Anton J. M. Peeters

The TRANSPARENT TESTA12 Gene of Arabidopsis Encodes a Multidrug Secondary Transporter-like Protein Required for Flavonoid Sequestration in Vacuoles of the Seed Coat Endothelium

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2

Variation of flowering time is found in the natural populations of many plant species. The underlying genetic variation, mostly of a quantitative nature, is presumed to reflect adaptations to different environments contributing to reproductive success. Analysis of natural variation for flowering time in Arabidopsis thaliana has identified several quantitative trait loci (QTL), which have yet to be characterized at the molecular level. A major environmental factor that determines flowering time is photoperiod or day length, the length of the light period, which changes across the year differently with geographical latitude. We identified the EDI locus as a QTL partly accounting for the difference in flowering response to the photoperiod between two Arabidopsis accessions: the laboratory strain Landsberg erecta (Ler), originating in Northern Europe, and Cvi, collected in the tropical Cape Verde Islands. Positional cloning of the EDI QTL showed it to be a novel allele of CRY2, encoding the blue-light photoreceptor cryptochrome-2 that has previously been shown to promote flowering in long-day (LD) photoperiods. We show that the unique EDI flowering phenotype results from a single amino-acid substitution that reduces the light-induced downregulation of CRY2 in plants grown under short photoperiods, leading to early flowering.

Regulatory network construction in Arabidopsis by using genome-wide gene expression quantitative trait loci

Accessions of a plant species can show considerable genetic differences that are analyzed effectively by using recombinant inbred line (RIL) populations. Here we describe the results of genome-wide expression variation analysis in an RIL population of Arabidopsis thaliana. For many genes, variation in expression could be explained by expression quantitative trait loci (eQTLs). The nature and consequences of this variation are discussed based on additional genetic parameters, such as heritability and transgression and by examining the genomic position of eQTLs versus gene position, polymorphism frequency, and gene ontology. Furthermore, we developed an approach for genetic regulatory network construction by combining eQTL mapping and regulator candidate gene selection. The power of our method was shown in a case study of genes associated with flowering time, a well studied regulatory network in Arabidopsis. Results that revealed clusters of coregulated genes and their most likely regulators were in agreement with published data, and unknown relationships could be predicted.

Plant hormones regulate fast shoot elongation under water: From genes to communities

Flooding affects the abundance and distribution of plant species worldwide. Many plants are damaged or even killed by flooding events due to the associated oxygen deprivation in cells. Stimulated shoot elongation is an important adaptive mode that can restore contact of leaves with the atmosphere above the water surface. This strongly im- proves inward diffusion of oxygen and the rate of photosynthesis. Fast elongation of sub- merged petioles of the model plant Rumex palustris involves the integrated action of the plant hormones ethylene, auxin, gibberellin, and abscisic acid. The closely related Rumex acetosa is unable to switch on petiole elongation when submerged. In a comparative study of these two Rumex species, we found that the response to the gaseous phytohormone ethylene, which accumulates in plant tissues during submergence, explains their contrasting elongation behavior. In order to study the importance of this shoot elongation response in the distributional patterns of plants in natural floodplains, we quantified the ethylene- induced elongation response of 22 plant species occurring in the Rhine River floodplain. These results were compared with the results of a multivariate analysis based on 84 veg- etation surveys performed in the same area. The species compositions of the surveys were grouped along two environmental gradients: flooding duration and soil dehydration after the floodwater subsided. If we superimpose the ethylene-induced elongation capacity on these vegetation data, it becomes clear that the capacity to elongate upon exposure to ethylene positively correlates with flooding duration and negatively with soil dehydration. Based on this analysis, we conclude that the capacity to elongate is an important selective trait in field distribution patterns of plants in flood-prone environments. Fast shoot elon- gation under water seems to be a favorable trait only in environments with shallow and prolonged flooding events, while costs associated with this response prevent its expression in sites with deep floods, sites with floods short in duration, or in sites in which flood water recedes rapidly. The approach outlined in this paper may be more widely applicable in ecological studies that aim to understand the functional relationship between plant traits and species distributions along environmental gradients.

ANTHOCYANINLESS2, a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis.

The ANTHOCYANINLESS2 (ANL2) gene was isolated from Arabidopsis by using the maize Enhancer–Inhibitor transposon tagging system. Sequencing of the ANL2 gene showed that it encodes a homeodomain protein belonging to the HD–GLABRA2 group. As we report here, this homeobox gene is involved in the accumulation of anthocyanin and in root development. Histological observations of the anl2 mutant revealed that the accumulation of anthocyanin was greatly suppressed in subepidermal cells but only slightly reduced in epidermal cells. Furthermore, the primary roots of the anl2 mutant showed an aberrant cellular organization. We discuss a possible role of ANL2 in the accumulation of anthocyanin and cellular organization of the primary root.

Auxin transport through PIN-FORMED 3 (PIN3) controls shade avoidance and fitness during competition

Plants grow in dense vegetations at the risk of being out-competed by neighbors. To increase their competitive power, plants display adaptive responses, such as rapid shoot elongation (shade avoidance) to consolidate light capture. These responses are induced upon detection of proximate neighbors through perception of the reduced ratio between red (R) and far-red (FR) light that is typical for dense vegetations. The plant hormone auxin is a central regulator of plant development and plasticity, but until now it has been unknown how auxin transport is controlled to regulate shade-avoidance responses. Here, we show that low R:FR detection changes the cellular location of the PIN-FORMED 3 (PIN3) protein, a regulator of auxin efflux, in Arabidopsis seedlings. As a result, auxin levels in the elongating hypocotyls are increased under low R:FR. Seedlings of the pin3-3 mutant lack this low R:FR-induced increase of endogenous auxin in the hypocotyl and, accordingly, have no elongation response to low R:FR. We hypothesize that low R:FR-induced stimulation of auxin biosynthesis drives the regulation of PIN3, thus allowing shade avoidance to occur. The adaptive significance of PIN3-mediated control of shade-avoidance is shown in plant competition studies. It was found that pin3 mutants are outcompeted by wild-type neighbors who suppress fitness of pin3-3 by 40%. We conclude that low R:FR modulates the auxin distribution by a change in the cellular location of PIN3, and that this control can be of great importance for plants growing in dense vegetations.

How to decide? Different methods of calculating gene expression from short oligonucleotide array data will give different results

BackgroundShort oligonucleotide arrays for transcript profiling have been available for several years. Generally, raw data from these arrays are analysed with the aid of the Microarray Analysis Suite or GeneChip Operating Software (MAS or GCOS) from Affymetrix. Recently, more methods to analyse the raw data have become available. Ideally all these methods should come up with more or less the same results. We set out to evaluate the different methods and include work on our own data set, in order to test which method gives the most reliable results.ResultsCalculating gene expression with 6 different algorithms (MAS5, dChip PMMM, dChip PM, RMA, GC-RMA and PDNN) using the same (Arabidopsis) data, results in different calculated gene expression levels. Consequently, depending on the method used, different genes will be identified as differentially regulated. Surprisingly, there was only 27 to 36% overlap between the different methods. Furthermore, 47.5% of the genes/probe sets showed good correlation between the mismatch and perfect match intensities.ConclusionAfter comparing six algorithms, RMA gave the most reproducible results and showed the highest correlation coefficients with Real Time RT-PCR data on genes identified as differentially expressed by all methods. However, we were not able to verify, by Real Time RT-PCR, the microarray results for most genes that were solely calculated by RMA. Furthermore, we conclude that subtraction of the mismatch intensity from the perfect match intensity results most likely in a significant underestimation for at least 47.5% of the expression values. Not one algorithm produced significant expression values for genes present in quantities below 1 pmol. If the only purpose of the microarray experiment is to find new candidate genes, and too many genes are found, then mutual exclusion of the genes predicted by contrasting methods can be used to narrow down the list of new candidate genes by 64 to 73%.

The many functions of ERECTA

The Arabidopsis thaliana accession Landsberg erecta contains an induced mutation in the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA. Landsberg erecta is commonly used as a genetic background in mutant screens and in natural variation studies. Therefore, the erecta mutation is present in many loss-of-function mutants and recombinant inbred lines. Information on how the absence of functional ERECTA affects the interpretation of obtained phenotypic results is scattered. In this report we inventoried ERECTA functions and highlight ERECTA as a pleiotropic regulator of developmental and physiological processes, as well as a modulator of responses to environmental stimuli.

Ethylene-Induced Differential Growth of Petioles in Arabidopsis. Analyzing Natural Variation, Response Kinetics, and Regulation

Plants can reorient their organs in response to changes in environmental conditions. In some species, ethylene can induce resource-directed growth by stimulating a more vertical orientation of the petioles (hyponasty) and enhanced elongation. In this study on Arabidopsis (Arabidopsis thaliana), we show significant natural variation in ethylene-induced petiole elongation and hyponastic growth. This hyponastic growth was rapidly induced and also reversible because the petioles returned to normal after ethylene withdrawal. To unravel the mechanisms behind the natural variation, two contrasting accessions in ethylene-induced hyponasty were studied in detail. Columbia-0 showed a strong hyponastic response to ethylene, whereas this response was almost absent in Landsberg erecta (Ler). To test whether Ler is capable of showing hyponastic growth at all, several signals were applied. From all the signals applied, only spectrally neutral shade (20 μmol m−2 s−1) could induce a strong hyponastic response in Ler. Therefore, Ler has the capacity for hyponastic growth. Furthermore, the lack of ethylene-induced hyponastic growth in Ler is not the result of already-saturating ethylene production rates or insensitivity to ethylene, as an ethylene-responsive gene was up-regulated upon ethylene treatment in the petioles. Therefore, we conclude that Ler is missing an essential component between the primary ethylene signal transduction chain and a downstream part of the hyponastic growth signal transduction pathway.

The Roles of Ethylene, Auxin, Abscisic Acid, and Gibberellin in the Hyponastic Growth of Submerged Rumex palustris Petioles

Rumex palustris responds to complete submergence with upward movement of the younger petioles. This so-called hyponastic response, in combination with stimulated petiole elongation, brings the leaf blade above the water surface and restores contact with the atmosphere. We made a detailed study of this differential growth process, encompassing the complete range of the known signal transduction pathway: from the cellular localization of differential growth, to the hormonal regulation, and the possible involvement of a cell wall loosening protein (expansin) as a downstream target. We show that hyponastic growth is caused by differential cell elongation across the petiole base, with cells on the abaxial (lower) surface elongating faster than cells on the adaxial (upper) surface. Pharmacological studies and endogenous hormone measurements revealed that ethylene, auxin, abscisic acid (ABA), and gibberellin regulate different and sometimes overlapping stages of hyponastic growth. Initiation of hyponastic growth and (maintenance of) the maximum petiole angle are regulated by ethylene, ABA, and auxin, whereas the speed of the response is influenced by ethylene, ABA, and gibberellin. We found that a submergence-induced differential redistribution of endogenous indole-3-acetic acid in the petiole base could play a role in maintenance of the response, but not in the onset of hyponastic growth. Since submergence does not induce a differential expression of expansins across the petiole base, it is unlikely that this cell wall loosening protein is the downstream target for the hormones that regulate the differential cell elongation leading to submergence-induced hyponastic growth in R. palustris.