in Tokak the leaf symptoms appeared only slightly. Hamidiye also tended to show more decrease in growth caused by Cd supply when compared to Tokak. The differences in sensitivity to Cd between Tokak and Hamidiye were not related to Cd concentrations in roots and shoots or Cd accumulation per plant. With the exception of catalase, activities of the enzymes involved in detoxification of reactive oxygen species (ROS) were markedly enhanced in Hamidiye by increasing Cd supply. By contrast, in Tokak there was either only a slight increase or no change in the activities of the antioxidative enzymes. Similarly, levels of ascorbic acid and especially non-protein SH-groups were increased in Hamidiye by Cd supply, but not affected in Tokak. The results indicate the existence of a large genotypic variation between barley cultivars for Cd tolerance. The differential Cd tolerance found in the barley cultivars was not related to uptake or accumulation of Cd in plants, indicating importance of internal mechanisms in expression of differential Cd tolerance in barley. As a response to increasing Cd supply particular increases in antioxidative mechanisms in the Cd-sensitive barley cultivar Hamidiye suggest that the high Cd sensitivity of Hamidiye is related to enhanced production and oxidative damage of ROS.     

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts.     

Variation in chemical composition and physical characteristics of cereal grains from different genotypes

Genotypes of cereal grains, including winter barley (n = 21), maize (n = 27), oats (n = 14), winter rye (n = 22), winter triticale (n = 21) and winter wheat (n = 29), were assayed for their chemical composition and physical characteristics as part of the collaborative research project referred to as GrainUp. Genotypes of one grain species were grown on the same site, except maize. In general, concentrations of proximate nutrients were not largely different from feed tables. The coefficient of variation (CV) for the ether extract concentration of maize was high because the data pool comprised speciality maize bred for its high oil content. A subset of 8 barley, 20 rye, 20 triticale and 20 wheat samples was analysed to differ significantly in several carbohydrate fractions. Gross energy concentration of cereal grains could be predicted from proximate nutrient concentration with good accuracy. The mean lysine concentration of protein was the highest in oats (4.2 g/16 g N) and the lowest in wheat (2.7 g/16 g N). Significant differences were also detected in the concentrations of macro elements as well as iron, manganese, zinc and copper. Concentrations of arsenic, cadmium and lead were below the limit of detection. The concentration of lower inositol phosphates was low, but some inositol pentaphosphates were detected in all grains. In barley, relatively high inositol tetraphosphate concentration also was found. Intrinsic phytase activity was the highest in rye, followed by triticale, wheat, barley and maize, and it was not detectable in oats. Substantial differences were seen in the thousand seed weight, test weight, falling number and extract viscoelasticity characteristics. The study is a comprehensive overview of the composition of different cereal grain genotypes when grown on the same location. The relevance of the variation in composition for digestibility in different animal species will be subject of other communications.     

Influence of waterlogging on some anti-oxidative enzymatic activities of two barley genotypes differing in anoxia tolerance

The experiment was conducted to investigate the formation of oxidative stress and the development of anti-oxidative enzymes in two barley genotypes differing in anoxia tolerance. Waterlogging led to significant reduction in root and shoot weight, green leaf area and tillers per plant, but tolerant Xiumai 3 was much less reduced than sensitive Gerdner. Malondialdehyde (MDA) content, an indicator of membrane lipid peroxidation, significantly increased in Gerdner when the plants were subjected to waterlogging, but remained little changed in Xiumai 3. Superoxide dismutase (SOD) activity was increased with waterlogging treatment and the sensitive cultivar had higher activity than the tolerant one during the experimental duration. At early stage of waterlogging treatment, both peroxidase (POD) and catalase (CAT) activities significantly increased in Xuimai 3, while obviously decreased in Gerdner. Moreover, both cultivars showed substantial increase in both POD and CAT with the progress of waterlogging exposure. Glutathione reductase (GR) activity was increased in both tolerant- and sensitive cultivars under waterlogging. It may be assumed from the current results that SOD activity appears to be not a constraining factor limiting the scavenging of ROS, and it is the change of POD and CAT activity under waterlogging that determine the status of oxidative stress. The difference between genotypes in waterlogging tolerance could be distinguished from the changed patterns of these enzymatic activities.     

Vascular transport of auxins and cytokinins in Ricinus

Analyses of vascular saps supplying source and sink organs havedemonstrated the presence of major endogenous hormones and/or theirprecursors. Indol-3yl-acetic acid, a number of gibberellins, cytokininsand abscisic acid, as well as the precursor for ethylene production havebeen found in these vascular saps, allowing the sites of hormonalsynthesis and putative target tissues to be deduced. Exogenously appliedhormones are also readily loaded into these vascular pathways and may betranslocated over considerable distances from a point of application.Observations such as these indicate a possible co-ordination systembetween source and sink regulated by the synthesis and transport ofendogenous hormones. It is widely accepted that the partitioning ofassimilates between photosynthetic source organs and utilising sinkorgans is regulated by endogenous plant hormones. The key intermediatesteps involved in assimilate transport, such as phloem loading andunloading, have been shown to be responsive to applied hormones,although the role of endogenous hormones in these processes remainsessentially unresolved. Results of the analyses of vascular saps fromRicinus communis, which have been obtained using a range ofphysicochemical methods, are compared and contrasted with those obtainedby the application of exogenous hormones or their precursors. Theseresults are evaluated critically and interpreted in the light of currentmodels of source:sink regulatory processes and the long-distancetransport of auxins and cytokinins in higher plants.     

Enzymes of starch metabolism in developing grains of high lysine barley mutant

The absolute activities of ADPG(UDPG)-pyrophosphorylase, starch phosphorylase, ADPG(UDPG)-starch synthetase, NDP-kinase and inorganic pyrophosphatase have been studied in high lysine mutant barley Notch-2 and its parent NP 113 grains during development. In general, mutant Notch-2 grains had higher average activities of UDPG-pyrophosphorylase and starch phosphorylase and lower activity of ADPG(UDPG)-starch synthetase per grain than the parent NP 113 during grain development. Activities of NDP-kinase, ADPG-pyrophosphorylase and inorganic pyrophosphatase differed only to a small extent between the mutant Notch-2 and NP 113. It is suggested that the lower activity of ADPG(UDPG)-starch synthetase might be responsible for the reduced accumulation of starch in the mutant Notch-2 grain as compared with parent NP 113 during development.     

An analysis of soluble starch synthase isozymes from the developing grains of normal and shx cv. Bomi barley (Hordeum vulgare)

Soluble starch synthase (SSS, EC 2.4.1.21) catalyzes formation of the α-1,4 bonds of amylopectin. It occurs in multiple isozymes which are either type I, primer-independent in the presence of citrate, or type II. always primer-dependent. To analyze the enzyme. a sensitive native gel assay was developed, monitoring ADP-[¹⁴C]glucose incorporation into insoluble α-glucan in the presence of either sodium citrate or glycogen primer or both. Using this system, we observed multiple type I and type II forms in developing grains of barley ( Hordeum vulgare L.) cv. Bomi, the relative activities of which vary with seed development. At least one form comigrates in native gels with starch branching enzyme. Assays of the shx mutant, which is severely reduced in starch accumulation and in type I SSS activity, indicate that one type I isozyme becomes primer-dependent.