We have previously reported on purification and characterization of beta-fructofuranosidase (beta-FFase) from Bifidobacterium adolescentis G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of B. longum NCC2705 and B. adolescentis ATCC 15703 were determined, and cscA gene in the both genome sequences encoding beta-FFase was predicted. Here, cloning of cscA gene encoding putative beta-FFase from B. adolescentis G1, its expression in E. coli and properties of the recombinant protein are described.

The fruit of date palm trees are an important part of the diet for a large portion of the Middle East and North Africa. The fruit is consumed both fresh and dry and can be stored dry for extended periods of time. Date fruits vary significantly across hundreds of cultivars identified in the main regions of cultivation. Most dried date fruit are low in sucrose but high in glucose and fructose. However, high sucrose content is a distinctive feature of some date fruit and affects flavor as well as texture and water retention. To identify the genes controlling high sucrose content, we analyzed date fruit metabolomics for association with genotype data from 120 date fruits. We found significant association of dried date sucrose content and a genomic region that contains 3 tandem copies of the beta-fructofuranosidase (invertase) gene in the reference Khalas genome, a low-sucrose fruit. High-sucrose cultivars including the popular Deglet Noor had a homozygous deletion of two of the 3 copies of the invertase gene. We show the deletion allele is derived when compared to the ancestral allele that retains all copies of the gene in 3 other species of Phoenix. The fact that 2 of the 3 tandem invertase copies are associated with dry fruit sucrose content will assist in better understanding the distinct roles of multiple date palm invertases in plant physiology. Identification of the recessive alleles associated with end-point sucrose content in date fruit may be used in selective breeding in the future.

Enzymes in the newly described rumen bacterium, Treponema zioleckii strain kT, capable of digesting Timothy grass fructan, inulin, and sucrose were identified and characterized. Two specific endolevanases and one non-specific beta-fructofuranosidase were found in a cell-free extract. The molecular weight of the endolevanases were estimated to be 60 and 36 kDa, whereas that of beta-fructofuranosidase, 87 kDa. The former of the specific enzymes was associated with the outer membrane, while the latter and the non-specific beta-fructofuranosidase, with the periplasm or cytosol. The K(m) and V(max) for Timothy grass fructan degradation by endolevanase were 0.27% and 15.75 microM fructose equivalents x mg protein(-1) x min(-1), those for sucrose and inulin digestion by beta-fructofuranosidase were 1.35 x 10(-3)M and 1.73 microM hexoses x mg protein(-1) x min(-1) and 1.77% and 1.83 microM hexoses x mg protein(-1) x min(-1), respectively.

Raffinose family oligosaccharides (RFOs) are implicated in plant regulatory mechanisms of abiotic stresses tolerance and, despite their antinutritional proprieties in grain legumes, little information is available about the enzymes involved in RFO metabolism in Fabaceae species. In the present study, the systematic survey of legume proteins belonging to five key enzymes involved in the metabolism of RFOs (galactinol synthase, raffinose synthase, stachyose synthase, alpha-galactosidase, and beta-fructofuranosidase) identified 28 coding-genes in Arachis duranensis and 31 in A. ipaënsis. Their phylogenetic relationships, gene structures, protein domains, and chromosome distribution patterns were also determined. Based on the expression profiling of these genes under water deficit treatments, a galactinol synthase candidate gene (AdGolS3) was identified in A. duranensis. Transgenic Arabidopsis plants overexpressing AdGolS3 exhibited increased levels of raffinose and reduced stress symptoms under drought, osmotic, and salt stresses. Metabolite and expression profiling suggested that AdGolS3 overexpression was associated with fewer metabolic perturbations under drought stress, together with better protection against oxidative damage. Overall, this study enabled the identification of a promising GolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in crops, whilst also contributing to the understanding of RFO metabolism in legume species.

Submergence impedes photosynthesis and respiration but facilitates aerenchyma formation in bermudagrass. Still, the regulatory genes underlying these physiological responses are unclear in the literature. To identify differentially expressed genes (DEGs) related to these physiological mechanisms, we studied the expression of DEGs in aboveground and underground tissues of bermudagrass after a 7 d treatment under control (CK), shallow submergence (SS), and deep submergence (DS). Results show that compared with CK, 12276 and 12559 DEGs were identified under SS and DS, respectively. Among them, the DEGs closely related to the metabolism of chlorophyll biosynthesis, light-harvesting, protein complex, and carbon fixation were down-regulated in SS and DS. Meanwhile, a large number of DEGs involved in starch and sucrose hydrolase activities, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation were down-regulated in aboveground tissues of bermudagrass in SS and DS. Whereas in underground tissues of bermudagrass these DEGs were all up-regulated under SS, only beta-fructofuranosidase and α-amylase related genes were up-regulated under DS. In addition, we found that DEGs associated with ethylene signaling, Ca2+-ROS signaling, and cell wall modification were also up-regulated during aerenchyma formation in underground tissues of bermudagrass under SS and DS. These results provide the basis for further exploration of the regulatory and functional genes related to the adaptability of bermudagrass to submergence.

MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are "intracellular organelle," "binding," "metabolic process," "pigmentation," and "biological regulation." Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

Poa crymophila Keng is highly adaptable to long-term low temperature and drought conditions, making it a desirable foraging grass of the Qinghai-Tibet Plateau. Here, the widely targeted metabolomics and comparative transcriptome analyses were utilized for the discovery of metabolites and genes in P. crymophila in response to cold and drought stresses. P. crymophila were exposed to -5°C for 24 h and recovered to 22°C for 48 h, as well as drought for 10 days followed by re-watering for 1 day. In total, 779 metabolic features were assigned to metabolites and 167,845 unigenes were generated. Seventeen compounds showed significant up-regulation (variable importance in project >1) under both stresses in the metabolic profiling, mainly annotated as carbohydrates, flavones, and phenylpropanoids. The genes which were positively correlated with these metabolites were assigned to pathways (sucrose-starch, raffinose, phenylpropanoid, and flavone metabolism) using the Mapman software package. Alpha-amylase, beta-fructofuranosidase, and sugar transport genes degraded the glucose and starch to small molecule sugars for the purpose of osmotic adjustment and to provide more energy for the growth of P. crymophila in an adverse environment. The induction of cinnamoyl-CoA reductase (CCR) and the MYB gene as well as the sharp increase in schizandrin, a kind of lignan, showed that this likely has the closest connection with the tolerance to both stresses. Four significantly induced flavone compounds are probably involved in reducing oxidative damage. Our results indicated that activation of the phenlypropanoid pathway plays the primary role in P. crymophila adapting to harsh environments. This study showed the mechanism of P. crymophila responding to both cold and drought stresses and showed the discovery of a new biological regulator against stresses.

Microarray analysis was performed on RNA isolated from guard cells that were manually dissected from leaves of Arabidopsis. By pooling our data with those of two earlier studies on Arabidopsis guard cell protoplasts, we provide a robust view of the guard-cell transcriptome, which is rich in transcripts for transcription factors, signaling proteins, transporters, and carbohydrate-modifying enzymes. To test the hypothesis that photosynthesis-derived sugar signals guard cells to adjust stomatal opening, we determined the profile of genes expressed in guard cells from leaves that had been treated with sucrose. The results revealed that expression of 440 genes changed in guard cells in response to sucrose. Consistent with this hypothesis, these genes encoded cellular functions for photosynthesis and transport of sugars, water, amino acids, and ions. Plants of T-DNA insertion lines for 50 genes highly responsive to sucrose were examined for defects in guard cell function. Twelve genes not previously known to function in guard cells were shown to be important in leaf conductance, water-use efficiency, and/or stomate development. Of these, three are of particular interest, having shown effects in nearly every test of stomatal function without a change in stomatal density: TPS5 (At4g17770), a TRAF domain-containing protein (At1g65370), and a WD repeat-containing protein (At1g15440).