Inhibition of Anion Transport in Corn Root Protoplasts

Abstract

MATERIALS AND METHODSThe effects of several amino-reactive disulfonic stilbene deriatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Clr, S042-, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4'-isothiocyano-2,2'-stilbenedisulfonic acid, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, 4,4'-diamino-2,2'-stilbenedisulfonic acid, and NAP-taurine inhibited Cl and S042-but not Pi and K+ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl1 or S04-2 uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl and S042-transport inhibitors.A growing interest has emerged on the nature of anion transport in several biological systems. For example, carrier proteins for phosphate, chloride, and sulfate transport have been recently identified and isolated from bacteria (1, 20), fungi (6), and human red blood cells (see reviews 10, 24). In the red blood cell studies (2-4, 8, 11, 17), several nonpermanent, amino-reactive reagents (e.g., SITS,2 DIDS, DADS, and NAP-taurine), which covalently bind to the plasmalemma, have been used to identify and characterize the band 3 membrane protein as the carrier for CF-and s042-transport.Virtually no information is available on the nature of the carrier proteins responsible for ion transport in plant cells, particularly root cells which are the major sites of entry for ions into the plants. 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. Delaware, 19801. 2Abbreviations: SITS, 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid; DIDS: 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid; DADS, 4,4'-diamino-2,2'-stilbenedisulfonic acid; NAP-taurine, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate; FCCP, (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone; DIFP, diisopropylfluorophosphate.A modification of our previously reported method (13) was used to isolate protoplasts from young corn roots. As outlined in Figure 1, 0.1% pectolyase Y-23 (16) substituted for pectinase and hemicellulase in the digestion enzyme mixture. 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...

Nitrite Uptake into Intact Pea Chloroplasts

“…The reported preferential alkaline pH for nitrite uptake into chloroplasts (3) also confficted with the characteristic acidic pH optima for anion transport (4,16,22). This anomaly of anion uptake at alkaline pH values has been recognized by other workers (4, 14,27,28) and was clarified by the proposed existence of relatively specific, pH sensitive, anion channels in biological membranes (4,14,27,28).…”

“…It could be assumed that mersalic acid, a water-soluble mercurial compound (16), did not rapidly penetrate to the stroma, since the reported inhibition of nitrite reductase activity ( 11) was not apparent in the present study. An alternative phosphate transport inhibitor, DABS (9), showed a very slight inhibition of nitrite uptake (Table IV).…”

“…When the recognized phosphate transport inhibitors, mersalic acid (15,16) and competitive pyridoxal-5'-P (4, 9) were included in the chloroplast incubation medium, no significant effect on net nitrite uptake into intact chloroplasts was observed (Table IV). It could be assumed that mersalic acid, a water-soluble mercurial compound (16), did not rapidly penetrate to the stroma, since the reported inhibition of nitrite reductase activity ( 11) was not apparent in the present study.…”

“…The Cl-/SO42-channel inhibitors, DIDS (4,16), and APMB (4) did not influence nitrite uptake into intact chloroplasts (Table IV). Both of these reagents are slow or nonmembrane penetrating (4).…”

Specific Labeling of the Phosphate Translocator in C3 and C4 Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2′ -Disulfo-4,4′ -Diisothiocyano] Diphenylethane)

“…Following the successful use of DIDS to study anion transport in mammalian tissue, Lin (25) (Fig. lb) of the adjacent three lanes of the gel in Figure la …”