Enzyme-Catalyzed Production of Potato Galactan-Oligosaccharides and Its Optimization by Response Surface Methodology.

This work shows an optimized enzymatic hydrolysis of high molecular weight potato galactan yielding pectic galactan-oligosaccharides (PGOs), where endo-ß-1,4-galactanase (galactanase) from Cellvibrio japonicus and Clostridium thermocellum was used. For this, response surface methodology (RSM) by central composite design (CCD) was applied. The parameters varied were temperature (°C), pH, incubation time (min), and enzyme/substrate ratio (U/mg). The optimized conditions for the production of low degree of polymerization (DP) PGOs were obtained for each enzyme by spectrophotometric assay and confirmed by chromatography. The optimal conditions predicted for the use of C. japonicus galactanase to obtain PGOs of DP = 2 were T = 51.8 °C, pH 5, E/S = 0.508 U/mg, and t = 77.5 min. For DP = 3, they were T = 21 °C, pH 9, E/S = 0.484 U/mg, and t = 12.5 min; and for DP = 4, they were T = 21 °C, pH 5, E/S = 0.462 U/mg, and t = 12.5 min. The efficiency results were 51.3% for substrate hydrolysis. C. thermocellum galactanase had a lower yield (35.7%) and optimized conditions predicted for PGOs of DP = 2 were T = 60 °C, pH 5, E/S = 0.525 U/mg, and time = 148 min; DP = 3 were T = 59.7 °C, pH 5, E/S = 0.506 U/mg, and time = 12.5 min; and DP = 4, were T = 34.5 °C, pH 11, E/S = 0.525 U/mg, and time = 222.5 min. Fourier transformed infrared (FT-IR) and nuclear magnetic resonance (NMR) characterizations of PGOs are presented.

Strategy for Structural Elucidation of Polysaccharides: Elucidation of a Maize Mucilage that Harbors Diazotrophic Bacteria.

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

Disassembly of the fruit cell wall by the ripening-associated polygalacturonase and expansin influences tomato cracking.

Fruit cracking is an important problem in horticultural crop production. Polygalacturonase (SlPG) and expansin (SlEXP1) proteins cooperatively disassemble the polysaccharide network of tomato fruit cell walls during ripening and thereby, enable softening. A Golden 2-like (GLK2) transcription factor, SlGLK2 regulates unripe fruit chloroplast development and results in elevated soluble solids and carotenoids in ripe fruit. To determine whether SlPG, SlEXP1, or SlGLK2 influence the rate of tomato fruit cracking, the incidence of fruit epidermal cracking was compared between wild-type, Ailsa Craig (WT) and fruit with suppressed SlPG and SlEXP1 expression (pg/exp) or expressing a truncated nonfunctional Slglk2 (glk2). Treating plants with exogenous ABA increases xylemic flow into fruit. Our results showed that ABA treatment of tomato plants greatly increased cracking of fruit from WT and glk2 mutant, but not from pg/exp genotypes. The pg/exp fruit were firmer, had higher total soluble solids, denser cell walls and thicker cuticles than fruit of the other genotypes. Fruit from the ABA treated pg/exp fruit had cell walls with less water-soluble and more ionically and covalently-bound pectins than fruit from the other lines, demonstrating that ripening-related disassembly of the fruit cell wall, but not elimination of SlGLK2, influences cracking. Cracking incidence was significantly correlated with cell wall and wax thickness, and the content of cell wall protopectin and cellulose, but not with Ca2+ content.

Effect of NaHCO3 treatments on the activity of cell-wall-degrading enzymes produced by Penicillium digitatum during the pathogenesis process on grapefruit.

BACKGROUND: This study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with sodium bicarbonate (NaHCO3 ), at two different pH values (5 and 8.3), on cell-wall-degrading enzyme activity over a period of 72 h. RESULTS: Treatment with NaHCO3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, compared with the control or the same treatment at pH 5. In contrast, pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5-treated plugs up to 72 h. CONCLUSION: The rapid increase in pectin methyl esterase activity under alkaline conditions is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact, at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. © 2018 Society of Chemical Industry.

Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii.

Sedum alfredii is one of a few plant species known to hyperaccumulate cadmium (Cd). Uptake, localization, and tolerance of Cd at cellular levels in shoots were compared in hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii. X-ray fluorescence images of Cd in stems and leaves showed only a slight Cd signal restricted within vascular bundles in the NHEs, while enhanced localization of Cd, with significant tissue- and age-dependent variations, was detected in HEs. In contrast to the vascular-enriched Cd in young stems, parenchyma cells in leaf mesophyll, stem pith and cortex tissues served as terminal storage sites for Cd sequestration in HEs. Kinetics of Cd transport into individual leaf protoplasts of the two ecotypes showed little difference in Cd accumulation. However, far more efficient storage of Cd in vacuoles was apparent in HEs. Subsequent analysis of cell viability and hydrogen peroxide levels suggested that HE protoplasts exhibited higher resistance to Cd than those of NHE protoplasts. These results suggest that efficient sequestration into vacuoles, as opposed to rapid transport into parenchyma cells, is a pivotal process in Cd accumulation and homeostasis in shoots of HE S. alfredii. This is in addition to its efficient root-to-shoot translocation of Cd.

Biochemistry and Cell Wall Changes Associated with Noni (Morinda citrifolia L.) Fruit Ripening.

Quality and compositional changes were determined in noni fruit harvested at five ripening stages, from dark-green to thaslucent-grayish. Fruit ripening was accompanied by acidity and soluble solids accumulation but pH diminution, whereas the softening profile presented three differential steps named early (no significant softening), intermediate (significant softening), and final (dramatic softening). At early step the extensive depolymerization of hydrosoluble pectins and the significantly increment of pectinase activities did not correlate with the slight reduction in firmness. The intermediate step showed an increment of pectinases and hemicellulases activities. The final step was accompanied by the most significant reduction in the yield of alcohol-insoluble solids as well as in the composition of uronic acids and neutral sugars; pectinases increased their activity and depolymerization of hemicellulosic fractions occurred. Noni ripening is a process conducted by the coordinated action of pectinases and hemicellulases that promote the differential dissasembly of cell wall polymers.

Co-culturing Chlorella minutissima with Escherichia coli can increase neutral lipid production and improve biodiesel quality.

Lipid productivity and fatty acid composition are important metrics for the production of high quality biodiesel from algae. Our previous results showed that co-culturing the green alga Chlorella minutissima with Escherichia coli under high-substrate mixotrophic conditions enhanced both culture growth and crude lipid content. To investigate further, we analyzed neutral lipid content and fatty acid content and composition of axenic cultures and co-cultures produced under autotrophic and mixotrophic conditions. We found that co-culturing C. minutissima with E. coli under high substrate conditions (10 g/L) increased neutral lipid content 1.9- to 3.1-fold and fatty acid content 1.5- to 2.6-fold compared to equivalent axenic C. minutissima cultures. These same co-cultures also exhibited a significant fatty acid shift away from trienoic and toward monoenoic fatty acids thereby improving the quality of the synthesized fatty acids for biodiesel production. Further investigation suggested that E. coli facilitates substrate uptake by the algae and that the resulting growth enhancement induces a nitrogen-limited condition. Enhanced carbon uptake coupled with nitrogen limitation is the likely cause of the observed neutral lipid accumulation and fatty acid profile changes.

Effects of timing and severity of salinity stress on rice (Oryza sativa L.) yield, grain composition, and starch functionality.

The aim of this work was to examine agronomic, compositional, and functional changes in rice (Oryza sativa L. cv. Nipponbare) grains from plants grown under low-to-moderate salinity stress in the greenhouse. Plants were grown in sodium chloride-containing soil (2 or 4 dS/m(2) electrical conductivity), which was imposed 4-weeks after transplant (called Seedling EC2 and EC4) or after the appearance of the anthers (called Anthesis EC2 and EC4). The former simulates field conditions while the latter permits observation of the isolated effect of salt on grain filling processes. Key findings of this study are the following: (i) Plants showed adaptive responses to prolonged salt treatment with no negative effects on grain weight or fertility. Seedling EC2 plants had more panicles and enhanced caryopsis dimensions, while surprisingly, Seedling EC4 plants did not differ from the control group in the agronomic parameters measured. (ii) Grain starch increased in Seedling EC4 (32.6%) and Anthesis EC2 (39%), respectively, suggesting a stimulatory effect of salt on starch accumulation. (iii) The salinity treatment of 2 dS/m(2) was better tolerated at anthesis than the 4 dS/m(2) treatment as the latter led to reduced grain weight (28.8%) and seed fertility (19.4%) and compensatory increases in protein (20.1%) and nitrogen (19.8%) contents. (iv) Although some salinity treatments led to changes in starch content, these did not alter starch fine structure, morphology, or composition. We observed no differences in reducing sugar and amylose content or starch granule size distribution among any of the treatments. The only alterations in starch were limited to small changes in thermal properties and glucan chain distribution, which were only seen in the Anthesis EC4 treatment. This similarity of compositional and functional features was supported by multivariate analysis of all variables measured, which suggested that differences due to treatments were minimal. Overall, this study documents the specific response of rice under defined conditions, and illustrates that the plasticity of plant response to mild stress is complex and highly context-dependent, even under greenhouse conditions in which other potential environmental stress impacts are minimized.

Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts.

Cell walls are barriers that impair colonization of host tissues, but also are important reservoirs of energy-rich sugars. Growing hyphae of necrotrophic fungal pathogens, such as Botrytis cinerea (Botrytis, henceforth), secrete enzymes that disassemble cell wall polysaccharides. In this work we describe the annotation of 275 putative secreted Carbohydrate-Active enZymes (CAZymes) identified in the Botrytis B05.10 genome. Using RNAseq we determined which Botrytis CAZymes were expressed during infections of lettuce leaves, ripe tomato fruit, and grape berries. On the three hosts, Botrytis expressed a common group of 229 potentially secreted CAZymes, including 28 pectin backbone-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes that might target pectin and hemicellulose side-branches, and 16 enzymes predicted to degrade cellulose. The diversity of the Botrytis CAZymes may be partly responsible for its wide host range. Thirty-six candidate CAZymes with secretion signals were found exclusively when Botrytis interacted with ripe tomato fruit and grape berries. Pectin polysaccharides are notably abundant in grape and tomato cell walls, but lettuce leaf walls have less pectin and are richer in hemicelluloses and cellulose. The results of this study not only suggest that Botrytis targets similar wall polysaccharide networks on fruit and leaves, but also that it may selectively attack host wall polysaccharide substrates depending on the host tissue.

Microplate assay for quantitation of neutral lipids in extracts from microalgae.

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC-MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 µg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

The impact of elevated CO2 concentration on the quality of algal starch as a potential biofuel feedstock.

Cultured microalgae are viewed as important producers of lipids and polysaccharides, both of which are precursor molecules for the production of biofuels. This study addressed the impact of elevated carbon dioxide (CO2) on Chlorella sorokiniana production of starch and on several properties of the starch produced. The production of C. sorokiniana biomass, lipid and starch were enhanced when cultures were supplied with 2% CO2. Starch granules from algae grown in ambient air and 2% CO2 were analyzed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The granules from algae grown in 2% CO2 were disk-shaped and contained mainly stromal starch; granules from cultures grown in ambient air were cup-shaped with primarily pyrenoid starch. The granules from cells grown in 2% CO2 had a higher proportion of the accumulated starch as the highly branched, amylopectin glucan than did granules from cells grown in air. The rate of hydrolysis of starch from 2% CO2-grown cells was 1.25 times greater than that from air-grown cells and 2-11 times higher than the rates of hydrolysis of starches from cereal grains. These data indicate that culturing C. sorokiniana in elevated CO2 not only increases biomass yield but also improves the structure and composition of starch granules for use in biofuel generation. These modifications in culture conditions increase the hydrolysis efficiency of the starch hydrolysis, thus providing potentially important gains for biofuel production.

Spatial imaging of Zn and other elements in Huanglongbing-affected grapefruit by synchrotron-based micro X-ray fluorescence investigation.

Huanglongbing (HLB) is a highly destructive, fast-spreading disease of citrus, causing substantial economic losses to the citrus industry worldwide. Nutrient levels and their cellular distribution patterns in stems and leaves of grapefruit were analysed after graft-inoculation with lemon scions containing 'Candidatus Liberibacter asiaticus' (Las), the heat-tolerant Asian type of the HLB bacterium. After 12 months, affected plants showed typical HLB symptoms and significantly reduced Zn concentrations in leaves. Micro-XRF imaging of Zn and other nutrients showed that preferential localization of Zn to phloem tissues was observed in the stems and leaves collected from healthy grapefruit plants, but was absent from HLB-affected samples. Quantitative analysis by using standard references revealed that Zn concentration in the phloem of veins in healthy leaves was more than 10 times higher than that in HLB-affected leaves. No significant variation was observed in the distribution patterns of other elements such as Ca in stems and leaves of grapefruit plants with or without graft-inoculation of infected lemon scions. These results suggest that reduced phloem transport of Zn is an important factor contributing to HLB-induced Zn deficiency in grapefruit. Our report provides the first in situ, cellular level visualization of elemental variations within the tissues of HLB-affected citrus.

Virus infection of Chlorella variabilis and enzymatic saccharification of algal biomass for bioethanol production.

Experiments were conducted to investigate the application of virus infection and amylolytic enzyme treatment on sugar release from Chlorella variabilis NC64A and bioethanol production from released sugars via Escherichia coli KO11 fermentation. Chlorella variabilis NC64A accumulated starch when it was cultured in a nitrogen-limited medium. The accumulated starch was not consumed during viral infection based on analysis of sugars released during infection. Both amylolytic enzyme addition and virus infection increased the hydrolysis of carbohydrates. Addition of amylolytic enzymes increased the release of glucose from algal biomass while virus addition increased the release of non-glucose neutral sugars. The combination of enzyme addition and virus infection also resulted in the highest ethanol production after fermentation. Acetic acid was generated as a co-product during fermentation in all sets of experiments. This study demonstrated that infection of microalgae with an algal virus resulted in disruption and hydrolysis of algal biomass to generate fermentable sugars.

Analysis of metal element distributions in rice (Oryza sativa L.) seeds and relocation during germination based on X-ray fluorescence imaging of Zn, Fe, K, Ca, and Mn.

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.

Efficient xylem transport and phloem remobilization of Zn in the hyperaccumulator plant species Sedum alfredii.

Sedum alfredii is one of a few species known to hyperaccumulate zinc (Zn) and cadmium (Cd). Xylem transport and phloem remobilization of Zn in hyperaccumulating (HP) and nonhyperaccumulating (NHP) populations of S. alfredii were compared. Micro-X-ray fluorescence (µ-XRF) images of Zn in the roots of the two S. alfredii populations suggested an efficient xylem loading of Zn in HP S. alfredii, confirmed by the seven-fold higher Zn concentrations detected in the xylem sap collected from HP, when compared with NHP, populations. Zn was predominantly transported as aqueous Zn (> 55.9%), with the remaining proportion (36.7-42.3%) associated with the predominant organic acid, citric acid, in the xylem sap of HP S. alfredii. The stable isotope (68)Zn was used to trace Zn remobilization from mature leaves to new growing leaves for both populations. Remobilization of (68)Zn was seven-fold higher in HP than in NHP S. alfredii. Subsequent analysis by µ-XRF, combined with LA-ICPMS (laser ablation-inductively coupled plasma mass spectrometry), confirmed the enhanced ability of HP S. alfredii to remobilize Zn and to preferentially distribute the metal to mesophyll cells surrounding phloem in the new leaves. The results suggest that Zn hyperaccumulation by HP S. alfredii is largely associated with enhanced xylem transport and phloem remobilization of the metal. To our knowledge, this report is the first to reveal enhanced remobilization of metal by phloem transport in hyperaccumulators.

Vascular occlusions in grapevines with Pierce's disease make disease symptom development worse.

Vascular occlusions are common structural modifications made by many plant species in response to pathogen infection. However, the functional role(s) of occlusions in host plant disease resistance/susceptibility remains controversial. This study focuses on vascular occlusions that form in stem secondary xylem of grapevines (Vitis vinifera) infected with Pierce's disease (PD) and the impact of occlusions on the hosts' water transport and the systemic spread of the causal bacterium Xylella fastidiosa in infected vines. Tyloses are the predominant type of occlusion that forms in grapevine genotypes with differing PD resistances. Tyloses form throughout PD-susceptible grapevines with over 60% of the vessels in transverse sections of all examined internodes becoming fully blocked. By contrast, tylose development was mainly limited to a few internodes close to the point of inoculation in PD-resistant grapevines, impacting only 20% or less of the vessels. The extensive vessel blockage in PD-susceptible grapevines was correlated to a greater than 90% decrease in stem hydraulic conductivity, compared with an approximately 30% reduction in the stems of PD-resistant vines. Despite the systemic spread of X. fastidiosa in PD-susceptible grapevines, the pathogen colonized only 15% or less of the vessels in any internode and occurred in relatively small numbers, amounts much too small to directly block the vessels. Therefore, we concluded that the extensive formation of vascular occlusions in PD-susceptible grapevines does not prevent the pathogen's systemic spread in them, but may significantly suppress the vines' water conduction, contributing to PD symptom development and the vines' eventual death.

Texture, composition and anatomy of spinach leaves in relation to nitrogen fertilization.

BACKGROUND: The postharvest quality and shelf life of spinach are greatly influenced by cultural practices. Reduced spinach shelf life is a common quandary in the Salinas Valley, California, where current agronomic practices depend on high nitrogen (N) rates. This study aimed to describe the postharvest fracture properties of spinach leaves in relation to N fertilization, leaf age and spinach cultivar. RESULTS: Force-displacement curves, generated by a puncture test, showed a negative correlation between N fertilization and the toughness, stiffness and strength of spinach leaves (P > 0.05). Younger leaves (leaves 12 and 16) from all N treatments were tougher than older leaves (leaves 6 and 8) (P > 0.05). Leaves from the 50 and 75 ppm total N treatments irrespective of spinach cultivar had higher fracture properties and nutritional quality than leaves from other N treatments (P > 0.05). Total alcohol-insoluble residues (AIR) and pectins were present at higher concentrations in low-N grown plants. These plants also had smaller cells and intercellular spaces than high-N grown leaves (P > 0.05). CONCLUSION: Observed changes in physicochemical and mechanical properties of spinach leaves due to excess nitrogen fertilization were significantly associated with greater postharvest leaf fragility and lower nutritional quality.

Mobility of Transgenic Nucleic Acids and Proteins within Grafted Rootstocks for Agricultural Improvement.

Grafting has been used in agriculture for over 2000 years. Disease resistance and environmental tolerance are highly beneficial traits that can be provided through use of grafting, although the mechanisms, in particular for resistance, have frequently been unknown. As information emerges that describes plant disease resistance mechanisms, the proteins, and nucleic acids that play a critical role in disease management can be expressed in genetically engineered (GE) plant lines. Utilizing transgrafting, the combination of a GE rootstock with a wild-type (WT) scion, or the reverse, has the potential to provide pest and pathogen resistance, impart biotic and abiotic stress tolerance, or increase plant vigor and productivity. Of central importance to these potential benefits is the question of to what extent nucleic acids and proteins are transmitted across a graft junction and whether the movement of these molecules will affect the efficacy of the transgrafting approach. Using a variety of specific examples, this review will report on the movement of organellar DNA, RNAs, and proteins across graft unions. Attention will be specifically drawn to the use of small RNAs and gene silencing within transgrafted plants, with a particular focus on pathogen resistance. The use of GE rootstocks or scions has the potential to extend the horticultural utility of grafting by combining this ancient technique with the molecular strategies of the modern era.

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa.

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a ß-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a ß-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.

Polysaccharide compositions of intervessel pit membranes contribute to Pierce's disease resistance of grapevines.

Symptom development of Pierce's disease (PD) in grapevine (Vitis vinifera) depends largely on the ability of the bacterium Xylella fastidiosa to use cell wall-degrading enzymes (CWDEs) to break up intervessel pit membranes (PMs) and spread through the vessel system. In this study, an immunohistochemical technique was developed to analyze pectic and hemicellulosic polysaccharides of intervessel PMs. Our results indicate that PMs of grapevine genotypes with different PD resistance differed in the composition and structure of homogalacturonans (HGs) and xyloglucans (XyGs), the potential targets of the pathogen's CWDEs. The PMs of PD-resistant grapevine genotypes lacked fucosylated XyGs and weakly methyl-esterified HGs (ME-HGs), and contained a small amount of heavily ME-HGs. In contrast, PMs of PD-susceptible genotypes all had substantial amounts of fucosylated XyGs and weakly ME-HGs, but lacked heavily ME-HGs. The intervessel PM integrity and the pathogen's distribution in Xylella-infected grapevines also showed differences among the genotypes. In pathogen-inoculated, PD-resistant genotypes PM integrity was well maintained and Xylella cells were only found close to the inoculation site. However, in inoculated PD-susceptible genotypes, PMs in the vessels associated with bacteria lost their integrity and the systemic presence of the X. fastidiosa pathogen was confirmed. Our analysis also provided a relatively clear understanding of the process by which intervessel PMs are degraded. All of these observations support the conclusion that weakly ME-HGs and fucosylated XyGs are substrates of the pathogen's CWDEs and their presence in or absence from PMs may contribute to grapevine's PD susceptibility.