Mutations in the SGR4, SGR5 and SGR6 Loci of Arabidopsis thaliana Alter the Shoot Gravitropism

Shoots of higher plants grow upward in response to gravity.To elucidate the molecular mechanism of this response, we haveisolated shoot gravitropism (sgr) mutants in Arabidopsis thaliana.In this report, we describe three novel mutants, sgr4-1, sgr5-1and sgr6-1 whose inflorescence stems showed abnormal gravitropicresponses as previously reported for sgr1, sgr2 and sgr3. Thesenew sgr mutations were recessive and occurred at three independentgenetic loci. The sgr4-1 mutant showed severe defect in gravitropismof both inflorescence stem and hypocotyl but were normal inroot gravitropism as were sgr1 and sgr2. The sgr5-1 and sgr6-1mutants showed reduced gravitropism only in inflorescence stemsbut normal in both hypocotyls and roots as sgr3. These resultssupport the hypothesis that some mechanisms of gravitropismare genetically different in these three organs in A. thaliana.In addition, these mutants showed normal phototropic responses,suggesting that SGR4, SGR5 and SGR6 genes are specifically involvedin gravity perception and/or gravity signal transduction forthe shoot gravitropic response. (Received November 21, 1996; Accepted February 17, 1997)     

Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana

Polar auxin transport inhibitors, including N-1-naphthylphthalamicacid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have variouseffects on physiological and developmental events, such as theelongation and tropism of roots and stems, in higher plants.We isolated NPA-resistant mutants of Arabidopsis thaliana, withmutations designated pir1 and pir2, that were also resistantto TIBA. The mutations specifically affected the root-elongationprocess, and they were shown ultimately to be allelic to aux1and ein2, respectively, which are known as mutations that affectresponses to phytohormones. The mechanism of action of auxintransport inhibitors was investigated with these mutants, inrelation to the effects of ethylene, auxin, and the polar transportof auxin. With respect to the inhibition of root elongationin A. thaliana, we demonstrated that (1) the background levelof ethylene intensifies the effects of auxin transport inhibitors,(2) auxin transport inhibitors might act also via an inhibitorypathway that does not involve ethylene, auxin, or the polartransport of auxin, (3) the hypothesis that the inhibitory effectof NPA on root elongation is due to high-level accumulationof auxin as a result of blockage of auxin transport is not applicableto A. thaliana, and (4) in contrast to NPA, TIBA itself hasa weak auxin-like inhibitory effect. (Received April 12, 1996; Accepted September 2, 1996)     

Cloning and Functional Expression of a Novel Geranylgeranyl Pyrophosphate Synthase Gene from Arabidopsis thaliana in Escherichia coli

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP)synthase from Arabidopsis thaliana has been identified and termedGGPS5. The gene has been sequenced and expressed in Escherichiacoli. The deduced amino acid sequence showed 64.5% and 57.5%identity with a putative GGPP synthase from Arabidopsis andCapsicum annuum, respectively. GGPP enzymatic activity was detectedin E. coli cells expressing the GGPS5 gene in two differentways. One was the direct measurement of GGPP synthase activityin cell extracts and the other was the yellow color productionof cells when the GGPS5 gene was co-expressed with crtB, crtI,crtX, crtY and crtZ genes derived from Erwinia uredovora. (Received May 20, 1996; Accepted December 14, 1996)     

VR-ACS6 is an Auxin-Inducible l-Aminocyclopropane-l-carboxylate Synthase Gene in Mungbean (Vigna radiata)

We have isolated four cDNA clones of ACC synthase from etiolatedmungbean seedlings treated with auxin. PVR-ACS2, pVR-ACS3 andpVR-ACS6 contained the same sequences as the previously reportedDNA fragments, pMAC2, pMAC3 (Botella et al. 1992b) and pMBAl(Kim et al. 1992), respectively. pVR-ACSl was identical withpAIM-1 (Botella et al. 1992a). VR-ACS6 was specifically induced in response to the auxin signal.The IAA-induction of VR-ACS6 was very rapid (within 30 min)and insensitive to cycloheximide treatment at concentrationsup to 100 µM. Significant accumulation of VR-ACS6 mRNAwas detected at 1 µM.IAA.The IAA-induced expression ofVR-ACS6 was suppressed by ABA and ethylene, but enhanced byBA. These characteristics of VR-ACS6 expression were well correlatedwith the physiological data of auxin-induced ethylene productionin mungbean hypocotyls. VR-ACS1 was strongly induced by cycloheximide, but was foundto be not auxin-specific. Inhibitors of either ethylene biosynthesis(AOA) or action (NBD) increased the basal level of VR-ACS1 mRNA. (Received May 7, 1996; Accepted November 25, 1996)     

Molecular Cytogenetics of the Genus Arabidopsis: In situ Localization of rDNA Sites, Chromosome Numbers and Diversity in Centromeric Heterochromatin

We have used in situ hybridization to determine the number ofsites of rDNA in species in the genus Arabidopsis. A. wallichii(2n = 16) has one major pair of sites and one minor pair ofsites, while A. pumila and A. griffithiana (both 2n = 32) havesix major and two minor rDNA sites. A. thaliana (2n = 10) isknown to have two pairs of rDNA sites. a highly repeated para-centromericsequence from A. thaliana, pAL1, is absent in the other threespecies. Hence the A.thaliana genome is not present (or thecentromeric DNA has evolved substantially) in the polyploidspecies A. pumila and A. griffithiana. Analysis of Arabidopsisspecies is a valuable complement to the large programmes forgenetic analysis of A. thaliana.Copyright 1993, 1999 AcademicPress Arabidopsis, centromeric DNA, maps (genetic), nuclear architecture, repetitive DNA, ribosomal DNA, rDNA, evolution, Brassicaceae, Crucifereae, in situ hybridization     

Plastid Distribution in Columella Cells of a Starchless Arabidopsis Mutant Grown in Microgravity

Wild-type and starchless Arabidopsis thaliana mutant seedlings(TC7) were grown and fixed in the microgravity environment ofa U.S. Space Shuttle spaceflight. Computer image analysis oflongitudinal sections from columella cells suggest a differentplastid positioning mechanism for mutant and wild-type in theabsence of gravity. (Received September 24, 1996; Accepted January 21, 1997)     

Identification of clones carrying minisatellite-like loci in an Arabidopsis thaliana YAC library

Minisatellite-like DNA elements occur in the Arabidopsis thalianagenome in low copy and are weakly polymorphic between ecotypes.YAC clones from the EG-Arabidopsis library were identified withhomology to minisatellite 33.15 and bacteriophage M13 repeatelements. Other highly repeated A. thaliana DNA elements tendnot to be found in YAC clones carrying the minisatellite elementssuggesting that the elements are dispersed in the Arabidopsisgenome in regions of low complexity. The minisatellite elementsare represented at low copy in the EG-YAC library reflectingtheir frequency in the Arabidopsis genome. Key words: Minisatellite elements, Arabidopsis thaliana, YAC library screening     

Structure and Expression of Mitochondrial Citrate Synthases from Higher Plants

Mitochondrial citrate synthase (EC 4.1.3.7 [EC] ) represents the firstenzyme of the tricarboxylic acid cycle, catalyzing the condensationof acetyl-CoA and oxaloacetate, finally yielding citrate andCoA. We report here the isolation of cDNA clones encoding citratesynthase from Nicotiana tabacum, Beta vulgaris and Populus.Nucleotide and deduced amino acid sequences were compared withpreviously published sequences of mitochondrial citrate synthasesfrom Arabidopsis thaliana and potato, as well as with the sequenceof glyoxysomal citrate synthase from pumpkin. Homologies betweenthe various plant mitochondrial enzymes were in the range from77.2% (potato vs. Arabidopsis) to 94.2% (potato vs. tobacco)on the nucleotide level (coding regions only), and in the rangefrom 70.1% to 90.4% (potato vs. Arabidopsis, and potato vs.tobacco, respectively) on the amino acid level. Identities ofthe mitochondrial isozymes to the pumpkin glyoxysomal enzymewere below 30% on the nucleotide and amino acid level. In Northernblot experiments citrate synthase mRNA was detected in all tissuesanalyzed. However, levels of expression showed tissue dependencydespite the fact that citrate synthase is usually considereda house-keeping enzyme. Whether these different levels of expressionreflect tissuespecifc variations with respect to basic metabolismawaits further analysis. (Received May 20, 1996; Accepted August 20, 1996)     

Haustoria of Cuscuta japonica, a Holoparasitic Flowering Plant, Are Induced by the Cooperative Effects of Far-Red Light and Tactile Stimuli

When seedlings of Cuscuta japonica were grown with Vigna radiata(the host plant) in a flower pot for 6 d under white light andthen irradiated with far-red or blue light (ca. 6 µmolphotons m^–2 s^–1), the seedlings parasitized V. radiata.However, no parasitism of the seedlings was observed under redor white light or in darkness. The parasitic behavior of seedlingsof C. japonica was observed even if an acrylic rod was usedas a substitute for the host plant. Upon incubation under far-redlight, the seedling twined tightly around the rod and developedhaustoria towards it. Haustoria also developed when apical andsubapical regions of seedlings were held between two glass platesthat were about 0.7 mm apart and were irradiated with far-redlight. However, no haustoria were induced by either the holdor irradiation alone. These results indicate that parasitismof Cuscuta japonica is controlled by the cooperative effectsof two physical signals, far-red light and appropriate tactilepressure. Our findings suggest that parasitism by the genusCuscuta involves a novel strategy. (Received April 10, 1996; Accepted August 21, 1996)     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

The Level of mRNA Transcribed from psaL, Which Encodes a Subunit of Photosystem I, Is Increased by Cytokinin in Darkness in Etiolated Cotyledons of Cucumber

A cDNA clone for an mRNA whose level increased within 2 h ofthe start of treatment with N⁶-benzyladenine in etiolated cotyledonsof cucumber was isolated by differential hybridization. ThecDNA was homologous to psaL, which encodes subunit XI (PSI-L)of photosystem I. The accumulation of psaL mRNA was specificallyinduced by cytokinins or light. (Received April 10, 1996; Accepted July 31, 1996)     

Mechanical Stress Elicits Nitric Oxide Formation and DNA Fragmentation in Arabidopsis thaliana

The effect of mechanical stress (centrifugation) on the inductionof nitric oxide (NO) formation and DNA fragmentation was investigatedin leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifugedleaves from wild-type and nitrate reductase (NR)nia1, nia2 doublemutant, defective in the assimilation of nitrate, were labelledwith 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualizein vivo NO production. After these treatments, DNA fragmentationwas detected by the terminal deoxynucleotidyl transferase-mediateddUTP nick end in situ labelling (TUNEL) method. Exposure toan NO-releasing compound, sodium nitroprusside (SNP) mimickedthe cell response to centrifugation (20 g). The involvementof endogenous NO as a signal in mechanical stress and in DNAfragmentation was confirmed by inhibition of NO production usinga nitric oxide synthase (NOS) inhibitor viz. N^G-monomethyl-L -arginine (L -NMMA). These results indicate that NOS-likeactivity was present in A. thaliana leaves and was increasedby mechanical stress. The effect of leaf-wounding on nitricoxide production was identical to that of centrifugation. Experimentswith A. thaliana NR mutant also showed that NO bursts were inducedby mechanical and wounding stresses and that NO was not a by-productof NR activity. A positive and significant correlation betweenNO production and DNA fragmentation was recorded for both centrifugedand non-centrifuged cells. Our results suggest that factorsother than NO contribute to DNA damage and cell death, and furthermore,that an inducible form of NOS is present in A. thaliana. Copyright2001 Annals of Botany Company Arabidopsis thaliana, cell death, DNA fragmentation, NO, plant stress, wounding     

Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by Column Chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B

Chlorophylls extracted from spinach leaves were made free fromcarotenoids, pheophytins, chlorophyllides and plastoquinonesby column chromatography with DEAE-Sepharose CL-6B. Chlorophylla and b were separated by column chromatography with SepharoseCL-6B. By a combined use of DEAE-Sepharose CL-6B and SepharoseCL-6B, pure chlorophyll a and b were prepared from the leavesin a short time. Bacteriochlorophyll a_p extracted from a photosyntheticbacterium Chromatium vinosum was made free from carotenoids,bacteriopheophytin, ubiquinone and lipids by column chromatographywith Sepharose CL-6B. (Received April 19, 1983; Accepted June 16, 1983)     

A COMPARATIVE STUDY OF THE MATURE SPERMATOZOA OF BULINUS AFRICANUS (KRAUSS, 1848) AND B. GLOBOSUS (MORELET, 1866) (GASTROPODA: PLANORBIDAE)

The morphology of the mature spermatozoa of the closely-relatedfreshwater pulmonates, Bulinus africanus and Bulinus globosus,was examined using scanning and transmission electron microscopy.A comparison showed no useful differences that could be usedto distinguish the species though they did differ in one respectfrom Bulinus tropicus, the only other bulinid for which spermmorphology is known. (Received 12 August 1996; accepted 6 December 1996)     

AGR,an Agravitropic Locus of Arabidopsis thaliana, Encodes a Novel Membrane-Protein Family Member

Mutations in the Agr locus of Arabidopsis thaliana impair theroot gravitropic response. Root growth of agr mutants is moderatelyresistant to ethylene and to an auxin transport inhibitor. Verticallyplaced agr roots grow into agar medium containing IAA or naphthalene-1-aceticacid, but not into medium containing 2,4-D. Positional cloningshowed that AGR encodes a root-specific member of a novel membrane-proteinfamily with limited homology to bacterial transporters. (Received September 4, 1998; Accepted September 21, 1998)     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

The signature of seeds in resurrection plants: a molecular and physiological comparison of desiccation tolerance in seeds and vegetative tissues

Desiccation-tolerance in vegetative tissues of angiosperms hasa polyphyletic origin and could be due to 1) appropriation ofthe seed-specific program of gene expression that protects orthodoxseeds against desiccation, and/or 2) a sustainable version ofthe abiotic stress response. We tested these hypotheses by comparingmolecular and physiological data from the development of orthodoxseeds, the response of desiccation-sensitive plants to abioticstress, and the response of desiccation-tolerant plants to extremewater loss. Analysis of publicly-available gene expression dataof 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitiveArabidopsis thaliana identified 13 LEAs and 4 anti-oxidantsexclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin)are not expressed in vegetative tissues in A. thaliana, buthave orthologues that are specifically activated in desiccatingleaves of Xerophyta humilis. A comparison of antioxidant enzymeactivity in two desiccation-sensitive species of Eragrostiswith the desiccation-tolerant E. nindensis showed equivalentresponses upon initial dehydration, but activity was retainedat low water content in E. nindensis only. We propose that theseantioxidants are housekeeping enzymes and that they are protectedfrom damage in the desiccation-tolerant species. Sucrose isconsidered an important protectant against desiccation in orthodoxseeds, and we show that sucrose accumulates in drying leavesof E. nindensis, but not in the desiccation-sensitive Eragrostisspecies. The activation of "seed-specific" desiccation protectionmechanisms (sucrose accumulation and expression of LEA6 and1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerantplants points towards acquisition of desiccation tolerance fromseeds.     

Natural variation in Arabidopsis lyrata vernalization requirement conferred by a FRIGIDA indel polymorphism

Species share homologous genes to a large extent, but it isnot yet known to what degree the same loci have been targetsfor natural selection in different species. Natural variationin flowering time is determined to a large degree by 2 genes,FLOWERING LOCUS C and FRIGIDA, in Arabidopsis thaliana. Here,we examine whether FRIGIDA has a role in differences in floweringtime between and within natural populations of Arabidopsis lyrata,a close outcrossing perennial relative of A. thaliana. We found2 FRIGIDA sequence variants producing potentially functionalproteins but with a length difference of 14 amino acids. Thesevariants conferred a 15-day difference in flowering time inan association experiment in 2 Scandinavian populations. Thedifference in flowering time between alleles was confirmed withtransformation to A. thaliana. Because the north European late-floweringpopulations harbor both late- and early sequence variants atintermediate frequencies and the late-flowering variant is mostfrequent in the southern early flowering European population,other genetic factors must be responsible for the floweringtime differences between the populations. The length polymorphismoccurs at high frequencies also in several North American populations.The occurrence of functional variants at intermediate frequenciesin several populations suggests that the variation may be maintainedby balancing selection. This is in contrast to A. thaliana,where independent loss-of-function mutations at the FRIGIDAgene are responsible for differences between populations andlocal adaptation.