A paradigm shift towards low-nitrifying production systems: the role of biological nitrification inhibition (BNI).

BACKGROUND: Agriculture is the single largest geo-engineering initiative that humans have initiated on planet Earth, largely through the introduction of unprecedented amounts of reactive nitrogen (N) into ecosystems. A major portion of this reactive N applied as fertilizer leaks into the environment in massive amounts, with cascading negative effects on ecosystem health and function. Natural ecosystems utilize many of the multiple pathways in the N cycle to regulate N flow. In contrast, the massive amounts of N currently applied to agricultural systems cycle primarily through the nitrification pathway, a single inefficient route that channels much of this reactive N into the environment. This is largely due to the rapid nitrifying soil environment of present-day agricultural systems. SCOPE: In this Viewpoint paper, the importance of regulating nitrification as a strategy to minimize N leakage and to improve N-use efficiency (NUE) in agricultural systems is highlighted. The ability to suppress soil nitrification by the release of nitrification inhibitors from plant roots is termed 'biological nitrification inhibition' (BNI), an active plant-mediated natural function that can limit the amount of N cycling via the nitrification pathway. The development of a bioassay using luminescent Nitrosomonas to quantify nitrification inhibitory activity from roots has facilitated the characterization of BNI function. Release of BNIs from roots is a tightly regulated physiological process, with extensive genetic variability found in selected crops and pasture grasses. Here, the current status of understanding of the BNI function is reviewed using Brachiaria forage grasses, wheat and sorghum to illustrate how BNI function can be utilized for achieving low-nitrifying agricultural systems. A fundamental shift towards ammonium (NH4(+))-dominated agricultural systems could be achieved by using crops and pastures with high BNI capacities. When viewed from an agricultural and environmental perspective, the BNI function in plants could potentially have a large influence on biogeochemical cycling and closure of the N loop in crop-livestock systems.

Variations in abundance of 2 repetitive sequences in Leymus and Psathyrostachys species.

The Ns genome of the genus Psathyrostachys is a component of the polyploid genome in the genus Leymus. Using fluorescence in situ hybridization (FISH), the occurrence and abundance of 2 tandem repetitive sequences from Leymus racemosus (Lam.) Tzvelev, pLrTaiI-1 (TaiI family) and pLrPstI-1 (1 class of 350-bp family), were assayed in 4 species of the genera Psathyrostachys and Leymus. The pLrPstI-1 sequence was absent in all 4 Psathyrostachys species. While P. fragilis and P. huashanica did not have the pLrTaiI-1 sequence, 15 accessions of P. juncea and 2 accessions of P. lanuginosa had pLrTaiI-1 sites ranging in number from 7 to 16 and from 2 to 21, respectively. The numbers of pLrTaiI-1 and pLrPstI-1 sites were 1-24 and 0-30, respectively, in L. ramosus; 2-31 and 5-36 in L. racemosus; 0-4 and 0 in L. mollis; 2-9 and 24-27 in L. secalinus. The FISH assay on pLrTaiI-1 was successfully converted to a sequence-tagged-site polymerase chain reaction (STS-PCR) test using a primer pair designed from the sequence of this repetitive DNA. Seventy-three accessions representing 27 Leymus species were assayed for the abundance of pLrTaiI-1 by STS-PCR. With a few exceptions of uniformity in some accessions, nearly all Leymus species observed were heterogeneous for the abundance of pLrTaiI-1 sequence and no Leymus species was totally devoid of this repetitive sequence. These findings may have significance for the understanding of phylogeny, nature of polyploidy, adaptive ranges, and breeding potential of Leymus species.

Centromere-specific repetitive sequences from Torenia, a model plant for interspecific fertilization, and whole-mount FISH of its interspecific hybrid embryos.

Torenia fournieri is a good model plant to study fertilization in plants because it is easy to observe the fertilization process due to the protruding nature of the embryo sac. To study the location and movement of chromosomes and their centromeres in early embryogenesis of interspecific hybrid plants, we isolated two families of centromere-specific tandem repetitive sequences from T. fournieri and T. bailonii, and named them the "TCEN-family" and "BCEN-family", respectively. Both sequences consisted of a repeat unit of 52 bp located in the pericentric and centric heterochromatins. All signals of both sequences were prominent, but their intensity varied among the chromosomes. DNA-blot hybridization indicated the presence of similar sequences of TCEN-family in T. concolor, N. caerulea, and "Summer Wave", whereas the BCEN-family was found only in T. bailonii, thus indicating the wide or specific distribution of their repetitive families observed. We also applied whole-mount FISH to the interspecific hybrid embryos by using TCEN- and BCEN-family sequences as probes. Our results suggest that whole-mount FISH with the species-specific centromere sequences as probes is an ideal method to analyze the dynamics of chromosomes and centromeres in interspecific fertilization and early embryogenesis.

Novel repeated DNA sequences in safflower (Carthamus tinctorius L.) (Asteraceae): cloning, sequencing, and physical mapping by fluorescence in situ hybridization.

Two novel repetitive DNA sequences, pCtKpnI-1 and pCtKpnI-2, were isolated from Carthamus tinctorius (2n = 2x = 24) and cloned. Both represent tandemly repeated sequences. The pCtKpnI-1 and pCtKpnI-2 clones constitute repeat units of 343-345 bp and 367 bp, respectively, with 63% sequence heterogeneity between the two. Fluorescence in situ hybridization (FISH) was employed on metaphase chromosomes of C. tinctorius using, simultaneously, pCtKpnI-1 and pCtKpnI-2 repeated sequences. The pCtKpnI-1 sequence was found to be exclusively localized at subtelomeric regions on most of the chromosomes. On the other hand, sequence of the pCtKpnI-2 clone was distributed on two nucleolar and one nonnucleolar chromosome pairs. The satellite, and the intervening chromosome segment between the primary and secondary constrictions, in the two nucleolar chromosome pairs were wholly constituted by pCtKpnI-2 repeated sequence. The pCtKpnI-2 repeated sequence, showing partial homology to intergenic spacer (IGS) of 18S-25S ribosomal RNA genes of an Asteraceae taxon (Centaurea stoebe), and the 18S-25S rRNA gene clusters were located at independent, but juxtaposed sites in the nucleolar chromosomes. Variability in the number, size, and location of the two repeated sequences provided identification of most of the chromosomes in the otherwise not too distinctive homologues within the complement. This article reports the start of a molecular cytogenetics program targeting the genome of safflower, a major world oil crop about whose genetics very little is known.

Characteristics and behaviour of the chromosomes of Leymus mollis and L. racemosus (Triticeae, Poaceae) during mitosis and meiosis.

Leymus mollis and L. racemosus (Triticeae; Poaceae) are important as genetic resources for wheat improvement, as they carry genes for salt tolerance and disease resistance. Even though these species share common Ns and Xm genomes, the genomic relationship between these two species is not yet clearly understood. In this study, we examined the genomes of the two species by FISH and GISH, using combinations of tandem-repetitive sequences and genomic DNAs. Comparative GISH showed that genomes in the genus Leymus were diverse. Nevertheless, chromosomes of these two species were able to undergo complete meiotic pairing in hybrids, suggesting that differences in the subtelomeric heterochromatin and sequences distinguishable by GISH do not affect meiotic pairing.

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes.

Although Tail-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using Tail and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of Tail and Ty3/gypsy sequences depending on the chromosome; Tail-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing Tail-tandem arrays or a two-step amplification mechanism of the Tail family where each Tail colony was amplified to form arrays independently after the insertion of Tail-family sequences along the entire centromere. Although sequence analysis of centromeric Tail repeats in wheat and subtelomeric Tail repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.

Exclusive localization of tandem repetitive sequences in subtelomeric heterochromatin regions of Leymus racemosus (Poaceae, Triticeae).

Two kinds of tandem repetitive sequences were isolated from Leymus racemosus (Lam.) Tzvelev. One of them was classified in the 350-bp family originally isolated from Secale. The other was a novel repetitive sequence family, named 'TaiI family', which consisted of a repeat unit of 570 bp. Fluorescence in-situ hybridization of the chromosomes of L. racemosus indicated that both families were located in subtelomeric heterochromatin and that the 350-bp family and TaiI family occupied different heterochromatin regions. In addition, even homologous chromosomes did not show the same patterns of TaiI and 350-bp families. The combination of these two families of repetitive sequences, together with Afa-family sequences and rDNAs, helps to identify the ten homologous chromosome pairs of L. racemosus. From these data, we proposed a karyotype of L. racemosus and compared it with other karyotypes already reported.

Tandem repetitive Afa-family sequences from Leymus racemosus and Psathyrostachys juncea (Poaceae)

Tandem repetitive Afa-family sequences of 340 bp are known to occur in wheat and related species of tribe Triticeae. We isolated six and three Afa-family sequences from Leymus racemosus and Psathyrostachys juncea, respectively, both of which are perennial species. The sequences account for 0.5% and 0.2% of L. racemosus and P. juncea genomes, respectively, and using in situ hybridization were located in subtelomeric and interstitial regions of L. racemosus chromosomes. These sequences are clustered with those of Elymus trachycaulus in the phylogenetic tree. Our findings indicate that the Afa-family sequences have been amplified at least twice in the lineage of L. racemosus, P. juncea, and E. trachycaulus.

High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity.

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.