A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Endoplasmic reticulum Ca2+-ATPase pump is up-regulated in calcium-transporting dental enamel cells: a non-housekeeping role for SERCA2b.

Dental enamel-forming cells face a major challenge to avoid the cytotoxic effects of excess calcium. We have characterized sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps (SERCA) in rat enamel cells to address the proposal that non-mitochondrial calcium stores play a dominant role in transcellular calcium transport. A single major isoform, SERCA2b, was detected during the protein-secretory and calcium-transport stages of enamel formation using reverse-transcriptase PCR, cDNA cloning, Northern analysis and immunoblotting. Most importantly, SERCA2b exhibited a specific 3-fold up-regulation to high expression levels during calcium transport, as determined by quantitative immunoblotting and ATPase assays. Sensitivity of the calcium-dependent ATPase to thapsigargin and three other SERCA inhibitors was characterized. These findings indicate that enamel cells are well-equipped to sequester calcium in endoplasmic reticulum stores and so protect against calcium toxicity, associate SERCA with transcellular calcium transport for the first time, and establish SERCA2b as a molecular and pharmacological target for future investigations of calcium transcytosis. The observed physiological regulation in enamel cells contradicts the widespread perception that SERCA2b is restricted to general housekeeping duties.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-Induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts.

Attack by the specialist herbivore, Manduca sexta, on its native host Nicotiana attenuata Torr. ex Wats. produces a dramatic ethylene release, a jasmonate burst, and a suppression of the nicotine accumulation that results from careful simulations of the herbivore's damage. Methyl-jasmonate (MeJA) treatment induces nicotine biosynthesis. However, this induction can be suppressed by ethylene as pretreatment of plants with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene receptors, restores the full MeJA-induced nicotine response in herbivore attacked plants (J. Kahl, D.H. Siemens, R.J. Aerts, R. Gäbler, F. Kühnemann, C.A. Preston, I.T. Baldwin [2000] Planta 210: 336-342). To understand whether this herbivore-induced signal cross-talk occurs at the level of transcript accumulation, we cloned the putrescine methyltransferase genes (NaPMT1 and NaPMT2) of N. attenuata, which are thought to represent the rate limiting step in nicotine biosynthesis, and measured transcript accumulations by northern analysis after various jasmonate, 1-MCP, ethephon, and herbivory treatments. Transcripts of both root putrescine N-methyltransferase (PMT) genes and nicotine accumulation increased dramatically within 10 h of shoot MeJA treatment and immediately after root treatments. Root ethephon treatments suppressed this response, which could be reversed by 1-MCP pretreatment. Moreover, 1-MCP pretreatment dramatically amplified the transcript accumulation resulting from both wounding and M. sexta herbivory. We conclude that attack from this nicotine-tolerant specialist insect causes N. attenuata to produce ethylene, which directly suppresses the nitrogen-intensive biosynthesis of nicotine.