Elevated CO2 concentration impacts cell wall polysaccharide composition of green microalgae of the genus Chlorella.

UNLABELLED: The effect of CO2 concentration on the relative content of starch, lipid and cell wall carbohydrates in microalgal biomass was investigated for the four following Chlorella strains: C. vulgaris (UTEX 259), C. sorokiniana (UTEX 2805), C. minutissima (UTEX 2341) and C. variabilis (NC64A). Each strain had a different response to CO2 concentration. The starch content was higher in UTEX259 and NC64A cultured with 2% CO2 in the air supply than in cells cultured with ca. 0·04% CO2 (ambient air), while starch content was not affected for UTEX 2805 and UTEX 2341. The lipid content was higher in Chlorella minutissima UTEX 2341 cultured in 2% CO2 than in cells cultured in ambient air, but was unchanged for the other three strains. All four Chlorella strains tended to have a higher percentage of uronic acids and lower percentage of neutral sugars in their cell wall polysaccharide complement when grown with 2% CO2 supply. Although the percentage of neutral sugars in the cell walls varied with CO2 concentration, the relative proportions of different neutral sugar constituents remained constant for both CO2 conditions. The results demonstrate the importance of considering the effects of CO2 on the cell wall carbohydrate composition of microalgae. SIGNIFICANCE AND IMPACT OF THE STUDY: Microalgae have the potential to produce products that will reduce society's reliance on fossil fuels and address challenges related to food and feed production. An overlooked yet industrially relevant component of microalgae are their cell walls. Cell wall composition affects cell flocculation and the recovery of intracellular products. In this study, we show that increasing CO2 level results in greater cell wall polysaccharide and uronic acid content in the cell walls of three strains of microalgae. The results have implications on the management of systems for the capture of CO2 and production of fuels, chemicals and food from microalgae.

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback.

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.

The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea.

Fruit ripening is characterized by processes that modify texture and flavor but also by a dramatic increase in susceptibility to necrotrophic pathogens, such as Botrytis cinerea. Disassembly of the major structural polysaccharides of the cell wall (CW) is a significant process associated with ripening and contributes to fruit softening. In tomato, polygalacturonase (PG) and expansin (Exp) are among the CW proteins that cooperatively participate in ripening-associated CW disassembly. To determine whether endogenous CW disassembly influences the ripening-regulated increase in necrotropic pathogen susceptibility, B. cinerea susceptibility was assessed in transgenic fruit with suppressed polygalacturonase (LePG) and expansin (LeExp1) expression. Suppression of either LePG or LeExp1 alone did not reduce susceptibility but simultaneous suppression of both dramatically reduced the susceptibility of ripening fruit to B. cinerea, as measured by fungal biomass accumulation and by macerating lesion development. These results demonstrate that altering endogenous plant CW disassembly during ripening influences the course of infection by B. cinerea, perhaps by changing the structure or the accessibility of CW substrates to pathogen CW-degrading enzymes. Recognition of the role of ripening-associated CW metabolism in postharvest pathogen susceptibility may be useful in the design and development of strategies to limit pathogen losses during fruit storage, handling, and distribution.

Transgenic expression of pear PGIP in tomato limits fungal colonization.

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.

Cell Wall Metabolism in Ripening Fruit (IX. Synthesis of Pectic and Hemicellulosic Cell Wall Polymers in the Outer Pericarp of Mature Green Tomatoes (cv XMT-22).

Discs of outer pericarp were excised from mature green tomato (Lycopersicon esculentum Mill.) fruit and kept in sterile tissue culture plates for 4 d, including 2 d of incubation with D-[U-13C]glucose. Cell walls were prepared and the water-soluble, pectic, and hemicellulosic polymers were extracted. Cell wall synthetic capacity was determined by gas chromatography-mass spectrometry analysis of incorporation of the heavy isotope label. The "outer" 2-mm pericarp region, which included the cuticle, had a lower cell wall synthetic capacity than the "inner" 2-mm region immediately below it (closer to the locules), based on the percentage of labeling of the neutral sugars. There were no significant differences in relative abundance of glycosidic linkages in the two tissue regions. Label was incorporated into neutral sugars and linkages typical for each polysaccharide class were identified in the cell wall preparations. Galacturonic acid and glucuronic acid were labeled to an extent similar to that of the neutral sugars in each tissue region.

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates.

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits).

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.

Structure and expression of an inhibitor of fungal polygalacturonases from tomato.

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.

Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes).

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.

Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L. cv Bartlett.

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.

Cell Wall Metabolism in Ripening Fruit : V. Analysis of Cell Wall Synthesis in Ripening Tomato Pericarp Tissue Using a d-[U-C]Glucose Tracer and Gas Chromatography-Mass Spectrometry.

A gas chromatographic-mass spectrometric technique utilizing d-[U-(13)C]glucose as a density label tracer was used to follow the synthesis of cell wall polysaccharides in pericarp discs that were excised from mature green tomato fruit (Lycopersicon esculentum) and allowed to ripen in culture. The biosynthetic capacity of discs from four different maturity stages was examined. Label was differentially incorporated into wall polysaccharides as the discs matured, indicating a change in the nature of wall polymers being synthesized. These differential rates of incorporation are consistent with descriptions of ripening-related cell wall compositional changes previously reported by other authors. Specific changes in wall biosynthesis noted include increased incorporation of xylosyl and mannosyl residues into hemicellulosic cell wall fractions as the discs mature and decreased incorporation of galactosyl residues into chelator-soluble pectins.

Induction and regulation of ethylene biosynthesis by pectic oligomers in cultured pear cells.

Pectic oligomers induced a rapid, transient increase in ethylene biosynthesis when added to pear cells in suspension culture. The rate of ethylene biosynthesis increased within 30 to 40 minutes after oligomer addition, reached a maximum between 90 and 120 minutes after addition, and then decreased to basal rates of synthesis. Both the rapid increase and decrease in biosynthesis appear to be precisely regulated components of the ethylene response to oligomers. Induction of ethylene biosynthesis by pectic oligomers resulted in a reduced sensitivity of cells to further ethylene induction. This reduction in sensitivity occurred within 90 minutes after an oligomer treatment, slightly preceding the decline in ethylene synthesis. The degree of insensitivity induced was proportional to the concentration of oligomer in the first treatment. Induced insensitivity to elicitors appears to represent a novel mechanism which may limit continued ethylene biosynthesis after ethylene induction. Ethylene was produced by pear cells throughout the cell growth cycle, as cells increased in density over a 6 day period. Endogenous ethylene biosynthesis was at a maximum during the first 4 days of rapid cell growth, then declined to half the peak rate through day 10. Pectic oligomers could induce an increase in ethylene biosynthesis above this background rate only after day 5, as endogenous biosynthesis declined. Changes in sensitivity to added oligomer during the growth cycle may result from insensitivity to elicitors induced by growth processes.

Induction and regulation of ethylene biosynthesis and ripening by pectic oligomers in tomato pericarp discs.

The effect of pectic oligomers and 1-aminocyclopropane carboxylic acid on ethylene biosynthesis and color change was studied in ripening tomato pericarp discs excised from mature-green tomato fruit (Lycopersicon esculentum Mill.). Pectic oligomers induced at least four distinct responses when added to pericarp discs: (a) a short-term, transient increase in ethylene biosynthesis; (b) a long-term, persistent increase in climacteric ethylene in discs excised from mature-green fruit; (c) an advance in ripening processes, as indicated by increased reddening of the disc surfaces; and (d) a darkening of the treated endocarp surface. Pectic oligomers appear to affect the ripening of exocarp and endocarp tissues by different mechanisms. In exocarp tissues, the acceleration of reddening by pectic oligomers might simply be a consequence of induced ethylene biosynthesis. In endocarp tissues, the acceleration of reddening appears to be a direct effect of oligomers on ripening processes. We suggest that the rate of ripening of endocarp tissues may be regulated, in part, by the release of pectic oligomers from the cell walls of adjacent exocarp tissues. Exocarp and endocarp tissues of pericarp discs appear to differ in their sensitivity to ethylene at each maturity stage, and to exhibit independent changes in sensitivity to ethylene as ripening progresses. The tissue-specific pattern of reddening in tomato pericarp may result from this differential sensitivity to endogenous ethylene concentrations.

Cell turgor changes associated with ripening in tomato pericarp tissue.

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 (<0.2 megapascals) were much less than expected, based on tissue osmotic potential (-0.6 to -0.7 megapascals). These low turgor values may indicate the presence of apoplastic solutes. In both intact fruit and cultured discs of pericarp tissue, a small increase in turgor preceded the onset of ripening, and a decrease in turgor occurred during ripening. Differences in the turgor of individual intact fruit occurred 2 to 4 days before parallel differences in their ripening behavior were apparent, indicating that changes in turgor may reflect physiological changes at the cell level that precede expression of ripening at the tissue level.

Comparison of ripening processes in intact tomato fruit and excised pericarp discs.

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L(*)a(*)b(*) color space, changing from green (a(*) < -5) to red (a(*) > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO(2) production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a(*) = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a(*) = -5), appeared as discs turned yellow-red (a(*) = 5), and increased another eight-fold as discs became full red (a(*) value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a(*) = 0) to red.

Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion.

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.

Cell Wall Metabolism in Ripening Fruit: IV. Characterization of the Pectic Polysaccharides Solubilized during Softening of ;Bartlett' Pear Fruit.

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.

Microfluorometry of pectic materials in the dehiscence zone of almond (Prunus dulcis [Mill.] DA Webb) fruits.

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.

The induction of ethylene production from pear cell culture by cell wall fragments.

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.

Muralytic Activities of Ruminococcus albus 8.

Ruminococcus albus 8 was cultured with isolated alfalfa cell walls as the carbon source. The culture broth was assayed for muralytic enzyme activities. The effect, with respect to the production of such muralytic enzymes, of growing the microorganism on different carbon sources was also investigated. Also, the rates of solubilization and utilization by R. albus of individual alfalfa cell wall sugars during a 96-h growth period were examined.