Fructan Synthesis in Excised Barley Leaves (Identification of Two Sucrose-Sucrose Fructosyltransferases Induced by Light and Their Separation from Constitutive Invertases).

Excised leaves of barley (Hordeum vulgare L.) exposed to continuous light accumulate large amounts of soluble carbohydrates. Carbohydrates were analyzed in deionized extracts by high-pressure liquid chromatography on an anion exchange column coupled with pulsed amperometric detection. During the first few hours of illumination, the main sugar to accumulate was sucrose. The levels of glucose and fructans (oligofructosylsucroses) increased later. The trisaccharide 1-kestose (1-kestotriose) predominated initially among the fructans. Later, 6-kestose (6-kestotriose) and tetra- and pentasaccharides accumulated also. Total extracts from barley leaves were chromatographed on a MonoQ column, and each fraction was assayed for enzymes of interest by incubation with 200 mM sucrose for 3 h, followed by carbohydrate analysis. Freshly excised leaves yielded two peaks of invertase, characterized by formation of fructose and glucose, but had almost no trisaccharide-forming activities. In leaves exposed to continuous light, two new enzyme activities appeared that generated fructan-related trisaccharides and glucose from sucrose. One of them was a sucrose-sucrose fructosyl-1-transferase (1-SST), producing 1-kestose exclusively: the peak fractions of this activity contained almost no invertase. The other was a sucrose-sucrose fructosyl-6-transferase (6-SST), producing 6-kestose. It comigrated with one of the constitutive invertases on MonoQ but was separated from it by subsequent chromatography on alkyl Superose. Nevertheless, the preparation retained invertase activity, suggesting that this enzyme may act both as fructosidase and fructosyltransferase. When incubated with 1-kestose in addition to sucrose, this enzyme formed less 6-kestose but instead produced large amounts of the tetrasaccharide bifurcose (1&6-kestotetraose), the main fructan tetrasaccharide accumulating in vivo. These results suggest that two inducible enzymes, 1-SST and 6-SST, act in concert to initiate fructan accumulation in barley leaves.

High-temperature forced-air treatment alters the quantity of flavor-related, volatile constituents present in navel and Valencia oranges.

A number of volatile compounds that contribute to orange flavor were quantified following high-temperature forced-air (HTFA) treatment of the fruit to determine if a relationship exists between the flavor loss that is observed following HTFA treatment and the volatile composition of the juice. Following different durations of HTFA treatment, fruit were stored for a period of 4 weeks and juiced and the juice subjected to headspace analysis using either a Tenax/Carbotrap column or a solid-phase microextraction device for trapping of the volatiles. alpha-Pinene, beta-myrcene, and limonene were reduced in amount by 60%, 58%, and 34%, respectively, over the course of the 5-h HTFA treatment. The influence of heat on the amount of decanal was less clear, although in one of the two fruit lots there was little change. The amount of ethanol was reduced by 70% after the initial hour of HTFA treatment and then steadily increased to exceed the initial amount during the remaining 4 h of the treatment. Taste evaluations of the fruit showed a reduction of flavor quality following 4 h or more of treatment. Percent acidity and soluble solids, two other very important determinants of flavor, were nearly unchanged by treatment. Alterations in the volatile constituents of oranges by HTFA treatment may be an important reason behind the negative impact of this treatment on flavor quality.

The influence of altered gravity on carbohydrate metabolism in excised wheat leaves.

We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment.

Dose-morality and large-scale studies for controlling codling moth (Lepidoptera: Tortricidae) eggs on 'd'Agen' plums by using methyl bromide.

Codling moth, Cydia pomonella (L.), eggs on 'd'Agen' cultivar of plums, Prunus salicina Lindl., were treated with methyl bromide to determine if a quarantine treatment could be developed so that the plums could be exported to Japan from the United States. Small-scale tests consisted of treating codling moth eggs on plums with various doses of methyl bromide at 20 degrees C for 2 h. Small-scale tests showed that 0- to 24-h-old eggs of codling moth on the plums were controlled by doses > 22.5 g/m3. Because testing showed that 48 g/m3 had no adverse effect on the quality of plums, this dose was chosen for large-scale testing to establish the quarantine dose. Large-scale tests consisted of treating plums at 18.5 degrees C for 2 h using methyl bromide at a dose of 48 mg/liter. Large-scale tests showed that the dose of 48 g/m3 killed all 0- to 24-h-old codling moth eggs exposed on plums in packing cartons without affecting the quality of the plums.

Regulation of sucrose-sucrose-fructosyltransferase in barley leaves.

The activity of sucrose-sucrose-fructosyltransferase (SST), a vacuolar enzyme strongly induced by light in excised leaves of barley (Hordeum vulgare L.), rapidly declined even in continuous light upon feeding of cycloheximide (CHI). The rate of decline was similar to that observed in light-treated leaves that were placed into darkness, in the presence or absence of CHI. The protease inhibitor leupeptin totally stopped the decline in SST activity in the dark and caused a substantial increase in the rate of induction of SST activity by light. Feeding of sucrose prevented or even reversed the SST activity decay induced by darkness in the absence of CHI but did not stabilize SST activity in the presence of CHI. The results suggest that SST is continuously subjected to rapid, constant proteolytic degradation in the vacuole, and that the enhancement of SST activity in the light or upon feeding sucrose in the dark is due exclusively to de novo protein synthesis.

Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.

Occurrence of alternative respiratory capacity in soybean and pea.

Capacity for the alternative respiratory pathway was assessed in leaf and root tissue of male-sterile and fertile soybean (Glycine max [L.] Merr.) plants and in leaf, embryonic axis, and epicotyl tissue as well as isolated mitochondria of pea (Pisum sativum L.) by measurement of oxygen uptake in the presence and absence of KCN and salicylhydroxamic acid. Male-sterile and fertile soybean tissues showed similar responses to the inhibitors, and both possessed a capacity for alternative respiration. We also found that tissue and isolated mitochondria from ;Progress No. 9' pea possessed alternative respiratory capacity similar to that of ;Alaska' pea.