ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings

A key feature of the development of a higher plant is the continuous formation of new organs from the meristems. Originally patterned during embryogenesis, the meristems must activate cell division de novo at the time of germination, in order to initiate post-embryonic development. In a mutagenesis screen aimed at finding new players in early seedling cell division control, we identified ELONGATA3 (ELO3) as a key regulator of meristem cell cycle activation in Arabidopsis. Our results show that plants carrying a hypomorphic allele of ELO3 fail to activate cell division in the meristems following germination, which leads to seedling growth arrest and lethality. Further analyses suggest that this is due to a failure in DNA replication, followed by cell cycle arrest, in the meristematic tissue. Interestingly, the meristem cell cycle arrest in elo3 mutants, but not the later leaf developmental defects that have been linked to the loss of ELO3 activities, can be relieved by the addition of metabolic sugars in the growth medium. This finding points to a new role by which carbohydrate availability promotes meristem growth. Furthermore, growth arrested elo3 mutants suffer a partial loss of shoot meristem identity, which provides further evidence that cell cycle activities can influence the control of tissue identity.     

Inflorescence abnormalities occur with overexpression of Arabidopsis lyrata FT in the fwa mutant of Arabidopsis thaliana

Arabidopsis thaliana is a quantitative long-day plant with the timing of the floral transition being regulated by both endogenous signals and multiple environmental factors. fwa is a late-flowering mutant, and this phenotype is due to ectopic FWA expression caused by hypomethylation at the FWA locus. The floral transition results in the activation of the floral development process, the key regulators being the floral meristem identity genes, AP1 (APETALA1) and LFY (LEAFY). In this study, we describe inflorescence abnormalities in plants overexpressing the Arabidopsis lyrata FT (AlFT) and A. thaliana FWA (AtFWA) genes simultaneously. The inflorescence abnormality phenotype was present in only a proportion of plants. All plants overexpressing both AlFT and AtFWA flowered earlier than fwa, suggesting that the inflorescence abnormality and earlier flowering time are caused independently. The inflorescence abnormality phenotype was similar to that of the double mutant of ap1 and lfy, and AP1 and LFY genes were down-regulated in the abnormal inflorescences. From these results, we suggest that not only does ectopic AtFWA expression inhibit AtFT/AlFT function to delay flowering but that overexpression of AtFWA and AlFT together inhibits AP1 and LFY function to produce abnormal inflorescences.     

Identification of candidate genes in Arabidopsis and Populus cell wall biosynthesis using text-mining, co-expression network analysis and comparative genomics

Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of biofuels from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidence supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database, and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional characterization in relation to cell wall biosynthesis.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-μm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm⁻¹, differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana

Fatty acid desaturases play important role in plant responses to abiotic stresses including cold, high temperature, drought, and osmotic stress. In this work, we provide the evidence that Fad6, a chloroplast desaturase, is required for salt tolerance during the early seedling development of Arabidopsis. Expression of Fad6 was responsive to salt and osmotic stress. Compared with the wild-type plants, the fad6 mutant showed reduced tolerance to salt stress, and accumulated more Na⁺ and less K⁺ under high NaCl stress condition. Furthermore, cellular oxidative damage was more severe in fad6 when treated with high concentrations of NaCl, as indicated by increased electrolyte leakage rate and malondialdehyde production, as well as by decreased activities of anti-oxidative enzymes. All these results suggest that Fad6 is required for salt resistance in Arabidopsis.     

Quantitative analysis of herbivore-induced cytosolic calcium by using a Cameleon (YC 3.6) calcium sensor in Arabidopsis thaliana

Ca²⁺ is a key player in plant cell responses to biotic and abiotic stress. Owing to the central role of cytosolic Ca²⁺ ([Ca²⁺]_cyt) during early signaling and the need for precise determination of [Ca²⁺]_cyt variations, we used a Cameleon YC 3.6 reporter protein expressed in Arabidopsis thaliana to quantify [Ca²⁺]_cyt variations upon leaf mechanical damage (MD), herbivory by 3rd and 5th instar larvae of Spodoptera littoralis and S. littoralis oral secretions (OS) applied to MD. YC 3.6 allowed a clear distinction between MD and herbivory and discriminated between the two larvae instars. To our knowledge this is the first report of quantitative [Ca²⁺]_cyt determination upon herbivory using a Cameleon calcium sensor.     

The Arabidopsis thaliana flower organ specification gene regulatory network determines a robust differentiation process

The Arabidopsis thaliana flower organ specification gene regulatory network (FOS-GRN) has been modeled previously as a discrete dynamical system, recovering as steady states configurations that match the genetic profiles described in primordial cells of inflorescence, sepals, petals, stamens and carpels during early flower development. In this study, we first update the FOS-GRN by adding interactions and modifying some rules according to new experimental data. A discrete model of this updated version of the network has a dynamical behavior identical to previous versions, under both wild type and mutant conditions, thus confirming its robustness. Then, we develop a continuous version of the FOS-GRN using a new methodology that builds upon previous proposals. The fixed point attractors of the discrete system are all observed in the continuous model, but the latter also contains new steady states that might correspond to genetic activation states present briefly during the early phases of flower development. We show that both the discrete and the continuous models recover the observed stable gene configurations observed in the inflorescence meristem, as well as the primordial cells of sepals, petals, stamens and carpels. Additionally, both models are subjected to perturbations in order to establish the nature of additional signals that may suffice to determine the experimentally observed order of appearance of floral organs. Our results thus describe a possible mechanism by which the network canalizes molecular signals and/or noise, thus conferring robustness to the differentiation process.     

Metabolomics-oriented isolation and structure elucidation of 37 compounds including two anthocyanins from Arabidopsis thaliana

In order to conduct metabolomic studies in a model plant for genome research, such as Arabidopsis thaliana (Arabidopsis), it is a prerequisite to obtain structural information for the isolated metabolites from the plant of interest. In this study, we isolated metabolites of Arabidopsis in a relatively non-targeted way, aiming at the construction of metabolite standards and chemotaxonomic comparison. Anthocyanins (5 and 7) called A8 and A10 were isolated and their structures were elucidated as cyanidin 3-O-[2-O-(β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-E-p-coumaroyl)-β-d-glucopyranoside]-5-O-[6-O-(malonyl)-β-d-glucopyranoside] and cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)-β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-E-p-coumaroyl)-β-d-glucopyranoside]-5-O-[β-d-glucopyranoside] from analyses of 1D NMR, 2D NMR (¹H NMR, NOE, ¹³C NMR, HMBC and HMQC), HRFABMS, FT-ESI-MS and GC-TOF-MS data. In addition, 35 known compounds, including six anthocyanins, eight flavonols, one nucleoside, one indole glucosinolate, four phenylpropanoids and a derivative, together with three indoles, one carotenoid, one apocarotenoid, three galactolipids, two chlorophyll derivatives, one steroid, one hydrocarbon, and two dicarboxylic acids, were also isolated and identified from their spectroscopic data.