Arabidopsis Thaliana Callus Research Articles

Effects of nitrogen deficiency and drought stresses on miRNA expressions in Arabidopsis thaliana

MicroRNAs are now known to have an important role in regulating gene expression of eukaryotic organisms. miRNA research in plants gained importance after the discovery that several stress factors alter certain miRNA expressions, which subsequently regulate their target gene expressions and affect development and growth of plants. In this study, two of the widely studied abiotic stress conditions for plants, nitrogen deficiency and drought were used individually and as a combined stress treatment on Arabidopsis thaliana callus tissues to observe the change of expressions in certain miRNAs, when multiple stressors are encountered. Combined stress strongly inhibited callus growth compared to other conditions, while strongly altering certain miRNA expressions. Compared to control, in 7-day stress treated callus, miR165a-3p,b, miR319a,b, miR396b-5p, miR399d and miR827 showed significant downregulation for all stress treatments, while 7-day N deficiency caused miR167 upregulation. Stress treatments for 7 days mostly downregulated miR167c-5p, miR319c, miR399a, miR399e expressions except for the N deficient samples. After 14 days of stress, miR165a-3p,b, miR396a-5p, miR399b, miR399d were downregulated. During 14-day drought and combined stress, miR399a and miR396b-5p expressions were upregulated, respectively. The differences observed in this study between stress responses of 7 and 14-day-long treatments are believed to be valuable to further elucidate the associated molecular mechanisms of miRNAs, determination and validation of miRNA targets, and how plants respond to stress conditions via various genetic regulations.

Auxin resistant 1 gene (AUX1) mediates auxin effect on Arabidopsis thaliana callus growth by regulating its content and distribution pattern

Callus sustained growth relies heavily on auxin, which is supplied to the culture medium. Surprisingly, there is a noticeable absence of information regarding the involvement of carrier-mediated auxin polar transport gene in callus growth regulation. Here, we delve into the role of the AUXIN RESISTANT 1 (AUX1) influx transporter in the regulation of callus growth, comparing the effects under conditions of light versus darkness. It was observed that callus growth was significantly enhanced under light illumination. This growth-stimulatory effect was accompanied by a decrease in the levels of free auxin within the callus cells when compared to conditions of darkness. In the aux1–22 mutant callus, which lacks functional AUX1, there was a substantial reduction in IAA levels. Nonetheless, the mutant callus exhibited markedly higher growth rates compared to the wild type. This suggests that the reduction in exogenous auxin uptake through the AUX1-dependent pathway may prevent the overaccumulation of growth-restricting hormone concentrations. The growth-stimulatory effect of AUX1 deficiency was counteracted by nonspecific auxin influx transport inhibitors. This finding shows that other auxin influx carriers likely play a role in facilitating the diffusion of auxin from the culture medium to sustain high growth rates. AUX1 was primarily localized in the plasma membranes of the two outermost cell layers of the callus clump and the parenchyma cells adjacent to tracheary elements. Significantly, these locations coincided with the regions of maximal auxin concentration. Consequently, it can be inferred that AUX1 mediates the auxin distribution within the callus.

Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration.

In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

Nanoscale Polyion Complex Vesicles for Delivery of Cargo Proteins and Cas9 Ribonucleoprotein Complexes to Plant Cells

DNA-free genome editing using Cas9 ribonucleoprotein is advantageous because it introduces fewer potential undesirable genetic modifications than comparable methods. Here, we show a direct protein delivery system for plants using a cell-penetrating peptide-displayed polyion complex vesicle, CPP-PICsome, as a biological nanocarrier. With this system, we demonstrate nuclear genome editing by delivery of a Cas9 ribonucleoprotein complex in Arabidopsis thaliana callus.

Role of strigolactones in adventitious rooting of Arabidopsis thaliana callus in vitro

Role of strigolactones in adventitious rooting of Arabidopsis thaliana callus in vitro

A cross-kingdom assay model for evaluating estrogenic activity: application of transgenic Arabidopsis thaliana callus

Phytoestrogens have attracted attention in the last few decades as potential therapeutic candidates targeting several ailments including osteoporosis and menopausal syndrome. Many herbal preparations possess phytoestrogenic activity without solid evidence due the lack of an easy and feasible assay protocol in evaluating their activity. In 2005, the first report of a cross-kingdom bioassay using transgenic Arabidopsis assay system was published by our group to evaluate the estrogenic activity of potent natural products. In the current investigation, calli induced from the Arabidopsis pER8:GUS line were utilized for the first time to evaluate the estrogenic activity of 17β-estradiol. Calli, induced on solid 1DK1 medium, showed significant GUS activity in a dose-dependent manner, and the minimum detectable concentration of 17β-estradiol was 0.0001 µM. Furthermore, calli from liquid media ɩ1N1B and ɩ1/2 1N1B produced by suspension culture exhibited significant sensitivity towards 17β-estradiol in a dose-dependent manner. However, calli of ɩ1NK5, ɩ1/2 1NK5, ɩ1DK1, or ɩ1/2 1DK1 media could not show reliable activity toward GUS expression, which was demonstrated by the irregular fluorometric levels. Calli production avoided gene variation from the sexual generational shift and thus ensured uniformity and accuracy of the results. The optimal methods of transgenic calli induction, production, and estrogenic activity assay were established. This method can be used to investigate new sources of phytoestrogens and to detect environmental estrogenic compounds. This is the first developed protocol specialized in evaluating animal function using plant callus.

Changes in Plastid and Mitochondria Protein Expression in Arabidopsis Thaliana Callus on Board Chinese Spacecraft SZ-8

Microgravity represents an adverse abiotic environment, which causes rearrangements in cellular organelles and changes in the energy metabolism of cells. Plastids and mitochondria are two subcellular energy organelles that are responsible for major metabolic processes, including photosynthesis, oxidative phosphorylation, s-oxidation, and the tricarboxylic acid cycle. In our previous study performed on board the Chinese spacecraft SZ-8, we evaluated the global changes exerted by microgravity on the proteome of Arabidopsis thaliana cell cultures by comparing the microgravity-exposed samples with the controls either under 1 g centrifugation in space or 1 g ground conditions. Here, we report additional data from this space experiment that highlights the plastid and mitochondria proteins that responded to space flight conditions. We observed that 43 plastidial proteins and 50 mitochondrial proteins changed their abundances under microgravity in space. The major changes in both plastids and mitochondria involved proteins that functions in a suite of redox antioxidant and metabolic pathways. These results suggested that these antioxidant and metabolic changes in plastids and mitochondria could be important components of the adaptive strategy in plants subjected to microgravity in space.

Differential protein expression profiling of Arabidopsis thaliana callus under microgravity on board the Chinese SZ-8 spacecraft.

Exposure of Arabidopsis callus to microgravity has a significant impact on the expression of proteins involved in stress responses, carbohydrate metabolism, protein synthesis, intracellular trafficking, signaling, and cell wall biosynthesis. Microgravity is among the main environmental stress factors that affect plant growth and development in space. Understanding how plants acclimate to space microgravity is important to develop bioregenerative life-support systems for long-term space missions. To evaluate the spaceflight-associated stress and identify molecular events important for acquired microgravity tolerance, we compared proteomic profiles of Arabidopsis thaliana callus grown under microgravity on board the Chinese spacecraft SZ-8 with callus grown under 1g centrifugation (1g control) in space. Alterations in the proteome induced by microgravity were analyzed by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry with isobaric tags for relative and absolute quantitation labeling. Forty-five proteins showed significant (p < 0.05) and reproducible quantitative differences in expression between the microgravity and 1g control conditions. Of these proteins, the expression level of 24 proteins was significantly up-regulated and that of 21 proteins was significantly down-regulated. The functions of these proteins were involved in a wide range of cellular processes, including general stress responses, carbohydrate metabolism, protein synthesis/degradation, intracellular trafficking/transportation, signaling, and cell wall biosynthesis. Several proteins not previously known to be involved in the response to microgravity or gravitational stimuli, such as pathogenesis-related thaumatin-like protein, leucine-rich repeat extension-like protein, and temperature-induce lipocalin, were significantly up- or down-regulated by microgravity. The results imply that either the normal gravity-response signaling is affected by microgravity exposure or that microgravity might inappropriately induce altered responses to other environmental stresses.

Hyperaccumulation of arsenic by callus, sporophytes and gametophytes of Pteris vittata cultured in vitro

The callus of Pteris vittata was induced from gametophytes generated from spores in vitro, and grew rapidly with periodical medium change. Arsenic tolerance and accumulation of P. vittata callus were compared with those of Arabidopsis thaliana callus. Cell death was not detected in P. vittata callus even at arsenate concentrations up to 2 mM; however, A. thaliana callus died at low (0.2 mM) arsenate concentrations. Meanwhile, P. vittata callus accumulated almost three times more As than A. thaliana callus when exposed to 0.2 mM arsenate. About 60% of the total As was removed when 7.5 g of P. vittata callus was cultured on 150 ml of half-strength MS liquid medium containing 450 microg As for 2 days. Furthermore, P. vittata callus, sporophytes, and gametophytes all grew well under 1 mM of arsenate and accumulated 1,250; 1,150 and 2,180 mg kg(-1 )dry weight As when grown on 2 mM arsenate for 15 or 30 days. The characteristics of non-differentiated cells, large biomass, ease of culture, good synchronization, and excellent As sequestering, make the callus of P. vittata a new ideal system to study the mechanisms of As hyperaccumulation and phytoremediation in As-contaminated groundwater.

A proteomic approach to analysing responses of Arabidopsis thaliana callus cells to clinostat rotation

Callus cells of Arabidopsis thaliana (cv. Landsberg erecta) were exposed for 8 h to a horizontal clinostat rotation (H, simulated weightlessness), a vertical clinostat rotation (V, clinostat control), or a stationary control (S) growth condition. The amount of glucose and fructose apparently decreased, while starch content increased in the H compared with the V- and S-treated cells. In order to investigate the influences of clinostat rotation on the cellular proteome further, the proteome alterations induced by horizontal and vertical clinostat rotation have been comparatively analysed by high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 80 protein spots showed quantitative and qualitative variations that were significantly (P <0.01) and reproducibly different between the clinorotated (H or V) and the stationary control samples. Protein spots excised from 2-D gels were analysed by microbe high performance liquid chromatography-ion trap-mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. 18 protein spots, which showed significant expression alteration only under the H condition compared with those under V and S conditions, were identified. Of these proteins, seven were involved in stress responses, and four protein spots were identified as key enzymes in carbohydrate metabolism and lipid biosynthesis. Two reversibly glycosylated cell wall proteins were down-regulated in the H samples. Other proteins such as protein disulphide isomerase, transcription initiation factor IIF, and two ribosomal proteins also exhibited altered expression under the H condition. The data presented in this study illustrate that clinostat rotation of Arabidopsis callus cells has a significant impact on the expression of proteins involved in general stress responses, metabolic pathways, gene activation/transcription, protein synthesis, and cell wall biosynthesis.

A proteomic analysis of organelles from Arabidopsis thaliana.

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.