Corn Root Protoplasts

Abstract

In recent years, plant protoplasts have become increasingly important in plant biology research (see reviews 5,23,25). For example, protoplasts have been used in the studies of intracellular localization of metabolic processes (2,3,18), cell wall biosynthesis (21), organelle isolation and characterization (2,3,13,18,19), and protoplast fusion in genetic research (4). Because of the lack of methodology in the large scale isolation of viable root protoplasts, no studies have yet been conducted on ion uptake in isolated protoplasts from root cells. Recent reports by Mettler and Leonard (15,16) Delaware, 19898. 2Prehminay results had been reported at the "Plant Membrane Transport Workshop," Toronto, Canada, July, 1979. showed that this treatment inhibited cell wall digestion by cellulase during the isolation procedure. As shown diagrammatically in Figure 1, the segments were then incubated in a cell wail digestion enzyme solution (3 g tissue/20 ml enzyme solution) consisting of 2% Cellulysin (Calbiochem), 1% hemicellulase, and 0.5% pectinase in a solution of 0.2 mM CaC12 and 0.6 M mannitol at pH 5.5 for 3.5 to 4.5 h with constant shaking (50 cycles/min) at 30 C. After the cuticle and some undigested materials were removed with a pair of forceps or a spatula, the remaining crude protoplast mixture was layered onto 20 ml of a solution containing 5% Ficoll (type 400), 25 mm Mes (pH 5.5), 0.2 mm CaCl2, and 0.5 mm DTT3 in 0.7 M mannitol. The mixture was then centrifuged at 400g for 10 min to pellet the starch grains and other large debris. The isolation procedure was continued by transferring the upper layer, which still contained most of the intact protoplasts, to another centrifuge tube where they were diluted with 15 to 20 ml of 0.7 M mannitol containing 25 mm Mes (pH 6.0), 0.2 mm CaCl2, and 0.5 mm DTT (protoplast suspension solution). Protoplasts were immediately spun down at 2,000g for 10 min and then washed once with an additional 20 ml of suspension solution.Final protoplast pellets were resuspended in 7 ml of the protoplast suspension solution to which 10%o Ficoll had been added, and 7 ml each of 8, 5, and 0%o Ficoll-containing protoplast suspension solutions were then layered on top in sequence. TheFicoll gradients were then centrifuged at 300g for 20 min. Based on light microscopic analysis, intact viable protoplasts were found at the interface of the 0 and 5% Ficoll gradients. These were removed with a pipet, washed once with 20 ml protoplast suspension solution, followed by a 10-min, 2,000g-pelleting centrifugation. Morphologically, intact protoplasts could also be found in other interfaces ofthe Ficoll gradient. However, vital staining tests showed that most of these protoplasts were not viable. With the exception of incubation, all procedures were performed at room temperature. All solutions used were sterilized by passing them through a 0.

Inhibition of Anion Transport in Corn Root Protoplasts

“…Also, the tissue incubation time in the digestion mixture was shortened to 1.5 to 2 h and the precentrifugation steps were omitted. The yield of 106 protoplasts/g root tissue and viability of the protoplasts were similar to those previously described (13). Transport rates for Pi and K+ were 2-fold higher and linear for longer periods of time (10-20 min longer) than in the previously described technique (Ref.…”

“…Transport rates for Pi and K+ were 2-fold higher and linear for longer periods of time (10-20 min longer) than in the previously described technique (Ref. 13; Table I and Fig. 2).…”

“…At present, the reason for the strong stimulatory effect of DADS on Pi uptake in the protoplast suspension is unknown. Previous studies with corn root segments (12) and protoplasts (13) suggested that Pi was taken up through a Pi/OH-antiporter. It was shown that Pi uptake decreased with increasing external pH.…”

“…The lack of such information is due, in part, to the lack of suitable technique for isolating functional protoplasts from roots which are, by their nature, more amenable to techniques that have been used to characterize membrane transport. Recently, we developed techniques for the large scale and rapid isolation of protoplasts from corn roots (13) which are capable of transporting several ions (K+, H2PO4 , and H+). In this study, several chemical modifiers which have been used to study anion transport into red blood cells were employed to study anion transport into corn root protoplasts with the intent of providing further insight into anion transport in plant cells.…”

Extensor and Flexor Protoplasts from Samanea Pulvini

“…Although protoplasts undergo severe stresses during isolation, in many systems they retain physiological responses observed in intact cells (9,12,15,19). Protoplasts from stomatal guard cells, for example, swell in response to light (24) and shrink in response to ABA (23) Solutions.…”