Low-Cost System for Maintaining Low Atmospheric CO2 Levels During Arabidopsis Cultivation.

Photosynthesis requires CO2 as the carbon source, and the levels of ambient CO2 determine the oxygenation or carboxylation of Ribulose-1,5-bisphosphate (RuBP) by RuBP carboxylase/oxygenase (Rubisco). Low CO2 levels lead to oxygenation and result in photorespiration, which ultimately causes a reduction in net carbon assimilation through photosynthesis. Therefore, an increased understanding of plant responses to low CO2 contributes to the knowledge of how plants circumvent the harmful effects of photorespiration. Methods for elevating CO2 above ambient concentrations are often achieved by external sources of CO2, but reducing CO2 below the ambient value is much more difficult as CO2 gas needs to be scrubbed from the atmosphere rather than added to it. Here, we describe a low-cost method of achieving low CO2 conditions for Arabidopsis growth.

A low-cost open-source imaging platform reveals spatiotemporal insight into leaf elongation and movement.

Plant organs move throughout the diurnal cycle, changing leaf and petiole positions to balance light capture, leaf temperature, and water loss under dynamic environmental conditions. Upward movement of the petiole, called hyponasty, is one of several traits of the shade avoidance syndrome (SAS). SAS traits are elicited upon perception of vegetation shade signals such as far-red light (FR) and improve light capture in dense vegetation. Monitoring plant movement at a high temporal resolution allows studying functionality and molecular regulation of hyponasty. However, high temporal resolution imaging solutions are often very expensive, making this unavailable to many researchers. Here, we present a modular and low-cost imaging setup, based on small Raspberry Pi computers that can track leaf movements and elongation growth with high temporal resolution. We also developed an open-source, semiautomated image analysis pipeline. Using this setup, we followed responses to FR enrichment, light intensity, and their interactions. Tracking both elongation and the angle of the petiole, lamina, and entire leaf in Arabidopsis (Arabidopsis thaliana) revealed insight into R:FR sensitivities of leaf growth and movement dynamics and the interactions of R:FR with background light intensity. The detailed imaging options of this system allowed us to identify spatially separate bending points for petiole and lamina positioning of the leaf.

Low-cost and efficient confocal imaging method for arabidopsis flower.

For an extensive period of time apical meristem (SAM) has been considered as a mysterious organ, due to its small, hidden and dynamic structure. Confocal imaging, combined with fluorescent reporters, enables researchers to unveil the mechanisms underlying cellular activities, such as gene expression, cell division, growth patterns and cell-cell communications. Recently, a series of protocols were developed for confocal imaging of inflorescence meristem (IM) and floral meristem (FM). However, the requirement of high configuration, such as the need of a water-dipping lens without coverslip and the specialized turrets associated with fixed-stage microscopes, impedes the wide adoption of these methods. We exploited an improved object slide and matching method aiming to decrease the configuration requirement. Following this protocol, various dry microscope lenses can be selected with flexibility for building 3D images of IM and FM.

Leaf economics and slow-fast adaptation across the geographic range of Arabidopsis thaliana.

Life history strategies of most organisms are constrained by resource allocation patterns that follow a 'slow-fast continuum'. It opposes slow growing and long-lived organisms with late investment in reproduction to those that grow faster, have earlier and larger reproductive effort and a short longevity. In plants, the Leaf Economics Spectrum (LES) depicts a leaf-level trade-off between the rate of carbon assimilation and leaf lifespan, as stressed in functional ecology from interspecific comparative studies. However, it is still unclear how the LES is connected to the slow-fast syndrome. Interspecific comparisons also impede a deep exploration of the linkage between LES variation and adaptation to climate. Here, we measured growth, morpho-physiological and life-history traits, at both the leaf and whole-plant levels, in 378 natural accessions of Arabidopsis thaliana. We found that the LES is tightly linked to variation in whole-plant functioning, and aligns with the slow-fast continuum. A genetic analysis further suggested that phenotypic differentiation results from the selection of different slow-fast strategies in contrasted climates. Slow growing and long-lived plants were preferentially found in cold and arid habitats while fast growing and short-lived ones in more favorable habitats. Our findings shed light on the role of the slow-fast continuum for plant adaptation to climate. More broadly, they encourage future studies to bridge functional ecology, genetics and evolutionary biology to improve our understanding of plant adaptation to environmental changes.

Assessment of heterosis in two Arabidopsis thaliana common-reference mapping populations.

Hybrid vigour, or heterosis, has been of tremendous importance in agriculture for the improvement of both crops and livestock. Notwithstanding large efforts to study the phenomenon of heterosis in the last decades, the identification of common molecular mechanisms underlying hybrid vigour remain rare. Here, we conducted a systematic survey of the degree of heterosis in Arabidopsis thaliana hybrids. For this purpose, two overlapping Arabidopsis hybrid populations were generated by crossing a large collection of naturally occurring accessions to two common reference lines. In these Arabidopsis hybrid populations the range of heterosis for several developmental and yield related traits was examined, and the relationship between them was studied. The traits under study were projected leaf area at 17 days after sowing, flowering time, height of the main inflorescence, number of side branches from the main stem or from the rosette base, total seed yield, seed weight, seed size and the estimated number of seeds per plant. Predominantly positive heterosis was observed for leaf area and height of the main inflorescence, whereas mainly negative heterosis was observed for rosette branching. For the other traits both positive and negative heterosis was observed in roughly equal amounts. For flowering time and seed size only low levels of heterosis were detected. In general the observed heterosis levels were highly trait specific. Furthermore, no correlation was observed between heterosis levels and the genetic distance between the parental lines. Since all selected lines were a part of the Arabidopsis genome wide association (GWA) mapping panel, a genetic mapping approach was applied to identify possible regions harbouring genetic factors causal for heterosis, with separate calculations for additive and dominance effects. Our study showed that the genetic mechanisms underlying heterosis were highly trait specific in our hybrid populations and greatly depended on the genetic background, confirming the elusive character of heterosis.

Re-assessment of biolistic transient expression: An efficient and robust method for protein localization studies in seedling-lethal mutant and juvenile plants.

Knowledge on the subcellular localization of target proteins in a plant mutant background is important for revealing the function of the genes investigated. However, in Arabidopsis and rice, mutant lethality is one major barrier to such studies. Here we describe an optimized bombardment-mediated transient expression approach for studying subcellular protein localization in Arabidopsis seedling of lethal mutants. The whole experiment comprises four stages: cultivation and preparation of plants, coating gold particles with plasmid DNA, delivery of DNA into plants via bombardment, plant incubation and gene expression analysis which include localization and dynamics, co-localization comparison with reporter proteins and functional analysis. The entire process takes about 3-10 days from plant cultivation to protein detection. It has a high efficiency and the results are reproducible. Additionally, this protocol is applicable for the transient expression of chimeric fluorescent fusion proteins in juvenile rice seedlings and leaf sheaths, saving time dramatically in comparison of generating transgenic rice plant.

Assessment and Refinement of Sample Preparation Methods for Deep and Quantitative Plant Proteome Profiling.

A major challenge in the field of proteomics is obtaining high-quality peptides for comprehensive proteome profiling by LC-MS. Here, evaluation and modification of a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissue are done. It was found that inclusion of filter-aided sample preparation (FASP) based on filter digestion improves all protein extraction methods tested. Ultimately, a detergent-free urea-FASP approach that enables deep and robust quantification of leaf and root proteomes is shown. For example, from 4-day-old leaf tissue, up to 11 690 proteins were profiled from a single sample replicate. This method should be broadly applicable to researchers working with difficult to process plant samples.

Chemical priming of immunity without costs to plant growth.

ß-Aminobutyric acid (BABA) induces broad-spectrum disease resistance, but also represses plant growth, which has limited its exploitation in crop protection. BABA perception relies on binding to the aspartyl-tRNA synthetase (AspRS) IBI1, which primes the enzyme for secondary defense activity. This study aimed to identify structural BABA analogues that induce resistance without stunting plant growth. Using site-directed mutagenesis, we demonstrate that the (l)-aspartic acid-binding domain of IBI1 is critical for BABA perception. Based on interaction models of this domain, we screened a small library of structural BABA analogues for growth repression and induced resistance against biotrophic Hyaloperonospora arabidopsidis (Hpa). A range of resistance-inducing compounds were identified, of which (R)-ß-homoserine (RBH) was the most effective. Surprisingly, RBH acted through different pathways than BABA. RBH-induced resistance (RBH-IR) against Hpa functioned independently of salicylic acid, partially relied on camalexin, and was associated with augmented cell wall defense. RBH-IR against necrotrophic Plectosphaerella cucumerina acted via priming of ethylene and jasmonic acid defenses. RBH-IR was also effective in tomato against Botrytis cinerea. Metabolic profiling revealed that RBH, unlike BABA, does not majorly affect plant metabolism. RBH primes distinct defense pathways against biotrophic and necrotrophic pathogens without stunting plant growth, signifying strong potential for exploitation in crop protection.

Differential Costs of Two Distinct Resistance Mechanisms Induced by Different Herbivore Species in Arabidopsis.

Plants respond to herbivory with the induction of resistance, mediated by distinct phytohormonal signaling pathways and their interactions. Phloem feeders are known to induce plant resistance via the salicylic acid pathway, whereas biting-chewing herbivores induce plant resistance mainly via the jasmonate pathway. Here, we show that a specialist caterpillar (biting-chewing herbivore) and a specialist aphid (phloem feeder) differentially induce resistance against Pieris brassicae caterpillars in Arabidopsis (Arabidopsis thaliana) plants. Caterpillar feeding induces resistance through the jasmonate signaling pathway that is associated with the induction of kaempferol 3,7-dirhamnoside, whereas aphid feeding induces resistance via a novel mechanism involving sinapoyl malate. The role of sinapoyl malate is confirmed through the use of a mutant compromised in the biosynthesis of this compound. Caterpillar-induced resistance is associated with a lower cost in terms of plant growth reduction than aphid-induced resistance. A strong constitutive resistance against P. brassicae caterpillars in combination with a strong growth attenuation in plants of a transfer DNA (T-DNA) insertion mutant of WRKY70 (wrky70) suggest that the WRKY70 transcription factor, a regulator of downstream responses mediated by jasmonate-salicylic acid signaling cross talk, is involved in the negative regulation of caterpillar resistance and in the tradeoff between growth and defense. In conclusion, different mechanisms of herbivore-induced resistance come with different costs, and a functional WRKY70 transcription factor is required for the induction of low-cost resistance.

An Assessment of Engineered Calcium Oxalate Crystal Formation on Plant Growth and Development as a Step toward Evaluating Its Use to Enhance Plant Defense.

The establishment of new approaches to control chewing insects has been sought not only for direct use in reducing crop loss but also in managing resistance to the pesticides already in use. Engineered formation of calcium oxalate crystals is a potential strategy that could be developed to fulfill both these needs. As a step toward this development, this study investigates the effects of transforming a non-calcium oxalate crystal accumulating plant, Arabidopsis thaliana, into a crystal accumulating plant. Calcium oxalate crystal accumulating A. thaliana lines were generated by ectopic expression of a single bacterial gene encoding an oxalic acid biosynthetic enzyme. Biochemical and cellular studies suggested that the engineered A. thaliana lines formed crystals of calcium oxalate in a manner similar to naturally occurring crystal accumulating plants. The amount of calcium oxalate accumulated in leaves also reached levels similar to those measured in the leaves of Medicago truncatula in which the crystals are known to play a defensive role. Visual inspection of the different engineered lines, however, suggested a phenotypic consequence on plant growth and development with higher calcium oxalate concentrations. The restoration of a near wild-type plant phenotype through an enzymatic reduction of tissue oxalate supported this observation. Overall, this study is a first to provide initial insight into the potential consequences of engineering calcium oxalate crystal formation in non-crystal accumulating plants.

An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions.

BACKGROUND: Hydroponics is a plant growth system that provides a more precise control of growth media composition. Several hydroponic systems have been reported for Arabidopsis and other model plants. The ease of system set up, cost of the growth system and flexibility to characterize and harvest plant material are features continually improved in new hydroponic system reported. RESULTS: We developed a hydroponic culture system for Arabidopsis and other model plants. This low cost, proficient, and novel system is based on recyclable and sterilizable plastic containers, which are readily available from local suppliers. Our system allows a large-scale manipulation of seedlings. It adapts to different growing treatments and has an extended growth window until adult plants are established. The novel seed-holder also facilitates the transfer and harvest of seedlings. Here we report the use of our hydroponic system to analyze transcriptomic responses of Arabidopsis to nutriment availability and plant/pathogen interactions. CONCLUSIONS: The efficiency and functionality of our proposed hydroponic system is demonstrated in nutrient deficiency and pathogenesis experiments. Hydroponically grown Arabidopsis seedlings under long-time inorganic phosphate (Pi) deficiency showed typical changes in root architecture and high expression of marker genes involved in signaling and Pi recycling. Genome-wide transcriptional analysis of gene expression of Arabidopsis roots depleted of Pi by short time periods indicates that genes related to general stress are up-regulated before those specific to Pi signaling and metabolism. Our hydroponic system also proved useful for conducting pathogenesis essays, revealing early transcriptional activation of pathogenesis-related genes.

Azolla domestication towards a biobased economy?

Due to its phenomenal growth requiring neither nitrogen fertilizer nor arable land and its biomass composition, the mosquito fern Azolla is a candidate crop to yield food, fuels and chemicals sustainably. To advance Azolla domestication, we research its dissemination, storage and transcriptome. Methods for dissemination, cross-fertilization and cryopreservation of the symbiosis Azolla filiculoides-Nostoc azollae are tested based on the fern spores. To study molecular processes in Azolla including spore induction, a database of 37 649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26 d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7 months. The unigene database entirely covered central metabolism and to a large degree covered cellular processes and regulatory networks. Analysis of genes engaged in transition to sexual reproduction revealed a FLOWERING LOCUS T-like protein in ferns with special features induced in sporulating Azolla fronds. Although domestication of a fern-cyanobacteria symbiosis may seem a daunting task, we conclude that the time is ripe and that results generated will serve to more widely access biochemicals in fern biomass for a biobased economy.

GPLEXUS: enabling genome-scale gene association network reconstruction and analysis for very large-scale expression data.

The accurate construction and interpretation of gene association networks (GANs) is challenging, but crucial, to the understanding of gene function, interaction and cellular behavior at the genome level. Most current state-of-the-art computational methods for genome-wide GAN reconstruction require high-performance computational resources. However, even high-performance computing cannot fully address the complexity involved with constructing GANs from very large-scale expression profile datasets, especially for the organisms with medium to large size of genomes, such as those of most plant species. Here, we present a new approach, GPLEXUS (http://plantgrn.noble.org/GPLEXUS/), which integrates a series of novel algorithms in a parallel-computing environment to construct and analyze genome-wide GANs. GPLEXUS adopts an ultra-fast estimation for pairwise mutual information computing that is similar in accuracy and sensitivity to the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE) method and runs ∼1000 times faster. GPLEXUS integrates Markov Clustering Algorithm to effectively identify functional subnetworks. Furthermore, GPLEXUS includes a novel 'condition-removing' method to identify the major experimental conditions in which each subnetwork operates from very large-scale gene expression datasets across several experimental conditions, which allows users to annotate the various subnetworks with experiment-specific conditions. We demonstrate GPLEXUS's capabilities by construing global GANs and analyzing subnetworks related to defense against biotic and abiotic stress, cell cycle growth and division in Arabidopsis thaliana.

Structural assessment of the impact of environmental constraints on Arabidopsis thaliana leaf growth: a 3D approach.

Light and soil water content affect leaf surface area expansion through modifications in epidermal cell numbers and area, while effects on leaf thickness and mesophyll cell volumes are far less documented. Here, three-dimensional imaging was applied in a study of Arabidopsis thaliana leaf growth to determine leaf thickness and the cellular organization of mesophyll tissues under moderate soil water deficit and two cumulative light conditions. In contrast to surface area, thickness was highly conserved in response to water deficit under both low and high cumulative light regimes. Unlike epidermal and palisade mesophyll tissues, no reductions in cell number were observed in the spongy mesophyll; cells had rather changed in volume and shape. Furthermore, leaf features of a selection of genotypes affected in leaf functioning were analysed. The low-starch mutant pgm had very thick leaves because of unusually large palisade mesophyll cells, together with high levels of photosynthesis and stomatal conductance. By means of an open stomata mutant and a 9-cis-epoxycarotenoid dioxygenase overexpressor, it was shown that stomatal conductance does not necessarily have a major impact on leaf dimensions and cellular organization, pointing to additional mechanisms for the control of CO(2) diffusion under high and low stomatal conductance, respectively.

Phytochrome signaling in green Arabidopsis seedlings: impact assessment of a mutually negative phyB-PIF feedback loop.

The reversibly red (R)/far-red (FR)-light-responsive phytochrome (phy) photosensory system initiates both the deetiolation process in dark-germinated seedlings upon first exposure to light, and the shade-avoidance process in fully deetiolated seedlings upon exposure to vegetational shade. The intracellular signaling pathway from the light-activated photoreceptor conformer (Pfr) to the transcriptional network that drives these responses involves direct, physical interaction of Pfr with a small subfamily of bHLH transcription factors, termed Phy-Interacting Factors (PIFs), which induces rapid PIF proteolytic degradation. In addition, there is evidence of further complexity in light-grown seedlings, whereby phyB-PIF interaction reciprocally induces phyB degradation, in a mutually-negative, feedback-loop configuration. Here, to assess the relative contributions of these antagonistic activities to the net phenotypic readout in light-grown seedlings, we have examined the magnitude of the light- and simulated-shade-induced responses of a pentuple phyBpif1pif3pif4pif5 (phyBpifq) mutant and various multiple pif-mutant combinations. The data (1) reaffirm that phyB is the predominant, if not exclusive, photoreceptor imposing the inhibition of hypocotyl elongation in deetiolating seedlings in response to prolonged continuous R irradiation and (2) show that the PIF quartet (PIF1, PIF3, PIF4, and PIF5) retain and exert a dual capacity to modulate hypocotyl elongation under these conditions, by concomitantly promoting cell elongation through intrinsic transcriptional-regulatory activity, and reducing phyB-inhibitory capacity through feedback-loop-induced phyB degradation. In shade-exposed seedlings, immunoblot analysis shows that the shade-imposed reduction in Pfr levels induces increases in the abundance of PIF3, and mutant analysis indicates that PIF3 acts, in conjunction with PIF4 and PIF5, to promote the known shade-induced acceleration of hypocotyl elongation. Conversely, although the quadruple pifq mutant displays clearly reduced hypocotyl elongation compared to wild-type in response to prolonged shade, immunoblot analysis detects no elevation in phyB levels in the mutant seedlings compared to the wild-type during the majority of the shade-induced growth period, and phyB levels are not robustly correlated with the growth phenotype across the pif-mutant combinations compared. These results suggest that PIF feedback modulation of phyB abundance does not play a dominant role in modulating the magnitude of the PIF-promoted, shade-responsive phenotype under these conditions. In seedlings grown under diurnal light-dark cycles, the data show that FR-pulse-induced removal of Pfr at the beginning of the dark period (End-of-Day-FR (EOD-FR) treatment) results in longer hypocotyls relative to no EOD-FR treatment and that this effect is attenuated in the pif-mutant combinations tested. This result similarly indicates that the PIF quartet members are capable of intrinsically promoting hypocotyl cell elongation in light-grown plants, independently of the effects of PIF feedback modulation of photoactivated-phyB abundance.

An assessment of transgenomics as a tool for identifying genes involved in the evolutionary differentiation of closely related plant species.

• Transgenomics is the process of introducing genomic clones from a donor species into a recipient species and then screening the resultant transgenic lines for phenotypes of interest. This method might allow us to find genes involved in the evolution of phenotypic differences between species as well as genes that have the potential to contribute to reproductive isolation: potential speciation genes. • More than 1100 20-kbp genomic clones from Leavenworthia alabamica were moved into Arabidopsis thaliana by transformation. After screening a single primary transformant for each line, clones associated with mutant phenotypes were tested for repeatability and co-segregation. • We found 84 clones with possible phenotypic effects, of which eight were repeatedly associated with the same phenotype. One clone, 11_11B, co-segregated with a short fruit phenotype. Further study showed that 11_11B affects seed development, with as much as one-third of the seeds aborted in some fruit. • Transgenomics is a viable strategy for discovering genes of evolutionary interest. We identify methods to reduce false positives and false negatives in the future. 11_11B can be viewed as a potential speciation gene, illustrating the value of transgenomics for studying the molecular basis of reproductive isolation.

Root developmental adaptation to phosphate starvation: better safe than sorry.

Phosphorus is a crucial component of major organic molecules such as nucleic acids, ATP and membrane phospholipids. It is present in soils in the form of inorganic phosphate (Pi), which has low availability and poor mobility. To cope with Pi limitations, plants have evolved complex adaptive responses that include morphological and physiological modifications. This review describes how the model plant Arabidopsis thaliana adapts its root system architecture to phosphate deficiency through inhibition of primary root growth, increase in lateral root formation and growth and production of root hairs, which all promote topsoil foraging. A better understanding of plant adaptation to low phosphate will open the way to increased phosphorus use efficiency by crops. Such an improvement is needed in order to adjust how we manage limited phosphorus stocks and to reduce the disastrous environmental effects of phosphate fertilizers overuse.

Application of proteomics to the assessment of the response to ionising radiation in Arabidopsis thaliana.

Ionising radiation (IR) affects cellular and tissue function. However, the biological effects and interactions induced by IR are unclear. The aim of this study was to decipher the proteomic patterns that influence these pathways. The proteomes of Arabidopsis thaliana roots and rosettes were analysed in response to sub-lethal IR doses (0, 10, and 40 Gy). For each dose, the dynamic response was observed at different time points (2, 24 and 72 h). To quantitatively measure the effect of IR on the proteome, total proteins were extracted and subjected to 2-DE, and the changes in the 2-DE protein profiles were analysed. Statistical analysis revealed a total of 172 proteins (145 in leaves and 27 in roots) that were differentially expressed. These proteins were subsequently analysed by MALDI-TOF/TOF MS and comparative database analysis, and 144 (118 in leaves and 26 in roots) proteins were identified. The changes in the protein profile were quantitatively more significant for the 40 Gy dose than for the 10 Gy dose. In addition, specific functional groups of proteins were identified based on the consistency of the dose- and time-responses. The molecular mechanisms involved in the response to IR and a comparison of the observed responses are discussed.

An assessment of morphogenetic fluctuation during reproductive phase change in Arabidopsis.

BACKGROUND AND AIMS: Reproductive phase change in Arabidopsis thaliana is characterized by two transitions in phytomer identity, the differentiation of the first elongate internode (bolting transition) and of the first flower (floral transition). An evaluation of the dynamics of these transitions was sought by examining the precision of the corresponding phytomer identity changes. METHODS: The length of the first elongate internode and the frequency of chimeric inflorescence structures, e.g. paraclades not subtended by a leaf (no-leaf/paraclades) and flowers subtended by a bract (bract/flowers), were measured in the Wassilewskija (Ws) accession and 47 early flowering mutants under a wide range of photoperiods. The impact of photoperiodic perturbations applied to Ws plants at different times of development was also evaluated. KEY RESULTS: In Ws, both types of characters were remarkably constant across photoperiods in spite of a high degree of interindividual variability. Bract/flowers were not normally produced in Ws, but they were observed in conditions that suggest enhanced light signalling, e.g. in response to continuous light perturbations and in mutants with reduced hypocotyl elongation. In contrast, no-leaf/paraclades were normally present in approx. 20 % of Ws plants, and their frequency was increased in conditions that suggest reduced light signalling, e.g. in mutants with altered specification of long-day responses. The length of the first elongate internode was unrelated to the rate of stem elongation and to the regulation of reproductive phase change. CONCLUSIONS: Bract/flowers and no-leaf/paraclades corresponded to opposite effects on the floral transition that reflected different dynamics of progression to flowering. In contrast, the length of the first elongate internode was only indirectly related to the regulation of reproductive phase change and was mainly dependent on global morphogenetic constraints. This paper proposes that morphogenetic variability could be used to identify critical phases of development and characterize the canalization of developmental patterns.