bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus
Authors: Wang Tzann‐Wei , Balsamo Ronald A. , Ratnayake Chandra , Platt Kathryn A. , Ting Julie T.L. , Huang Anthony H.C.
Published: March 2003

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15‐residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS‐PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of ∼ 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N‐terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of low‐density organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule‐filled plastids and lipid‐containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low‐density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil‐body oleosins share common epitopes at the generally non‐conserved C‐terminal domain with the sporophytic tapetum oleosins.
Alternative processing of the maize Ac transcript in Arabidopsis
Authors: Martin D.J. , Firek S. , Moreau E. , Draper J.
Published: March 2003

The successful application of the maize transposable element system Ac/Ds as a genome mutagen in heterologous plant species has recently proved the versatility and power of this technique in plant molecular biology. However, the frequency of Ac/Ds transposition is considerably lower in Arabidopsis thaliana than in most other dicot plant species that have been studied. Since previous research has established that transcripts derived from monocot genes can be alternatively processed in dicot plants, we have investigated both the efficiency of intron splicing and polyadenylation of the maize Ac transposase pre‐mRNA in Arabidopsis thaliana, Nicotiana tabacum, Nicotiana plumbaginifolia and Zea mays. In this paper, we demonstrate that intron 4 is alternatively spliced within Arabidopsis, using cryptic 5′ and 3′ splice sites within the intron sequence, leading to a heterogeneous population of full length transposase transcript. Furthermore, analysis of transposase transcript polyadenylation revealed that at least four alternative poly(A) sites were utilized between introns 2 and 3, resulting in truncated transposase transcripts. Finally, by Northern blotting, we established that the truncated transposase transcript was the most abundant form of transposase message in Arabidopsis. In contrast to these findings, the alternative splicing and premature polyadenylation of Ac message in Arabidopsis was unparalleled in the other species examined. We suggest that the poor frequency of transposition of Ac in Arabidopsis may be in part due to the low quantity of correctly processed transposase transcript available in this species.
Isolation and characterization of chromosome‐specific DNA sequences from a chromosome arm genomic library of common wheat
Authors: Liu Bao , Segal Gregorio , Vega Juan Manuel , Feldman Moshe , Abbo Shahal
Published: March 2003

Isolation, physical mapping and polymorphism of chromosome‐specific DNA sequences in wheat are reported. Following the microdissection of the long arm of chromosome 5B (5BL) of common wheat, its DNA was amplified by degenerate oligonucleotide‐primed PCR and directly cloned into plasmid vectors. Characterization of the chromosome arm library showed that ∼55% of the inserts are of low‐copy nature. Southern analysis using aneuploid lines of common wheat revealed that five of 11 low‐copy inserts analyzed map to chromosome arm 5BL; four of these are 5BL‐specific. By deletion mapping, the 5BL‐specific sequences were located to sub‐ chromosome arm regions. Based on the hybridization patterns of three 5BL‐specific sequences to DNA from a diverse collection of goat‐grass (Aegilops) and wheat (Triticum) species, it was concluded that these sequences emerged at different times in the course of evolution of this group of plant species.
Isolation of a novel class of bZIP transcription factors that interact with ABA‐responsive and embryo‐specification elements in the Dc3 promoter using a modified yeast one‐hybrid system
Authors: Kim Soo Young , Chung Hwa‐Jee , Thomas Terry L.
Published: March 2003

Dc3 is a carrot lea class gene that is abundantly expressed during somatic and zygotic embryogenesis. Its expression is normally embryo‐specific and also can be induced by abscisic acid. The regulatory elements mediating the embryo‐specific expression of Dc3 reside within the proximal promoter region (−117 to +26), which is also essential for ABA‐induced expression. In this study, an optimized version of the yeast one‐hybrid system has been used to clone factors that bind to the promoter region of the Dc3 gene. Twenty‐five million yeast transformants were screened in a single experiment, and nine independent cDNA clones were isolated from a sunflower library that encode proteins that specifically bind to functional cis‐regulatory elements in the Dc3 promoter. Analysis of these clones showed that they are derived from three different mRNA species that encode two basic leucine zipper proteins. The basic regions of these proteins, named DPBF‐1 and 2 (Dc3 Promoter‐Binding Factor‐1 and 2), respectively, are nearly identical to each other and are similar to the plant G‐box binding factor GBF‐4. Outside the basic region, however, DPBF‐1 and 2 diverge significantly from each other and from other known factors. Both factors have transcriptional activity in yeast, and bind to DNA as dimers. Unlike other plant bZIP factors, DPBF‐1 and 2 recognize sequences containing the ACACNNG core. Cloning of these factors demonstrates the power of the one‐hybrid approach when optimally applied.
Nitrogen and hormonal responsiveness of the 22 kDa α‐zein and b‐32 genes in maize endosperm is displayed in the absence of the transcriptional regulator Opaque‐2
Authors: Müller Martin , Dues Gisbert , Balconi Carlotta , Salamini Francesco , Thompson Richard D.
Published: March 2003

The maize (Zea mays L.) b‐ZIP transcriptional activator Opaque‐2 (O2) regulates the synthesis of major endosperm proteins. In the o2 homozygote, 22 kDa zein prolamins and the b‐32 ribosome‐inactivating protein are greatly reduced in level. An in vitro endosperm culture system has been studied in which o2 endosperm synthesizes 22 kDa zein and b‐32 in response to nitrogen supplements. An increase in 22 kDa zein mRNA concentration is also seen, implying an effect at the level of transcription or differential RNA turnover. The nitrogen‐dependent induction of 22 kDa zein synthesis in cultured o2 endosperm was further investigated by analysing transient expression of reporter constructs. The highest response to nitrogen was exhibited by the intact 22 kDa zein promoter. Removal of individual O2 binding sites either reduced or increased overall promoter activity, but always decreased the nitrogen‐dependent stimulation of activity. This effect was observed equally in wild‐type and o2 mutant endosperm. It is concluded that a factor other than O2 is responsible for activating the 22 kDa zein promoter under high‐nitrogen culture conditions. Despite its occurrence in the absence of O2 protein, the nitrogen response is mediated through binding at O2 binding sites. An induction of 22 kDa zein and b‐32 synthesis in cultured o2 endosperm could also be achieved on nitrogen‐free media by the addition of abscisic acid or methyl jasmonate.
Novel DNA‐binding characteristics of a protein associated with DNA polymerase‐α in pea
Authors: Burton Sara K. , Van't Hof Jack , Bryant John A.
Published: March 2003

DNA polymerase‐α‐primase may be isolated from pea shoot tip cells as a large (1.25 × 106 Da) multi‐protein complex. The complex exhibits several enzyme activities and also binds to DNA. One of the DNA‐binding activities has been purified as a 42 kDa polypeptide. The binding of this polypeptide to linear DNA fragments and to open circular plasmids has been studied by electron microscopy. The protein binds to restriction enzyme‐generated cohesive ends of linear fragments and also exhibits some interstitial binding. Binding at the ends of linear molecules is very markedly reduced if the molecules are previously treated with S1 nuclease. The protein also binds to open circular plasmids; the number of binding sites is increased by exposing the plasmids to γ‐irradiation prior to the DNA‐protein interaction. In these experiments, the number of protein units bound is directly related to the radiation dose. With both linear and open circular molecules, binding of the protein to the DNA leads to an apparent shortening of the DNA molecule. These observations, taken with the finding that the protein does not bind to completely single‐stranded DNA, lead to the suggestion that the protein binds to double‐stranded‐single‐stranded (ds‐ss) junctions in DNA and that binding causes the DNA to wrap round the protein.
Tracing the ontogeny of stomatal clusters in Arabidopsis with molecular markers
Authors: Serna Laura , Fenoll Carmen
Published: March 2003

The origin and process by which the mosaic of the different cell types is established during the development of the leaf epidermis in Arabidopsis are largely unknown, although the recent characterization of two mutants which develop stomatal clusters (four lips (flp) and too many mouths (tmm)) has opened up the possibility for genetic dissection of the stomata spacing. By using growth conditions which limit gas exchange with the open atmosphere, stomatal clusters that look like phenocopies of flp and tmm have been induced, suggesting that stomata spacing is under environmental as well as genetic control in Arabidopsis. The origin of these clusters has been addressed by following promoter activity for genes that are markers for competence for cell division (cdc2aAt), mitotic activity (cyc1aAt), and guard mother cell and developing guard cell identity (rha1). Their different expression patterns in the various cell types during epidermal differentiation and the asynchrony in the development of the various stomata that constitute each cluster suggest that these stomatal clusters derive from a single protodermal cell through a process that involves changes in cell fate in a subset of subsidiary cells. It was also found that guard cells express cdc2aAt and cyc1aAt, supporting the idea that they may remain competent for cell division.
Myb26: a MYB‐like protein of pea flowers with affinity for promoters of phenylpropanoid genes
Authors: Uimari Anne , Strommer Judith
Published: March 2003

Plant Myb proteins represent a group of transcription factors which have a DNA‐binding domain similar to that found in the products of the animal myb proto‐oncogenes. Members of the Myb family regulate the biosynthesis of phenylpropanoids, including anthocyanin and phlobaphene pigments, in several species. In this study, PCR with degenerate primers was used to analyse the presence of myb‐like genes in pea (Pisum sativum L.). A fragment representing a flower bud‐expressed gene, designated myb26, was recovered, and a full length cDNA was isolated from a pea flower bud cDNA library. The predicted protein is 217 amino acids long and its Myb‐domain and carboxy terminal region show extensive homology to the snap‐dragon proteins Myb305 and Myb340, both of which regulate phenylpropanoid biosynthesis. Expression of myb26 is flower‐specific and parallels the expression of flavonoid biosynthetic genes, increasing as the flower bud matures and intensifies in colour. However, myb26 represents neither of the two known regulatory genes for anthocyanin production in pea, nor does it complement mutants of the myb‐like anthocyanin regulatory gene an2 in petunia. Myb26 was expressed in E. coli as a fusion protein. It was shown that in vitro Myb26 recognizes the c‐Myb and P‐box‐like binding sites representing cis‐elements in the promoter regions of several phenylpropanoid biosynthetic genes. The results suggest that myb26 is a previously undefined gene involved in regulation of some aspect of phenylpropanoid production in pea.
Chromosome micro‐dissection and region‐specific libraries from pachytene chromosomes of maize (Zea mays L.)
Authors: Stein  , Ponelies  , Musket  , McMullen  , Weber
Published: March 2003

For the first time, pachytene chromosomes have been used for micro‐dissection, amplification and cloning using the short arm of maize chromosome 6. For this purpose, methods for the preparation of unstained pachytene chromosomes were developed. In two separate experiments, 10 and 12 segments of the satellite region were isolated utilizing glass needles and DNA was amplified by linker‐adaptor‐PCR. After amplification, maize‐specific DNA was verified by genomic Southern hybridizations. Chromosomal in situ suppression hybridization confirmed that the adaptor PCR products originated from the micro‐dissected 6S region. The adaptor PCR products were cloned into a plasmid vector and the chromosome region‐specific libraries were characterized.
Transposase binding site methylation in the epigenetically inactivated Ac derivative Ds‐cy
Authors: Wang Lihua , Kunze Reinhard
Published: March 2003

The authors have determined the C‐methylation pattern of the non‐autonomous transposable element Ds‐cy, which is an epigenetically inactivated, transcriptionally silent derivative of the maize Activator (Ac) element. Like Ac, Ds‐cy is hypermethylated at the 3′‐end. However, in Ds‐cy the 5′‐end is also hypermethylated, including all subterminal binding sites for the Ac‐encoded transposase protein. As Ds‐cy transposes in the presence of an active Ac in the genome, the authors conclude that methylation of the 5′‐end TPase binding sites does not interfere with transposition, but correlates with inactivity of the Ac promoter. The authors discuss the implications of these results for the chromatid selectivity of Ac/Ds transposition and the mechanism of Ac‐induced chromosome breakage events.
Gene silencing from plant DNA carried by a Geminivirus
Authors: Kjemtrup Susanne , Sampson Kim S. , Peele Charles G. , Nguyen Long V. , Conkling Mark A. , Thompson William F. , Robertson Dominique
Published: March 2003

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open‐reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3‐5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti‐sense orientation, and a 403 bp 3' fragment. TGMV::su‐induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti‐sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.
Glucocorticoid‐inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death
Authors: McNellis Timothy W. , Mudgett Mary Beth , Li Karen , Aoyama Takashi , Horvath Diana , Chua Nam‐Hai , Staskawicz Brian J.
Published: March 2003

Pathogenic strains of Pseudomonas syringae pv. tomato carrying the avrRpt2 avirulence gene specifically induce a hypersensitive cell death response in Arabidopsis plants that contain the complementary RPS2 disease resistance gene. Transient expression of avrRpt2 in Arabidopsis plants having the RPS2 gene has been shown to induce hypersensitive cell death. In order to analyze the effects of conditional expression of avrRpt2 in Arabidopsis plants, transgenic lines were constructed that contained the avrRpt2 gene under the control of a tightly regulated, glucocorticoid‐inducible promoter. Dexamethasone‐induced expression of avrRpt2 in transgenic lines having the RPS2 gene resulted in a specific hypersensitive cell death response that resembled a Pseudomonas syringae‐induced hypersensitive response and also induced the expression of a pathogenesis‐related gene (PR1). Interestingly, high level expression of avrRpt2 in a mutant rps2–101C background resulted in plant stress and ultimately cell death, suggesting a possible role for avrRpt2 in Pseudomonas syringae virulence. Transgenic RPS2 and rps2 plants that contain the glucocorticoid‐inducible avrRpt2 gene will provide a powerful new tool for the genetic, physiological, biochemical, and molecular dissection of an avirulence gene‐specified cell death response in both resistant and susceptible plants.
Transcription pattern of a FIM homologue in Impatiens during floral development and reversion
Authors: Pouteau Sylvie , Nicholls David , Tooke Fiona , Coen Enrico , Battey Nicholas
Published: March 2003

Flowering and reversion in Impatiens are characterised by gradual transitions of organ identity and constitute a unique system for the molecular and physiological study of floral organogenesis. The authors have isolated an Impatiens homologue of the FIM gene of Antirrhinum (UFO in Arabidopsis), Imp‐FIM, and analysed its expression in three states of the terminal meristem: vegetative, floral, and reverted. In floral meristems, Imp‐FIM transcription is associated with petal identity, as in Antirrhinum and Arabidopsis, but this is achieved through a novel transcription pattern, characterised by a high level of transcript within petal primordia. This novel transcription pattern could contribute to the more diffuse boundaries between organ types in Impatiens. In vegetative meristems, Imp‐FIM is expressed in the axils of leaf primordia which are arranged in a spiral. A similar pattern is observed in reverted meristems in which leaf primordia are initiated in a whorled arrangement. This result indicates that the maintenance of floral phyllotaxis is not associated with a specific pattern of Imp‐FIM transcription. Transcription of Imp‐FIM in a non‐reverting line is no different from that in the reverting line. Therefore, the lack of floral commitment in the reverting line does not seem to be responsible for Imp‐FIM transcription within petals. The novel transcription pattern in petals, together with features of Impatiens that are reminiscent of fim and ufo mutant phenotypes suggest an evolutionary divergence for Imp‐FIM regulation in this species.
Functional integration of non‐native carotenoids into chloroplasts by viral‐derived expression of capsanthin–capsorubin synthase in Nicotiana benthamiana
Authors: Kumagai Monto H. , Keller Yves , Bouvier Florence , Clary David , Camara Bilal
Published: March 2003

The biosynthesis of leaf carotenoids in

Nicotiana benthamianawas altered by forced re‐routing of the pathway to the synthesis of capsanthin, a non‐native chromoplast‐specific xanthophyll, using an RNA viral vector containing capsanthin–capsorubin synthase (

Ccs) cDNA. The cDNA encoding

Ccswas placed under the transcriptional control of a tobamovirus subgenomic promoter. Leaves from transfected plants expressing

Ccsdeveloped an orange phenotype and accumulated high levels of capsanthin (up to 36% of total carotenoids). This phenomenon was associated with thylakoid membrane distortion and reduction of grana stacking. In contrast to the situation prevailing in chromoplasts, capsanthin was not esterified and its increased level was balanced by a concomitant decrease of the major leaf xanthophylls, suggesting an autoregulatory control of chloroplast carotenoid composition. Capsanthin was exclusively recruited into the trimeric and monomeric light‐harvesting complexes of photosystem II (PSII) and shown to significantly contribute to the light‐harvesting capacity. On a chlorophyll basis, the concentrations of PSI and PSII reaction centres were not modified. This demonstration that higher plant antenna complexes can accommodate non‐native carotenoids provides compelling evidence for functional remodelling of photosynthetic membranes toward a better photoreactivity by rational design of the incorporated carotenoid structures.
Temporal and photoregulated expression of five tomato phytochrome genes
Authors: Hauser Bernard A. , Cordonnier‐Pratt Marie‐Michèle , Pratt Lee H.
Published: March 2003

Quantitative measurements of the absolute amounts of mRNAs transcribed from each of five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE, PHYF) throughout the life cycle of a tomato plant and in response to changes in ambient light conditions are reported here. From their lowest level in unimbibed seed, all five transcripts increase by from 10‐ to 1000‐fold during the first 24 h following the onset of imbibition, both in continuous darkness and in a greenhouse. In a greenhouse and on a whole‐plant basis, all continue to increase throughout day 6, after which all but PHYE decline over the next week to a plateau at about one‐half of the maximal value. PHYE mRNA differs in that it continues to increase in abundance during the first 2–3 weeks and thereafter remains at that maximal level. In adult plants, on a whole‐plant basis and in decreasing order of abundance, PHYA, PHYB1, PHYE, PHYB2, and PHYF transcripts were present at approximately 120, 40, 40, 15, and 8 μmol μg–1 of poly(A)+‐enriched RNA, respectively. The data are consistent with the demonstrated roles of phytochromes A and B1 during seedling development and lead to the suggestion that phytochrome E might have a more important role in mature plants. Somewhat unexpectedly, PHYA and PHYB2 expression patterns are very similar. In seedlings, PHYA and PHYB2 exhibit the greatest increase in expression following a light‐to‐dark transition, as well as the greatest decrease following a dark‐to‐light transition. PHYA and PHYB2 are also similar in that both exhibit comparable variation on a natural diurnal cycle, while PHYB1 also exhibits variation but with a markedly different phase. The diurnal variation in expression of PHYA, PHYB1 and PHYB2 is consistent with the possibility that one or more of the phytochromes they encode is important with respect not only to photoperiodic behavior but also to the regulation of other events whose photosensitivity varies during a diurnal cycle.
Inversions in the Chlamydomonas chloroplast genome suppress a petD 5′ untranslated region deletion by creating functional chimeric mRNAs
Authors: Higgs David C. , Kuras Richard , Kindle Karen L. , Wollman Francis‐André , Stern David B.
Published: March 2003

FUD6 is a non‐photosynthetic Chlamydomonas mutant that lacks the cytochrome b6/f complex, due to a 236 bp deletion that removes the promoter and part of the 5′ untranslated region (UTR) of the chloroplast petD gene, which encodes subunit IV of the complex. Two photosynthetic revertants of FUD6 that synthesized wild‐type levels of subunit IV were found to contain related inversions of the chloroplast genome that resulted from recombination between small inverted repeats. These inversions created a functional chimeric petD gene that includes the promoter and part of the 5′ UTR of the newly identified ycf9‐psbM transciption unit, fused to the petD 5′ UTR upstream of the FUD6 deletion. Accumulation of the ycf9‐psbM dicistronic transcript was disrupted in the revertants, but monocistronic psbM mRNA accumulated normally. The FUD6 revertants demonstrate the ability of the Chlamydomonas chloroplast genome to undergo a large inversion without a deleterious effect on chloroplast function, reminiscent of events that have led to the evolutionary divergence of chloroplast genomes.
Characterization of exon skipping mutants of the COP1 gene from Arabidopsis
Authors: Simpson Craig G. , McQuade Clare , Lyon Jackie , Brown John W. S.
Published: March 2003

The removal of introns from pre‐mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1–1 was a G→A change 4 nt upstream from the 3′ splice site of intron 5, while the mutation in cop1–2 was a G→A at the first nucleotide of intron 6, abolishing the conserved G within the 5′ splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1–8 was G→A in the final nucleotide of intron 10 abolishing the conserved G within the 3′ splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.
Sucrose‐specific signalling represses translation of the Arabidopsis ATB2 bZIP transcription factor gene
Authors: Rook Fred , Gerrits Nathalie , Kortstee Anne , Van Kampen Muriel , Borrias Mies , Weisbeek Peter , Smeekens Sjef
Published: March 2003

The Arabidopsis bZIP transcription factor gene ATB2 has been shown previously to be expressed in a light‐regulated and tissue‐specific way. Here we describe the precise localization of ATB2 expression, using transgenic lines containing an ATB2 promoter–GUS reporter gene construct. The observed expression pattern suggests a role for ATB2 in the control of processes associated with the transport or utilization of metabolites. Remarkably, expression of the ATB2–GUS reporter gene construct was specifically repressed by sucrose. Other sugars, such as glucose and fructose, alone or in combination, were ineffective. Repression was observed at external sucrose concentrations exceeding 25 mM. Transcript levels of both the endogenous ATB2 gene and the ATB2–GUS reporter gene were not repressed by sucrose, suggesting that sucrose affects mRNA translation. This translational regulation involves the ATB2 leader sequence because deletion of the leader resulted in loss of sucrose repression. Our results provide evidence for a sucrose‐specific sugar sensing and signalling system in plants.
The 3′ untranslated region of a rice α‐amylase gene mediates sugar‐dependent abundance of mRNA
Authors: Chan Ming‐Tsair , Yu Su‐May
Published: March 2003

A decrease in transcript stability is one of the important mechanisms that control the sugar repression of α‐amylase gene expression in rice suspension cells. In this study, we investigated the function of the 3′ untranslated region (3′UTR) of a rice α‐amylase gene, αAmy3, in relation to sugar‐dependent accumulation of mRNA. By examining the transient expression of chimeric genes in rice protoplasts, we were able to demonstrate that the αAmy3 3′UTR mediated the sugar‐dependent repression of fused heterologous gene expression. The same kinetics of accumulation of αAmy3 mRNA and reporter mRNA carrying the αAmy3 3′UTR in response to glucose deprivation were observed, suggesting that the αAmy3 3′UTR is sufficient, and probably the major determinant for controlling the abundance of these transcripts. Functional analysis of two subdomains of αAmy3 3′UTR by insertion into a sugar‐inducible chimeric gene confirmed their roles in sugar repressibility. The regulatory sequences in the αAmy3 3′UTR may act as potent determinants of mRNA stability in response to sugar availability. This finding has important implications for studying the regulatory mechanism of sugar repression in eukaryotes.
Non‐toxic concentrations of cadmium inhibit systemic movement of turnip vein clearing virus by a salicylic acid‐independent mechanism
Authors: Citovsky Vitaly , Ghoshroy Soumitra , Tsui Frank , Klessig Daniel
Published: March 2003

Systemic movement of plant viruses is a central event in viral infection. To better understand this process, the heavy metal cadmium was used to inhibit systemic spread of turnip vein clearing virus (TVCV), a tobamovirus, in tobacco plants. Study of the mechanism by which cadmium exerts this inhibitory effect may provide insights into the essential steps of the TVCV systemic movement pathway. Our results demonstrated that cadmium treatment did not affect TVCV transport from the inoculated non‐vascular tissue into the plant vasculature but blocked viral exit into uninoculated non‐vascular tissues. Thus, TVCV virions may enter and exit the host plant vascular system by two different mechanisms. We also showed that cadmium‐treated plants still supported systemic spread of an unrelated tobacco etch virus (TEV), suggesting multiple pathways for systemic infection. Finally, cadmium‐induced arrest in TVCV systemic infection was shown to occur by a salicylic acid‐independent mechanism.
Phylogenetic transfer of organelle genes to the nucleus can lead to new mechanisms of protein integration into membranes
Authors: Michl Doris , Karnauchov Ivan , Berghöfer Jürgen , Herrmann Reinhold G. , Klösgen Ralf Bernd
Published: March 2003

Subunits CFo‐I and CFo‐II of ATP synthase in chloroplast thylakoid membranes are two structurally and functionally closely related proteins of bitopic membrane topology which evolved from a common ancestral gene. In higher plants, CFo‐I still originates in plastid chromosomes (gene:

atp

F), while the gene for CFo‐II (

atp

G) was phylogenetically transferred to the nucleus. This gene transfer was accompanied by the reorganization of the topogenic signals and the mechanism of membrane insertion. CFo‐I is capable of integrating correctly as the mature protein into the thylakoid membrane, whereas membrane insertion of CFo‐II strictly depends on a hydrophobic targeting signal in the transit peptide. This requirement is caused by three negatively charged residues at the N‐terminus of mature CFo‐II which are lacking from CFo‐I and which have apparently been added to the protein only after gene transfer has occurred. Accordingly, the CFo‐II transit peptide is structurally and functionally equivalent to typical bipartite transit peptides, capable of also translocating hydrophilic lumenal proteins across the thylakoid membrane. In this case, transport takes place by the Sec‐dependent pathway, despite the fact that membrane integration of CFo‐II is a Sec‐independent, and presumably spontaneous, process.
The role of actin in root hair morphogenesis: studies with lipochito‐oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug
Authors: Miller Deborah D. , De Ruijter Norbert C. A. , Bisseling Ton , Emons Anne mie C.
Published: March 2003

Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament configuration during bulge formation, root hair initiation, sustained tip growth, growth termination, and in full‐grown hairs. Lipochito‐oligosaccharides (LCOs) were used to interfere with growth (

De Ruijter

et al

. 1998

,

Plant J.

13, 341–350), and cytochalasin D (CD) was used to interfere with actin function. Actin filament bundles lie net‐axially in cytoplasmic strands in the root hair tube. In the subapex of growing hairs, these bundles flare out into fine bundles. The apex is devoid of actin filament bundles. This subapical actin filament configuration is not present in full‐grown hairs; instead, actin filament bundles loop through the tip. After LCO application, the tips of hairs that are terminating growth swell, and a new outgrowth appears from a site in the swelling. At the start of this outgrowth, net‐axial fine bundles of actin filaments reappear, and the tip region of the outgrowth is devoid of actin filament bundles. CD at 1.0 μ
m
, which does not affect cytoplasmic streaming, does not inhibit bulge formation and LCO‐induced swelling, but inhibits initiation of polar growth from bulges, elongation of root hairs and LCO‐induced outgrowth from swellings. We conclude that elongating net‐axial fine bundles of actin filaments, which we call FB‐actin, function in polar growth by targeting and releasing Golgi vesicles to the vesicle‐rich region, while actin filament bundles looping through the tip impede vesicle retention.
Characterization of nuclear import of potato spindle tuber viroid RNA in permeabilized protoplasts
Authors: Woo Young‐Min , Itaya Asuka , Owens Robert A. , Tang Li , Hammond Rosemarie W. , Chou Huei‐Chi , Lai Michael M. C. , Ding Biao
Published: March 2003

Most viroids replicate in the nuclei of infected plant cells. Nuclear import of the incoming RNA thus represents a key control point for establishment of a systemic infection. However, little is known about the mechanisms by which viroids are transported into the nucleus. We have characterized nuclear import of infectious, fluorescein‐labeled potato spindle tuber viroid (F‐PSTVd) in permeabilized tobacco BY2 cells. Import was observed for F‐PSTVd but not for mRNA fragments of the same size or two viroids believed to replicate in the chloroplasts. Import of F‐PSTVd was inhibited by addition of a 10‐fold excess of non‐fluorescent PSTVd but not by similar amounts of control RNAs. Import was not inhibited by pre‐incubation with GTP‐γ‐S or GDP‐β‐S, however. Disruption of microtubules and actin filaments with oryzalin or cytochalasin D did not inhibit F‐PSTVd import. Taken together, our results indicate that (i) PSTVd possesses a sequence and/or structural motif for nuclear import and (ii) the import is a cytoskeleton‐independent process that is mediated by a specific and saturable receptor. Insensitivity to GTP‐γ‐S and GDP‐β‐S treatment suggests that PSTVd import is not coupled to Ran GTPase cycle, which mediates nuclear transport of many proteins and nucleic acids. To our knowledge, our studies are the first to examine the mechanisms of nuclear transport of RNA in plants.
The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana
Authors: Ephritikhine Geneviève , Pagant Silvère , Fujioka Shozo , Takatsuto Suguru , Lapous Danielle , Caboche Michel , Kendrick Richard E. , Barbier‐Brygoo Hélène
Published: March 2003

In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (
Ephritikhine et al. 1999
; Plant J. 18, 303–314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3β‐hydroxyl,Δ5,6 precursors to 3‐oxo,Δ4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroid‐dependent growth of seedlings in both light and dark conditions.
RNA maturation of the rice SPK gene may involve trans‐splicing
Authors: Kawasaki Tsutomu , Okumura Satoru , Kishimoto Naoki , Shimada Hiroaki , Higo Kenichi , Ichikawa Norio
Published: March 2003

A gene encoding a calcium‐dependent seed‐specific protein kinase (SPK) is abundantly expressed in developing rice seeds (Kawasaki, T et al. Gene (1993) 129, 183–189). Rice genomic clones encoding SPK were isolated using the entire cDNA fragment as a probe. Physical mapping of these genomic clones indicated that the genomic region corresponding to the entire cDNA was divided into two different regions, SPK‐A and SPK‐B, located on different rice chromosomes. The results of RACE–PCR analyses showed that the respective tran‐ scripts from SPK‐A and SPK‐B contained additional sequences which were not found in the SPK cDNA, and that these sequences were removed like introns during maturation of the SPK mRNA. These results suggest that two different RNAs were independently transcribed from SPK‐A and SPK‐B and joined, possibly by trans‐splicing.
Expression analysis of two gene subfamilies encoding the plasma membrane H+‐ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme
Authors: Moriau Luc , Michelet Baudouin , Bogaerts Pierre , Lambert Laurence , Michel Alain , Oufattole Mohammed , Boutry Marc
Published: March 2003

The plasma membrane H+‐ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+‐ATPase) genes. Four of these are classified into two gene subfamilies, pma1‐2‐3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5′ end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma–gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell‐type specific manner. Expression in these organs was confirmed at the protein level, using subfamily‐specific antibodies. (ii) pma4–gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+‐ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+‐ATPases with different enzymatic properties.
Control of gluconeogenesis by isocitrate lyase in endosperm of germinating castor bean seedlings
Authors: Runquist Mårten , Kruger Nicholas J.
Published: March 2003

The aim of this work was to quantify the contribution of isocitrate lyase to the control of gluconeogenesis in endosperm from 4‐day‐old castor bean seedlings. The approach was based on metabolic control analysis following selective inhibition of enzyme activity. Both 3‐nitropropionate and itaconate decreased the proportion of either [1‐14C]acetate or [2‐14C]acetate converted to sucrose, and increased the proportion metabolized through the tricarboxylic acid cycle. Kinetic analysis of isocitrate lyase activity from endosperm revealed that itaconate is a pure uncompetitive inhibitor (Ki′ = 11.9 ±0.98 μm) with respect to isocitrate. In contrast, 3‐nitropropionate is a slow, tight‐binding inhibitor. The half‐time for inhibition of isocitrate lyase by 3‐nitropropionate was 5–10 min, whereas the half‐time for reactivation was in excess of 10 h. Incubating endosperm in 3‐nitropropionate resulted in a concentration‐dependent decrease in isocitrate lyase activity that remained stable in tissue extracts for at least 4 h. From a comparison of the extent of in situ inactivation of isocitrate lyase by 3‐nitropropionate and the effect of this compound on the rate of sucrose production from [2‐14C]acetate, the flux control coefficient of isocitrate lyase on gluconeogenesis from acetate in castor bean endosperm was calculated to be 0.66 ± 0.09. It is concluded that isocitrate lyase activity is quantitatively important in the control of gluconeogenic flux, and suggested that developmental changes in the amount of this enzyme may be an important factor in determining the conversion of lipid to sugar in young castor bean seedlings.
Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA‐ and cytochrome P450 gene‐mediated selection
Authors: Koprek Thomas , McElroy David , Louwerse Jeanine , Williams‐Carrier Rosalind , Lemaux Peggy G.
Published: March 2003

Efficient negative selection systems are increasingly needed for numerous applications in plant biology. In recent years various counter‐selectable genes have been tested in six dicotyledonous species, whereas there are no data available for the use of negative selection markers in monocotyledonous species. In this study, we compared the applicability and reliability of two different conditional negative selection systems in transgenic barley. The bacterial codA gene encoding cytosine deaminase, which converts the non‐toxic 5‐fluorocytosine (5‐FC) into the toxic 5‐fluorouracil (5‐FU), was used for in vitro selection of germinating seedlings. Development of codA‐expressing seedlings was strongly inhibited by germinating the seeds in the presence of 5‐FC. For selecting plants in the greenhouse, a bacterial cytochrome P450 mono‐oxygenase gene, the product of which catalyses the dealkylation of a sulfonylurea compound, R7402, into its cytotoxic metabolite, was used. T1 plants expressing the selectable marker gene showed striking morphological differences from the non‐transgenic plants. In experiments with both negative selectable markers, the presence or absence of the transgene, as predicted from the physiological appearance of the plants under selection, was confirmed by PCR analysis. We demonstrate that both marker genes provide tight negative selection; however, the use of the P450 gene is more amenable to large‐scale screening under greenhouse or field conditions.
The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas
Authors: Schroda Michael , Blöcker Dagmar , Beck Christoph F.
Published: March 2003

The Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, β2TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A–RBCS2 promoter fusion as compared to the RBCS2 promoter alone.
Identification and analysis of proteins that interact with the Avena fatua homologue of the maize transcription factor VIVIPAROUS 1
Authors: Jones Huw D. , Kurup Smita , Peters Nicholas C. B. , Holdsworth Michael J.
Published: March 2003

The Avena fatua (wild oat) homologue of VIVIPAROUS 1 (AfVP1) has been implicated in controlling the maintenance of embryo dormancy in mature imbibed seeds, but the detailed mechanisms by which this transcription factor family activates embryo maturation pathways and simultaneously represses germination are not known. A two‐hybrid screen in yeast identified three proteins that interacted specifically with AfVP1 (AfVP1 interacting proteins; AfVIPs). AfVIPs 2 and 3 interacted with the C‐terminus of AfVP1, which contains the B2 + B3 domains, previously shown to bind DNA, whereas AfVIP1 interacted with the isolated B3 domain. Using purified proteins in in vitro experiments, all three AfVIPs were shown also to interact with the Arabidopsis homologue ABSCISIC ACID INSENSITIVE 3 (ABI3). The three AfVIPs were expressed in both dormant and non‐dormant embryos, but the abundance of AfVIP1 and 3 transcripts was greater in germinated than dormant seeds, whereas transcripts of AfVIP2 (and AfVP1) were more highly expressed in dormant embryos. The AfVIP3 protein has homology to a human cell‐crisis gene with a predicted role in the cell cycle; AfVIP2 contains a ring‐type zinc finger motif. These homologies, together with analysis of expression studies, suggest that these proteins may play specific roles in AfVP1‐mediated regulation of the dormancy to germination transition in A. fatua seeds.
Functional rescue of a bacterial dapA auxotroph with a plant cDNA library selects for mutant clones encoding a feedback‐insensitive dihydrodipicolinate synthase
Authors: Vauterin Marc , Frankard Valérie , Jacobs Michel
Published: March 2003

Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5≈ 10 μM), whereas the bacterial enzymes are less (50‐fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine‐sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS‐deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.