The class III peroxidase multigenic family in rice and its evolution in land plants

Plant peroxidases (class III peroxidases, E.C. 1.11.1.7) are secreted glycoproteins known to be involved in the mechanism of cell elongation, in cell wall construction and differentiation, and in the defense against pathogens. They usually form large multigenic families in angiosperms. The recent completion of rice (Oryza sativa japonica c.v. Nipponbare) genome sequencing allowed drawing up the full inventory of the genes encoding class III peroxidases in this plant. We found 138 peroxidase genes distributed among the 12 rice chromosomes. In contrast to several other gene families studied so far, peroxidase genes are twice as numerous in rice as in Arabidopsis. This large number of genes results from various duplication events that were tentatively traced back using a phylogenetic tree based on the alignment of conserved amino acid sequences. We also searched for peroxidase encoding genes in the major phyla of plant kingdom. In addition to gymnosperms and angiosperms, sequences were found in liverworts, mosses and ferns, but not in unicellular green algae. Two rice and one Arabidopsis peroxidase genes appeared to be rather close to the only known sequence from the liverwort Marchantia polymorpha. The possible relationship of these peroxidases with the putative ancestor of peroxidase genes is discussed, as well as the connection between the development of the class III peroxidase multigenic family and the emergence of the first land plants.     

Biosynthesis of cholestanol in higher plants

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.     

Generalist insects behave in a jasmonate-dependent manner on their host plants,leaving induced areas quickly and staying longer on distant parts

Plants are sessile, so have evolved sensitive ways to detect attacking herbivores and sophisticated strategies to effectively defend themselves. Insect herbivory induces synthesis of the phytohormone jasmonic acid which activates downstream metabolic pathways for various chemical defences such as toxins and digestion inhibitors. Insects are also sophisticated animals, and many have coevolved physiological adaptations that negate this induced plant defence. Insect behaviour has rarely been studied in the context of induced plant defence, although behavioural adaptation to induced plant chemistry may allow insects to bypass the host''s defence system. By visualizing jasmonate-responsive gene expression within whole plants, we uncovered spatial and temporal limits to the systemic spread of plant chemical defence following herbivory. By carefully tracking insect movement, we found induced changes in plant chemistry were detected by generalist Helicoverpa armigera insects which then modified their behaviour in response, moving away from induced parts and staying longer on uninduced parts of the same plant. This study reveals that there are plant-wide signals rapidly generated following herbivory that allow insects to detect the heterogeneity of plant chemical defences. Some insects use these signals to move around the plant, avoiding localized sites of induction and staying ahead of induced toxic metabolites.     

MIZ1-regulated hydrotropism functions in the growth and survival of Arabidopsis thaliana under natural conditions

Background and Aims

Root hydrotropism is a response to water-potential gradients that makes roots bend towards areas of higher water potential. The gene MIZU-KUSSEI1 (MIZ1) that is essential for hydrotropism in Arabidopsis roots has previously been identified. However, the role of root hydrotropism in plant growth and survival under natural conditions has not yet been proven. This study assessed how hydrotropic response contributes to drought avoidance in nature.

Methods

An experimental system was established for the study of Arabidopsis hydrotropism in soil. Characteristics of hydrotropism were analysed by comparing the responses of the miz1 mutant, transgenic plants overexpressing MIZ1 (MIZ1OE) and wild-type plants.

Key Results

Wild-type plants developed root systems in regions with higher water potential, whereas the roots of miz1 mutant plants did not show a similar response. This pattern of root distribution induced by hydrotropism was more pronounced in MIZ1OE plants than in wild-type plants. In addition, shoot biomass and the number of plants that survived under drought conditions were much greater in MIZ1OE plants.

Conclusions

These results show that hydrotropism plays an important role in root system development in soil and contributes to drought avoidance, which results in a greater yield and plant survival under water-limited conditions. The results also show that MIZ1 overexpression can be used for improving plant productivity in arid areas.     

Increase in recombination rate in Arabidopsis thaliana plants sharing gaseous environment with X-ray and UVC-irradiated plants depends on production of radicals

X-ray and UVC are the two physical agents that damage DNA directly, with both agents capable of inducing double-strand breaks. Some of our recent work has demonstrated that local exposure to UVC results in a systemic increase in recombination frequency, suggesting that information about exposure can be passed from damaged to non-damaged tissue. Indeed, we recently showed that plants sharing the same enclosed environment with UVC-irradiated plants exhibit similar increase in homologous recombination frequency as irradiated plants. Here, we further tested whether yet another DNA-damaging agent, X-ray, is capable of increasing recombination rate (RR) in neighboring plants grown in a Petri dish. To test this, we grew plants exposed to X-ray or UVC irradiation in an enclosed environment next to non-exposed plants. We found that both X-ray and UVC-irradiated plants and neighboring plants exhibited comparable increases in the levels of strand breaks and the RR. We further showed that pre-exposure of plants to radical scavenger DMSO substantially alleviates the radiation-induced increase in RR and prevents formation of bystander signal. Our results suggest that the increase in RR in bystander plants can also be triggered by X-ray and that radicals may play some role in initiation or maintenance of this signal.     

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

In the past, we showed that exposure to abiotic and biotic stresses changes the homologous recombination frequency (HRF) in somatic tissue and in the progeny. In current work we planned to answer the following question: do stress intensity/duration and time during exposure influence changes in somatic HRF and transgenerational changes in HRF? Here, we tested the effects of exposure to UV-C, cold and heat on HRF at 7, 14, 21 and 28 days post germination (dpg). We found that exposure at 14 and 21 dpg resulted in a higher increase in HRF as compared to exposure at 7 dpg; longer exposure to UV-C resulted in a higher frequency of HR, whereas prolonged exposure to cold or heat, especially at later developmental stages, had almost no effect on somatic HRF. Exposure at 7 dpg had a positive effect on somatic growth of plants; plants exposed to stress at this age had larger leaves. The analysis of HRF in the progeny showed that the progeny of plants exposed to stress at 7 dpg had an increase in somatic HRF and showed larger sizes of recombination spots on leaves. The progeny of plants exposed to UV-C at 7 dpg and the progeny of plants exposed to cold or heat at 28 dpg had larger leaves as compared to control plants. To summarize, our experiments showed that changes in somatic and transgenerational HRF depend on the type of stress plants are exposed to, time of exposure during development and the duration of exposure.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-013-0197-z) contains supplementary material, which is available to authorized users.     

Interactome analysis reveals versatile functions of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 in RNA processing within the nucleus and cytoplasm

Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3) shares an RNA chaperone function with E. coli cold shock proteins and regulates freezing tolerance during cold acclimation. Here, we screened for AtCSP3-interacting proteins using a yeast two-hybrid system and 38 candidate interactors were identified. Sixteen of these were further confirmed in planta interaction between AtCSP3 by a bi-molecular fluorescence complementation assay. We found that AtCSP3 interacts with CONSTANS-LIKE protein 15 and nuclear poly(A)-binding proteins in nuclear speckles. Three 60S ribosomal proteins (RPL26A, RPL40A/UBQ2, and RPL36aB) and the Gar1 RNA-binding protein interacted with AtCSP3 in the nucleolus and nucleoplasm, suggesting that AtCSP3 functions in ribosome biogenesis. Interactions with LOS2/enolase and glycine-rich RNA-binding protein 7 that are cold inducible, and an mRNA decapping protein 5 (DCP5) were observed in the cytoplasm. These data suggest that AtCSP3 participates in multiple complexes that reside in nuclear and cytoplasmic compartments and possibly regulates RNA processing and functioning.     

The structure of "defective in induced resistance" protein of Arabidopsis thaliana, DIR1, reveals a new type of lipid transfer protein

Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1-1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1-lipid complex were both determined by fluorescence and X-ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side-by-side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways.     

Molecular control of seasonal flowering in rice,arabidopsis and temperate cereals

Background

Rice (Oryza sativa) and Arabidopsis thaliana have been widely used as model systems to understand how plants control flowering time in response to photoperiod and cold exposure. Extensive research has resulted in the isolation of several regulatory genes involved in flowering and for them to be organized into a molecular network responsive to environmental cues. When plants are exposed to favourable conditions, the network activates expression of florigenic proteins that are transported to the shoot apical meristem where they drive developmental reprogramming of a population of meristematic cells. Several regulatory factors are evolutionarily conserved between rice and arabidopsis. However, other pathways have evolved independently and confer specific characteristics to flowering responses.

Scope

This review summarizes recent knowledge on the molecular mechanisms regulating daylength perception and flowering time control in arabidopsis and rice. Similarities and differences are discussed between the regulatory networks of the two species and they are compared with the regulatory networks of temperate cereals, which are evolutionarily more similar to rice but have evolved in regions where exposure to low temperatures is crucial to confer competence to flower. Finally, the role of flowering time genes in expansion of rice cultivation to Northern latitudes is discussed.

Conclusions

Understanding the mechanisms involved in photoperiodic flowering and comparing the regulatory networks of dicots and monocots has revealed how plants respond to environmental cues and adapt to seasonal changes. The molecular architecture of such regulation shows striking similarities across diverse species. However, integration of specific pathways on a basal scheme is essential for adaptation to different environments. Artificial manipulation of flowering time by means of natural genetic resources is essential for expanding the cultivation of cereals across different environments.     

Proteomics of calcium-signaling components in plants

Calcium functions as a versatile messenger in mediating responses to hormones, biotic/abiotic stress signals and a variety of developmental cues in plants. The Ca(2+)-signaling circuit consists of three major "nodes"--generation of a Ca(2+)-signature in response to a signal, recognition of the signature by Ca2+ sensors and transduction of the signature message to targets that participate in producing signal-specific responses. Molecular genetic and protein-protein interaction approaches together with bioinformatic analysis of the Arabidopsis genome have resulted in identification of a large number of proteins at each "node"--approximately 80 at Ca2+ signature, approximately 400 sensors and approximately 200 targets--that form a myriad of Ca2+ signaling networks in a "mix and match" fashion. In parallel, biochemical, cell biological, genetic and transgenic approaches have unraveled functions and regulatory mechanisms of a few of these components. The emerging paradigm from these studies is that plants have many unique Ca2+ signaling proteins. The presence of a large number of proteins, including several families, at each "node" and potential interaction of several targets by a sensor or vice versa are likely to generate highly complex networks that regulate Ca(2+)-mediated processes. Therefore, there is a great demand for high-throughput technologies for identification of signaling networks in the "Ca(2+)-signaling-grid" and their roles in cellular processes. Here we discuss the current status of Ca2+ signaling components, their known functions and potential of emerging high-throughput genomic and proteomic technologies in unraveling complex Ca2+ circuitry.     

Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses

Background

Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca²⁺-dependent and Ca²⁺-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt).

Scope

Here we show that CO₂-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca²⁺ elevations. Moreover, Ca²⁺ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca²⁺-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca²⁺ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca²⁺ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO₂ enhance the sensitivity of stomatal closing mechanisms to intracellular Ca²⁺, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca²⁺-sensing proteins, giving rise to the question, how can specificity in Ca²⁺ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca²⁺ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Box 1. Improved resolution of stimulus-induced stomatal movements in guard cells by tracking of individual stomatal apertures

Arabidopsis guard cells have become a prime model system for analysing signal transduction, since early research combining genetic and ion channel analyses in this system (Ichida et al., 1997; Pei et al., 1997, 1998; Roelfsema and Prins, 1997). Arabidopsis stomata are small relative to other stomatal model systems and stomatal apertures of various plant types including Arabidopsis are known to show variability in the size of individual stomatal complexes and also variability in the opening apertures of stomata of similar size in a given leaf (Gorton et al., 1988; Mott and Buckley, 2000; Mott and Peak, 2007). Thus stomatal aperture measurements are expected to show a clear degree of statistical variation. Use of blind experiments, in which the genotype and, when possible, the stimulus being applied to guard cells is unknown to the experimenter (Murata et al., 2001) has been employed by several laboratories, has become a standard in the field and has aided in addressing the above limitations of the range of stomatal aperture sizes found under any given condition.Research in our laboratory has shown that a major additional improvement in experiments can be made, by adding imaging of the same individual stomatal apertures over time (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Siegel et al., 2009), while performing blind experiments. In such ‘stomatal tracking’ experiments the lower side of a leaf is attached to a glass coverslip in an extracellular incubation medium (Webb et al., 2001; Young et al., 2006). The mesophyll and upper leaf epidermis are removed surgically for better optical resolution of stomatal apertures in the intact lower leaf epidermis (Young et al., 2006). For stimulus-induced stomatal closing analyses, a field of well-opened stomata is located and images are captured (e.g. using Scion Image software) for later analyses and data storage. The bottom (dry side) of coverslips can be marked with colour marker pens to label grids in the regions where apertures where imaged, for finding these same stomata subsequently if needed. Images of the same stomatal apertures are taken over time and can be stored for later analyses of individual stomatal apertures and for deposition of image files. While this approach has been used as a standard for imposed Ca²⁺ oscillation studies (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Fig. 4), we have found that this method also substantially improves stomatal movement response analyses to any given stimulus (Siegel et al., 2009; see Figs 1 and 4 and, Box Fig. 1). For example, while individual stomata are known to have diverse apertures (e.g. Box Fig. 1C), the relative responses of wide open stomata and smaller stomatal apertures to ABA or to CO₂ were comparable (Fig. 1 and Box Fig. 1; Siegel et al., 2009). Note that this method has previously been proposed and used in Vicia faba (Gorton et al., 1988), for which stomata exhibit relatively weak ABA and CO₂ responses, compared with, for example, Arabidopsis. We propose that this simple image-capturing approach, together with blind analyses, be used as a standard for stomatal response research in arabidopsis. Our research experience with this method shows that this approach will aid in greatly improving resolution and robustness and in defining the functions of individual Ca²⁺-independent and Ca²⁺-dependent components and mechanisms in stomatal response analyses. Open in a separate windowBox Fig. 1.ABA-induced stomatal closing of individually tracked stomatal apertures. (A) Average individually tracked stomatal apertures in the presence of 50 µm Ca²⁺ (open triangles) and in the presence of 200 nm free Ca²⁺ (open squares) in the bath solution from three experiments are shown and were normalized to the stomatal apertures at time = 0. (B, C) ABA-induced stomatal closing in the presence of 50 µm Ca²⁺ in five individually tracked stomatal apertures. In (A; open triangles) normalized stomatal apertures of the same stomata depicted in (B) and (C) are shown. Methods used in these experiments tracking individual stomatal apertures are described in Siegel et al. (2009). ABA-induced stomatal closing experiments are reproduced from Siegel et al. (2009) with permission of the publisher.     

Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

An Efficient Method for Quantitative,Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.     

Assessing the regulation of leaf redox status under water stress conditions in Arabidopsis thaliana: Col-0 ecotype (wild-type and vtc-2), expressing mitochondrial and cytosolic roGFP1

Using Arabidopsis plants Col-0 and vtc2 transformed with a redox sensitive green fluorescent protein, (c-roGFP) and (m-roGFP), we investigated the effects of a progressive water stress and re-watering on the redox status of the cytosol and the mitochondria. Our results establish that water stress affects redox status differently in these two compartments, depending on phenotype and leaf age, furthermore we conclude that ascorbate plays a pivotal role in mediating redox status homeostasis and that Col-0 Arabidopsis subjected to water stress increase the synthesis of ascorbate suggesting that ascorbate may play a role in buffering changes in redox status in the mitochondria and the cytosol, with the presumed buffering capacity of ascorbate being more noticeable in young compared with mature leaves. Re-watering of water-stressed plants was paralleled by a return of both the redox status and ascorbate to the levels of well-watered plants. In contrast to the effects of water stress on ascorbate levels, there were no significant changes in the levels of glutathione, thereby suggesting that the regeneration and increase in ascorbate in water-stressed plants may occur by other processes in addition to the regeneration of ascorbate via the glutathione. Under water stress in vtc2 lines it was observed stronger differences in redox status in relation to leaf age, than due to water stress conditions compared with Col-0 plants. In the vtc2 an increase in DHA was observed in water-stressed plants. Furthermore, this work confirms the accuracy and sensitivity of the roGFP1 biosensor as a reporter for variations in water stress-associated changes in redox potentials.     

mAtNOS1 regulates mitochondrial functions and apoptosis of human neuroblastoma cells

mAtNOS1 is a novel gene recently reported in mammalian cells with functions that are not fully understood. The present study generated human neuroblastoma SHSY cells over- and underexpressing mAtNOS1 and shows that mAtNOS1 is involved in regulating mitochondrial nitric oxide, mitochondrial transmembrane potential, protein tyrosine nitration, cytochrome c release, and apoptosis of those cells.     

Transcriptional regulation and functional properties of <Emphasis Type="Italic">Arabidopsis</Emphasis> Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants

Phosphate mobilization into the plant is a complex process requiring numerous transporters for absorption and translocation of this major nutrient. In the genome of Arabidopsis thaliana, nine closely related high affinity phosphate transporters have been identified but their specific roles remain unclear. Here we report the molecular, histological and physiological characterization of Arabidopsis pht1;4 high affinity phosphate transporter mutants. Using GUS-gene trap and in situ hybridization, Pht1;4 was found mainly expressed in inorganic phosphate (Pi) limiting medium in roots, primarily in the epidermis, the cortex and the root cap. In addition to this, expression was also observed at the lateral root branch points on the primary root and in the stele of lateral roots, suggesting a role of Pht1;4 in phosphate absorption and translocation from the growth medium to the different parts of the plant. Pi-starved pht1;4 plantlets exhibited a strong reduction of phosphate uptake capacity (40). This phenotype appears only related to the pht1;4 mutation as there were no obvious changes in the expression of other Pht1 family members in the mutants background. However, after 10 days of growth on phosphate deficient or sufficient medium, the Pi content in the mutants was not significantly different from that of the corresponding wild type controls. Furthermore, the mutants did not display any obvious growth defects or visible phenotypes when grown on a low phosphate containing medium. The work described here offers a first step in the complex genetic dissection of the phosphate transport system in planta.     

Perspectives on Systematic Analyses of Gene Function in Arabidopsis thaliana: New Tools, Topics and Trends

Since the sequencing of the nuclear genome of Arabidopsis thaliana ten years ago, various large-scale analyses of gene function have been performed in this model species. In particular, the availability of collections of lines harbouring random T-DNA or transposon insertions, which include mutants for almost all of the ~27,000 A. thaliana genes, has been crucial for the success of forward and reverse genetic approaches. In the foreseeable future, genome-wide phenotypic data from mutant analyses will become available for Arabidopsis, and will stimulate a flood of novel in-depth gene-function analyses. In this review, we consider the present status of resources and concepts for systematic studies of gene function in A. thaliana. Current perspectives on the utility of loss-of-function and gain-of-function mutants will be discussed in light of the genetic and functional redundancy of many A. thaliana genes.