bluuebunny/crossref_metadata_2025 huggingface dataset:
Antisense expression of chaperonin 60β in transgenic tobacco plants leads to abnormal phenotypes and altered distribution of photoassimilates
Authors: Zabaleta Eduardo , Oropeza Araceli , Assad Nacyra , Mandel Alejandra , Salerno Graciela , Herrera‐Estrella Luis
Published: March 2003

Chaperonins are a class of molecular chaperone, present in bacteria, mitochondria and chloroplasts, that are involved in protein folding and assembly in many organisms. Plastid α and β chaperonins have been suggested to be involved specifically in the assembly of Ribulose bisphosphate carboxylase/oxygenase. However, to date there is no direct evidence to confirm the in vivo role of plastid chaperonin 60 polypeptides as molecular chaperones. This paper reports on the production, by means of antisense technology, of transgenic tobacco plants with reduced levels of chaperonin 60β (Cpn60β). Antisense cpn60β plants showed drastic phenotypic alterations including slow growth, delayed flowering, stunting and leaf chlorosis. The most extreme effect appeared to be lethality suggesting that cpn60β functions are essential for viability. Cpn60β antisense plants accumulated Rubisco to specific activities equal to or higher than that of controls and had high plastid starch contents. These observations are discussed with respect to the suggestion that chaperonin is required for the assembly of active Rubisco in vivo. In addition, metabolic alterations in the antisense transgenic plants such as reduced soluble carbohydrate content as well as higher levels of starch in chloroplasts, suggest that Cpn60β may be required for import, assembly or membrane insertion of several chloroplast membrane proteins. These results are in agreements with the proposed role of Cpn60β as a molecular chaperone.
K+ channels of stomatal guard cells: bimodal control of the K+ inward‐rectifier evoked by auxin
Authors: Blatt Michael R. , Thiel Gerhard
Published: March 2003

The influence of the auxins indole‐3‐acetic acid (IAA) and 1‐napthylene acetic acid (NAA) on K+ channels and their control was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes to record membrane potentials and to monitor K+ channel currents under voltage clamp during exposures to 0.1–100 µM IAA and NAA. Following impalements, challenge with either IAA or NAA in the presence of 10 mM KCl resulted in the concerted modulation of at least four different currents with distinct kinetic characteristics and concentration dependencies. Equivalent concentrations of benzoic acid were wholly without effect. Most striking, current carried by inward‐rectifying K+ channels (IK,in) exhibited a bimodal response to both IAA and NAA which was reversed on washing the auxins from the bathing medium. The steady‐state current was augmented 1.3‐ to 2‐fold at concentrations between 0.1 and 10 µM and antagonized at concentrations near 30 µM and above. Auxin agonism of IK,in was time‐ and voltage‐independent. By contrast, IK,in inactivation at the higher auxin concentrations was marked by a voltage‐dependence and slowing of the kinetics for current activation. Inactivation of IK,in by the auxins was relieved when cytoplasmic pH (pHi) was clamped near 7.0 in the presence of 30 mM Na+‐butyrate. In addition to the control of IK,in, current carried by a second class of (outward‐rectifying) K+ channels rose in a monotonic and largely voltage‐independent manner with auxin concentrations about 10 µM and above, and IAA and NAA also activated an inward‐going current with a voltage dependence characteristic of guard cell anion channels. Further changes in background current were consistent with a limited activation of the H+‐ATPase. Over the concentration range examined, the auxins evoked membrane hyperpolarizations and depolarizations of up to ±12–19 mV, depending on the free‐running membrane potential prevailing before auxin additions. Prolonging exposures to 100 µM auxin beyond 3–5 min frequently elicited rapid transitions to voltages near EK as well as regenerative action potentials. However, in every case the voltage response was a predictable consequence of auxin action on the K+ channels and, at 100 µM auxin, on the anion current. These results demonstrate a control of K+ channel activity by auxin, consistent with the roles of these channels in mediating K+ flux for stomatal movements; the data associate a bimodal characteristic with the activity of IK,in, implicating pHi as a putative intermediate in its control, and offer strong evidence for a multiplicity of signal cascades evoked by auxin; finally, they highlight a coordinate modulation of transport activities by auxin, thereby drawing a close analogy to the pattern of stimulus‐response coupling in abscisic acid.
Developmental control of small heat shock protein expression during pea seed maturation
Authors: DeRocher Amy Elizabeth , Vierling Elizabeth
Published: March 2003

Plants synthesize four classes of small heat shock proteins (sHSPs); two classes are targeted to the plastid and endoplasmic reticulum, respectively, and two are found in the cytoplasm. In this paper, we describe a new role for the two classes of cytoplasmic HSPs in maturing embryos of developing seeds. The expression of each class of sHSPs was examined in pea seeds grown under non‐stress conditions using Western and Northern analysis. Class I and class II cytoplasmic sHSPs are coordinately expressed in the embryo and accumulate to levels seen in moderately heat‐stressed leaves. Their induction in cotyledons coincides with the mid‐maturation phase of seed development, and induction in axes roughly coincides with abscission of the seed from the ovary wall. Both classes of sHSPs persisted in cotyledons for 4 days after the onset of imbibition, but disappeared from axes shortly after germination. Neither class of cytoplasmic sHSP is expressed in non‐embryonic organs associated with the seed. The timing and organ specificity of sHSP expression is paralleled by the expression of the corresponding mRNAs. Neither the plastid nor the endoplasmic reticulum sHSPs were consistently expressed during seed development, but both could be induced by heat‐stressing the developing seed. Developmental regulation of the cytoplasmic sHSPs is evidence that these proteins function not only in responding to heat‐stress but also during seed development and/or germination.
Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm
Authors: Mu Chen , Harn Chee , Ko Yuan‐Tih , Singletary George W. , Keeling Peter L. , Wasserman Bruce P.
Published: March 2003

Soluble starch synthase (SSS) I was purified 361‐fold from hand‐dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min−1 mg−1. A key to this purification protocol was the introduction of a size‐exclusion chromatography step, a size‐based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl‐Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.
Production of the Escherichia coli betaine‐aldehyde dehydrogenase, an enzyme required for the synthesis of the osmoprotectant glycine betaine, in transgenic plants
Authors: Holmström Kjell‐Ove , Welin Björn , Mandal Abul , Kristiansdottir Ingileif , Teeri Teemu H. , Lamark Trond , Strøm Arne R. , Palva E. Tapio
Published: March 2003

In several organisms osmotic stress tolerance is mediated by the accumulation of the osmoprotective compound glycine betaine. With the ambition to transfer the betaine biosynthetic pathway into plants not capable of synthesizing this osmoprotectant, the Escherichia coli gene betB encoding the second enzyme in the pathway, betaine‐aldehyde dehydrogenase was introduced into Nicotiana tabacum. The betB structural gene was fused to the promoter of ats1a, a gene coding for the small subunit of Rubisco in Arabidopsis thaliana. Two types of constructs were made, either encoding the N‐terminal transit peptide for chloroplast targeting or without the targeting signal for cytoplasmic localization of the BetB polypeptide. Analysis of transgenic N. tabacum plants harboring these constructs showed that in both cases the transgenes were expressed. Northern analysis of the plants demonstrated the accumulation of betB‐related mRNA of the correct size. The production and processing of the corresponding polypeptides could be demonstrated by immunoblotting using polyclonal antisera raised against the BetB polypeptide. The transit peptide encoded by ats1a was able to direct BetB to the chloroplast, as suggested by the presence of the correctly processed BetB polypeptide in the chloroplast fraction. High betaine‐aldehyde dehydrogenase activity was detected in transgenic plants, both in those where the chimeric gene product was targeted to the chloroplast and those where it remained in the cytoplasm. The transgenic tobacco acquired resistance to the toxic intermediate, betaine aldehyde, in the betaine biosynthetic pathway indicating that the bacterial enzyme is biologically active in its new host. Furthermore, these transgenic plants were able to convert exogenously supplied betaine aldehyde efficiently to glycine betaine.
In vitro transcription of plant RNA polymerase I‐dependent rRNA genes is species‐specific
Authors: Fan Hao , Yakura Kimitaka , Miyanishi Masako , Sugita Mamoru , Sugiura Masahiro
Published: March 2003

An in vitro transcription system derived from tobacco (Nicotiana tabacum) cultured cell (BY‐2) nuclei supports transcription of the RNA polymerase I‐dependent tobacco rRNA gene. The transcription initiation site determined in vitro was found at residue A which corresponds to that lying within the consensus sequence surrounding plant pre‐rRNA initiation sites. The tobacco rRNA gene is actively transcribed in the tobacco nuclear extract while the broad bean rRNA gene is inactive in the heterologous system, indicating that plant RNA polymerase I‐dependent transcription is species‐specific.
An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7‐sterol‐C‐5‐desaturase from yeast
Authors: Gachotte Daniel , Meens Roger , Benveniste Pierre
Published: March 2003

The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)‐mutagenized population of Arabidopsis thaliana which consisted of 22 000 M2 plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples.

STE 1 was characterized by the accumulation of three Δ7‐sterols concomitantly with the decrease of the three corresponding Δ5‐sterols which are the end products of the sterol pathway in wild‐type leaves. The structure of these Δ7‐sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC‐MS) and proton nuclear magnetic resonance spectrometry (1H‐NMR). The accumulation of Δ7‐sterols suggested that the mutant is deficient in the activity of the Δ7‐sterol‐C‐5‐desaturase.

Genetic analysis showed that the accumulation of Δ7‐sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild‐type. The resulting STE 1 plants had wild‐type morphology and set seeds normally, suggesting that the Δ7‐sterols in STE 1 are good surrogates of physiologically active Δ5‐sterols to sustain normal development.

STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Δ7‐sterol‐C‐5‐desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root‐derived calli showed an increase in Δ5‐sterols and a concomitant decrease in Δ7‐sterols in comparison with STE 1 untransformed root‐derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of Δ7‐sterols in the STE 1 mutant is due to a deficiency of the Δ7‐sterol‐C‐5‐desaturation step in the plant sterol biosynthesis pathway.
FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis
Authors: Kilby Nigel J. , Davies Gareth J. , Snaith Michael R. , Murray James A.H.
Published: March 2003

FLP site‐specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP‐expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly‐oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP‐mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP‐mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase‐expressing and target‐containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat‐shock promoter. Heat‐shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS‐positive. A variety of clonal sectors were produced in plants derived from seed that was heat‐shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat‐shock of unopened flower heads which produced large sectors. It was concluded that heat‐shock‐induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat‐shock.
One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA‐binding domain
Authors: Boehm Ulrich , Heinlein Manfred , Behrens Ute , Kunze Reinhard
Published: March 2003

The nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β‐glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino‐terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N‐terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA‐binding domain of TPase. A substitution of two amino acids in this segment (HiS191→Arg and Arg193→His) results in a total loss of DNA‐binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS‐deficient 71 kDa TPase derivative is co‐imported into the nucleus in the presence of wildtype TPase.
Characterization and partial purification of two pre‐tRNA 5′‐processing activities from Daucus carrota (carrot) suspension cells
Authors: Franklin Scott E. , Zwick Michael G. , Johnson Jerry D.
Published: March 2003

Two distinct RNase P‐like activities which cleave leader sequences from pre‐tRNA molecules to give mature 5′ ends have been identified in carrot suspension‐culture cells. An Escherichia coli pre‐tRNAPhe and a tobacco pre‐tRNATyr were transcribed in vitro then used as substrates for processing reactions in a cell‐free extract. The pre‐tRNATyr transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5′ and 3′ processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5′ ends of both pre‐tRNAs. Subsequent fractionation of the 5′ end‐processing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCl, when assayed with the tobacco pre‐tRNATyr substrate. When the same fractions were assayed with the E. coli pre‐tRNAPhe, only the 0.1 M KCl fraction exhibited activity. Both of the active fractions display sensitivity to micrococcal nuclease (MN) and proteinase K indicating each is a ribonucleoprotein, a result not seen with other plant RNase Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.
The AXR1 and AUX1 genes of Arabidopsis function in separate auxin‐response pathways
Authors: Timpte Candace , Lincoln Cynthia , Pickett F. Bryan , Turner Jocelyn , Estelle Mark
Published: March 2003

The recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin‐regulated gene SAUR‐AC1 in mutant and wild‐type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin‐induced accumulation of SAUR‐AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR‐AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an interaction between the AXR1 and AUX1 genes. In mature plants, the aux1‐7 mutation acts to partially suppress the morphological defects conferred by the axr1‐12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR‐AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.
Regulation of node number in day‐neutral Nicotiana tabacum: a factor in plant size
Authors: McDaniel Carl N. , Hartnett Laura K. , Sangrey Karla A.
Published: March 2003

Employing genotypes of day‐neutral tobacco that exhibit a wide range in the number of nodes produced, it has been established that node number, and plant size, in tobacco is regulated, in large part, by two endogenous signals and one developmental state, competence. All genotypes have the same level of a root signal that maintains a vegetative pattern during early growth. The number of nodes produced before the formation of the terminal flower, as well as plant size, is a function of the strength of the floral stimulus from the leaves and the competence of the terminal meristem to respond to the floral stimulus by initiating the terminal flower.
Phytoene synthase from Narcissus pseudonarcissus: functional expression, galactolipid requirement, topological distribution in chromoplasts and induction during flowering
Authors: Schledz Michael , Al‐Babili Salim , Lintig Johannes v. , Haubruck Heinz , Rabbani Said , Kleinig Hans , Beyer Peter
Published: March 2003

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full‐length overexpressed enzyme exhibited very reduced catalytic activity compared with an overexpressed N‐truncated form, with its transit sequence removed by site‐directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though association took place. In this latter case, free galactose was capable of substituting for galactolipids, resulting in enzymatic activity. It is concluded that galactolipids are involved in catalytic activity, but do not serve as a membrane anchor. Antibodies raised against the recombinant enzyme made it possible to distinguish between a membrane‐bound and a soluble, protein‐complexed inactive form of phytoene synthase, present in the chromoplast stroma. These findings and data on phytoene synthase mRNA and protein expression presented here are discussed in terms of a possible regulatory role in color formation during chromoplast (flower) development.
Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency†
Authors: Yi Ying , Guerinot Mary Lou
Published: March 2003

Reduction of Fe(III) to Fe(II) by Fe(III) chelate reductase is thought to be an obligatory step in iron uptake as well as the primary factor in making iron available for absorption by all plants except grasses. Fe(III) chelate reductase has also been suggested to play a more general role in the regulation of cation absorption. In order to experimentally address the importance of Fe(III) chelate reductase activity in the mineral nutrition of plants, three Arabidopsis thaliana mutants (frd1‐1, frd1‐2 and frd1‐3), that do not show induction of Fe(III) chelate reductase activity under iron‐deficient growth conditions, have been isolated and characterized. These mutants are still capable of acidifying the rhizosphere under iron‐deficiency and accumulate more Zn and Mn in their shoots relative to wild‐type plants regardless of iron status. frd1 mutants do not translocate radiolabeled iron to the shoots when roots are presented with a tightly chelated form of Fe(III). These results: (1) confirm that iron must be reduced before it can be transported, (2) show that Fe(III) reduction can be uncoupled from proton release, the other major iron‐deficiency response, and (3) demonstrate that Fe(III) chelate reductase activity per se is not necessarily responsible for accumulation of cations previously observed in pea and tomato mutants with constitutively high levels of Fe(III) chelate reductase activity.
A single base change prevents import of cytosolic tRNAAla into mitochondria in transgenic plants
Authors: Dietrich André , Maréchal‐Drouard Laurence , Carneiro Vera , Cosset Anne , Small Ian
Published: March 2003

Plant mitochondria do not contain a full set of tRNA genes, and the additional tRNAs needed for protein synthesis (including tRNAAla) are imported from the cytosol. The import process appears to be highly specific for certain tRNAs, and it has been suggested that the cognate aminoacyl‐tRNA synthetases may be responsible for this specificity. In order to test this, we have grown transgenic tobacco plants expressing Arabidopsis thaliana tRNAAla carrying a U70 to C70 mutation, which we have previously shown blocks aminoacylation by the plant alanyl‐tRNA synthetase. Unlike the wild‐type tRNAAla, the mutant tRNA is not present in the mitochondrial tRNA fraction. This is the first report of a tRNA mutation which prevents mitochondrial import and strongly supports the hypothesis that aminoacyl‐tRNA synthetases are involved in this process in plants. Insertion of four bases into the anticodon loop of tRNAAla does not prevent mitochondrial import, implying that the tRNA might not need to participate in translation to be imported.
Nitrite reductase expression is regulated at the post‐transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana
Authors: Crété Patrice , Caboche Michel , Meyer Christian
Published: March 2003

Higher plant nitrite reductase (NiR) is a monomeric chloroplastic protein catalysing the reduction of nitrite, the product of nitrate reduction, to ammonium. The expression of this enzyme is controlled at the transcriptional level by light and by the nitrogen source. In order to study the post‐transcriptional regulation of NiR, Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed with a chimaeric NiR construct containing the tobacco leaf NiR1 coding sequence driven by the CaMV 35S RNA promoter. Transformed plants did not show any phenotypic difference when compared with the wild‐type, although they overexpressed NiR activity in the leaves. When these plants were grown in vitro on media containing either nitrate or ammonium as sole nitrogen source, NiR mRNA derived from transgene expression was constitutively expressed, whereas NiR activity and protein level were strongly reduced on ammonium‐containing medium. These results suggest that, together with transcriptional control, post‐transcriptional regulation by the nitrogen source is operating on NiR expression. This post‐transcriptional regulation of tobacco leaf NiR1 expression was observed not only in the closely related species N. plumbaginifolia but also in the more distant species A. thaliana.
Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts
Authors: Suzuki Hideki , Ingersoll John , Stern David B. , Kindle Karen L.
Published: March 2003

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild‐type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.
Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition
Authors: Jarvis Paul , Belzile François , Page Tania , Dean Caroline
Published: March 2003

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin‐containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi‐dominant. The iae1 and iae2 mutants show 550‐ and 70‐fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re‐insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re‐insertions following germinal excision of 22%. The iae mutants represent a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.
Inefficient and incorrect processing of the Ac transposase transcript in iae1 and wild‐type Arabidopsis thaliana
Authors: Jarvis Paul , Belzile François , Dean Caroline
Published: March 2003

As part of the analysis of the Arabidopsis mutant iae1‐1 (increased Ac excision), quantitative studies of the Ac transposase transcript were conducted. The primary transcript of Ac contains three small introns (introns 1–3; mean size 89 bp) and one larger intron (intron 4; 387 bp). We analysed the splicing of intron 3 and intron 4 in wild‐type Arabidopsis and the iae1‐1 mutant. Our results demonstrate that the splicing of Ac introns 3 and 4 is inefficient (splicing efficiencies 57 and 30% respectively) compared with that of an intron of an endogenous Arabidopsis gene (PHYB intron 1; splicing efficiency 90%). The poor splicing efficiency of Ac intron 4 was found to correlate with aberrant processing. Steady state levels of total Ac transcript were higher in the iae1‐1 mutant than wild‐type, but the same aberrant processing occurred. The inefficient processing of Ac in Arabidopsis prompted us to construct an Ac element lacking introns (Ac::cDNA) in an attempt to increase transposition frequencies. Autonomous activity of the Ac::cDNA element was undetectable in Arabidopsis, despite its ability to transpose at high frequency in response to a strong transposase source (35S::transposase) in trans, and the demonstrable autonomy of the same element in tobacco. A number of smaller transcripts were detected in Arabidopsis lines containing Ac::cDNA or Ac. Analysis of these smaller transcripts revealed a high frequency of premature polyadenylation in exon 2 and splicing of cryptic introns.
Pollen‐specific expression of DEFH125, a MADS‐box transcription factor in Antirrhinum with unusual features
Authors: Zachgo Sabine , Saedler Heinz , Schwarz‐Sommer Zsuzsanna
Published: March 2003

MADS‐box genes encode transcription factors that regulate different processes of early and late floral development. A novel type of MADS‐box gene, DEFH125, was isolated from a stamen specific cDNA library from Antirrhinum majus. The DEFH125 protein shows extensive similarity over the entire length to AGL17, a root‐specific MADS‐box protein of Arabidopsis. By sharing amino acid deviations from the consensus MADS‐box sequence not found in other MADS‐box families, these two proteins constitute a novel MADS‐box subfamily. However, in contrast to members of other subfamilies the overall structural similarity between the DEFH125 and AGL17 proteins does not coincide with a similarity of expression patterns and functions. The DEFH125 gene is expressed at detectable levels only in the third whorl when the meiotic division of the pollen mother cell is already accomplished. The DEFH125 protein has been located in the cytoplasm of the vegetative cell within the maturing pollen. Surprisingly, after pollination, the DEFH125 protein is also found in nuclei of cells within the transmitting tract of the carpel. The intriguing role of DEFH125, the first MADS‐box transcription factor of this type, in aspects of fertilization, such as pollen maturation, pollen tube formation or pollen tube guidance in the carpel, is discussed.
adg2‐1 represents a missense mutation in the ADPG pyrophosphorylase large subunit gene of Arabidopsis thaliana
Authors: Wang Shue‐Mei , Chu Becky , Lue Wei‐Ling , Yu Tien‐Shin , Eimert Klaus , Chen Jychian
Published: March 2003

Arabidopsis mutants affecting ADPG pyrophosphorylase (ADGase) activity can be divided into two complementation groups, adg1 and adg2. Previous biochemical studies of adg2‐1 mutant indicated that mutant plants do not accumulate ADGase large subunit protein and that ADGase small subunits assemble as homotetramers. This suggested that the ADG2 gene may encode the large subunit of ADGase. In this paper, it is shown that adg2‐1 mutant plants accumulate near wild‐type levels of transcripts encoding both the large and small subunits of ADGase. However, by RFLP analysis and complementation of adg2‐1 with the ADGase large subunit gene, we show that the adg2‐1 mutant does represent a mutation of the ADGase large subunit gene. Sequence analysis of the adg2‐1 allele revealed a missense mutation. The results therefore suggest either that the missense mutation affects the stability of the ADGase large subunit protein or that it prevents assembly of the large subunit into holoenzyme.
Alteration of anion channel kinetics in wild‐type and abi1‐1 transgenic Nicotiana benthamiana guard cells by abscisic acid
Authors: Grabov Alexander , Leung Jeffrey , Giraudat Jerome , Blatt Michael R.
Published: March 2003

The influence of the plant water‐stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild‐type Nicotiana benthamiana L. and of transgenic plants expressing the dominant‐negative (mutant) Arabidopsis abi1‐1 protein phosphatase. Intact guard cells were impaled with double‐barrelled micro‐electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCI and 15 mM tetraethylammonium chloride (TEA‐CI) to eliminate K+ channel currents. Under these conditions, the free‐running voltage was situated close to 0 mV (+9 ± 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tail current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9‐anthracene carboxylic acid and niflumic acid, and by its voltage‐dependent kinetics. Pronounced activation of ICl was recorded on stepping from a conditioning voltage of −250 mV to voltages between −30 and +50 mV, and the current deactivated with a voltage‐dependent halftime at more negative voltages (τ⋍ 0.3 sec at −150 mV). Challenge with 20 µM ABA increased the steady‐state current conductance, gCl, near 0 mV by 1.2‐ to 2.8‐fold and at −150 mV by 4.5‐ to sixfold with a time constant of 40 ± 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near −50 mV, introducing two additional voltage‐sensitive kinetic components to these current relaxations. Neither the steady‐state and kinetic characteristics of ICl, nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad‐range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three‐ to four‐fold. Quantitatively similar current characteristics were recorded from guard cells of abi1‐1 transgenic N. bentamiana, indicating that the abi1‐1 protein phosphatase does not influence the ànion current or its response to ABA directly. These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long‐lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.
Mutants of Arabidopsis thaliana with altered cell wall polysaccharide composition
Authors: Reiter Wolf‐Dieter , Chapple Clint , Somerville Chris R.
Published: March 2003

To analyze the synthesis, structure and function of the plant cell wall by a genetic approach, 5200 chemically mutagenized Arabidopsis plants were screened for changes in the monosaccharide composition of hydrolyzed cell wall material by gas chromatography of alditol acetates. This screening procedure identified 23 mutant lines representing 11 different loci designated mur1 to mur11. The mur lines fall into essentially three groups: (1) complete absence of a monosaccharide, (2) significant reduction in the amount of a single monosaccharide, and (3) complex alterations in the relative amounts of several monosaccharides. All mutants in the first category represent alleles of the mur1 locus, and are deficient in the de novo synthesis of fucose. Mutants with reductions in a single monosaccharide have been identified for fucose (mur2, mur3), arabinose (mur4, mur5, mur6, mur7), and rhamnose (mur8). Mutants with complex changes in monosaccharide composition are represented by the mur9, mur10 and mur11 loci. Most of the mutant lines did not show obvious morphological or physiological alterations; however, lines mur1, mur9 and mur10 co‐segregated with reduced vigor or dwarfism of the plants. These results demonstrate the feasibility of identifying plants with altered cell wall compositions via a biochemical screening procedure. The availability of these mutants provides novel opportunities to study the functions of cell wall polysaccharides, gain insight into the biosynthesis of cell wall material, and clone cell wall‐related genes.
Genetics of homology‐dependent gene silencing in Arabidopsis; a role for methylation
Authors: Davies Gareth J. , Sheikh Mazhar A. , Ratcliffe Oliver J. , Coupland George , Furner Ian J.
Published: March 2003

Ninety‐eight independent transformed (T1) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T2 generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology‐ dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self‐fertilization of plants hemizygous for the B and C‐inserts suggested that these CHS‐silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C‐insert homozygotes was reduced to undetectable levels.

Outcrosses of B‐ and C‐insert homozygotes to wild‐type plants resulted in F1 plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild‐type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F1 plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C‐insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild‐type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C‐insert‐induced silencing and retained methylation and partial silencing after the silencing T‐DNA was lost.
Storage of environmental signals in flax. Their morphogenetic effect as enabled by a transient depletion of calcium
Authors: Verdus Marie‐Claire , Thellier Michel , Ripoll Camille
Published: March 2003

It is possible to induce the formation of epidermal meristems in the hypocotyl of non‐injured and non‐hormone‐treated plantlets of flax, by combining various sorts of physical stimulations with a transient depletion of calcium. The characteristic times for the decrease of the tissue concentration of calcium during calcium depletion and for the recovery of the normal tissue concentration of calcium after resupplying the latter ion, are close to 1 day. The stimuli may correspond to wind or drought or even to the manipulation stress occurring when the plantlets are transferred from their germination to their growth vessel. Meristem production is increased by combining several physical stimulations. When calcium depletion is delayed relative to the application of the physical stimulation(s), the production of meristems is delayed accordingly. This means that the signal induced by the physical stimulation(s) may be stored within the plants, without apparent effect, until a calcium depletion finally allows the stored signal to take effect (formation of meristems). For storage periods of up to 8 days no loss of the potency of the stored signal was observed. A few other examples of storage of morphogenetic signals in plants have been described in the literature. The mechanism involved in signal storage is still not clearly understood. However, it seems that the sensing and/or storing of the signals require that the plant tissues are sufficiently rich in calcium, whereas the licensing of the plants for the translation of signals into the final response (meristem production) is done by a transient calcium depletion.
In vitro dephosphorylation inhibits the activity of soybean lysine‐ketoglutarate reductase in a lysine‐regulated manner
Authors: Miron Daphna , Ben‐Yaacov Sari , Karchi Hagai , Galili Gad
Published: March 2003

In plant seeds, the essential amino acid lysine auto‐regulates its own level by modulating the activity of its catabolic enzyme lysine‐ketoglutarate reductase via an intracellular signaling cascade, mediated by Ca2+ and protein phosphorylation/dephosphorylation. In the present report, it has been further tested whether the activity of soybean lysine‐ketoglutarate reductase, as well as that of saccharopine dehydrogenase, the second enzyme in the pathway of lysine catabolism, are modulated by direct phosphorylation of the bifunctional polypeptide containing both of these linked activities. Incubation of purified lysine‐ketoglutarate reductase/ saccharopine dehydrogenase with casein kinase II resulted in a significant phosphorylation of the bifunctional enzyme. Moreover, in vitro dephosphorylation of the bifunctional polypeptide with alkaline phosphatase significantly inhibited the activity of lysine‐ketoglutarate reductase, but not of its linked enzyme saccharopine dehydrogenase. The inhibitory effect of alkaline phosphatase on lysine‐ketoglutarate reductase activity was dramatically stimulated by binding of lysine to the enzyme. Our results suggest that in plant seeds, active lysine‐ketoglutarate reductase is a phospho‐protein, and that its activity is modulated by opposing actions of protein kinases and phosphatases. Moreover, this modulation is subject to a compound regulation by lysine.
Zygote implantation to cultured ovules leads to direct embryogenesis and plant regeneration of wheat
Authors: Kumlehn Jochen , Brettschneider Reinhold , Lörz Horst , Kranz Erhard
Published: March 2003

Direct embryogenesis and plant regeneration were obtained by implantation of individual wheat (Triticum aestivum L.) zygotes into cultured ovules of wheat or barley. The zygotes were isolated mechanically from emasculated spikes, 3–9 h after hand‐pollination. In 13 independent experiments, a total of 186 zygotes were implanted into excised ovules obtained from emasculated spikes which had been treated previously with 2,4‐dichlorophenoxyacetic acid to induce parthenocarpic, embryoless ovary development. On average, 17.2% of the implanted zygotes gave rise to dorsiventrally differentiated embryos. The embryos resembled those growing in planta with no obvious deviation from the zygotic embryogenesis pathway. In contrast to previously described regeneration systems from individual zygotes of higher plants, this is the first study in which direct embryo formation is reproducibly obtained without intermediate tissue dedifferentiation. Most embryos germinated when transferred to regeneration medium, and later formed phenotypically normal, fully fertile plants. Regenerants were confirmed to be derived from the implanted zygotes by means of AFLP and/or morphological analyses. Although zygote implantation has long been established as a useful method in sexual animal reproduction, an equivalent technique for plants is described here for the first time. Since the zygotes enter the embryogenic pathway directly, the genome is presumably as stable as during embryogenesis in planta. With this new approach, isolated wheat zygotes are accessible to micromanipulation without affecting their subsequent embryonic development.
Re‐evaluation of the role of a transmitting tract‐specific glycoprotein on pollen tube growth
Authors: Sommer‐Knudsen Jens , Lush W. Mary , Bacic Antony , Clarke Adrienne E.
Published: March 2003

It has been proposed that a stylar glycoprotein, the transmitting tissue‐specific (TTS) protein isolated from Nicotiana tabacum, serves both as a growth stimulant, by providing a source of nutrients, and as an attractant for pollen tubes during their growth through the style. Working with a galactose‐rich style glycoprotein (GaRSGP) that is the N. alata homologue (97% homology) of the TTS protein, a series of experiments, similar to those done with the TTS protein, was performed. Evidence was found for inhibition of pollen tube growth at high concentrations, but no evidence was found for stimulation of growth at concentrations up to 2 mg ml–1. No effect as a pollen tube attractant was detected. The discrepancies in the features and functionality between these homologous glycoproteins in the closely related Nicotiana species warrants further investigation before a general function is assigned to these molecules.
Early stages of the apoptotic pathway in plant cells are reversible
Authors: O’brien Iona E. W. , Baguley Bruce C. , Murray Brian G. , Morris Bret A. M. , Ferguson Ian B.
Published: March 2003

Chromatin condensation and nDNA fragmentation, indicators of apoptosis in mammalian cells, occur in plant cells during senescence and following induction by chemical agents. In Nicotiana plumbaginifolia cells, camptothecin, okadaic acid, salicylic acid, hydrogen peroxide, and the calcium ionophore A23187 induced chromatin condensation and nDNA fragmentation. Exposure of cells to low concentrations or removal of the chemical agent resulted in an initial phase of chromatin condensation, followed by its reversal. A further feature of apoptosis in mammalian cells, annexin V binding, indicative of phosphotidylserine exposure, was also confirmed in relation to the other events in the apoptotic pathway. With respect to flow cytometric characteristics, apoptosis triggered by a variety of chemicals occurs in plant cells in a manner closely related to that in mammalian cells. However, the extent of chromatin condensation is substantially greater, and in the early stages is reversible.