Synthesis Of Kestoses Lab Report

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Synthesis Of Kestoses Lab Report



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These data provide new insights in the biochemical diversity of wild and cultivated bananas, and shed light on potential roles that fructans may fulfill across species, during plant development and adaptation to changing environments. Furthermore, the promiscuous behavior of banana fruit invertases sucrose and fructose as acceptor substrates besides water provides a new avenue to boost future work on structure-function relationships on these enzymes, potentially leading to the development of genuine banana fructosyltransferases that are able to increase fructan content in banana fruits. Fructans are fructose-based oligo- and polysaccharides derived from sucrose by the action of fructosyltransferases, which evolved from vacuolar invertases VIs Van den Ende et al. Plant inulin-type fructans are produced by the combined action of sucrose: sucrose 1-fructosyl transferase 1-SST and fructan: fructan 1-fructosyl transferase 1-FFT Van den Ende, All these enzymes belong to family GH32, in which structure-function relationships have been thoroughly investigated Van den Ende et al.

In addition to their function as reserve carbohydrates, fructans might fulfill other, perhaps more specific, roles in plant adaptation to environmental stresses drought resistance, frost tolerance and in osmoregulation Asega and de Carvalho, Fructan remobilization in the stem of wheat plants under drought stress is an important contributor to grain filling Joudi et al. Antioxidant and signaling functions have been recently suggested for fructans Van den Ende, ; Matros et al. Banana is also a fructan accumulator Der Agopian et al. Typically, biotic and abiotic stresses stand as major constraints in banana production. Interestingly, bananas are quite sensitive to drought. However, genotypes with the B genome seem to be more tolerant than those solely based on the A genome Ravi et al.

Inulin-type fructans, like FOS, are known to confer benefits to human health by selectively promoting the growth of bifidobacteria in the colon, which has been associated with increased mineral absorption, modification of lipid metabolism, enhancement of the systemic defense function, and prevention of colon cancer and inflammatory bowel disease Sangeetha et al. Fructan-containing plant species are found in a limited number of families, such as Liliaceae, Poaceae, and Asteraceae Van den Ende, Instead, fructans can be easily extracted and processed from bulbs, tubers and tuberous roots of plants with higher harvest indexes Sims, The edible bananas originated from natural inter- sub specific- and inter-specific crosses between M.

Within cultivated banana, Musa spp. These correspond to the genetic constitutions of wild Musa species M. Bananas are one of the most consumed fruits worldwide and represent an important source of revenue for tropical countries, where they are also one of their main staple foods Moshfegh et al. As such, they constitute a fundamental source of energy, vitamins and minerals for tropical countries Wall, Different cultivars are available worldwide, with well-known agronomic characteristics and organoleptic properties, such as color, size, texture, sweetness and flavor Aurore et al. Although it has been reported that banana fruits contain small fructans, with significant differences in their concentrations due to cultivar identity, stage of ripening and processing L'Homme et al.

The aim of this study was to characterize the variation of type and content of small WSC sucrose, glucose, and fructose and fructans present in different vegetative organs and fruits of 11 accessions of Musa with different genomic constitution. Such insights may boost future work to enhance fructan content in banana plants, which could be useful for the plants' stress responses as well as to increase the nutritional properties of banana fruit.

In this study 11 banana accessions I to XI comprising eight cultivars and three fertile wild species [ M. W, elevation Table 1. List of Musa accessions used in the present study. Leaves, fruits and rhizomes were harvested from healthy plants between and am during the warmest dry season from March to May There was a 1. Leaf samples were harvested from the last expanded leaf from physiologically mature plants. Leaf samples were transported to the lab in coolers, and once there, frozen with liquid nitrogen. Fruits were harvested at maturation stage 6 Dadzie and Orchard, Ripe fruits were peeled, and the pulp was sliced and frozen in liquid nitrogen. Rhizomes were cut in halves; portions of the central cylinder were extracted and frozen in liquid nitrogen.

All frozen samples were lyophilized prior to extraction. Carbohydrate extraction was conducted from two plants per accession with three replicates each. Lyophilized samples 0. Extracts were pooled and then lyophilized again. High performance anion exchange chromatography with integrated pulsed amperometric detection HPAEC-IPAD was used to analyze the soluble carbohydrate composition in extracts from lyophilized samples of leaf, rhizome and fruit pulp of the three wild diploid species M.

Interestingly, significant variations in the contents of these WSC were detected Tables 2 , 3. Table 2. Table 3. In the pulp of ripe fruits, this fraction fluctuated even stronger, from 0 to Overall, the rhizomes contained much less of this fraction Table 2. The presumed 1,1-nystose accumulated mainly in the rhizomes, with contents varying between On the contrary, the content of 1-kestotriose was higher in the pulp of ripe fruits as compared to leaves and rhizomes, with the lowest content Intriguingly, 1-kestotriose only accumulated in the rhizome of five of the eleven accessions, at varying concentrations Table 2. When the levels of the small WSC sucrose, glucose and fructose are compared, a clear genotypic variation is detected Table 3.

The lowest sucrose content 6. The lowest glucose content was detected in accession VI 2. Rhizomes showed lower levels and even greater variation in the di— and monosaccharides, sucrose In the pulp of ripe fruits, sucrose varied from To examine the variations in ripe fruit of glucose, fructose, and sucrose levels among accessions in more detail, a ternary diagram was employed Figure 1 , where their location revealed the organ's pattern, given by the contribution percentages of each sugar.

Figure 1. Ternary diagram illustrating variations in sucrose, glucose and fructose contents in fruits collected from mature plants of 11 Musa accessions. Roman numbers of different accessions correspond to those in Table 1. Each point represents the proportional composition of carbohydrates in each individual accession. The distance from a point to the side of the triangle is proportional to the relative importance of the carbohydrate in the sample. Figure 2 shows the carbohydrate patterns for M. Very similar patterns were obtained for M. In addition to glucose, fructose and sucrose, a comparison with a chicory fructan profile showed that banana fruit also contained 1-kestotriose, inulobiose, and inulotriose.

Surprisingly, no 1,1-nystose, 1,1,1-kestopentaose and higher DP inulin-type fructans were detected Figure 2. Rhizomes and leaves contained lower amounts of WSC, and showed even more complex patterns Figure 2. To unravel the identities of the peaks sequentially eluting after 1-kestotriose in banana fruit, we compared the patterns to references containing malto-oligosaccharides MOS, maltose and maltotriose, breakdown products of starch, being very abundant in fruits , raffinose family oligosaccharides the RFOs raffinose and stachyose and the three kestoses: 1-kestotriose, 6-kestotriose, and 6G-kestotriose neokestose Figure 3.

While the banana fruit shows peaks that elute at the same retention time as the three kestoses, no MOS and RFOs could be detected in banana fruit Figure 3. The peak eluting after 16 min, that was first thought to represent maltose, is not co-eluting with maltose Figure S3. The presence of the three kestoses in banana fruit was further confirmed by mild acid hydrolysis Figure S4 , enzymatic hydrolysis and co-elution experiments Figure S5. The identity of inulobiose, a major peak in banana fruit, was further confirmed by its sensitivity to hot alkaline treatment Figure S6. The identity of 4 peaks remains unknown see red arrows in Figure 3. Figure 2. A pattern of chicory root inulin is provided alongside as a reference, containing glucose Glc , fructose Fru , sucrose Suc , 1-kestotriose 1-Kes , inulobiose F 2 , 1,1-nystose Nys , inulotriose F 3 , 1,1,1-kestopentaose GF 4 and higher molecular weight inulin-type fructans.

Figure 3. Besides the three kestoses, M. In conclusion, both non-reducing GF n and reducing inulo-n-oses or F n types fructans are present in the mature fruit of the three species analyzed, M. Therefore, data of the presumptive 1,1-nystose and higher molecular weight inulins were left out from the principal component analysis PCA. Of these, the first two accounted for The seven most important pairs of variables contributing to describe the first principal component PC1 were glucose and sucrose in leaves, glucose and fructose in rhizomes, and glucose, fructose and sucrose in fruit, whereas the most important variables for PC2 were 1-kestotriose and sucrose in leaves and 1-kestotriose and glucose in rhizomes.

PC3 was defined by the variable 1-kestotriose in leaves and pulp from ripe fruit Supplementary Table S1. The PCA plot, built using the Eigen-values of the two first principal components, displayed two clearly defined, major groups Figure 4. Figure 4. Principal component analysis PCA of the four WSC, sucrose, glucose, fructose, and 1-kestotriose, present in three organs of 11 Musa accessions. The non-reducing GF n type of fructans 1-kestotriose, 6-kestotriose and 6G-kestotriose older name, neokestose , as well as the reducing inulo- n -oses or F n type fructans inulobiose F 2 and, to a lesser extent, inulotriose F 3 , could be clearly detected in the pulp of ripe fruit, and to a lower degree, also in the leaves and rhizomes of three of the wild Musa accessions, which are in the ancestry of the cultivated bananas.

Thus, the most important finding is that no 1,1-nystose and inulins of higher molecular weight were present in wild banana samples, demonstrating the inaccuracy of relying only on HPLC-RID data for fructan species identification. Total contents of 1-kestotriose varied not only in regard to the organ being studied, but also among the analyzed genotypes. To our knowledge, this is the first report of the presence of GF n and F n type of small fructans in the leaves and rhizomes of banana plants. In contrast, only Further work needs to be done to determine the exact contents of 6-kestotriose, 6G-kestotriose, inulobiose, and inulotriose in the different organs of the cultivated accessions.

In particular, little is known about the natural occurrence and physiological roles of inulo- n -oses in plants. Van den Ende et al. These authors rather proposed the presence of a single, soluble VI with intrinsic 1-SST side activity. Plant VIs can produce the three kestoses in a certain ratio from high sucrose De Coninck et al. Moreover, it can be speculated that one or more VI-type of enzymes can produce inulobiose from sucrose donor substrate and fructose acceptor substrate , and inulotriose from sucrose donor substrate and inulobiose acceptor substrate Figure 5. This need to be further corroborated by enzyme purification and in depth characterization of the purified enzyme or enzymes involved. Figure 5. Model of vacuolar invertase action in banana fruit, linked to the identity of FOS.

Typically, vacuolar invertases use water as acceptor substrate, producing glucose and fructose. However, increased fructose and sucrose levels may lead to the replacement of water as acceptor substrate by sucrose and fructose. In particular, inulobiose may be produced from sucrose donor substrate and fructose acceptor substrate. The produced inulobiose can be used as acceptor substrate to produce inulotriose.

Furthermore, the three kestoses 1-kestotriose, 6-kestotriose, and 6G-kestotriose can be produced when sucrose acts as an acceptor substrate. Previous studies presented contrasting data with regard to fructan contents in banana fruits. For instance, in banana cv. The presence of 1,1-nystose Der Agopian et al. These data contrast with our findings, pointing at lower amounts in fruits and greater variation among the analyzed genotypes. For instance, the two wild diploid accessions M. The importance of these two species resides in their close genetic relations to present varieties of diploid and triploid bananas and plantains d'Hont et al.

In bananas, the physiological and biochemical roles of fructans remain to be fully elucidated, but they may function as a temporal storage or reserve of energy under adverse environmental conditions Van den Ende, In cereals e. This remobilization is thought to contribute to the final grain yield, especially when crops are subject to adverse abiotic stress Joudi et al.

Additionally, fructans may be involved in hydroxyl radical scavenging Peshev et al. Each point represents the proportional composition of carbohydrates in each individual accession. The distance from a point to the side of the triangle is proportional to the relative importance of the carbohydrate in the sample. Very similar patterns were obtained for M. In addition to glucose, fructose and sucrose, a comparison with a chicory fructan profile showed that banana fruit also contained 1-kestotriose, inulobiose, and inulotriose. The peak eluting after 16 min, that was first thought to represent maltose, is not co-eluting with maltose Figure S3.

The presence of the three kestoses in banana fruit was further confirmed by mild acid hydrolysis Figure S4 , enzymatic hydrolysis and co-elution experiments Figure S5. The identity of inulobiose, a major peak in banana fruit, was further confirmed by its sensitivity to hot alkaline treatment Figure S6. A pattern of chicory root inulin is provided alongside as a reference, containing glucose Glc , fructose Fru , sucrose Suc , 1-kestotriose 1-Kes , inulobiose F 2 , 1,1-nystose Nys , inulotriose F 3 , 1,1,1-kestopentaose GF 4 and higher molecular weight inulin-type fructans. Besides the three kestoses, M. In conclusion, both non-reducing GF n and reducing inulo-n-oses or F n types fructans are present in the mature fruit of the three species analyzed, M.

Therefore, data of the presumptive 1,1-nystose and higher molecular weight inulins were left out from the principal component analysis PCA. Of these, the first two accounted for The seven most important pairs of variables contributing to describe the first principal component PC1 were glucose and sucrose in leaves, glucose and fructose in rhizomes, and glucose, fructose and sucrose in fruit, whereas the most important variables for PC2 were 1-kestotriose and sucrose in leaves and 1-kestotriose and glucose in rhizomes.

PC3 was defined by the variable 1-kestotriose in leaves and pulp from ripe fruit Supplementary Table S1. Principal component analysis PCA of the four WSC, sucrose, glucose, fructose, and 1-kestotriose, present in three organs of 11 Musa accessions. The non-reducing GF n type of fructans 1-kestotriose, 6-kestotriose and 6G-kestotriose older name, neokestose , as well as the reducing inulo- n -oses or F n type fructans inulobiose F 2 and, to a lesser extent, inulotriose F 3 , could be clearly detected in the pulp of ripe fruit, and to a lower degree, also in the leaves and rhizomes of three of the wild Musa accessions, which are in the ancestry of the cultivated bananas.

Thus, the most important finding is that no 1,1-nystose and inulins of higher molecular weight were present in wild banana samples, demonstrating the inaccuracy of relying only on HPLC-RID data for fructan species identification. Total contents of 1-kestotriose varied not only in regard to the organ being studied, but also among the analyzed genotypes. To our knowledge, this is the first report of the presence of GF n and F n type of small fructans in the leaves and rhizomes of banana plants. In contrast, only Further work needs to be done to determine the exact contents of 6-kestotriose, 6G-kestotriose, inulobiose, and inulotriose in the different organs of the cultivated accessions.

In particular, little is known about the natural occurrence and physiological roles of inulo- n -oses in plants. Van den Ende et al. These authors rather proposed the presence of a single, soluble VI with intrinsic 1-SST side activity. Plant VIs can produce the three kestoses in a certain ratio from high sucrose De Coninck et al. This need to be further corroborated by enzyme purification and in depth characterization of the purified enzyme or enzymes involved. Model of vacuolar invertase action in banana fruit, linked to the identity of FOS. Typically, vacuolar invertases use water as acceptor substrate, producing glucose and fructose.

However, increased fructose and sucrose levels may lead to the replacement of water as acceptor substrate by sucrose and fructose. In particular, inulobiose may be produced from sucrose donor substrate and fructose acceptor substrate. The produced inulobiose can be used as acceptor substrate to produce inulotriose. Furthermore, the three kestoses 1-kestotriose, 6-kestotriose, and 6G-kestotriose can be produced when sucrose acts as an acceptor substrate. Previous studies presented contrasting data with regard to fructan contents in banana fruits. For instance, in banana cv. The presence of 1,1-nystose Der Agopian et al. These data contrast with our findings, pointing at lower amounts in fruits and greater variation among the analyzed genotypes.

For instance, the two wild diploid accessions M. The importance of these two species resides in their close genetic relations to present varieties of diploid and triploid bananas and plantains d'Hont et al. In bananas, the physiological and biochemical roles of fructans remain to be fully elucidated, but they may function as a temporal storage or reserve of energy under adverse environmental conditions Van den Ende, In cereals e. This remobilization is thought to contribute to the final grain yield, especially when crops are subject to adverse abiotic stress Joudi et al. Additionally, fructans may be involved in hydroxyl radical scavenging Peshev et al.

Similar functions may be proposed for fructans in banana. For instance, accessions with high levels of sucrose and low glucose and fructose contents e. Biochemical diversity in fruits' small WSC contents might be explained by the activity of enzymes involved in starch degradation and sucrose metabolism during fruit ripening Fils-Lycaon et al. SPS is a key enzyme in sucrose accumulation in ripening bananas Do Nascimento et al. In this regard, the activity of VIs is probably one of the key elements in the regulation of sucrose to hexose balance during banana ripening Fils-Lycaon et al. Furthermore, 1-kestotriose levels in leaves and rhizomes, and glucose levels in rhizomes added the most weight to the second component, for the final separation of accessions into the different groups.

In summary, PCA analysis highlighted genuine differences among the three wild diploid accessions with different WSC profiles, providing some clues on the nature of these differences. Particularly interesting is the grouping of the two accessions with B genomes M. Overall, the enormous variation in the 1-kestotriose contents within the 11 accessions highlights the necessity of deeper studies on the biochemistry of fructan biosynthesis within and between the different PCA subgroups, to further exploit banana biodiversity for breeding purposes, aiming to enrich fructan contents in fruits. Our knowledge on structure-function relationships in GH32 Van den Ende et al.

Moreover, further studies are needed to elucidate the physiological roles of the different fructan types detected in the different banana organs. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Ir. Co-injection of banana fruit sugars with maltose. Maltose G 2 does not co-elute with a peak of M.

Mild acid hydrolysis of chicory and banana fruit sugars. Two lower chromatograms: sucrose and inulin-type fructans from chicory C. Two upper chromatograms: the three kestoses in banana fruit M. Identity of the three kestoses in banana fruit: enzymatic hydrolysis with 1-FEH and co-elution. Treatment of the three kestoses and M. The identity of the kestoses in banana fruit was further confirmed by co-elution. Alkali treatment of banana fruit sugars.

Both inulobiose F 2 and an unknown peak X are sensitive to hot alkali treatment. This treatment generated massive amounts of gluconic acid, influencing the retention time and peak characteristics of 1-kestotriose 1-Kes. The levels of 1-Kes, 6-kestotriose 6-Kes and 6G-kestotriose 6G-Kes were not affected under this treatment. Contribution of different variables to vectors and Eigen-values in the multivariate data analysis PCA of WSCs in three organs from the eleven Musa accessions. Read article at publisher's site DOI : Hereditas , , 06 Feb Plant Cell Environ , 41 1 , 08 Nov Cited by: 11 articles PMID: Front Plant Sci , , 22 Sep This data has been text mined from the article, or deposited into data resources.

To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. J Chromatogr A , , 01 Jun Cited by: 13 articles PMID: Front Plant Sci , , 01 Mar J Exp Bot , 62 6 , 31 Dec Pereira A , Maraschin M. J Ethnopharmacol , , 13 Nov Cited by: 39 articles PMID: Roberfroid MB. Cited by: articles PMID: Contact us. Europe PMC requires Javascript to function effectively. Recent Activity. Search life-sciences literature Over 39 million articles, preprints and more Search Quick link: Coronavirus articles and preprints. Recent history Saved searches. Miranda-Ham ML 1 ,. Search articles by 'Lizbeth A Castro-Concha'. Castro-Concha LA 1 ,. Ku-Cauich JR 1 ,. Search articles by 'Rudy Vergauwen'.

Vergauwen R 2 ,. Search articles by 'Timmy Reijnders'. Reijnders T 2 ,. Van den Ende W 2 ,. Escobedo-GraciaMedrano RM 1. Affiliations 5 authors 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract The water soluble carbohydrates WSC glucose, fructose, and sucrose are well-known to the great public, but fructans represent another type of WSC that deserves more attention given their prebiotic and immunomodulatory properties in the food context. High performance anion exchange chromatography with integrated pulsed amperometric detection HPAEC-IPAD showed the presence of 1-kestotriose [GF2], inulobiose [F2], inulotriose [F3], 6-kestotriose and 6G-kestotriose neokestose fructan species in the pulp of mature fruits of different accessions, but the absence of 1,1-nystose and 1,1,1 kestopentaose and higher degree of polymerization DP inulin-type fructans.

Free full text. Front Plant Sci. Published online Jun 9. PMID: Carlos I. Miranda-Ham , 1 Lizbeth A. Lizbeth A. Rosa M. Author information Article notes Copyright and License information Disclaimer. Reviewed by: Nabil I. Received Mar 13; Accepted May The use, distribution or reproduction in other forums is permitted, provided the original author s or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

This article has been cited by other articles in PMC. TIF K. Figure S3: Co-injection of banana fruit sugars with maltose. Figure S4: Mild acid hydrolysis of chicory and banana fruit sugars. Figure S5: Identity of the three kestoses in banana fruit: enzymatic hydrolysis with 1-FEH and co-elution. Figure S6: Alkali treatment of banana fruit sugars. DOCX 19K. Keywords: banana, fructo-oligosaccharides FOS , fruit, metabolite-phenotyping, rhizome, water soluble carbohydrates WSC. Materials and methods Plant material In this study 11 banana accessions I to XI comprising eight cultivars and three fertile wild species [ M.

Table 1 List of Musa accessions used in the present study. Open in a separate window. Figure 1. Figure 2. Figure 3. PCA reveals banana groups in terms of biochemical diversity in carbohydrate metabolites PCA analysis on the small WSC glucose, fructose, sucrose, and 1-kestotriose showed that Figure 4. Discussion Biochemical diversity of WSC in different organs from 11 accessions of banana The non-reducing GF n type of fructans 1-kestotriose, 6-kestotriose and 6G-kestotriose older name, neokestose , as well as the reducing inulo- n -oses or F n type fructans inulobiose F 2 and, to a lesser extent, inulotriose F 3 , could be clearly detected in the pulp of ripe fruit, and to a lower degree, also in the leaves and rhizomes of three of the wild Musa accessions, which are in the ancestry of the cultivated bananas.

Figure 5. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Click here for additional data file.

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