BACKGROUND AND AIMS:ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focuses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana.METHODS:We isolated homozygous knockout individual mutants (pfk1, pfk3, pfk6 and pfk7) and two double mutants (pfk1/7 and pfk3/6), and characterized their growth and metabolic phenotypes.KEY RESULTS:In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and showed a clear visual and metabolic phenotype with reduced shoot growth, early flowering and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates.CONCLUSIONS:We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently in source-sink relationships in A. thaliana, while PFK3 and PFK6 only play a minor role under normal growth conditions.

Twenty-four-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the "phospho-dawn." Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1-overexpressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1-overexpression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating posttranslational mechanisms in the transmission of circadian timing information in plants.

Abstract Background Trastuzumab and anthracyclines are conventional chemotherapies used in breast cancer. Unfortunately, they are associated with a decrease in left ventricular function potentially leading to heart failure (HF). In order to prevent this, randomised controlled trials (RCTs) assess the preventive effect of concomitant beta-blocker (BB), angiotensin receptor blocker (ARB) and angiotensin converting enzyme inhibitor (ACEI) therapy during chemotherapy. Purpose To assess the preventive effect of BB, ARB or ACEIs on left ventricular ejection fraction (LVEF) during trastuzumab and anthracycline treatment in patients without HF. Methods Our primary outcomes were the effect of BBs or ARB/ACEIs during 1) trastuzumab and 2) anthracycline treatment. Secondary outcomes were the distinct effects of 1) BBs and 2) ARB/ACEIs in either trastuzumab or anthracycline treatments. Through the search term “(RCTs), prevention, cancer chemotherapy and cardiotoxicity” in PubMed, studies were selected, excluding those without randomising to a BB, ARB/ACEI and a placebo control group during chemotherapy. Means of the LVEF and the standard deviation (SD) post-chemotherapy were applied. Meta-analyses estimated the standardised mean difference (SMD) in the LVEF. Heterogeneity was calculated as the I2. Results A total of 7 studies (Table 1) were included in the analysis. Between 93 and 100% were woman. Age varied from 41 to 51 years. Treatment time varied from 12 to 52 weeks. Concomitant BB or ARB/ACEI therapy during trastuzumab treatment was not associated with the LVEF, significantly (Fig. 1A; p=0.07). Oppositely, in the anthracycline regime the LVEF remained significant higher in the concomitant BB and ARB/ACEI groups as compared to controls (Fig. 1B). BB and ARB/ACEI separation in the analysis showed both to influence the LVEF positively independent of chemotherapy (P=0.03 & p=0.005). Table 1 Study reference Year Chemotherapies Preventive drugs Pituskin et al., “Multidisciplinary Approach to Novel Therapies in Cardio-Oncology Research (MANTICORE 101-Breast): A Randomized Trial for the Prevention of Trastuzumab-Associated Cardiotoxicity.” 2017 Trastuzumab Perindopril. bisoprolol Gulati et al., “Prevention of Cardiac Dysfunction during Adjuvant Breast Cancer Therapy (PRADA).” 2016 Trastuzumab Candesartan, metoprolol Boekhout et al., “Angiotensin II-Receptor Inhibition With Candesartan to Prevent Trastuzumab-Related Cardiotoxic Effects in Patients With Early Breast Cancer: A Randomized Clinical Trial.” 2016 Trastuzumab Candesartan Janbabai et al., “Effect of Enalapril on Preventing Anthracycline-Induced Cardiomyopathy.” 2017 Anthracycline Enalapril Nabati et al., “Cardioprotective Effects of Carvedilol in Inhibiting Doxorubicin-Induced Cardiotoxicity.” 2017 Anthracycline Carvedilol Tashakori Beheshti et al., “Carvedilol Administration Can Prevent Doxorubicin-Induced Cardiotoxicity: A Double-Blind Randomized Trial.” 2016 Anthracycline Carvedilol Kaya et al., “Protective Effects of Nebivolol against Anthracycline-Induced Cardiomyopathy: A Randomized Control Study.” 2013 Anthracycline Nebivolol Figure 1 Conclusions Concomitant BB and ARB/ACEI therapy both favoured maintenance of the LVEF during trastuzumab and anthracyclines regimens as compared to controls.

A Levanase, LevB, from Bacillus subtilis 168, was expressed as a His6-tagged protein in Escherichia coli. The enzyme was purified and characterised for its activity and substrate specificity. LevB has a pH optimum of 6.0-6.5 and a maximum observed specific activity of 3Umg−1 using levan from Erwinia herbicola as substrate. Hydrolysis products were analysed by HPAEC, TLC, and NMR using chicory root inulin, mixed linkage fructans purified from ryegrass (Lolium perenne) and levan from E. herbicola as substrates. This revealed that LevB is an endolevanase that selectively cleaves the (β-2,6) fructosyl bonds and does not hydrolyse inulin. Ryegrass fructans and bacterial levan was hydrolysed partially releasing oligosaccharides, but together with exoinulinase, LevB hydrolysed both ryegrass fructans and bacterial levan to near completion. We suggest that LevB can be used as a tool to achieve more structural information on complex fructans and to achieve complete degradation and quantification of mixed linkage fructans.

Arbuscular mycorrhizal fungi (AMF) have a key role in plant phosphate (Pi) uptake by their efficient capture of soil phosphorus (P) that is transferred to the plant via Pi transporters in the root cortical cells. The activity of this mycorrhizal Pi uptake pathway is often associated with downregulation of Pi transporter genes in the direct Pi uptake pathway. As the total Pi taken up by the plant is determined by the combined activity of mycorrhizal and direct pathways, it is important to understand the interplay between these, in particular the actual activity of the pathways. To study this interplay we modulated the delivery of Pi via the mycorrhizal pathway in Pisum sativum by two means: (1) Partial downregulation by virus-induced gene silencing of PsPT4, a putative Pi transporter gene in the mycorrhizal pathway. This resulted in decreased fungal development in roots and soil and led to reduced plant Pi uptake. (2) Changing the percentage of AMF-colonized root length by using non-, half-mycorrhizal or full-mycorrhizal split-root systems. The combination of split roots, use of ³²P and ³³P isotopes and partial silencing of PsPT4 enabled us to show that the expression of PsPT1, a putative Pi transporter gene in the direct pathway, was negatively correlated with increasing mycorrhizal uptake capacity of the plant, both locally and systemically. However, transcript changes in PsPT1 were not translated into corresponding, systemic changes in actual direct Pi uptake. Our results suggest that AMF have a limited long-distance impact on the direct pathway.

The transcription factor PHR1 (PHOSPHATE STARVATION RESPONSE 1; encoded by gene At4g28610) is central for adaptation to phosphate deficiency in Arabidopsis (Arabidopsis thaliana). A rapid turnover of phosphate pools in the leaves is essential for energy transfer and metabolism within photosynthesis, and consequently, we hypothesized that PHR1 is needed for adaptation to high-light stress during P deficiency. We analyzed three Arabidopsis plant lines: wild-type, a transgenic PHR1 overexpressor line and a knockout mutant, phr1. The plants were grown under phosphate-limiting and sufficient conditions and exposed to different light conditions. Photosynthetic activity and light stress of the leaves were characterized by analyzing accumulation of carbohydrates, chlorophyll fluorescence, immunoblot detection of photosystem subunits and anthocyanin accumulation. Compared to the wild-type and the overexpressor line, the phr1 mutant has decreased levels of phosphate, anthocyanins and carbohydrates during combined P deficiency and light stress. The stressed mutant also has strongly decreased photosystem II (PSII) quantum efficiency, and shows degradation of the core units of PSII demonstrating extensive irreversible photodamage. We conclude that PHR1 is needed for the metabolic balance, for retaining P(i) levels and for inducing anthocyanin production, and during P deficiency PHR1 is vital for adaptations to avoid permanent damage to photosystems during high-light conditions.

A proper concentration and turnover of inorganic phosphate (Pi) is essential to maintain cellular processes. Consequently, plants have mechanisms to control Pi homeostasis and to alleviate Pi limitation. The MYB-related transcription factor, PHR1, is important for gene induction during Pi starvation. PHR1 belongs to a family, characterised by the presence of a GARP- and a coiled coil domain. We propose that this family, with 15 members in Arabidopsis thaliana (L.) Heynh., be termed the GCC-family. In this study, transgenic plants overexpressing one member, GCC7, and a T-DNA knockout mutant, gcc7, are characterised. We find overexpressor plants to accumulate more Pi in shoots, irrespective of the Pi supply. Therefore, GCC7 was characterised in relation to Pi starvation. We conclude that GCC7 is not strictly required for a P-starvation response since the gcc7 mutant responds to Pi limitation. However, overexpression of GCC7 strongly interferes with the P-starvation response, abolishing induction of several P-responsive genes including AT4, IPS1 and several P-transporter genes, whereas GCC7 does not directly interfere with the PHR1 (GCC1) dependent regulation of miR399d. Thus GCC7 influences P-accumulation and P-dependent gene regulation, but GCC7 has a function entirely different from PHR1.

BACKGROUND:Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.RESULTS:Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component.CONCLUSIONS:Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.

MicroRNAs (miRNAs) are short RNA chains (20-24 bp) which are emerging as important regulators of gene expression. miRNAs are encoded by specific genes, and in Arabidopsis, 190 genes have presently been identified. It has been shown that miR399 is essential for the phosphate starvation response, and recent studies have shown transcriptional changes in a number of additional miRNAs in response to a shortage of phosphate. In this study, global profiles of the miRNA in shoots of Arabidopsis plants grown on limited phosphate or full nutrient in combination with sucrose feed were analysed using the miRCURY LNA microRNA Array system. Furthermore, changes in miRNA transcript were compared between a mutant lacking the transcription factor phosphate starvation responses 1 (PHR1) and wild-type plants. The global analysis identified miRNAs belonging to nine families to respond to P deprivation, sucrose or PHR1. Among these, miR399d, miR827, miR866, miR391 and miR163 were most prominently induced upon P starvation, whereas miR169b/c was strongly induced in previously starved plants when provided with sufficient P and more so when combined with an addition of sucrose. This study shows that array analysis is in general agreement with data obtained by other high-throughput technologies. The array data were confirmed by real-time reverse transcriptase-polymerase chain reaction analyses of selected pri-miRNAs. Our data corroborate the implication that several miRNAs are involved in the P-starvation response and further identify miR866 and miR163 as new candidates of miRNAs associated with the regulation of the P-starvation response.

Inorganic phosphate (Pi) is an essential nutrient for plants, and the low bioavailability of Pi in soils is often a limitation to growth and development. Consequently, plants have evolved a range of regulatory mechanisms to adapt to phosphorus-starvation in order to optimise uptake and assimilation of Pi. Recently, significant progress has been made in elucidating these mechanisms. The coordinated expression of a large number of genes is important for many of these adaptations. Several global expression studies using microarray analysis have been conducted in Arabidopsis thaliana. These studies provide a valuable basis for the identification of new regulatory genes and promoter elements to further the understanding of Pi-dependent gene regulation. With focus on the Arabidopsis transcriptome, we extract common findings that point to new groups of putative regulators, including the NAC, MYB, ethylene response factor/APETALA2, zinc-finger, WRKY and CCAAT-binding families. With a number of new discoveries of regulatory elements, a complex regulatory network is emerging. Some regulatory elements, e.g. the transcription factor PHR1 and the microRNA (miRNA) miR399 and associated factors are well documented, yet not fully understood, whereas other suggested components need further characterisation. Here, we evaluate the contribution of the regulatory elements to the P-responses and present a model comprising factors directly or indirectly involved in transcriptional regulation and the role of miRNAs as regulators and long-distance signals. A striking feature is a series of feedback loops and parallel mechanisms that can modify and attenuate responses. We suggest that these mechanisms are instrumental in providing an accurate response and in keeping P-homeostasis.

This article was submitted without an abstract, please refer to the full-text PDF file.

Onions are excellent sources of bioactive compounds including fructo-oligosaccharides (FOS) and polyphenols. An onion by-product was characterised in order to be developed as a potentially bioactive food ingredient. Our main aim was to investigate whether the potential health and safety effects of this onion by-product were shared by either of two derived fractions, an extract containing the onion FOS and polyphenols and a residue fraction containing mainly cell wall materials. We report here on the effects of feeding these products on markers of potential toxicity, protective enzymes and gut environment in healthy rats. Rats were fed during 4 weeks with a diet containing the products or a control feed balanced in carbohydrate. The onion by-product and the extract caused anaemia as expected in rodents for Allium products. No other toxicity was observed, including genotoxicity. Glutathione reductase (GR) and glutathione peroxidase (GPx1) activities in erythrocytes increased when rats were fed with the onion extract. Hepatic gene expression of Gr, Gpx1, catalase, 5-aminolevulinate synthase and NAD(P)H:quinone oxidoreductase was not altered in any group of the onion fed rats. By contrast, γ-glutamate cysteine ligase catalytic subunit gene expression was upregulated but only in rats given the onion residue. The onion by-products as well as the soluble and insoluble fractions had prebiotic effects as evidenced by decreased pH, increased butyrate production and altered gut microbiota enzyme activities. In conclusion, the onion by-products have no in vivo genotoxicity, may support in vivo antioxidative defence and alter the functionality of the rat gut microbiota.

Global gene expression was analyzed in Arabidopsis (Arabidopsis thaliana) by microarrays comprising 21,500 genes. Leaf segments derived from phosphorus (P)-starved and P-replenished plants were incubated with or without sucrose (Suc) to obtain tissues with contrasting combinations of P and carbohydrate levels. Transcript profiling revealed the influence of the two factors individually and the interactions between P- and sugar-dependent gene regulation. A large number of gene transcripts changed more than 2-fold: In response to P starvation, 171 genes were induced and 16 repressed, whereas Suc incubation resulted in 337 induced and 307 repressed genes. A number of new candidate genes involved in P acquisition were discovered. In addition, several putative transcription factors and signaling proteins of P sensing were disclosed. Several genes previously identified to be sugar responsive were also regulated by P starvation and known P-responsive genes were sugar inducible. Nearly 150 genes were synergistically or antagonistically regulated by the two factors. These genes exhibit more prominent or contrasting regulation in response to Suc and P in combination than expected from the effect of the two factors individually. The genes exhibiting interactions form three main clusters with different response patterns and functionality of genes. One cluster (cluster 1) most likely represents a regulatory program to support increased growth and development when both P and carbohydrates are ample. Another cluster (cluster 3) represents genes induced to alleviate P starvation and these are further induced by carbohydrate accumulation. Thus, interactions between P and Suc reveal two different signaling programs and novel interactions in gene regulation in response to environmental factors. cis-Regulatory elements were analyzed for each factor and for interaction clusters. PHR1 binding sites were more frequent in promoters of P- regulated genes as compared to the entire Arabidopsis genome, and E2F and PHR1 binding sites were more frequent in interaction clusters 1 and 3, respectively.

Plants have evolved a number of adaptive strategies to cope with fluctuations in phosphorus (P) supply. The current knowledge of the transcriptional regulation of the P-starvation response in plants is limited. However, one MYB-related transcription factor, PHR1, is known to be involved in the P-starvation response. In this paper, we characterize a T-tagged phr1 knockout mutant and a series of transgenic plant lines which over-express PHR1 in wild type (WT) and phr1 mutant background. The knockout mutant has an altered phosphate (P-i) allocation between root and shoot; accumulates less anthocyanins, sugars and starch than P-starved WT; has a lower AGPase activity; and is impaired in induction of a subset of P-i starvation-induced genes. Expression of PHR1 in the phr1 mutant rescues the responsiveness to P-starvation and leads to WT levels of sugars and starch during P-i starvation conditions, confirming the involvement of PHR1 in adjusting carbon metabolism. Over-expression of PHR1 further resulted in a dramatic increase in the microRNA miR399d, and this resulted in changes in the transcript level for the target gene PHO2. Furthermore, over-expression of PHR1 in both WT and phr1 mutant results in strongly increased content of P-i irrespective of P regime. This shows that targeting a key regulatory element in the P-i starvation regulatory network represents a useful approach for molecular breeding of plants towards more efficient P-i uptake and assimilation.

Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, DK–1871 Frederiksberg C, Denmark (R.M., M.M., L.N., T.H.N.); Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Kemitorvet, DK–2800 Lyngby, Denmark (H.J.); and Department of Agricultural Sciences, Crop Science, Royal Veterinary and Agricultural University, 2630 Taastrup, Denmark (R.M.)

Expression of selected genes in relation to phosphate (Pi) starvation and sugar sensing was studied in leaves of Arabidopsis. Excised leaf segments with different P status were supplied with combinations of Pi and sugars. Sugar‐inducible genes, encoding β‐amylase (β‐AMY) and chalcone synthase (CHS), were also induced by P deficiency, and were more strongly regulated by sugars when leaf segments originated from P‐starved plants. Furthermore, transcript levels of the P‐starvation‐inducible genes ACP5 (encoding an acid phosphatase), RNS1 (encoding a ribonuclease), and IPS1 (unknown function) increased in response to exogenously applied sugars. Supply of Pi to the leaf segments reversed both P‐starvation‐induced and sugar‐induced gene expression. These interactions reveal a close relationship between P and sugar sensing. To differentiate between hexokinase‐dependent and hexokinase‐independent sugar sensing the effect of the glucose analogue 2‐deoxyglucose and gene expression in the hexokinase‐1 deficient mutant, gin2‐1, were studied. Both β‐AMY and CHS were induced by supplying sucrose to excised leaves but not by 2‐deoxyglucose, confirming that these genes are regulated by hexokinase‐independent sugar sensing. In the gin2‐1 mutant both β‐AMY and CHS responded clearly to P starvation excluding that hexokinase‐1 mediates the response to P. Similarly, the P‐responding genes, IPS1 and RNS1 were repressed by addition of Pi also in the gin2‐1 mutant. In conclusion, several phosphate starvation‐induced genes are also sugar‐induced and hexokinase‐independent sugar sensing in Arabidopsis is strongly intensified by phosphate starvation.

Carbohydrate metabolism was investigated in barley leaves subjected to drought or osmotic stress induced by sorbitol incubation. Both drought and osmotic stress resulted in accumulation of hexoses, depletion of sucrose and starch, and 5 - 10-fold increase in the level of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P-2). These changes were paralleled by an increased activity ratio of fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase (F2KP). The drought-induced changes in carbohydrate content and Fru-2,6-P2 metabolism were reversed upon re-watering. This reveals a reversible mechanism for modi. cation of the F2KP enzyme activity. This suggests that F2KP might be involved in altering carbohydrate metabolism during osmotic stress. However, labelling with [C-14]-CO2 showed that sucrose synthesis was not inhibited, despite the increased Fru-2,6-P-2 levels, and demonstrated that increased flux into the hexose pools probably derived from sucrose hydrolysis. Similar effects of osmotic stress were observed in leaf sections incubated in the dark, showing that the changes did not result from altered rates of photosynthesis. Metabolism of [C-14]- sucrose in the dark also revealed increased flux into hexoses and reduced flux into starch in response to osmotic stress. The activities of a range of enzymes catalysing reactions of carbohydrate metabolism in general showed only a marginal decrease during osmotic stress. Therefore, the observed changes in metabolic flux do not rely on a change in the activity of the analysed enzymes. Fructose-2,6-bisphosphate metabolism responds strongly to drought stress and this response involves modi. cation of the F2KP activity. However, the data also suggests that the sugar accumulation observed during osmotic stress is mainly regulated by another, as yet unidentified mechanism.

ABSTRACT Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.

Expression of phosphate starvation inducible genes was studied during recovery from phosphate starvation of Arabidopsis thaliana. Genes analysed were ACP5 (encoding an acid phosphatase), RNS1 (encoding an RNase), At4 and IPS1 (both of unknown function), Pht1;7 and Pht2;1 (encoding phosphate transporters). Plants grown at limiting phosphate exhibited reduced growth rate and accumulated anthocyanins, soluble sugars, and starch. Re‐supply of phosphate resulted in dramatic uptake of phosphate, increased growth rate and decreased levels of anthocyanins, soluble sugars, and starch in leaf tissue. In both shoots and roots re‐supplied with Pi, transcript levels decreased rapidly, and first changes were observed within 30 min. These alterations in gene expression occurred before the content of carbohydrates decreased, indicating that transcriptional regulation was due to phosphate sensing, and not a secondary effect of carbon accumulation. The data reveal different response rates for individual genes and demonstrate that roots and shoots can differ with respect to both timing and genes responding. In general, the changes in transcriptional activity in roots preceded the changes in shoots. Furthermore, transcriptional regulation was observed in isolated roots and shoots. This implies that roots do not strictly require a signal from the shoot, and vice versa, in order to respond to phosphate starvation.

Complex microbial communities may serve as ideal and ecologically relevant toxicity indicators. We here report an assessment of frequently used methods in microbial ecology for their feasibility to detect toxic effects of the environmentally important surfactant linear alkylbenzene sulfonate (LAS) on microbial communities in lake water and treated waste water. The two microbial communities were evaluated for changes in community structure and function over a period of 7 weeks in replicated aquatic microcosms amended with various levels of LAS (0, 0.1, 1, 10 or 100 mgl−1) and inorganic nutrients. In general, the two communities behaved similarly when challenged with LAS. Following lag periods of 1–3 weeks, LAS was degraded to non-toxic substances. Denaturing gradient gel electrophoresis of 16S rRNA gene fragments and [3H]leucine incorporation were the most sensitive assays with effect levels of 0–1 and 1–10 mg LAS l−1, respectively. Community-level physiological profiles and pollution-induced community tolerance determinations using Biolog microplates demonstrated less sensitivity with effect levels of 10–100 mg LAS l−1. Total cell counts and net uptake of inorganic N and P were unaffected even at 100 mg LAS l−1. Interestingly, different microbial communities developed in some replicate microcosms, indicating the importance of stochastic events for community succession. We conclude that microbial community-level toxicity testing holds great promise and suggest a polyphasic approach involving a range of independent methods targeting both the structure and function of the tested microbial communities.