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Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ~20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

Arabidopsis thaliana was the first plant to be sequenced in 2000 and is widely used as a model organism for flowering plants. This has led to the generation of large data sets, such as sequences (DNA, RNA and proteins), gene predictions, protein information and microarray data. A number of different databases have been developed to store, distribute and analyse these data. These range from large repositories that contain a wide range of data (e.g. sequences, gene models, germplasm data) and small specialised databases containing one type of data (e.g. protein localisation). In addition tools have been developed to enable users to access, download and analyse the data. These include tools to analyse sequences, gene function, metabolomic pathways and microarray data. A number of these databases and tools will be described.

Together, auxin and cytokinin regulate many of the processes that are critical to plant growth, development, and environmental responsiveness. We have previously shown that exogenous auxin regulates cytokinin biosynthesis in Arabidopsis thaliana. In this work, we show that, conversely, the application or induced ectopic biosynthesis of cytokinin leads to a rapid increase in auxin biosynthesis in young, developing root and shoot tissues. We also show that reducing endogenous cytokinin levels, either through the induction of CYTOKININ OXIDASE expression or the mutation of one or more of the cytokinin biosynthetic ISOPENTENYLTRANSFERASE genes leads to a reduction in auxin biosynthesis. Cytokinin modifies the abundance of transcripts for several putative auxin biosynthetic genes, suggesting a direct induction of auxin biosynthesis by cytokinin. Our data indicate that cytokinin is essential, not only to maintain basal levels of auxin biosynthesis in developing root and shoot tissues but also for the dynamic regulation of auxin biosynthesis in response to changing developmental or environmental conditions. In combination with our previous work, the data suggest that a homeostatic feedback regulatory loop involving both auxin and cytokinin signaling acts to maintain appropriate auxin and cytokinin concentrations in developing root and shoot tissues.

Reports that low-intensity microwave radiation induces heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg(-1) for 6-well plates) that minimises temperature differentials between sham and exposed conditions (< or =0.1 degrees C). Parallel measurement and simulation studies of SAR distribution within this exposure system are presented. We compared five Affymetrix gene arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against five gene arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run). No genes showed consistent expression changes across all five comparisons, and all expression changes appeared modest after normalisation (< or =40% up- or down-regulated). The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131). We conclude that the pattern of gene expression in L4/adult C. elegans is substantially unaffected by low-intensity microwave radiation; the minor changes observed in this study could well be false positives. As a positive control, we compared RNA samples from N2 worms subjected to a mild heat-shock treatment (30 degrees C) against controls at 26 degrees C (two gene arrays per condition). As expected, heat-shock genes are strongly up-regulated at 30 degrees C, particularly an hsp-70 family member (C12C8.1) and hsp-16.2. Under these heat-shock conditions, we confirmed that an hsp-16.2::GFP transgene was strongly up-regulated, whereas two non-heat-inducible transgenes (daf-16::GFP; cyp-34A9::GFP) showed little change in expression.

Ripening is a tightly controlled and developmentally regulated process involving networks of genes, and metabolites that result in dramatic changes in fruit colour, texture and flavour. Molecular and genetic analysis in tomato has revealed a series of regulatory genes involved in fruit development and ripening, including MADS box and SPB box transcription factors and genes involved in ethylene synthesis, signalling and response. Volatile metabolites represent a significant part of the plant metabolome, playing an important role in plant signalling, defence strategies and probably in regulatory mechanisms. They also play an important role in fruit quality. In order to acquire a better insight into the biochemical and genetic control of flavour compound generation and links between these metabolites and the central regulators of ripening, five pleiotropic mutant tomato lines were subjected to volatile metabolite profiling in comparison with wild-type Ailsa Craig. One hundred and seventeen volatile compounds were identified and quantified using SPME (Solid Phase Microextraction) headspace extraction followed by Gas Chromatography–Mass Spectrometry (GC–MS) and the data were subjected to multivariate comparative analysis. We find that the different mutants each produce distinct volatile profiles during ripening. Through principal component analysis the volatiles most dramatically affected are those derived from fatty-acids. The results are consistent with the suggestion that specific isoforms of lipoxygenase located in the plastids and the enzymes that provide precursors and downstream metabolites play a key role in determining volatile composition.

The fermentable carbohydrate composition of wort and the manner in which it is utilized by yeast during brewery fermentation have a direct influence on fermentation efficiency and quality of the final product. In this study the response of a brewing yeast strain to changes in wort fermentable carbohydrate concentration and composition during full-scale (3275 hl) brewery fermentation was investigated by measuring transcriptome changes with the aid of oligonucleotide-based DNA arrays. Up to 74% of the detectable genes showed a significant (p

Commercial brewing yeast strains are exposed to a number of potential stresses including oxidative stress. The aim of this investigation was to measure the physiological and transcriptional changes of yeast cells during full-scale industrial brewing processes with a view to determining the environmental factors influencing the cell's oxidative stress response. Cellular antioxidant levels and genome-wide transcriptional changes were monitored throughout an industrial propagation and fermentation. The greatest increase in cellular antioxidants and transcription of antioxidant-encoding genes occurred as the rapidly fermentable sugars glucose and fructose were depleted from the growth medium (wort) and the cell population entered the stationary phase. The data suggest that, contrary to expectation, the oxidative stress response is not influenced by changes in the dissolved oxygen concentration of wort but is initiated as part of a general stress response to growth-limiting conditions, even in the absence of oxygen. A mechanism is proposed to explain the changes in antioxidant response observed in yeast during anaerobic fermentation. The available data suggest that the yeast cell does not experience oxidative stress during industrial brewery handling. This information may be taken into consideration when setting parameters for industrial brewery fermentation.

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.

Recent studies in the model plant Arabidopsis provide new insight into the regulation of root architecture, a key determinant of nutrient- and water-use efficiency in crops. Lateral root (LR) primordia originate from a subset of pericycle founder cells. Sophisticated mass-spectroscopy-based techniques have been used to map the sites of biosynthesis of auxin and its distribution in Arabidopsis seedlings, highlighting the importance of the phytohormone during LR initiation and emergence. Key components of the cell cycle and signal-transduction pathway(s) that promote and attenuate auxin-dependent LR initiation have recently been identified. Additional signals, such as abscisic acid and nitrate, also regulate LR emergence, raising intriguing questions about the cross-talk between their transduction pathways.

Plants sense and respond to endogenous signals and environmental cues to ensure optimal growth and development. Plant cells must integrate the myriad transduction events into a comprehensive network of signalling pathways and responses. The phytohormone auxin occupies a central place within this transduction network, frequently acting in conjunction with other signals, to co-ordinately regulate cellular processes such as division, elongation and differentiation. As a non-cell autonomous signal, auxin also interacts with other signalling pathways to regulate inter-cellular developmental processes. As part of this especially themed edition of Plant Molecular Biology, we will review examples of 'cross-talk' between auxin and other signalling pathways. Given the current state of knowledge, we have deliberately focused our efforts reviewing auxin interactions with other phytohormone and light signalling pathways. We conclude by discussing how new genomic approaches and the Arabidopsis genome sequence are likely to impact this area of research in the future.

Introduction Non-traumatic thunderclap headache (TCH, a sudden-onset severe headache) is a concerning presenting symptom as it may indicate serious pathologies including subarachnoid haemorrhage (SAH). Secondary causes must be comprehensively excluded before primary headache is diagnosed. Objectives To assess the management of TCH against a standard of acute neuroimaging (CT/MRI) followed by lumbar puncture (LP) where this is negative, with referral for further investigation if initial tests are non-diagnostic. Methods Retrospective audit of electronic records of patients aged >15 years presenting with headache over one year at a District General Hospital, with casenote review to identify true TCH. Results 58 probable cases of TCH were identified from 444 attendances coded ‘headache’ over one year, with acute neuroimaging in 145 cases showing four SAHs, six infarcts, five tumours, a cyst and one hydrocephalus. LP was performed in 20% of cases with non-diagnostic imaging, identifying two SAHs, two bacterial and one viral meningitis. No evidence of onward referral or further imaging after non-diagnostic LP was found. Conclusions Emergency department doctors appear to employ an appropriately low threshold for neuroimaging in TCH, however there is little evidence that LPs are sufficiently performed where imaging is negative, or that further investigation is arranged following negative LP.

Lateral root development in Arabidopsis provides a model for the study of hormonal signals that regulate postembryonic organogenesis in higher plants. Lateral roots originate from pairs of pericycle cells, in several cell files positioned opposite the xylem pole, that initiate a series of asymmetric, transverse divisions. The auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) arrests lateral root development by blocking the first transverse division(s). We investigated the basis of NPA action by using a cell-specific reporter to demonstrate that xylem pole pericycle cells retain their identity in the presence of the auxin transport inhibitor. However, NPA causes indoleacetic acid (IAA) to accumulate in the root apex while reducing levels in basal tissues critical for lateral root initiation. This pattern of IAA redistribution is consistent with NPA blocking basipetal IAA movement from the root tip. Characterization of lateral root development in the shoot meristemless1 mutant demonstrates that root basipetal and leaf acropetal auxin transport activities are required during the initiation and emergence phases, respectively, of lateral root development.

We describe various types of outliers seen in Affymetrix GeneChip data. We have been able to utilise the data in the Gene Expression Omnibus to screen GeneChips across a range of scales, from single probes, to spatially adjacent fractions of arrays, to whole arrays, to whole experiments. In this review we describe a number of causes for why some reported intensities might be misleading on GeneChips.

International audience; Lateral roots originate deep within the parental root from a small number of founder cells at the periphery of vascular tissues and must emerge through intervening layers of tissues. We describe how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells. Auxin induces the expression of a previously uncharacterized auxin influx carrier LAX3 in cortical and epidermal cells directly overlaying new primordia. Increased LAX3 activity reinforces the auxin-dependent induction of a selection of cell-wall-remodelling enzymes, which are likely to promote cell separation in advance of developing lateral root primordia.

Background Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe-set, consisting of up to 16 probe-pairs. Signal intensities across probe-pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.

Calcium (Ca) and magnesium (Mg) are essential plant nutrients and vital for human and animal nutrition. Biofortification of crops has previously been suggested to alleviate widespread human Ca and Mg deficiencies. In this study, new candidate genes influencing the leaf accumulation of Ca and Mg were identified in young Brassica napus plants using associative transcriptomics of bionomics datasets. A total of 247 and 166 SNP markers were associated with leaf Ca and Mg concentration, respectively, after false discovery rate correction and removal of SNPs with low second allele frequency. Gene expression markers at similar positions were also associated with leaf Ca and Mg concentration, including loci on chromosomes A10 and C2, within which lie previously identified transporter genes ACA8 and MGT7. Further candidate genes were selected from seven loci and the mineral composition of whole Arabidopsis thaliana shoots were characterized from lines mutated in orthologous genes. Four and two mutant lines had reduced shoot Ca and Mg concentration, respectively, compared to wild type plants. Three of these mutations were found to have tissue specific effects; notably reduced silique Ca in all three such mutant lines. This knowledge could be applied in targeted breeding, with the possibility of increasing Ca and Mg in plant tissue for improving human and livestock nutrition.