Cell wall biochemistry and biomechanics of harvested white asparagus shoots as affected by temperature

The effects of temperature on the dynamics of changes in shoot mechanical properties, cell wall components, relevant soluble sugars and respiration activity of harvested white asparagus spears were investigated during a 7-day storage period. All functional cell wall components of asparagus spears increased closely temperature dependent. The content of soluble glucose declined with a similar temporal dynamics and to a comparable degree, indicating a major carbon flow of this storage sugar into cell walls (60–70%). Irrespective of temperature, the contents of stored soluble fructose and sucrose remained more or less constant. Lower temperatures reduced cell wall development but do not significantly affect the relative carbon flow from storage sugars into cell walls or maintenance respiration. Compared with cell walls, maintenance respiration is by far the smaller carbon sink in stored asparagus spears. Temperature differentially affects the absolute amount and the relative contribution of the different cell wall components and the temporal dynamics of changes in structural carbohydrate and lignin content. At higher temperatures, secondary cell wall thickening resulted mainly from a large increase in cellulose content. The pronounced increase in the fractions of cellulose and especially lignin may stress the important role of lignin in cell wall strengthening. While the fraction of cell wall proteins decreased, those of hemicellulose and the pectic components were not influenced.     

Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce

Mechanical wounding by cuts or punctures caused a brief increase in ethylene production by iceberg lettuce ( Lactuca sativa L.) leaf tissue. Wounding increased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, which was a function of the degree of injury. Wound-induced PAL activity appeared after 4 h and reached maximum activity in about 24 h before slowly declining to normal levels in about a week. A signal for PAL induction was transmitted at about 0.5 cm h⁻¹ from the site of injury to cells up to 2.5 cm away. Treatment with 100 μ2-aminoethoxyvinylglycine prevented wound-induced ethylene production but did not affect induced PAL activity. Injury increased the concentration of several soluble phenolic compounds that were easily oxidized to brown substances by polyphenol oxidase (EC 1.10.3.2) isolated from lettuce tissue. Wounding also increased peroxidase (EC 1.11.1.7) activity and lignin content, with cell wall lignification localized in wounded and adjacent cells. Although wounding alone did not induce russet spotting, it did greatly increase susceptibility to ethylene-induced russet spot development. In the presence of 3 μ1⁻¹ ethylene, the russet spot score increased as the degree of injury increased.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

低氧气调包装对去壳雷笋褐变和木质化的影响

研究了在厚度为0.04 mm的聚乙烯(PE)袋内充气成分为2%O2、5%CO2和93%N2于10℃下贮藏时气调袋装去壳雷笋褐变和木质化进程.与对照相比,低氧气调包装(MAP)抑制了去壳雷笋贮藏前期丙二醛(MDA)的生成,显著抑制了过氧化物酶(POD)(P＜0.05)和苯丙氨酸解氨酶(PAL)活性(P＜0.01),最终显著抑制了笋肉的褐变(P＜0.01),显著抑制了木质素(P＜0.05)和纤维素的合成,从而延长了保鲜期.去壳雷笋的褐变可能由POD和PAL活性作用引起.POD和PAL活性的增加也可能诱导了木质素的合成.     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

A preliminary investigation of L-phenylalanine ammonialyase activity in asparagus: Distribution and response to storage, excision and incubation

The level of L-phenylalanine ammonia-lyase (PAL) (E.C. 4.3.1.5 [EC] )activity was greatest in the basal portion of freshly harvestedasparagus spears and decreased toward the tips. Basal disksdeveloped large increases in L-phenylalanine ammonia-lyase activityin response to excision and incubation. The level of PAL activitymeasured in basal tissue of intact spears increased with storage,while the ability of the same tissue to respond to excisionand incubation with increased PAL activity was lost. (Received March 23, 1971; )     

Long-term effects of asparagus virus 2 infection on growth and productivity in asparagus

An asparagus field trial was established with clonal plants to determine the long-term effects of asparagus virus 2 (AV2) infection on spear production. Yield data, analysed by ANOVA, showed that AV2 infection caused significant (P < 0.05) decreases in spear yield which became more pronounced as the trial progressed. Mean marketable spear yields were reduced by 14%, 28%, 20%, 48% and 57% and reject spear yields were increased by 93%, 105%, 207%, 352% and 167%, during harvest years 1–5 respectively. Marketable spear yields from AV2–free plants increased annually to yr 5, but for AV2–infected plants, yields increased to yr 3 and decreased annually thereafter. Spears from AV2–infected plants were thinner than those from AV2–free plants, resulting in more reject thin spears by 109%, 88%, 220%, 499% and 216% during harvest yr 1–5, respectively. Further, data collected in yr 4 and 5 showed that AV2 infection had caused a 31 % reduction in mean spear diameter and reductions of 27% and 22% respectively, in diameter and height of fern stalks. Clearly, plants with smaller fern stalks were less able to accumulate carbohydrate reserves and therefore produced fewer, smaller spears and fern stalks the following spring. This may result in annual cycles of diminishing productivity in which the size and number of spears and fern stalks decrease with each successive year. The type and timespan of symptoms caused by AV2 infection in this trial are similar to those reported for asparagus decline syndrome and therefore it is likely that AV2 infection is a factor contributing to asparagus decline.     

Physiological and biochemical events leading to vitrification of plants cultured in vitro

Water content, peroxidase activity and isoperoxidases, phenylalanine ammonia-lyase activity and phenolic content were comparatively analyzed in tissues of normal and vitreous plants cultured in vitro. The release of ethylene in flask atmospheres by normal and vitrifying plants was also measured. On the basis of the results, it is hypothesized that vitrification results from a burst of ethylene controlled by the peroxidase-IAA-oxidase system. An initiating stress (e.g. excess of cytokinins or of NH₄⁺ ions) would mediate the enhancement of the activity of soluble and membrane-bound peroxidases through a rapid modification of the phenolic level. The excess of ethylene in the atmosphere of stressed plants would retroinhibit its own biosynthesis and as a consequence decrease the activities of PAL and acidic peroxidases, thus hindering lignification processes. A parallel decrease in cellulose synthesis due to a diverted conversion of sugars to amino acids is expected (from data in the literature). Deficiency of both cellulose and lignin would allow more water uptake due to reduced wall pressure and bring about the hyperhydric malformations.     

Activities of some enzymes of lignin formation in reaction wood of Thuja orientalis,Metasequoia glyptostroboides and Robinia pseudoacacia

The activities of the following five enzymes which are involved in the formation of lignin have been compared in reaction wood and in opposite wood: phenylalanine ammonia lyase (EC 4.3.1.5), caffeate 3-O-methyltransferase (EC 2.1.1.-), p-hydroxycinnamate: CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-) and peroxidase (EC 1.11.1.7). The activities of the four first-named enzymes in the compression wood of Thuja orientalis L. and Metasequoia glyptostroboides Hu et Cheng were 2.8±1.4-fold and 2.6±1.5-fold higher than those in opposite wood, respectively, whereas peroxidase had the same level of activity in either type of wood. On the other hand, no differences were observed in the activities of the five enzymes between tension and opposite woods of Robinia pseudoacacia L. These findings are well in accord with the chemical structure of lignin in the compression and tension woods of the three species studied: high content of lignin rich in condensed units in compression wood, and little difference in lignin between tension and opposite woods.Abbreviations CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - OMT caffeate O-methyltransferase (EC 2.1.1-) - PAL phenylalanine ammonia lyase (EC 4.3.1.5) - PCL p-hydroxycinnamate: CoA ligase (EC 6.2.1.12) - PO peroxidase (EC 1.11.1.7)     

Carry-over of thigmomorphogenetic characteristics in calli derived fromBryonia dioica internodes

Calli have been initiated in vitro from young internodes (control and rubbed) ofBryonia dioica, where previously it had been shown, using intact plants, that rubbing induced limited growth through enhanced lignification. Calli derived from rubbed internodes were somewhat more compact and showed biochemical changes, i.e. enhanced activity of total peroxidase and isoperoxidases, enhanced production of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene, enhanced tissue capacity to convert ACC into ethylene, enhanced activity of phenylalanine ammonia-lyase (PAL) and higher content of lignin, which characterized rubbed internodes. Differences in ethylene metabolism between the two types of calli tended to fade from the third week onwards of initial culture, whereas lignin content, peroxidase activity and peroxidase isoenzyme pattern appeared to be more persistant rubbing-induced markers for several subcultures. The results point to the persistance of environmentally induced changes in gene expression.     

Condensed lignins are synthesized in poplar leaves exposed to ozone

Poplar (Populus tremula x alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L(-1)). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L(-1)). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.     

Identification and Characterization of an mRNA Encoding a Proline-Rich Protein that Rapidly Declines in Abundance in the Tips of Harvested Asparagus Spears

We previously isolated a cDNA clone, pTIP13, whose homologousmRNA rapidly declined in abundance in the tips of harvestedasparagus (Asparagus officinalis L.) spears [King and Davies(1992) Plant Physiol. 100: 1661]. In order to identify factorsregulating the postharvest deterioration of asparagus, we havenow sequenced the pTIP13 cDNA, derived the encoded amino acidsequence and determined the cellular location of pTIP13 mRNAby in situ hybridization. pTIP13 encodes a derived protein thatis rich in proline (22.3%), but also has a high content of lysine(15.2%) and threonine (14.1%). The proline residues are locatedin motifs at the amino-terminal region of the protein. The carboxyl-terminalregion of the derived protein has a high leucine content andshares >64% amino acid identity with derived proteins identifiedfrom cDNA clones to cell wall protein precursor mRNAs obtainedfrom soybean hypocotyls, alfalfa roots, and tomato fruit. GenomicSouthern analysis suggests that pTIP13 is encoded by a single-copygene in asparagus. pTIP13 mRNA was localized to specific celltypes in the young bracts of the asparagus spear tip. The resultsprovide new information on the complexity of tissue responsesin the tips of asparagus spears following harvest. (Received February 5, 1996; Accepted May 16, 1996)     

Nitric oxide affecting root growth,lignification and related enzymes in soybean seedlings

This study analyzed the involvement of nitric oxide (NO) in the root lignification of soybean seedlings. To this end, changes in root cell viability; phenylalanine ammonia-lyase (PAL) and soluble and cell wall bound peroxidase (POD) activities and lignin and hydrogen peroxide (H₂O₂) contents of soybean roots treated with the NO-donor sodium nitroprusside (SNP) and its relationships with root growth were evaluated. Seedlings were cultivated in a nutrient solution supplemented with 5 to 1,000 μM SNP for 24 h. At an extremely low concentration (5 μM), SNP induced root growth and increased lignification and activities of related enzymes (PAL and cell wall-bound POD). At a high concentration (1,000 μM), SNP reduced root growth and lignification (PAL activity and H₂O₂ and lignin contents) and caused a loss of cell viability. Application of potassium ferrocyanide (an analog of SNP that cannot release NO) and PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoleline-1-oxyl-3-oxide, a scavenger of NO) revealed that the inhibitory/stimulatory effects on root lignification may be due to NO itself. These results indicate that NO, depending on its concentration, may act as a stress factor, due to its toxic action, or as a signal molecule, inducing soybean root growth and lignification.     

Thigmomorphogenesis inBryonia dioica: Changes in soluble and wall peroxidases,phenylalanine ammonia-lyase activity,cellulose, lignin content and monomeric constituents

Rubbing young internodes ofBryonia dioica results in a reduced elongation and an increased diameter of the internodes. In the present study activities of some enzymes involved in the lignification process and levels of lignification were compared in rubbed and non-rubbed internodes. Rubbing caused an increase in the activities of phenylalanine ammonia-lyase and soluble and ionically- and covalently-bound cell wall peroxidases. Sensitivity of the covalently-bound wall peroxidase assay was markedly increased if syringaldazine was used as a substrate. Mechanical perturbation induced an increase in lignin, lignin monomer (sinapylic, coniferylic and p-coumarylic alcohols) content and the number of lignifying vessels. Conversely, rubbing resulted in a decrease in cellulose content. The hypothetical interpretation of the thigmomorphogenetic response through cell wall lignification and hence rigidification is consistent with all the presented results. A comparison is possible between this accelerated lignification and induced lignification as a mechanism of disease resistance. the thigmomorphogenetic response inBryonia dioica can be considered as a mechanism of resistance in order to withstand further environmental mechanical perturbation.Research partly supported by the Belgian FRFC grant 2.9009 to T.G. and by the French CNRS (LA 45, RCP 474).     

Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess

Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed.     

Elicitor-Induced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum L.) Leaves

The substrate-specific induction of wheat (Triticum aestivum L. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Botrytis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-cou-maryl and coniferyl alcohol dehydrogenase activities were detectable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase activity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extracts. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase activity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissues of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase activity.     

Isoforms of glutamine synthetase in asparagus spears: the cytosolic enzyme increases after harvest

Two distinct forms of glutamine synthetase (GS) have been identified in the spear tip tissues of harvested asparagus (Asparagus officinalis L. cv. Limbras 10). The GS activities were separated by anion exchange chromatography. They have distinct kinetic properties and contain polypeptides of different sizes, and the abundances of the GS isoforms change differently after harvest. Plastid GS has a 44 kD polypeptide, and during the post-harvest period the abundance of this polypeptide declined dramatically. After 5 d, the activity of plastid GS had declined to just 20% of that at harvest. Cytosolic GS has a 40 kD polypeptide and is the major constituent of the GS activity present at harvest (73% of total). After harvest, cytosolic GS activity declined by half and then, at 3 or 4 d after harvest, rose to 80% of the cytosolic GS activity present at harvest. The nitrogen metabolism of asparagus spears is significantly altered as the tissues deteriorate rapidly after harvest. We demonstrate that cytosolic GS activity increases during the post-harvest period and is likely to be a critical feature of the physiology of the tip of a harvested asparagus spear.