De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin.     

Preparation and properties of mitochondria from cowpea nodules

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O₂ uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD⁺-malic enzyme which was 12-fold lower in the mitochondria from nodules.     

Four isoforms of glutamine synthetase in light-grown soybeans

Glutamine synthetase (GS) activity recovered from linear sucrose gradients was associated with the cytosol of cells isolated from etiolated soybean hypocotyls whereas light-grown tissue contained increased GS activity localized in both the cytosol and chloroplasts. DEAE-cellulose chromatography indicated two GS isoforms in etiolated hypocotyls whereas light-grown hypocotyls and primary leaves contained four isoforms. Only one GS isoform was recovered from both etiolated and light-grown cotyledons.     

Antioxidant enzymes and isoflavonoids in chilled soybean (Glycine max (L.) Merr.) seedlings

Changes of activity antioxidant enzymes and of levels of isoflavonoids were studied in the roots and hypocotyls of the etiolated soybean (Glycine max (L.) Merr. var. Essor) seedlings, submitted to cold. Prolonged exposure to 1 degrees C inhibited hypocotyl and root elongation and limited their growth after seedlings were transferred to 25 degrees C. Roots were more sensitive to chilling than hypocotyls. At 1 degrees C a gradual increase in MDA concentration in roots but not in hypocotyls was observed. An increase in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activity in hypocotyls was observed both at 1 degrees C and after transfer of plants to 25 degrees C. In roots, CAT activity increased after 4 days of chilling, while SOD activity only after rewarming. L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity decreased in roots of chilled seedlings, but did not change in hypocotyls until activity increased after transfer to 25 degrees C. The content of genistein and daidzein increased after 24 h of treatment by low temperature and then decreased with prolonged chilling in hypocotyls and remained high in roots. However, it should be noted that genistin level (genistein glucoside) in chilled hypocotyls is 10 times higher than in roots, despite falling tendency. The role of antioxidant enzymes and isoflavonoids in preventing chilling injury in hypocotyls and roots of soybean seedlings is discussed.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Auxin-activated nadh oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

Summary The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14–17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.     

Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid

Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used ¹⁴C-aspartate, which was then separated from the ¹⁴C-ureidosuccinate product by Dowex 50 (H⁺ form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5′-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.     

表油菜素内酯对黄瓜幼苗下胚轴过氧化物酶和IAA氧化酶活性的影响

黄瓜幼苗用0.1～1 ppm表油菜素内酯(epiBR)处理1～3d后,下胚轴中过氧化物酶活性明显低于对照;随着处理浓度的增加和处理时间的延长,与对照之间的差别愈趋增大。当浓度高于1ppm时,过氧化物酶的活性不再继续降低。表油菜素内醋对过氧化物酶活性的这种抑制作用需经约1O h的滞后期。IAA氧化酶的活性变化与过氧化物酶相似,epiBR处理时间愈长酶活性增加愈趋缓慢。 经IAA处理的下胚轴,过氧化物酶和IAA氧化酶的活性变化与对照无明显差异。这提示油菜素内酯与IAA促进生长可能是通过不同的作用方式。     

Comparison of the effects of white light and the growth retardant paclobutrazol on the ethylene production in bean hypocotyls

At a concentration of 17 µmol·L^–1, paclobutrazol (PP), a triazole plant growth retardant, effectively reduced the elongation and increased the thickness of hypocotyls in 6-day-old Phaseolus vulgaris L. cv. Juliska seedlings, both in the light and in the dark. PP treatment did not increase the cell number in transverse sections of hypocotyls. The diameter of hypocotyls was uniform from the zone of intensive elongation along the whole hypocotyl in etiolated plants, but those grown in the light exhibited an additional lateral expansion at the base. Ethylene evolution was not reduced by PP in etiolated hypocotyls, and did not differ significantly in the elongating apical and fully grown basal zones. PP reduced the ethylene release by the growing zones in green hypocotyls, but not in the basal parts, which resulted in an increasing ethylene gradient towards the hypocotyl base. The level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was much higher in retardant-treated hypocotyls than in the controls, which was due in part to the reduced malonylation. The swelling of the hypocotyl bases could be eliminated by inhibitors of ethylene biosynthesis or action, or could be induced by 10 µmol·L^–1ACC in control plants in the light. None of these treatments had a significant effect on the lateral expansion of hypocotyls in etiolated seedlings. PP treatment induced a similar effect to that of white light in etiolated seedlings, and amplified the effect of light in green plants with respect to the ACC distribution, and consequently, the ethylene production in the hypocotyls of 6-day-old bean seedlings. It can be concluded that the lateral expansion of hypocotyl bases in PP-treated green plants is controlled by ethylene.     

Hypocotyl-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.     

Effect of polyamines on ethylene production

The polyamines putrescine, cadaverine, spermidine and spermine reduced the amount of ethylene produced by senescing petals of Tradescantia but they did not prevent anthocyanin leakage from these same petals. These polyamines also inhibited auxin-mediated ethylene production by etiolated soybean hypocotyls to less than 7 % of the control. The basic amino acids lysine and histidine reduced the amount of auxin-induced ethylene produced by soybean hypocotyls by ca 50 %. In the hypocotyls, methionine was unable to overcome the inhibition caused by the polyamines. The polyamines spermidine and spermine inhibited ethylene production induced by the application of 1-aminocyclopropane-1-carboxylic acid and they also reduced the endogenous content of this amino acid in the treated tissues.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

Inhibition of plasma membrane redox activities and elongation growth of soybean

NADH-ferricyanide oxido-reductase (EC 1,6,99,3) of purified plasma membrane vesicles isolated by aqueous two-phase partition from segments of etiolated soybean [ Glycine max (L.) Merr. cv. Williams] hypocotyls was used as a measure of plasma membrane redox activity. Elongation growth of hypocotyl segments floated on the solutions was determined in parallel. Cis -platinum (II) diammine dichloride ( cis -platin), adriamycin and p -nitrophenylacetate, agents known to inhibit cell proliferation and plasma membrane redox activities in mammalian cells inhibited both NADH-ferricyanide oxido-reductase of the isolated membrane vesicles and elongation growth of intact hypocotyl segments. Auxin(2,4-dichlorophenoxyacetic acid)-induced growth of the isolated segments was inhibited preferentially at drug concentrations where control growth was affected only slightly. The findings suggest a connection between plasma membrane redox reactions and the control of elongation growth in plants.     

Changes in Levels of Auxin and Abscisic Acid and the Evolution of Ethylene in Squash Hypocotyls after Treatment with Brassinolide

Brassinolide stimulated an increase in fresh weight of segmentsof hypocotyls of etiolated squash (Cucurbita maxima Duch. cv.Houkou-Aokawaamaguri). Brassinolide-treated segments containedhigher levels of IAA than water-treated segments and also showeda tendency towards decreased levels of ABA. However, treatmentwith brassinolide did not have much effect on the productionof ethylene. (Received May 30, 1988; Accepted May 22, 1989)     

Circadian rhythms in crassulacean acid metabolism: phase relationships between gas exchange,leaf water relations and malate metabolism in Kalanchoë daigremontiana

Hypocotyls of 5-d-old etiolated soybean seedlings (Glycine max (L.) Merr. cv. Altona) were treated with (a) dithiothreitol (DTT) or one of the sulfhydryl-binding reagents N-ethylmaleimide (NEM), p-hydroxymercuribenzoate (PMB) und p-chloromercuribenzene sulfonic acid (PMBS), (b) one of the sulfhydryl reagents in combination with DTT, (c) sulfhydryl reagent subsequent to treatment with DTT, and (d) PMBS followed by DTT. Glyceollin was extracted 24 and 48 h after initiation of treatment. The order of decreasing glyceollin-eliciting activity was PMBSDTT>PMBNEM. Elicitor effectiveness of sulfhydryl reagents and their reactivity with either L-cysteine or sulfhydryl groups in soybean hypocotyls were not strictly correlated. Mixtures of sulfhydryl reagent and DTT, pretreatment of hypocotyls with DTT and subsequent application of either PMB or PMBS, as well as application of PMBS prior to DTT induced less glyceollin than sulfhydryl reagents alone. In contrast, such pretreatment did not appreciably alter glyceollin accumulation elicited by NEM. The results indicate that glyceollin synthesis can be regulated by interaction with sulfhydryl groups located mainly at the outer surface of the plasmalemma.Abbreviations DTT DL-dithiothreitol - NEM N-ethylmaleimide - PMB p-hydroxymercuribenzoate (sodium salt) - PMBS p-chloromercuribenzene sulfonic acid     

Phototropic stimulation induces the conversion of glucosinolate to phototropism-regulating substances of radish hypocotyls

The distribution of natural growth inhibitors, the raphanusanins (isomers of 3-(methylthio)methylene-2-pyrrolidinethione) and their precursors (4-methylthio-3-butenyl glucosinolate (MTBG) and 4-methylthio-3-butenyl isothiocyanate (MTBI), between illuminated and shaded halves of radish hypocotyls during phototropic curvature was analyzed using a physicochemical assay. Phototropic stimulation rapidly decreased MTBG content, and abruptly increased contents of MTBI and raphanusanins in the illuminated halves of radish hypocotyls within 30 min after the onset of unilateral illumination. Content in the shaded halves was similar to that in dark controls. When MTBG, MTBI, and raphanusanins at endogenous levels were applied unilaterally to etiolated hypocotyls, MTBI and raphanusanins caused hypocotyls to bend but MTBG showed no activity. Blue illumination promoted myrosinase (thioglucosidase) activity, which releases MTBI from MTBG, in hypocotyls after 10 min, although enzyme activity in dark controls did not change. These results suggest that phototropic stimulation promotes myrosinase activity in the illuminated side of radish hypocotyls, releasing bioactive MTBI from inactive MTBG and simultaneously producing bioactive raphanusanins.     

Expression of flavonoid 6-hydroxylase candidate genes in normal and mutant soybean genotypes for glycitein content

Soybean seeds accumulate large amounts of isoflavones (genistein, daidzein and glycitein), secondary metabolites known for their phytoestrogenic activities. Isoflavone composition depends on the seed part and glycitein is almost found exclusively in hypocotyls. Moreover, two major phenotypes are encountered in soybean cultivars, with either low (35 %) or high (55 %) levels of glycitein in their hypocotyls. This trait was under a quasi-mendelian heredity, implicating at most one or two genes. A CYP71D9 cDNA displaying a flavonoid 6-hydroxylase (F6H) activity had previously been isolated from elicitor-induced soybean (Glycine max L.) cells. This enzyme allows the synthesis of the glycitein flavanone intermediate (6,7,4′- trihydroxyflavanone) by catalyzing the A-ring hydroxylation of liquiritigenin. In this study, the CYP71D9 gene (F6H1) and two other candidates (F6H2 and F6H3) were studied using contrasted soybean cultivars for glycitein content (0, 35, 55 and 80 %). Their expression was observed in chitosan elicited leaves. They encode P450 proteins of 496, 469 and 481 amino acids respectively and were expressed in leaves with or without elicitation. The expression patterns of these three genes were performed in cotyledons and hypocotyls at different developmental stages. F6H1 and F6H2 were not expressed in the developing seed. F6H3 was only expressed in hypocotyls. Its expression levels did not correlate with hypocotyls glycitein content, but it was not expressed in the null mutant for glycitein. Thus, this F6H3 gene is a good potential candidate for glycitein biosynthesis in soybean seed.     

Chromatin RNA polymerase activity from soybean hypocotyls treated with gibberellic acid and AMO-1618

Chromatin isolated from control, AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine carboxylate] and gibberellic acid (GA) treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid-insoluble RNA. Gibberellic acid, sprayed on intact soybean hypocotyls, is shown to have enhanced the level of chromatin RNA polymerase activity while chromatin isolated from hypocotyls pretreated with AMO-1618 exhibits a lower polymerase activity relative to the control. Chromatin extracted from the treated or untreated seedlings are all sensitive to the inhibition (in varying degrees) by the presence of actinomycin D, pyrophosphate, or ribonuclease. Thus enhanced (or decreased) RNA-synthesizing capacity of chromatin in response to chemical treatments may be due to enhanced (or decreased) synthesis of RNA polymerase.