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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.

This work shows an optimized enzymatic hydrolysis of high molecular weight potato galactan yielding pectic galactan-oligosaccharides (PGOs), where endo-β-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.

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.

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.

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.

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.

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.

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. © 2014 © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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.

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.

Xylem-dwelling pathogens become systemic, suggesting that microorganisms move efficiently in the xylem. To better understand xylem pathways and how bacteria move within the xylem, vessel connectivity between stems and leaves of Vitis vinifera cv. Chardonnay and Muscadinia rotundifolia cv. Cowart was studied. Three methods were used: (1) the light-producing bacterium, Yersinia enterocolitica, (Ye) strain GY5232 was loaded into petioles and followed using X-ray film, (2) fluorescent beads were loaded and followed by microscopy, and (3) low-pressure air was pumped into leaves and extruded bubbles from cuts in submerged leaves were followed. Bacteria, beads, and air moved through long and branched xylem vessels from the petiole into the veins in leaves of both varieties. From the stem, bacteria and air traveled into primary and secondary veins of leaves one, two, and three nodes above the loading point of the bacteria or air. Particles and air could move unimpeded through single xylem vessels or multiple vessels (conduits) connected possibly through broken pit membranes from within the stem axis into leaf blades. Bacteria were also able to move long distances within minutes from stem to leaf passively without having to cross pit membranes. Such complex, open xylem conduits have not been well documented before; these findings will help elucidate mechanisms involved in the systemic spread of pathogens. Key words: grapevine; Muscadinia rotundifolia; particle movement; pit membrane; Vitis vinifera; xylem vessel connectivity.

The cloning and purification of the major family 10 xylanase (Xyn10A) from Acidothermus cellulolyticus 11B is described. Xylan cleavage patterns have shown that Xyn10A is an endoxylanase and its half-life at 90 [degree]C is approximately 1.5 h in the presence of xylan.

Polyclonal antibodies are raised against a modified xanthan gum polymer that is similar to the Xylella fastidiosa exopolysaccharide (EPS) polymer, which is quantified by using enzyme-linked immunosorbent assay. Immunolocalization microscopy is used for examining the distribution of Xanthomonas fastidiosa EPS in biofilms formed in vitro and in planta.

ABSTRACT: 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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]