Kenneth C. Gross

Changes in cell wall neutral sugar composition during fruit ripening: a species survey

Abstract Non-cellulosic neutral sugar composition of cell walls from seventeen fruit types were analysed during ripening. Galactose was the major non-cellulosic neutral sugar in cell walls of cucurbit and solanaceous fruit, xylose was the predominant non-cellulosic neutral component of berries, and arabinose was the major non-cellulosic component of pome fruits. The major non-cellulosic neutral sugar residue in cell walls of stone fruits varied. In nectarine and peach, plum, and apricot, the major sugar was arabinose, galactose, and xylose, respectively. In 15 of the 17 types of fruit, a net loss of non-cellulosic neutral sugar residues occurred during ripening. No net loss occurred in plums and cucumbers. A net loss of cell wall galactose and/or arabinose occurred in 14 of the types of fruit. Xylose was the major neutral sugar residue lost from walls of apricot during ripening. In general, berry cell walls were comparatively low in galactose and arabinose content.

Hidden sources of galactose in the environment

A galactose-restricted diet free of lactose is lifesaving in patients with galactose-1-phosphate uridyl transferase (GALT) deficiency, but does not prevent long-term complications such as developmental delay, abnormal speech, poor growth and, in females, ovarian failure. Lactose, found in dairy products and as an extender in drugs, has been considered the primary source of galactose in the diet. Two recent publications reported that small amounts of galactose are present in many fruits and vegetables. We report the presence of considerable amounts of free galactose in some legumes (dried beans and peas) and the presence of bound galactose in many food plants. Galactose, in various glycosidic linkages, such as α-1,6, β−1,3 and β−1,4, and as a component of lipids, is ubiquitous in animals and plants. The bioavailability of α−1,6 and β−1,3 linked galactose in foods is unknown. However, α-galactosidases found in plant and animal tissues may release galactose in α−1,6 linkage, and from digalactosyldiacylglycerol. Galactose in β−1,4 linkage and as monogalactosyldiacylglycerol may be released by β-galactosidases in animal and plant tissues. Foods fermented by micro-organisms for preparation or preservation purposes may contain free galactose. The role of free and bound galactose in cereals, fruits, legumes, nuts, organ meats, seeds, and vegetables in the poor outcome seen in some patients with GALT deficiency is unknown. It is certain that no patients with GALT deficiency have ever ingested a galactose-free diet.

Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice

We purified osmoregulated periplasmic glucans (OPGs) from Salmonella enterica serovar Typhimurium and found them to be composed of 100 % glucose with 2-linked glucose as the most abundant residue, with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structural genes for OPG biosynthesis, opgG and opgH, form a bicistronic operon, and insertion of a kanamycin resistance gene cassette into this operon resulted in a strain devoid of OPGs. The opgGH mutant strain was impaired in motility and growth under low osmolarity conditions. The opgGH mutation also resulted in a 2 log increase in the LD50 in mice compared to the wild-type strain SL1344. Inability to synthesize OPGs had no significant impact on the organisms lipopolysaccharide pattern or its ability to survive antimicrobial peptides-, detergent-, pH- and nutrient-stress conditions. We observed that the opgGH-defective strain respired at a reduced rate under acidic growth conditions (pH 5.0) and had lower ATP levels compared to the wild-type strain. These data indicate that OPGs of S. Typhimurium contribute towards mouse virulence as well as growth and motility under low osmolarity growth conditions.

N-Terminal Amino Acid Sequence of Persimmon Fruit [beta]-Galactosidase

[beta]-Galactosidase (EC 3.2.1.23) from persimmon fruit was purified 114-fold with a 15% yield using Sephadex G-100 gel filtration, CM-Sephadex ion exchange, and Sephacryl S-200 gel filtration chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The estimated molecular mass of the native [beta]-galactosidase by Sephacryl S-200 was 118 kD. After sodium dodecyl sulfate-PAGE of the enzyme electroeluted from native gels, two subunits with estimated molecular masses of 34 and 44 kD were observed, suggesting that the native enzyme was an aggregate of several subunits. Amino acid composition and N-terminal amino acid sequences of the two major subunits were different.

Effect of ethylene on mRNA abundance of three β-galactosidase genes in wild type and mutant tomato fruit

Abstract Recent studies indicate that β-galactosidases may play an important role in fruit development and ripening. The temporal expression patterns of a recently discovered family of tomato β-galactosidase (TBG) genes suggest that accumulation of some TBG mRNAs may be regulated by ethylene. Because of the dramatic changes in mRNA abundance patterns that TBG4, TBG5 and TBG6 exhibit at the onset of fruit ripening (when ethylene production begins), we analyzed the effects of ethylene exposure on expression of these genes at 35 days after pollination (Mature Green) in wild type and three tomato ripening-impaired mutants: ripening inhibitor ( rin ), non ripening ( nor ) and Never ripe ( Nr ). RNA gel blot analysis showed that 48 h of ethylene treatment increased accumulation of TBG4 mRNA up to 20-fold, except in the nor mutant, suggesting that the NOR gene product is required for ethylene up-regulation of TBG4. Experiments in which fruit were exposed to ethylene for either 2 or 24 h indicated that the observed TBG4 up-regulation is an indirect response to the ethylene treatment, rather than a primary (or direct) response. Ethylene treatment caused the levels of TBG5 mRNA to decrease 63–93% in wild type and mutant fruit at the Mature Green stage, but had no effect after the onset of ripening. Ethylene dramatically decreased the mRNA abundance of TBG6 in wild type and all mutants at the Mature Green stage. It was shown previously that the TBG4-encoded enzyme has β-galactosidase and exo-galactanase activity. In order to elucidate its potential cell wall modifying activity, the TBG5-encoded enzyme was expressed in yeast. β-Galactosidase and exo-galactanase activity of the TBG5-encoded enzyme were confirmed through a quantified release of galactosyl residues from p -nitrophenyl-β- d -galactopyranoside and from tomato fruit cell wall fractions containing β(1→4)- d -galactan, respectively. The TBG6-encoded product was not successfully expressed in yeast. We present a summary of the potential function of the TBGs studied here and their mRNA accumulation in relation to the climacteric production of ethylene in tomato fruit.

Cloning and characterization of a rhamnogalacturonan hydrolase gene from Botrytis cinerea

Rhamnogalacturonan hydrolase (Rgase A) cleaves α1→2 linkages between rhamnosyl and galacturonosyl residues in pectin. A 1.9 kb RGase A cDNA clone (BCRHGA) was isolated from a B. cinerea cDNA library using a PCR‐amplified Aspergillus aculeatus RGase A probe. Its 1.7 kb open reading frame had 62% identity at the amino acid level with A. aculeatus RGase A. Northern blots of B. cinerea total RNA probed with BCRHGA revealed a 2 kb band, suggesting the cDNA clone is full or nearly‐full length. To determine mRNA expression of the gene, B. cinerea was grown in media containing 0.5% apple pectin, 0.5% rhamnogalacturonan‐I and 1% glucose carbon sources. Northern analysis revealed the BCRHGA gene was expressed on all carbon sources, but with different patterns of expression. B. cinerea RGase A appeared to be coded for by a single or low copy number gene based on Southern analysis.

Enzymatic activity and substrate specificity of recombinant tomato β-galactosidases 4 and 5

The open reading frames of tomato β-galactosidase (TBG) 4 and 5 cDNAs were expressed in yeast, and the enzymes properties and substrate specificities were investigated. The two enzymes had peak activities between pH 4–4.5 and 37–45°C. TBG4 specifically hydrolyzed β-(1→4) and 4-linked galactooligosaccharides. TBG5 had a strong preference to hydrolyze β-(1→3) and β-(1→6)-linked galactooligosaccharides. Exo-β-galactanase activity of the TBG enzymes was measured by determining the release of galactosyl residues from native tomato cell wall fractions throughout fruit development and ripening. Both TBGs released galactose from all of the fractions and stages tested. TBG4 activity was highest using chelator soluble pectin and alkali soluble pectin at the turning stage of ripening. Using aminopyrene trisulfonate labeled substrates, TBG4 was the only enzyme with strong exo-β-(1→4)-galactanase activity on 5xa0mer or greater galactans. TBG4 and TBG5 were both able to degrade galactosylated rhamnogalacturonan. Neither enzyme was able to degrade galactosylated xyloglucan.

Changes in cell wall galactosyl and soluble galactose content in tomato fruit stored in low oxygen atmospheres

Abstract Mature green tomato ( Lycopersicon esculentum Mill.) fruit were stored in atmospheres of low oxygen (3.05 kPa; 3%) or air (control) at 20°C. Their subsequent ripening behavior was compared in an effort to evaluate the temporal relationship among ethylene production, respiration, cell wall galactosyl residue loss and the increase in free galactose that occurs during ripening. An increase in ethylene evolution and respiration, red color development, and loss in fruit firmness were delayed in fruit held in low oxygen compared to control fruit held in air which showed normal ripening-related changes. Both the ripening-related decrease in cell wall galactosyl residues and the increase in soluble galactose were suppressed for 9 days in fruit held in low oxygen. However, after 9 days of storage, these processes did commence and changed significantly by 13 days of storage. These results suggest that either only relatively low baseline levels of ethylene were necessary for it to exert its effect on these cell wall-related fruit ripening processes to occur or that the cell wall-related changes observed were ethylene independent. It is clear that the levels of ethylene generally produced during the respiratory climacteric were not necessary to begin the processes associated with cell wall modification, such as the large net loss of galactosyl residues from the cell wall during tomato fruit ripening.

Effects of hypoxia on respiration and the onset of senescence in cut carnation flowers (Dianthus caryophyllus L.)

Abstract Oxygen concentrations ranging between 1.62 and 5.01 kPa O2 reduced the rate of CO2 evolution in cut carnation flowers (Dianthus caryophyllus L.) and, more importantly, delayed the onset of senescence. The suppressive effects of low O2 on respiration could not be ascribed to the restriction of mitochondrial terminal oxidases. An O2 concentration of 2.02 kPa totally suppressed the onset of the climacteric rise in CO2 evolution and increased the vase life of flowers twofold over that of flowers treated with STS, indicating that the retarding effects of hypoxia on the onset of senescence transcend its inhibitory effects on the action of C2H4. Hypoxia diminished the effects of exogenously applied C2H4 on the rate of CO2 evolution and appeared to retard the rate at which the usual morphological symptoms associated with petal senescence occurred. Rapid wilting, concomitant with the climacteric, was a facet of C2H4 action and coincided with a decrease in water uptake. The latter could not be ascribed to the obstruction of vascular bundles but rather to the inability of petals to absorb water. It is suggested that the sudden increase in water loss may be the result of an C2H4-induced increase in hydraulic conductivity of the petals.

Unconjugated Man5GlcNAc occurs in vegetative tissues of tomato

Unconjugated Man alpha 1-->6(Man alpha 1-->3)Man alpha 1-->6(Man alpha 1-->3)Man beta 1-->4GlcNAc (Man5Glc NAc) delayed ripening when added exogenously at 10 ng g-1 fr. wt to both whole tomato fruit and excised pericarp tissue discs. In addition, Man5GlcNAc was one of 10 unconjugated N-glycans purified from tomato pericarp tissue. When applied exogenously at a concentration of 10 nM, Man5GlcNAc prevented the delay of ripening induced by tunicamycin, an inhibitor of its de novo synthesis. Since it was possible that this unconjugated N-glycan might be specifically related to ripening, we checked to see if it also occurred in fully expanded leaf and stem tissues using high-pH anion-exchange chromatography with pulsed amperometric detection. This method allowed us to detect 1 ng of oligosaccharide and to quantify accurately amounts in the range 60-600 ng per injection, significantly more sensitive than the amine-bonded HPLC method previously used to separate and quantify unconjugated N-glycans in tomato fruit pericarp tissue. Four high-mannosyl type unconjugated N-glycans were detected in tomato vegetative tissues, including Man5GlcNAc. These results support a potential role of Man5GlcNAc and/or other unconjugated N-glycans in plant developmental processes, in addition to its apparent role as a modulator of senescence of fruit. However, an initial experiment showed no effect of Man5GlcNAc on senescence of tomato leaf discs, as measured by chlorophyll loss.