Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies,Bemisia argentifolii,and Erwinia rhapontici

Isomaltulose [alpha-D-glucopyranosyl-(1,6)-D-fructofuranose] and trehalulose [alpha-D-glucopyranosyl-(1,1)-D-fructofuranose] are commercially valuable sucrose-substitutes that are produced in several microorganisms by the palI gene product, a sucrose isomerase. Trehalulose also occurs in the silverleaf whitefly, Bemisia argentifoli, as the major carbohydrate in the insect's honeydew. To determine if the enzyme that synthesizes trehalulose in whiteflies was similar to the well-characterized sucrose isomerase from microbial sources, the properties of the enzymes from whiteflies and the bacterium, Erwinia rhapontici, were compared. Partial purification of both enzymes showed that the enzyme from whiteflies was a 116 kD membrane-associated polypeptide, in contrast to the enzyme from E. rhapontici, which was soluble and 66 kD. The enzyme from E. rhapontici converted sucrose to isomaltulose and trehalulose in a 5:1 ratio, whereas the enzyme from whiteflies produced only trehalulose. Unlike the E. rhapontici enzyme, the whitefly enzyme did not convert isomaltulose to trehalulose, but both enzymes catalyzed the transfer of fructose to trehalulose using sucrose as the glucosyl donor. The results indicate that trehalulose synthase from whiteflies is structurally and functionally distinct from the sucrose isomerases described in bacteria. The whitefly enzyme is the first reported case of an enzyme that converts sucrose to exclusively trehalulose.     

Effect of high temperature on the metabolic processes affecting sorbitol synthesis in the silverleaf whitefly,Bemisia argentifolii

Whiteflies accumulate the polyhydric alcohol, sorbitol, when exposed to temperatures greater than about 30 degrees C. Feeding experiments using artificial diets containing labeled sucrose showed that more of the label was incorporated into whitefly bodies and less was excreted in the honeydew when feeding was conducted at 41 compared with 25 degrees C. Analysis of the components of the honeydew showed that more of the excreted label was in glucose and fructose and less in trehalulose at 41 degrees C than at 25 degrees C. A similar effect of temperature on honeydew composition occurred for whiteflies feeding on cotton leaves. Measurement of the activities of glycolytic, pentose-phosphate and polyol pathway enzymes at 30 and 42 degrees C showed that NADPH-dependent ketose reductase/sorbitol dehydrogenase (NADPH-KR/SDH), sucrase, glucokinase and glucose-6-phosphate dehydrogenase activities were stimulated to a greater extent at 42 degrees C than trehalulose synthase and fructokinase. NAD(+)-sorbitol dehydrogenase (NAD(+)-SDH) activity was inhibited at 42 degrees C. We propose that high temperature alters metabolic activity in a way that increases the availability of fructose and stimulates pentose-phosphate pathway activity, providing both the substrate and coenzyme for sorbitol synthesis. High temperature also increases the activity of NADPH-KR/SDH, the enzyme in whiteflies that synthesizes sorbitol, but inhibits the activity of NAD(+)-SDH, the enzyme that degrades sorbitol.     

Presence of trehalulose and other oligosaccharides in hemipteran honeydew, particularly Aleyrodidae

Abstract. Production by a bacterium of the disaccharide trehalulose was reported 30 years ago. The association between the fabrication of trehalulose and Insecta was found more recently. It was initially discovered in the honeydew, excreta, of the whitefly Bemisia tabaci feeding on Euphorbia pulcherrima where it was the predominant sugar. In the present study, B. tabaci -produced trehalulose was again found in honeydew at significant levels, this time when the whitefly fed on 10 different plants. In seven of 10 Bemisia /host combinations, trehalulose accounted for more than 30% of the total carbohydrates found in their honeydew and was the principal oligosaccharide. Trehalulose constituents, glucose and fructose, were also present at lower levels, as were other oligosaccharides such as melezitose. Feeding by B. tabaci on the three other plant hosts also resulted in the production of trehalulose at relatively high levels (6.1–16.5%). Other whitefly species examined had little (e.g. B. afer and Aleurothrixus spp.), or no trehalulose (e.g. Trialeurodes spp. and Siphoninus phillyreae ), in their honeydew. Trehalulose was also found in the honeydew of two aphid and one scale insect species. In insects whose honeydew had low levels or no trehalulose, sucrose, its constituents, or larger sugars predominated. The trisaccharide bemisiose was also found in the honeydew of half the whitefly species examined. Bemisiose was discovered here for the first time in the honeydew of three aphid species and three species of scale insects. Reasons for the production of trehalulose are often linked to high levels of dietary sucrose in whiteflies. This is probably true in our case because cotton and cucurbits, at least, are known to contain a great deal of sucrose. Although other functional possibilities were explored, the data suggest that trehalulose, being less susceptible to hydrolysis than sucrose, is involved in osmoregulation.     

The longevity effects of trehalulose feeding by parasitoids

Abstract.  Insects, particularly phloem-feeding Sternorrhyncha, are known to produce sugars in their honeydew (excreta) that are not found in their host plants. Of these, Bemisia tabaci , the sweet potato whitefly, is the only insect known to produce trehalulose [α- d -glucose (1,1) d -fructose] as a major component of its honeydew. The present study aims to determine whether trehalulose is comparable to sucrose as a nutrient source for three whitefly parasitoids ( Encarsia formosa , Encarsia pergandiella and Eretmocerus eremicus ). In addition, the study also examines trehalulose feeding effects on longevity for a parasitoid of muscoid Diptera, Nasonia vitripennis . Parasitoids are provided diets of either sucrose or trehalulose in varying concentrations (from 0.1% to 70%) or a water control. Sucrose and trehalulose are not significantly different in affecting survival when compared at the same concentration. This was true for all Bemisia parasitoids and N. vitripennis. Certain specific diets are significantly different in pairwise combination tests. There is a significant effect of species, diet type and the interaction of these two factors on the longevity of the three different Bemisia parasitoid species; however, within species, there is no significant increase in longevity observed for either carbohydrate diet. This result contrasts with expectations for the effects of host-modified carbohydrates on longevity. The implications are that, although carbohydrate feeding is essential for these parasitoids, these host-provided sources of carbohydrates are equally capable of extending longevity.     

Plant nitrogen status rapidly alters amino acid metabolism and excretion in Bemisia tabaci

The effect of plant nitrogen (N) status on the content and distribution of free amino acids in the bodies and honeydew of silverleaf whiteflies Bemisia tabaci (Gennadius) Biotype B (= B. argentifolii Bellows and Perring) was determined. Whiteflies fed for 4 days on cotton leaves that received high or low N fertility. For low-N plants, photosynthesis and leaf total N levels were decreased, and a much-reduced amount of free amino acids was recovered in phloem sap. Low N fertility did not affect whitefly total N content, but did markedly decrease the free amino acid content. Glutamine, alanine and proline accounted for over half of the insect free amino acid pool for both N treatments. On a relative basis, adjustments in glutamine levels in response to plant N status were much larger compared to the other amino acids. Large amounts of amino N, especially asparagine, were excreted from whiteflies fed on high-N plants whereas amino N excretion essentially ceased for whiteflies fed on low N plants. The distribution of amino acids in the insects and honeydew was not closely related to the phloem sap amino acids. However, total amino acid excretion was quite indicative of the plant N status and the quality of the insect diet. The results indicated that whitefly free amino acid pools and excretion of amino N were rapidly altered by plant N status.     

High Performance Liquid Chromatography Analysis of Carbohydrates of Cotton-Phloem Sap and of Honeydew Produced by Bemisia tabaci Feeding on Cotton

Phloem sap from cotton (Gossypium hirsutum L.) was collected from young and mature leaves by the aphid-stylet technique. Exudate was analyzed for carbohydrates by HPLC using sensitive pulsed amperometric detection. The predominant carbohydrate present (>90%) was identified as sucrose. A second, unidentified compound that was not one of the more commonly translocated sugars was detected in mature leaves. Carbohydrates in honeydew produced by the sweet-potato whitefly (Bemisia tabaci [Genn.]) feeding on cotton were sucrose, glucose, fructose, trehalulose, and a series of oligosaccharides.     

A thermoprotective role for sorbitol in the silverleaf whitefly,Bemisia argentifolii

Accumulation of polyols in insects is well known as a cold-hardening response related to overwintering or to protection against cold shock. The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) is a major insect pest in tropical and subtropical regions where heat stress and desiccation pose formidable threats to survival. We found that sorbitol levels increased ten-fold when whiteflies were exposed to elevated temperatures. Sorbitol levels rose from 0.16nmolwhitefly(-1) at 25 degrees C to 1.59nmolwhitefly(-1) at 42 degrees C. Sorbitol levels fluctuated diurnally under glasshouse and field conditions increasing ten-fold from morning to early afternoon. Feeding experiments on artificial diets showed that both temperature and dietary sucrose concentration were key factors influencing sorbitol accumulation. Cell free extracts prepared from adult whiteflies catalyzed NADPH-dependent fructose reduction, but were unable to reduce glucose with either NADPH or NADH. Radiotracer experiments with labeled glucose and fructose showed that fructose was the immediate precursor of sorbitol. Thus, sorbitol synthesis in the whitefly is apparently unconventional, involving conversion of fructose by a novel NADPH-dependent ketose reductase. We propose that sorbitol accumulation is a mechanism for thermoprotection and osmoregulation in the silverleaf whitefly, allowing the insect to thrive in environments conducive to thermal and osmotic stress.     

甘蓝与黄瓜寄主上B型烟粉虱和温室白粉虱蜜露糖分、氨基酸和挥发物组分的比较分析

粉虱蜜露是粉虱寄生性天敌搜索寄主的主要利它素源。应用离子色谱分别对甘蓝与黄瓜上B型烟粉虱(Bemisia tabaci B-biotype)蜜露以及黄瓜上温室白粉虱Trialeurodes vaporariorum蜜露的接触性利它素糖和氨基酸组分和含量进行了比较研究。结果表明：2种粉虱在不同寄主植物上的蜜露均富含糖和氨基酸,其中糖含量占绝对优势,甘蓝上B型烟粉虱蜜露、黄瓜上B型烟粉虱蜜露和黄瓜上温室白粉虱蜜露中的糖含量分别是相应氨基酸含量的42.5、2.6和5.4倍,其中糖类物质中又以寡糖含量占绝对优势,分别占89.3%、81.7%和88.2%。不同寄主植物和粉虱种类显著影响蜜露中糖和氨基酸的组成和含量。其中,甘蓝上B型烟粉虱蜜露中的寡糖以二糖占优势,占97.3%;二糖中又以蔗糖异构糖和松二糖占优势,分别占52.7%和35.4%。黄瓜上B型烟粉虱蜜露和温室白粉虱蜜露寡糖中以三糖和四糖占优势,分别占73.1%和85.4%;优势糖水苏（四）糖和松三糖分别占40.3%和 26.2%及49.9%和27.0%。甘蓝上B型烟粉虱蜜露中氨基酸以丙氨酸占优势,含量为66.5%;而黄瓜上B型烟粉虱及温室白粉虱蜜露中氨基酸以甘氨酸含量最高,分别占氨基酸总量的38.2%和51.7%。应用GC-MS对甘蓝上B型烟粉虱蜜露和黄瓜上温室白粉虱蜜露挥发物组分的鉴定结果显示,两种粉虱蜜露中共同含有的主要挥发物为邻苯二甲酸二（2-乙基）己酯。     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field‐grown sugarcane

Sugarcane plants were developed that produce the sucrose isomers trehalulose and isomaltulose through expression of a vacuole‐targeted trehalulose synthase modified from the gene in ‘Pseudomonas mesoacidophila MX‐45’ and controlled by the maize ubiquitin (Ubi‐1) promoter. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mm , with near‐complete conversion of sucrose in the most mature internodes. Plants remained vigorous, and trehalulose production in selected lines was retained over multiple vegetative generations under glasshouse and field conditions.     

Microorganisms influence the composition of honeydew produced by the silverleaf whitefly, Bemisia argentifolii

Sugars in the honeydew produced by the silverleaf whitefly, Bemisia argentifolii, and in fermentation of sucrose using homogenates of these insects were analyzed by high performance liquid chromatography. Results suggest that the unusual disaccharide, trehalulose, found in large quantity in honeydew of B. argentifolii, is produced by obligate intracellular microorganisms residing in this insect's mycetomes. Some larger oligosaccharides in this honeydew may be produced by certain Bacillus spp. residing in or on the insects but these bacteria are not involved in an obligate relationship with the whitefly.     

Isobemisiose: an unusual trisaccharide abundant in the silverleaf whitefly, Bemisia argentifolii

The major soluble carbohydrates in the silverleaf whitefly, Bemisia argentifolii, were glucose, alpha,alpha-trehalose and an unknown sugar. Analysis of the unknown sugar and its chemical and enzymatic digestion products by high-performance liquid chromatography (HPLC) showed that it was probably a trisaccharide, consisting entirely of glucose, and containing both alpha,alpha-trehalose and isomaltose moieties. Matrix-assisted laser desorption mass spectrometry, mass spectrometry and 13C and 1H nuclear magnetic resonance spectroscopy confirmed that the sugar was a trisaccharide with the following structure: O-alpha-D-glucopyranosyl-(1-->6)-O-alpha-D-glucopyranosyl-(1<-->1)-alpha-D-glucopyranoside. This trisaccharide, found primarily in the bodies of B. argentifolii and not in their honeydew, is structurally similar to bemisiose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1<-->1)-alpha-D-glucopyranoside], a sugar first identified in Bemisia honeydew. Consequently, the common name isobemisiose is proposed for the newly identified sugar. Isobemisiose, which has not been previously reported to occur in nature, constituted as much as 46% (w/w) of the ethanol-soluble sugars in adult B. argentifolii, equivalent to approximately 10% of their dry weight. It was also found in similar quantities in immature B. argentifolii. Isobemisiose was detected in two other whitefly species and in several species of aphids, but at lesser concentrations than in B. argentifolii. Labeling and pulse-chase experiments using [14C]sucrose supplied to B. argentifolii in an artificial diet revealed that label accumulated in and was chased from isobemisiose more slowly than for either glucose or trehalose. Incubation of isobemisiose with cell-free extracts of B. argentifolii demonstrated that these whiteflies contained the necessary complement of enzymes to fully degrade isobemisiose to glucose. These labeling and digestion experiments indicate that isobemisose is probably a storage carbohydrate in B. argentifolii.     

Effect of acarbose on the increased plasma concentration of uric acid induced by sucrose ingestion

Sucrose is converted fructose and glucose, which may increase plasma uric acid concentration (pUA) through increased purine degradation and/or decreased uric acid (UA) excretion. To investigate effects of acarbose, an inhibitor of alpha-glucosidase, on the increased pUA from sucrose administration, we measured pUA and urinary UA excretion in 6 healthy subjects before and after administering sucrose, with and without co-administration of acarbose. Sucrose raised pUA by 10% (p < 0.01). However, excretion and fractional clearance of UA were unchanged. Sucrose and acarbose coadministration also increased pUA, but less than did sucrose alone (sucrose: 4.9 to 5.4 mg/dl; sucrose + acarbose, 4.7 to 4.9 mg/dl, p < 0.05) without changes in urinary excretion and fractional clearance of UA. Acarbose appears to attenuate the rise in pUA by sucrose ingestion by inhibiting sucrose absorption.     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

Effect of Acarbose on the Increased Plasma Concentration of Uric Acid Induced by Sucrose Ingestion

Sucrose is converted fructose and glucose, which may increase plasma uric acid concentration (pUA) through increased purine degradation and/or decreased uric acid (UA) excretion. To investigate effects of acarbose, an inhibitor of alpha-glucosidase, on the increased pUA from sucrose administration, we measured pUA and urinary UA excretion in 6 healthy subjects before and after administering sucrose, with and without co-administration of acarbose. Sucrose raised pUA by 10% (p < 0.01). However, excretion and fractional clearance of UA were unchanged. Sucrose and acarbose coadministration also increased pUA, but less than did sucrose alone (sucrose: 4.9 to 5.4 mg/dl; sucrose + acarbose, 4.7 to 4.9 mg/dl, p < 0.05) without changes in urinary excretion and fractional clearance of UA. Acarbose appears to attenuate the rise in pUA by sucrose ingestion by inhibiting sucrose absorption.     

Changes in Starch Formation and Activities of Sucrose Phosphate Synthase and Cytoplasmic Fructose-1,6-bisphosphatase in Response to Source-Sink Alterations

Short term experiments were conducted with vegetative soybean plants (Glycine max L. Merr. `Ransom' or `Arksoy') to determine whether sourcesink manipulations, which rapidly changed the `demand' for sucrose and partitioning of photosynthetically fixed carbon into starch, were associated with alterations in activities of sucrose-P synthase and/or cytoplasmic fructose-1,6-bisphosphatase in leaf extracts. When demand for sucrose from a particular source leaf was increased by defoliation of other source leaves, starch accumulation was restricted and activities of both enzymes were markedly enhanced. When demand for sucrose from source leaves was limited by excision, starch accumulation in the detached leaves was increased while activity of sucrose-P synthase declined sharply. The consistent responsiveness of sucrose-P synthase activity to changes in demand for sucrose supports the contention that regulation of sucrose-P synthase is an integral component of the system which controls sucrose biosynthesis and partitioning of carbon between starch and sucrose biosynthesis in the light.     

Carbon partitioning and sucrose metabolism in tomato plants growing under salinity

The different growth responses under salinity in relation to the carbon partitioning and sucrose metabolism in both sink and source organs have been studied in a salt-tolerant (cv. Pera) and in a salt-sensitive (cv. Volgogradskij) tomato genotype ( Lycopersicon esculentum Mill.). After 3 weeks of salinization, the plant dry weight was reduced by 12–34% in cv. Pera and by 45–58% in cv. Volgogradskij. Photosynthesis was positively correlated to plant growth in cv. Pera but not in cv. Volgogradskij. In this salt-sensitive genotype, both photosynthesis and growth were negatively correlated with fructose, glucose and sucrose accumulation in both mature and young leaves, suggesting a blockage in their use for growth. The transient accumulation of sucrose and hexoses in the young leaves of cv. Pera was linked to increases in all soluble sucrolytic activities, mainly acid invertase (EC 3.2.1.25) and sucrose synthase (EC 2.4.1.13), which was related to sink activity and growth capacity. The sucrose-phosphate synthase activity (EC 2.4.1.14) was related to the ability of mature leaves to regulate assimilate production, accumulation and export. The salt-tolerant cv. Pera accumulated a higher amount of total carbohydrates, but cv. Volgogradskij showed the highest soluble fraction under salinity. The carbohydrate availability and the photosynthetic rate do not seem to be the first limiting factors for plant growth under saline conditions, but the different behavior observed in both genotypes concerning the distribution and use of photoassimilates could help to explain their different salt-tolerance degrees.     

Cardinium infection alters cotton defense and detoxification metabolism of its whitefly host

Field monitoring revealed that the infection ratio of the bacterial symbiont Cardinium in the whitefly (Bemisia tabaci MED) was relatively low in northern China. However, the role of this symbiont and the symbiont–whitefly–host plant interaction mechanism are poorly understood. We investigated the influence of Cardinium on the competitiveness of the host whitefly and the physiological interaction between the host plants and host whiteflies. Cardinium-infected whiteflies were displaced by uninfected whiteflies after 5 generations, which showed that Cardinium infection reduced whitefly competitiveness. The defense response genes of cotton significantly decreased under infestation by infected whiteflies compared to uninfected whiteflies. The expression of detoxification metabolism genes, especially the uridine 5ʹ-diphospho-glucuronyltransferase and P450 genes, in infected whiteflies significantly decreased. These results demonstrated that Cardinium could inhibit the defense response of the host plant and decrease the detoxification metabolism ability of the host whitefly. The reduced competitiveness of infected whiteflies may be associated with the inhibition of the whitefly detoxification metabolism by Cardinium, resulting in the reduced performance of infected whiteflies. However, Cardinium infection can suppress plant defenses, which may benefit both infected and uninfected whiteflies when they coexist. This research illustrates the symbiont–whitefly–host plant interaction mechanism and the population dynamics of the whitefly.