Boron deficiency effects on peroxidase, hydroxyproline, and boron in cell walls and cytoplasm of Helianthus annuus L. hypocotyls

Boron-deficient sunflower hypocotyls have, on a fresh weightbasis, more cytoplasmic and cell wall peroxidase, more cytoplasmichydroxyproline but equal cell wall hydroxyproline, and slightlyless cell wall boron, than controls. Incubation of either Bdeficientor control cell wall suspensions with Sclerotium rolfsii culturenitrate released 80% of the peroxidase activity, but only 14to 30% of the hydroxyproline. This differential extraction suggeststhat the hydroxyproline-containing protein of cell walls isnot identical with peroxidase. Boron deficiency increased thesusceptibility of cell walls to degradation by fungal enzymes,as measured by release of peroxidase and hydroxyproline, butnot by reduction in dry weight. ¹Scientific article No. A1847, Contribution No. 4756 of theUniversity of Maryland Agricultural Experiment Station. Thisresearch represents part of a thesis for the M.S. degree, Universityof Maryland, by J.B.S., Jr. This paper is dedicated to ProfessorHugh G. Gauch on the occasion of his sixtieth birthday. (Received December 5, 1972; )     

Expression of Cell Wall Proteins in Seeds and During Early Seedling Growth of Araucaria araucana is a Response to Wound Stress and is Developmentally Regulated

Araucaria araucana seeds and seedlings respond to wounding after48 h with a 3- to 4-fold increase of hydroxyproline-rich glycoproteins(HRGP) in the cell walls of the embryo and with a 15-fold increasein the cell walls of the megagametophyte. The megagametophytewalls accumulate six times more hydroxyproline per µgof cell wall protein than the embryo in this wound response.Tissue immunoprints of different parts of seeds and seedlingsobtained with polyclonal antibodies raised against HRGP fromcarrot roots or soybean seed coats indicate that the responseis due to an increase in a protein similar to the ones seenin carrot roots or soybean seed coats. Western blots of embryoand megagametophyte cell wall proteins subjected to SDS-PAGEshow three bands that cross-react with these antibodies. Ina native cationic gel system followed by Western blot analysis,only two bands react with these antibodies. Expression of suchproteins in Araucaria araucana seeds seems to be developmentallyregulated and tissue specific, since they are present mainlyin the megagametophyte and the root cap of the embryo. Key words: Araucaria araucana, seeds, seedlings, cell walls, hydroxyproline-rich glycoproteins     

Hydroxyproline and Peroxidases in Cell Walls of Pisum sativum: Regulation by Ethylene

Purified cell-wall preparations from the epicotyl of etiolatedPisum sativum contain covalently bound peroxidases and hydroxyproline-richproteins. Towards the end of cell elongation there is a largerise in these wall components and thereafter a continuing slowrise which is associated with increasing age of tissue. Ethyleneat concentrations of 0.1 ppm or more increases both peroxidaseactivity and hydroxyproline levels in the walls, the greatestresponse occurring in immature tissue including the apical hook.Growth of these tissues is highly sensitive to ethylene whichcauses an inhibition of elongation in extending cells and anenhanced lateral cell expansion. We suggest that the effectsof ethylene on wall-bound peroxidase and hydroxyproline areimplicated in the ethylene regulation of cell growth. The covalently bound wall peroxidase was found to be extremelystable and to contain unique isoenzymes which do not occur ineither the cytoplasm or in the peroxidase which is ionicallybound to walls. Ethylene increases peroxidase activity in boththe cytoplasmic and the ionically bound wall fractions, butthere is little or no increase in their hydroxyproline content.The possible relationships between covalently bound wall peroxidaseand hydroxyproline are discussed and we speculate that thisperoxidase may be involved in the hydroxylation of proline inthe walls.     

Proline, Hydroxyproline, and Lily Pollen Tube Elongation

Cytoplasm of freshly-harvested, ungerminated Lilium longiflorum,cv. Ace pollen contains 0.14 per cent soluble and 0.35 per centprotein-bound proline (pro). Their metabolic fates in germinationand tube elongation are not known with certainty. Here is reportedconversion of pro to hydroxyproline (hyp)—containing constituentsas well as distribution and isolation of these constituents.Colorimetry revealed pro and hyp in wall, trichloroacetic acid(TCA)—precipitable, and TCA-soluble cytoplasmic fractions.A balance sheet summarizing quantitative changes in pro andhyp for these fractions revealed that TCA—precipitablecytoplasmic pro could be a precursor to wall-bound pro and asubstrate for hydroxylation yielding cytoplasmic and wall-boundhyp. To determine whether hyp was a component of tube and/orgrain walls, pollen was allowed to germinate 1.5 h and thentransferred to sorbitol medium which prevented further tubeelongation. Hyp was absent from walls of transferred pollen.Electron microscope autoradiography of tubes exposed to ²H-prosuggested that a pro- and/or hyp-containing constituent waslocalized in the growing tip. Light microscope autoradiographyof intact tubes labelled with ¹⁴C-pro showed that the constituentwas distributed throughout the pollen tubes. Gel filtrationof hyp-containing material enzymically released from walls supportedthe view that they contained hyp-glycopeptides.     

Abnormal Stomatal Behaviour and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: EFFECT OF ABSCISIC ACID AND AUXIN ON STOMATAL BEHAVIOUR AND PEROXIDASE ACTIVITY

The wilty tomato mutant flacca and the normal variety RheinlandsRuhm were compared in terms of: (1) potassium transport intoand out of the guard cells, (2) cell wall properties which includeprotein, hydroxyproline and peroxidase activity, and (3) activityof indol-3yl-acetic acid oxidase. Also studied were the effectsof auxin on stomatal behaviour and peroxidase activity whenapplied to normal plants during development, and the short-termeffect of abscisic acid on the resistance of flacca stomatato closure under plasmolysis. Potassium transport, wall protein and hydroxyproline all seemedto be equal in mutant and normal plants. Peroxidase activitywas higher in the soluble and wall fractions of the mutant,and decreased toward normal in the mutant treated with abscisicacid. More stomata were open and peroxidase activity was higherin normal plants treated with auxin during development. Thepercentage of open stomata under plasmolysis was lower and theiraperture size was smaller in the epidermal strips taken fromabscisic-acid-treated mutant plants than from control mutantplants.     

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)     

Endoreduplication Occurs during Pod Wall Development in Temperate Grain Legumes

Differentiation to a specialized function in plant tissue isassociated with an increase in the DNA content of the cellsover the diploid state. Using flow cytometry, ploidy levelswere assessed during pod development in three white lupin genotypesunder three environmental conditions and in other lupin speciesand temperate grain legumes in one environmental condition.Endoreduplication was found in the pod walls of all genotypesstudied. Higher ploidy levels coincided with maximal growthof the pod. DNA replication was not related to the proportionof the pod that comprised walls. Endoreduplication also occurredin other temperate grain legumes with at least two DNA duplicationsduring the development of the pod walls. The biological significanceof endoreduplication in the pod walls of grain legumes is unknown,but could be related to the storage function of these organs.Copyright2000 Annals of Botany Company Lupinus species, Pisum sativum, Phaseolus vulgaris, Vicia faba, pod wall development, ploidy level, flux cytometry, Bis-benzimide Hoechst 33258     

Anatomy of Ethylene-induced Petal Abscission in Pelargonium x hortorum

When viewed under the light microscope, the abscission zoneat the petal base of Pelargonium x hortorum consisted of smallcells which, when stained with Toluidine Blue, possessed denselystained cells walls. After treatment with 1 µl l^-1 ethyleneat 22°C, the force required to separate the petals fromthe receptacle declined after a lag phase of only 30 min, withseparation complete 60-90 min later depending upon the stageof development of the flower. Transmission electron micrographsof the petal abscission zones showed evidence of cell wall degradation,particularly in the middle lamella. These cells also containedextensive rough endoplasmic reticulum and numerous Golgi bodiesribosomes. When abscission was complete, cells at the fractureface showed evidence of breakdown of cellular compartmentalization,often with little sign of an intact tonoplast. Scanning electronmicrographs of recently-abscissed surfaces showed that the epidermalcells surrounding the abscisson zone were turgid and rounded,whereas those of the mesophyll cells were partially collapsed.The micrographic evidence is consistent with the hypothesisthat ethylene-induced separation is caused by rapid enzymaticof the cell walls.Copyright 1993, 1999 Academic Press Abscission, cell walls, ethylene, flower, Pelargonium x hortorum     

Factors Affecting Urease Activity in the Lichen Evernia prunastri (L.) Ach

In the lichen Evernia prunastri increased urease activity inthe presence of urea is enhanced by phosphate buffer and respirablereserves and decreased by desiccation. Although stimulatory,exogenous urea is not essential and may act as both an enzymeactivator and inducer. The previously observed decline in ureaseactivity on prolonged urea treatment, attributed to in vivoenzyme inactivation by phenolic lichen substances, is prevented,but not reversed, by in vitro additions of dithiothreitol. Evernia prunastri urease activity, enzyme inactivation, enzyme induction desiccation lichen     

Herbaspirillum, an endophytic diazotroph colonizing vascular tissue 3Sorghum bicolor L. Moench

Leaves of Sorghum bicolor were examined at 5 d and 14 d afterinoculation with the N₂-fixing endophytic bacteria Herbaspirillumseropedicae and Herbaspirillum rubrisubalbicans. Plants inoculatedwith H. rubrisubalbicans expressed symptoms of ‘red stripedisease’ i.e. red stripes along the secondary veins ofthe leaf blade close to the inoculation point and spreadingup the leaves. Infected leaves showed dense colonization byH. rubrlsubalbicans in regions showing red stripe symptoms at5 d after inoculation. The infection was confined within thevascular system, in particular, the protoxylem and associatedlacunae, which were often completely filled with bacteria, withsome of the latter expressing nitrogenase. The bacteria wererecognized using H. rubrisubalbicans-speciflc antibodies andimmunogold labelling, which also showed that the antibody reactedwith material on the surface of the bacteria, and that thismucus was released into the lumen of the xylem. At 14 d afterinoculation, disease symptoms were slightly more severe, withboth meta- and protoxylem being even more heavily colonizedin parts of the leaf showing red stripes. However, a stronghost defence response was also apparent at this stage, withgums lining the walls of the vessels and enclosing the bacteria,although the latter were still actively dividing. At the edgesof visible disease symptoms, plant gums filled the xylem; bacteriahad formed distinct colonies within these gums, with some ofthe colonies associated with the xylem walls. Plants inoculatedwith H. seropedicae either did not express the disease or showedvery mild symptoms close to the inoculation point. In the lattercase, H. seropedicae were localized within protoxylem vesselsand the metaxylem was partly occluded with plant-derived gums.By contrast with H. rubrisubalbicans, H. seropedicae was alsolocalized in leaves at 14 d without disease symptoms and didnot always appear to elicit a host response, i.e. they colonizedthe walls of metaxylem, with the xylem vessels themselves remainingunoccluded and largely free of gums. The fine line separatingplant pathogens, endophytes and symbioses is discussed in lightof these results. Key words: Herbaspirillum, Sorghum bicolor, nitrogen fixation, endophyte, xylem     

The Use of C14-Proline by Growing Cell: Its Conversion to Protein and Hydroxyproline

C¹⁴-proline is readily absorbed by growing tissue cultures ofcarrot root phloem and of potato tuber in experiments carriedout under aseptic conditions. The C¹⁴-proline rapidly entersinto the protein of the tissue, appearing there in as shorta period as 15 minutes, and, thereafter, the amount incorporatedinto the protein bears a linear relation to time. Virtuallyall the C¹⁴ appears in the protein hydrolysate in the form ofproline and hydroxyproline. It is shown that the conversionfrom proline to hydroxyproline occurs after the C¹⁴-prolineis combined into the protein and that this conversion proceedsprogressively with time. The ratio of C¹⁴ as proline to C¹⁴as hydroxyproline declined progressively from a value of 4.0after 30 minutes of contact and seemed to become stabilizedeventually at 0.7. C¹⁴-hydroxyproline, which can be absorbedby the tissue, seems not to be incorporated into the proteinas such. The protein moiety which contains the C¹⁴-hydroxyprolinefrom C¹⁴-proline represents a stable protein which is not metabolizedand whose carbon does not ‘turn over’. This inertprotein seems to be characteristic of cells which are in rapiddivision under the influence of coconut milk or are synthesizingprotein in response to other stimuli such as the events at acut tissue surface. The protein in question seems to be presentmainly in the cytoplasm rather than in its paniculate inclusions.These results are compatible with earlier views which requirethat part of the protein in the cell ‘turns over’its carbon, whereas another part does not do so.     

Plantlet Regeneration from Suspension and Protoplast Cultures of the Tropical Pasture Legume Stylosanthes guyanensis (Aubl.) Sw

Hypocotyl- and leaf-derived suspension cultures of Stylosanthesguyanensis were established and plants were regenerated fromthem, even after several sub-cultures. Protoplasts, enzymaticallyisolated from cell aggregates of a highly morphogenetic suspensionculture, synthesized new cell walls, divided and developed intocalli. Plantlets were regenerated from these calli after successivetransfers to solid maintenance and shoot induction media. Stylosanthes guyanensis (Aubl.) Sw, suspension culture, protoplasts, shoot regeneration, plantlet regeneration     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity     

Ultrastructural Observations on Proliferating Storage Cells of Mature Cotyledons of Phaseolus vulgaris L. Cultured in vitro

Explants of mature cotyledons of Phaseolus vulgaris form callusrapidly when cultured in vitro with their adaxial surfaces embeddedin a solidified nutrient medium containing coconut milk, kinetinand 2,4-D. Proliferation is confined to the highly polyploidstorage cells and commences near the adaxial epidermis, whichis soon ruptured by the callus developing internally. Callusformation progresses to the abaxial tissue and within 3–4weeks sub-culturing is possible. The in vitro grown storage cells undergo thinning of their walls,loss of food reserves, hypertrophy, development of various new-wallsand nuclear activation leading to division. The induction ofnuclear and cell divisions within this mature storage tissuecontrasts with normal germination in which these cells undergorapid senescence after depletion of their food reserves. Nuclear division in early callus growth is apparently mainlyamitotic. It is preceded by the development of multiple nucleoli.The nuclear envelope also becomes more complex and deeply lobed;leading to formation of a nuclear isthmus and final separationinto two nuclei. No chromosomes are visible during nuclear fragmentation.Amitosis is accompanied by freely-forming walls, which may developadjacent to a nuclear isthmus and perhaps participate in nuclearfragmentation. Large labyrinthine wall bodies frequently occuron these walls. Mitoses are only observed in already dividedstorage cells. A cell plate forms between the two daughter nuclei,and microtubules are present at its margins in contrast to freely-formingwalls where none are evident. Phaseolus vulgaris L., bean, in-vitro culture, cotyledon, ultrastructure     

Cell Wall Autolysis During Kiwifruit Development

Cell walls prepared from developing kiwifruits showed autolyticactivity. The proteins extracted from active walls were alsoable to release carbohydrates from inactive cell walls. Analysisof the sugars released, using both procedures, showed that uronicacids were the major component, especially during the firsthours of incubation, although neutral sugars such as glucoseand galactose were also present. Most of the carbohydrates autolyticallyreleased from the cell wall eluted in the void volume on a BioGel P2 column. However, carbohydrates released from inactivecell walls by the protein extract mostly eluted in the monosaccharideuronic acid and glucose peaks. The autolytic activity of isolatedcell walls, as well as the glycosylhydrolase activity of theproteins extracted from the cell walls, showed important changesduring fruit development. The differences between autolyticactivity and the glycosylhydrolase activity against the cellwall suggest that the glycosylhydrolases ‘in muro’are subjected to some regulatory mechanism which disappearswith their extraction. Finally, the role of glycosylhydrolases,such as polygalacturonases and galactosidases, in relation tocell wall changes in fruits, is discussed.Copyright 1998 Annalsof Botany Company Actinidia deliciosa; autolysis; cell wall enzymes; fruit growth; glycosylhydrolases; kiwifruit.     

Fine Structure of Hydrolysed Primary Walls in Tracheary Elements of Petiolar Xylem in Eucalyptus delegatensis

The hydrolysed lateral primary walls of tracheary elements ofthe petiolar xylem of Eucalyptus delegatensis were examinedby electron microscopy. Vessel-vessel and vessel—tracheidhydrolysed walls were strikingly different in appearance fromtracheid—tracheid walls. The difference seemed to be inthe degree to which the primary walls were hydrolysed. The observationssuggest the wall hydrolysis to be an ordered and controlledprocess. Eucalyptus delegatensis, hydrolysed wall, petiolar xylem, tracheary elements     

The Relations of Haemoglobin and Lignin-like Compounds to Acetylene Reduction in Symbiotic Casuarina

Nodules from six Casuarina symbioses (Casuarina cunninghamiana,C. equisetifolia, and C. glauca inoculated with each of twodifferent Frankia sources) were evaluated for (i) concentrationsof haemoglobin (measured as CO-reactive haem) at 124, 144 and165 d after inoculation, (ii) acetylene reduction and (iii)occurrence of lignin-like compounds in cell walls of the nodulesat 165 d after inoculation. Haemoglobin concentration and theoccurrence of lignin-like compounds were related to acetylenereduction at final harvest. Concentration of haemoglobin innodules ranged from 1.56 to 22.27 nmol haem (g FW)^-1. Therewere marked plant species-Frankia interactions. At final harvestplant growth was greatest in C. cunninghamiana inoculated withFrankia inoculum designated SI, while haemoglobin concentrationwas intermediate in this symbiosis. Acetylene reduction activitywas detected in all the symbioses except C. equisteifolia inoculatedwith SI, and was highest in C. glauca inoculated with SI. Haemoglobinconcentration per fresh weight nodule and acetylene reductionper plant were positively correlated (r= 0.84, n= 12). The highestlevels of cumulative nitrogen fixation were always associatedwith the highest haemoglobin concentrations and the highestrates of acetylene reduction activity. Nitrogen content waslowest in C. equisetifolia inoculated with SI (23.89±3.07mg) and highest in C. cunninghamiana inoculated with the sameinoculum (217.2±22.5 mg). Irrespective of symbiotic performance,lignin-like compounds were found in the cell walls of the nodulesin all symbioses in this study. The cells containing these compoundswere mainly localized in the infected areas of the nodule. Theroles of haemoglobin and occurrence of lignin-like compoundsin relation to nitrogen fixation in Casuarina symbioses arediscussed.     

THE CHEMICAL COMPOSITION OF THE CELL WALL OF CHLAMYDOMONAS GYMNOGAMA AND THE CONCEPT OF A PLANT CELL WALL PROTEIN

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.     

Junction Complexes between Sieve Tubes in the Secondary Phloem of Myrtaceae

Junction complexes of unusual structure form between neighbouringsieve tubes in the secondary phloem of Eucalyptus species. Thick-walledribs support thin-walled ‘sieve areas’. In longitudinalsections the structures have a ‘concertina’- likeappearance. They are relatively large, up to 0.2 mm in length.Electron micrographs confirmed that the structures consistedof thin-walled areas perforated with pores, supported by muchthicker ribs. The structures provide a vast surface area fortransfer of metabolites between sieve tubes compared with thatof lateral wall sieve areas of other plants. Hydrolysis of parenchymacell walls occurs during the development of the junction complexes.The structures are only found when sieve tubes are in closeproximity and it is the redifferentiation and partitioning ofintervening parenchyma cells which result in junction complexformation. A survey for the presence of the structures in thephloem of other genera in the family Myrtaceae was made andthey were found in Tristania and Angophora but were not observedin Acmena and Metrosideros. Eucalyptus, sieve tubes, lateral walls, ultrastructure