Jasmonic acid promotes germination and lipase activity in non‐stratified apple embryos

The effects of (−)jasmonic acid (JA) on germination of embryos isolated from dormant seeds of apple ( Malus domestica Borb. cv. Antonówka) cultured in darkness or at 12‐h photoperiod were studied, as well as its effects on the activity of alkaline lipase (AlkL, EC 3.1.1.3) in these embryos. The maximum sensitivity of germination to JA occurred on days 3 and 4 of embryo culture. Both germination and enzyme activity were stimulated by JA, its effect being additive to that of light. Inhibitors of lipoxygenase inhibited embryo germination and AlkL activity, both effects being partially reversed by JA treatment. We suggest that (1) JA is implicated in an endogenous complex controlling apple seed germination, and that (2) it acts independently of the mechanism triggered by light.     

Interaction of jasmonic acid with some plant growth regulators in the control of apple (Malus domestica) embryo germination

Embryos isolated from dormant apple seeds were treated with jasmonic acid (JA), gibberellin A₃ (GA₃), abscisic acid (ABA) and hydrogen cyanide in darkness and in light. The chemicals were present in the culture medium continuously and simultaneously or applied for 2 days and in different sequences. All treatments stimulated embryo germination except ABA, which was strongly inhibitory. Additive effects of JA with light and with GA₃ on embryo germination were observed, whereas ABA interacted synergically with JA, HCN and light. ABA and GA₃ were most effective when applied early during embryo incubation, but the late JA treatment was more stimulatory. It is concluded that JA does not act on the regulatory pathway that is initiated by light and which leads to embryo germination through gibberellin accumulation and alkaline lipase activation. ABA and HCN appear to be involved in the control of this pathway. JA and ABA may be involved in the control of alkaline lipase activity, independently of this regulatory chain.Abbreviations ABA abscisic acid - GA₃ gibberellin A₃ - JA jasmonic acid     

Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

Increases in jasmonic acid caused by indole-3-acetic acid and auxin herbicides in cleavers (Galium aparine)

The effects of indole-3-acetic acid and auxin herbicides on endogenous jasmonic acid (JA) concentrations were studied in relation to changes in ethylene and abscisic acid (ABA) levels in cleavers (Galium aparine). When plants were root-treated with increasing concentrations of indole-3-acetic acid (IAA), ethylene biosynthesis was stimulated in response to the accumulation of endogenous IAA in the shoot tissue. Within 25h of treatment, stimulated ethylene formation was accompanied by increases in immunoreactive concentrations of JA and ABA, which reached maxima of 4.5-fold and 26-fold of the control, respectively, at 100 microM of applied IAA. Corresponding effects were obtained using synthetic auxins and when the ethylene-releasing compound ethephon was applied exogenously. This represents the first report, to our knowledge, of an auxin-mediated increase in JA levels. The increase in JA may be triggered by ethylene.     

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.     

Cyanide controls enzymes involved in lipid and sugar catabolism in dormant apple embryos during culture

The activities of alkaline lipase (EC 3.1.1.3, AlkL), isocitrate lyase (EC 4.1.3.1. ICL), pyruvate kinase (EC 2.7.1.40. PK) and glucose–6–phosphate dehydrogenase (EC 1.1.1.49, G6PDH) were determined in cultured, dormant embryos of apple (Malus domestica Borb. cv. Antonówka), pretreated with gaseous HCN. The C₆/C₁ , ratio was estimated in the same material. The activities of AlkL and ICL were not stimulated by HCN pretreatment until the period of maximum stimulation of germination. The activity of G6PDH was inhibited by cyanide only late during the culture of embryos. Therefore, the changes in these activities are considered to be the result and not the cause of enhanced germination. On the other hand, also PK, active very early during the culture of embryos, was modified as a result of the treatment. The cyanide-induced changes in activity of this enzyme in cotyledons (inhibition followed by stimulation) were similar to those in the whole embryo, whereas its changes in the embryonal axis (stimulation followed by inhibition) resembled CN-induced changes in PK in axes of apple seeds submitted to cold stratification (Bogatek and Lewak 1988). The estimation of C₆/C₁ ratios partly confirmed these observations. A role of HCN-induced modifications of PK activity in embryonal dormancy is proposed.     

Influence of jasmonic acid as potential activator of induced resistance against Karnal bunt in developing spikes of wheat

Induction of defense response against Karnal bunt (KB) by suppressing the pathogenesis was observed upon exogenous application of jasmonic acid (JA) as evident from decrease in the coefficient of infection and overall response value in both susceptible and resistant varieties of wheat. The ultra-structural changes during disease progression showed the signs of programmed cell death (PCD). However, JA strengthened the defense barrier by enhancing the lignifications of cell walls as observed in spikes of both varieties by histochemical analysis. Compared to the plants inoculated with pathogen alone, plants of resistant line (RJP) first treated with JA followed by inoculation with pathogen showed more lignifications and extracellular deposition of other metabolites on cells, which is supposed to prevent mycelial invasions. Contrary to this, susceptible (SJP) lines also showed lignifications but the invasion was more compared to resistant line. Induction of protease activity was higher in resistant variety than its corresponding susceptible variety. The protease activity induced during the colonization of the pathogen and its proliferation inside the host system gets inhibited by JA treatment as demonstrated by the quantitative and in-gel protease assay. The results indicate the role of JA signalling in inhibiting the proteases due to expression of certain protease inhibitor genes. SDS-PAGE analysis shows differential gene expression through induction and/or suppression of different proteins in wheat spikes of resistant and susceptible varieties under the influence of JA. Thus, exogenously applied JA provides the conditioning effect prior to the challenge of infection and induces defense against KB probably by maintaining a critical balance between proteases and protease inhibitors and/or coordinating induction of different families of new proteins.     

Jasmonic acid affects dormancy and sugar catabolism in germinating apple embryos

Embryos isolated from dormant seeds of apple (Malus domestica Borb., cv. Antonówka) were cultured in darkness in the presence of jasmonic acid (JA, 20 μM) for 7 d in parallel to control non-treated ones. Soluble sugars were quantified and some sugar-catabolysing enzyme activities were determined in axes and in cotyledons of the embryos during the culture. JA treatment stimulated growth of the axis and sucrose hydrolysis in this organ. In contrast, JA inhibited the growth of isolated cotyledons. In intact embryos, JA treatment inhibited the growth of the lower cotyledon (being in contact with wet medium) thus alleviating the growth asymmetry of cotyledons. In both cotyledons JA stimulated hydrolysis of sucrose during the period preceding growth. The effect persisted in the upper cotyledon for the whole experimental period, whereas in the lower one the treatment provoked a sharp rise in soluble sugar levels observed relatively late during the experiment. The later effect correlated with the stimulation of isocitrate lyase activity in the lower cotyledon by JA. The results suggest the induction of the gluconeogenetic pathway by JA in the lower cotyledon of cultured dormant apple embryos. They also provide evidence for the site of JA action in the regulatory complex controlling embryonic dormancy in apple.     

Stomatal responses to jasmonic acid, linolenic acid and abscisic acid in wild-type and ABA-deficient tomato plants

Wild-type and abscisic acid (ABA) -deficient (sitiens) tomato plants were used to analyse the effects of abscisic acid (ABA), butyric acid (BA), jasmonic acid (JA) and linolenic acid (LA) on assimilation and transpiration rates in detached leaves taking up those substances into the transpiration stream. BA did not affect assimilation and transpiration rates. ABA decreased assimilation and transpiration in both wild-type and ABA-deficient mutants. JA reduced the assimilation rate in both lines but induced a significant reduction of transpiration in the wild type only. The response to LA in both lines was slower than that to JA.     

The embryonic axis controls lipid catabolism in cotyledons of apple seeds during germination

Activities of alkaline lipase (AlkL, EC 3.1.1.3), isocitrate lyase (ICL, EC 4.1.3.1), glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and pyruvate kinase (PK, EC 2.7.1.40) were determined in embryos of apple ( Malus domestica Borb. cv. Antonówka) during culture in darkness or at 12 h photoperiod; in both cases either in the presence of gibberellin A₃ (GA₃) or AMO 1618 (inhibitor of GA synthesis). AlkL and ICL were stimulated by light and GA₃; light stimulation was reversed by AMO. G6PDH and PK were not affected by culture conditions. Almost all the activity of all enzymes was found in the cotyledons; only PK was distributed between axis and cotyledons. GA-like activity was found almost exclusively in the embryo axis. Cultured isolated cotyledons lost their sensitivity to light and AMO, but AlkL and ICL were still stimulated by GA₃. Translocation of GA from axis to cotyledons during the culture of embryos is postulated.     

Genome‐wide association study reveals novel players in defense hormone crosstalk in Arabidopsis

Jasmonic acid (JA) regulates plant defenses against necrotrophic pathogens and insect herbivores. Salicylic acid (SA) and abscisic acid (ABA) can antagonize JA‐regulated defenses, thereby modulating pathogen or insect resistance. We performed a genome‐wide association (GWA) study on natural genetic variation in Arabidopsis thaliana for the effect of SA and ABA on the JA pathway. We treated 349 Arabidopsis accessions with methyl JA (MeJA), or a combination of MeJA and either SA or ABA, after which expression of the JA‐responsive marker gene PLANT DEFENSIN1.2 (PDF1.2) was quantified as a readout for GWA analysis. Both hormones antagonized MeJA‐induced PDF1.2 in the majority of the accessions but with a large variation in magnitude. GWA mapping of the SA‐ and ABA‐affected PDF1.2 expression data revealed loci associated with crosstalk. GLYI4 (encoding a glyoxalase) and ARR11 (encoding an Arabidopsis response regulator involved in cytokinin signalling) were confirmed by T‐DNA insertion mutant analysis to affect SA–JA crosstalk and resistance against the necrotroph Botrytis cinerea. In addition, At1g16310 (encoding a cation efflux family protein) was confirmed to affect ABA–JA crosstalk and susceptibility to Mamestra brassicae herbivory. Collectively, this GWA study identified novel players in JA hormone crosstalk with potential roles in the regulation of pathogen or insect resistance.     

Roles of jasmonic acid in the development of sweet cherries as measured from fruit or disc samples

The effect of jasmonic acid (JA) on callus formation was investigated ondiscs taken from the pulp of sweet cherry fruit (Prunusavium L.). The discs were sampled at 16 days after full bloom(DAFB),22 DAFB, and 29 DAFB and cultured on B5 medium involving different combinationsof 1-naphthaleneacetic acid (NAA), N⁶-benzyl adenine (BA), and JA.Only at 16 DAFB, 1.0 M JA concentration increased callusweightgain relative to discs incubated without hormonal additives, although JAinhibited, or had no effect on callus formation, at 22 and 29 DAFB. The weightof the callus, which was subcultured, was also increased by 0.45–1.0M JA, without hormonal additives. Although the number of cellsincreased until 15 DAFB, after this time it did not change. These resultsdemonstrate that endogenous JA may be related to cell division in sweet cherryfruit. The interactions between JA and abscisic acid (ABA) were alsoinvestigated. Discs from pulp at 20 DAFB (immaturity), 32 DAFB (beforematuration), and 48 DAFB (maturation) were placed in petri dishes containing 10ml 0.4 M mannitol with JA or ABA. In addition, at 48DAFB, JA or ABA solutions had been absorbed by the fruit for 7 days via theshoot. ABA treatment did not influence endogenous JA concentrations in discs,with few exceptions. Although the ABA concentration in the fruit increased to2.2 times that of the control by ABA the 7 day treatment, endogenous JA failedto increase. Thus, ABA may not influence the JA pathway in sweet cherry fruit.Although the increase of endogenous ABA was observed in discs at earlier timesafter JA treatment, ABA concentration decreased in the fruit treated for 7 dayswith JA. This implies that the concentration of JA may influence ABA levels. JAtreatment did not influence anthocyanin accumulation, in spite of the increaseof JA in the fruit by the treatment. JA may not play a role in anthocyaninaccumulation in sweet cherry fruit.     

Cadmium stress affects seed germination and seedling growth in <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench by changing the activities of hydrolyzing enzymes

Seed germination, one of the most important phases in the life cycle of a plant, is highly responsive to existing environment. Hydrolyzing enzymes play a major role in the mobilization of food reserves by hydrolyzing carbohydrates, proteins and fats. This paper reports on the effect of Cd toxicity on seed germination and the activities of hydrolyzing enzymes, like acid phosphatases (ACPs), proteases and α-amylases in Sorghum bicolor (L.) Moench. The metal uptake by embryonic axes and seeds was quantified. We found that sorghum could tolerate up to 0.5 mM Cd. At concentrations above 3.0 mM, seed germination was adversely affected with a complete cessation of seedling growth. All investigated hydrolyzing enzymes exhibited a significant decrease in activity with increasing Cd concentrations. The isozyme profiles indicated the loss of one or two isozymes of ACP, induction of a new isozyme for total protease (at 3.0 mM Cd) and a decline in the intensity of α-amylase isozymes. SEM studies revealed that Cd affected a change in root hair density. SEM investigations also confirmed the assay results of the inhibition of starch mobilization from endosperm. This suggested an inhibition of the hydrolysis of reserve carbohydrates and translocation of hydrolyzed sugars, ultimately resulting in decreased germination and disruption of seedling growth. Because sorghum is an important dryland crop, its response to the presence of Cd in agro-ecosystems and Cd-induced phytotoxicity during seed germination and seedling growth needs critical investigation.     

Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4 . Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling.     

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.     

Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage.     

Desiccation tolerance in developing soybean seeds: The role of stress proteins

The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of certain "late embryogenesis abundant" (LEA) proteins suggests that these proteins reduce desiccation-induced cellular damage. The aim of the present work was to test this hypothesis. Exogenous abscisic acid (ABA) was used to elevate the level of heal-soluble LEA-like proteins in axes from immature (30 days after flowering: mid-development) seeds of soybean ( Glycine max [L.] Merrill cv. Chippewa 64). As the LEA-like proteins accumulated in response to ABA, the leakage of all elements after desiccation and subsequent rehydration markedly declined. Both LEA-like protein accumulation and the decline in desiccation-induced electrolyte leakage were apparently dependent on the presence of ABA. Both effects of ABA were inhibited by cycloheximide. Light microscopy revealed a marked effect of the ABA on cellular integrity following desiccation. Osmotic stress also caused a decrease in desiccation-induced electrolyte leakage and stimulated the accumulation of LEA-like proteins. Our data are consistent with the hypothesis that the LEA-like proteins contribute to the increase in desiccation tolerance in response to ABA, and are consistent with a general protective role for these proteins in desiccation tolerance.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of low-temperature stress on abscisic acid,jasmonates, and polyamines in apples

The effect of low temperatures on polyamines, jasmonates, abscisic acid (ABA), and antioxidant activities was investigated in apple fruitlets. Although endogenous ABA concentrations were not significantly different between untreated control fruit kept at −2°C and those kept at 20°C, endogenous jasmonic acid (JA), putrescine, and spermidin concentrations at −2°C were generally higher than those at 20°C. Endogenous ABA concentrations increased in n-propyl dihydrojasmonate (PDJ)—or spermine-treated fruit in comparison to the untreated control at 20 and −2°C. The applications of PDJ or spermine decreased low-temperature injuries such as splitting and spotting in fruit. Although the IC₅₀ of 1,1-diphenil-2-pycrylhydrazyl (DPPH)-radical scavenging activities was not significantly different among the treatments, the IC₅₀ of O₂ ⁻-scavenging activities in PDJ-treated or Spm-treated fruit at 5 days after the low-temperature treatment was lower than in the untreated control at 20 and −2°C. The expression of MdCHS increased in Spm-treated fruit. The concentrations of ascorbic acid, catechin, chlorogenic acid, epi-catechin, and phloridzin in Spm-treated fruit were higher than in the untreated control at −2 or 20°C. These facts suggest that ABA, jasmonates and polyamines may be associated with low-temperature stress tolerance in apple fruitlets.     

中华真地鳖中肠主要消化酶的活性研究

以中华真地鳖EupolyphagasinensisWalker为研究材料,测定人工饲养和野生地鳖虫在不同生长阶段消化酶的活性以及温度及pH对人工饲养地鳖虫中肠消化酶活性的影响。结果表明,在地鳖虫生长发育过程中,蛋白酶和脂肪酶活性随发育而逐渐增强,淀粉酶活性却随发育而逐渐减弱。在低龄若虫、高龄若虫和成虫阶段,人工饲养地鳖虫蛋白酶活力比野生地鳖虫低,人工饲养地鳖虫淀粉酶和脂肪酶活力比野生地鳖虫高;在30～60℃的范围内,人工饲养地鳖虫蛋白酶、淀粉酶的适宜温度范围为40～50℃,脂肪酶的适宜温度范围为35～45℃;蛋白酶、淀粉酶和脂肪酶的适宜pH范围分别为6.5～7.5,5.6～6.4和7.5～8.5。