Isolation of leaf bundle sheath protoplasts from C4 dicot species and intracellular localization of selected enzymes

Abstract

A new source of cell wall digestive enzymes, Onozuka Cellulase RS (from Trichoderma viride), was evaluated for the effectiveness of leaf digestion and the release of bundle sheath protoplasts (BSP) in a variety of C₄ monocots and dicots. Several dicot species were identified from which BSP, as well as mesophyll protoplasts (MP), can readily be isolated by using the RS cellulase. Assays of marker enzymes, plus chlorophyll measurements, permitted analysis of protoplast yield and purity. BSP and MP have been purified (90–100%) from Atriplex spongiosa (NAD-malic enzyme (ME) subgroup) and from Falveria trinervia (NADP-ME subgroup), with final yields ranging from 6–24%.

Enzyme localization studies indicate that a substantial amount of NADP-malate dehydrogenase (DH) in F. trinervia occurs not only in the mesophyll chloroplasts, but also in the bundle sheath chloroplasts. Similar activities of an apparent NADP-utilizing malate dehydrogenase (but not activated by dithioerythritol, DTE) occurs in the mitochondria of bundle sheath cells of A. spongiosa. Aspects of the potential role of this enzyme in photosynthetic metabolism are further discussed.