Patrick Armengaud

Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments ☆

One of the main objectives in the analysis of microarray experiments is the identification of genes that are differentially expressed under two experimental conditions. This task is complicated by the noisiness of the data and the large number of genes that are examined simultaneously. Here, we present a novel technique for identifying differentially expressed genes that does not originate from a sophisticated statistical model but rather from an analysis of biological reasoning. The new technique, which is based on calculating rank products (RP) from replicate experiments, is fast and simple. At the same time, it provides a straightforward and statistically stringent way to determine the significance level for each gene and allows for the flexible control of the false‐detection rate and familywise error rate in the multiple testing situation of a microarray experiment. We use the RP technique on three biological data sets and show that in each case it performs more reliably and consistently than the non‐parametric t‐test variant implemented in Tusher et al.s significance analysis of microarrays (SAM). We also show that the RP results are reliable in highly noisy data. An analysis of the physiological function of the identified genes indicates that the RP approach is powerful for identifying biologically relevant expression changes. In addition, using RP can lead to a sharp reduction in the number of replicate experiments needed to obtain reproducible results.

The Potassium-Dependent Transcriptome of Arabidopsis Reveals a Prominent Role of Jasmonic Acid in Nutrient Signaling

Full genome microarrays were used to assess transcriptional responses of Arabidopsis seedlings to changing external supply of the essential macronutrient potassium (K+). Rank product statistics and iterative group analysis were employed to identify differentially regulated genes and statistically significant coregulated sets of functionally related genes. The most prominent response was found for genes linked to the phytohormone jasmonic acid (JA). Transcript levels for the JA biosynthetic enzymes lipoxygenase, allene oxide synthase, and allene oxide cyclase were strongly increased during K+ starvation and quickly decreased after K+ resupply. A large number of well-known JA responsive genes showed the same expression profile, including genes involved in storage of amino acids (VSP), glucosinolate production (CYP79), polyamine biosynthesis (ADC2), and defense (PDF1.2). Our findings highlight a novel role of JA in nutrient signaling and stress management through a variety of physiological processes such as nutrient storage, recycling, and reallocation. Other highly significant K+-responsive genes discovered in our study encoded cell wall proteins (e.g. extensins and arabinogalactans) and ion transporters (e.g. the high-affinity K+ transporter HAK5 and the nitrate transporter NRT2.1) as well as proteins with a putative role in Ca2+ signaling (e.g. calmodulins). On the basis of our results, we propose candidate genes involved in K+ perception and signaling as well as a network of molecular processes underlying plant adaptation to K+ deficiency.

The effect of potassium nutrition on pest and disease resistance in plants

Providing a fast growing world population with sufficient food while preserving ecological and energy resources of our planet is one of the biggest challenges in this century. Optimized management of chemical fertilizers and pesticides will be essential for achieving sustainability of intensive farming and requires both empirical data from field trials and advanced fundamental understanding of the molecular processes controlling plant growth. Genes involved in plant responses to nutrient deficiency and pathogen/herbivore attack have been identified, but we are lacking information about the cross-talk between signalling pathways when plants are exposed to a combination of abiotic and biotic stress factors. The focus of this review is on the relationship between the potassium status of plants and their susceptibility to pathogens and herbivorous insects. We combine field evidence on potassium-disease interaction with existing knowledge on metabolic and physiological factors that could explain such interaction, and present new data on metabolite profiles and hormonal pathways from the model plant Arabidopsis thaliana. The latter provides evidence that facilitated entry and development of pathogens or insects in(to) potassium-deficient plants as a result of physical and metabolic changes is counteracted by an increased defence. A genetic approach should now be applied to establish a causal relationship between disease susceptibility on the one hand and individual enzymatic and signal components on the other. Once identified, these can be used to design agricultural strategies that support the nutritional status of the crops while exploiting their inherent potential for defence.

Multilevel Analysis of Primary Metabolism Provides New Insights into the Role of Potassium Nutrition for Glycolysis and Nitrogen Assimilation in Arabidopsis Roots

Potassium (K) is required in large quantities by growing crops, but faced with high fertilizer prices, farmers often neglect K application in favor of nitrogen and phosphorus. As a result, large areas of farmland are now depleted of K. K deficiency affects the metabolite content of crops with negative consequences for nutritional quality, mechanical stability, and pathogen/pest resistance. Known functions of K in solute transport, protein synthesis, and enzyme activation point to a close relationship between K and metabolism, but it is unclear which of these are the most critical ones and should be targeted in biotechnological efforts to improve K usage efficiency. To identify metabolic targets and signaling components of K stress, we adopted a multilevel approach combining transcript profiles with enzyme activities and metabolite profiles of Arabidopsis (Arabidopsis thaliana) plants subjected to low K and K resupply. Roots and shoots were analyzed separately. Our results show that regulation of enzymes at the level of transcripts and proteins is likely to play an important role in plant adaptation to K deficiency by (1) maintaining carbon flux into amino acids and proteins, (2) decreasing negative metabolic charge, and (3) increasing the nitrogen-carbon ratio in amino acids. However, changes in transcripts and enzyme activities do not explain the strong and reversible depletion of pyruvate and accumulation of sugars observed in the roots of low-K plants. We propose that the primary cause of metabolic disorders in low-K plants resides in the direct inhibition of pyruvate kinase activity by low cytoplasmic K in root cells.

EZ-Rhizo: integrated software for the fast and accurate measurement of root system architecture

The root system is essential for the growth and development of plants. In addition to anchoring the plant in the ground, it is the site of uptake of water and minerals from the soil. Plant root systems show an astonishing plasticity in their architecture, which allows for optimal exploitation of diverse soil structures and conditions. The signalling pathways that enable plants to sense and respond to changes in soil conditions, in particular nutrient supply, are a topic of intensive research, and root system architecture (RSA) is an important and obvious phenotypic output. At present, the quantitative description of RSA is labour intensive and time consuming, even using the currently available software, and the lack of a fast RSA measuring tool hampers forward and quantitative genetics studies. Here, we describe EZ-Rhizo: a Windows-integrated and semi-automated computer program designed to detect and quantify multiple RSA parameters from plants growing on a solid support medium. The method is non-invasive, enabling the user to follow RSA development over time. We have successfully applied EZ-Rhizo to evaluate natural variation in RSA across 23 Arabidopsis thaliana accessions, and have identified new RSA determinants as a basis for future quantitative trait locus (QTL) analysis.

Effects of N,P,K and S on metabolism: new knowledge gained from multi-level analysis

Deficiency of mineral nutrients such as nitrate, phosphate, potassium and sulphate strongly affects the type and amount of metabolites produced by crops with knock-on effects on nutritional quality of the crop, its processing properties and disease resistance. Owing to the multitude of biochemical reactions underlying metabolism and the high degree of connectivity between biochemical pathways, predicting the output of the metabolic network in response to a change in nutrient input is an enormous challenge. Recently several studies have taken a systemic approach monitoring the response of plants to withdrawal and/or re-supply of mineral nutrients at the level of transcripts, metabolites and enzyme activities. These multi-level studies have provided important new insight into how plants re-prioritise different metabolic pathways during nutrient shortage and how they integrate metabolism with growth. On the basis of the obtained information we can formulate specific hypotheses about the causal relationships between changes in individual transcripts, proteins and metabolites.

Adjustment of growth and central metabolism to a mild but sustained nitrogen-limitation in Arabidopsis

We have established a simple soil-based experimental system that allows a small and sustained restriction of growth of Arabidopsis by low nitrogen (N). Plants were grown in a large volume of a peat-vermiculite mix that contained very low levels of inorganic N. As a control, inorganic N was added in solid form to the peat-vermiculite mix, or plants were grown in conventional nutrient-rich solids. The low N growth regime led to a sustained 20% decrease of the relative growth rate over a period of 2 weeks, resulting in a two- to threefold decrease in biomass in 35- to 40-day-old plants. Plants in the low N regime contained lower levels of nitrate, lower nitrate reductase activity, lower levels of malate, fumarate and other organic acids and slightly higher levels of starch, as expected from published studies of N-limited plants. However, their rosette protein content was unaltered, and total and many individual amino acid levels increased compared with N-replete plants. This metabolic phenotype reveals that Arabidopsis responds adaptively to low N by decreasing the rate of growth, while maintaining the overall protein content, and maintaining or even increasing the levels of many amino acids.

Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus

Abstract Potassium and phosphorus are important macronutrients for crops but are often deficient in the field. Very little is known about how plants sense fluctuations in K and P and how information about K and P availability is integrated at the whole plant level into physiological and metabolic adaptations. This chapter reviews recent advances in discovering molecular responses of plants to K and P deficiency by microarray experiments. These studies provide us not only with a comprehensive picture of adaptive mechanisms, but also with a large number of transcriptional markers that can be used to identify upstream components of K and P signalling pathways. On the basis of the available information we discuss putative receptors and signals involved in the sensing and integration of K and P status both at the cellular and at the whole plant level. These involve membrane potential, voltage‐dependent ion channels, intracellular Ca and pH, and transcription factors, as well as hormones and metabolites for systemic signalling. Genetic screens of reporter lines for transcriptional markers and metabolome analysis of K- and P-deficient plants are likely to further advance our knowledge in this area in the near future.

Analysis of the Root System Architecture of Arabidopsis Provides a Quantitative Readout of Crosstalk between Nutritional Signals

Root architectures of Arabidopsis thaliana were quantified in multiple combinations of N, P, K, S, and light to understand how plants integrate multiple nutritional signals. Combined with mutant phenotypes, transcriptional profiles, and shoot ion contents, the comprehensive data set facilitates the dissection of the nutrient signaling network and identifies roles for receptor-transporters. As plant roots forage the soil for food and water, they translate a multifactorial input of environmental stimuli into a multifactorial developmental output that manifests itself as root system architecture (RSA). Our current understanding of the underlying regulatory network is limited because root responses have traditionally been studied separately for individual nutrient deficiencies. In this study, we quantified 13 RSA parameters of Arabidopsis thaliana in 32 binary combinations of N, P, K, S, and light. Analysis of variance showed that each RSA parameter was determined by a typical pattern of environmental signals and their interactions. P caused the most important single-nutrient effects, while N-effects were strongly light dependent. Effects of K and S occurred mostly through nutrient interactions in paired or multiple combinations. Several RSA parameters were selected for further analysis through mutant phenotyping, which revealed combinations of transporters, receptors, and kinases acting as signaling modules in K–N interactions. Furthermore, nutrient response profiles of individual RSA features across NPK combinations could be assigned to transcriptionally coregulated clusters of nutrient-responsive genes in the roots and to ionome patterns in the shoots. The obtained data set provides a quantitative basis for understanding how plants integrate multiple nutritional stimuli into complex developmental programs.

Potassium deficiency induces the biosynthesis of oxylipins and glucosinolates in Arabidopsis thaliana

BackgroundMineral fertilization and pest control are essential and costly requirements for modern crop production. The two measures go hand in hand because plant mineral status affects plant susceptibility to pests and vice versa. Nutrient deficiency triggers specific responses in plants that optimize nutrient acquisition and reprogram metabolism. K-deficient plants illustrate these strategies by inducing high-affinity K-uptake and adjusting primary metabolism. Whether and how K deficient plants also alter their secondary metabolism for nutrient management and defense is not known.ResultsHere we show that K-deficient plants contain higher levels of the phytohormone jasmonic acid (JA), hydroxy-12-oxo-octadecadienoic acids (HODs) and 12-oxo-phytodienoic acid (OPDA) than K-sufficient plants. Up-regulation of the 13-LOX pathway in response to low K was evident in increased transcript levels of several biosynthetic enzymes. Indole and aliphatic glucosinolates accumulated in response to K-deficiency in a manner that was respectively dependent or independent on signaling through Coronatine-Insensitive 1 (COI1). Transcript and glucosinolate profiles of K-deficient plants resembled those of herbivore attacked plants.ConclusionsBased on our results we propose that under K-deficiency plants produce oxylipins and glucosinolates to enhance their defense potential against herbivorous insects and create reversible storage for excess S and N.