Biochemical and Physiological Responses of Arabidopsis thaliana Leaves to Moderate Mechanical Stimulation

Mechanical stimulation of plants can be caused by various abiotic and biotic environmental factors.Apart from the negative consequences,it can also cause positive changes,such as acclimatization of plants to stress conditions.Therefore,it is necessary to study the physiological and biochemical mechanisms underlying the response of plants to mechanical stimulation.Our aim was to evaluate the response of model plant Arabidopsis thaliana to a moderate force of 5 N(newton)for 20 s,which could be compared with the pressure caused by animal movement and weather conditions such as heavy rain.Mechanically stimulated leaves were sampled 1 h after exposure and after a recovery period of 20 h.To study a possible systemic response,unstimulated leaves of treated plants were collected 20 h after exposure alongside the stimulated leaves from the same plants.The effect of stimulation was assessed by measuring oxidative stress parameters,antioxidant enzymes activity,total phenolics,and photosynthetic performance.Stimulated leaves showed increased lipid peroxidation 1 h after treatment and increased superoxide dismutase activity and phenolic oxidation rate after a 20-h recovery period.Considering photosynthetic performance after the 20-h recovery period,the effective quantum yield of the photosystem II was lower in the stimulated leaves,whereas photochemical quenching was lower in the unstimulated leaves of the treated plants.Nonphotochemical quenching was lower in the stimulated leaves 1 h after treatment.Our study suggested that plants sensed moderate force,but it did not induce pronounced change in metabolism or photosynthetic performance.Principal component analysis distinguished three groups–leaves of untreated plants,leaves analysed 1 h after stimulation,while stimulated and unstimulated leaves of treated plants analysed 20 h after treatment formed together the third group.Observed grouping of stimulated and unstimulated leaves of treated plants could indicate signal transduction from the stimulated to distant leaves,that is,a systemic response to a local application of mechanical stimuli.

Glucosinolates and Their Hydrolysis Products in Arabidopsis thaliana Influence Performance and Feeding Choice of Pieris rapae and Spodoptera exigua

Glucosinolates and their hydrolysis products, found in plants of the order Brassicales, are well-known for their defensive properties against insect herbivores. Arabidopsis thaliana (Col-0) genetic lines with mutations that modify the type of glucosinolates (i.e. myb28myb29 and cyp79B2cyp79B3 are deficient in the production of aliphatic and indolyl glucosinolates, respectively) make it possible to test for the specific effects of these secondary chemicals on insect herbivores. The Pad3 mutant (deficient in camalexin), which has a role in resistance to pathogens, was also tested. Likewise, the effects of different glucosinolate hydrolysis products can be evaluated using genetically modified (GM) lines of the wild type Col-0 ecotype, which naturally produces isothiocyanates. These GM lines include the nitrile-producing 35S: ESP and the double knockout tgg1tgg2, which virtually lacks hydrolysis products. In both no-choice and choice experiments, the crucifer specialist Pieris rapae was virtually unaffected by differences in the type of glucosinolates or hydrolysis products. In contrast, the generalist insect Spodoptera exigua had statistically significant increases in pupae/adult weight and faster developmental times when reared on mutants deficient in the production of aliphatic and indolyl glucosinolates and their hydrolysis products. There were no differences in the performance of either insect species when reared on wild type Col-0 or Pad3. Results from feeding choice trials showed that Pieris rapae had no statistically significant preference for any of the genetic lines. In contrast, Spodoptera exigua had a significant feeding preference for the double mutant tgg1tgg2. This study provides evidence that variation in the type of glucosinolates and their hydrolysis products can influence insect performance and feeding choices, and that responses are species-specific.

Physiological Analysis of Two Arabidopsis thaliana Mutants in Response to CO2

[Objective] The 15urpose was to seek for the different phenotypes between wild type and Arabidopsis Mutants in response to CO2. [Method] The epidermis bioassays and seed germination test were carried out to analyze the physiological characteristics of two Arabidopsis mu- tants and their wild type. [Result] There existed distinct differences in stomata apertures, water loss and leaf temperature compared with wild type except for stomata density. In addition, seed germination test on the medium indicated that cdfl was insensitive to ABA, mannitol and NaCI, but cdsl performed contrary to cdil. [ Conclusion] There are some different physiological characteristics between wild type and mutants.

A Rapid DNA Extraction Method for PCR Detection of Arabidopsis thaliana

[Objective] The aim was to introduce a rapid DNA extraction method for PCR detection of Arabidopsis thaliana.[Method] Through the improvement of conventional DNA extraction method,a rapid Arabidopsis thaliana DNA extraction method was obtained.With randomly selected Arabidopsis thaliana transgenic strains and mutants as samples,the method was verified.[Result] After electrophoresis,UV absorption detection,it was found that DNA samples are complete and less pollution,and the result of PCR amplification objective fragment was good which proved DNA is suitable as a template for PCR reaction.After PCR detection,positive plants gene amplified bands were clear,without false-positive,and the test results were satisfactory.[Conclusion] The method is suitable for rapid extraction of Arabidopsis thaliana DNA and PCR detection.

Effects of Drought Stress Simulated by PEG on Glucosinolates Content in Arabidopsis thaliana

[Objective] The paper aimed to study effects of drought stress simulated by PEG on glucosinolates content in Arabidopsis thaliana.[Method] Drought stress was simulated by PEG-6000,ecological seeds of Arabidopsis thaliana were cultivated by the control group and drought treatment group respectively,Physical signs of Arabidopsis thaliana and contents of glucosinolates were determined after 0,4,5,6,7 d treatment.[Result] The results showed that leaf water content of rosette leaves was obviously decreased,leaf relative conductivity （characterized by membrane permeability） and the concentration of MDA increased,the extent of damage increased with the increased time.Content of total glucosinolate,aliphatic glucosinolate and indole glucosinolate increased got their maximum after 5 days treatment,and rapidly decreased after 6 and 7 days of treatment,even much lower than the control group.Each kind of glucosinolate changed with difference from each other.4MSOB which made the most proportion of the total glucosinolate changed consistently with the total glucosinolate and difference significant.As a whole,aliphatic glucosinolates were more sensitive to drought than indole glucosinolate.The proportion of some kind glucosinolate,like 4MSOB varied had correlation with the content change.[Conclusion] Drought stress had an effects on the contents of total glucosinolate,aliphatic glucosinolate and indole glucosinolate,which made the glucosinolate participated in defense response of plant to the outside of drought stress,but long-term drought stress in leaves was not conducive to the accumulation of glucosinolates.

Ultrastructural Studies on the Sieve Elements in Root Protophleom of Arabidopsis thaliana

The ultrastructures of the root protophleom sieve element at different developmental stages of Arabidopsis thaliana L. were investigated using the technique of high pressure freezing and freeze substitution fixing specimen. The results show that in the development of the sieve elements, the nuclei undergo typical characteristics of the programmed cell death (PCD): the nuclear envelopes form emboli, the chromatin condenses and aggregates towards the nuclear envelope, which degrades and fully disappears later. Before the nucleus degradation, neither the nuclear envelope undulation, nucleus lobe nor marked dilation (or bleb) of perinuclear space could be observed. In the cytoplasm of the mature sieve element, there are starch-like granules separately sheathed with a layer of membrane and usually with mitochondria around. These gnanules seem to provide substrates to mitochondria in their function. Small vacuoles originate from endoplasmic reticulum (ER), and no bigger vacuole was found.

Effects of Salt Stress on Epidermal Cell Expansion in Leaves of Arabidopsis thaliana

[Objective] The aim of this study was to investigate the effects of salt stress on cell expansion in Arabidopsis thaliana rosette leaves.[Method] Arabidopsis seedlings were treated by sodium chloride at the concentration of 0,100 or 150 mmol/L. At the 7th and 14th d of treatment,with nail enamel printing mark method and computer software,the leaf blades area and abaxial epidermal pavement cells area was measured and compared using statistical analysis in Excel. [Result] The growth of Arabidopsis rosette leaves was inhibited under salt stress. Leaves treated for 7 or 14 d expanded less compared with controls. The salt-mediated decrease in leaf expansion is associated with a decrease in abaxial pavement cell expansion. [Conclusion] The decreased leaf and epidermal cell expansion under salt stress is the most important characteristic of plant physiological response to salt stress.

Cytological Studies on the Development of Sieve Element and Floral Nectary Tissue in Arabidopsis thaliana

Ultrastructural changes in the sieve element and dense cells of nectariferous tissue during the development of floral nectary in Arabidopsis thaliana L. were investigated with transmission electron microscopy. Samples were prepared with high pressure freezing and freeze substitution techniques. The ultrastructure of dense cell was similar to that of sieve element at its early developmental stage. With the concurrent agglutination of chromatin in the nucleus, the abnormal location of organelles and the high density of cytoplasm, the ultrastructural characteristics in die dense cells of the nectariferous tissue and in the sieve element are matched with those of the programmed cell death in animal and plant reported in recent years. The disorganization of nucleus and most organelles in the differentiation of sieve elements and dense cells is closely associated with the transportation and modification of pre-nectar and the transference of nectar. This suggests that the cytological changes in sieve element and nectariferous tissue are closely associated with the nectary functional activities.

Different Ecological FLC Gene in the Arabidopsis thaliana Sequence Analysis

[Objective]Analysis of FLC sequence that Vernalization-related genes in Arabidopsis.[Method]Advance through natural populations of Arabidopsis QTL analysis of vernalization response was found on chromosome 5 of Arabidopsis thaliana have a flowering-related QTL,this test is to use sequence analysis to determine whether it is with the FLC gene homology.[Result]Arabidopsis thaliana,Italy and Sweden in the 27th,No.461,p.501,p.638,p.738,No.884 different base.While these bases are different,but the codon encoding the first nine amino acids out of the first 167 amino acids,the first 246 amino acids,due to codon degeneracy,encode proteins that are the same.[Conclusion] Arabidopsis has a rich genetic diversity,the FLC gene is highly conserved sequence length,base variable sites rich degenerate codons encoding the same amino acids they are not affect the growth of Arabidopsis thaliana.This indicates that the Arabidopsis genome sequence will be the environment.

Regulation of Heat Shock Factor AtHsfA1a on Ascorbate Peroxidase under Heat Stress in Arabidopsis thaliana

[Objective] This study was conducted to investigate the regulation of heat shock factor AtHsfA1a on ascorbate peroxidase under heat stress in Arabidopsis thaliana. [Method] After heat stress treatment on transgenetic A. thaliana with silenced endogenetic AtHsfA1a gene and wild A. thaliana plants as materials, the change in activity of APX enzyme was analyzed by spectrophotometry, the expression level of APX gene was investigated by real-time fluorescent quantitative PCR, and the binding condition of AtHsfAla with the promoter region of APX gene was analyzed by chromatin immunoprecipitation assay. [Result] The activity and mRNA level of APX in plants with silenced endogenetic AtHsfAla gene were higher than those in wild plants. Fragments of the promoter region of APX gene were not screened from the plants with silenced endogenetic AtHsfA1a gene, but found in wild plants. [Conclusion] This study provides a theoretical basis for the understanding of the important role of AtHsfAla in resistance to stress in plant, and is of great significance to the revealing of mechanism of resistance to stress in plant.

Cloning and Expression Analysis of the High Mobility B Group Genes in Arabidopsis thaliana

[Objective] The aim was to better research the function and action mode of High Mobility Group B （HMGB） proteins in higher plants. [Method] At2G33450,At5G23405 and At5G23420 genes of HMGB protein family in Arabidopsis thaliana were cloned by the use of RT-PCR method,and the expression of these three proteins in E.coli and Arabidopsis thaliana were detected by using SDS-PAGE,Northern blot and subcellular localization methods. [Result] The results showed that the molecular weights of the three proteins were 17.5,17.0 and 27.0 kD respectively,and the expression levels of the proteins in Arabidopsis thaliana were At5G23420At5G23405At2G33450. In addition,all the three proteins were located in nucleus. [Conclusion] The study will provide a basis for the further research on the biological function of HMGB proteins in higher plants.

Study on the Callus Induction of Wild Arabidopsis thaliana Leaves

[Objective] The callus induction of wild Arabidopsis thaliana leaves was studied.[Method] As explants,leaf segments of wild A.thaliana were inoculated in MS medium including 6-BA and NAA with different concentration,and studying the callus induction and regenerated plant of A.thaliana.[Result]6-BA was necessary for callus induction,but higher concentration resulted into vitrification easily;single use of NAA was beneficial to rooting,and bud differentiation was easy in the medium with NAA and 6-BA;the optimum medium of callus induction was MS ＋0.5 mg/L 6-BA ＋0.10 mg/L NAA,with the callus induction frequency of 100%.[Conclusion]Our study could lay a foundation for the genetic transformation and cell culture of A.thaliana.

Quercetin Promotes Auxin Transport in Arabidopsis thaliana

Study on the role of quereentin in polar auxin transportation. Arabidopsis was cultured on medium supplemented with quereetin to observe the growth of hypoeotyls, ^14C-IAA transport assays were conducted to measure the auxin transport activity. The results showed that Arabidopsis mutant auxl which had been deficient in auxin influx transportion obviously recovered the ability after eultured on the medium with quercetin. The polar auxin transport was promoted by the addition of quereetin. These results indicated that quereetin could promote polar auxin transport in vivo.

Predicting Arabidopsis thaliana Gene Function by Transitiving Co-expression in Shortest-path

The present paper predicted the function of unknow genes by analyzing the co-expression data of Arabidopsis thaliana from biological pathway based on the shortest-path algorithm. This paper proposed that transitive co-expression among genes can be used as an important attribute to link genes of the same biological pathway. The genes from the same biological pathway with similar functions are strongly correlated in expression. Moreover,the function of unknown genes can be predicted by the known genes where they are strongly correlated in expression lying on the same shortest-path from the biological pathway. Analyzing the Arabidopsis thaliana from the biological pathway,this study showed that this method can reliably reveal function of the unknown Arabidopsis thaliana genes and the approach of predicting gene function by transitiving co-expression in shortest-path is feasible and effective.

Mechanisms of Salt Tolerance in Transgenic Arabidopsis thaliana Carrying a Peroxisomal Ascorbate Peroxidase Gene from Barley

Ascorbate peroxidases （APX）, localized in the cytosol, peroxisome, mitochondria, and chloroplasts of plant cells, catalyze the reduction of H2O2 to water by using ascorbic acid as the specific electron donor. To determine the role of peroxisomal type ascorbate peroxidase （pAPX）, an antioxidant enzyme, in protection against salt-induced oxidative stress, transgenic Arabidopsis thaliana plant carrying a pAPX gene （HvAPX1） from barley （Hordeum vulgate L.） was analyzed. The transgenic line pAPX3 was found to be more tolerant to salt stress than the wild type. Irrespective of salt stress, there were no significant differences in Na^＋, K^＋, Ca^2＋, and Mg^2＋ contents and the ratio of K^＋ to Na^＋ between pAPX3 and the wild type. Clearly, the salt tolerance in pAPX3 was not due to the maintenance and reestablishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation （measured as the levels of malondialdehyde） accumulation under salt stress was higher in the wild type than in pAPX3. The mechanism of salt tolerance in transgenic pAPX3 can thus be explained by reduction of oxidative stress injury. Under all conditions tested, activities of superoxide, glutathione reductase, and catalase were not significantly different between pAPX3 and the wild type. In contrast, the activity of APX was significantly higher in the transgenic plant than in wild type under salt stress. These results suggested that in higher plants, HvAPX1 played an important role in salt tolerance and was a candidate gene for developing salttolerant crop plants.

Regulatory Function of Polyamine Oxidase-Generated Hydrogen Peroxide in Ethylene-Induced Stomatal Closure in Arabidopsis thaliana

Hydrogen peroxide （H2O2） is an important signaling molecule in ethylene-induced stomatal closure in Arabidopsis thaliana. Early studies on the sources of H2O2 mainly focused on NADPH oxidases and cell-wall peroxidases. Here, we report the involvement of polyamine oxidases （PAOs） in ethylene-induced H2O2 production in guard cells. In Arabidopsis epidermal peels, application of PAO inhibitors caused the failure of ethylene to induce H2O2 production and stomatal closure. Results of quantitative RT-PCR analysis and pharmacological experiments showed that AtPAO2 and AtPAO4 transcripts and activities of PAOs were both induced by ethylene. In transgenic Arabidopsis plants over-expressing AtPAO2 and AtPAO4, stomatal movement was more sensitive to ethylene treatment and H2O2 production was also significantly induced. The increased H2O2 production in the transgenic lines compared to the wild-type plants suggests that AtPAO2 and AtPAO4 probably are involved in ethylene-induced H2O2 production. Several factors which induce stomatal closure such as dehydration and high salinity all enhanced the expression of AtPAO2 and AtPAO4 to different degrees. Moreover, GFP- AtPAOs fusion protein localized in the nucleus, cytoplasm, and cell wall of the guard cells. Therefore, our results strongly indicated that PAO is a source of H2O2 generation in Arabidopsis guard cells and plays crucial roles in stomatal movement.

Abiotic Stresses and Phytohormones Regulate Expression of FAD2 Gene in Arabidopsis thaliana

Modification of unsaturated fatty acid （FA） levels has been found to accompany multiple abiotic stress acclimations in many plants. Delta 12 fatty acid desaturase （FAD2） plays a critical role in the synthesis of polyunsaturated FAs in plant cells by converting oleic acid （18：1） to linoleic acid （18：2）. To better understand the relationship between polyunsaturated FAs metabolism and stress adaptation, the expression of FAD2 gene and changes in the FA compositions under various abiotic stresses and phytohormone treatments in Arabidopsis thaliana was investigated in this study. A 1 423-bp promoter of the FAD2 gene was cloned and characterized from Arabidopsis. Several putative hormone- and stress- inducible cis-elements were identified in the cloned promoter, which include salt- and pathogen-inducible GT-1 motifs, low-temperature-responsive MYC element, dehydration-responsive MYB element, and GA signaling related WRKY71OS element. To investigate the fine regulation of FAD2 gene, a recombinant FAD2 promoter-GUS construct was introduced into Arabidopsis plants. Histochemical study showed that the promoter was ubiquitously active and responsive not only to exogenous phytohormones including ABA, 24-eBL, and SA but also to darkness, temperature, salt, and sucrose stresses in Arabidopsis seedlings. Consistent with the expression change, treatments with exogenous 24-eBL, ABA, SA, and NaCl resulted in reduction in polyunsaturated FAs in Arabidopsis seedlings. These findings suggest that the FAD2 gene with a wide variety of putative response elements in its promoter is responsive to multiple phytohormones and abiotic stresses and therefore may play an important role in stress responses of Arabidopsis during plant growth and seed development.

Bio-toxicity Effect of Polycyclic Aromatic Hydrocarbons Pyrene on Arabidopsis thaliana

[Objective] The aim was to study the effects of polycyclic aromatic hydrocarbons（PAHs）pyrene on the physiology and biochemistry of plant.[Method] Taking model plant A.thaliana as material,the effects of 4-ring PAHs pyrene stress on A.thaliana growth were studied,as well as the changes of antioxidant enzyme activity and ultrastructure in leaves.[Result] Under pyrene stress,the root growth of A.thaliana was enhanced under low concentration but was inhibited under high concentration.Trichomes of A.thaliana leaves distorted with the increase of pyrene concentration.Under 0.25 mmol/L of pyrene stress for 28 d,the photosynthetic process of A.thaliana was inhibited,and chlorophyll content in A.thaliana leaves decreased significantly.With the increase of pyrene concentration,the activity of peroxidase（POD）,superoxide dismutase（SOD）and ascorbate peroxidase（APX）in A.thaliana leaves increased significantly,while catalase（CAT）activity went up firstly and then went down.In addition,average H2O2 content in A.thaliana leaves under different pyrene concentrations were higher than that of control.Meanwhile,damaged ultrastructures of few chloroplasts in A.thaliana leaves under pyrene stress were observed,and photosynthesis was inhibited significantly.[Conclusion] The study could provide references for the toxic mechanism of PAHs on plant and plant selection in the phytoremediation of PAHs contaminated soil.

Induction of defence gene expression by oligogalacturonic acid requires in-creases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings expressing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca2+]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H2O2. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca2+]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.

Effects of temperature on UV-B-induced DNA damage and photorepair in Arabidopsis thaliana

DNA damage in the form of cyclobutane pyrimidine dimers(CPDs) and (6-4) photoproducts(6-4PPs) induced by UV-B radiation in Arabidopsis thaliana at different temperatures was investigated using ELISA with specific monoclonal antibodies. CPDs and 6-4PPs increased during 3 h UV-B exposure, but further exposure led to decreases. Contrary to the commonly accepted view that DNA damage induced by UV-B radiation is temperature-independent because of its photochemical nature, we found UV-B-induction of CPDs and 6-4PPs in Arabidopsis to be slower at a low than at a high temperature. Photorepair of CPDs at 24℃ was much faster than that at 0℃ and 12℃, with 50% CPDs removal during 1 h exposure to white light. Photorepair of 6-4PPs at 12℃ was very slow as compared with that at 24℃, and almost no removal of 6-4PPs was detected after 4 h exposure to white light at 0℃. There was evidence to suggest that temperature-dependent DNA damage and photorepair could have important ecological implications.