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)

Abstract

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-'3C]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. l h e "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. lhere 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. Calacturonic acid and glucuronic acid were labeled to an extent similar to that of the neutral sugars in each tissue region.

CESA TRAFFICKING INHIBITOR Inhibits Cellulose Deposition and Interferes with the Trafficking of Cellulose Synthase Complexes and Their Associated Proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1

“…We monitored the relative incorporation of 13 C into the Glc detected in the cellulose fraction, using gas chromatography-mass spectrometry analysis of alditol acetates generated after hydrolysis of the cellulose fraction. Selective ion monitoring mode, as described previously (Greve and Labavitch, 1991;Huysamer et al, 1997), indicated that CESTRIN significantly decreased the amount of [ 13 C]Glc that was incorporated into the cellulose fraction; this reduction was greater when more CESTRIN was applied (P , 0.00001 for 15 mM, P = 0.000124 for 8 mM) in our short-term treatment (Table II). In addition, a less profound but still significant reduction was observed in the 2 N trifluoroacetic acid (TFA)-hydrolyzed cell wall fraction (i.e.…”

“…Cell wall materials were isolated into AIR (Günl et al, 2010). The AIR remaining insoluble after a 1-h 2 N TFA hydrolysis at 121°C (cellulose fraction) was collected by centrifugation, dissolved by stirring overnight in 15 N TFA at room temperature, and then hydrolyzed in 5 N TFA for 1 h. The 2 N and 15 N TFA fractions were derivatized into alditol acetates as described previously (Greve and Labavitch, 1991;Huysamer et al, 1997). Gas chromatographymass spectrometry analysis was carried out as described previously in the selective ion monitoring mode to determine the relative incorporation of [ 13 C] Glc.…”

“…Gas chromatographymass spectrometry analysis was carried out as described previously in the selective ion monitoring mode to determine the relative incorporation of [ 13 C] Glc. Secondary ions of 187 atomic mass units (from the 12 C precursor) and 191 atomic mass units (from the 13 C precursor) from the alditol acetates for Glc were used for quantification as described previously (Greve and Labavitch, 1991;Huysamer et al, 1997).…”

A Reevaluation of the Key Factors That Influence Tomato Fruit Softening and Integrity

“…Subsequent steps in preparing crude cell wall samples (alcohol-insoluble solids [AISs]) were also as in Huysamer et al (1997), ending with two washes of the wall pellets with acetone and drying in a vacuum oven. Starch was removed by incubating 200 mg of AIS with 40 units each of a-amylase (from porcine pancreas, Sigma-Aldrich) and pullulanase (from Bacillus acidopullulyticus, Novozymes Biologicals) in 20 mL of Tris-HCl pH 7.0, 0.1% NaN 3 , for 24 h. Neutral sugar composition of the AIS was determined by gas-liquid chromatography as in Campbell et al (1990).…”

“…Cell walls were prepared from 50 g of diced outer pericarp of AC and DFD fruit (MG and RR stages) by boiling the pericarp pieces in 95% ethanol for 30 min to prevent autolytic activity, as described in Huysamer et al (1997). Subsequent steps in preparing crude cell wall samples (alcohol-insoluble solids [AISs]) were also as in Huysamer et al (1997), ending with two washes of the wall pellets with acetone and drying in a vacuum oven.…”