Suppressed expression of choline monooxygenase in sugar beet on the accumulation of glycine betaine.

Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of choline. Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation.

Sodium-dependent uptake of glutamate by novel ApGltS enhanced growth under salt stress of halotolerant cyanobacterium Aphanothece halophytica.

Glutamate is a major free amino acid in cyanobacteria, but its transport properties remain largely unknown. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica, contained a sodium dependent glutamate transporter (ApGltS). The deduced amino acid sequence of ApGltS exhibited low homology (18-19% identity) to GltS from Synechocystis sp. PCC 6803 (slr1145) and Escherichia coli. The predicted ApGltS consisted of 476 amino acid residues with a molecular weight of 50,976 Da. As analysed by hydropathy profiling, ApGltS contains 11 transmembrane segments. The ApgltS gene was isolated and expressed in E. coli ME9107, which is deficient in glutamate uptake. ME9107, expressing ApGltS, took up glutamate and its rates increased with increasing concentrations of NaCl. Kinetics studies revealed that ApGltS is a high-affinity glutamate transporter with a K(m) of about 5 µM. The presence of 0.5 M NaCl in the assay medium increased V(max) by about 3-fold. Competition experiments revealed that glutamate, glutamine, aspartate, and asparagine inhibited glutamate uptake. The level of mRNA for ApgltS was higher in A. halophytica grown at high salinity. Under high salinity conditions supplemented with glutamate, A. halophytica showed a significant increase in intracellular glycine betaine.

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet.

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.

Isolation and characterization of proline/betaine transporter gene from oil palm.

Oil production from oil palm is adversely affected by drought and salt. Under drought and salt stress, proline content increases in oil palm; the mechanism for this is unknown. Here, an 8319-nucleotide sequence including cDNA, genomic DNA and the promoter region of proline transporter gene from oil palm Elaeis guineensis was determined. The transporter gene exhibited high similarity to Bet/ProT genes from several plants, but the highest homology was found with rice ProT1. The exon-intron structure of genomic DNA was unique, and numerous stress-response cis-elements were found in the promoter region. Expression of cDNA EgProT1 in Escherichia coli mutant exhibited uptake activities for glycinebetaine and choline as well as proline. Under salt-stressed conditions, exogenously applied glycinebetaine was taken up into the root more rapidly than the control. These data indicate that oil palm has a unique Pro/T1 gene. Nucleotide sequence data for the cDNA and genomic DNA of proline transporter gene from Elaeis guineensis are available in the DDJB database under accession numbers AB597035 and AB597036, respectively.

Preferential accumulation of betaine uncoupled to choline monooxygenase in young leaves of sugar beet--importance of long-distance translocation of betaine under normal and salt-stressed conditions.

It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.