Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems

Most of the activity of an α-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic α-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the α-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this α-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the β-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.     

A Xyloglucan-Specific Endo-1,4-[beta]-Glucanase Isolated from Auxin-Treated Pea Stems

A xyloglucan-specific endo-1,4-[beta]-glucanase was isolated from the apoplast fraction of auxin-treated pea (Pisum sativum) stems, in which both the rate of stem elongation and the amount of xyloglucan solubilized were high. The enzyme was purified to apparent homogeneity by sequential cation-exchange chromatographies, affinity chromatography, and gel filtration. The purified enzyme gave a single protein band on sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and the molecular size was determined to be 77 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 70 kD by gel filtration. The isoelectric point was about 8.1. The enzyme specifically cleaved the 1,4-[beta]-glucosyl linkages of the xyloglucan backbone to yield mainly nona- and heptasaccharides but did not hydrolyze carboxymethylcellulose, swollen cellulose, and (1->3, 1->4)-[beta]-glucan. By hydrolysis, the average molecular size of xyloglucan was decreased from 50 to 20 kD with new reducing chain ends in the lower molecular size fractions. This suggests that the enzyme has endo-1,4-[beta]-glucanase activity against xyloglucan. In conclusion, a xyloglucan-specific endo-1,4-[beta]-glucanase with an activity that differs from the activities of cellulase and xyloglucan endotransglycosylase has been isolated from elongating pea stems.     

Endo-1,4-{beta}-Glucanase in the Cell Wall of Stems of Auxin-Treated Pea Seedlings

Endo-1,4-ß-glucanase induced by treatment of pea seedlingswith 2,4-D was extracted from a preparation of the walls ofepicotyl cells. The ß-glucanase was purified by chromatographyon DEAE-cellulose, affinity chromatography on Con A-Sepharoseand SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The activityof ß-glucanase was retained after removal of SDS andextraction from polyacrylamide gels. The band of a protein (46kDa), that corresponded to the activity of endo-1,4-ß-glucanase,was injected directly into mice for preparation of antiserumand the protein was also subjected to amino acid sequencingafter blotting onto a membrane. Western blot analysis showedthat the antiserum obtained bound to a 46-kDa polypeptide andrecognized endo-1,4-ß-glucanase. The N-terminal sequenceof the 46-kDa polypeptide revealed some homology to abscissionendo-1,4-ß-glucanases of bean and avocado fruit. (Received September 29, 1993; Accepted January 20, 1994)     

The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis of Agrobacterium tumefaciens-Induced Crown Galls on Tomato Stems

We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis.     

Morphometric and Serologic Comparisons of a Number of Populations of Cyst Nematodes

Thirty-five populations of Heterodera glycines and populations of 15 other Heterodera, Globodera, and Punctodera species were studied morphometrically and some were compared serologically. There was a wide range of each measurement within each nematode population. Except for one soybean cyst nematode population from Indiana, which was a tetraploid and considerably larger than the others, morphometric measurements overlapped. In a discriminant function comparison most of the populations were closely grouped but at least three were rather distinctly separated. Morphometrically H. fici, H. cruciferae, H. schachtii, and H. trifolii were closely associated with H. glycines. Serology indicated a close relationship between H. glycines, H. lespedezae, H. trifolii, H. schachtii, and the Heterodera sp. from Rumex, while H. betulae appeared to be more distantly related.     

Some Factors Affecting Survival of Desiccation by Infective Juveniles of Orrina phyllobia

The survival of desiccation by J4 Orrina phyllobia was examined at controlled relative humidities. When nematodes were transferred from water to air at 10% relative humidity (rh), 80% died within 30 minutes. When nematodes were transferred from water to air with rh at 70% or greater for ca. 15 minutes prior to being transferred to 10% rh, more than 90% of them survived desiccation. This phenomenon is referred to as preconditioning and occurred at much faster rates (2-30 minutes) than has been observed for other nematode species (24 hours). Differences in preconditioning rates may be due to technique-dependent variations in boundary layer resistance around nematodes during desiccation.     

Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiologicalagent of Chagas disease, a public health concern with an increasing incidence rate.This increase is due, among other reasons, to the parasite''s drug resistancemechanisms, which require nicotinamide adenine dinucleotide (NAD⁺).Furthermore, this molecule is involved in metabolic and intracellular signallingprocesses necessary for the survival of T. cruzi throughout its lifecycle. NAD⁺ biosynthesis is performed by de novo andsalvage pathways, which converge on the step that is catalysed by the enzymenicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commissionnumber: 2.7.7.1). The identification of the NMNAT of T.cruzi is important for the development of future therapeutic strategiesto treat Chagas disease. In this study, a hypothetical open reading frame (ORF) forNMNAT was identified in the genome of T. cruzi. The correspondingputative protein was analysed by simulating structural models. The ORF was amplifiedfrom genomic DNA by polymerase chain reaction and was further used for theconstruction of a corresponding recombinant expression vector. The expressedrecombinant protein was partially purified and its activity was evaluated usingenzymatic assays. These results comprise the first identification of an NMNAT inT. cruzi using bioinformatics and experimental tools and hencerepresent the first step to understanding NAD⁺ metabolism in theseparasites.     

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.     

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate retina responsible for continuous generation of 11-cis-retinal from its all-trans-isomer (1-3). Because 11-cis-retinal is the chromophore of rhodopsin and cone visual pigments (4), disabling mutations in genes encoding proteins of the retinoid cycle can cause a spectrum of retinal diseases affecting sight (3). Moreover, the efficiency of the mammalian visual system and health of photoreceptors and retinal pigment epithelium (RPE)² decrease significantly with age. Even in the presence of a functional retinoid cycle, A2E, retinal dimer (RALdi), and other toxic all-trans-retinal condensation products (5-7) can accumulate as a consequence of aging (8). Under experimental conditions, these compounds can produce toxic effects on RPE cells (9-11). Patients affected by age-related macular degeneration, Stargardt disease, or other retinal diseases associated with accumulation of surrogate markers, such as A2E, all develop retinal degeneration (12). Thus, elucidating the fundamental causes of these age-dependent changes is of increasing importance. Encouragingly, our understanding of both retinoid metabolism outside the eye and production of 11-cis-retinal unique to the eye has accelerated recently (Scheme 1) (1-3), and genetic mouse models are readily available to study these processes and their potential aberrations in vivo (13). Thus, a central question can be addressed, namely what initiates the death of photoreceptor cells and the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual cycle. After diffusion from the RPE, the visual chromophore, 11-cis-retinal, combines with rhodopsin and then is photoisomerized to all-trans-retinal. Most of the all-trans-retinal dissociates from opsin into the cytoplasm, where it is reduced to all-trans-retinol by RDHs, including RDH8. The fraction of all-trans-retinal that dissociates into the disc lumen is transported by ABCA4 into the cytoplasm (23) before it is reduced. All-trans-retinol then is translocated to the RPE, esterified by LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are associated with human macular degeneration, Stargardt disease, and age-related macular degeneration (55, 56).Several mechanisms associated with retinoid metabolism may contribute to different retinopathies (1). For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or chromophore in retinoid isomerase knock-out (Rpe65^-/-) mice leads to rapid degeneration of cone photoreceptors and slowly progressive death of rods (14). Such mice do not produce toxic condensation products from all-trans-retinal. Instead, their retinopathies have been attributed to continuous activation of visual phototransduction (15) due to either the basal activity of opsin (16-18) or disordered vectorial transport of cone visual pigments without bound chromophore (19). Paradoxically, an abnormally high flux of retinoids through the retinoid cycle can also lead to retinopathy in other mouse models (20, 21). Animal models featuring anomalies in the retinoid cycle illustrate the importance of chromophore regeneration and provide an approach to elucidating mechanisms involved in human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8 (retinol dehydrogenase 8), one of the main enzymes that reduces all-trans-retinal in rod and cone outer segments (22), and Abca4 (ATP-binding cassette transporter 4), which transports all-trans-retinal from the inside to the outside of disc membranes (23), rapidly accumulate all-trans-retinal condensation products and exhibit accentuated RPE/photoreceptor dystrophy at an early age (24). Although these studies suggest retinoid toxicity, it is still unclear if the elevated levels of retinal and/or its condensation products, such as A2E, are the cause of this retinopathy or merely a nonspecific reflection of impaired retinoid metabolism. Here, we report that spent chromophore, all-trans-retinal, is most likely responsible for photoreceptor degeneration in Rdh8^-/-Abca4^-/- mice. Toxic effects of all-trans-retinal include caspase activation and mitochondria-associated cell death.     

A Nitrogen-Fixing Endophyte of Sugarcane Stems (A New Role for the Apoplast)

The intercellular spaces of sugarcane (Saccharum officinarum L.) stem parenchyma are filled with solution (determined by cryoscanning microscopy), which can be removed aseptically by centrifugation. It contained 12% sucrose (Suc; pH 5.5.) and yielded pure cultures of an acid-producing bacterium (approximately 104 bacteria/mL extracted fluid) on N-poor medium containing 10% Suc (pH 5.5). This bacterium was identical with the type culture of Acetobacter diazotrophicus, a recently discovered N2-fixing bacterium specific to sugarcane, with respect to nine biochemical and morphological characteristics, including acetylene reduction in air. Similar bacteria were observed in situ in the intercellular spaces. This demonstrates the presence of an N2-fixing endophyte living in apoplastic fluid of plant tissue and also that the fluid approximates the composition of the endophytes's optimal culture medium. The apoplastic fluid occupied 3% of the stem volume; this approximates 3 tons of fluid/ha of the crop. This endogenous culture broth consisting of substrate and N2-fixing bacteria may be enough volume to account for earlier reports that some cultivars of sugarcane are independent of N fertilizers. It is suggested that genetic manipulation of apoplastic fluid composition may facilitate the establishment of similar symbioses with endophytic bacteria in other crop plants.     

Development of Four Populations of Meloidogyne hapla on Two Cultivars,of Cucumber at Different Temperatures

The infectivity and development of four populations of Meloidogyne hapla were compared, at three temperatures, on tomato and two varieties of cucumber. A population from Canada produced few root-galls on cucumber and, except at 24 C, no larvae developed into adult females and produced egg masses. In contrast, a population with 45 chromosomes from America produced many galls on cucumber and small proportions of larvae became females and produced egg masses at 20 and 24 C. At 18 C this population produced no egg masses on cucumber, but a population from Britain and one from America with 17 chromosomes produced more egg masses at this temperature than at 20 or 24 C. Dissection of the galls showed that on cucumber many larvae died or their growth and development was slowed.     

Coupling of Solute Transport and Cell Expansion in Pea Stems

As cells expand and are displaced through the elongation zone of the epicotyl of etiolated pea (Pisum sativum L. var Alaska) seedlings, there is little net dilution of the cell sap, implying a coordination between cell expansion and solute uptake from the phloem. Using [¹⁴C]sucrose as a phloem tracer (applied to the hypogeous cotyledons), the pattern of label accumulation along the stem closely matched the growth rate pattern: high accumulation in the growing zone, little accumulation in nongrowing regions. Several results suggest that a major portion of phloem contents enters elongating cells through the symplast. We propose that the coordination between phloem transport and cell expansion is accomplished via regulatory pathways affecting both plasmodesmata conductivity and cell expansion.     

Gaseous Requirements for Postparasitic Development of Romanomermis culicivorax

The development of postparasitic stages of Romanomermis culicivorax was studied under various concentrations of oxygen and carhon dioxide. The nematode developed poorly if only nitrogen was supplied; only one-third molted and all died eventually. In the presence of 5% CO₂ - 95% N₂, development was normal; most nematodes molted and oviposited with respective mean developmental times of 32 and 50 d. Addition of 0.2% O₂ stimulated development; molting and oviposition commenced at days 18 and 41, respectively. There was an additional stimulation of development by increasing amounts of O₂ up to 1%, but concentrations greater than 1% produced no additional stimulation. Carbon dioxide was required for development after exsheathment under anaerobic conditions or O₂ concentrations less than 1%. Oxygen or CO₂ were not required for embryological development or egg hatch. It is suggested that post-parasitic stages function as facultative anaerobes,