Preparation and composition of mesophyll, epidermis and fibre cell walls from leaves of perennial ryegrass (lolium perenne) and italian ryegrass (lolium multiflorum)

Abstract

AbstractCells of mesophyll, epidermis and residual fibrous material were obtained from leaves of Italian and perennial ryegrass harvested at different stages of maturity by mechanical disruption of leaf tissue. Mesophyll cells were selectively removed by filtration through 0.045 mm nylon mesh and remaining non‐mesophyll cells centrifuged in metrizamide solutions (56–58% wt to vol.) of known density (1.308–1.329 g cm3 at 5°C) to obtain a pure epidermis cell fraction and a residual fibre fraction. Whole mesophyll cells contributed 63–72%, epidermis 12–15% and the fibre fraction 15–24% to the total leaf dry matter. Fibre values were higher in late‐cut samples. Cell walls were prepared from mesophyll and epidermis cells by disruption and washing to remove cell contents. Fibre cells were judged free of cell contents and received no further treatment. Examination of cell wall preparations by light and electron microscopy showed that both mesophyll and epidermis preparations were essentially free from contaminating material. Mesophyll cell walls were uniformly thin (200 nm) while those of epidermis ranged from 2000–3000 nm at the outer face, thinning to 300 nm or less at the inner surface. An electron‐light layer (cuticle) of approximately 200 nm thickness was present covering the outer face of the epidermis. The fibre fraction largely consisted of sclerenchyma, but contained, in addition, other vascular cells, detached annular rings and heavily silicified leaf hairs. Analysis of cell walls accounted for 85–90% of dry matter. Cellulose was the major component of all cell walls examined (approximately 40% of dry matter) with xylose residues accounting for a further 11% of mesophyll, 13.5–17.5% of epidermis and 21–25% of fibre cell walls. Arabinose was low in fibre cells but was present in much higher proportions in mesophyll and epidermis walls. The ratio of arabinose to xylose was approximately 1:1.5 for mesophyll, 1:2.5 for epidermis and 1:7.0 for the fibre fraction. The molar ratio acetyl to xylose remained fairly constant at 1:4 regardless of the grass, cell type or maturity of the sample. The uronic acid content of epidermis was higher than that in other cell types and showed an increase with increasing maturity of the grass, reaching over 9% in late‐cut samples. Total phenolic material represented 2–3% of mesophyll and epidermis cell walls and 6% of fibre walls. Ferulic acid alone was released from the primary cell walls by saponification and p‐coumaric and ferulic acids from the secondary‐thickened fibre walls. Crude protein values (NX6.25) were high in mesophyll cell wall preparations and low in epidermis and fibre cell walls. Amino acid patterns were similar for both grasses and cell types but hydroxyproline was found in greater amounts in fibre cell walls than in either epidermis or mesophyll.