Arabidopsis Thaliana Rosette Research Articles

Quantitative and spatially resolved detection of multiplexed microRNA from plant tissue via hybridization to hydrogel-bound DNA probes in nanoliter well arrays

Understanding complex regulatory networks in plant systems requires elucidating the roles of various gene regulators under a spatial landscape. MicroRNA are key regulators that impart high information value through their tissue specificity and stability when using expression patterns for evaluating network outcomes. However, current techniques that utilize spatial multiplexing and quantitation of microRNA are limited to primarily mammalian systems. Here, we present a method to spatially resolve and quantify multiple endogenous microRNA in situ using ethanol fixed, paraffin embedded model plant species. This method utilizes target-specific microRNA capture along with universal ligating and labelling, all within functionalized hydrogel posts containing DNA probes in nanoliter well arrays. We demonstrate the platform’s multiplexing capabilities through analyzing three endogenous microRNA in Arabidopsis thaliana rosettes which provide useful answers to fundamental plant growth and development from the unique expression patterns. The spatial tissue technique is also validated using non-spatial small RNA assays to demonstrate the versatility of the well array platform. Our new platform expands the toolkit of spatial omics technologies for plants.

Identification of gene function based on models capturing natural variability of Arabidopsis thaliana lipid metabolism

Lipids play fundamental roles in regulating agronomically important traits. Advances in plant lipid metabolism have until recently largely been based on reductionist approaches, although modulation of its components can have system-wide effects. However, existing models of plant lipid metabolism provide lumped representations, hindering detailed study of component modulation. Here, we present the Plant Lipid Module (PLM) which provides a mechanistic description of lipid metabolism in the Arabidopsis thaliana rosette. We demonstrate that the PLM can be readily integrated in models of A. thaliana Col-0 metabolism, yielding accurate predictions (83%) of single lethal knock-outs and 75% concordance between measured transcript and predicted flux changes under extended darkness. Genome-wide associations with fluxes obtained by integrating the PLM in diel condition- and accession-specific models identify up to 65 candidate genes modulating A. thaliana lipid metabolism. Using mutant lines, we validate up to 40% of the candidates, paving the way for identification of metabolic gene function based on models capturing natural variability in metabolism.

Lipo-chitooligosaccharide and thuricin 17 act as plant growth promoters and alleviate drought stress in Arabidopsis thaliana.

Lipo-chito-oligosaccharide (LCO-from Bradyrhizobium japonicum) and thuricin 17 (Th17-from Bacillus thuringiensis) are bacterial signal compounds from the rhizosphere of soybean that have been shown to enhance plant growth in a range of legumes and non-legumes. In this study, an attempt to quantify phytohormones involved in the initial hours after exposure of Arabidopsis thaliana to these compounds was conducted using UPLC-ESI-MS/MS. A petri-plate assay was conducted to screen for drought stress tolerance to PEG 8000 infusion and plant growth was studied 21-days post-stress. Arabidopsis thaliana plants grown in trays with drought stress imposed by water withhold were used for free proline determination, elemental analysis, and untargeted proteomics using LC-MS/MS studies. At 24 h post-exposure to the signal compounds under optimal growth conditions, Arabidopsis thaliana rosettes varied in their responses to the two signals. While LCO-treated rosettes showed a decrease in total IAA, cytokinins, gibberellins, and jasmonic acid, increases in ABA and SA was very clear. Th17-treated rosettes, on the other hand, showed an increase in IAA and SA. Both treatments resulted in decreased JA levels. Under severe drought stress imposed by PEG 8000 infusion, LCO and Th17 treatments were found to significantly increase fresh and dry weight over drought-stressed control plates, indicating that the presence of the signaling compounds decreased the negative effects experienced by the plants. Free proline content increased in LCO- and Th17-treated plants after water-withhold drought stress. Elemental analysis showed a significant increase in carbon percentage at the lower concentration of Th17. Untargeted proteomics revealed changes in the levels of drought-specific ribosomal proteins, glutathione S-transferase, late embryogenesis proteins, vegetative storage proteins 1 and 2, thaumatin-like proteins, and those related to chloroplast and carbon metabolism. The roles of some of these significantly affected proteins detected under drought stress are discussed.

Arabidopsis thaliana rosette habit is controlled by combined light and energy signaling converging on transcriptional control of the TALE homeobox gene ATH1.

In the absence of light signals, Arabidopsis plants fail to develop the rosette habit typical for this species. Instead, plants display caulescent growth due to elongation of rosette internodes. This aspect of photomorphogenic development has been paid little attention and molecular events involved, downstream of photoreceptor signaling, remain to be identified. Using a combination of genetic and molecular approaches, we show that Arabidopsis rosette habit is a photomorphogenic trait controlled by induction of ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) as downstream target of multiple photoreceptors. ATH1 induction prevents rosette internode elongation by maintaining the shoot apical meristem (SAM) rib zone area inactive and requires inactivation of photomorphogenesis inhibitors, including PHYTOCHROME INTERACTING FACTOR (PIF) proteins. ATH1 activity results in tissue-specific inhibition of PIF expression, establishing double-negative feedback-regulation at the SAM. Light-requirement for ATH1 expression can be overcome by high sugar availability to the SAM. Both sugar and light signals that induce ATH1 and, subsequently, rosette habit are mediated by TOR kinase. Collectively, our data reveal a SAM-specific, double-negative ATH1-PIF feedback loop at the basis of rosette habit. Upstream, TOR kinase functions as central hub integrating light and energy signals that control this for Arabidopsis quintessential trait.

Effects of Environmental Stresses on the Growth of Arabidopsis thaliana Rosette Leaves

Abstract Plant growth is constantly affected by biotic and abiotic stresses, which are especially expressed in plant leaves. Therefore, leaf phenotype is considered to be an important indicator of phenotypic plasticity in plants. The effects of various growth environmental factors on the final size of Arabidopsis thaliana rosette leaves and the number of leaves were analyzed in orthogonal tests using image analysis, and growth curves were estimated statistically. Finally, the optimum growth environment for A. thaliana Col-0 was determined. In this study, temperature, humidity, and light intensity were chosen as factors and studied at the three levels each (temperature: 22 °C, 25 °C, 28 °C; humidity: 50%, 65%, 80%; light intensity: 92 μmol·m−2·s−1; 184 μmol·m−2·s−1; 278 μmol·m−2·s−1). The results showed that light intensity was a major factor in the final leaf size, whereas for the number of plant leaves the most important was temperature. According to the major and minor order of environmental factors, the following scheme appeared to be optimal for A. thaliana growth: temperature 22 °C, humidity 50%, illumination intensity 184 μmol·m−2·s−1.

Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes.

Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning.

Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance.

While the roles of a few specific lipids in plant freezing tolerance are understood, the effect of many plant lipids remains to be determined. Acclimation of plants to non-freezing cold before exposure to freezing temperatures improves the outcome of plants, compared to plants exposed to freezing without acclimation. Arabidopsis thaliana plants were subjected to one of three treatments: (1) “control”, i.e., growth at 21 °C, (2) “non-acclimated”, i.e., 3 days at 21 °C, 2 h at −8 °C, and 24 h recovery at 21 °C, and (3) “acclimated”, i.e., 3 days at 4 °C, 2 h at −8 °C, and 24 h recovery at 21 °C. Plants were harvested at seven time points during the treatments, and lipid levels were measured by direct-infusion electrospray ionization tandem mass spectrometry. Ion leakage was measured at the same time points. To examine the function of lipid species in relation to freezing tolerance, the lipid levels in plants immediately following the freezing treatment were correlated with the outcome, i.e., ion leakage 24-h post-freezing. Based on the correlations, hypotheses about the functions of specific lipids were generated. Additionally, analysis of the lipid levels in plants with mutations in genes encoding patatin-like phospholipases, lipoxygenases, and 12-oxophytodienoic acid reductase 3 (opr3), under the same treatments as the wild-type plants, identified only the opr3-2 mutant as having major lipid compositional differences compared to wild-type plants.

Plastid retrograde regulation of miRNA expression in response to light stress

BackgroundMicroRNAs (miRNAs) are a class of endogenous noncoding RNAs that play a pivotal role in the regulation of plant development and responses to the surrounding environment. Despite the efforts made to elucidate their function in the adaptation of plants to many abiotic and biotic stresses, their role in high light (HL) stress is still vague. HL stress often arises upon plant exposure to full sunlight. Subsequent changes in nuclear gene expression are triggered by chloroplast-derived retrograde signals.ResultsIn this study, we show that HL is involved in miRNA-dependent regulation in Arabidopsis thaliana rosettes. Microtranscriptomic screening revealed a limited number of miRNAs reacting to HL. To explain the miRNA regulation mechanisms at the different biogenesis stages, chemical and genetic approaches were applied. First, we tested the possible role of plastoquinone (PQ) redox changes using photosynthetic electron transport chain inhibitors. The results suggest that increased primary transcript abundance (pri-miRNAs) of HL-regulated miRNAs is dependent on signals upstream of PQ. This indicates that such signals may originate from photosystem II, which is the main singlet oxygen (1O2) source. Nevertheless, no changes in pri-miRNA expression upon a dark–light shift in the conditional fluorescent (flu) mutant producing 1O2 were observed when compared to wild-type plants. Thus, we explored the 1O2 signaling pathway, which is initiated independently in HL and is related to β-carotene oxidation and production of volatile derivatives, such as β-cyclocitral (β-CC). Pri-miRNA induction by β-CC, which is a component of this 1O2 pathway, as well as an altered response in the methylene blue sensitivity 1 (mbs1) mutant support the role of 1O2 signaling in miRNA regulation.ConclusionsWe show that light stress triggers changes in miRNA expression. This stress response may be regulated by reactive oxygen species (ROS)-related signaling. In conclusion, our results link ROS action to miRNA biogenesis, suggesting its contribution to inconsistent pri- and mature miRNA dynamics.

Genetic architecture of variation in Arabidopsis thaliana rosettes.

Rosette morphology across Arabidopsis accessions exhibits considerable variation. Here we report a high-throughput phenotyping approach based on automatic image analysis to quantify rosette shape and dissect the underlying genetic architecture. Shape measurements of the rosettes in a core set of Recombinant Inbred Lines from an advanced mapping population (Multiparent Advanced Generation Inter-Cross or MAGIC) derived from inter-crossing 19 natural accessions. Image acquisition and analysis was scaled to extract geometric descriptors from time stamped images of growing rosettes. Shape analyses revealed heritable morphological variation at early juvenile stages and QTL mapping resulted in over 116 chromosomal regions associated with trait variation within the population. Many QTL linked to variation in shape were located near genes related to hormonal signalling and signal transduction pathways while others are involved in shade avoidance and transition to flowering. Our results suggest rosette shape arises from modular integration of sub-organ morphologies and can be considered a functional trait subjected to selective pressures of subsequent morphological traits. On an applied aspect, QTLs found will be candidates for further research on plant architecture.

An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome

BackgroundRosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses.ResultsTo take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species.ConclusionsWe provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.

Cloning and Functional Analysis of the Soybean GmRIQ2 Promoter

The RIQ gene can optimize the structure of light-harvesting complex II by regulating the degree of granum stacking, and ultimately participates in plant light stress responses. The GmRIQ2 gene contributes to plant photoprotection and may be involved in negative regulation of yield. To understand the regulatory mechanisms of GmRIQ2 expression, we isolated the GmRIQ2 promoter (PGmRIQ2) from the KenFeng 16 soybean genome. Sequence analysis showed that several cis-acting elements were involved in hormone responsiveness, seed-specific regulation, anaerobic induction and light responsiveness. PGmRIQ2 was expressed in soybean roots. GUS activity was detected in Arabidopsis thaliana rosette leaves, stems and roots, indicating that PGmRIQ2 tended to drive expression in rosette leaf vascular tissue and had spatiotemporal expression characteristics at the early growth stage. Three promoter deletion fragments of 450 bp, 284 bp and 194 bp were obtained by 5′ deletion. These three fragments all drove GUS gene expression in Arabidopsis thaliana rosette leaves, stems and roots. GUS gene expression decreased with deletion fragment truncation. In addition, the PGmRIQ2 auxin (IAA), abscisic acid (ABA) and methyl jasmonate (MeJA) response elements were located at base pairs −1661~−450, −450~−284 and −284~−194, respectively, and the promoter was induced by IAA, ABA, MeJA and light.

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery.

SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.

Assessing Protein Synthesis and Degradation Rates in Arabidopsis thaliana Using Amino Acid Analysis.

Plants continually synthesize and degrade proteins, for example, to adjust protein content during development or during adaptation to new environments. In order to estimate global protein synthesis and degradation rates in plants, we developed a relatively simple and inexpensive method using a combination of 13 CO2 labeling and mass spectrometry-based analyses. Arabidopsis thaliana plants are subjected to a 24-hr 13 CO2 pulse followed by a 4-day 12 CO2 chase. Soluble alanine and serine from total protein and glucose from cell wall material are analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and their 13 C enrichment (%) is estimated. The rate of protein synthesis during the 13 CO2 pulse experiment is defined as the rate of incorporation of labeled amino acids into proteins normalized by a correction factor for incomplete enrichment in free amino acid pools. The rate of protein degradation is estimated as the difference between the rate of protein synthesis and the relative growth rate calculated using the 13 C enrichment of glucose from cell wall material. Degradation rates are also estimated from the 12 CO2 pulse experiment. The following method description includes setting up and performing labeling experiments, preparation and measurement of samples, and calculation steps. In addition, an R script is provided for the calculations. 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Setting up the 13 CO2 labeling system and stable isotope labeling of Arabidopsis thaliana rosette leaves Basic Protocol 2: Extraction of soluble amino acids for GC-TOF-MS analysis Basic Protocol 3: Preparation of amino acids from total protein for GC-TOF-MS analysis Basic Protocol 4: Preparation of sugars from cell wall material for GC-TOF-MS analysis Basis Protocol 5: GC-TOF-MS analysis of 13 C-labeled samples and estimation of 13 C enrichment (%) Basis Protocol 6: Estimation of protein synthesis and degradation rates.

Natural variation in Arabidopsis thaliana rosette area unveils new genes involved in plant development

Growth is a complex trait influenced by multiple genes that act at different moments during the development of an organism. This makes it difficult to spot its underlying genetic mechanisms. Since plant growth is intimately related to the effective leaf surface area (ELSA), identifying genes controlling this trait will shed light on our understanding of plant growth. To find new genes with a significant contribution to plant growth, here we used the natural variation in Arabidopsis thaliana to perform a genome-wide association study of ELSA. To do this, the projected rosette area of 710 worldwide distributed natural accessions was measured and analyzed using the genome-wide efficient mixed model association algorithm. From this analysis, ten genes were identified having SNPs with a significant association with ELSA. To validate the implication of these genes into A. thaliana growth, six of them were further studied by phenotyping knock-out mutant plants. It was observed that rem1.2, orc1a, ppd1, and mcm4 mutants showed different degrees of reduction in rosette size, thus confirming the role of these genes in plant growth. Our study identified genes already known to be involved in plant growth but also assigned this role, for the first time, to other genes.

Developmentally controlled changes during Arabidopsis leaf development indicate causes for loss of stress tolerance with age

Leaf senescence is the final stage of leaf development and is induced by the gradual occurrence of age-related changes (ARCs). The process of leaf senescence has been well described, but the cellular events leading to this process are still poorly understood. By analysis of progressively ageing, but not yet senescing, Arabidopsis thaliana rosette leaves, we aimed to better understand processes occurring prior to the onset of senescence. Using gene expression analysis, we found that as leaves mature, genes responding to oxidative stress and genes involved in stress hormone biosynthesis and signalling were up-regulated. A decrease in primary metabolites that provide protection against oxidative stress was a possible explanation for the increased stress signature. The gene expression and metabolomics changes occurred concomitantly to a decrease in drought, salinity, and dark stress tolerance of individual leaves. Importantly, stress-related genes showed elevated expression in the early ageing mutant old5 and decreased expression in the delayed ageing mutant ore9. We propose that the decreased stress tolerance with age results from the occurrence of senescence-inducing ARCs that is integrated into the leaf developmental programme, and that this ensures a timely and certain death.

Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana.

Availability of plant-specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measured in vitro, often under non-physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximal in vivo catalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome-b6f complex, ATP-citrate synthase, sucrose-phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition-specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements from Arabidopsis thaliana rosette with and fluxes through canonical pathways in a constraint-based model of leaf metabolism. In comparison to findings in Escherichia coli, we demonstrate weaker concordance between the plant-specific in vitro and in vivo enzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximal in vivo catalytic rates, and available quantitative metabolomics data are below reported values and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome-wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation.

Interspecific Signaling Between the Parasitic Plant and the Host Plants Regulate Xylem Vessel Cell Differentiation in Haustoria of Cuscuta campestris.

The genus Cuscuta is stem parasitic angiosperms that parasitize a wide range of vascular plants via de novo formation of a distinctive parasitic organ called a haustorium. In the developing haustorium, meristematic cells, which are initiated from the stem cortical tissue, differentiate into haustorial parenchyma cells, which elongate, penetrate into the host tissues, and finally connect with the host vasculature. This interspecific vasculature connection allows the parasite to uptake water and nutrients from the host plant. Although histological aspects of haustorium development have been studied extensively, the molecular mechanisms underlying vasculature development and the interspecific connection with the host vasculature remain largely unknown. To gain insights into the interspecific cell-to-cell interactions involved in haustorium development, we established an in vitro haustorium induction system for Cuscuta campestris using Arabidopsis thaliana rosette leaves as the host plant tissue. The in vitro induction system was used to show that interaction with host tissue was required for the differentiation of parasite haustorial cells into xylem vessel cells. To further characterize the molecular events occurring during host-dependent xylem vessel cell differentiation in C. campestris, we performed a transcriptome analysis using samples from the in vitro induction system. The results showed that orthologs of genes involved in development and proliferation of vascular stem cells were up-regulated even in the absence of host tissue, whereas orthologs of genes required for xylem vessel cell differentiation were up-regulated only after some haustorial cells had elongated and contacted the host xylem. Consistent results were obtained by another transcriptome analysis of the haustorium development in C. campestris undergoing parasitization of an intact host plant. These findings suggest the involvement of host-derived signals in the regulation of non-autonomous xylem vessel differentiation and suggest that its connection to the host xylem during the haustorium development activates a set of key genes for differentiation into xylem vessel cells.

Comparative analysis of nonlinear growth curve models for Arabidopsis thaliana rosette leaves

As a model organism, modeling and analysis of the phenotype of Arabidopsis thaliana (A. thaliana) leaves for a given genotype can help us better understand leaf growth regulation. A. thaliana leaves growth trajectories are to be nonlinear and the leaves contribute most to the above-ground biomass. Therefore, analysis of their change regulation and development of nonlinear growth models can better understand the phenotypic characteristics of leaves (e.g., leaf size) at different growth stages. In this study, every individual leaf size of A. thaliana rosette leaves was measured during their whole life cycle using non-destructive imaging measurement. And three growth models (Gompertz model, logistic model and Von Bertalanffy model) were analyzed to quantify the rosette leaves growth process of A. thaliana. Both graphical (plots of standardized residuals) and numerical measures (AIC, R2 and RMSE) were used to evaluate the fitted models. The results showed that the logistic model fitted better in describing the growth of A. thaliana leaves compared to Gompertz model and Von Bertalanffy model, as it gave higher R2 and lower AIC and RMSE for the leaves of A. thaliana at different growth stages (i.e., early leaf, mid-term leaf and late leaf).

Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System.

Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster ( Mito et al., 2005 ), but the presented protocol has been developed specifically for Arabidopsis thaliana ( Sura et al., 2017 ).

A \u201cDo-It-Yourself\u201d phenotyping system: measuring growth and morphology throughout the diel cycle in rosette shaped plants

BackgroundImprovements in high-throughput phenotyping technologies are rapidly expanding the scope and capacity of plant biology studies to measure growth traits. Nevertheless, the costs of commercial phenotyping equipment and infrastructure remain prohibitively expensive for wide-scale uptake, while academic solutions can require significant local expertise. Here we present a low-cost methodology for plant biologists to build their own phenotyping system for quantifying growth rates and phenotypic characteristics of Arabidopsis thaliana rosettes throughout the diel cycle.ResultsWe constructed an image capture system consisting of a near infra-red (NIR, 940 nm) LED panel with a mounted Raspberry Pi NoIR camera and developed a MatLab-based software module (iDIEL Plant) to characterise rosette expansion. Our software was able to accurately segment and characterise multiple rosettes within an image, regardless of plant arrangement or genotype, and batch process image sets. To further validate our system, wild-type Arabidopsis plants (Col-0) and two mutant lines with reduced Rubisco contents, pale leaves and slow growth phenotypes (1a3b and 1a2b) were grown on a single plant tray. Plants were imaged from 9 to 24 days after germination every 20 min throughout the 24 h light–dark growth cycle (i.e. the diel cycle). The resulting dataset provided a dynamic and uninterrupted characterisation of differences in rosette growth and expansion rates over time for the three lines tested.ConclusionOur methodology offers a straightforward solution for setting up automated, scalable and low-cost phenotyping facilities in a wide range of lab environments that could greatly increase the processing power and scalability of Arabidopsis soil growth experiments.