Amino Acids that Confer Transport of Raffinose and Maltose Sugars in the Raffinose Permease (RafB) of Escherichia coli as Implicated by Spontaneous Mutations at Val-35, Ser-138, Ser-139, Gly-389 and Ile-391

In order to identify amino acid residues in the Escherichia coli raffinose-H⁺ permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and α-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants appeared red. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35→Ala (MT1); one mutant had Ile-391→Ser (MT2); and two mutants had Ser-138→Asp, Ser-139→Leu and Gly-389→Ala (MT3). Transport studies of [³H]-maltose showed that cells harboring MT1, MT2 and MT3 had greater uptake (P ≤ 0.05) than cells harboring wild-type RafB. However, [¹⁴C]-raffinose uptake was reduced in all mutant cells (P ≤ 0.05) with MT1, MT2 and MT3 mutants compared to cells harboring wild-type RafB. Kinetic analysis showed enhanced apparent K _m values for maltose and reduced V _max/ K _m ratios for raffinose compared to wild-type values. The apparent K _i value of maltose for RafB indicates a competitive relationship between maltose and raffinose. Maltose “uphill” accumulation was greater for mutants (P ≤ 0.05) than for cells with wild-type RafB. Thus, we implicate residues in RafB that are responsible for raffinose transport and suggest that the substituted residues in RafB dictate structures that enhance transport of maltose.     

Changes in the sugarcane metabolome with stem development. Are they related to sucrose accumulation?

Sucrose content increases with internode development down the stem of sugarcane. In an attempt to determine which other changes in metabolites may be linked to sucrose accumulation gas chromatography-mass spectrometry was used to obtain metabolic profiles from methanol/water extracts of four samples of different age down the stem of cultivar Q117. Extracts were derivatized with either N-methyl-N-(trimethylsilyl) trifluoracetamide (TMS) or N-methyl N-(tert-butyldimethylsilyl) trifluoroacetamide (TBS) separately in order to increase the number of metabolites that could be detected. This resulted in the measurement of 121 and 71 metabolites from the TMS and TBS derivatization, respectively. Fifty-five metabolites were identified using commercial and publicly available libraries. Statistical analysis of the metabolite profiles resulted in clustering of tissue types. Particular metabolites were correlated with the level of sucrose accumulation, which as expected increased down the stem. Metabolites, such as tricarboxylic acid cycle intermediates and amino acids, were more abundant in the M2 sample (meristem to internode 2) that was actively growing and decreased in an apparently coordinated developmentally programmed manner in more mature internodes down the stem. However, other metabolites such as trehalose and raffinose showed positive correlations with sucrose concentration. Here we discuss the technique used to measure metabolites in sugarcane and the changes in metabolite abundance down the sugarcane stem.     

Sorbitol accumulation during natural and accelerated ageing of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) seeds

The aim of the present study was to determine the effect of accelerated ageing on the composition and content of the soluble carbohydrates in pea seeds of six genotypes differing in the composition of raffinose family oligosaccharides. A gradual decrease in the concentration of higher homologues of raffinose was observed along with seed ageing. At the same time the seeds lost vigor, viability and germination capacity. No increase in the concentration of reducing sugars was recorded, but sorbitol accumulated in pea embryos. Sorbitol accumulation may indicate seed quality deterioration during storage.     

Vitrification of domestic cat (Felis catus) ovarian tissue: Effects of three different sugars

We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.     

Isolation and structural analysis of ajugose from Vigna mungo L

The hexasaccharide ajugose, alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside, generally uncommon in legumes, was detected in the seeds of Vigna mungo L. by TLC and paper chromatography. Ajugose was then isolated by silica gel chromatography and its structure was established by acid and enzymatic hydrolysis, fast atom bombardment mass spectrometry and both one- and two-dimensional 1H and 13C NMR techniques.     

Characterisation of an alpha-galactosidase with potential relevance to ripening related texture changes

alpha-Galactosidase (EC 3.2.1.22) from ripe papaya (Carica papaya L.) fruit was fractionated by a combination of ion exchange and gel filtration chromatography into three forms, viz., alpha-galactosidase 1, 2 and 3. The predominant isoform, alpha-gal 2, was probably a tetramer with a native molecular mass of about 170 kDa and 52 kDa-sized subunits and an estimated pI of 7.3. The subunit's N-terminal amino acid sequence shared high identity (97%) with the deduced sequence of a papaya cDNA clone encoding a putative alpha-galactosidase PAG2 as well as with an Ajuga reptans L. GGT1 clone encoding a galactan: galactan galactosyltransferase (66%). During ripening, alpha-galactosidase activity increased concomitantly with firmness loss and this increase was largely ascribed to alpha-gal 2. The protein level of alpha-gal 2 as estimated by immunoblot was low in developing fruits and generally increased with ripening. alpha-Galactosidase 2 also had the ability to markedly catalyse increased pectin solubility and depolymerisation while the polymers were still structurally attached to the cell walls mimicking, in part, the changes that occur during ripening. The close correlation between texture changes, alpha-gal 2 activity and protein levels as well as capability to modify intact cell walls suggest that the enzyme might contribute to papaya fruit softening during ripening. The purported mechanism of alpha-gal 2 action as a softening enzyme was discussed in terms of its functional capacity as a glycanase or perhaps, as a transglycosylase.     

Heterologous expression and functional characterization of two hybrid poplar cell-wall invertases

The expression of two hybrid poplar cell-wall invertases (EC 3.2.1.26; PaxgINV1 and PaxgINV2) were previously shown to be spatially and temporally regulated in the vegetative tissues. The expression of PaxgINV1 was linked to processes relating to dormancy, while PaxgINV2 expression was prominent in tissues undergoing growth and expansion. In an effort to further elucidate the physiological roles of these key cell wall enzymes, PaxgINV1 and PaxgINV2 were heterologously expressed in the methylotrophic yeast Pichia pastoris. Three-dimensional predictive models of the poplar invertases revealed a structural channel containing both the conserved β-fructofuranosidase and cell-wall invertase motifs, suggesting that this channel is the putative active site of these enzymes. Recombinant PaxgINV1 and PaxgINV2 had pH optima of 4.8 and 5.6 and temperature optima of 45 and 40°C, respectively. Functional characterization revealed the ability for both enzymes to hydrolyze the fructose residue of sucrose, raffinose, stachyose and verbascose, with PaxgINV2 having higher specific activity for each of the substrates tested. The K _m values of sucrose/raffinose/stachyose were 1.7/1.8/5.0 mM for PaxgINV1 and 1.6/1.7/1.9 mM for PaxgINV2, respectively. Activity analyses in the presence of various metal cations showed that PaxgINV2 was strongly inhibited by Cu²⁺, Zn²⁺ and Hg²⁺, while PaxgINV1 was only weakly inhibited by these cations. The results from this study, coupled with previous expression data, suggest that PaxgINV1 and PaxgINV2 have distinct roles with respect to the physiology and development of hybrid poplar, specifically phloem unloading and processes related to dormancy and bud break.     

Grafting helps improve photosynthesis and carbohydrate metabolism in leaves of muskmelon

The most important quality for muskmelon (Cucumis melo L.) is their sweetness which is closely related to the soluble sugars content. Leaves are the main photosynthetic organs in plants and thus the source of sugar accumulation in fruits since sugars are translocated from leaves to fruits. The effects of grafting muskmelon on two different inter-specific (Cucurbita maxima×C. moschata) rootstocks was investigated with respect to photosynthesis and carbohydrate metabolism. Grafting Zhongmi1 muskmelon on RibenStrong (GR) or Shengzhen1 (GS) rootstocks increased chlorophyll a, chlorophyll b and chlorophyll a+b content and the leaf area in middle and late developmental stages of the plant compared to the ungrafted Zhongmi1 check (CK). Grafting enhanced the net photosynthesis rate, the stomatal conductance, concentration of intercellular CO(2) and transpiration rate. Grafting influenced carbohydrates contents by changing carbohydrate metabolic enzymes activities which was observed as an increase in acid invertase and neutral invertase activity in the functional leaves during the early and middle developmental stages compared to CK. Grafting improved sucrose phosphate synthase and stachyose synthase activities in middle and late developmental stages, thus translocation of sugars (such as sucrose, raffinose and stachyose) in GR and GS leaves were significantly enhanced. However, compared with CK, translocation of more sugars in grafted plants did not exert feedback inhibition on photosynthesis. Our results indicate that grafting muskmelon on inter-specific rootstocks enhances photosynthesis and translocation of sugars in muskmelon leaves.     

Changes of soluble sugars and ATP content during DMSO droplet freezing and PVS3 droplet vitrification of potato shoot tips

The potato's great genetic diversity needs to be maintained for future agricultural applications and can be preserved at ultra-low temperatures. To decipher detailed physiological processes, the aim of the study was to analyze the regrowth in 28 gene bank accessions and to reveal metabolite changes in a subset of four accessions that showed pronounced differences after shoot tip cryopreservation using DMSO droplet freezing and PVS3 droplet vitrification. Regrowth varied in all 28 genotypes ranging from 5% (‘Kagiri’) to 100% (‘Karakter’) and was higher after PVS3 droplet vitrification (71 ± 19%) than after cryopreservation using DMSO (54 ± 17%). Sucrose, glucose, and fructose were analyzed and showed significant increases after pre-culture in combination with PVS3 or DMSO and liquid nitrogen treatment and were reduced during regeneration. In contrast, adenosine triphosphate (ATP) reached its minimum concentration after cryoprotection and liquid nitrogen treatment and recovered most quickly after PVS3 droplet vitrification. A shortening of the explant pre-culture period reduced dramatically the regrowth after PVS3 vitrification. However, correlations between the shoot tip regrowth and sugar concentration were absent and significant at a low extent with ATP (r = 0.4, P < 0.01). Interestingly, several sub-cultivations of the donor plants from the previous stock affected negatively the regrowth. In conclusion, the cryopreservation protocol, genotypes, pre-culture period and number of sub-cultures affect the regrowth ability of explants, which was best estimated by the ATP concentration after low-temperature treatment. Due to the superior performance of PVS3, the routine potato cryopreservation at the Gatersleben gene bank was changed to PVS3 droplet vitrification.