A Comparative Study of the Changes in Root Growth, Induced by Coumarin, Auxin, Ethylene, Kinetin and Gibberellic Acid

The effect of coumarin, IAA, ethylene, kinetin and gibberellic acid on roots of maize and wheat was investigated. Sterile attached and detached roots and isolated elongation zones were used. In some experiments a semi-sterile procedure was followed. The effects of the different regulators separately or in various combinations together with coumarin were studied on the root growth with regard to division, elongation and swelling of the cells. The ethylene production in isolated elongation zones was measured after treatment with coumarin, IAA, PCIB, kinetin, colchicine and dinitrophenol. The results show the following: 1) Each substance produces a specific morphologic pattern. 2) Changes in polarity were demonstrated for auxin-induced swelling in cell divisions and cell expansion and for coumarin-induced swelling in cell divisions. Other cell expansion in swollen parts was due to cylindric cells increasing in width while retaining their cylindric form. 3) Coumarin-induced inhibition could not be counteracted by IAA, PCIB, carbon dioxide, kinetin, gibberellic acid or Cycocel. 4) The ethylene production in isolated elongation zones increases noticeably after kinetin treatment, less strongly after auxin treatment and least after coumarin treatment. The production of ethylene does not seem to be correlated with the morphogenetic effect of the different substances. 5) The isolated elongation zones reacted to a) IAA and kinetin with an increase in length in some cases and b) gibberellic acid with a reduction of their width. 6) The inhibitory effect of coumarin on the growth in length of the elongation zones was diminished by kinetin. The swelling produced by coumarin in these zones was reduced by gibberellic acid. The effects just mentioned of kinetin and gibberellic acid were considered as indirect ones. - From the present findings it was concluded that concomitant effects of auxin, ethylene, cytokinins and gibberellins are not obligatory for coumarin to exert its morphogenetic effects on root growth. In discussing the results some pitfalls in studies of growth reactions after application of hormones to roots containing meristem were emphasized.     

Electron microscopy of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to oxygen shortage

This paper examines the ultrastructure of cortical cells in maize root tips during the early stages in lysigenous aerenchyma formation, promoted by oxygen-deficient nutrient solution. The aim was to determine whether changes in fine structure were compatible with oxygen starvation as the primary cause of cell degeneration and death. There was an initial collapse of some cortical cells, indicating loss of turgor, and the cytoplasm became more electron dense. Mitochondria and endoplasmic reticulum appeared normal at this early stage though the tonoplast lost its integrity. Subsequently the cytoplasm became less electron dense than surrounding healthy cells, and underwent further degeneration while the plasmalemma retracted from the cell wall. Cell walls remained unaltered until this stage, but some then became thin and electron transparent. No cells of the stele were found to degenerate. These observations, which do not readily accord with the hypothesis that oxygen starvation was the cause of cell death, are compared with detailed studies of cell degeration in other cell types. An alternative mechanism for the stimulation of cortical cell lysis in poorly oxygenated roots involving the hormone ethylene, is discussed.     

Change in Size and Cell Number during the Development of Lateral Root Primordia in Zea mays L.

Increase in cell number, anlage volume and length have beeninvestigated during the overall period of lateral root primordiumdevelopment in excised primaries and in attached roots of Zeamays L. Each of these aspects of anlage growth was found toincrease more or less exponentially during the interval betweenprimordium initiation and subsequent emergence as a lateralin both batches of roots. Values were then determined for celldoubling time (Td), the size of the proliferative fraction (Pf),and for anlage volume (Tv) and length (Tt) doubling times duringthe overall period of primordium development and at intervalsduring this period in both the excised and attached roots. Thepattern of change which took place in Td, Tv, Tl and Pf duringlateral primordium development was found to be similar in bothbatches of roots. However, the overall period of anlage developmentwas shorter in the excised roots than in the attached ones.Moreover, when laterals grew out of the excised roots they didso with fewer cells than comparable laterals emerging from theattached roots. Zea mays L., maize, root primordia, lateral emergence, cell doubling time     

Transfer Cells in Roots of Phaseolus coccineus: Ultrastructure and Possible Function in Exclusion of Sodium from the Shoot

A study was made of ultrastructural aspects and ion distributionin roots of Phaseolus coccineus as affected by NaCl and Na₂SO₄salinity. In the proximal region of the root, xylem parenchymacells are differentiated as transfer cells with well developedwall protuberances adjacent to the half-bordered pits of thevessels. The cytoplasm of these transfer cells contains cisternaeof rough endoplasmic reticulum, the number of which was increasedgreatly when the plants were grown in the presence of NaCl orNa₂SO₄. The cisternae of the endoplasmic reticulum are oftenassociated closely with the plasmalemma and interconnected withit by fibrillar bridges. Wall protuberances occur also in the exodermis and epidermisof the more apical region of the root. Their function is stillunknown. P. coccineus excludes Na, but not Cl, from the leaves by retainingit particularly in the proximal region of the root. X-ray microanalysisof unfixed, frozen, hydrated specimens revealed that the transfercell-type xylem parenchyma cells in salt-treated roots accumulatedNa relative to both the adjoining xylem vessels and the corticalcells and showed very high Na/K and Na/Cl ratios. It is suggestedthat the xylem parenchyma cells can reabsorb Na from the vessels,probably in exchange for K, and that Na exclusion from the shootis at least partly mediated by this process. The implicationof this for regulation of salt transport in salt sensitive glycophytesis discussed.     

The Effect of Coumarin on Growth, Production of Dry Matter, Protein and Nucleic Acids in Roots of Maize and Wheat and the Interaction of Coumarin with Metabolic Inhibitors

Effects of coumarin on fresh weight, dry matter, protein and nucleic acid content per cell in attached roots of maize and wheat and in whole excised elongation zones of maize were determined. The inhibition in cell length exerted by coumarin did not correspond to an inhibition of the net synthetic capacity. Coumarin treatment increased the cell surface, the production of dry matter and the protein content per cell. The dry matter and the protein content per unit surface was slightly increased or unaffected. The effect of coumann on cell shape seemed to be independent of that on dry matter production and net protein synthesis. The same was found in excised elongation zones. —The net DNA-synthesis per cell was slightly increased in attached roots by coumann treatment, but this effect was probably not correlated with the morphogenetic changes. Inhibition of DNA-synthesis with hydroxyurea did not alter the coumarin induced changes in cell shape. —The net RNA-synthesis per cell was slightly decreased after coumarin treatment, but the net RNA-synthesis per cell and the morphogenetic effects exerted by coumarin were not related with each other. Inhibition of m-RNA-synthesis with actinomycin D did not prevent the effects of coumarin on cell division, cell expansion, dry matter production and net protein synthesis. The same was true for inhibitors of protein synthesis, puromycin and p-fluorophenyl-alanine. The findings are in support of the view that coumarin affects already existing structures or enzymes. —Comparisons between coumarin and the uncouplers, DNP and dicoumarol, showed that the effects of coumarin were not, solely, due to uncoupling. SH-protecting agents, BAL, DTE and glutathione, did, with few exceptions, not reduce the morphogenetic effects of coumarin.     

Preservation of the Tonoplast in Metaphloem Sieve Elements of Embryonic Roots of Zea mays L.

Decortication of embryonic roots of 4- to 5-day-old Zea seedlingsand subsequent chemical fixation permitted comparison of cutand uncut developing sieve elements In a decorticated root wheresieve tubes are not severed, metaphloem sieve elements in latestates of development and some mature sieve elements exhibita highly vacuolate condition When roots are cut or diced inthe course of fixation intact vacuoles are not observed in latestages of sieve-element ontogeny The degree of callose formationat sites of developing sieve-plate pores and in the pores ofmature sieve elements varies greatly with both decorticationand non-decortication treatments Nuclei were not observed insieve elements at the electron microscope level, but they wereseen at the light microscope level in serial sections of sieveelements in the late to mature developmental stages representedAlthough the occurrence and distribution of plastids, mitochondria,endoplasmic reticulum, dictyosomes and nbosomes also vanes insieve elements of decorticated roots, disruption or surgingof sieve-element contents is greater for sieve tubes that aresevered during fixation treatment A discussion is presentedrelating effects of trauma on observed developmental stagesand sieve-element structure Zea mays L, maize, corn, phloem, Sieve elements, tonoplast, ultrastructure     

Responses of the Root Systems of Sunflower and Maize to Unidirectional Stem Flexure

Plants of two contrasting species of herbaceous annuals, thedicot sunflower (Helianthus annuusL.) and the monocot maize(Zea maysL.), grown in the glasshouse were subjected to regularunidirectional stem flexure. Differences in morphology and mechanicalproperties of roots and shoots were then investigated. Rootsystems were divided into quadrants around the axis of stimulationand differences in root morphology and mechanics between thezones were investigated. There were considerable differencesbetween roots in the leeward and windward zones compared withroots perpendicular to the axis of stimulation. First-orderlateral roots in both species were thicker, more rigid and morenumerous. These results suggest that plant roots respond locallyto mechanical stimulation. There were, however, also differencesin the responses of the two species. In sunflower, the tap rootand stem base became elliptical in cross section with the majoraxis lying in the plane of stimulation. The lateral roots offlexed sunflowers in both the leeward and windward zones showedsimilar growth responses: roots were thicker, more numerousand weighed more than those in the perpendicular zones. However,only leeward roots showed significant differences in their mechanicalproperties; roots were more rigid, stronger and stiffer. Incontrast, the leeward roots of maize were thicker and more numerous,with a greater biomass than the windward roots. However, onlyroots in the windward zone were stiffer than those in the perpendicularzone. These differences between sunflower and maize are relatedto their contrasting anchorage mechanics.Copyright 1998 Annalsof Botany Company Anchorage, biomechanics, adaptive growth, roots, thigmomorphogenesis,Helianthus annuusL.,Zea maysL.     

Ultrastructure and anatomy of nematode-induced syncytia in roots of susceptible and resistant sugar beet

Summary Using susceptible and resistant sugar beet lines, comparative analyses of root histology and ultrastructure were made during invasion by nematodes and the induction and formation of specific feeding structures (syncytia).The resistant line carried the resistance geneHs1^pro–1.Nematodes were able to invade and induce functional syncytia in roots of resistant and susceptible lines. However, syncytia in resistant roots were smaller and less hypertrophied. The vacuolar system of syncytia in susceptible plants contained many small vacuoles. In resistant plants vacuoles were larger but less numerous. Smooth endoplasmic reticulum prevailed in syncytial protoplasts of susceptible plants, whereas almost only rough endoplasmic reticulum occurred in syncytia in resistant plants. The most conspicuous and hitherto undescribed trait of syncytia in resistant roots was the initial appearance of loose, and later compact, aggregations of the endomembrane system which composed most of the endoplasmicreticulum system of syncytia at later stages. Syncytia in resistant plants usually degraded before the nematodes reached their adult stage. The appearance of membrane aggregations and the other resistance-specific features are discussed in relation to their possible effects on syncytium function and role in nematode resistance.Abreviations DAI days after inoculation - ER endoplasmic reticulum - ISC initial syncytial cell - J2 second-stage juvenile - MA membrane aggregations - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Ammonium Tolerance and Carbohydrate Status in Maize Cultivars

Four maize (Zea mays L.) hybrids were grown hydroponically for4 weeks with 20 mM ammonium or nitrate as the sole nitrogensource. Dry matter production was strongly depressed by ammoniumnutrition in the hybrid Helga relative to plants grown on nitrate,and moderately decreased in the hybrid Melina. Ammonium hadno inhibitory effect on total yield in the other two hybrids(Ramses and DK 261). The relative growth rate (RGR) of rootsand shoots of the sensitive hybrid Helga decreased significantlyunder ammonium nutrition during the first 2 weeks of the experiment,while at the end of the experiment nitrogen form had no effecton the RGR in any of the four hybrids. The strong reductionin RGR of Helga in the early seedling stage was correlated withthe accumulation of twice the concentration of free ammoniumin the shoot tissue relative to the other hybrids. Helga wastherefore unable to sufficiently detoxify ammonia in the roots.Root concentrations of water soluble carbohydrates (WSC) inHelga and Melina in the early seedling stage did not differunder ammonium and nitrate nutrition. In contrast, Ramses andDK 261 both had elevated WSC concentrations in ammonium-fedroots. It is hypothesized that a sufficient supply of carbonskeletons for ammonium assimilation in the roots is requiredfor maximum growth under high ammonium concentrations, and thatthere is genotypic variability in this physiological trait. Ammonium; carbohydrates; growth rate; maize; nitrate; roots; Zea mays L     

Vacuolar symplast formation is due to highly permeable gap junctions between the tonoplast and endoplasmic reticulum membrane

An NMR method with a pulsed magnetic field gradient was applied to study changes in water permeability of the vacuolar symplast in maize (Zea mays L.) seedling roots treated with various inhibitors of cell metabolism. The results were qualitatively analogous to literature data on conductivity changes of intercellular gap junctions in animal cells exposed to similar treatments. Electron microscopy examination of root cells provided evidence for the existence of membrane contacts between the endoplasmic reticulum and the tonoplast. It is supposed that vacuoles of neighboring plant cells are interconnected through highly dynamical gap junctions between the tonoplast and the endoplasmic reticulum membrane.     

The Development of Protein Bodies in the Storage Tissues1

Homogenates of the developing endosperms of maize, wheat, andbarley and of developing cotyledons of peas have been separatedon sucrose density gradients. Protein bodies have been recognizedby their content of storage proteins, their density, and theirappearance under the electron microscope. The distribution ofthe endoplasmic reticulum was followed by measuring NADH-cytochromec reductase. Glutamate dehydrogenase, catalase, and triose phosphatedehydrogenase—respectively marker enzymes for mitochondria,microbodies, and plastids—were also present in the gradients.Differences were found between the different species with respectto the association between the endoplasmic reticulum and thestorage proteins. There was no evidence for any vacuolar markerenzymes associated with the cereal protein bodies. The proteinbodies of wheat and barley appear to be similar to each otherbut differ from maize in that the latter have much more endoplasmicreticulum associated with them. In contrast the protein bodiesof peas co-sediment with vacuolar marker enzymes.     

Purification, sequencing and functions of calreticulin from maize

The most abundant proteins in the lumen of the endoplasmic reticulum(ER) are thought to be molecular chaperones, some of which mightalso be involved in calcium storage and release. We have purifiedcalreticulin from maize by ion exchange and reverse-phase chromatography.Identity with plant and animal calreticulins was confirmed byN-terminal amino acid sequencing and it was shown to bind calciumwith a calcium overlay technique. An antiserum raised to thepurified protein was used to screen an expression library andthe full coding sequence for maize calreticulin was determinedfrom the clones selected. The sequence shows 96% identity tobarley calreticulin and 55% identity to animal calreticulins.The three major functional regions are conserved, as are targetingand retention features. When visualized by indirect immunofluorescencemicroscopy, calreticulin was found to be confined to the ERand nuclear envelope of maize root cells. It was distributedthroughout the ER compartment and we found no evidence of calreticulin-enriched areas of ER, such as might be associated with specializedcalcium storage domains. Increasing or decreasing extracellularcalcium did not induce measurable changes in calreticulin levels.In addition, maize calreticulin, as well as other recognizedchaperones, was shown to bind to denatured protein and couldbe eluted specifically by nucleoside trisphosphates. Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L     

Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.     

Brefeldin A-induced disassembly of the Golgi apparatus is followed by disruption of the endoplasmic reticulum in plant cells

Brefeldin A (BFA), a fungal fatty acid derivative, is a potentagent for disrupting the Golgi apparatus in plant and animalcells. We have examined its action using marker antibodies whichrecognize an epitope in the plant Golgi apparatus (JIM 84),and for proteins held in the endoplasmic reticulum by the HDELER-retention signal (2E7), in combination with double immunolabelling.In maize root cells, disruption of the ER occurs after breakdownof the Golgi apparatus is initiated. The redistribution of theGolgi is shown to be predominantly separate from that of theER, and as with the Golgi, the action of BFA on the ER is alsoreversible. The mode of action of BFA on the ER and Golgi ofplant cells is compared with that described for animal cells. Key words: Zea mays L, Brefeldin A, plant cells, endoplasmic reticulum, Golgi apparatus     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

Stages in the Initiation of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Reciprocal transfers of Nicotiana tabacum cv. Xanthi nc. leafexplants were made daily between root inducing medium (RIM)and shoot inducing medium (SIM), SIM and a basal medium containingno growth regulators (BM), and RIM and BM. It was found thatthe explants became determined for shoot production after 6d, while roots were produced after only 1 d on RIM before transferto BM. The competence of the explant to produce roots was greatlyreduced by culture on BM prior to culture on RIM. There wasfar less reduction in shoot numbers with preculture on BM. Explantswere found to be only weakly canalized for both caulogenesisand rhizogenesis for the first 2 d after determination. Thereafterthey became strongly canalized. Transfers were also made fromBM to SIM and back to BM, which revealed that the explants becamecompetent for caulogenesis in the absence of cytokinins priorto determination. The period for which SIM is required can bereduced to only 1 d. Key words: Nicotiana tabacum, in vitro, organogenesis, competence, determination     

Effects of Hydroxyurea on the Ultrastructure of Giant Cells in Galls Induced by Meloidogyne javanica

Hydroxyurea (HU) at concentrations of 10 or 20 mg/liter was included in a medium on which excised tomato roots infected with the root-knot nematode Meloidogyne javanica were grown. In the HU, treated roots, giant cells were small and contained large vacuoles. Giant cell nuclei were amoeboidal with relatively small nucleoli in treated roots, compared with giant cells of nontreated galls. In treated-root giant cells, the cytoplasm was diffuse and few organelles such as mitochondria, dictyosomes, and endoplasmic reticulum were detected; also, walls of giant cells were thin with less extensive ingrowths than in nontreated roots. We conclude that HU suppressed normal giant cell formation interfering with its function as a feeding cell.     

Cyclopropyl sterol and phospholipid composition of membrane fractions from maize roots treated with fenpropimorph

Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Δ⁵-sterols disappeared almost completely and were replaced by 9β, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Δ⁵-sterols.     

Ultrastructural Changes in Maturing Sporangia of Albugo Candida

Maturation of sporangia in Albugo sp. involved considerableinternal differentiation. There was a burst of activity in sporangiasoon after their formation when the numbers of mitochondria,and the amounts of endoplasmic reticulum increased. Perinuclearvesicles and smooth surfaced cisternae differentiated into well-developedgolgi apparatus which remained secretory until completion ofmaturation. The cisternae of rough endoplasmic reticulum arrangedthemselves in parallel stacked arrays. Maturing sporangia hadautophagic vacuoles containing various cell organelles. Nucleardegeneration and mitosis proceeded simultaneously. All activitiesdeclined towards the end of sporangial maturation: Golgi dictyosomesbecame quiescent and numbers of mitochondria and amounts ofendoplasmic reticulum decreased. There was a three-fold increasein the thickness of the sporangial wall during maturation.     

Differentiation and Ultrastructure of the Protophloem Sieve Elements of Minor Veins in the Maize Leaves: Changes in the Protoplast

Protophloem sieve element differentiation in the minor veins of the maize ( Zea mays L. ) leaves was first evidenced as an increase of the wall thickness, which began in the comers of the cell and then extended to other parts of the wall, and the appearance of long rough endoplasmic reticulum cistemae distributed throughout the cytoplasm, and then the presence of characteristic crystalloid inclusions within the plastids. As differentiation progressed, long cisternae of rough endoplasmic reticulum appeared to transform into shorter forms and eventually aggregated into small stacks, losing their ribosomes during the process. The nuclei degenerated, although frequently persisted until very late in differentiation the stages of maturation, as darkly stained amorphous aggregates surrounded by double nuclear envelope or only inner membrane of nuclear envelope. Subsequently, the nuclear envelope collapsed and became discontinuous. At the beginning of nuclear degeneration the perinuclear spaces were partly dilated and sometimes the outer nuclear envelope in the dilated portions then ruptured, and was accompanied by the disappearance of the cytoplasmic portion near it. During the peried of nuclear degeneration, in addition to the endoplasmic reticulum, plastids and mitochondria underwent structural modification, while components such as ribosomes, cytoplasmic ground substances, vacuoles and dictyosomes disintegrated and disappeared. At maturity, the surviving protoplasmic components, including plasmalemma, mitochondria, small stacked smooth endoplasmic reticulum and P-type plastids with crystalloids, became parietal in position. As differentiation of adjacent metaphloem sieve elements proceeded, the protoplasmic components of the mature protophloem sieve elements progresively degenerated and finally obliterated.