Accumulation of Maltose during Photosynthesis in Protoplasts Isolated from Spinach Leaves Treated with Mannose

Abstract

When mannose was included in the enzyme incubation medium during the preparation of protoplasts from leaves of spinach, maltose was an early product of protoplast photosynthesis and, after 12 minutes, accounted for up to 15% of the 'C incorporated from 'CO2. Maltose was not detected in protoplasts prepared in the normal enzyme medium. Rapid separation of cytoplasm and chloroplasts following exposure to "CO2 showed that maltose was present in both fractions. Direct measurements of [I4Clmaltose uptake indicated transport across the chloroplast envelope at rates similar to the transport of glucose. The source of maltose and site of its initial formation are discussed.Maltose occurs in only small amounts in leaves (18) but has been detected as a "C-labeled product of "4CO2 assimilation in several species (9,19,20,22,23,30 Photosynthetic Carbon Assimilation. Protoplasts were added to a medium containing 500 mm sorbitol, 1 mm CaC12, 25 mm Tricine (pH 7.6), and 5 mm NaH14CO3 (20 Ci/mol) to give 50 ,ug Chl/ml.The mixture was illuminated in an O2 electrode (5) at 20 C and photosynthesis was measured by 02 evolution and by incorporation of 14C (below). The reaction was terminated by the addition of boiling absolute ethanol to give a final concentration of 801% (v/v). This total extract was centrifuged, and the pellet reextracted twice in 80%1o ethanol and twice in warm H20 to give an ethanol/ H20-soluble fraction (13). The remaining pellet was incubated with amyloglucosidase solution (3), and the starch content was estimated by release of ['4C]glucose. Samples of the total extract, the ethanol/H20-soluble fraction, and the supernatant following amyloglucosidase digestion ofthe pellet were counted on planchets using a Nuclear Chicago gas flow counter, together with appropriate standards. Free sugars and a base line component (which includes charged compounds and oligosaccharides) in the ethanol/ H20-soluble extract, and glucose in the amyloglucosidase extract were separated by paper chromatography in ethyl acetate-pyridine-H20 (8:2:1, v/v) and radioactivity was determined with a Berthold LB 280 scanner. In addition to the earlier methods used for maltose identification (13), extracts were heated with borohydride which reduces maltose to maltitol (18), followed by maltase treatment. Maltitol, sorbitol, and glucose released from it by maltase were identified by chromatography against "4C-labeled standards and by GLC. The identity of sucrose was confirmed as before (26).Protoplast Fractionation. Protoplasts were incubated in a medium containing 400 mm sorbitol, 1 mm CaCl2, 25 mm Tricine (pH 7.6), and 5 mm NaH'4C03 (60 Ci/mol). Protoplasts were illuminated and photosynthesis was measured by 02 evolution and 14C incorporation (27). After 12 min illumination, or at 2-min intervals for time-course experiments, 1OO-AtL protoplast samples of extract (5 jig Chl) were taken and the chloroplastic and cytoplasmic fractions were rapidly separated by centrifugation filtration through nylon mesh and silicone oil 19:1), using the ...

Respiration of Sugars in Spinach (Spinacia oleracea), Maize (Zea mays), and Chlamydomonas reinhardtii F-60 Chloroplasts with Emphasis on the Hexose Kinases

“…Both the uptake of maltose (Herold et al, 1981) and its formation from hydrolytic starch breakdown Peavey et al, 1977) have been demonstrated in isolated spinach chloroplasts. Levi and Preiss (1978) and Okita et al (1979) found no evidence that maltase is present in significant amounts in chloroplasts.…”

“…Inasmuch as maltose is not found in the chloroplast except in trace amounts during starch degradation, we suggest that its metabolism is catalyzed by maltose phosphorylase and perhaps a low leve1 of maltase. Maltose could be exported from the chloroplast by a translocator (Herold et al, 1981). In contrast, an appreciable activity of maltose metabolism is associated with the Chlamydomonas chloroplast ( Table I).…”

Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

“…Herold et al (1981), Beck (1985), and Rost et al (1996) reported that the chloroplast envelope is permeable for maltose but not for maltodextrins. Rost et al (1996) demonstrated that the uptake of D -Glc does not compete with maltose transport.…”

Export of Carbon from Chloroplasts at Night

“…However, known hexose-phosphate transporters in plastids have low activity (Batz et al, 1992) and are generally considered important primarily in starch accumulation Overlach et al, 1993;Schott et al, 1995;Häusler et al, 1998). The Glc/maltose transporter described by Schäfer et al (1977) and Herold et al (1981) could explain the nighttime export.…”

“…Amylolytic starch breakdown leads to maltose and Glc, both of which can be transported, presumably on the Glc transporter (Schäfer et al, 1977;Herold et al, 1981). A number of studies have concluded that the phosphorolytic pathway is the primary pathway for starch breakdown Levi and Preiss, 1978;Stitt and Heldt, 1981a).…”