Cell Wall Cementing Materials of Grass Leaves

Abstract

Treatment of grass leaves with either a purified pectin lyase of Aspergillus japonicus or a purified xylanase of Trichoderma viride could lead to the isolation of some single leaf cells. However, a mixture of pectin lyase and xylanase brought about more rapid isolation of single cells than did either of the two enzymes alone, indicating a synergistic effect. Analysis of the components released from oat cell walls by the enzymes indicated that both homogalacturonans with a high degree of esterification and a kind of glucuronoarabinoxylan with ferulic acid ester may play a role in cell wall cementing in grass leaves.The enzymic maceration of plant tissues, in which cells are freed from one another, is the first reaction step of enzymic protoplast isolation. Breakdown of intercellular cementing materials leads to separation of individual cells. Endo-types of PG' (2,3,25), PL (5,16,22), and pectate lyase (8,10,20) Purified Enzymes. Purified PG and PL were obtained from the culture medium of Aspergillus japonicus by the methods described previously (17,18). Xylanase (fraction P4) was purified from a cellulase preparation of Trichoderma viride by the method described in a previous paper (15).Assay of Macerating Activity. The lower epidermis of oat leaves was removed, and the leaves (140 mg) were placed in a 50-ml Erlenmeyer flask containing 0.8 M mannitol, 20 mm Mes buffer (pH 5.5), 10 mM cysteine, enzymes, and water to a final volume of 5.0 ml. Mannitol was added as an osmotic stabilizer. The flasks were shaken on a rotary shaker at 120 rpm and 25°C. After 2 h the mixture was filtered through Miracloth, and the OD ofthe filtrate was measured at 660 nm. A linear relationship exists between the OD at 660 nm and the number of single cells counted in a hemocytometer.Treatment of Oat Cell Walls by Enzyme. The cell walls (100 mg) were placed in 150-ml Erlenmeyer flasks containing 50 ml of 0.1 M ammonium acetate buffer (pH 5.5) and 370 gg of PL or 1.65 mg ofxylanase. The flasks were shaken on a rotary shaker at 100 rpm and 30°C. After 3 h the reaction mixtures were filtered through Toyo No. SC paper and the residues were repeatedly washed with 0.1 M ammonium acetate buffer (pH 5.5). The filtrate and washings were combined and lyophilized.Gel Filtration. Gel filtration was carried out on a Sephadex G-100 column (1.9 x 100 cm) equilibrated with 0.1 M ammonium acetate buffer (pH 5.5). Samples were applied in 2.0 ml to the column, and the elution was carried out with 0.1 M ammonium acetate buffer (pH 5.5) at a flow rate of 20 ml/h. Analysis of Sugars. The uronide content was estimated by the m-hydroxydiphenyl method using galacturonic acid as a standard (4) by TLC using ethyl acetate-pyridine-acetic acid-water (5:5:1:3, by vol) and the organic phase from a mixture of 100 g of phenol, 100 ml of water, and 1 ml of 85% HCOOH (23)

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

“…Another endo-xylanase in a cellulase preparation of T. viride (fraction P-6 in a previous paper [15]) could solubilize arabinoxylans in cell walls but could not macerate gramineous tissues (13,15). This nonmacerating xylanase was not a deleterious enzyme to the rice cells as shown in Table I.…”

“…The degradation products of Gramineae cell walls by the xylanase were oligoarabinoxylosides with or without phenolic acids (13). However, as shown in Table I, an autoclaved preparation of the reaction filtrate was not detrimental to the cells.…”

“…These enzymes were homogeneous on disc electrophoresis and free from other enzyme activities. Non-macerating xylanase was a partially purified preparation of fraction P-6 as described in the previous papers (13,15 …”

“…These enzymes were homogeneous on disc electrophoresis and free from other enzyme activities. Non-macerating xylanase was a partially purified preparation of fraction P-6 as described in the previous papers (13,15 (Table I). This indicated that the osmotic condition employed in this experiment to isolate rice protoplasts did not damage the cells with respect to cell division.…”

The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies

“…In this scheme, release of HG would be incidental to solubilization of hsGAX. For EXPB1 acting on grass CWs, these actions would also weaken the middle lamella, which in grasses is composed of GAX and HG (Ishii, 1984), thereby promoting pollen tube penetration between cells of the maize stigma and style (Valdivia et al, 2007(Valdivia et al, , 2009. This model may apply specifically to the clade of b-expansins that include group-1 grass pollen allergens, but additional work is needed to test its applicability to other groups of b-expansins and to other types of GAX-containing CWs (Sampedro et al, 2015).…”