Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, “the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance”, has instigated lots of debates. In this study, according to the previous research of Pprl’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. radiodurans R1 strain, pprf function-deficient mutant (YR1), and pprl function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence microscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells exhibit the tightly packed ring-like morphology, while the pprl function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprl completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprl partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a Pprl C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensitive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance. Supported by the National Basic Research Program of China (Grant No. 2004CB19604), the National Natural Science Foundation of China (Grant No. 30330020), and the National Science fund for Distinguished Young Scholars (Grant No. 30425038)     

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes -galactosidase, and a polyadenylation 3-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5 promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypcotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged.Transgenic plants with a 600 bp promoter construct (–0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a –500 bp promoter had levels of expression similar to the –3.0 kb construct. The –0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 × 10^–6 to 5 × 10^–5 M 2,4-D and was responsive to as little as 5 × 10^–8 M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.co-first authors     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of the SSLP-based method for detection of rare apomictic events in a sexual AtSERK1 transgenic Arabidopsis population

Here we present a screening method to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of single sequence length polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1) gene expressed under the control of the AtLTP1 promoter in sexual Arabidopsis plants. In one of the three tested F2 transgenic populations expressing the AtLTP1::AtSERK1 construct we observed two plants with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for a number of heterozygous loci, it was concluded that these two putative apomictic plants either lost their clonal ability in the next generation or resulted from incidental recombination events displaying the genotype of the parent. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Evidence for a role of AtCAD 1 in lignification of elongating stems of Arabidopsis thaliana

The cinnamyl alcohol dehydrogenase (AtCAD) multigene family in Arabidopsis is composed of nine genes. Our previous studies focused on the two isoforms AtCAD C and AtCAD D which show a high homology to those related to lignification in other plants. This study focuses on the seven other Arabidopsis CAD for which functions are not yet elucidated. Their expression patterns were determined in different parts of Arabidopsis. Only CAD 1 protein can be detected in elongating stems, flowers, and siliques using Western-blot analysis. Tissue specific expression of CAD 1, B1, and G genes was determined using their promoters fused to the GUS reporter gene. CAD 1 expression was observed in primary xylem in accordance with a potential role in lignification. Arabidopsis T-DNA mutants knockout for the different genes CAD genes were characterized. Their stems displayed no substantial reduction of CAD activities for coniferyl and sinapyl alcohols as well as no modifications of lignin quantity and structure in mature inflorescence stems. Only a small reduction of lignin content could be observed in elongating stems of Atcad 1 mutant. These CAD genes in combination with the CAD D promoter were used to complement a CAD double mutant severely altered in lignification (cad c cad d). The expression of AtCAD A, B1, B2, F, and G had no effect on restoring a normal lignin profile of this mutant. In contrast, CAD 1 complemented partly this mutant as revealed by the partial restoration of conventional lignin units and by the decrease in the frequency of β-O-4 linked p-OH cinnamaldehydes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Immunochemically detectable phytochrome is present at normal levels but is photochemically nonfunctional in the hy 1 and hy 2 long hypocotyl mutants of Arabidopsis

The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158–165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.     

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

Modulation of flavonoid metabolism in Arabidopsis using a phage-derived antibody

The utility of recombinant antibodies for immunomodulation of plant metabolism was tested using the Arabidopsis flavonoid biosynthetic pathway as a target. Two genes encoding antibodies against chalcone isomerase (CHI) were isolated from a human synthetic single chain variable fragment (scFv) library and expressed in the cytoplasm of transgenic plants. In one line, low-level expression of the scFv resulted in reduced visible pigmentation in seedlings as well as lower accumulation of phenolics in plants throughout development. Surprisingly, other transgenic lines with much higher expression of the same antibody showed no phenotype. Protein mobility shift assays indicated that the scFv is bound to the target enzyme, but only in the affected plants. This work shows that recombinant scFv antibody technology offers a viable approach to disrupting protein activity in plants, but that further refinement is required before it will be of general utility for metabolic engineering and other antibody-based applications in plants.