Next-generation ABACUS biosensors reveal cellular ABA dynamics driving root growth at low aerial humidity.

The plant hormone abscisic acid (ABA) accumulates under abiotic stress to recast water relations and development. To overcome a lack of high-resolution sensitive reporters, we developed ABACUS2s-next-generation Förster resonance energy transfer (FRET) biosensors for ABA with high affinity, signal-to-noise ratio and orthogonality-that reveal endogenous ABA patterns in Arabidopsis thaliana. We mapped stress-induced ABA dynamics in high resolution to reveal the cellular basis for local and systemic ABA functions. At reduced foliar humidity, root cells accumulated ABA in the elongation zone, the site of phloem-transported ABA unloading. Phloem ABA and root ABA signalling were both essential to maintain root growth at low humidity. ABA coordinates a root response to foliar stresses, enabling plants to maintain foraging of deeper soil for water uptake.

Time-series transcriptomics reveals a BBX32-directed control of acclimation to high light in mature Arabidopsis leaves.

The photosynthetic capacity of mature leaves increases after several days' exposure to constant or intermittent episodes of high light (HL) and is manifested primarily as changes in chloroplast physiology. How this chloroplast-level acclimation to HL is initiated and controlled is unknown. From expanded Arabidopsis leaves, we determined HL-dependent changes in transcript abundance of 3844 genes in a 0-6 h time-series transcriptomics experiment. It was hypothesized that among such genes were those that contribute to the initiation of HL acclimation. By focusing on differentially expressed transcription (co-)factor genes and applying dynamic statistical modelling to the temporal transcriptomics data, a regulatory network of 47 predominantly photoreceptor-regulated transcription (co-)factor genes was inferred. The most connected gene in this network was B-BOX DOMAIN CONTAINING PROTEIN32 (BBX32). Plants overexpressing BBX32 were strongly impaired in acclimation to HL and displayed perturbed expression of photosynthesis-associated genes under LL and after exposure to HL. These observations led to demonstrating that as well as regulation of chloroplast-level acclimation by BBX32, CRYPTOCHROME1, LONG HYPOCOTYL5, CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA-105 are important. In addition, the BBX32-centric gene regulatory network provides a view of the transcriptional control of acclimation in mature leaves distinct from other photoreceptor-regulated processes, such as seedling photomorphogenesis.

Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells.

Phototoxicity is a significant constraint for live cell fluorescence microscopy. Excessive excitation light intensities change the homeostasis of the observed cells. Erroneous and misleading conclusions may be the problematic consequence of observing such light-induced pathophysiology. In this study, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Tracking PC3 cells at different light doses and intensities, we show how motility can be used to reliably assess subtle positive and negative effects of illumination. We further show that the effects are a factor of intensity rather than light dose. Mitotic delay was not a sensitive indicator of phototoxicity. For early detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment.

Spatial chloroplast-to-nucleus signalling involving plastid-nuclear complexes and stromules.

Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastid-nuclear complexes, which provide close physical contact between these organelles. Plastid-nuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H2O2 to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid-nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid-nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state, the two modes of connection contribute to varying extents. We hypothesize that this association is dynamic and that there may be a link between plastid-nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.

ROS-dependent signalling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes.

Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signalling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidising equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signalling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (1O2) and hydrogen peroxide (H2O2), initiate distinct signalling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signalling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signalling pathway. How plants translate a H2O2 message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells.

Development of pGEMINI, a Plant Gateway Destination Vector Allowing the Simultaneous Integration of Two cDNA via a Single LR-Clonase Reaction.

Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious. To our knowledge, this study reports on the first development of a vector allowing the simultaneous integration of two independent cDNAs via a single LR-clonase reaction. This vector "pGEMINI" represents a powerful molecular tool offering the ability to study the role of multi-cDNA constructs on plant development, and opens up the process of gene stacking and the study of gene combinations through transient or stable transformation procedures.

Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism.

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.Multiple plastid-derived signals have been proposed but not shown to move to the nucleus to promote plant acclimation to fluctuating light. Here the authors use a fluorescent hydrogen peroxide sensor to provide evidence that H2O2 is transferred directly from chloroplasts to nuclei to control nuclear gene expression.

The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants.

Exposure of photosynthetic cells of leaf tissues of Arabidopsis thaliana (Arabidopsis) to high light intensities (HL) may provoke a rapid rise in hydrogen peroxide (H2O2) levels in chloroplasts and subcellular compartments, such as peroxisomes, associated with photosynthetic metabolism. It has been hypothesized that when H2O2 is contained at or near its site of production then it plays an important role in signaling to induce acclimation to HL. However, should this discrete containment fail and H2O2 levels exceed the capacity of antioxidant systems to scavenge them, then oxidative stress ensues which triggers cell death. To test this hypothesis, the spatiotemporal accumulation of H2O2 needs to be quantified in different subcellular compartments. In this chapter, preliminary experiments are presented on the use of Arabidopsis seedlings transformed with a nuclear-encoded cytosol-located yellow fluorescent protein-based sensor for H2O2, called HyPer. HyPer allows ratiometric determination of its fluorescence at two excitation wavelengths, which frees quantification of H2O2 from the variable levels of HyPer in vivo. HyPer fluorescence was shown to have the potential to provide the necessary spatial, temporal, and quantitative resolution to study HL responses of seedlings using confocal microscopy. Chlorophyll fluorescence imaging was used to quantify photoinhibition of photosynthesis induced by HL treatment of seedlings on the microscope staging. However, several technical issues remain, the most challenging of which is the silencing of HyPer expression beyond the seedling stage. This limited our pilot studies to cotyledon epidermal cells, which while not photosynthetic, nevertheless responded to HL with 45% increase in cytosolic H2O2.

Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases: the ToFZY gene family.

The flavin monooxygenases (FMO) encoded by plant YUCCA genes are thought to catalyze a rate-limiting step in the tryptamine pathway for indole-3-acetic acid biosynthesis. Recent experiments with different plant models have indicate that YUCCA genes play essential roles in growth and development through their contribution to the local pool of free auxin. In this study we have characterized five new genes that encode YUCCA-like FMOs in the tomato genome (ToFZY2 to ToFZY6), including gene structure, conserved motifs and phylogenetic analyses. As a first step towards clarifying the individual functions of ToFZY genes, we have used quantitative real-time RT-PCR to conduct a systematic comparison of the steady-state mRNA levels of 6 ToFZY genes, in 33 samples representing major organs and the entire tomato life cycle. We followed an absolute quantification strategy which allowed us to cross-compare transcript levels among different ToFZY genes in a given spatiotemporal coordinate. Our results indicate that expression of ToFZY genes is temporally and spatially regulated, and that the distinctive expression pattern of each ToFZY gene partially overlaps with other members of the multigenic family. We compare our data with previous results in other plant species and make some predictions about the role of tryptamine pathway in tomato growth and development.

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis.

Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.

Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process.

BACKGROUND: The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. RESULTS: In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. CONCLUSION: This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.