Hexose efflux from the peeled grape berry

Abstract

Context and purpose of the study ‐ After the onset of grape berry ripening, phloem unloading follows an apoplasmic route into the mesocarp tissue. In the apoplast, most of the unloaded sucrose is cleaved by cell wall invertases, and imported into the cells as glucose and fructose. Alternatively, sucrose can be imported directly from the apoplast and cleaved into glucose and fructose, either in the cytoplasm or vacuoles. In low‐sucrose cultivars, such as Shiraz, glucose and fructose are the dominant sugars in vacuoles. Transport of sugars across the plasma membrane and tonoplast is a complex process, not fully understood. Some of the elements of the sugar transport mechanism may work in a reverse mode. The purpose of this study was to indirectly observe the nature of the transport mechanism by creating conditions inducing hexose efflux from a peeled berry.

Material and methods ‐ Potted plants of cv. Shirazwere grown in a glass‐house (25/16°C), from the end of anthesis onward. The experimental method was derived from the “berry‐cup” technique: a peeled berry, still attached to the plant, was immersed in a MES buffer (2‐(N‐morpholino)ethanesulfonic acid, pH 5,5)) solution that was collected every 30 minutes over a 3 hour period. The experiment was repeated weekly during the ripening phase. Additionally, during the period of intensive sugar accumulation (one to three weeks after veraison), three treatments were imposed: (i) a comparison of sugar unloading from berries detached or attached to the vine, (ii) the addition of the membrane‐ impermeant sulfhydryl‐specific cytotoxin p‐chloromercuribenzenesulfonic (PCMBS, 1mM) to the buffer solution, (iii) exposing the berry to cold (10°C), room temperature (27°C) or warm (40°C) buffer. Collected samples were analyzed for glucose and fructose concentration.

Results ‐ During five weeks of ripening, the rate of hexose (mg of glucose+fructose per g of berry fresh weight) efflux from the peeled berry into the buffer solution increased.There was no difference in efflux rate between attached or detached berries, however efllux rates were temperature dependent. The non‐penetrating enzyme inhibitor, PCMBS, depressed glucose and fructose efflux at the first sampling date, but not two weeks later. The inhibitory effect of PCMBS on fructose efflux was different from glucose, however for both hexoses the reversible nature of PCMBS was confirmed. During ripening, the glucose to fructose ratio within the collected buffer was significantly lower than in the grape juice, and had the opposite trend. These results lead us to the conclusion that the origin of the collected hexoses was vacuolar, and that the hexose efflux mechanism is differently sensitive to PCMBS at the two stages of ripening.