Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death （PCD）. However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 （/br11-1）, which fails to generate reactive oxygen intermediates （ROIs）, is incapable of initiating PCD when the mutant is challenged by fumonisin B l （FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 （LCB 1） subunit of serine palmitoyltransferase （SPT）, which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.     

Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro

D-type cyclin-dependent kinases (Cdk4 and Cdk6) regulate the G1 to S phase progression of the mammalian cell cycle. It has been suggested that Cdk4 and Cdk6 may have distinct functions in vivo, even though they are indistinguishable biochemically. Here we show that although these Cdks phosphorylate multiple residues in pRB, they do so with different residue selectivities in vitro; Thr821 and Thr826 are preferentially phosphorylated by Cdk6 and Cdk4, respectively. This raises the possibility different substrate specificities lead to their different roles in the regulation of cellular events. Furthermore, our results indicate the new concept that Cdk itself contributes to substrate recognition.     

Properties of two different Na+/H+ antiport systems in alkaliphilic Bacillus sp. strain C-125.

Na+/H+ antiport was studied in alkaliphilic Bacillus sp. strain C-125, its alkali-sensitive mutant 38154, and a transformant (pALK2) with recovered alkaliphily. The transformed was able to maintain an intracellular pH (pHin) that was lower than that of external milieu and contained an electrogenic Na+/H+ antiporter driven only by delta psi (membrane potential, interior negative). The activity of this delta psi-dependent Na+/H+ antiporter was highly dependent on pHin, increasing with increasing pHin, and was found only in cells grown at alkaline pH. On the other hand, the alkali-sensitive mutant, which had lost the ability to grow above pH 9.5, lacked the delta psi-dependent Na+/H+ antiporter and showed defective regulation of pHin at the alkaline pH range. However, this mutant, like the parent strain, still required sodium ions for growth and for an amino acid transport system. Moreover, another Na+/H+ antiporter, driven by the imposed delta pH (pHin > extracellular pHout), was active in this mutant strain, showing that the previously reported delta pH-dependent antiport activity is probably separate from delta psi-dependent antiporter activity. The delta pH-dependent Na+/H+ antiporter was found in cells grown at either pH 7 or pH 9. This latter antiporter was reconstituted into liposomes by using a dilution method. When a transmembrane pH gradient was applied, downhill sodium efflux was accelerated, showing that the antiporter can be reconstituted into liposomes and still retain its activity.     

Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases

A bacterium Sphingomonas sp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia coli cells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by beta-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.     

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3?Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0?bp (one SNP for every 78.3?bp in introns and for every 106.7?bp in exon regions). Nucleotide diversity in C. japonica (???=?0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8?%) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species?? range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Developmental changes in crossover frequency in Arabidopsis

Parental genomes are generally rearranged by two processes during meiosis: one is the segregation of homologous chromosomes and the other is crossing over between such chromosomes. Although the mechanisms underlying chromosome segregation and crossing over are well understood because of numerous genetic and molecular investigations, their contributions to the rearrangement of genetic information have not yet been analysed at a genome-wide level in Arabidopsis thaliana. We established 343 CAPS or SSLP markers to identify polymorphisms between two different Arabidopsis ecotypes, Col and Ler, which are distributed at an average distance of approximately 400kb between pairs of markers throughout the entire genome. Using these markers, crossover frequencies and chromosome segregation were quantified with respect to sex and age. Our large-scale analysis demonstrated that: (i) crossover frequencies during pollen formation were 1.79 and 1.37 times higher than those during megaspore formation in early and late flowers, respectively (P<0.001); (ii) the crossover frequencies during pollen formation were not significantly different between early and late flowers of main shoots (P>0.05), whereas the frequencies increased 1.30 times with shoot age during megaspore formation (P<0.001); (iii) the effect of aging depended on the developmental age of the individual shoot rather than on the age of the whole plant; and (iv) five chromosomes were randomly selected and mixed during meiosis.