Physiological measurements of nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.

Nitrate transport and signalling

Association of a Soybean Raffinose Synthase Gene with Low Raffinose and Stachyose Seed Phenotype

A novel α-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-α-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-α-1, 4-Gal-α-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-α-1,4 linkage, a novel galactosidic link formed by microbial α-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.

Cloning and characterization of a novel α‐galactosidase from Bifidobacterium breve 203 capable of synthesizing Gal‐α‐1,4 linkage

Properties of a novel α-galactosidase from Streptomyces sp. S27 and its potential for soybean processing

Two distinct extracellular bifunctional proteins with β-L-arabinopyranosidase/α-D-galactopyranosidase activities were purified from the culture filtrate of Fusarium oxysporum 12S. The molecular masses of the enzymes were estimated to be 55 (Fo/AP1) and 73 kDa (Fo/AP2) by SDS-PAGE. They hydrolyzed both p-nitrophenyl β-L-arabinopyranoside and p-nitrophenyl α-D-galactopyranoside with different specificities. Fo/AP1 also showed low activity towards α-D-galactopyranosyl oligosaccharides such as raffinose. Interestingly, both enzymes hydrolyzed larch wood arabinogalactan (releasing arabinose) but not carob galactomannan, which has α-D-galactopyranosyl side chains. When larch wood arabinogalactan was incubated with excess Fo/AP1 or Fo/AP2, both enzymes released approximately 10% of the total arabinose in the substrate. cDNAs encoding Fo/AP1 and Fo/AP2 (Foap1 and Foap2) were isolated by in vitro cloning. The coding sequences of Foap1 and Foap2 genes were 1,647 and 1,620 bp in length and encode polypeptides of 549 and 540 amino acids, respectively. The N-terminal halves of both proteins had high similarity to putative conserved domains of the melibiase superfamily (Pfam account number 02065). The deduced amino acid sequences of the two enzymes indicate that they belong to glycosyl hydrolase family 27. Moreover, the C-terminal regions of both proteins contain a putative family 35 carbohydrate-binding module.

Identification of two GH27 bifunctional proteins with β-L-arabinopyranosidase/α-D-galactopyranosidase activities from Fusarium oxysporum

Two putative α-galactosidase genes from rice (Oryza sativa L. var. Nipponbare) belonging to glycoside hydrolase family 27 were cloned and expressed in Escherichia coli. These enzymes showed α-galactosidase activity and were purified by Ni Sepharose column chromatography. Two purified recombinant α-galactosidases (α-galactosidase II and III; α-Gal II and III) showed a single protein band on SDS–PAGE with molecular mass of 42 kDa. These two enzymes cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose, and stachyose, but also liberated the galactosyl residues attached to the O-6 position of the mannosyl residue at the reducing-ends of mannobiose and mannotriose. In addition, these enzymes clipped the galactosyl residues attached to the inner-mannosyl residues of mannopentaose. Thus, α-Gal II catalyzes efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.

https://www.tandfonline.com/doi/pdf/10.1271/bbb.60554

Prukutty A Prema

Papers

Expression of Rice (Oryza sativa L. var. Nipponbare) α-Galactosidase Genes in Escherichia coli and Characterization