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• Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.

Plant growth and productivity are affected by various abiotic stresses such as heat, drought, cold, salinity, etc. The mechanism of salt tolerance is one of the most important subjects in plant science as salt stress decreases worldwide agricultural production. In our present study we used cDNA-AFLP technique to compare gene expression profiles of a salt tolerant and a salt-sensitive cultivar of foxtail millet (Seteria italica) in response to salt stress to identify early responsive differentially expressed transcripts accumulated upon salt stress and validate the obtained result through quantitative real-time PCR (qRT-PCR). The expression profile was compared between a salt tolerant (Prasad) and susceptible variety (Lepakshi) of foxtail millet in both control condition (L₀ and P₀) and after 1 h (L₁ and P₁) of salt stress. We identified 90 transcript-derived fragments (TDFs) that are differentially expressed, out of which 86 TDFs were classified on the basis of their either complete presence or absence (qualitative variants) and 4 on differential expression pattern levels (quantitative variants) in the two varieties. Finally, we identified 27 non-redundant differentially expressed cDNAs that are unique to salt tolerant variety which represent different groups of genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, mRNA splicing, seed development and storage, etc. The expression patterns of seven out of nine such genes showed a significant increase of differential expression in tolerant variety after 1 h of salt stress in comparison to salt-sensitive variety as analyzed by qRT-PCR. The direct and indirect relationship of identified TDFs with salinity tolerance mechanism is discussed.

Sheath blight of rice, caused by Rhizoctonia solani Kühn AG-1 IA [teleomorph: Thanatephorus cucumeris (Frank) Donk], is one of the major diseases of rice ( Oryza sativa L.) worldwide. Sclerotia produced by R. solani AG-1 IA are crucial for their survival in adverse environments and further dissemination when environmental conditions become conducive. Differentially expressed genes during three stages of sclerotial metamorphosis of R. solani AG-1 IA were investigated by utilizing complementary DNA amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 258 transcript derived fragments (TDFs) were obtained and sequenced, among which 253 TDFs were annotated with known functions through BLASTX by searching the GenBank database and 19 annotated TDFs were assigned into 19 secondary metabolic pathways through searching the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database. Moreover, the results of quantitative real-time PCR (qRT-PCR) analysis showed that the expression patterns of eight representative annotated TDFs were positively correlated with sclerotial metamorphosis. Sequence annotation of TDFs showed homology similarities to several genes encoding for proteins belonging to the glycosyltransferases B (GTB) and RNA recognition motif (RRM) superfamily and to other development-related proteins. Taken together, it is concluded that the members of the GTB and RRM superfamilies and several new genes involved in proteolytic process identified in this study might serve as the scavengers of free radicals and reactive oxygen species (ROS) and thus play an important role in the sclerotial metamorphosis process of R. solani AG-1 IA.

Background Buckwheat, consisting of two cultivated species Fagopyrum tataricum and F. esculentum, is the richest source of flavonoid rutin. Vegetative tissues of both the Fagopyrum species contain almost similar amount of rutin; however, rutin content in seed of F. tataricum are ~50 folds of that in seed of F. esculentum . In order to understand the molecular basis of high rutin content in F. tataricum, differential transcript profiling through cDNA-AFLP has been utilized to decipher what genetic factors in addition to flavonoid structural genes contribute to high rutin content of F. tataricum compared to F. esculentum. Results Differential transcript profiling through cDNA-AFLP in seed maturing stages (inflorescence to seed maturation) with 32 primer combinations generated total of 509 transcript fragments (TDFs). 167 TDFs were then eluted, cloned and sequenced from F. tataricum and F. esculentum . Categorization of TDFs on the basis of their presence/absence (qualitative variation) or differences in the amount of expression (quantitative variation) between both the Fagopyrum species showed that majority of variants are quantitative (64%). The TDFs represented genes controlling different biological processes such as basic and secondary metabolism (33%), regulation (18%), signal transduction (14%), transportation (13%), cellular organization (10%), and photosynthesis & energy (4%). Most of the TDFs except belonging to cellular metabolism showed relatively higher transcript abundance in F. tataricum over F. esculentum . Quantitative RT-PCR analysis of nine TDFs representing genes involved in regulation, metabolism, signaling and transport of secondary metabolites showed that all the tested nine TDFs (Ubiquitin protein ligase, ABC transporter, sugar transporter) except MYB 118 showed significantly higher expression in early seed formation stage (S7) of F. tataricum compared to F. esculentum . qRT-PCR results were found to be consistent with the cDNA-AFLP results. Conclusions The present study concludes that in addition to structural genes, other classes of genes such as regulators, modifiers and transporters are also important in biosynthesis and accumulation of flavonoid content in plants. cDNA-AFLP technology was successfully utilized to capture genes that are contributing to differences in rutin content in seed maturing stages of Fagopyrum species. Increased transcript abundance of TDFs during transition from flowers to seed maturation suggests their involvement not only in the higher rutin content of F. tataricum over F. esculentum but also in nutritional superiority of the former.

The ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.), a typical pioneer species which associates with young pine trees colonizing disturbed sites, is a common root symbiont found at heavy metal contaminated sites. Three Cd-sensitive and three Cd-tolerant isolates of S. luteus, isolated respectively from non-polluted and a heavy metal-polluted site in Limburg (Belgium), were used for a transcriptomic analysis. We identified differentially expressed genes by cDNA-AFLP analysis. The possible roles of some of the encoded proteins in heavy metal (Cd) accumulation and tolerance are discussed. Despite the high conservation of coding sequences in S. luteus, a large intraspecific variation in the transcript profiles was observed. This variation was as large in Cd-tolerant as in sensitive isolates and may help this pioneer species to adapt to novel environments.

Plants exploit a broad range of defense mechanisms to effectively combat invasion by pathogens or herbivores. Each environmental stress activates multiple signal transduction pathways to ensure an effective spatial and temporal defense response. A detailed transcriptome analysis using the cDNA-AFLP technique was performed to identify genes that are differentially expressed in oilseed rape (Brassica napus cv. Westar) leaves upon treatment with methyl jasmonate, mechanical wounding, or feeding by diamondback moth larvae (Plutella xylostella). In total, 16 different primer combinations were used, generating cDNA fragments ranging from 50 bp to 500 bp in size. This technique generated an average of 60 amplification products per reaction and therefore a total number of 5,600 fragments per treatment. Out of 16,800 bands, 124 showed qualitative differences among the treated and their respective control samples, including 95 up-regulated and 29 down-regulated bands. Expression of a selected subset of differentially expressed genes was confirmed by Northern blot analysis. Sequencing of fragments grouped many of the expressed genes in the categories of signaling and wound or pathogen response with examples like Jacalin, Strictosidine synthase and MD-2-LPS homologs. Genes with altered expression in distal tissue included those involved in cellular housekeeping functions, suggesting modified resource allocation needed to respond to different stress conditions. Differences in local and systemic response as well as among the three different challenges were observed. Several new transcripts were identified that may play a role in insect attack and other signal transduction pathways.

cDNA amplified fragment length polymorphism (cDNA-AFLP) is a powerful transcript-profiling tool widely used in diverse plant species. When applied to a new biological system, however, existing protocols usually require substantial modifications. Furthermore, the usage of radioactive isotope in typical protocols excludes their application in many labs. Latex, as the cytoplasm of rubber-producing cells sees a critical role in elucidating rubber biosynthesis and its regulation in rubber tree (Hevea brasiliensis). This paper describes a detailed step-by-step silver-staining cDNA-AFLP procedure, which is suitable to latex transcript profiling analysis. Theoretical analysis revealed that with the combination of two restriction enzyme pairs (ApoI/MseI and TaqI/MseI), approximately 94% of latex whole transcriptome could be visualized. After varying multiple parameters, including the amounts of primary and secondary template usage, pre-amplification cycle number and gel development, we obtained a high-quality silver-staining fingerprint. In the ApoI/MseI system, an average of 88.6 discernable bands (100-1,000 bp) was produced for each selective primer pair, and 97.2 bands for another system (TaqI/MseI). TaqI/MseI was the first pair of 4-bp cutters used in cDNA-AFLP analysis and proved to be efficient and reliable. The sensitivity and reliability of our method were further verified by an application example in detecting differential gene expression in the latex of Hevea tree.

Common bacterial blight (CBB), incited by Xanthomonas axonopodis pv. phaseoli (Xap), is a serious seed-borne disease of common bean (Phaseolus vulgaris L.) in both temperate and tropical production zones. The line HR45 is highly resistant to Xap infection on leaves and pods in both field and greenhouse. To understand the molecular mechanisms underlying CBB resistance in HR45, cDNA-amplified fragment length polymorphism (AFLP) technique was used to identify the genes that are differentially expressed in the leaves of HR45 at different time-periods after inoculation. Selective amplifications with 34 primer combinations allowed the visualization of 2,448 transcript-derived fragments (TDFs) in infected leaves, and 259 (10.6%) of them were differentially expressed TDFs (DE-TDFs). Seventy-seven of the DE-TDFs were cloned and sequenced. Thirty-nine of the 77 (50.6%) DE-TDFs representing bean transcripts were not previously reported in any EST database. The expression patterns of 10 representative DE-TDFs were further confirmed by real-time RT-PCR. BLAST analysis suggested that 40% (31 of 77) of the DE-TDFs were homologous to the genes related to metabolism, photosynthesis, and cellular transport, whereas 28% (22 of 77) of the DE-TDFs showed homology to the genes involved in defence response, response to stimulus, enzyme regulation, and transcription regulation. Thus, the 22 pathogenesis-related DE-TDFs were selected as potential functional candidate genes (FCGs) in association with CBB resistance. Meanwhile, six of the DE-TDFs (1FCG and five other DE-TDFs) were in silico mapped to the distal region of the bean linkage group B6 (the genomic location containing the major CBB resistance QTL in HR45) and, therefore, were considered as positional candidate genes (PCGs). This study represents a first step towards the discovery of bean genes expressed upon Xap infection. This information will be useful for elucidating the molecular basis of the resistance response process and identifying the genes that underlie the CBB-resistance.

To understand the molecular genetic basis underlying drought tolerance in grasses, the cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was applied for identification of genes responding to drought stress in a xerophytic adapted plant, Festuca mairei. A total of 11,346 transcript derived fragments (TDFs) were detected, and 464 (4.1%) TDFs were identified as differentially expressed fragments (DEFs) during the drought treatment of the plant. The expression patterns of these DEFs included up-regulated (~30%), down-regulated (~54.3%), and the remainder (~16.7%) showing transient changes. The differential expression patterns of 171 DEFs were further confirmed by macroarray hybridization analysis. Sequences had been obtained for 163 DEFs, and 62 sequences had no significant hits to sequences currently in public databases. Predicted functions of remaining 101 sequences were subdivided into 17 categories. Down-regulated genes were highly represented by metabolism and cellular biogenesis. Up-regulated DEFs were enriched in genes involved in transcription, defense, cell cycle and DNA processing. Analysis of the 163 DEFs provides a first glimpse into the transcripts of F. mairei during drought stress treatment. The combination of data from studies on genetic model plants and on diverse plant species will enhance understanding of the drought tolerance mechanisms in plants.