bluuebunny/crossref_metadata_2025 huggingface dataset:
A self‐fertile mutant of Phalaris produces an S protein with reduced thioredoxin activity
Authors: Li Xinmin , Nield Jan , Hayman David , Langridge Peter
Published: March 2003

Gametophytic self‐incompatibility in the Phalaris coerulescens is controlled by two unlinked genes, S and Z. Isolation of the S gene from the pollen of this grass species indicated that the C terminus has significant hemology with thioredoxin H proteins. The protein from the C terminus, expressed in Escherichia coli, exhibits thioredoxin‐like activity. This paper demonstrates that the C terminus of the S protein from an S complete mutant shows significant reduction in thioredoxin activity when compared with the wild‐type form. Both pollen and stigma have lost self‐incompatibility in this mutant. Close examination of the lesions, which were found only in the C terminus of the mutant gene suggests that the substitution of a serine by an arginine is responsible for the reduced enzymatic activity. The association between reduced activity and the loss of the self‐incompatibility provides evidence for a role of thioredoxin activity in the self‐incompatibility reaction of this species.
Oxidative stress in Agrobacterium‐induced tumors on Kalanchoe plants
Authors: Jia Shi Rong , Kumar Prakash P. , Kush Anil
Published: March 2003

The induction of tumor formation by wild‐type Agrobacterium tumefaciens was found to be associated with the apparent rate of production of superoxide anions (O2°−) in the infected tissue of Kalanchoe plants. The transcript levels of MnSOD and Cu/ZnSOD and the specific activity of superoxide dismutase (SOD) during different stages of tumor induction support this. A. tumefaciens strain with mutations in the cytokinin biosynthetic gene produced delayed, smaller and abnormal tumors which underwent significantly lower levels of superoxide stress compared with normal tumors, produced by the wild‐type strain. These suppressed tumors developed into normal tumors with the concurrent production of O2°− when supplemented in vitro with exogenous phytohormones. Phytohormone‐induced callus from non‐infected stem tissues maintained in vitro in the presence of phytohormones produced O2°− in the same order of magnitude as the tumor cells. Transgenic plants expressing three‐ to five‐fold higher levels of SOD activity exhibited a 55% reduction in the apparent rate of O2°− production compared with that of the non‐transgenic plants when challenged with wild‐type Agrobacterium. These results suggest the possible role of phytohormones in creating superoxide stress which, in turn, may cause deregulation of cell division, oncogenicity and ultimately tumor formation.
Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport
Authors: Maathuis Frans J.M. , Sanders Dale
Published: March 2003

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in planta. Such questions can be addressed through the use of well‐defined transport mutants. Csi52, a caesium‐insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch‐clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb+ uptake experiments reveal a reduced uptake in csi52 in both the high‐ and low‐affinity uptake range. Patch‐clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild‐type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wild‐type and mutant. Rb+ uptake studies show that in both wild‐type and csi52 the high‐affinity uptake pathway is considerably less sensitive to Cs+ than the low‐affinity pathway with K1/2 values for Cs+ of around 1.3 and 0.2 mM, respectively. Furthermore, K+ starvation leads to a larger relative increase in high‐affinity K+ uptake in the mutant than the wild‐type. The results demonstrate the Cs+ sensitivity of each individual uptake pathway is comparable in wild‐type and csi52 but the high‐affinity pathway is less Cs+ sensitive (in both wild‐type and csi52). Therefore, the larger shift toward high‐affinity uptake in the mutant compared with the wild‐type under K+‐starvation conditions will endow the mutant with a higher degree of overall Cs+ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K+ uptake.
Distribution of tobamovirus movement protein in infected cells and implications for cell‐to‐cell spread of infection
Authors: Padgett Hal S. , Epel Bernard L. , Kahn Theodore W. , Heinlein Manfred , Watanabe Yuichiro , Beachy Roger N.
Published: March 2003

The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.
CLA1, a novel gene required for chloroplast development, is highly conserved in evolution
Authors: Mandel M. Alejandra , Feldmann Kenneth A. , Herrera‐Estrella Luis , Rocha‐Sosa Mario , León Patricia
Published: March 2003

An albino mutant designated cla1‐1 (for ‘cloroplastos alterado’, or ‘altered chloroplasts’) has been isolated from a T‐DNA‐generated library of Arabidopsis thaliana. In cla11 plants, chloroplast development is arrested at an early stage. cla1‐1 plants behave like wild‐type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected. Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T‐DNA insertion is responsible for the mutant phenotype. RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady‐state RNA levels of some nuclear‐ and chloroplast‐encoded photosynthetic genes. Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.
Detailed description of four YAC contigs representing 17 Mb of chromosome 4 of Arabidopsis thaliana ecotype Columbia
Authors: Schmidt Renate , West Joanne , Cnops Gerda , Love Karina , Balestrazzi Alma , Dean Caroline
Published: March 2003

The detailed arrangement of 563 YAC clones comprising four contigs covering ∼17 Mbp of chromosome 4 is presented. YAC clones were positioned relative to each other and to markers by taking into account marker and end fragment hybridization data and the sizes of all YAC clones. This analysis made it possible to estimate physical distances between the majority of chromosome 4 markers. It also identified a relatively large number of YAC clones containing chimaeric inserts. The YAC contig map of the Columbia ecotype presents an important resource for map‐based cloning experiments, rapid mapping of DNA sequences and large‐scale genomic sequencing programs.
Cloning and expression of solanidine UDP‐glucose glucosyltransferase from potato
Authors: Moehs Charles P. , Allen Paul V. , Friedman Mendel , Belknap William R.
Published: March 2003

A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato. The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms. The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP‐glucosyltransferases. The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato. SGT mRNA and protein accumulated in tubers in response to wounding. The time course for SGT mRNA accumulation paralleled that of 3‐hydroxy‐3‐methylglutaryl‐coenzymeA isoform 1 (hmg1) mRNA. Steady‐state SGT mRNA levels also increased transiently upon wounding of leaves.
Domain exchanges between KNAT3 and KNAT1 suggest specificity of the kn1‐like homeodomains requires sequences outside of the third helix and N‐terminal arm of the homeodomain
Authors: Serikawa Kyle A. , Zambryski Patricia C.
Published: March 2003

Domain exchange constructs that traded regions surrounding the homeodomain were constructed for two kn1‐like genes, KNAT1 and KNAT3, and introduced into Arabidopsis thaliana under the control of the 35S CaMV promoter. The kn1‐like homeodomain proteins all have the homeodomain located near the C‐terminus of the protein, and also share a second conserved domain (the ELK domain) immediately N‐terminal to the homeodomain. Progeny were scored for the appearance of the KNAT1 overexpression phenotype. A construct containing the KNAT3 N‐terminus and the KNAT1 ELK‐ and homeodomain resulted in a KNAT1 overexpression phenotype, indicating that specificity mainly resides within the ELK‐ and homeodomain region. Further exchanges demonstrated that specificity probably does not arise from a single region within the ELK and/or homeodomain but rather requires sequences both N‐terminal and C‐terminal to residue 23 of the homeodomain. Further, in contrast to some animal homeodomains, KNAT1 does not utilize the residues of the N‐terminal arm of the homeodomain for specificity.
Intracellular localization of GBF proteins and blue light‐induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells
Authors: Terzaghi William B. , Bertekap Robert L. , Cashmore Anthony R.
Published: March 2003

The G‐box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G‐box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBFS. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB‐M) cell cultures indicated that over 90% of proteins detected with anti‐GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G‐box binding activity was cytoplasmic. DMA affinity chromatography demonstrated that each protein detected with anti‐GBF1 specifically bound the G‐box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB‐M protoplasts. Of GUS:GBF1 proteins, 50–62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark‐grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.
Induction of plant cell division by beet curly top virus gene C4
Authors: Latham Jonathan R. , Saunders Keith , Pinner Marion S. , Stanley John
Published: March 2003

Beet curly top virus (BCTV) is a small DNA virus that causes tumorigenic growths (enations) in infected plants by inducing division of phloem parenchyma cells (hyperplasia). It has previously been shown that BCTV C4 plays an important role in symptom development in sugarbeet and Nicotiana benthamiana, and it has been suggested that this gene is responsible for the induction of hyperplasia. Using in situ hybridization, we show that BCTV infection is closely associated with the vascular system in these hosts, although hyperplastic cells associated with wild‐type virus infection frequently do not contain detectable levels of viral DNA. Extensive hyperplasia was not observed in plants infected with a C4 mutant, demonstrating a role for C4 in virus‐induced cell proliferation. Ectopic expression of C4 in transgenic N. benthamiana resulted in abnormal plant development and the production of tumorigenic growths, confirming that this gene alone is sufficient to initiate cell division in permissive cells when removed from the context of the viral genome.
Analyses of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa nodules
Authors: Pathirana M. Sudam , Samac Deborah A. , Roeven Robert , Yoshioka Hirofumi , Vance Carroll P. , Gantt J. Stephen
Published: March 2003

Phosphoenolpyruvate carboxylase (PEPC) plays a crucial role in the assimilation of CO2 during symbiotic N2 fixation in legume root nodules. In this study, an alfalfa PEPC gene (PEPC‐7), whose transcripts are found at elevated levels in nodules relative to either leaves or roots, has been isolated and characterized. The intron/exon structure of this gene is identical to that of most other plant PEPC genes except for the presence of an additional intron in the 5′ untranslated region. In situ RNA hybridization studies showed that PEPC transcripts were present in the nodule meristem, the infection zone, the nitrogen‐fixing zone, and the parenchyma. PEPC transcripts were also found in vascular tissue of roots and nodules and in the pulvinus of petioles. In transgenic alfalfa, a chimeric reporter gene was expressed in these same regions except that little expression was found in the nodule meristem. Analyses of promoter deletions suggest that the region between −634 and −536 is of particular importance in directing transcriptional activity to the infected zone of nodules. Within this region is a mirror repeat sequence that is potentially capable of forming an H‐DNA structure. These results indicate that PEPC‐7 has a central role in nitrogenfixing nodules and that regulation of transcription is an important determinant of its activity.
Identification of a high‐affinity binding protein for N‐acetylchitooligosaccharide elicitor in the plasma membrane of suspension‐cultured rice cells by affinity labeling
Authors: Ito Yuki , Kaku Hanae , Shibuya Naoto
Published: March 2003

A high‐affinity binding protein for the N‐acetylchito‐oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross‐linking in the plasma membrane of suspension‐cultured rice cells. Both a [125I]‐labeled photolabile 2‐(4‐azidophenyl)ethylamino conjugate ([125I]‐GN8‐AzPEA) and a [125I]‐labeled 2‐ (4‐aminophenyl)ethylamino conjugate ([125]‐GN8‐APEA) of N‐acetylchito‐octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two‐phase partitioning, and covalently cross‐linked to the elicitor binding site by irradiation with UV light or treatment with the cross‐linking agent glutaraldehyde, respectively. Autoradiography of the SDS‐PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half‐maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross‐linking, respectively. The labeling of the 75 kDa protein was inhibited by N‐acetylchito‐oligasaccharides in a size‐dependent manner, and N‐acetylchito‐octaose (GlcNAc)8 showed a half‐maximal inhibition at concentrations of the order of 10 nM. However, neither chito‐octaose (GlcN)8, cellopentaose nor α‐1,4 linked N‐acetylgalactosamine octamer (GalNAc)8 at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high‐affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.
Phytochrome‐induced intercellular signalling activates cab::luciferase gene expression
Authors: Bischoff Friedrich , Millar Andrew J. , Kay Steve A. , Furuya Masaki
Published: March 2003

The phytochrome‐induced expression pattern of the chlorophyll a/b binding protein (cab) gene was studied using a cab2::luciferase reporter transgene in the tobacco cotyledon. The role of developmentally regulated competence, cooperativity among cells and signal propagation was investigated by red‐light microbeam irradiation of distinct areas of the cotyledon. Even with a minimal fluence, the response was not restricted to the irradiated cells. Following irradiation of the cotyledon base, the luciferase activity revealed a robust propagation of activating signals to areas that had not received a light stimulus. The acropetal outspread formed distinct expression patterns depending on the site of the irradiation. The combination of imaging luciferase activity in living seedlings and microbeam microscopy provides significant experimental evidence of how cellular light perception and intercellular signalling contribute to the cab gene expression pattern.
Developmental and environmental regulation of tissue‐ and cell‐specific expression for a pea protein farnesyltransferase gene in transgenic plants
Authors: Zhou Dafeng , Qian Daqi , Cramer Carole L. , Yang Zhenbiao
Published: March 2003

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras‐related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl‐transferase (FTase) gene was examined using transgenic expression of the β‐glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light‐grown seedlings, and hypocotyls of dark‐grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark‐grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.
Analysis of SCARECROW expression using a rapid system for assessing transgene expression in Arabidopsis roots
Authors: Malamy Jocelyn E. , Benfey Philip N.
Published: March 2003

Although the introduction of foreign genes into Arabidopsis has become routine, the production of transgenic Arabidopsis plants still requires several months. A transgene expression system (TES) has been developed that allows characterization of gene expression patterns and the effects of foreign genes in the Arabidopsis root in 2–4 weeks. The method is based on regeneration of stably transformed roots directly from callus tissue. TES has been used to study the expression of the SCARECROW gene, which is involved in establishing radial patterning in the root. The 2.5 kb region directly upstream of the SCARECROW coding region was found to be sufficient to confer cell‐type specific expression. Furthermore, this promoter is active in the scr mutant background, indicating that factors essential for cell‐type specific expression are present even in the absence of correct radial patterning. Finally, TES was used to demonstrate that the SCARECROW gene under control of this promoter complements the root organization defect of the scr mutant. These experiments demonstrate the utility of the TES system for studying gene expression in roots in wild‐type and mutant backgrounds and for molecular complementation of root mutant phenotypes. It is possible that the method will also be applicable to other organs.
Promoter elements required for phloem‐specific gene expression from the RTBV promoter in rice
Authors: Yin Yanhai , Chen Lili , Beachy Roger
Published: March 2003

Previous studies indicated that a DNA fragment comprising nucleotides (nt) −164 to +45 of the RTBV promoter is sufficient to drive phloem‐specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt −3 to +5) and Box II (nt −53 to −39) were identified by DNA‐protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem‐specific gene expression, among which are a direct repeat (ASL Box, nt −98 to −79) and a GATA motif (nt −143 to −135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss‐of‐competition in electrophoretic mobility shift assays. A DNA fragment comprising nt −164 to −32, which contains Box II, the ASL Box and the GATA motif, conferred phloem‐specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the −103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue‐specific gene expression. Similar studies in the −164 to +45 promoter fragment indicated that the −164 to −103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the −103 to −32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem‐specific gene expression.
Maturation and secretion of a serine proteinase is associated with events of late microsporogenesis
Authors: Taylor Andrew A. , Horsch Andrea , Rzepczyk Annette , Hasenkampf Clare A. , Riggs C. Daniel
Published: March 2003

An antiserum against meiotic proteins which bind to DNA cellulose was generated as a tool to assist the identification and purification of microsporogenesis‐specific proteins. In immunoblotting experiments, this antiserum identified three meiotic proteins which are differentially expressed in anthers during microsporogenesis. One of these proteins was purified and characterized by biochemical and immunological techniques. This 82 kDa protein is synthesized as a preproprotein, acquires glycans as it moves through the endoplasmic reticulum and Golgi body, and is secreted into the anther locule. Immunocytochemical experiments demonstrate that the protein is expressed primarily in tapetal cells, and reaches peak concentrations as the microsporocytes reach the tetrad stage. Zymogram analyses and protein sequence comparisons indicate that the protein is a member of the serine proteinase family. The possible roles of the proteinase in microsporogenesis and pollen development are discussed.
Expression of a dominant negative form of cpSRP54 inhibits chloroplast biogenesis in Arabidopsis
Authors: Pilgrim Marsha L. , Va Klaas‐Jan , Wijk  , Parry Devin H. , Sy Donna A. C. , Hoffman Neil E.
Published: March 2003

The chloroplast homolog of the 54 kDa subunit of signal recognition particle is required for the in vitro targeting of chlorophyll a/b binding proteins (LHCP) to the thylakoid membrane. To explore the function of cpSRP54 in vivo, plants that are mutated in cpSRP54 function were generated. Dominant negative forms of cpSRP54 altered in single amino acids within the conserved guanine nucleotide binding domain were expressed in Arabidopsis. Transformed plants contained less than 30% of the wild‐type level of cpSRP54 protein. As a consequence of the reduced cpSRP54 protein content, the first emerging leaves were yellow and contained immature chloroplasts. Although the chlorophyll (chl) content of the leaves was reduced by 75%, the chl a/b ratio was unaffected, indicating a role of cpSRP54 in the biogenesis of proteins besides LHCP. Many chloroplast proteins were less abundant in the first emerging leaves, including non‐pigmented proteins, thylakoid proteins known to be targeted by alternative pathways, and soluble proteins. These observations indicate that the cpSRP54 mutation also has a pleiotropic effect on chloroplast biogenesis. Whereas the level of cpSRP54 remained low as the plants aged, leaves emerging subsequently had a wild‐type appearance, suggesting that the adult plants compensated for the reduction in cpSRP54 protein.
Transcription of Arabidopsis and wheat Cab genes in single tobacco transgenic seedlings exhibits independent rhythms in a developmentally regulated fashion
Authors: Kolar Claudia , Fejes Erzsébet , Ádám Éva , Schäfer Eberhard , Kay Steven , Nagy Ferenc
Published: March 2003

Transcription of Cab genes has been previously shown to be regulated by a circadian oscillator coupled to the red light‐absorbing plant photoreceptor phytochrome in various plant species. In addition, it has recently been suggested that rhythmic expression of the Cab genes could also be affected by a phytochrome‐independent circadian oscillator in a developmentally regulated fashion. This study has shown that a red light‐insensitive oscillator and a phytochrome‐coupled circadian clock indeed co‐regulate the oscillating expression of individual Cab genes at the level of transcription at an early developmental stage. The study involved analysing the expression patterns of transgenes, containing short fragments of the Arabidopsis thaliana Cab2 or the wheat Cab‐1 promoter fused to the firefly luciferase reporter gene, by a video‐imaging system in single, etiolated tobacco seedlings. Germination and red/far‐red light treatments applied between 12 and 36 h after sowing lead to the appearance of two independent circadian rhythms. These rhythms co‐exist, both exhibiting period lengths close to 25 h but phased differently. However, repeated red‐light treatments given 60 h or later after sowing synchronize these free‐running rhythms and induce a single new circadian oscillation. These data indicate that both oscillators regulate the expression of the Cab genes studied at the level of transcription and that the cis‐acting element(s) of the wheat Cab‐1 and A. thaliana Cab2 genes mediating these responses are located on short, 250 bp promoter regions. Furthermore, these red‐light induced rhythms are also inducible by far‐red light treatments alone. Therefore, in tobacco, the phytochrome‐coupled oscillator is regulated, at least partially, by the very low fluence response of phytochrome A.
Mode of phytochrome B action in the photoregulation of seed germination in Arabidopsis thaliana
Authors: Shinomura Tomoko , Hanzawa Hiroko , Schäfer Eberhard , Furuya Masaki
Published: March 2003

Arabidopsis thaliana seeds imbibed for a short duration show phytochrome B (PhyB)‐specific photo‐induction of germination. Using this system, the relationship was determined between the amount of PhyB in seeds and photon energy required for PhyB‐specific germination in two transgenic Arabidopsis lines transformed with either the Arabidopsis PhyB cDNA (ABO) or the rice PhyB cDNA (RBO). Immunochemical detection of PhyB apoprotein (PHYB) showed that the expression level of PHYB in ABO seeds was at least two times higher than that in the wild‐type seeds, but in RBO seeds the PHYB level was indistinguishable from that in wild‐type seeds. The photon fluence required for induction and photoreversible inhibition of germination was examined using the Okazaki large spectrograph. At the wavelengths of 400–710 nm, the ABO seeds required significantly less photon fluence than wild‐type seeds for induction of germination, whereas the RBO seeds required similar fluence to wild‐type seeds. A critical threshold wavelength for either induction or inhibition of germination of ABO seeds shifted towards the longer wavelengths relative to wild‐type seeds. By assuming that PhyA and PhyB are similar in their photochemical parameters, amounts of Pfr at each wavelength were calculated. The photon fluence required for 50% germination was equivalent to the fluence generating a Pfr/Ptot ratio of 0.21–0.43 in wild‐type seeds, and of 0.035–0.056 in ABO seeds. These results indicate that PhyB‐specific seed germination is not strictly a function of the Pfr/Ptot ratio, but is probably a function of the absolute Pfr concentration.
Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis
Authors: Trethewey Richard N. , Geigenberger Peter , Riedel Kerstin , Hajirezaei Mohammad‐Reza , Sonnewald Uwe , Stitt Mark , Riesmeier Jörg W. , Willmitzer Lothar
Published: March 2003

The original aim of this work was to increase starch accumulation in potato tubers by enhancing their capacity to metabolise sucrose. We previously reported that specific expression of a yeast invertase in the cytosol of tubers led to a 95% reduction in sucrose content, but that this was accompanied by a larger accumulation of glucose and a reduction in starch. In the present paper we introduced a bacterial glucokinase from Zymomonas mobilis into an invertase‐expressing transgenic line, with the intention of bringing the glucose into metabolism. Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent invertase line and which did not accumulate glucose. Unexpectedly, there was a further dramatic reduction in starch content, down to 35% of wild‐type levels. Biochemical analysis of growing tuber tissue revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids, two‐ to threefold increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three‐ to fivefold increases in carbon dioxide production. These changes occur in the lines expressing invertase, and are accentuated following introduction of the second transgene, glucokinase. We conclude that the expression of invertase in potato tubers leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that heterologous overexpression of glucokinase enhances this change in partitioning.
Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide Nod signals
Authors: Schultze Michael , Staehelin Christian , Brunner Frédéric , Genetet Isabelle , Legrand Michel , Fritig Bernard , Kondorosi Éva , Kondorosi Ádám
Published: March 2003

The ability of 16 chitinases from seven different plant species to hydrolyze a collection of several structurally related lipochitooligosaccharides (LCOs) of

Rhizobium

was analyzed. It was found that the enzymes differed to a large extent in their activity on different LCOs. Differences were attributed to (i) the relative activity on different LCOs as substrate (e.g. sulfated versus non‐sulfated LCOs); (ii) the relative cleavage site preference on a given LCO molecule (hydrolysis of either the second, third or fourth glycosidic bond from the non‐reducing end of the molecule); and (iii) the stereochemistry of the reaction (retention or inversion of the anomeric configuration). A graphic representation of the different substrate specificities resulted in a ‘fingerprint’ that is characteristic for a given enzyme or a family of related enzymes. By comparing the LCO‐fingerprint of unknown enzymes with those obtained for already characterized proteins, it is possible to identify new glycosyl hydrolases. The high diversity of substrate specificity found among plant chitinases may reflect variations in the natural substrates of the enzymes, such as substitutions on the chitin moiety of fungal cell walls or, in plants, the presence of putative endogenous substrates related to LCOs.
Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY‐2 glyoxysomes
Authors: Flynn C. Robb , Mullen Robert T. , Trelease Richard N.
Published: March 2003

In this study of the type 2 peroxisomal targeting signal (PTS2) pathway, we examined the apparent discontinuity and conservation of residues within the PTS2 nonapeptide and demonstrated that this topogenic signal is capable of directing heteromultimeric protein import in plant cells. Based on cumulative data showing that at least 26 unique, putative PTS2 nonapeptides occur within 12 diverse peroxisomal‐destined proteins, the current (‐R/K‐L/V/I‐X5‐H/Q‐L/A‐) as well as the original (‐R‐L‐X5‐H/Q‐L‐) PTS2 motif appear to be oversimplified. To assess the functionality of residues within the motif, rat liver thiolase (rthio) and various chimeric chloramphenicol acetyltransferase (CAT) proteins were expressed transiently in suspension‐cultured tobacco (

Nicotiana tabacum

L.) cv Bright Yellow cells (BY‐2), and their subcellular location was determined by immunofluorescence microscopy. Hemagglutinin (HA)‐epitope‐tagged‐CAT subunits, lacking a PTS2 (CAT‐HA), were ‘piggybacked’ into glyoxysomes by PTS2‐bearing CAT subunits (rthio‐CAT), whereas signal‐depleted CAT‐HA subunits that were modified to prevent oligomerization did not import into glyoxysomes. These results provided direct evidence that signal‐depleted subunits imported into peroxisomes were targeted to the organelle as oligomers (heteromers) by a PTS2. Mutational analysis of residues within PTS2 nonapeptides revealed that a number of amino acid substitutions were capable of maintaining targeting function. Furthermore, functionality of residues within the PTS2 nonapeptide did not appear to require a context‐specific environment conferred by adjacent residues. These results collectively suggest that the functional PTS2 is not solely defined as a sequence‐specific motif, i.e. ‐R/K‐X6‐H/Q‐A/L/F‐, but defined also by its structural motif that is dependent upon the physiochemical properties of residues within the nonapeptide.
Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high‐efficiency expression library
Authors: Galaud Jean‐Philippe , Carrière Marguerite , Pauly Nicolas , Canut Hervé , Chalon Pascale , Caput Daniel , Pont‐Lezica Rafael F.
Published: March 2003

We constructed a high‐efficiency expression library from

Arabidopsis

cDNA clones by introducing a poly (dC) stretch at the 5′ end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high‐efficiency expression library with antibodies raised against total proteins from

Arabidopsis

plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane‐associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for

Arabidopsis

, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from

Arabidopsis

cells submitted to osmotic stress allows the selection of genes over‐ and under‐expressed in stress conditions.
A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis
Authors: Næsted Henrik , Fennema Marko , Hao Lin , Andersen Mathias , Janssen Dick B. , Mundy John
Published: March 2003

The dhlA gene of Xanthobacter autotrophicus GJ10 encodes a dehalogenase which hydrolyzes dihalo‐ alkanes, such as 1,2‐dichloroethane (DCE), to a halo‐ genated alcohol and an inorganic halide (
Janssen et al. 1994
, Annu. Rev. Microbiol. 48, 163–191). In Xanthobacter, these alcohols are further catabolized by alcohol and aldehyde dehydrogenase activities, and by the product of the dhlB gene to a second halide and a hydroxyacid. The intermediate halogenated alcohols and, in particular, the aldehydes are more toxic than the haloalkane substrates or the pathway products. We show here that plants, including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild‐type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhlA. The dhlA marker therefore provides haloalkane dehalogenase reporter activity and substrate dependent negative selection in transgenic plants.
The late pollen‐specific actins in angiosperms
Authors: Kandasamy Muthugapatti K. , McKinney Elizabeth C. , Meagher Richard B.
Published: March 2003

The actin gene family of Arabidopsis has eight functional genes that are grouped into two ancient classes, vegetative and reproductive, and into five subclasses based on their phylogeny and mRNA expression patterns. Progress in deciphering the functional significance of this diversity is hindered by the lack of tools that can distinguish the highly conserved subclasses of actin proteins at the biochemical and cellular level. In order to address the functional diversity of actin isovariants, we have used Arabidopsis recombinant actins as immunogens and produced several new anti‐actin monoclonal antibodies. One of them, MAb45a, specifically recognizes two closely related reproductive subclasses of actins. On immunoblots, MAb45a reacts strongly with actins expressed in mature pollen but not with actins in other Arabidopsis tissues. Moreover, immunocytochemical studies show that this antibody can distinguish actin filaments in pollen tubes from those in most vegetative tissues. Peptide competition analyses demonstrate that asparagine at position 79 (Asn79) within an otherwise conserved sequence is essential for MAb45a specificity. Actins with the Asn79 epitope are also expressed in the mature pollen from diverse angiosperms and Ephedra but not from lower gymnosperms, suggesting that this epitope arose in an ancestor common to angiosperms and advanced gymnosperms more than 220 million years ago. During late pollen development in angio‐ sperms there is a switch in expression of actins from vegetative to predominantly reproductive subclasses, perhaps to fulfil the unique functions of pollen in fertilization.
Plant interstitial telomere motifs participate in the control of gene expression in root meristems
Authors: Tremousaygue Dominique , Manevski Alexandra , Bardet Claude , Lescure Nicole , Lescure Bernard
Published: March 2003

The promoters of Arabidopsis eEF1A genes contain a telomere motif, the telo‐box, associated with an activating sequence, the tef‐box. Database searches indicated the presence of telo‐boxes in the 5′ region of numerous genes encoding components of the translational apparatus. By using several promoter constructs we demonstrate that the telo‐box is required for the expression of a β‐glucoronidase gene in root primordia of transgenic Arabidopsis. This effect was observed when a telo‐box was inserted upstream or downstream from the transcription initiation site, and occurred in synergy with the tef‐box. These results clearly indicate that interstitial telomere motifs in plants are involved in control of gene expression. South‐western screening of a λZAP library with a double‐stranded Arabidopsis telomere motif resulted in characterization of a protein related to the conserved animal protein Purα. The possibility of a regulation process similar to that achieved by the Rap1p in Saccharomyces cerevisiae is discussed.
Identification of a novel peptide motif that mediates cross‐linking of proteins to cell walls
Authors: Domingo Concha , Saurí Asunción , Mansilla Elena , Conejero Vicente , Vera Pablo
Published: March 2003

A cDNA clone representing a member of a novel class of cell wall proteins was isolated from tobacco plants. We have designated this protein NtTLRP for tyrosine‐ and lysine‐rich protein. It is structurally related to the previously identified TLRP from tomato plants, sharing a high amino‐acid sequence similarity at the C‐terminal region. This region contains what appears to be a novel peptide motif which we call CD for cysteine‐rich domain, and which is common to several other cell‐wall proteins. By using a functional test in transgenic plants, we demonstrate that the presence of the CD domain is per se sufficient to cross‐link previously soluble proteins to the cell wall. We present evidence that NtTLRP is cross‐linked and specifically localizes to the cell wall of lignified cells. The highly localized deposition of NtTLRP in these cells indicates that this class of cell‐wall proteins may have a specialized function in the formation of xylem tissue.
Transcriptional activation of a heat shock gene promoter in sunflower embryos: synergism between ABI3 and heat shock factors
Authors: Rojas Anabel , Almoguera Concepción , Jordano Juan
Published: March 2003

Transient expression analyses in sunflower embryos demonstrated that ABI3, a seed‐specific transcription factor from Arabidopsis, activated chimaeric genes with the Ha hsp17.7 G4 promoter. Nucleotide substitutions at crucial positions of heat shock cis‐elements established that they are required for the transcriptional activation involving ABI3. Trans‐activation with Lp‐HSFA1, a heat shock factor from tomato, reproduced the activation patterns of wild‐type and mutant promoters observed with ABI3. In addition, ABI3 and Lp‐HSFA1 syner‐gistically activated the Ha hsp17.7 G4 promoter, but only when it contained the intact proximal and distal heat shock cis‐elements. The activation domain of Lp‐HSFA1 was necessary for promoter activation. An amino terminal deletion of ABI3 had dominant negative effects on activation by Lp‐HSFA1. We failed to detect a substantial transcriptional activation by ABI3 in the absence of either functional heat shock factors or heat shock elements (HSEs). Furthermore, the wild‐type, but not the mutant HSEs (from − 136 to − 49 in Ha hsp17.7 G4) were sufficient, in the context of a − 46 CaMV 35S promoter, to support activation by Lp‐HSFA1, or Lp‐HSFA1 and ABI3. These results demonstrate, for the first time, transcriptional activation of a heat shock protein promoter by ABI3. We also suggest that ABI3 functions as a transcriptional co‐activator through heat shock factors.
RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica
Authors: Bittner‐Eddy Peter D. , Crute Ian R. , Holub Eric B. , Beynon Jim L.
Published: March 2003

Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd‐1, designated RPP13‐Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS‐LRR type R protein with a putative amino‐terminal leucine zipper. The RPP13‐Rld allele, cloned from the accession Rld‐2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (>95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved β‐strand/β‐turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS‐LRR type R proteins, suggesting functional significance.
Allocation of raffinose family oligosaccharides to transport and storage pools in Ajuga reptans: the roles of two distinct galactinol synthases
Authors: Sprenger Norbert , Keller Felix
Published: March 2003

Raffinose family oligosaccharides (RFOs) are important phloem transport and storage carbohydrates for many plants. Ajuga reptans, a frost‐hardy evergreen labiate, ideally combines these two physiological roles and served as our model plant to study the regulation and importance of RFO metabolism. Galactinol is the galactosyl donor for the synthesis of raffinose (RFO‐trisaccharide) and stachyose (RFO‐tetrasaccharide), and its synthesis by galactinol synthase (GolS) is the first committed step of the RFO biosynthetic pathway. Two cDNAs encoding two distinct GolS were isolated from A. reptans source and sink leaves, designated GolS‐1 and GolS‐2, respectively. Warm‐ and cold‐grown sink and source leaves were compared, revealing both isoforms to be cold‐inducible and GolS‐1 to be source leaf‐specific; GolS‐1 expression correlated positively with GolS activity. Conversely, GolS‐2 expression was comparatively much lower and its contribution to the total extractable GolS activity is most probably only minor. These observations, together with results from phloem exudation and leaf shading experiments suggest that GolS‐1 is mainly involved in the synthesis of storage RFOs and GolS‐2 in the synthesis of transport RFOs. Furthermore, in situ hybridization studies showed GolS‐1 to be primarily expressed in the mesophyll, the site of RFO storage, and GolS‐2 in the phloem‐associated intermediary cells known for their role in RFO phloem loading. A model depicting the spatial compartmentation of the two GolS isoforms is proposed.