Changes in Two Forms of Membrane-Associated Cellulase during Ethylene-Induced Abscission

Only one form of membrane-associated cellulase was found previously in the lower petiolar pulvinus of Phaseolus vulgaris (cv Red Kidney). The cellulase has an isoelectric point (pI) of 4.5 (DE Koehler, LN Lewis 1979 Plant Physiol 63: 677-679). This enzyme was detected in abscission zones collected before the onset of abscission (control tissue), and was thought to represent a pre-secretory form of another cellulase, the abscission cellulase, which has a basic pI and is secreted during abscission. We now show that this acidic, membrane-associated cellulase is a glycoprotein, tightly bound to the membrane, with maximum activity at pH 5.1, and that it is not immunologically related to the abscission cellulase. Furthermore, when bean explants are induced to abscise with ethylene, the activity of the acidic cellulase declines rapidly to 50% of control levels in the first day. When abscission is fully developed, the membranes contain a basic form of cellulase with a pI of 8.0 to 9.0 and only trace levels of the acidic cellulase. The basic form is not a high mannose glycoprotein; it has maximum activity in a broad pH range (4.0-8.0) and is antigenically related to the abscission cellulase, which is induced during abscission and transported to the cell wall. Antibody raised against the abscission cellulase recognized two proteins in a crude membrane fraction from abscising tissue. One of those proteins comigrated with the abscission cellulase, and the other was 1 to 2 kilodaltons larger. Thus, during abscission, the acidic membrane-associated cellulase rapidly declines before the appearance of the abscission cellulase. We conclude that there is no conversion from the acidic cellulase to the basic cellulase and suggest that the acidic and basic cellulase isoenzymes are proteins derived from two different genes.     

Anatomical Changes and Immunolocalization of Cellulase during Abscission as Observed on Nitrocellulose Tissue Prints

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.     

Distribution of an Indoleacetic Acid-oxidase-inhibitor in the Storage Root of Daucus carota

Indoleacetic acid (IAA)-oxidase from both secondary phloem and xylem was dependent on 2,4-dichlorophenol for activity, and was enhanced by addition of Mn²⁺. The pH optimum was 6.0 from both tissues. IAA-oxidase and its inhibitors were distributed differently in the secondary phloem and secondary xylem of carrot root. In the phloem a high IAA-oxidase activity was distributed uniformly along the radius but in the xylem a somewhat lower concentration decreased from the cambium. IAA-oxidase inhibitor in the phloem increased exponentially from a very low concentration near the cambium, whereas in the xylem an appreciable concentration was present near the cambium, decreasing linearly with distance from the cambium. Longitudinal gradients in the xylem parallel studies by other workers with the greatest IAA-destroying capacity present in older tissues. In the xylem inhibitor decreased and IAA-oxidase increased from the root apex. In the phloem IAA-oxidase was uniform, whereas the inhibitor increased in older tissue.

The IAA-oxidase inhibitors in phloem and xylem may be different. In the xylem the IAA-oxidase inhibitor may be a lignin precursor present in young cells which disappears as lignification proceeds. In the phloem IAA-oxidase reacting with endogenous IAA appears to form a physiologically active product.

     

Characteristics of lysosomes in the rat placental cells

Six acid hydrolases, cytochrome oxidase, and alkaline phosphatase were demonstrated in 0.25 m sucrose homogenates of rat chorioallantoic placenta. The acid hydrolases were: acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, and acid deoxyribonuclease, showing optimum activity near pH 4.5; cathepsin, with optimum activity near pH 3.6. The free acid hydrolases present in cytoplasmic extracts expressed 20–40% of their total activity. “Total” activity was defined as the enzyme activity observed in the presence of Triton X-100, while “free” activity denoted enzyme activity measured under similar assay conditions except in the presence of sucrose and absence of Triton X-100. The decreased activity or latency in the assays for the free activity of acid phosphatase, acid deoxyribonuclease, and cathepsin persisted after incubation at pH 5 and 37 ° up to an hour. In contrast, this latency did not persist after incubation of the β-glycosidases. Additionally, the free activity of all the designated enzymes of the cytoplasmic extract was in excess of the nonsedimentable activity observed.     

Endogenous and exogenous ethylene induces needle abscission and cellulase activity in post-harvest balsam fir (Abies balsamea L.)

Post-harvest needle loss is a major problem for balsam fir and other Christmas tree species. Recent evidence has implicated ethylene as a signal responsible for post-harvest needle abscission, but enzymological changes remain unknown. The objective of this study was to identify and quantify cellulase activity associated with endogenous and exogenous ethylene-induced abscission. An experiment was designed with three treatments (control, endogenous ethylene, or exogenous ethylene) with five replicates. Key response variables include needle retention duration, xylem pressure potential, ethylene evolution rate, and cellulase activity. Two complimentary methods were used to assess cellulase activity: a cellulose plate digestion and zymography. The results confirm ethylene as a signal for post-harvest abscission and identify ethylene-induced cellulase. Ethylene evolution was typically between 15 and 16 μL g⁻¹ h⁻¹, but there was no difference among the three treatments. However, exogenous ethylene significantly decreased needle retention by 60% and resulted in a sixfold decrease in xylem pressure potential. In addition, cellulase activity increased by 8- and 12-fold in endogenous and exogenous ethylene-induced abscission, respectively, compared to the control. Identification of ethylene-induced cellulase activity has increased our understanding of the post-harvest needle abscission process and confirms ethylene’s role as a signal molecule.     

The Effects of pH, Ionic Strength, and Ethylene on the Extraction of Cellulase from Abscission Zones of Citrus Leaf Explants

The solubility of cellulase extracted from the abscission zones of citrus leaf explants (Citrus sinensis L. Osbeck) in sodium phosphate buffer depends on the pH of the extracting solution and, to a lesser extent, on the ionic strength. By increasing molarity from 0.01 to 0.16, the solubility of cellulase increased from 51% to 89% at pH 6.1 and from 70% to 98% at pH 7. In all cases, residual cellulase was further extracted from the pellet by buffer containing 1 m NaCl. Most of the enzymic activity was found in tissues proximal to the separation line, and activity of the cellulase which was soluble in phosphate buffer was closely correlated with abscission at both pH values. When extraction of cellulase at pH 6.1 with phosphate buffer was followed by a reextraction of the pellet with buffer containing 1 m NaCl, the activity of the cellulase soluble in the fortified buffer was also correlated with abscission. Pretreatment of explants with ethylene increased the solubility of cellulase in the phosphate buffer regardless of the pH used at the first extraction.     

The Vascular Pattern of Italian Ryegrass (Lolium multiflorum Lam.) 2. Development of the Seedling Axis to Maturity

The vascular system present in a grass seedling axis persistsin a functional state at the base of a maturing plant, but undergoesa number of modifications. Two strands of phloem, accompanied by some internal xylem, differentiatein association with the bicollateral mesocotyl trace at rightangles to the existing phloem, resulting in a tetrarch bundle.Lateral seminal roots are themselves tetrarch and the vascularinsertion of a seminal root on to the mesocotyl is a distinctivethree-dimensional feature. At the base of the mesocotyl thetetrarch bundle merges with the tetrarch bundle of the primaryseminal root via a transition zone. The four phloem poles uniteand then diverge again; the central xylem strand splits intothree and then reunites, the two tissues being intimately interlockedby this rearrangement. The additional vascular tissue of the mesocotyl extends up intothe coleoptilar node and becomes involved in the vascular attachmentof nodal roots at this point. Additional vascular tissue continuesto differentiate in the periphery of the maturing stem and ishere termed the ‘peripheral plexus’. In the seedling, the xylem of the ‘bridge’ linkingthe mesocotyl trace with the scutellar trace is associated withxylem transfer cells and also contains tracheids with distinctive,thin-barred scalariform thickening. These transfer cells disappearas the plant matures but numerous tracheids with thin-barredscalariform thickening are then to be found. The possible significanceof transfer cells in the coleoptilar node is discussed.     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.     

Comparative Studies on Differentiation of Regenerated Tissue in Broussonetia papyrifera

This paper describes the differentiation process of regenerated tissue after ordinary girdling or after removal of a section of xylem from the stem, and the disparity in differentiation of the regenerated tissues after being differently treateds in Broussonetia papyrifera. After ordinary girdling for 3–4 weeks, new bark regenerated in the xylem. During the process of rind' formation, many specks of meristematic tissue were formed in the callus, from which vascular tissue clusters were developed. In addition, the new periderm appeared almost at the same time as the new vascular cambium was seen. When a section of xylem was removed from the stem, numerous calli developed rapidly on the inner surface of the bark. Meanwhile, the vascular cambium appeared in the immature phloem. Soon after, discontinued meristematic tissue bands also occurred in the callus. These meristematic tissues then connected with each other to form a concave oblate cambial ring which developed xylem inward and phloem outward. About 2–3 weeks later, the concave oblate trunk grew lengthwisely connecting with the upper anct lower portions of the normal stem. By then, the tree continued to grow. The inner surface tissue of the bark, after the xylem was removed, differentiated about one week earlier than the tissue on the surface of the xylem after girdling.     

Patterned cell determination in a plant tissue: the secondary phloem of trees

The secondary vascular tissues (xylem and phloem) of woody plants originate from a vascular cambium and develop as radially oriented files of cells. The secondary phloem is composed of three or four cell types, which are organised into characteristic recurrent cellular sequences within the radial cell files of this tissue. There is a gradient of auxin (indole acetic acid) across both the cambium and the immediately postmitotic cells within the xylem and phloem domains, and it is believed that this morphogen, probably in concert with other morphogenic factors, is closely associated with the determination and differentiation of the different cells types in each tissue. A hypothesis is developed that, in conjunction with the positional values conferred by the graded radial distribution of morphogen, cell divisions at particular positions within the cambium are sufficient to determine not only each of the phloem cell types but also their recurrent pattern of differentiation within each radial cell file.     

INFLUENCE OF PHLOEM BLOCKAGE ON CAMBIAL GROWTH OF SUGAR MAPLE

Circular patches of bark were surgically isolated on the sides of sugar maple (Acer saccharum Marsh.) trees at breast height at various times during the dormant and growing seasons. Subsequently, samples of wood and attached bark were taken from isolated and control sites to determine the effects of isolation of the bark on cambial activity and xylem and phloem development. In control sites cambial activity and xylem and phloem development occurred normally. Isolation of bark during the dormant season (in November, February, or March) prevented initiation of cambial activity and xylem and phloem development in isolated areas of half of the trees. Varying degrees of cambial activity (periclinal divisions) occurred in the remaining isolated areas, but normal cambial activity and xylem and phloem development were prevented. Isolation of bark after initiation of cambial activity and phloem differentiation, but prior to initiation of xylem differentiation, resulted in the formation of very narrow xylem and phloem increments with atypically short vessel members and sieve-tube members, respectively. The xylem increments consisted primarily of parenchyma cells. Isolation of bark after initiation of xylem differentiation resulted in curtailment of secondary wall formation in the last-formed part of many increments. The last-formed vessel members of all these xylem increments were atypically short. Similarly, the last formed sieve-tube members of corresponding phloem increments were atypically short. The atypically short cells in the xylem and phloem of isolated areas reflected the effect of isolation on the cambial region, viz., the subdivision of all fusiform cells into strands of cells. Ultimately, the strands of short fusiform cells lapsed into maturity, leaving only strands of parenchymatous elements between xylem and phloem.     

Abscission: role of cellulase

Cellulase (β-1,4-glucan-glucanohydrolase EC 3.2.1.4) activity increased during abscission and was localized in the cell separation layer of Phaseolus vulgaris L. cv. Red Kidney (bean), Gossypium hirsutum L. cv. Acala 4-42 (Cotton) and Coleus blumei Benth. Princeton strain (Coleus) abscission zone explants. Cellulase activity was optimum at pH 7, was reduced by one-half after heating to 55° for 10 min, and was associated with the soluble components of the cell. Explants treated with aging retardants (indoleacetic acid, ⁶N-benzyladenine, and coumarin), CO₂, actinomycin D or cycloheximide had less cellulase activity than untreated controls. Ethylene increased cellulase activity of aged explants after a 3-hr lag period but had no effect on cellulase activity of freshly excised explants. It was concluded that 1 of the roles of ethylene in abscission is to regulate the production of cellulase which in turn is required for cell separation.     

Ganglioside biosynthesis. Characterization of uridine diphosphate galactose: GM2 galactosyltransferase in golgi apparatus from rat liver.

An enzyme that transfers galactose from UDP-Gal to ganglioside GM2 (Tay-Sachs ganglioside) was concentrated 50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents or phospholipids as dispersing agents. Of the numerous detergents tested, sodium taurocholate and Triton CF-54 were most effective in stimulating the reaction. Cardiolipin alone was more effective than any of the detergents tested in stimulating enzyme activity. The pH optimum for the reaction varied with the nature of the dispersing agent. With sodium taurocholate, Triton CF-54 and cardiolipin, the pH optima were 6.2, 5.9, and 5.6, respectively. The enzyme had a nearly absolute requirement for Mn2+, with maximum activity being attained at a concentration of 15 mM Mn2+. Other divalent or trivalent cations were either less effective than Mn2+ or inhibited the transferase reaction. The Km values calculated for UDP-Gal and GM2 were 1.1 X 10(-4) M and 9.9 X 10(-5) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane and not part of the luminal contents. The newly synthesized GM2, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.     

Stem anatomy of Dolichos lablab Linn (Fabaceae): Origin of cambium and reverse orientation of vascular bundles

Secondary growth in the stem of Dolichos lablab is achieved by the formation of eccentric successive rings of vascular bundles. The stem is composed of parenchymatous ground tissue and xylem and phloem confined to portions of small cambial segments. However, development of new cambial segments can be observed from the obliterating ray parenchyma, the outermost phloem parenchyma and the secondary cortical parenchyma. Initially cambium develops as small segments, which latter become joined to form a complete cylinder of vascular cambium. Each cambial ring is functionally divided into two distinct regions. The one segment of cambium produces thick-walled lignified xylem derivatives in centripetal direction and phloem elements centrifugally. The other segment produces only thin-walled parenchyma on both xylem and phloem side. In mature stems, some of the axial parenchyma embedded deep inside the xylem acquires meristematic activity and leads to the formation of thick-walled xylem derivatives centrifugally and phloem elements centripetally. The secondary xylem comprises vessel elements, tracheids, fibres and axial parenchyma. Rays are uni-multiseriate in the region of cambium that produces xylem and phloem derivatives, while in some of the regions of cambium large multiseriate, compound, aggregate and polycentric rays can be noticed.     

Experimental induction of vascular tissue in an undifferentiated plant callus

1. By the implantation of wedges containing indol-3-ylacetic acid and sucrose into blocks of undifferentiated bean-callus tissue it has been possible to induce the formation of xylem and phloem cells. 2. The differentiation has been investigated cytologically and measured chemically. 3. The optimum concentrations of the nutrients in the wedge, which gave differentiation closely resembling the vascular development found in the stem of the intact plant, was 0.1mg. of indol-3-ylacetic acid/l. and 2% sucrose. 4. The ratios of the xylose/arabinose concentrations of the tissues increased in the differentiated callus tissue compared with those of the undifferentiated tissue. A similar increase has been found for the ratios determined for xylem tissue compared with those for cambium. 5. The lignin content of the differentiated tissue compared with the undifferentiated tissue was greater in both the callus and stem tissue. 6. Chemical analysis of lignin showed that in the differentiated callus tissue it consisted of sub-units based on p-hydroxybenzaldehyde and vanillin. This was compared with the lignin obtained from undifferentiated callus tissue and that obtained from the tissues of the intact stem. 7. The results of the investigation have been discussed with reference to the problems of cell growth and differentiation and related to the changing patterns of the ultrastructure of the cell during its development.     

Regulation of Vascular Development by CLE Peptide-receptor Systems

Cell division and differentiation of stem cells are controlled by non-cell-autonomous signals in higher organisms. The plant vascular meristem is a stem-cell tissue comprising procambial cells that produce xylem cells on one side and phloem cells on the other side. Recent studies have revealed that TDIF (tracheary element differentiation inhibitory factor)/CLE41/CLE44 peptide signal controls the procambial cell fate in a non-cell-autonomous manner. TDIF produced in and secreted from phloem cells is perceived by TDR/PXY, a leucine-rich repeat receptor kinase located in the plasma membrane of procambial cells. This signal suppresses xylem cell differentiation of procambial cells and promotes their proliferation. In addition to TDIF, some other CLE peptides play roles in vascular development. Here, we summarize recent advances in CLE signaling governing vascular development.     

EFFECTS OF TREE AGE ON SECONDARY XYLEM AND PHLOEM ANATOMY IN STEMS OF GREAT BASIN BRISTLECONE PINE (PINUS LONGAEVA)

Xylem and phloem tissue samples were collected from various-aged Great Basin bristlecone pine (Pinus longaeva D. K. Bailey) stems in southern Utah and southeastern California to determine whether the vascular cambia of older trees produce fewer xylem rays, shorter-lived xylem and phloem ray cells, fewer phloem sieve cells, and a thinner phloem. Increment cores were examined to determine whether ‘aged’ cambia produced narrower tracheids that might reduce water translocation. Sapwood thickness was measured and sapwood growth layers were counted on these cores. Regression and Classification and Regression Tree (CART) analyses of sample data found no age-related changes in cambial products. Phloem and xylem production appeared normal at all ages, with no evidence of cambial malfunction.