bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid construction of a transcription map for a cosmid contig of Arabidopsis thaliana genome using a novel cDNA selection method
Authors: Seki Motoaki , Hayashida Nobuaki , Kato Nobuko , Yohda Masafumi , Shinozaki Kazuo
Published: March 2003

Significant progress has been made on the random sequencing of cDNAs (ESTs) and the genetic and physical mapping of the Arabidopsis thaliana genome. New techniques are now required to identify and map the expressed genes efficiently on A. thaliana chromosomes. A novel method to construct a transcription map of expressed genes or cDNAs in specific regions of the genome using DNA‐latex particles has been developed. The region‐specific DNA fragments prepared from six cosmid clones that constitute a contig covering the abi1 locus on chromosome 4 were covalently bound to latex particles. The DNA‐latex particles were used for the selection of region‐specific cDNAs. Sequence analysis of the cDNA clones revealed that ABI1, RPS2, casein kinase I (CK1), nucleosome assembly protein I (NAP) cDNAs and T20837 EST, which are situated within the contig near abi1 locus, were selected. These results indicate that the cDNAs in the specific region of the genome were faithfully selected with this method. Sequence analysis also indicated that 11 selected cDNAs were derived from novel genes located near the abi1 locus and that four of the selected cDNAs encode putative proteins that have sequence similarity to cationic peroxidase, phosphatidylserine decarboxylase 2 (PSD2), trans‐caffeoyl CoA 3‐O‐methyltransferase (CCoAMT), and proteasome subunit XC3.
The viviparous8 mutation delays vegetative phase change and accelerates the rate of seedling growth in maize
Authors: Evans Matthew M.S. , Poethig R. Scott
Published: March 2003

Post‐embryonic shoot development in plants can be divided into a juvenile vegetative, an adult vegetative, and a reproductive phase, which are expressed in different domains on the shoot axis. The number and position of the phytomers in each phase are determined by the time at which a plant begins and ceases making phytomers of a particular phase and the rate at which phytomers are made during that phase. The viviparous8 (vp8) mutation of maize increases the number of juvenile vegetative phytomers and decreases the number of adult vegetative phytomers by affecting both of these processes. vp8 increases the number of juvenile vegetative phytomers by increasing the rate of leaf initiation early in shoot development and delaying the juvenile‐to‐adult transition (vegetative maturation). It reduces the number of adult phytomers because the delay in vegetative maturation is not matched by a corresponding delay in flowering time; vp8 plants produce a tassel at the same time as wild‐type plants. Thus, Vp8 normally controls the production of a factor that functions both to repress the rate of growth early in shoot development and to promote vegetative maturation, but which has no major role in floral induction. vp8 dramatically enhances the phenotypes of the dwarf and Teopod mutants and requires a functional Glossy15 gene to prolong the expression of juvenile epidermal traits. Evidence suggesting that vp8 does not affect phase change by reducing the level of abscisic acid is discussed.
Internal AU‐rich elements modulate activity of two competing 3′ splice sites in plant nuclei
Authors: Merritt Hanne , McCullough Andrew J. , Schuler Mary A.
Published: March 2003

In vivo analyses using an autonomously replicating Agrobacterium/geminivirus vector have enabled identification of AU‐rich intronic elements critical for 5′ and 3′ splice site selection in dicot plant nuclei and development of a model for pre‐mRNA intron recognition in plant nuclei. To determine the minimal length, spacing and nucleotide compositions constraining recognition of the 3′ boundary of an intron, two or four nucleotide substitutions have been introduced into the two AU‐rich elements located between 50 and 66 nucleotides upstream from the 3′ splice site of maize Adh 1 intron 3. In each case tested, substitutions in the distal left element (−62 to −66) inactivate the downstream 3′ splice site at −1 more effectively than substitutions in the proximal right element (−50 to −55). Guanosine or cytosine substitutions in either element reduce recognition of the −1 site significantly; adenosine substitutions have a less severe effect. Mutations in both of these AU elements additively block recognition of the downstream 3′ splice site. The strong additive effect of these mutations supports a model in which short sets of AU islands bind interactive factors and cooperatively modulate usage of the downstream splice site. In contrast to the uridine requirements documented for the 3′ terminus of plant introns, adenosines are partially interchangeable with uridines within this internal region of the intron.
A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean
Authors: Gijzen Mark
Published: March 2003

The Ep locus severely affects the amount of peroxidase enzyme in soybean seed coats. Plants containing the dominant Ep allele accumulate large amounts of peroxidase in the hourglass cells of the sub‐epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in the hourglass cells and are much reduced in total seed coat peroxidase activity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was constructed from developing seed coats and an abundant 1.3 kb peroxidase transcript was cloned. The corresponding structural gene was also isolated from a genomic library. Sequence analysis shows that the seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pl of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorphisms that distinguished EpEp and epep genotypes. Co‐segregation of the polymorphisms in an F2 population from a cross of EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lacks 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically reduced amounts of peroxidase transcript compared with EpEp plants. The peroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indicate that the lack of peroxidase accumulation in seed coats of homozygous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.
Gibberellins are required for embryo growth and seed development in pea
Authors: Swain Stephen M. , Reid James B. , Kamiya Yuji
Published: March 2003

The gibberellin (GA) biosynthesis mutants lh‐1 and lh‐2 have been used to examine the physiological role of GAs in pea seed development. The LH protein is required for the three‐step oxidation of ent‐kaurene to ent‐kaurenoic acid early in the GA biosynthesis pathway. The allele‐specific interaction of lh‐1 and lh‐2 with chemical inhibitors of these three steps suggests that LH encodes the multi‐functional GA biosynthesis enzyme ent‐kaurene oxidase. Unlike the lh‐2 mutation which reduces seed weight and decreases seed survival by ∼50% compared with wild‐type plants, the lh‐1 allele has a transient effect on embryo and seed growth and only slightly increases seed abortion. These seed phenotypes parallel the effects of the two mutant alleles on GA levels in young seeds. Detailed examination of the growth of lh‐1 seeds reveals homeostatic regulation of GA‐promoted embryo and seed growth. Although GA‐deficient seeds grow more slowly than WT seeds, decreased assimilate availability to the developing seeds is not the primary reason for the altered seed development. Instead, GAs act to promote some process(es) required for embryo and seed growth and only indirectly influence the distribution of assimilates. How GA deficiency causes seed abortion is not known but it may simply be a consequence of reduced seed or embryo growth rate. These results demonstrate that even relatively small changes in the levels of GAs in young seeds can alter seed development and suggest that the available GA‐related mutants may represent only a subset of all possible mutants with reduced GA levels or GA signalling.
A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants
Authors: Haake Volker , Zrenner Rita , Sonnewald Uwe , Stitt Mark
Published: March 2003

Antisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several ‘antisense’ potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32–43%, 56–71%, 79–83% and 91–97% in A‐70, A‐3, A‐51 and A‐2. Separation on a Q‐Sepharose–FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose‐1,7‐biphosphatase and plastid fructose‐1,6‐bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A‐70, A‐3, A‐51 and A‐2. The transformants contained increased triose phosphates, and very low ribulose‐1,5‐bisphosphate and glycerate‐3‐phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A‐70 and A‐3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A‐70, was decreased by 41% in A‐3, and was severely inhibited in plants with under 20% of wild‐type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.
Effects of mevinolin on cell cycle progression and viability of tobacco BY‐2 cells
Authors: Hemmerlin Andréa , Bach Thomas J.
Published: March 2003

Mevinolin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY‐2 cells. After treatment with 5 μM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of α‐like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin‐treated TBY‐2 cells was clearly arrested before the aphidicolin‐inducible block. By the aid of a double‐blocking technique, it was shown that the mevinolin‐induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above‐mentioned induction of cell death.
Systemic and local induction of an Arabidopsis thionin gene by wounding and pathogens
Authors: Vignutelli Alberto , Wasternack Claus , Apel Klaus , Bohlmann Holger
Published: March 2003

The Arabidopsis Thi2.1 thionin gene was cloned and sequenced. The promoter was fused to the uidA gene and stably transformed into Arabidopsis to study its regulation. GUS expression levels correlated with the steady‐state levels of Thi2.1 mRNA, thus demonstrating that the promoter is sufficient for the regulation of the Thi2.1 gene. The sensitivity of the Thi2.1 gene to methyl jasmonate was found to be developmentally determined. Systemic and local expression could be induced by wounding and inoculation with Fusarium oxysporum f sp. matthiolae. A deletion analysis of the promoter identified a fragment of 325 bp upstream of the start codon, which appears to contain all the elements necessary for the regulation of the Thi2.1 gene. These results support the view that thionins are defence proteins, and indicate the possibility that resistance of Arabidopsis plants to necrotrophic fungal pathogens is mediated through the octadecanoid pathway.
Translation initiation factors are differentially regulated in cereals during development and following heat shock
Authors: Gallie Daniel R. , Le Hanh , Tanguay Robert L. , Browning Karen S.
Published: March 2003

The level of protein synthetic activity varies greatly in plants during development or following many environmental stresses. In order to determine whether these global changes in protein synthesis involve changes in the expression of the translational machinery itself, the expression patterns of the initiation factors (eIFs) during cereal seed development, germination, and in leaves following a heat shock were investigated. The expression patterns of initiation factors during seed development fell into four classes: those whose levels were high during early to mid‐development but decreased during late seed development (eIF4 A, eIFiso4E, eIFiso4G, and most eIF3 subunits); those whose levels increased from early to mid‐development followed by a decrease during late seed development (eIF4B, eIF4E, eIF2α, eIF2β, and PABP); those whose levels steadily increased throughout seed development (eIF4G); and those whose levels remained constant (the p34 subunit of eIF3). In contrast to these observations, the expression patterns of the factors appeared to be coordinately regulated during early germination although differences were observed at later stages. Following a heat shock, the changes in initiation factor expression in wheat leaves fell into two classes, those whose level increased (eIF4G, eIFiso4G, eIF3, and PABP) and those whose level remained unchanged (eIF4 A, eIF4B, eIF4E, eIFiso4E). These observations reveal the complexity of the expression patterns of the initiation factors during seed development, germination, and following thermal stress and may have mechanistic importance for the selective translation of certain mRNAs under these conditions.
KAS IV: a 3‐ketoacyl‐ACP synthase fromCuphea sp. is a medium chain specific condensing enzyme
Authors: Dehesh Katayoon , Edwards Patricia , Fillatti JoAnne , Slabaugh Mary , Byrne James
Published: March 2003

cDNA clones encoding a novel 3‐ketoacyl‐ACP synthase (KAS) have been isolated from

Cuphea

. The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic

Brassica

plants over‐expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic

Brassica

seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co‐expression of the

Cuphea

KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co‐expression of the

Cuphea

KAS along with an 18:0/18:1‐ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the

Cuphea

KAS reported here has a different acyl‐chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.
Two dehydration‐inducible transcripts from the resurrection plantCraterostigma plantagineumencode interacting homeodomain‐leucine zipper proteins
Authors: Frank Wolfgang , Phillips Jonathan , Salamini Francesco , Bartels Dorothea
Published: March 2003

The molecular dissection of desiccation tolerance in the resurrection plant

Craterostigma plantagineum

led to the isolation of two dehydration‐stress inducible homeodomain‐leucine zipper genes (

CPHB‐1

and

‐2

). When the coding region of

CPHB‐1

was used as bait in the yeast two‐hybrid system, the ability of CPHB‐1 to form homodimers was demonstrated. The two‐hybrid system was also used to isolate CPHB‐2, which heterodimerises with CPHB‐1. Both transcripts are inducible by dehydration in leaves and roots, but steady state levels vary in response to exogenously applied ABA. Although expression of

CPHB‐1

is not inducible by ABA, the transcript level of

CPHB‐2

increases during ABA‐treatment. Both genes are expressed at very early stages of dehydration and thus may be involved in the regulation of gene expression during dehydration.

CPHB‐1

and ‐

2

differential expression in response to ABA suggests that they act in different branches of the dehydration‐induced signalling network.

In vitro

binding studies revealed that CPHB‐1 specifically binds to the pseudopalindromic sequence CAAT(C/G)ATTG. Using this element for

in vitro

binding studies with nuclear proteins from dehydrated leaves, an inducible DNA‐protein complex was identified.
Characterization of the TAC box, acis‐element within an elicitor‐inducible sesquiterpene cyclase promoter
Authors: Newman Jeffrey D. , Yin Shaohui , Chappell Joseph
Published: March 2003

The first unique step in the synthesis of the tobacco phytoalexin capsidiol is cyclization of farnesyl pyrophosphate catalyzed by 5‐epi‐aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated that the elicitor‐inducibility of this enzyme activity corresponded to the transcriptional activation of at least one gene,

EAS4

, of a rather complex gene family consisting of > 10 members. To investigate the mechanism(s) controlling expression of this gene, fragments of the

EAS4

promoter were examined for binding by proteins in nuclear and whole‐cell extracts. A strong protein binding site (TAC box; ACTCTACAGTACTC) was identified between –245 and –232 by the electrophoretic mobility shift assay, and DNAase I and methylation interference footprinting. Several distinctly migrating bands representing protein–TAC box complexes were also observed in the mobility shift assays, and the relative abundance of these bands varied in extracts from cells at different stages of EAS induction. The TAC box binding factor (TacBBF) was purified > 450‐fold from crude whole‐cell extracts by a combination of DNA affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding properties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding

in vitro

and increased both basal and elicitor‐inducible gene expression 2.5‐fold in transgenic tobacco plants harboring promoter–GUS fusions, consistent with the notion that this

cis

‐element functions as a silencer or repressor of EAS gene expression.
Functional analysis of regulatory sequences controllingPR‐1 gene expression in Arabidopsis
Authors: Lebel Edouard , Heifetz Peter , Thorne Leigh , Uknes Scott , Ryals John , Ward Eric
Published: March 2003

The Arabidopsis PR‐1 gene is one of a suite of genes induced co‐ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6‐dichloroisonicotinic acid (INA). We have characterized

cis

‐acting regulatory elements in the PR‐1 promoter involved in INA induction using deletion analysis, linker‐scanning mutagenesis, and

in vivo

footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10‐fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10‐bp linker‐scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The –640 linker‐scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the –610 linker‐scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF‐κB. Furthermore, several inducible

in vivo

footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This

in vivo

signature of protein–DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.
Use of the IGF BAC library for physical mapping of theArabidopsis thalianagenome
Authors: Mozo Teresa , Fischer Sabine , Meier‐Ewert Sebastian , Lehrach Hans , Altmann Thomas
Published: March 2003

In order to generate a physical map of the

Arabidopsis thaliana

genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the

Arabidopsis

–IGF–BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied ‘double‐end clone‐limited/sampling without replacement’ hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map‐based gene cloning approaches and further genome analysis.
Regulation of gibberellin biosynthesis genes during flower and early fruit development of tomato
Authors: Rebers Mariken , Kaneta Tsuyoshi , Kawaide Hiroshi , Yamaguchi Shinjiro , Yang Young‐Yell , Imai Ryozo , Sekimoto Hiroyuki , Kamiya Yuji
Published: March 2003

Gibberellins (GAs) are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In the GA‐biosynthetic pathway, the reactions catalyzed by GA 20‐oxidases have been implicated as site of regulation. To study the regulation of GA biosynthesis in flower and early fruit development, we isolated three tomato GA 20‐oxidase cDNA clones,

Le20ox‐1, ‐2 and ‐3

. The three genes showed different organ‐specific patterns of mRNA accumulation. Analysis of the transcript levels of the three

GA 20‐oxidase

genes, as well as those of

copalyl diphosphate synthase

(

LeCPS

) and

GA 3β‐hydroxylase (Le3OH‐2)

during flower bud and early fruit development, revealed temporally distinct patterns of mRNA accumulation. Up until anthesis, transcripts were observed for

LeCPS, Le20ox‐1, ‐2 and Le3OH‐2

, with an accumulation of

Le20ox‐1

mRNA. In contrast to the high level of

Le3OH‐2

transcripts in the fully open flower, mRNA levels of

Le20ox‐1, ‐2 and LeCPS

were reduced at this stage. After anthesis,

LeCPS

and

Le20ox‐1

transcripts increased again. In addition,

Le20ox‐3

transcripts increased whereas the transcripts of

Le3OH‐2

decreased to an undetectable level.

In situ

hybridization results demonstrated that during early stages of bud development,

Le20ox‐2

transcripts were localized in the tapetum and placenta. The presented results supply novel data about localization of GA biosynthesis gene transcripts, and indicate that transcript levels of GA biosynthesis genes are all highly regulated during flower bud development.
Regional insertional mutagenesis of genes onArabidopsis thalianachromosome V using theAc/Dstransposon in combination with a cDNA scanning method
Authors: Ito Takuya , Seki Motoaki , Hayashida Nobuaki , Shibata Daisuke , Shinozaki Kazuo
Published: March 2003

For regional insertional mutagenesis of

Arabidopsis thaliana

genes, we combined a cDNA scanning method (Hayashida

et al

.

Gene

1995; 165:155–161) and an

Ac/Ds

transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on

A. thaliana

chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub‐libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub‐library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non‐redundant groups of cDNAs were obtained. Forty‐four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed

Ds

from a donor

Ds–GUS–

T‐DNA line, Ds4391–20, already mapped to the CIC7E11/8B11 region. We obtained

Ds

‐transposed lines and recovered their

Ds

‐flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot‐blot analysis indicated that 20% of the lines contained transposed

Ds

in the CIC7E11/8B11 region, suggesting that this

Ac/Ds

transposon system is effective for regional insertional mutagenesis. To isolate

Ds

insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800

Ds

‐containing lines using

Ds

‐specific and gene‐specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain

Ds

insertion mutations, and that five lines contain

Ds

mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the

Ac/Ds

transposon gives a powerful tool for regional insertional mutagenesis not only in

Arabidopsis

but also in other plants or crops whose genomes are not sequenced.
The dormancy‐related peroxiredoxin anti‐oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells
Authors: Stacy Robin A. P. , Nordeng Tommy W. , Culiáñez‐Macià Francisco A. , Aalen Reidunn B.
Published: March 2003

Protection against desiccation‐induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by‐products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation‐induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free‐radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol‐requiring anti‐oxidants with one conserved cysteine residue (1‐Cys), and displays in vitro anti‐oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy‐related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non‐dormant seeds. Our data identify the location of this seed‐specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C‐terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.
Suppression of pea nuclear topoisomerase I enzyme activity by pea PCNA
Authors: Van Hop Duong , Gaikwad Amos , Yadav Badam Singh , Reddy Malireddy Kodandarami , Sopory Sudhir , Mukherjee Sunil Kumar
Published: March 2003

Proliferating cell nuclear antigen (PCNA), a highly conserved DNA polymerase accessory protein of eukary‐ otic kingdom, has not been studied thoroughly in bio‐ chemical terms in plants. We describe the isolation of the cDNA encoding PCNA from the pea cDNA library using the PCR approach. The cDNA was used for expression of pea PCNA in bacteria as a fusion protein (GST.PCNA) with the GST tag at the amino terminal end. The GST.PCNA stimulated the partially purified pea DNA polymerases approximately 30‐fold. The stimulation was due to the oligomeric form of GST.PCNA. The pea PCNA interacted with the recombinant type I pea topoiso‐ merase as well as the native pea nuclear topoisomerase I and repressed the DNA relaxation activities. However, the DNA binding activity of Topo I remained undisturbed in the presence of high amounts of PCNA, thereby signify‐ ing that the catalysis of Topo I was probably affected by PCNA.
Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation
Authors: Ye Guang‐Ning , Stone Deborah , Pang Sheng‐Zhi , Creely Wendi , Gonzalez Kathleen , Hinchee Maud
Published: March 2003

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post‐infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3–5 days post‐infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5–11 days post‐infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.
Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell‐type specific expression in plants
Authors: Kaltenbach Melanie , Schröder Gudrun , Schmelzer Elmon , Lutz Verena , Schröder Joachim
Published: March 2003

We investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68–78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3′,5′‐hydroxylase, but 3′‐hydroxylated products were also detected. The substrate specificity was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted flavanones as well as flavones, dihydroflavonols and flavonols, and both performed 3′‐ as well as 3′5′‐hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein and RNA showed that the F3′5′H was present in dark‐grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4‐hydroxylase and flavanone 3β‐hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3′5′H substrates. Immunohistochemical localization of F3′5′H had not been performed before. A comparison of CHS and F3′5′H in cotyledons and flower buds from C. roseus identified CHS expression preferentially in the epidermis, while F3′5′H was only detected in the phloem. The cell‐type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.
Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis
Authors: Hartmann Ulrike , Höhmann Susanne , Nettesheim Klaus , Wisman Ellen , Saedler Heinz , Huijser Peter
Published: March 2003

Flowering time mutants represent genetic functions in control of the floral transition, an important developmental phase switch in the life cycle of higher plants. Many such mutants have been identified and characterized, particular in Arabidopsis. Here we describe the identification and initial characterization of a new early flowering mutant of Arabidopsis. The corresponding gene, SVP (SHORT VEGETATIVE PHASE), was cloned through transposon tagging and represents a new member of the MADS‐box gene family of transcription factors. Analysis of its transcriptional activity revealed the presence of differently sized transcripts that were confined to vegetative tissues and floral primordia and absent from developed flowers and siliques. The function of SVP as a repressor of the floral transition is discussed.
The Arabidopsis Cdc2a‐interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin‐dependent kinase activity in vitro
Authors: Lui Helen , Wang Hong , DeLong Catherine , Fowke Larry C. , Crosby William L. , Fobert Pierre R.
Published: March 2003

Cyclin‐dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle. To study protein–protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two‐hybrid system. Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a carboxy‐terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK‐binding domain of the mammalian inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins. At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1‐associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1. These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions.
Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1
Authors: Liu Zongrang , Hossain Gazi Showkat , Islas‐Osuna Maria A. , Mitchell David L. , Mount David W.
Published: March 2003

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV‐induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium‐mediated transformation of the uvh1‐1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1‐1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1‐1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3′ splice site and introducing an in‐frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.
The two largest chloroplast genome‐encoded open reading frames of higher plants are essential genes
Authors: Drescher Anja , Ruf Stephanie , Calsa Tercilio , Carrer Helaine , Bock Ralph
Published: March 2003

The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild‐type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild‐type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild‐type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.
Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate
Authors: Munnik Teun , Meijer Harold J. G. , Ter Riet Bas , Hirt Heribert , Frank Wolfgang , Bartels Dorothea , Musgrave Alan
Published: March 2003

In mammalian cells, phospholipase D (PLD) and its product phosphatidic acid (PA) are involved in a number of signalling cascades, including cell proliferation, membrane trafficking and defence responses. In plant cells a signalling role for PLD and PA is also emerging. Plants have the extra ability to phosphorylate PA to produce diacylglycerol pyrophosphate (DGPP), a newly discovered phospholipid whose formation attenuates PA levels, but which could itself be a second messenger. Here we report that increases in PA and its conversion to DGPP are common stress responses to water deficit. Increases occur within minutes of treatment and are dependent on the level of stress. Part of the PA produced is due to PLD activity as measured by the in vivo transphosphatidylation of 1‐butanol, and part is due to diacylglycerol kinase activity as monitored via 32P‐PA formation in a differential labelling protocol. Increases in PA and DGPP are found not only in the green alga Chlamydomonas moewusii and cell‐suspension cultures of tomato and alfalfa when subjected to hyperosmotic stress, but also in dehydrated leaves of the resurrection plant Craterostigma plantagineum. These results provide further evidence that PLD and PA play a role in plant signalling, and provide the first demonstration that DGPP is formed during physiological conditions that evoke PA synthesis.
Developmental and light‐regulated post‐translational control of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase levels in potato
Authors: Korth Kenneth L. , Jaggard Daniel A. W. , Dixon Richard A.
Published: March 2003

In plants, the first committed step in the cytosolic pathway for biosynthesis of isoprenoids is catalysed by 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR). We have added an eight amino‐acid‐residue epitope tag to a potato (Solanum tuberosum L.) HMGR isoform and expressed this novel protein (HMGR–FLAG) in transgenic plants. Despite high levels of transcript accumulation in all leaf stages of transgenic plants, high levels of HMGR–FLAG protein were found only in apical meristematic tissue, suggesting post‐translational regulation of potato HMGR affected by plant development. Protein immunoblots, and determination of enzymatic activity and transcript accumulation in the HMGR–FLAG transgenic and the non‐transgenic parental plant lines, show that HMGR levels decrease dramatically in the dark. Again, the mechanism of this control occurs at a post‐translational level. After 2.5 h in darkness, levels of HMGR–FLAG are approximately one‐half of those in plants in the light; protein levels recover rapidly when dark‐treated plants are returned to the light. In non‐transgenic plants, hmg transcript levels are reduced in the dark, whereas dark treatments do not affect transgene hmg transcripts expressed under the control of a constitutive promoter. Furthermore, transcripts for HMGR–FLAG remain associated with polyribosomes in dark‐treated tissues. Addition of inhibitors of cysteine proteases during microsomal protein extraction is required for recovery of immunoreactive HMGR–FLAG. The epitope‐tagged isozyme has been used to show for the first time that a regulated decrease in plant HMGR activity correlates closely with a loss of the HMGR protein. We have used whole plants to demonstrate that developmental and light‐regulated control of HMGR occurs post‐translationally in vivo.
The alternative oxidase in roots of Poa annua after transfer from high‐light to low‐light conditions
Authors: Millenaar Frank F. , Roelofs Roeland , Gonzàlez‐Meler Miquel A. , Siedow James N. , Wagner Anneke M. , Lambers Hans
Published: March 2003

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high‐light to low‐light conditions, and at the same time from long‐day to short‐day conditions for four days. As a result of the low‐light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O‐isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low‐light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.
Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa
Authors: Horie Tomoaki , Yoshida Kazuya , Nakayama Hideki , Yamada Katsuyuki , Oiki Shigetoshi , Shinmyo Atsuhiko
Published: March 2003

It is thought that Na+ and K+ homeostasis is crucial for salt‐tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+‐Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni‐OsHKT1). We also isolated two cDNAs homologous to Ni‐OsHKT1 from salt‐tolerant indica rice, cv Pokkali (Po‐OsHKT1, Po‐OsHKT2). The predicted amino acid sequence of Ni‐OsHKT1 shares 100% identity with Po‐OsHKT1 and 91% identity with Po‐OsHKT2, and they are 66–67% identical to wheat HKT1. Low‐K+ conditions (less than 3 mm) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mm Na+. We further characterized the ion‐transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+‐uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+‐limiting conditions. When OsHKTs were expressed in Na+‐sensitive yeast, OsHKT1 rendered the cells more Na+‐sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+‐ and K+‐coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.
Requirement for 3‐ketoacyl‐CoA thiolase‐2 in peroxisome development, fatty acid β‐oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings
Authors: Germain Véronique , Rylott Elizabeth L. , Larson Tony R. , Sherson Sarah M. , Bechtold Nicole , Carde Jean‐Pierre , Bryce James H. , Graham Ian A. , Smith Steven M.
Published: March 2003

3‐ketoacyl‐CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid β‐oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identified a T‐DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modified. Long‐chain (C16 to C20) fatty acyl‐CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long‐chain thiolase activity. In addition, extracts from kat2 seedlings have significantly decreased activity with aceto‐acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germination and seedling growth, indicating that KAT2 has broad substrate specificity. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal β‐oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies.