Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Cumulative effect of low and high atrazine concentrations on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Atrazine belongs to the widely used herbicides blocking the electron transport chain in chloroplasts, thus resulting in the generation of active oxygen species. In the present work, we demonstrated that, at low concentrations mimicking residual amounts, atrazine enhanced the susceptibility of Arabidopsis plants to further treatments with the same herbicide applied at the recommended field rate. Arabidopsis thaliana plants were treated three times (at five-day intervals) with 1 µM atrazine. Five days after the last treatment, the plants were sprayed with 5 mM atrazine. Atrazine increased the levels of lipid peroxidation products, hydrogen peroxide, and ion leakage, and caused changes in the activities of antioxidant enzymes, such as superoxide dismutase, guaiacol peroxidase, and catalase.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 243–249.Original English Text Copyright © 2005 by Ivanov, Alexieva, Karanov.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

A meiotic time-course for<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

A meiotic time-course for Arabidopsis pollen mother cells has been established based on BrdU pulse-labelling of nuclear DNA in the meiotic S-phase. Labelled flower buds were sampled at intervals and the progress of labelled cells through meiosis assessed by anti-BrdU antibody detection. The overall duration of meiosis from the end of meiotic S-phase to the tetrad stage, at 18.5°C, was 33 h, which is about three times longer than the mitotic cell cycle in seedlings. The onset of leptotene was defined by reference to the loading of the axis-associated protein Asy1, and this permitted the detection of a definite G2 stage, having a maximum duration of 9 h. It is likely, from two independent sources of evidence, that the meiotic S-phase has a duration similar to that of G2. The durations of leptotene and zygotene/pachytene are 6 h and 15.3 h, respectively, but the remaining meiotic division stages are completed very rapidly, within 3 h. The establishment of a meiotic time-course provides a framework for determining the relative timing and durations of key molecular events of meiosis in Arabidopsis in relation to cytologically defined landmarks. In addition, it will be important in a broader developmental context for determining the timing of epigenetic mechanisms that are known or suspected to occur during meiosis.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

Glucose delays seed germination in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Here we report that glucose delays germination of Arabidopsis thaliana (L.) Heynh. seeds at concentrations below those known to inhibit early seedling development. This inhibition acts on embryo growth and is independent of hexokinase (HXK) function. Hormones and hormone inhibitors were applied to the germination media and several hormone biosynthesis and signalling mutants were tested on glucose media to investigate a possible role of abscisic acid (ABA), gibberellin and ethylene in the glucose-induced germination delay. Results indicate that the germination inhibition by glucose cannot be antagonized by ethylene or gibberellin and is independent of the HXK1/ABA/ABI4 signalling cascade. These findings suggest that there is a separate regulatory pathway independent of ABI2/ABI4/ABI5. Thus, in a relatively short time frame sugars utilize different signalling cascades to inhibit germination and post-germination growth, underlining the complexity of sugar responses.Abbreviations ABA Abscisic acid - ABI ABA insensitive - ACC 1-Aminocyclopropane-1-carboxylic acid - BR Brassinosteroid - CAB Chlorophyll a/b-binding protein - FUS3 Fusca3 - GA Gibberellin - GA ₃ Gibberellic acid - HXK Hexokinase - LEC1 Leafy cotyledon1 - RBCS Ribulose-1,5-bisphosphate carboxylase small subunit - WT Wild type     

Effects of salicylic acid on paraquat tolerance in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Foliar spraying ofArabidopsis thaliana (Columbia ecotype) plants with a 1.0-mM salicylic acid (SA) solution significantly improved their tolerance to subsequent paraquat (PQ)-induced oxidative damage. Leaf injuries, including losses of chlorophyll, protein, and fresh weight, were reduced. Our analysis of antioxidant enzymes in the leaves showed that SA pre-treatment effectively retarded rapid decreases in the activities of Superoxide dismutase (SOD), catalase, and ascorbate peroxidase that are normally associated with PQ exposure. In addition, guaiacol peroxidase activity was remarkably increased. In a native gel assay of peroxidase (POD) isozymes, staining activity of the POD1 isozyme, which disappeared in plants exposed only to 10 μM PQ, was significantly recovered by the 1.0-mM SA pre-treatment POD2 isozyme activity was also pronounced in all SA-treated plants compared with the control. A 12-h SA pre-treatment, without subsequent PQ stress, also caused a small increase in the endogenous H₂O₂ content that accompanies the symptoms of mild leaf injuries. This enhanced level occurred in parallel with a slight SOD increase and a catalase decrease. From our results, it can be assumed that, due to the small increase in SOD as well as catalase inactivation via SA pre-treatment, a moderate increase in H₂O₂ levels may occur. In turn, a large induction of guaiacol peroxidase leads to enhanced PQ tolerance inA. thaliana plants.     

The Effect of Paraquat and Hypothermia on Norflurason-Resistant Mutants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and Derived Cell Cultures

A tolerance to paraquat (PQ) of plants and cell cultures of Arabidopsis thaliana mutants, nfz18 and nfz24, obtained by chemical mutagenesis and selected by their tolerance to norflurason was demonstrated. This tolerance to PQ was manifested in less active peroxidation of lipids (POL), which was assessed by the content of thiobarbituric acid-reactive substances, and in a less degree of plant and callus damages, which was accompanied by a higher activity of superoxide dismutase and other antioxidant enzymes. A capability of norflurason-tolerant mutants to cross-adaptation toward PQ and activation of antioxidant enzymes indicate a genetically determined activation of the antioxidant systems, resulting in improved mutant tolerance to these inducers of oxidative stress. The nfz24 mutant was much more sensitive to hypothermia than wild-type plants and nfz18 mutants, which was expressed in a higher level of POL in plants and calluses and in a more rapid decrease in the suspension cell viability of this mutant. A similarity in the responses of plants and derived heterotrophic cultures to PQ and hypothermia indicates that, in these A. thaliana mutants, adaptation to these types of stresses occurs mainly at the cellular level. Possible reasons of increased sensitivity to hypothermia of the nfz24 mutant, which was more tolerant to the inducers of oxidative stress, PQ and norflurason, are discussed.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 421–429.Original Russian Text Copyright © 2005 by Volkova, Burgutin, Soldatova, Ezhova, Lapshin.     

Phenotypic alterations in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> plants caused by<Emphasis Type="Italic"> Rhodococcus fascians</Emphasis> infection

Arabidopsis thaliana (L.) Heynh. plants were challenged with Rhodococcus fascians at several developmental stages and using different inoculation procedures. A variety of morphological alterations was scored on the infected plants; some of them resembled phenotypes of A. thaliana mutants in their shoot apical meristem (SAM) organization. Infection with R. fascians did not affect SAM organization in wild type nor in SAM mutants. Anatomical studies on the new organs formed after infection with R. fascians demonstrated extensive bacterial colonization. Colonization and concomitant production of specific signals are the likely cause of malformations.     

Single-embryo RT-PCR assay to study gene expression dynamics during embryogenesis in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We have developed a single-embryo RT-PCR protocol for studying gene expression during plant embryogenesis. Four genes,glyceraldhyde-3-phosphate dehydrogenase (GAPC), shoot-meristemless (STM), monopteros (MP), andshaggy-like kinase etha (ASKη), fromArabidopsis thaliana were used to test the sensitivity and reliability of this method by analyzing the differential signal intensities of their RT-PCR products. The method could detect genes expressed during embryogenesis at a single-embryo level and, therefore, can be used to identify phenotypes. When in vitro, embryogenesis also is used to control the time course of zygote development exactly. The single-embryo RT-PCR protocol becomes a powerful method to survey the dynamics of specific gene expression.     

Overexpression of Organellar and Cytosolic <Emphasis Type="Italic">AtHSP90</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Impairs Plant Tolerance to Oxidative Stress

Three AtHSP90 isoforms, cytosol-localized AtHSP90.2, chloroplast-localized AtHSP90.5, and endoplasmic reticulum (ER)-localized AtHSP90.7 genes, were constitutively overexpressed in Arabidopsis thaliana to study their functional mechanisms under oxidative stress. Overexpression of AtHSP90 genes reduced germination of transgenic seeds under oxidative stress. When exposed to 10 mM H₂O₂, AtHSP90 transgenic seedlings displayed lower activities of superoxide dismutase, catalase, and peroxidase; higher content of malondialdehyde; and higher levels of protein damage than detected in the wild type. This indicated that overexpression of AtHSP90.2, AtHSP90.5, and AtHSP90.7 in Arabidopsis impaired plant tolerance to oxidative stress. Moreover, overexpression of chloroplast- and ER-localized AtHSP90 resulted in lower resistance to oxidative stress than that of cytosolic AtHSP90. This suggested that HSP90.2, HSP90.5, and HSP90.7 localized in different cellular compartments were involved in different functional mechanisms during oxidative stress.     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

An improved,simple, hydroponic method for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis thaliana is one os the most studied plant model systems. Completing the genomic sequence ofA. thaliana has provided new opportunities for physiological and biochemical studies. While its small size is advantageous for genetic studies, the plant's low biomass makes it difficult to obtain enough plant material for biochemical and physiological research. The small size and rosette leaf structure, combined with the sensitivity of the apical meristem to flooding, make hydroponic growth of this model plant difficult. A few systems for hydroponic culture ofArabidopsis have been described. Gibeaut et al. (1997) introduced the use of rockwool forArabidopsis hydroponic culture. We have improved this system by introducing small-volume plastic containers with improved plugs to support the rockwool. This method is simpler than the original setup and provides improved germination and growth. The smaller containers enable the use of this system in growth chambers or small growth rooms for a large number of parallel experiments.     

Sugar-induced adventitious roots in<Emphasis Type="Italic"> Arabidopsis</Emphasis> seedlings

The effects of sugars on root growth and on development of adventitious roots were analyzed in Arabidopsis thaliana. Seeds were sown on agar plates containing 0.0–5.0% sugars and placed vertically in darkness (DD) or under long day (LD, 16 h:8 h) conditions, so that the seedlings were constantly attached to the agar medium. In the sucrose-supplemented medium, seedlings showed sustained growth in both DD and LD. However, only dark-grown seedlings developed adventitious roots from the elongated hypocotyl. The adventitious roots began to develop 5 days after imbibition and increased in number until day 11. They could, however, be initiated at any position along the hypocotyl, near the cotyledon or the primary root. They were initiated in the pericycle in the same manner as ordinary lateral roots. Sucrose, glucose and fructose greatly stimulated the induction of adventitious roots, but mannose or sorbitol did not. Sucrose at concentrations of 0.5–2.0% was most effective in inducing adventitious roots, although 5.0% sucrose suppressed induction. Direct contact of the hypocotyl with the sugar-supplemented agar medium was indispensable for the induction of adventitious roots. Electronic Publication     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis