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.     

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     

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     

<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     

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.     

A rapid<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

Identification of a Golgi-localised GRIP domain protein from<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

A family of Golgi-localised molecules was recently described in animals and fungi possessing extensive coiled regions and a short (~40 residues) conserved C-terminal domain, called the GRIP domain, which is responsible for their location to this organelle. Using the model plant Arabidopsis thaliana, we identified a gene (AtGRIP) encoding a putative GRIP protein. We demonstrated that the C-terminal domain from AtGRIP functions as a Golgi-targeting sequence in plant cells. Localisation studies in living cells expressing the AtGRIP fused to a DsRed2 fluorescent probe, showed extensive co-location with the Golgi marker -mannosidase I in transformed tobacco protoplasts. GRIP-like sequences were also found in genomic databases of rice, maize, wheat and alfalfa, suggesting that this domain may be a useful Golgi marker for immunolocalisation studies. Despite low sequence identity amongst GRIP domains, the plant GRIP sequence was able to target to the Golgi of mammalian cells. Taken together, these data indicate that GRIP domain proteins might be implicated in a targeting mechanism that is conserved amongst eukaryotes.Abbreviations -ManI -Mannosidase I - AtGRIP Arabidopsis GRIP domain protein - GCC Golgi-localized coiled-coil protein - GFP Green fluorescent protein - MES 2-(N-Morpholino)ethanesulfonic acid - TGN Trans-Golgi network     

Changes in gravitational forces induce modifications of gene expression in<Emphasis Type="Italic"> A. thaliana</Emphasis> seedlings

By comparing the expression patterns of selected genes from Arabidopsis thaliana (L.) Heynh. grown either at 1 g or on a clinostat (horizontally or vertically inverted, 1 rpm), and either used directly or after hypergravity stimulation, we have shown that the pattern of expression did not proceed in a stereotypical manner. Rather, the selected genes fell into different classes. These classes include (i) those insensitive to the gravitational conditions, (ii) those that are regulated in an opposite manner by hypergravity and clinostat conditions, (iii) those that are desensitised to hypergravity by long-term culture on a clinostat, and (iv) those enhanced by such a treatment. Our data suggest that rapid reorientation of gene expression is likely to occur in response to changes in the gravitational conditions.     

Electron tomographic analysis of post-meiotic cytokinesis during pollen development in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.Abbreviations AGP Arabinogalactan protein - MT Microtubule     

Photosynthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Mutants with Reduced Chloroplast Number

We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2–15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005–1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.     

Activation of the <Emphasis Type="Italic">WUS</Emphasis> gene induces ectopic initiation of floral meristems on mature stem surface in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Nitric oxide is induced by wounding and influences jasmonic acid signaling in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Nitric oxide (NO) has been associated with plant defense responses during microbial attack, and with induction and/or regulation of programmed cell death. Here, we addressed whether NO participates in wound responses in Arabidopsis thaliana (L.) Heynh.. Real-time imaging by confocal laser-scanning microscopy in conjunction with the NO-selective fluorescence indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) uncovered a strong NO burst after wounding or after treatment with JA. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive responses. Furthermore, we were able to detect NO in plants (here induced by wounding) by means of electron paramagnetic resonance measurements using diethyldithiocarbamate as a spin trap. When plants were treated with NO, Northern analyses revealed that NO strongly induces key enzymes of jasmonic acid (JA) biosynthesis such as allene oxide synthase (AOS) and lipoxygenase (LOX2). On the other hand, wound-induced AOS gene expression was independent of NO. Furthermore, JA-responsive genes such as defensin (PDF1.2) were not induced, and NO induction of JA-biosynthesis enzymes did not result in elevated levels of JA. However, treatment with NO resulted in accumulation of salicylic acid (SA). In transgenic NahG plants (impaired in SA accumulation and/or signaling), NO did induce JA production and expression of JA-responsive genes. Altogether, the presented data demonstrate that wounding in Arabidopsis induces a fast accumulation of NO, and that NO may be involved in JA-associated defense responses and adjustments.Abbreviations AOS Allene oxide synthase - cPTIO Carboxy-2-phenyl-4,4,5,5-tetramethylimidazolinone-3-oxide-1-oxyl - DAF-2 DA 4,5-Diaminofluorescein diacetate - DETC Diethyldithiocarbamate - EPR Electron paramagnetic resonance - iNOS Inducible nitric oxide synthase - JA Jasmonic acid - JIP Jasmonic acid-induced protein - LOX2 Lipoxygenase 2 - NO Nitric oxide - OPR3 12-Oxophytodienoate reductase - PDF1.2 Plant defensin - ROS Reactive oxygen species - SA Salicylic acid - SNP Sodium nitroprusside     

Two naturally occurring deletion mutants of 12S seed storage proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the -subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively. Sequence analyses of the genomic DNA flanking the deletion sites have demonstrated that both deletions occurred at the genomic level. These two mutants are referred to as CRB12 and CRC13 with the delta sign indicating a deletion and the number indicating amino acids deleted. Alignment of these two mutant sequences with that of soybean A3B4 subunit, for which the crystal structure was determined recently, have revealed that the CRC13 deletion is located in a hypervariable/disordered region, and will probably not affect the structure of the hexameric globulin. The CRB12 deletion, however, is located in a binding region that is thought to be important for the hexamer formation. However, CRB12 appears to accumulate normally as judged by its band intensity relative to the other SSP subunits on the protein gels. Thus it seems that the seed can, to a certain extent, tolerate some mutations in its storage proteins.     

Performing abstraction: two ways of modelling<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

What is the best way to analyse abstraction in scientific modelling? I propose to focus on abstracting as an epistemic activity, which is achieved in different ways and for different purposes depending on the actual circumstances of modelling and the features of the models in question. This is in contrast to a more conventional use of the term ‘abstract’ as an attribute of models, which I characterise as black-boxing the ways in which abstraction is performed and to which epistemological advantage. I exemplify my claims through a detailed reconstruction of the practices involved in creating two types of models of the flowering plant Arabidopsis thaliana, currently the best-known model organism in plant biology. This leads me to distinguish between two types of abstraction processes: the ‘material abstracting’ required in the production of Arabidopsis specimens and the ‘intellectual abstracting’ characterising the elaboration of visual models of Arabidopsis genomics. Reflecting on the differences between these types of abstracting helps to pin down the epistemic skills and research commitments used by researchers to produce each model, thus clarifying how models are handled by researchers and with which epistemological implications.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey