Wounding Triggers Callus Formation via Dynamic Hormonal and Transcriptional Changes
Momoko IkeuchiAkira IwaseBart RymenAlice LambolezMikiko KojimaYumiko TakebayashiJefri HeymanShunsuke WatanabeMitsunori SeoLieven De VeylderHitoshi SakakibaraKeiko Sugimoto
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Abstract:
Wounding is a primary trigger of organ regeneration, but how wound stress reactivates cell proliferation and promotes cellular reprogramming remains elusive. In this study, we combined transcriptome analysis with quantitative hormonal analysis to investigate how wounding induces callus formation in Arabidopsis (Arabidopsis thaliana). Our time course RNA-seq analysis revealed that wounding induces dynamic transcriptional changes, starting from rapid stress responses followed by the activation of metabolic processes and protein synthesis and subsequent activation of cell cycle regulators. Gene ontology analyses further uncovered that wounding modifies the expression of hormone biosynthesis and response genes, and quantitative analysis of endogenous plant hormones revealed accumulation of cytokinin prior to callus formation. Mutants defective in cytokinin synthesis and signaling display reduced efficiency in callus formation, indicating that de novo synthesis of cytokinin is critical for wound-induced callus formation. We further demonstrate that type-B ARABIDOPSIS RESPONSE REGULATOR-mediated cytokinin signaling regulates the expression of CYCLIN D3;1 (CYCD3;1) and that mutations in CYCD3;1 and its homologs CYCD3;2 and 3 cause defects in callus formation. In addition to these hormone-mediated changes, our transcriptome data uncovered that wounding activates multiple developmental regulators, and we found novel roles of ETHYLENE RESPONSE FACTOR 115 and PLETHORA3 (PLT3), PLT5, and PLT7 in callus generation. All together, these results provide novel mechanistic insights into how wounding reactivates cell proliferation during callus formation.Keywords:
Callus
Two Arabidopsis mutants atmyb123 and atkor1 were identified from the T-DNA insertion knockout mutant lines SAIL_005260 and SAIL_2_G11,respectively,and then a double mutant atmyb123/atkor1 was established by crossing method.The two mutants are lacking expression for ATMYB123 and ATKOR1 genes,respectively,which two were found to be tightly related to root development in Arabidopsis thaliana.The results obtained here showed that lack of ATMYB123 gene in expression led to a slow growth of plant rosettes and a yellow skin of seeds in Arabidopsis,while lack of ATKOR1 gene in expression had no marked effects on these two factors.Any one of the two genes ATMYB123 and ATKOR1 knockout extremely repressed the root development in Arabidopsis,especially the knockout of ATKOR1 gene,the mutant atkor1 showed only one third of length of roots as compared to wild type(WT).Interestingly,the double mutant atmyb123/atkor1 exhibited the characteristics of the single mutant atmyb123 has in plant rosette morphology and seed skins but presented intermediated root length between the two single mutants.In addition,the growth trend of roots among the three mutants had no fundamental changes when the plants were cultivated under different pH,NaCl treatments and GA concentration conditions,which imply that these three factors were not concerned in the root shortening event induced by lack of any one of ATMYB123 or/and ATKOR1 proteins in A.thaliana.These results suggest that both ATMYB123 and ATKOR1 genes participate in the root development of Arabidopsis and a specific relationship in functions exist between the two proteins,ATMYB123 and ATKOR1.The transcription factor ATMYB123 might act as a major regulator of ATKOR1 protein for participating the control of root development in Arabidopsis.
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Lateral root
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The effect of cytokinins was studied in three systems: the alga Chlorella, callus cultures and etiolated cucumber cotyledons. In Chlorella cultures: 1) A range of concentrations of 6BA and IP had no effect on growth; 2) Low concentrations of an anticytokinin had no effect on growth, whereas higher concentrations appeared to be inhibitory. 3) Characterisation of the Chlorella species suggested that it was surrounded by an impermeable sporopollenin layer which hindered the uptake of cytokinin. 4) The uptake of radioactive adenine occurred readily, whereas the uptake of radioactive 6BA was very slow in both growing and saturated cultures of Chlorella. 5) Extracts isolated from Chlorella and the medium in which Chlorella was growing contained cytokinin-like activity in two bioassays. 6) HPLC analyses of these extracts showed that there were fractions which eluted at the positions of IP and IPA. In callus cultures: A.1) A carrot callus was grown from the secondary phloem of the storage root of carrot. 2) This callus, which was grown on 2,4-D and kinetin, produced roots and shoots when subcultured onto IAA and kinetin. 3) Growth on 2,4-D alone was independent of the presence of kinetin. 4) Growth was inhibited in the presence of an anticytokinin, suggesting that the callus produced a cytokinin. B.1) A tobacco callus was grown from a young leaf of tobacco. 2) This callus habituated to cytokinin independence following subculture onto lower concentrations of kinetin. 3) Subculture of the habituated callus onto a higher concentration of kinetin resulted in the production of roots and shoots. C.1) Cytokinin-dependent soybean and tobacco callus cultures were obtained from the Botany Department, University of Otago. 2) Analysis of the total proteins from suspension and callus cultures of soybean by 1-D polyacrylamide gel electrophoresis showed one small 6BA-induced change in the proteins from the suspension cultures. In etiolated cucumber cotyledons: 1) 6BA caused the expansion of excised etiolated cucumber cotyledons after a 24h-hour incubation in the dark in a solution containing 6BA, in comparison to cotyledons incubated in water only. 2) The cotyledons curved upwards and in the light microscope the cells of the vascular bundles and the lower epidermis exhibited greater expansion than the upper epidermis. 3) Electron microscopic examination showed that the central vacuole of palisade cells from cotyledons treated with 6BA had expanded and that the cytoplasm had probably lost water and was compressed by the vacuole against the cell wall. 4) In contrast to other research, there was no apparent increase in polysome formation in 6BA-treated cotyledons in comparison to untreated cotyledons examined in the electron microscope. 5) A number of protein extraction methods were tried before a method was found which produced a protein extract suitable for both 1-D and 2-D polyacrylamide gel electrophoresis analyses. 6) 1-D and 2-D polyacrylamide gel electrophoresis showed that a number of proteins either increased or decreased following…
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Plant Physiology
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GUS reporter system
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尽管高活动性组 B (HMGB ) 蛋白质被识别了从许多在对改变环境条件的植物回答的植物种类，他们的重要性和功能的角色大部分是未知的。这里，我们调查了在对环境刺激的植物回答从黄瓜(Cucumis sativus L.) 孤立的 CsHMGB 的功能的角色。在正常生长条件下面或当使遭到了到冷应力时，在植物生长的差别都没在表示 CsHMGB 上在野类型、转基因的 Arabidopsis thaliana 之间被发现。由对比，当在高盐或脱水压力条件下面成长时，转基因的 Arabidopsis 植物与野类型的植物相比显示了延迟的萌芽。转基因的植物的萌芽被 abscisic 酸(骆驼毛的织物) 的增加推迟，暗示 CsHMGB 影响通过一个骆驼毛的织物依赖者方法的萌芽。CsHMGB 的表示影响了仅仅萌芽阶段，并且 CsHMGB 没在压力条件下面影响转基因的植物的幼苗生长。几萌芽应答的基因的抄本层次被 CsHMGB 的表示在 Arabidopsis 调制。总起来说，这些结果建议在 Arabidopsis 的 CsHMGB 的那宫外的表情调制几萌芽应答的基因的表示，并且从而在不同压力条件下面影响 Arabidopsis 植物的萌芽。
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The research was to determine the best callus of Arabidopsis thaliana for tissue culture.The callus of Arabidopsis thaliana was induced on the different medium and auxin concentration.The medium NB could improve the callus rate of Arabidopsis thaliana.It was found that the callus was the best on the medium of NB+2,4-D 2.0mg/L+6-BA 1.0mg/L.
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Research Article: The effects of aluminum toxicity on the protein expression of Arabidopsis thaliana
Wild type and mutant Arabidopsis thaliana plants were used to investigate the protein expression of plants exposed to elevated concentrations of aluminum. Plants were grown in a growth chamber at 25°C, 16-hour day, and 8-hour dark for eight weeks before subjecting them to different concentrations of AlCl3 solution. Time course experiments showed that Arabidopsis plants respond to aluminum toxicity by altering their protein expression. Our data indicate that aluminum toxicity caused Arabidopsis plants to express a protein in the range of 97 kD, and a medium-molecular weight protein in the range of 45 kD. Our data also indicate that responses by Arabidopsis plants to Al toxicity are not similar to responses to stresses that cause the induction of HSP70.
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Abstract: Polyclonal antisera against a fusion protein of β‐galactosidase and the 20 C‐terminal amino acids of the Arabidopsis thaliana sucrose carrier AtSUC2 were used to determine the cellular localization of the AtSUC2 protein. Using fluorescence‐labelling on sections from different organs of Arabidopsis the AtSUC2 protein was immunolocalized exclusively in companion cells. The presented data indicate that phloem loading in Arabidopsis may be catalyzed by the AtSUC2 sucrose carrier which transports sucrose into the companion cells. No evidence for a participation of the second Arabidopsis sucrose transporter AtSUC1 has been obtained.
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Ozet. Arabidopsis thaliana (Arabidopsis)’nin kromozom sayisinin az olmasi, bu bitkinin genetik yapisinin diger bitki turlerine gore daha kolay calisilmasina olanak saglamakta, ayrica diger bitkilerde patojenlere karsi gozlenen ana savunma mekanizmalari bu bitkide de bulunmaktadir. Bu acidan, konukcu bitkilerin patojen saldirilarina karsi savunma mekanizmalarini calisma konusunda Arabidopsis bitkisi ideal bir model sistem olusturmaktadir. Bu derlemenin amaci, patojenlere karsi bitki savunma mekanizmasinin genetigini anlamada Arabidopsis’in bir sablon olarak nasil kullanildigini, bitki ekotiplerinin patojen izolatlarina karsi gosterdigi farkliliklari (duyarlilik ve dayaniklilik) ortaya koyacak inceleme metodlarini aciklamaktadir. Anahtar Kelimeler: Arabidopsis thaliana, Peronospora parasitica, hastaliklara dayaniklilik, mutant bitkiler A Model Plant In Host-Pathogen Interaction: Arabidopsis thaliana Abstract. Because of its small genome, genetic studies are performed easier in Arabidopsis thaliana (Arabidopsis) compared to other plants and it exhibits all of the major kinds of defense responses described in other plants. Thus, Arabidopsis provides to be an ideal model system for studies of host defense responses to pathogen attack. This review details methods to isolate and utilize phytopathogens useful as probes in understanding the genetics of plant defense responses in Arabidopsis. As well, it will be defined how screening systems can be set up to detirmine differential responses (resistance and susceptibility) of Arabidopsis ecotypes to pathogen isolates. Key Words: Arabidopsis thaliana, Peronospora parasitica, disease resistance, mutant plants
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The article presents a study on the mutual influence of mutant genes in productivity of Arabidopsis plant ( Arabidopsis thaliana (L.) Heynh.), in different growing models of plant communities. It is shown that under the joint cultivation of five genetically pure lines of Arabidopsis is occurring mutual inhibition and aid, which leads decreasing or increasing of seed production plants.
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[Objective]The aim was to study the action mode of high mobility group B(HMGB)proteins of higher eukaryotic cells in the transcriptional regulation of plant stress responses.[Method]The At2G33450 gene encoding AT2G34450 protein in Arabidopsis thaliana was cloned.Then,binary vectors carrying the gene were transformed into Arabidopsis thaliana and the over-expression strains were selected to detect the influences of environmental stimuli on transgenic Arabidopsis.[Result]Under salt or drought stress,the transgenic Arabidopsis plants which over-expressed At2G33450 displayed retarded germination and subsequent growth compared with wild-type plants.[Conclusion]At2G33450 protein of HMGB protein family played an important role in the growth and development of Arabidopsis plants under various stress conditions.
Heterologous expression
Heterologous
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