A DIFFERENTIAL MECHANICAL TECHNIQUE FOR TISSUE SEPARATION IN DICOTYLEDONOUS C-4 PLANTS

A differential mechanical technique for tissue separation, based on th e different physical resistance to grinding between mesophyll (M) and bundle sheath (BS) cells, was tested on dicotyledonous C-4 plants Atri plex canscens, Atriplex halimus, Gomphrena globosa, Amaranthus retrofl exus, Amaranthus caudatus and Portulaca oleracea. A metal sieve (35 me sh) was placed inside a mortar and pieces of leaves (0.5 cm(2)) were g round in an aqueous medium on the sieve to obtain a homogenate. The ho mogenate was at first collected below the sieve and was then filtered through six layers of muslin. Microscopic examination showed that the filtrate was enriched by the M cells and the residue was enriched by B S cells, few of which were broken. The BS cell fraction was then vigor ously ground and filtered; this second filtrate was named the BS cell fraction and the first filtrate was named the M cell fraction. Ribulos e 1,5-bisphosphate carboxylase (EC 4.1.1.39) (RuBPC) and phosphoenolpy ruvate carboxylase (EC 4.1.1.31) (PEPC) were assayed, and chlorophyll determinations and protein estimations were made on both fractions. As expected, PEPC showed higher activities in the M fractions; contrary to expectation RuBPC was present in M cell fractions in the six dicoty ledonous C-4 plants tested. The relative high RuBPC activities found i n the M fraction could not be explained in terms of bundle sheath cont amination.