Salt Tolerance in Suspension Cultures of Sugar Beet

Abstract

ABSTRACICell suspension cultures of sugar beet were grown at various salinities (0-200 millimolar NaCI). Their tolerance to Na' was comparable to that of the intact plant. Tonoplast vesicles were prepared by sucrose density gradient centrifugation of microsomal membranes and shown to be highly purified. The vesicles were subjected to a pH jump in the presence of acridine orange and the rate of recovery of fluorescence after addition of Na' was used as a measure of Na'-dependent H' efflux. In the presence of K+ and valinomycin, the Naf/H' antiport showed saturation kinetics.Increasing Na' in the growth medium did not change the apparent K.for Nat, but increased V,,, to about twice the control value, suggesting a specific induction of antiport synthesis by salt.In a previous study (3) we characterized a Na+/H+ antiport in tonoplast vesicles isolated from storage tissue of red beet and sugar beet. Since the vacuolar accumulation of sodium is characteristic of salt-tolerant species (7,8) this tonoplast Na+/H+ antiport may be one of the principal physiological factors conferring salt tolerance on the plant. Further work on the expression and regulation of this transport system is therefore needed. A recent report ( 14) shows that the marked increase in capacity for Na+ accumulation which develops during washing of sliced storage tissue of red beet (11) is accompanied by a progressive decrease in apparent Km of the tonoplast Na+/H+ antiport.In the present work, we use sugar beet cell suspensions to investigate the regulation of antiport activity in response to extracellular Na+, and present evidence for a specific induction of antiport activity by salt. Bartlesville, OK). The homogenization medium consisted of 5% PVP, 0.5% BSA, 1 mm PMSF,3 30 mM Tris, 5 mM DDT, 5 mM EGTA, 5 mM MgSO4, 0.5 mm butylated hydroxytoluene, 0.5 mM dibucaine, and 0.25 M mannitol, adjusted to pH 8.0 with H2SO4. The isolation was performed at 4°C throughout. The homogenate was filtered through four layers of cheesecloth and centrifuged for 20 min at 8,000g to remove debris and mitochondria. Pellets were discarded and the supernatants were centrifuged for 35 min at 80,000g in a Beckman type 35 rbtor. The supernatant was aspirated and the microsomal pellet was resuspended with a Teflon pestle homogenizer in a medium containing 1.1 M glycerol, 1 mM Tris-EDTA, 0.5 mM dibucaine, 0.5 mM butylated hydroxytoluene, 3 mM DDT, 10 mM Tris-Mes (pH 7.5), and 0.15 M KI. The membranes were again sedimented at 80,000g for 35 min, resuspended in suspension medium without KI, and layered on sucrose gradients. For linear sucrose gradients, the KI-treated membranes were resuspended in 4 ml suspension medium and layered on a 33 ml linear gradient of 10 to 45% (w/w) sucrose in suspension medium. After centrifugation at 80,000g for 2 h in a Beckman SW 27.1 rotor, the gradient was divided into 22 to 24 fractions for subsequent assay. MATERIALS AND METHODSFor routine preparation of tonoplast vesicles, the KI-treated microsomal pellet was resuspended in 20 ml suspension ...

Ion channels in vacuoles from halophytes and glycophytes

“…Vacuoles were isolated by osmotic shock of protoplasts obtained from cell suspension cultures of sugar beet (Beta vulgaris) and tomato (Lycopersicum esculatum) [6]. Cells were incubated at 30°C for 1.5-2 h in a medium containing 2°70 cellulase Y-C, 0.1% pectolyase Y-23 (both enzymes from Seishin Pharmaceutical Co., Ltd, Tokyo, Japan), 0.5% BSA, 0,5 mM CaC12, 0.4 M sorbitol, and 25 mM Mes-KOH buffer, pH 5.5.…”

“…One such secondary active transport, the Na+/H + antiport in beet, has been fairly well characterized [5,6]. But there is as yet no definite information about a possible K+/H + antiport system and the clear necessity to postulate porters for the inward fluxes of anions such as Cland NO~ is still just that: a postulate.…”

Modulation of Na+/H+ Antiporter Activity by Extreme pH and Salt in the Halotolerant Alga Dunaliella salina

“…However, a vacuolar Na+/H+ antiporter was found and characterized in several plant species, and it was reported that an increase in salt concentration can induce an increased antiporter activity (1,4,7 It is demonstrated that the activity of the plasma membrane Na+/H+ antiporter is overproduced in cells adapted to ammonia or to high NaCl and depressed by adaptation to LiCl.…”

“…However, a vacuolar Na+/H+ antiporter was found and characterized in several plant species, and it was reported that an increase in salt concentration can induce an increased antiporter activity (1,4,7).…”

Beneficial Effects of Potassium on Growth, Water Relations, Mineral Accumulation and Oxidative Damage of Beta vulgaris in Sodic-alkaline Condition

“…Furthermore, K + content in living cells were maintained at a higher level, and Na + toxic effect in the cytoplasm was somewhat avoided. In terms of Na + influx and K + efflux from vacuoles, some studies reported that over-expression of tonoplast sodium/proton exchangers (NHXs) improved salt tolerance in Beta vulgaris (Blumwald and Poole, 1987;Xia et al, 2002;Adler et al, 2010), and tonoplast-located K + channels or transporters facilitating K + release from vacuoles into the cytoplasm (Wang and Wu, 2010;Chérel et al, 2014). The ability of Swiss chard to control the cytoplasmic K + concentration in Alkalinity+2 mM K and Alkalinity+0 mM K conditions could possibly have been regulated by the over-expression of NHXs or tonoplast-located K + channels and transporters, however, these phenomena need to be further investigated.…”