Dissolution Of Middle Lamella Research Articles

Ultrastructural changes during nectar secretion from extrafloral nectaries of Pithecellobium dulce Benth

The structural changes in the secretory cells are important to understand the ontogeny and nectar secretion process from the nectaries. In this study, we investigated the ultrastructural changes during different developmental/secretion stages of extrafloral nectaries (EFNs) of Pithecellobium dulce. The dense cytoplasm with active biosynthesis mechanisms such as ribosomes, mitochondria, large nucleus, and plastids with accumulated starch grains characterized the pre-secretion stage of young nectariferous cells. During the secretory phase, the cytoplasm showed distinct changes associated with endomembrane transport such as the predominant occurrence of Golgi, secretory vesicles, and ER resulting in the subsequent appearance of secretions in the intercellular and subcuticular spaces. Cell wall loosening following the dissolution of middle lamellae leading to the formation of subcuticular spaces was evident during advanced stages of nectar secretion. The characteristic cytoplasmic and apoplastic changes associated with cell death were noticed during the post-secretory stages. The structural evidence from the present study suggests the occurrence of two modes of secretion (merocrine and holocrine) during the early and late stages of secretion in the EFNs of P. dulce.

Constant storage temperature delays firmness decreasing and pectin solubilization of apple during post‐harvest storage

The aim of this study was to evaluate the effect of storage temperature fluctuation (temperature variation cycles about 6 hr) on the firmness and pectin solubilization of “Fuji” and “Golden Delicious” during storage. Results showed that apples in 5 ± 0°C group showed the lowest firmness decreasing level (15.86% for “Fuji”, 24.76% for “Golden Delicious”) coupled with the minimum ethylene production, and the slowest inter-converting of chelate-soluble pectin and sodium carbonate-soluble pectin to water-soluble pectin compared with that of 5 ± 5°C and 5 ± 1°C. Moreover, the enzymatic activities (PG, PME, β-Gal, α-L-Af) were at the lowest level in 5 ± 0°C temperature fluctuation groups both of “Fuji” and “Golden Delicious”. Ultrastructural observations also revealed that constant temperature could restrain the solubilization of pectin and the dissolution of middle lamella. Besides, the impact of 5± 1°C on “Golden Delicious” was more significant relative to “Fuji”. In practical applications, the temperature supervision on “Fuji” could be appropriately relaxed compared with “Golden Delicious” during short-term storage. Novelty impact statement Constant storage temperature retarded the firmness decreasing by delaying the solubilization of cell wall pectin and suppressed the activities of cell wall degrading enzymes of “Fuji” and “Golden Delicious” cultivars. Constant storage temperature Constant storage temperature retarded the dissolution of the middle lamella between the cell walls of “Fuji” and “Golden Delicious” cultivars. “Golden Delicious” cultivar was more sensitive to the variation of storage temperature compared with “Fuji” cultivar.

Expression Kinetics of Regulatory Genes Involved in the Vesicle Trafficking Processes Operating in Tomato Flower Abscission Zone Cells during Pedicel Abscission.

The abscission process occurs in a specific abscission zone (AZ) as a consequence of the middle lamella dissolution, cell wall degradation, and formation of a defense layer. The proteins and metabolites related to these processes are secreted by vesicle trafficking through the plasma membrane to the cell wall and middle lamella of the separating cells in the AZ. We investigated this process, since the regulation of vesicle trafficking in abscission systems is poorly understood. The data obtained describe, for the first time, the kinetics of the upregulated expression of genes encoding the components involved in vesicle trafficking, occurring specifically in the tomato (Solanum lycopersicum) flower AZ (FAZ) during pedicel abscission induced by flower removal. The genes encoding vesicle trafficking components included soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), SNARE regulators, and small GTPases. Our results clearly show how the processes of protein secretion by vesicle trafficking are regulated, programmed, and orchestrated at the level of gene expression in the FAZ. The data provide evidence for target proteins, which can be further used for affinity purification of plant vesicles in their natural state. Such analyses and dissection of the complex vesicle trafficking networks are essential for further elucidating the mechanism of organ abscission.

Regulation of apricot ripening and softening process during shelf life by post-storage treatments of exogenous ethylene and 1-methylcyclopropene

Abstract Post-storage applications of exogenous ethylene and 1-methylcyclopropene (1-MCP) were performed in cold-stored apricot to regulate the ripening and softening process during shelf life. The results showed that post-storage ethylene treatment could accelerate SSC accumulation, organic acids degradation, color change and ripening process of cold-stored apricot, and could effectively improve the consumer acceptability of the fruit. Post-storage 1-MCP treatment strongly suppressed ethylene production in apricot during shelf life, causing the suppression of expression levels of pectin related genes encoding for pectin degraded enzymes, including polygalacturonase, β-galactosidase and pectin methyl esterase. Microscopic observation provided a direct evidence that post-storage treatment of 1-MCP delayed pectin degradation and middle lamella dissolution of apricot, and delayed the softening process during shelf life. The regulation of ripening and softening process of apricot with post-storage treatment of ethylene or 1-MCP could be effective for improving consumer acceptability or delaying fruit softening of the fruit.

Laticifer development and its growth mode in Allamanda blanchetii A. DC. (Apocynaceae)

Recent studies have shown the laticifers of Apocynaceae, previously classified as nonarticulated, indeed are articulated, anastomosing laticifers whose transverse walls dissolve rapidly and entirely, although doubts about their growth mode still persist. To better understand the mode of laticifer growth and differentiation in this family, we studied its development in Allamanda blanchetii using anatomical and ultrastructural analyses. Our results showed that laticifers are formed by a row of cells that join each other through dissolution of the transverse walls from the center to the periphery. The laticifers originate from ground meristem and procambium; the laticifers in the different tissues connect through lateral fusion, generating a laticifer network. The laticifers occur in the cortex, pith, and vascular system of the shoot, mesophyll, and vascular bundles of the leaves. There is no apical growth, and all the organelles observed in the apices of the laticifers play a role in the production of latex or in the dissolution of the terminal walls between the cells that compose the laticifer. The latex is composed of many metabolites produced mainly in the endoplasmic reticulum and plastids. Mitochondria are abundant, and dictyosomes are scarce. The vacuome is prominent from the start of laticifer differentiation, and many small vacuoles and vesicles transport the secretion from cytosol into the central vacuole, where an emulsion of substances is stored. The articulated, anastomosing laticifers of A. blanchetii have no subcellular mechanism for production of a cell wall in a polarized manner or dissolution of middle lamella of the cells which surround the laticifer tip, proving that there is no intrusive growth in this secretory structure.

Slow softening of Kanzi apples (Malus × domestica L.) is associated with preservation of pectin integrity in middle lamella

Kanzi is a recently developed apple cultivar that has an extremely low ethylene production, and maintains its crispiness during ripening. To identify key determinants of the slow softening behaviour of Kanzi apples, a comparative analysis of pectin biochemistry and tissue fracture pattern during different ripening stages of Kanzi apples was performed against Golden Delicious, a rapid softening cultivar. While substantial pectin depolymerisation and solubilisation was observed during softening in Golden Delicious apples, no depolymerisation or increased solubilisation was observed in Kanzi apples. Moreover, tissue failure during ripening was mainly by cell breakage in Kanzi apples and, in contrast, by cell separation in Golden Delicious apples. Kanzi apples had lower activity of beta-galactosidase, with no decline in the extent of branching of the pectin chain. A sudden decrease in firmness observed during senescence in Kanzi apples was not due to middle lamella dissolution, as tissue failure still occurred by cell breakage.

Enhanced production of pectinase by Saccharomyces cerevisiae isolate using fruit and agro-industrial wastes: Its application in fruit and fiber processing

The present study is focused on the utilization of fruit and agro-industrial wastes for the production of yeast pectinases using response surface optimization techniques. Orange peel (OP), groundnut oil cake (GC), MnSO4 and incubation period (IP) were found as significant parameters for pectinase production. The central composite design of media optimization indicated that OP (5), GC (4), MnSO4 (0.08%, w/v) and IP (48 h) increased the pectinase production by 9-fold. Crude pectinase promoted the enzymatic peeling of oranges and processing of raw banana fibers. Topographical changes of enzyme treated orange and mango peels were studied using AFM which provides a new tool to unravel the role of pectin aggregation in the dissolution of middle lamella during enzymatic hydrolysis. Changes in the fingerprint regions of enzyme treated fruit substrates were observed along with the disappearance of the impurities such as waxes and pectin using FTIR spectral analysis.

Relating consumer preferences to textural attributes of cooked beans: Development of an industrial protocol and microstructural observations

The effects of temperature, duration of cooking and different cooking media (salt solutions) on bean-texture attributes were evaluated by response surface methodology. Sensory evaluation and scanning electron microscopy (SEM) were also carried out to clarify the properties of cooked beans. The sensory evaluation results showed that consumers most prefer beans with a soft texture. A 24 full-factorial central composite design (CCD) was used to optimize the process conditions. For each response, a second-order polynomial model was developed using multiple linear regression analysis. Applying the desirability function method, the most suitable combination of process conditions to obtain the texture found most acceptable were a cooking temperature of 75 °C, a cooking time of 63 min, NaCl concentration of 1.88 g/l and CaCl2 concentration of 0.12 g/l. RSM analysis indicated good correlation between experimental and predicted values. SEM studies of cooked bean tissues further confirmed that cell-wall separation was mainly the result of pectin solubilization and middle lamella dissolution. An important finding obtained in this study was determining the optimum salt concentration needed to soften the grain in a minimum period of time; this is important from an economic as well as from an industrial point of view.

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars

Activity and expression of polygalacturonase (PG), a hydrolytic enzyme involved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum), were investigated at different ripening stages of the pepper cultivars Mandi and Talanduo. Molecular cloning of CaPG was carried out by constructing a cDNA library from three stages of fruit ripening. Morphological determination, PG assay, RT-PCR, and ultrastructural studies were used to quantify changes in CaPG gene expression in the pericarp from green, color change and fully ripened stages. We found that CaPG gene expression, PG activity and striking changes in the structure of the cell wall occurred with the transition of ripening stages. CaPG gene expression was high (obvious PCR products) in mature and ripened stages of both cultivars; however, the CaPG gene was not expressed in preclimacteric fruits or vegetative tissues. We conclude that developmental regulation of CaPG gene expression is instrumental for sweet pepper fruit ripening; its expression during development leads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall.

Comparative study on the delignification of Azadirachta indica (L) Del., wood by Chrysosporium asperatum and Trichoderma harzianum

Structural alterations induced in response to degradation by two white rot Basidiomycetes on the secondary xylem of Azadirachta indica (L) Del., was compared. In vitro decay test was employed to investigate the pattern of delignification of Azadirachta wood by Trichoderma harzianum and Chrysosporium asperatum. Wood samples inoculated with both the strains were analyzed for different periods viz. 30, 60, 90 and 120 days after fungal inoculation. Initially there was no appreciable percent weight loss of the wood blocks but later on (after 60 days) it increased rapidly and was found similar for both the strains (43–46% of wood mass). Samples inoculated with both the strains showed dual pattern of degradation i.e. selective delignification in the initial stage followed by simultaneous rot during advance stage of decay. Separation of the cells due to dissolution of middle lamella was the characteristic feature of both strains but in the advanced stage of decay, formation of erosion troughs were conspicuous in all the cell types. Other features such as cell wall thinning, rounded pit erosion, formation of erosion channels and bore holes were also observed frequently. Initially, fungal invasion started through the vessel lumen, followed by all the cell types of the xylem. From the vessels, mycelia entered into the adjacent rays and parenchyma cells through the pits. In advanced stage, degradation was so pronounced that rays were partially or even completely destroyed while many cells including vessels were either deformed or destroyed due to loss of rigidity of their walls. Structural alterations induced in response to C. asperatum and T. harzianum attack is described in details.

Cellular evidence of allelopathic interference of benzoic acid to mustard ( Brassica juncea L.) seedling growth

Cellular changes in the roots of mustard ( Brassica juncea L.) grown in soil treated with 1.09, 1.46 and 1.83 mg benzoic acid per g soil, a known allelochemical, were analyzed after 7 days. The recoverable concentration of 1.09, 1.46 and 1.8 mg benzoic acid per g soil (measured by high performance liquid chromatography) was 68, 150 and 250 μg benzoic acid per g soil, respectively. The benzoic acid treatments suppressed root growth by 30.5%, 58.8% and 81.1% with increasing concentrations. Transmission electron microscopy studies of roots showed irregular shaped cells arranged in a disorganized manner and disruption of cell organelles at cellular level. Root cells showed dissolution of middle lamella (at 68 and 150 μg benzoic acid per g soil) but intact middle lamella with increased wall deposits was observed with 250 μg benzoic acid per g soil. Damage to the mustard root at cellular level was evidenced by the changes in cell morphology and internal organization.

Hydrolytic activity and ultrastructural changes in fruit skins from two prickly pear (Opuntia sp.) varieties during storage.

The activity of four cell wall hydrolases, pectinmethylesterase (PME), polygalacturonase (PG), cellulase, and beta-galactosidase (beta-Gal), was measured in fruit skins of two prickly pear varieties, Naranjona and Charola, during storage at 18 degrees C and 85-95% relative humidity (RH). In Naranjona (Opuntia ficus indica), of short postharvest life (ca. 2 weeks), PG, cellulase, and beta-Gal increased their activity more than twice, whereas PME activity tended to increase only slightly during storage. In Charola (Opuntia sp.), of long postharvest life (ca. 2 months), only beta-Gal increased its activity (77%), showing a high PG activity from the beginning of storage. Transmission electron microscopy observations showed middle lamella dissolution at the end of storage for both varieties. Naranjona showed a higher cell wall enzymatic activity than Charola, in agreement with their storability differences. Our results suggest that PG and cellulase in Naranjona and PG and beta-Gal in Charola are the main enzymes responsible for cell wall hydrolytic and ultrastructural changes in skins of stored prickly pears.

Effect of cooking on banana and plantain texture.

The effect of temperature and duration of cooking on plantain and banana fruit texture and cytpoplasmic and cell wall components was investigated. The firmness of both banana and plantain pulp tissues decreased rapidly during the first 10 min of cooking in water above 70 degrees C, although plantain was much firmer than banana. Cooking resulted in pectin solubilzation and middle lamella dissolution leading to cell wall separation (as observed by SEM). Dessert banana showed more advanced and extensive breakdown than plantain. Although dessert banana had a higher total pectin content than plantain, the former had smaller-sized carboxyethylenediaminetetraacetic acid (CDTA) soluble pectic polymers which are associated with plant tissues that have a propensity to soften. Plantain had higher levels of starch and amylose than banana but this was associated with a firmer fruit texture rather than a softening due to cell swelling during starch gelatinization. Different cooking treatments showed that cooking in 0.5% of CaCl(2) solution and temperatures below 70 degrees C had significant effects on maintenance of pulp firmness.

Effects of Calcium Compounds on Fruit Puffing and the Ultrastructural Characteristics of the Subepidermal Cell Walls of Puffy and Calcium-induced Non-puffy Satsuma Mandarin Fruits.

The alleviating effects of calcium compounds on fruit puffing were investigated in satsuma mandarin. The ultrastructural changes in the subepidermal cell walls of puffy fruits and the inhibiting strategies of calcium against these changes were also observed under transmission electron microscope. Although puffing occurred in both treated and non-treated fruits under certain environmental conditions, pre-harvest applications of CS-2H (CaSO4·2H2O, CaCl2) and CS-1B (CaCO3 microcrystal suspension) significantly reduced it and with the exception of peel puffing these chemicals did not affect other fruit characteristics. The subepidermal cells of non-treated puffy fruits had bigger intercellular spaces in the cell corners and schizogenous intercellular spaces between adjacent walls. Occasionally, cell corners and abutting walls did not possess these spaces, but the disintegration of middle lamella therein was distinct. The disintegration caused loosening of the intercellular connection and swelling of walls. The sporadic destruction of the cell wall was also found near the plasma membrane. The fibrillar materials of loose cell walls separated distinctly in the longitudinal direction ; this separation was crosswise near the plasma membrane. In a few cases, the middle lamella and its adjoining walls had disintegrated. The application of calcium compound (CS-2H) prevented these ultrastructural disorders so that intercellular spaces remained small, schizogenous intercellular spaces did not develop, and the dissolution of middle lamella was checked considerably. The cell corners retained their middle lamella or sometimes developed small intercellular spaces. The fibrillar materials in the cell walls were compact and their destruction was not evident. Cell wall swelling was rarely observed, and cellular separation was not found.

Apparent diffusion coefficient determination of sodium hydroxide through potato skin and flesh under different temperatures and concentration conditions

The aims of the present study were to analyze the most important chemical reactions between sodium hydroxide and potato constituents involved in potato chemical peeling, and to measure the apparent diffusivity of NaOH in potato skin and flesh separately, as a function of temperature and NaOH concentration, selected according to potato chemical peeling process. Chemical reaction of the suberin of the potato skin with NaOH was proposed as the most important one to promote chemical peeling. Whereas starch hydrolysis, middle lamella dissolution and cell wall disruptions were proposed as the principal chemical reactions in flesh. Experiments to determine NaOH apparent dijfusivities in skin and flesh were performed following a cell diffusion method at 25, 40, 50, 60 and 70 °C temperatures and concentrations of 4, 12 and 20 g NaOH/100 g of solution. Finally, correlation equations were determined to describe apparent diffusivity dependence on temperature and concentration.

Hard-to-cook defect in black beans: the contribution of proteins to salt soaking effects

Softening effects of chelating and ion-exchange agents in bean soaking solutions have been attributed to improved solubilization of pectates. The present results indicate a mechanism based on protein destabilization also contributes to softening. Solubility and calorimetric data established softening to be correlated with increased protein denaturation of isolated protein bodies and not with pectin solubilization during cooking, although pH 10, but not pH 7, carbonate buffer did extract significant ( p ≤ 0·05) quantities of both pectin and protein. Carbonate ions reduced onset temperature of protein body denaturation by > 10°C. Rates of protein and pectin solubilization in hardened beans were faster then softening rates, suggesting that softening was not totally attributable to thermal degradation of macromolecular components or middle lamella dissolution.

Pedicel abscission of soybean: cytological and ultrastructural changes induced by auxin and ethephon

Indole-3-acetic acid delayed the onset of pedicel abscission of soybean, Glycine max (L.) Merrill, by 20 h, and in some experiments, significantly reduced abscission over a 7-day treatment period. Cytological and ultrastructural development of the abscission zone could be divided into two phases: the first associated with longer cells and fewer multigrained amyloplasts in the incipient separation layer; the second with shorter cells (indicative of secondary cell division), increased numbers of amyloplasts, and significant changes in the percent volume of cytoplasm, vacuoles, and wall tissue. Middle lamella dissolution preceded cell separation. Indole-3-acetic acid delayed but did not disrupt the sequence of events leading to abscission. Ethephon treatment hastened abscission but both delayed and disrupted typical abscission events, especially secondary cell division, which occured less frequently. In addition, cellular collapse and greater swelling of cells were observed in the separation layer of ethephon-treated tissue. Key words: abscission, ethylene, Glycine max, indole-3-acetic acid, pedicel, raceme.

Enzymatic and anatomical changes in abscission zone cells of apple fruits induced by Ethephon

The enhancement of fruit abscission zone formation with ethephon treatment caused an increase in soluble proteins, endo-cellulase, exo-polygalacturonase and peroxidase activities. Exo-cellulase and endo-polygalacturonase did not show any relationship with apple abscission. The separation of cells initiated in the cortex region and progressed towards vascular tissue. Cell separation in the cortex appeared to be due to dissolution of middle lamella but vascular tissues ruptured mechanically.

Histochemical and Ultrastructural Studies of Fruit Abscission in the Olive after Treatment with 2-Chloroethyl-tris-(2-methoxy ethoxy)-silane1

Abstract Following the application of 2-chloroethyl-tris-(2-methoxyethoxy)-silane (CGA 13586), an ethylenereleasing compound, to olive (Olea europaea L.) shoots, 2 abscission zones were observed. One occurred at the proximal, the other at the distal end of the pedicel. Actual separation occurred mostly at the distal end of the pedicel. Changes observed during the development of the abscission zone included: cell plasmolysis, cell wall and middle lamella dissolution, starch grain accumulation and a general breakdown of cells at the abscission zone.

Ultrastructural observationof lemon fruit abscission

Ultrastructural changes in the abscission zone between ovary wall and floral disc were followed during abscission of young lemon ( Citrus limon (L.) Burm. f. cultivar ‘Villafranca’) fruit explants. Although some cell breakage was observed by light and scanning electron microscopy as well as cell wall dissolution, only middle lamella dissolution was observed by transmission electron microscopy, strongly suggesting that pectic substance dissolution is the primary cause of cell separation. Prior to cell separation the middle lamella of the cell wall stained deeply, the amount of rough endoplasmic reticulum (ER) profiles increased, and the number of Golgi bodies also increased. However no change in mitochondria or chloroplast structure was observed before cell separations, and no microbodies were observed. In Ethephon (2-chloroethylphosphonic acid)-treated explants, essentially similar cytoplasmic and cell wall changes occurred during the course of abscission, but the changes were more rapid.