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Suppression of the expression of a ripening-related expansin gene, LeExp1, in tomato enhanced fruit firmness and overexpression of LeExp1 resulted in increased fruit softening. Because of the incompletely understood relationship between fresh fruit texture and the consistency of processed products, we examined the effects of LeExp1 overexpression on the processing characteristics of tomato fruit. As determined by Bostwick consistency and by controlled strain rheometry, juices and pastes prepared from transgenic tomatoes with suppressed LeExp1 expression had a higher viscosity than preparations from control fruits. However, the viscosity of juice and paste prepared from fruit overexpressing LeExp1 was significantly greater than products from controls or lines with reduced LeExp1. Bostwick consistency increased by 9% (juice) and 6% (paste) in lines with suppressed LeExp1 expression but increased by 27.5% (juice) and 19.5% (paste) in lines overexpressing LeExp1, relative to controls. Determined by laser diffraction, the particles in juice and paste prepared from transgenic fruits with reduced LeExp1 expression were smaller, and preparations from fruits overexpressing LeExp1 had a size distribution indicating more large particles. Analysis of cell wall polysaccharides size indicated that LeExp1 overexpression enhanced depolymerization of water soluble pectins as well as tightly bound matrix glycans. LeExp1 overexpression may allow increased cell wall hydration, resulting in expanded particle size and increased viscosity of products. Because either LeExp1 suppression or overexpression leads to improved consistency, the interactions that contribute to optimal product rheological properties are complex.

α-L-Arabinofuranosidases (α-Af) are plant enzymes that have the capacity to release terminal arabinofuranosyl residues from a wide variety of pectic and hemicellulosic polymers, as well as different glycoconjugates. Our interest in α-Af is related to its potential role in ripening-related loss of arabinose from tomato fruit cell walls. Using both control (cv. VF 36) and ACC synthase antisense (A11.1) tomatoes (Lycopersicon esculentum Mill.), we demonstrate that tomato α-Af activity is present during the entire ontogeny of the fruit. Immature 10-day-old fruit displayed 6-fold more α-Af activity on a per gram fresh weight basis, than mature green fruit. In VF 36 fruit, α-Af activity increased 45% from mature green 4 (48 days post anthesis) to light red stages (55 days) when fruit ripened on the vine. In contrast, no similar increase was detected in ACC synthase antisense fruit that do not ripen in the same time frame. However, when A11.1 fruit were detached at 48 days after anthesis and treated continuously with 100 mL·L-1 ethylene the fruit ripened and α-Af increased, as in ripening normal fruit. The α-Af activity pattern is similar to that reported for tomato β-galactosidases. The increasing α-Af activity during ripening and the decreased activity in antisense ACC synthase fruit after reaching the mature green stage suggest a role for ethylene in the ripening-related synthesis or activation of this enzyme. Fil: Sozzi, Gabriel Oscar. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Fraschina, Adela Ana. Universidad de Buenos Aires; Argentina Fil: Navarro, Agustín A.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Cascone, Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Greve, L. Carl. University of California; Estados Unidos Fil: Labavitch, John M.. University of California; Estados Unidos

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit. In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009–1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

The pressure microprobe was used to determine whether the turgor pressure in tomato (Lycopersicon esculentum Mill., variety "Castelmart") pericarp cells changed during fruit ripening. The turgor pressure of cells located 200 to 500 micrometers below the fruit epidermis was uniform within the same tissue (typically +/- 0.02 megapascals), and the highest turgors observed (

Recent work has shown that pectic oligosaccharides extracted from tomatoes are capable of promoting ripening in mature green tomato fruit explants (11). A water soluble, ethano-insoluble extract of autolytically-inactivated tomato pericarp tissue will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs from mature green fruit. Treatment of Na2CO3− soluble but not chelator-soluble pectin from tomato with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit. The endogeneous concentration of these oligomers is low, but in excess of that necessary to promote ethylene biosynthesis in this system (29). A major difficulty in assigning a regulatory role to such pectic oligomers is that at the onset of ripening in tomato fruit little evidence of polygalacturonase activity exists. However our work demonstrating the presence of neutral sugar-rich acidic oligomers suggests that other enzymatic activities capable of degrading pectic substances exist in developing tomatoes. The recent report (22) of a rhamnogalacturonase-type activity in developing fruit supports this supposition. The possibility of a role for pectic oligomers in fruit ripening regulation will be discussed.

Knowledge of mineral localization within rice grains is important for understanding the role of different elements in seed development, as well as for facilitating biofortification of seed micronutrients in order to enhance seeds' values in human diets. In this study, the concentrations of minerals in whole rice grains, hulls, brown rice, bran and polished rice were quantified by inductively coupled plasma mass spectroscopy. The in vivo mineral distribution patterns in rice grains and shifts in those distribution patterns during progressive stages of germination were analyzed by synchrotron X-ray microfluorescence. The results showed that half of the total Zn, two thirds of the total Fe, and most of the total K, Ca and Mn were removed by the milling process if the hull and bran were thoroughly polished. Concentrations of all elements were high in the embryo regions even though the local distributions within the embryo varied between elements. Mobilization of the minerals from specific seed locations during germination was also element-specific. High mobilization of K and Ca from grains to growing roots and leaf primordia was observed; the flux of Zn to these expanding tissues was somewhat less than that of K and Ca; the mobilization of Mn or Fe was relatively low, at least during the first few days of germination.

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.

A polygalacturonase inhibitor glycoprotein with an apparent molecular mass of 43 kD was purified from pear (Pyrus communis L. cv Bartlett) fruit. Chemical deglycosylation of this protein decreased the molecular mass to 34 kD. Gas chromatographic analysis suggests that N-linked glycosylation accounts for the majority of sugar moieties. Partial amino acid sequence analysis of the purified polygalacturonase inhibitor protein provided information used to amplify a corresponding cDNA by polymerase chain reactions. Multiple cloned products of these reactions were sequenced and the same open reading frame was identified in all of the products. It encodes a 36.5-kD polypeptide containing the amino acid sequences determined by protein sequencing and predicts a putative signal sequence of 24 amino acids and seven potential N-glycosylation sites. The expression of polygalacturonase inhibitor is regulated in a tissue-specific manner. Activity and mRNA level were much higher in fruit than in flowers or leaves.

En este estudio se determinaron los principales cambios en la composición de la pared celular y la degradación de pectinas solubles en agua relacionados con el ablandamiento postcosecha de frutos de papaya (Carica papaya L. cv. ‘Maradol’). Se estudiaron frutos en estado de madurez 1/2 (50-75 % de coloración amarillo-naranja en la cáscara), los cuales se retrasaron en la maduración con 300 nL L-1 de 1-metilciclopropeno (1-MCP) o se adelantaron con 2.5 g L-1 de ácido 2-cloroetil fosfónico (etefón). Posterior a los tratamientos, los frutos se almacenaron en condiciones de simulación de mercadeo (20 ± 2 °C y HR 85 %) durante 6 d. Se evaluaron los cambios en firmeza, composición de la pared celular y despolimerización de las pectinas solubles en agua. La aplicación de 1-MCP inhibió totalmente el ablandamiento de los frutos. Los frutos testigo perdieron 69 % de la firmeza a los 3 d de almacenamiento y los frutos con etefón perdieron 84 % en el mismo periodo. El ablandamiento en los frutos testigo y etefón se acompañó de la solubilización de ácidos urónicos y azúcares totales de las fracciones CDTA, Na2CO3, KOH 4% y KOH 24 %. La despolimerización extensiva de pectinas solubles en agua y la acumulación de oligómeros derivados de pectinas sólo fue detectada en los tratamientos testigo y etefón, lo que muestra una relación con el ablandamiento de los frutos y sugire la posible acción de la poligalacturonasa.

The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination.

The rate and composition of cell wall polysaccharide synthesis during development and growth-inhibiting water deficits were investigated in leaves of grape (Vitis vinifera L.) The rate of leaf expansion was monitored as plant water status was manipulated by modulating the supply of irrigation water to potted plants over several days. The corresponding wall synthesis was determined by incubating leaf tissue with [ 14 C] glucose and quantifying incorporation into wall components. (...)

The pressure microprobe has been used to measure cell turgor and, in addition, to sample vacuolar tissues. In carrot, a rapid initial loss of tissue firmness (instron technique) occurred when the tissue was heater (cooked), and this could be entirely attributed to a loss in cell turgor. Turgor was well-correlated to firmness over the range of turgor measurements (0–0.8 MPa). In cherry and other fruits, turgor is typically 1 to 2 orders of magnitude lower than that expected based on cell osmotic potential, indicating the presence of apoplastic solutes. Cherry fruit firmness and cell turgor were well-correlated during the first 2 h of hydration at 20C, but, as fruit began to crack, tissue decreased, whereas turgor continued to increase.

Acid hydrolysis-generated pectic oligomers have been shown to affect ripening of tomato fruit by inducing both acceleration of reddening and increased ethylene biosynthesis (Campbell & Labavitch, 1991 Plant Physiol 97:706-713). In the present work, homogeneous size classes of these oligomers were demonstrated to have different impacts on ethylene production of tomato fruit pericarp discs. Endogenous oligomeric material of the same size classes was isolated from ripening tomato tissues and also tested for biological activity. They promoted some aspects of ripening as shown by increased ACC and ethylene production, which suggests that pectic oligomers are potential regulators of the ripening process in tomatoes. A metabolic origin for these oligomers is suggested by the fact that they are produced by in vitro polygalacturonase I treatment of polygalacturonic acid or tomato pectin.

Discs from outer pericarp of mature green (MG) and light red (LR) tomatoes were incubated with 13C6-glucose as precursor to cell wall constituents, to determine biosynthetic capacity of the outer 2mm (including cuticle) and adjacent inner 2mm of tissue. Cell wall material was fractionated into pectic and hemicellulosic classes by sequential extraction, and alditol acetates and partially-methylated alditol acetates were prepared. Neutral sugars (NS), glycosidic linkage compositions and incorporation of label were determined by GC-FID and GC-MS. Rhamnose, arabinose and galactose accounted for ca. 90% of both labeled and total NS in the pectic fractions (sugar ratios within ripeness stage were the same for labeled and total NS). Xylose and glucose accounted for ca. 70% of both labeled and total NS in the hemicellulosic fraction (sugar ratios within ripeness stage were different between labeled and total NS). In the crude cell wall, galactose and glucose contents were significantly higher in the inner than in outer tissues for both MG and LR tomatoes. Loss of galactose during ripening was higher from outer tissues. These results show compositional differences between inner and outer tissues, and suggest that ripening-related wall synthesis may give rise to pectic polymers similar in NS composition to existing polysaccharides, and hemicellulosic polymers which may differ in composition.

Higher plant inhibitors of fungal polygalacturonases are potential contributors to plant defense. To test this hypothesis we have raised antibodies against the `Bartlett' pear fruit polygalacturonase inhibitor (PGIP) and cloned a pear fruit PGIP cDNA. The pear PGIP cDNA was isolated by polymerase chain reactions based on our amino acid and nucleotide sequence information. Sequence analysis predicts a gene product of 34.5 kD with an isoelectric point of 6.02 in agreement with our biochemical data. Seven potential glycosylation sites are consistent with the glycoprotein character of these PGIPs. Southern blot analysis suggests the presence of 1 or 2 genes in the pear genome. Northern blot analysis indicates the presence of a transcript of 1.5 kb. Western blot analysis shows cross-reactivity of the anti-pear PGIP antibody to various dicot species as well as corn.

The pressure microprobe was used to measure cell turgor (ψp) in tomato pericarp tissue, and also to sample vacuolar fluid for the measurement of cell osmotic potential (ψs) in a nanoleter freezing point osmometer. In fresh tissue, cell ψs agreed well with the ψs of frozen-thawed whole tissue measured with a vapor pressure osmometer. Under a wide range of ripeness conditions however, and for both intact fruit and discs of fruit tissue, fruit cell turgor was consistently lower than expected, based on the values of cell ψs. When tissue discs were hydrated in aerated distilled water, disc fresh weight increased substantially (20 - 50+%), and both cell turgor and tissue ψs increased. Cell ψs however, remained relatively constant. These and other observations suggest that the turgor increase during hydration was largely due to losses of solute from the apoplastic space, partly by direct losses from the tissue, and partly by cell solute accumulation.

Commercially grown Granny Smith apples were stored at 0°C in air or 1% O2, and 2 sets of samples were taken every 4 weeks over a 28 week period. One set was immediately analysed for weight loss, firmness, color, soluble solids, pH and titratable acidity. Alcohol-insoluble substances were analysed for starch, water-soluble uronides, water-insoluble uronides, cellulose and neutral sugars. The second set of samples was kept in air at 20°C for an additional week, during which respiration and ethylene production rates were measured, prior to the above analyses. Storage in 1% O2 led to the improved maintenance of firmness, reduced respiration and ethylene production rates in ambient air, and a reduced content of water-soluble uronides, suggesting a reduced degree of hydrolysis. The correlation between firmness and water-soluble uronide content was not very strong. The predominant neutral sugars present in the wall were arabinose and galactose, and activities of putative hydrolyses that may be involved in the metabolism of polymers containing these sugars will be discussed.

Infiltration of apricot (Prunus armeniaca L.), Patterson cultivar fruits, which are susceptible to rapid softening, with calcium chloride before processing resulted in definite firming of the canned apricots. Non-susceptible fruits treated with citrate buffers (pH 3.7) showed dramatic post-process softening. In individual, untreated, early, green fruit, firmness after processing was directly correlated with the bound calcium:citrate ratio. Based on a chelation hypothesis, it was proposed that softening was accelerated when chelators such as organic acid anions removed structural calcium from the cell wall once cell membranes were lysed by heating.

Reproducible determinations of cell wall uronic acid content are obtained by first dissolving wall samples in sulfuric acid and then performing assays on aliquots of the cell wall solution. The colorimetric assay utilizing m-hydroxydiphenyl is recommended because of its low background readings in the presence of large quantities of neutral sugars.

An endopolygalacturonase has been purified from a crude commercial preparation that is derived from cultures of Aspergillus niger. The polygalacturonase has a pH optimum of 4.5 and is relatively heat labile, losing activity when heated to 45°C. The purified preparation acts on the α-1, 4-galacturonosyl linkages present in polygalacturonic acid. It does not act on linkages present in a variety of substrates which contain virtually all of the glycosidic bond types described for the cell walls of monocots, dicots, and various fruits.

The glycosidic linkage compositions of intact and, in some cases, enzyme-degraded polysaccharides extracted from the cell walls of oat coleoptiles and subsequently purified have been examined. A major component is shown to be a glucuronoarabinoxylan similar in structure to those described for a variety of other monocots. The noncellulosic glucan component is a β-linked polymer containing both 1,4- and 1,3-linked glucosyl residues in a ratio of 2 to 1. Analysis of the oligosaccharide produced by ‘lichenase’ digestion of this β-glucan suggests that the the 1,3- and 1,4-glucosyl linkages repeat in regular fashion. A small amount of xyloglucan polysaccharides like those described for cell walls of dicots was also detected.

A beta-1,4-xylanase has been purified from the mixture of carbohydrate-degrading enzymes found in a commercial preparation from cultures of Trichoderma viride. Purification from the desalted enzyme mixture is accomplished either by preparative isoelectric focusing or in two-column chromatographic steps. The xylanase has maximal activity at pH 5.0 and a molecular weight of approximately 13,000 daltons. The enzyme loses activity when heated to above 45 degrees C. The xylanase degrades xylans from larch and pear cell walls in an apparently endo-fashion.

A method was devised for quantitation of methylated indoleacetic (MeIAA) using a nitrogen-phosphorus (N-P) detector gas chromatograph with sensitivity greater than 5 pg MeIAA. Parameters are described for use conditions of the N-P detector.

Cell wall-degrading enzymes were extracted from the cell wall free space of mesocarp tissue from immature almonds [Prunus dulcis(Mill.)D.A. Webb, ‘Nonpareil’]. The activities of several of these enzymes were found to correlate with the development of gum ducts in this tissue. Polygalacturonase (EC 3.2.1.15) and 1,3-β-D-glucanase (EC 3.2.1.39) activities rose sharply at, or just prior to, the early schizogenous stage of duct initiation, while increases in α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), α-arabinosidase (EC 3.2.1.55), and α-mannosidase (EC 3.2.1.24) activities were correlated with the later lysigenous stage of duct formation. Cell wall analysis of almond mesocarp tissue sampled the week preceding gum duct formation determined that the predominant noncellulosic sugars present in the mesocarp cell walls are arabinose, galactose, xylose, and glucose, with smaller amounts of rhamnose and mannose also present. The walls also contain a high percentage of galacturonic acid and trace amounts of glucuronic acid. Methylation analysis of the cell walls confirmed that many of the specific glycosidic linkages that are cleaved by the enzymes tested are present in the mesocarp cell walls immediately prior to gum duct formation.

As the pistachio (Pistacia vera L. cv. Kerman) nut matured, kernel moisture, respiration rate, and total protein content decreased, while kernel dry weight increased. At optimum maturity, ether-extractable fat and total sugar contents reached a peak. Either or both of these constituents may be useful as a maturity index, in addition to ease of hull separation, to determine optimum harvest date for pistachio nuts. Nut quality was acceptable for harvest during a 2- to 3-week period bracketing the time when the hull separates easily from the shell. Compositional analyses of hulls indicated some limitations on their potential use as animal feed.

A cell free culture filtrate from 6-day cultures of Fusarium oxysporum f. sp. cubense was processed to give: (1) a heterogeneous enzyme mixture, (2) purified polygalacturonase (PG), (3) partially-purified polygalacturonate lyase and (4) β-1,4-xylanase. When introduced into explanted castor bean leaves each of these preparations was able to promote the formation of vascular system-obstructing gels. Exposure of castor bean leaves to ethylene (3 ppm) also triggered gel formation. Explanted leaves produced ethylene in response to the enzyme mixture and PG. Vascular gel formation did not occur when ethylene production in response to enzymes was prevented.

Turnover of cell wall polysaccharides and effects of auxin thereon were examined after prelabeling polysaccharides by feeding pea (Pisum sativum var. Alaska) stem segments (14)C-glucose, then keeping the tissue 7 hours in unlabeled glucose with or without indoleacetic acid. There followed an extraction, hydrolysis, and chromatography procedure by which labeled monosaccharides and uronic acids were released and separated with consistently high recovery. Most wall polymers, including galacturonan and cellulose, did not undergo appreciable turnover. About 20% turnover of starch, which normally contaminates cell wall preparations but which was removed by a preliminary step in this procedure, occurred in 7 hours. Quantitatively, the principal wall polymer turnover process observed was a 50% decrease in galactose in the pectinase-extractable fraction, including galactose attached to a pectinase-resistant rhamnogalacturonan. Other pectinase-resistant galactan(s) did not undergo turnover. No turnover was observed in arabinans, but a doubling of radioactivity in arabinose of the pectinase-resistant, hot-acid-degradable fraction occurred in 7 hours, possibly indicating conversion of galactan into arabinan. None of the above changes was affected by indoleacetic acid, but a quantitatively minor turnover of a pectinase-degradable xyloglucan was found to be consistently promoted by indole-acetic acid. This was accompanied by a reciprocal increase in water-soluble xyloglucan, suggesting that indoleacetic acid induces conversion of wall xyloglucan from insoluble to water-soluble form. The results indicate a highly selective pattern of wall turnover processes with an even more specific influence of auxin.

The chemical composition and sensory attributes of pistachio nuts (Pistacia vera L.) were studied in relation to genotype, production area, maturity, moisture content, degree of shell staining, and storage conditions. ‘Kerman’ kernals were rated higher in firmness and sweetness, and lower in crispness, bitterness, and rancidity than those of the ‘Red Aleppo’, ‘Trabonella’, and ‘Bronte’ cultivars. Differences in composition and flavor of ‘Kerman’ pistachios harvested from 3 production areas were small. Nuts harvested at near optimum maturity were superior in quality to those harvested earlier or later. Drying nuts to 4–6% moisture levels resulted in the best quality. Shell staining did not influence eating quality of kernels but detrimentally affected shell appearance. Dried pistachio nuts can be kept for 12 months at 20°C. Moisture content influenced crispness and firmness while total sugars content was related to sweetness and overall flavor intensity.

The ability of concanavalin A, soybean agglutinin and lectins from Pisum sativum and Bandeiraea simplicifolia to mediate the agglutination of protoplasts prepared from Nicotiana glauca, Zea mays, and Lactuca sativa was assessed. Pea lectin failed to mediate agglutination; the other lectins agglutinated the three cell types tested. A microtiter assay was used to assess the activity of the lectins. The three active lectins had different activities against each of the protoplast types tested.

An alpha-L- arabinofuranosidase has been purified from the extracellular broth of cultures of Ruminococcus albus 8. The purification procedure utilized gel filtration, (NH4)2SO4 precipitation, and isoelectric focusing. The purified enzyme appeared to be homogeneous when chromatographed on disc and analytical isoelectric focusing gels. The estimated molecular weight of the native protein was 305,000 to 310,000. Sodium dodecyl sulfate-gel electrophoresis analysis suggested that the native protein is a tetramer composed of 75,000-molecular-weight subunits. The enzyme appeared to have no metal cofactor requirement but was sensitive to several sulfhydryl reagents. The pH optimum with p-nitrophenyl-alpha-L-arabinofuranoside as the substrate was 6.9 and the Km was 1.3 mM. Several lines of evidence indicated that the enzyme is a glycoprotein. When assayed against alfalfa cell wall material, the enzyme hydrolyzed only small amounts of arabinose from the substrate. When assayed together with hemicellulolytic or pectinolytic enzymes against the same substrate, the arabinosidase significantly enhanced the hydrolytic action of the glycanases .

INTRODUCTION ....... 385 WALL TURNOVER AND CELL ELONGATION 387 Physiological and Biochemical Considerations 388 The stress-bearing portion of the cell wall •.•..•.•.... 390 Turnover of Monocot Cell Wall Components 391 Turnover of Dicot Cell Wall Components ..... 393 WALL TURNOVER DURING TERMINAL DEVELOPMENT 396 Seed Germination 396 Fruit Ripening 397 Woll structure in pome fruits 398 Ripening-ossociated c ell wall changes 398 CONCLUDING REMARKS ; 400

Fractionation of pectic polysaccharides from the juice of ripening `Bartlett' pears (Pyrus communis) gave two general types of polyuronides. The major type was a homogalacturonan (HGA) whose molecular weight decreased upon ripening. The other type comprised heteropolymers composed of various amounts of arabinose, rhamnose, and galactose. Treatment of the major arabinose-containing heteropolymeric fraction of high molecular weight (400,000) with a pear exo-polygalacturonase to degrade contaminating HGA gave a polyuronide which was inert to tomato endopolygalacturonase. Glycosyl-linkage analysis of this arabinosyl-polyuronide gave results expected from a rhamnogalacturonan I-like polysaccharide with large, highly branched araban side chains (RG-I). A linkage between HGA and RG-I was not found. RG-I, in ripening pears, appeared to be degraded with the initial loss of much of its arabinose.

The spatial distribution of the rate of deposition of uronic acids in the elongation zone of Zea mays L. Crow WF9 × Mo 17 was determined using the continuity equation with experimentally determined values for uronide density and growth velocity. In spatial terms, the uronide deposition rate has a maximum of 0.4 micrograms per millimeter per hour at s = 3.5 mm (i.e., at the location 3.5 mm from the root tip) and decreases to 0.1 mg mm−1 h−1 by s = 10 mm. In terms of a material tissue element, a tissue segment located initially from s = 2.0 to s = 2.1 mm has 0.14 μg of uronic acids and increases in both length and uronic acid content until it is 0.9 mm long and has 0.7 μg of uronide when its center is at s = 10 mm. Simulations of radioactive labeling experiments show that 15 min is the appropriate time scale for pulse determinations of deposition rate profiles in a rapidly growing corn root.

Third-generation navel orangeworm (Amyelois transitella) damage to almond (Prunus dulcis Mill.) kernels is a serious problem in the California almond industry. An early harvest is one means of reducing losses and increasing crop quality. A successful early harvest was accomplished 2 to 3 weeks before the normal harvest date, with no perceptible effect on kernel quality. Percent hullsplit was correlated with nut removal, providing a field guide to acceptable maturity. Within defined limits, yield, drying rate, and hullability of early harvested almonds were acceptable.

Mature `Bartlett9 pear ( Pyrus communis ) fruits were ripened at 20 C. Fruits at different stages of ripeness were homogenized, and extracts of the low speed pellet (crude cell wall) were prepared. These extracts contained polygalacturonase, pectin esterase, and activity against seven p -nitrophenyl glycoside substrates. Polygalacturonase, α-galactosidase, and α-mannosidase increased in activity as the fruit ripened. Cellulase and activities against pear wall xylan and arabinan were absent from the extracts.

The canning of slightly immature ‘Patterson’ apricot fruits leads to an immediate softening of the halves to the extent that many are of unacceptable texture. This softening occurs in conjunction with a substantial solubilization of uronic acid-, arabinose-, and galactose-rich (presumably pectic) polysaccharide material into the canning liquid. The solubilization is not accompanied by pectin depolymerization suggesting that some process other than acid-catalyzed polymer hydrolysis is responsible for fruit softening during processing.

Auxin promotes the liberation of a xlyoglucan polymer from the cell walls of elongating pea (Pisum sativum) stem segments. The released polymer can be isolated from the polysaccharide fraction of the water-soluble portion of tissue homogenates, thus providing as assay for this kind of metabolism. Promotion of xyloglucan metabolism by auxin begins within 15 minutes of hormone presentation. The effect increases with auxin concentration in a manner similar to the hormone effect on elongation. However, the xyloglucan effect of auxin occurs perfectly normally when elongation is completely blocked by mannitol. Metabolic inhibitors and Ca(2+), on the other hand, inhibit auxin promotion of elongation and of xyloglucan metabolism in parallel. The results suggest that the changes in xyloglucan reflect the means by which auxin modifies the cell wall to cause elongation.

We examined the middle lamella and the primary and secondary walls in almond pericarp dehiscence zone cells using a fluorescent cytochemical method which permitted specific, quantitative detection of pectic cell wall materials. Glycol methacrylate-embedded sections were stained with coriphosphine and pectin-specific fluorescent emissions at 630 nm were quantified using green excitation (546 nm). Examination of sectioned material extracted with purified pecto-lytic enzyme preparations was used to demonstrate the relative specificity of the staining reaction for pectic substances.

`Bartlett9 pear ( Pyrus communis ) fruits were picked at the mature, green stage and ripened at 20 C. Fruits at different stages of ripeness (based on flesh firmness) were homogenized, and the sugar and uronic acid contents of cell wall and soluble polysaccharides were determined. Substantial amounts of galacturonic acid and arabinose were lost from the wall fraction as the fruit ripened. Most of this cell wall material was recovered, in an 80% (volume/volume) ethanol-insoluble form, from the soluble fraction of tissue homogenates. Structural analysis of ethanol-precipitable material indicates that it is an acidic (pectic) polymer-bearing side groups containing variously-linked arabinosyl residues.

Development of the idea that carbohydrates digested from cell walls could carry the information necessary to “activate” host defense responses to pathogens began in the 1970s. Early work (Ayers et al., 1976) demonstrated that a glucan derived from cell walls of the soybean pathogen Phytophthora megasperma f. sp. glycinea could elicit the synthesis of phytoalexins by soybean tissues. Subsequent studies have identified a heptasaccharide subfragment of the hyphal wall glucan, presumably generated by the action of soybean-produced glucan hydrolases, as the most active component (Sharp et al., 1984). Studies in recent years have identified similar responses in several cell culture and tissue systems. The production of phytoalexins and other presumed “defense” compounds in response to elicitors derived from host, as well as pathogen, cell walls has been described (Darvill and Albersheim, 1984; Lamb et al., 1986). Most recently these studies have utilized the techniques of molecular genetics to follow responses at the level of gene transcription (e.g., Bell et al., 1986). Genome activation with a lag time as short as 5 to 10 minutes has been shown (Lawton and Lamb, 1987). Questions about the means by which cells perceive the presence of elicitors (Schmidt and Ebel, 1987) and then mobilize their responses remain. However, it is clear that the biochemical confrontation at the infection site often includes many enzymes that can generate elicitor-active wall fragments.

Whereas disease, pest and herbicide resistance have served as valuable entry points for plant biotechnology in general, the emphasis with crop plants is clearly shifting towards modifying traits that enhance quality, nutritional content and productivity. In tree crops this would involve specifically perturbing the expression patterns of endogenous genes associated with the trait under investigation. New tools such as enhanced gene silencing technology, EST database development, and total genome sequencing will likely accelerate the potential impact of this kind of work. Established plant transformation methods will continue to play crucial roles in evaluating newly discovered endogenous genes and in characterizing their function in whole plants. Examples of some of the areas in which we are currently integrating these kinds of approaches include the manipulation of sugar and ethylene metabolism in apples (Malus ×domestica Borkh.), of oil/fat composition in walnut (Juglans regia L.) and of growth architecture in citrus. In almonds [Prunus dulcis (Mill.) D.A. Webb] we are studying self-incompatibility, a trait with potentially significant implications regarding productivity.

Macerase, a pectinase-containing enzyme mixture, was used to digest cell walls isolated from cultured pear cells. Following digestion, the reaction mixture was boiled to inactivate enzymes. Addition of soluble aliquots of the mixture to suspension cultures of pear cells led to a rapid and transient production of ethylene by the cells.