bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcript profiling in rice (Oryza sativa L.) seedlings using serial analysis of gene expression (SAGE)
Authors: Matsumura Hideo , Nirasawa Shizuko , Terauchi Ryohei
Published: March 2003

Serial analysis of gene expression (SAGE) was applied for profiling expressed genes in rice seedlings. In the SAGE method, a 9–11 bp fragment (tag) represents each transcript, and frequency of a tag in the sample directly reflects the abundance of the respective mRNA. We studied 10 122 tags derived from 5921 expressed genes in rice (Oryza sativa L.) seedlings, among which only 1367 genes (23.1%) matched the rice cDNA or EST sequences in the DNA database. SAGE showed that most of the highly expressed genes in rice seedlings belong to the category of housekeeping genes (genes encoding ribosomal proteins or proteins responsible for metabolism and cell structure). Unexpectedly, the most highly expressed gene in rice seedlings was a metallothionein (MT) gene, and together with three other messages for MT, it accounts for 2.7% of total gene expression. To our knowledge, this is the first quantitative study of global gene expression in a higher plant. We further applied the SAGE technique to identify differentially expressed genes between anaerobically treated and untreated rice seedlings. Additionally, we show that a longer cDNA fragment can be easily recovered by PCR using the SAGE tag sequence as a primer, thereby facilitating the analysis of unknown genes identified by tag sequence in SAGE. In combination with micro‐array analysis, SAGE should serve as a highly efficient tool for the identification and isolation of differentially expressed genes in plants.
Diurnal regulation of Hsp70s in leaf tissue
Authors: Li Qin‐Bao , Haskell Dale , Zhang Chun , Sung Dong‐Yul , Guy Charles
Published: March 2003

Steady‐state mRNA levels for three Hsp70s were found to be regulated by a distinctive light/dark mechanism in spinach leaves. Messenger RNAs for the chloroplast stromal and two cytosolic forms displayed a diurnal expression pattern under isothermal conditions that appeared to be independent of circadian control. While protein blot data showed relatively constant Hsp70 protein levels, the higher Hsp70 mRNA levels in the light paralleled the diurnal cycle of total cell protein synthesis. Fractionation studies showed that the major cytosolic Hsp70 cognate group was associated with polysomes. Therefore, the variation of Hsp70 mRNAs is consistent with the diurnal metabolic activity of plant photosynthetic cells in which the demand of protein biogenesis for chaperone function and tissue temperature are highest during the day.
Matrix polysaccharide precursors in Arabidopsis cell walls are synthesized by alternate pathways with organ‐specific expression patterns
Authors: Seitz Beate , Klos Christoph , Wurm Marita , Tenhaken Raimund
Published: March 2003

The expression pattern of the single‐copy gene UDP‐glucose dehydrogenase (Ugd) was analysed in transgenic Arabidopsis plants by promoter::GUS and GFP fusions, Western blots, activity assays and histochemical activity staining. The enzyme oxidizes UDP‐glucose to UDP‐glucuronic acid and thus directs carbohydrates irreversibly into a cell wall‐specific pool of nucleotide sugars. UDP‐glucuronic acid is the central intermediate in the interconversion pathway to other nucleotide sugars, including the UDP‐derivatives of arabinose, xylose, apiose and galacturonic acid which account for half the biomass of a typical Arabidopsis leaf cell wall. These activated sugars are needed as substrates for the biosynthesis of matrix polysaccharide polymers. In plants up to 5 days old the Ugd gene is strongly expressed in young roots, but very little in hypocotyls. Older plants show a more uniform expression pattern with a preference for the vascular system. A complex expression pattern was observed in flowers with high activity in the stamen, stigma and nectaries. Meristems in the leaf axil of rosette and inflorescence leaves exhibit a high level of activity of the Ugd gene. Although many of the growing tissues show high activity levels of the Ugd gene, others such as the hypocotyl and the cotyledons of young seedlings do not. Instead these tissues efficiently incorporate 3H‐inositol into their cell walls. This indicates the biosynthesis of UDP‐glucuronic acid through an alternative pathway via the oxidation of inositol to glucuronic acid and subsequent activation to the nucleotide sugar. The data strongly suggest two alternative pathways for matrix polysaccharide precursors with spatial and developmental regulation.
Stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system
Authors: Love John , Scott Amie C. , Thompson William F.
Published: March 2003

We show that the tightly regulated tetracycline‐sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559–569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL‐tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER‐targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER‐targeted GFP fluorescence throughout the plant, indicating that the tTA–Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml−1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de‐repression of the Top10::mGFP5 transgene, with a visible ER‐targeted GFP signal appearing from 24 to 48 h after de‐repression.
Cell‐to‐cell movement of TMV RNA is temperature‐dependent and corresponds to the association of movement protein with microtubules
Authors: Boyko Vitaly , Ferralli Jacqueline , Heinlein Manfred
Published: March 2003

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell‐to‐cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell‐to‐cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature‐dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.
rgf1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development†
Authors: Maitz Monika , Santandrea Geraldina , Zhang Zhiyong , Lal Shailesh , Hannah L. Curtis , Salamini Francesco , Thompson Richard D.
Published: March 2003

The maize cob presents an excellent opportunity to screen visually for mutations affecting assimilate partitioning in the developing kernel. We have identified a defective kernel mutant termed rgf1, reduced grain filling, with a final grain weight 30% of the wild type. In contrast with most defective endosperm mutants, rgf1 shows gene dosage‐dependent expression in the endosperm. rgf1 kernels possess a small endosperm incompletely filling the papery pericarp, but embryo development is unaffected and the seeds are viable. The mutation conditions defective pedicel development and greatly reduces expression of endosperm transfer layer‐specific markers. rgf1 exhibits striking morphological similarities to the mn1 mutant, but maps to a locus approximately 4 c
m away from mn1 on chromosome 2 of maize. Despite reduced starch accumulation in the mutant, no obvious lesion in starch biosynthesis has been detected. Free sugar levels are unaltered in rgf1 endosperm. Rates of sugar uptake, measured over short (8 h) periods in cultured kernels, are increased in rgf1 compared to the wild type. rgf1 and wild‐type kernels, excised at 5 DAP and cultured in vitro also develop differently in response to variations in sugar regime: glucose concentrations above 1% arrest placentochalazal development of rgf1 kernels, but have no effect on cultured wild‐type kernels. These findings suggest that either uptake or perception of sugar(s) in endosperm cells at 5–10 DAP determines the rgf1 kernel phenotype.
Comparative physical mapping of segments of the genome of Brassica oleracea var. alboglabra that are homoeologous to sequenced regions of chromosomes 4 and 5 of Arabidopsis thaliana
Authors: O'neill Carmel M. , Bancroft Ian
Published: March 2003

Due to their relatedness to Arabidopsis thaliana (Arabidopsis), the cultivated Brassica species represent the first group of crops with which to evaluate comparative genomics approaches to understanding biological processes and manipulating traits. We have constructed a high‐quality binary BAC library (JBo) from genomic DNA of Brassica oleracea var. alboglabra, in order to underpin such investigations. Using the Arabidopsis genome sequence and clones from the JBo library, we have analysed aspects of gene conservation and microsynteny between a 222 kb region of the genome of Arabidopsis and homoeologous segments of the genome of B. oleracea. All 19 predicted genes tested were found to hybridize to clones in the JBo library, indicating a high level of gene conservation. Further analyses and physical mapping with the BAC clones identified allowed us to construct clone contig maps and analyse in detail the gene content and organization in the set of paralogous segments identified in the genome of B. oleracea. Extensive divergence of gene content was observed, both between the B. oleracea paralogous segments and between them and their homoeologous segment within the genome of Arabidopsis. However, the genes present show highly conserved collinearity with their orthologues in the genome of Arabidopsis. We have identified one example of a Brassica gene in a non‐collinear position and one rearrangement. Some of the genes not present in the discernible homoeologous regions appear to be located elsewhere in the B. oleracea genome. The implications of our findings for comparative map‐based cloning of genes from crop species are discussed.
Salicylic acid has a role in regulating gene expression during leaf senescence
Authors: Morris Karl , ‐Mackerness Soheila A. ‐H. , Page Tania , John C. Fred , Murphy Alex M. , Carr John P. , Buchanan‐Wollaston Vicky
Published: March 2003

Leaf senescence is a complex process that is controlled by multiple developmental and environmental signals and is manifested by induced expression of a large number of different genes. In this paper we describe experiments that show, for the first time, that the salicylic acid (SA)‐signalling pathway has a role in the control of gene expression during developmental senescence. Arabidopsis plants defective in the SA‐signalling pathway (npr1 and pad4 mutants and NahG transgenic plants) were used to investigate senescence‐enhanced gene expression, and a number of genes showed altered expression patterns. Senescence‐induced expression of the cysteine protease gene SAG12, for example, was conditional on the presence of SA, together with another unidentified senescence‐specific factor. Changes in gene expression patterns were accompanied by a delayed yellowing and reduced necrosis in the mutant plants defective in SA‐signalling, suggesting a role for SA in the cell death that occurs at the final stage of senescence. We propose the presence of a minimum of three senescence‐enhanced signalling factors in senescing leaves, one of which is SA. We also suggest that a combination of signalling factors is required for the optimum expression of many genes during senescence.
Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves
Authors: Hamada Susumu , Onouchi Hitoshi , Tanaka Hirokazu , Kudo Mitsuko , Liu Yao‐Guang , Shibata Daisuke , Machida Chiyoko , Machida Yasunori
Published: March 2003

The vegetative growth of Arabidopsis thaliana can be divided into two phases. The transition from the juvenile (early) phase to the adult (later) phase is associated with changes in several morphological features of leaves, such as the shape of leaf blades, the number of trichomes and patterns of venation. In a screening of mutants with altered morphological identities of leaves, we found one which we named juvenile leafless and misshapen shoot apical meristem (jam). The mutation represented a new allele of the WUSCHEL (WUS) gene, and, in its presence, plants produced no juvenile leaves. Analysis of the morphology of mutant plants revealed that all the rosette leaves had characteristics of adult leaves. The formation of the first rosette leaf in the wus(jam) mutant was markedly delayed, and occurred at the almost same time as formation of the third or fourth leaf in wild‐type plants. In the wild‐type, these leaves correspond to the first adult leaves. Analysis by RT–PCR showed that transcripts of WUS accumulated in shoot apices and roots, but not in cotyledons and leaves. The present results suggest that the WUS gene controls the morphological traits of rosette leaves either directly or indirectly. In view of the predicted function of the WUS gene, namely maintenance of stem cells within the shoot apical meristem, we suggest that the lack of juvenile leaves in the mutant might have been caused by interruption of leaf initiation during the juvenile phase or by halting of an entire process of formation of juvenile leaves.
Identification of a MADS‐box gene, FLOWERING LOCUS M, that represses flowering
Authors: Scortecci Katia C. , Michaels Scott D. , Amasino Richard M.
Published: March 2003

The timing of flowering is important for the reproductive success of plants. Here we describe the identification and characterization of a new MADS‐box gene, FLOWERING LOCUS M (FLM), which is involved in the transition from vegetative to reproductive development. FLM is similar in amino‐acid sequence to FLC, another MADS‐box gene involved in flowering‐time control. flm mutants are early flowering in both inductive and non‐inductive photoperiods, and flowering time is sensitive to FLM dosage. FLM overexpression produces late‐flowering plants. Thus FLM acts as an inhibitor of flowering. FLM is expressed in areas of cell division such as root and shoot apical regions and leaf primordia.
The CENTROMERE1 (CEN1) region of Arabidopsis thaliana: architecture and functional impact of chromatin
Authors: Haupt Wolfgang , Fischer Thilo C. , Winderl Sabine , Fransz Paul , Torres‐Ruiz Ramón A.
Published: March 2003

We have analysed the centromere 1 (CEN1) of Arabidopsis thaliana by integration of genetic, sequence and fluorescence in situ hybridisation (FISH) data. CEN1 is considered to include the centromeric core and the flanking left and right pericentromeric regions, which are distinct parts by structural and/or functional properties. CEN1 pericentromeres are composed of different dispersed repetitive elements, sometimes interrupted by functional genes. In contrast the CEN1 core is more uniformly structured harbouring only two different repeats.

The presented analysis reveals aspects concerning distribution and effects of the uniformly shaped heterochromatin, which covers all CEN1 regions. A lethal mutation tightly linked to CEN1 enabled us to measure recombination frequencies within the heterochromatin in detail. In the left pericentromere, the change from eu‐ to heterochromatin is accompanied by a gradual change in sequence composition but by an extreme change in recombination frequency (from normal to 53‐fold decrease) which takes place within a small region spanning 15 kb. Generally, heterochromatin is known to suppress recombination. However, the same analysis reveals that left and right pericentromere, though similar in sequence composition, differ markedly in suppression (53‐fold versus 10‐fold). The centromeric core exhibits at least 200‐fold if not complete suppression. We discuss whether differences in (fine) composition reflect quantitative and qualitative differences in binding sites for heterochromatin proteins and in turn render different functional properties. Based on the presented data we estimate the sizes of Arabidopsis centromeres. These are typical for regional centromeres of higher eukaryotes and range from 4.4 Mb (CEN1) to 3.55 Mb (CEN4).
1‐Deoxy‐d‐xylulose 5‐phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening
Authors: Rodríguez‐Concepción Manuel , Ahumada Iván , Diez‐Juez Ester , Sauret‐Güeto Susanna , Lois Luisa María , Gallego Francesca , Carretero‐Paulet Lorenzo , Campos Narciso , Boronat Albert
Published: March 2003

The recently discovered 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1‐deoxy‐d‐xylulose 5‐phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non‐limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin‐treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase‐1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.
The tomato Rme1 locus is required for Mi‐1‐mediated resistance to root‐knot nematodes and the potato aphid
Authors: De Ilarduya Oscar Martinez , Moore Ann E. , Kaloshian Isgouhi
Published: March 2003

The tomato Mi‐1 gene confers resistance against root‐knot nematodes (Meloidogyne spp.) and a biotype of the potato aphid (Macrosiphum euphorbiae). Four mutagenized Mi‐1/Mi‐1 tomato populations were generated and screened for altered root‐knot nematode resistance. Four independent mutants belonging to two phenotypic classes were isolated. One mutant was chosen for further analyzes; rme1 (for resistance to Meloidogyne) exhibited levels of infection comparable with those found on susceptible controls. Molecular and genetic data confirmed that rme1 has a single recessive mutation in a locus different from Mi‐1. Cross‐sections through galls formed by feeding nematodes on rme1 roots were identical to sections from galls of susceptible tomato roots. In addition to nematode susceptibility, infestation of rme1 plants with the potato aphid showed that this mutation also abolished aphid resistance. To determine whether Rme1 functions in a general disease‐resistance pathway, the response against Fusarium oxysporum f.sp. lycopersici race 2, mediated by the I‐2 resistance gene, was studied. Both rme1 and the wild type plants were equally resistant to the fungal pathogen. These results indicate that Rme1 does not play a general role in disease resistance but may be specific for Mi‐1‐mediated resistance.
Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra‐radical hyphae
Authors: David‐Schwartz Rakefet , Badani Hana , Smadar Wininger , Levy Avraham A. , Galili Gad , Kapulnik Yoram
Published: March 2003

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast‐neutron‐mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro‐Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc– phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth‐pattern characteristics similar to those of wild‐type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc– characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal‐inoculated wild‐type plants in the same growth medium. During evaluation of the pre‐infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild‐type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre‐mycorrhizal infection stages.
Regulation of plant water loss by manipulating the expression of phospholipase Dα
Authors: Sang Yongming , Zheng Suqin , Li Weiqi , Huang Bingru , Wang Xuemin
Published: March 2003

Phospholipase D (PLD) has been implicated in various processes, including signal transduction, membrane trafficking, and membrane degradation. Multiple forms of PLD with distinct biochemical properties have been described in the cell. In Arabidopsis, PLDα and PLDγ, but not PLDβ, were detected in guard cells, and antisense suppression resulted in a specific loss of PLDα. The abrogation of PLDα rendered plants less sensitive to abscisic acid and impaired stomatal closure induced by water deficits. PLDα‐depleted plants exhibited accelerated transpirational water loss and a decreased ability to tolerate drought stress. Overexpression of PLDα enhanced the leaf's sensitivity to abscisic acid. These findings provide molecular and physiological evidence that PLDα plays a crucial role in regulating stomatal movement and plant‐water status.
The MADS‐box gene DEFH28 from Antirrhinum is involved in the regulation of floral meristem identity and fruit development
Authors: Müller Bettina M. , Saedler Heinz , Zachgo Sabine
Published: March 2003

DEFH28 is a novel MADS‐box gene from Antirrhinum majus. Phylogenetic reconstruction indicates that it belongs to the SQUA‐subfamily of MADS‐box genes. Based on its expression pattern and the phenotype of transgenic plants it is predicted that DEFH28 exerts a dual function during flower development, namely control of meristem identity and fruit development. Firstly, DEFH28 is expressed in the inflorescence apical meristem and might control, together with SQUAMOSA (SQUA), floral meristem identity in Antirrhinum. Also, DEFH28 is sufficient to switch inflorescence shoot meristem to a floral fate in transgenic Arabidopsis thaliana plants. Secondly, DEFH28 is expressed in carpel walls, where it may regulate carpel wall differentiation and fruit maturation. Support for this later role comes from overexpression of DEFH28 throughout the silique in transgenic Arabidopsis plants where it altered the identity of the replum and valve margin cells so that they adopted a valve cell identity. This late aspect of the DEFH28 function is identical to the FRUITFULL (FUL) function of Arabidopsis as demonstrated in gain‐of‐function plants. FUL, like DEFH28, belongs to the SQUA‐subfamily of MADS‐box genes. DEFH28 most likely represents the ortholog of FUL. Promoter analysis shows that the control mechanism conferring a carpel wall specific expression has been conserved between Antirrhinum and Arabidopsis during evolution. Although the overall flower development between Antirrhinum and Arabidopsis is very similar, their carpels mature into different types of fruits: capsules and siliques, respectively. Therefore, it is suggested that the role of DEFH28 in control of carpel wall differentiation reflects a conserved molecular mechanism integrated into two very different carpel developmental pathways.
Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression
Authors: Jach Guido , Binot Elke , Frings Sabine , Luxa Kerstin , Schell Jeff
Published: March 2003

The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER‐targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.
Distinct cell‐specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination
Authors: Yamaguchi Shinjiro , Kamiya Yuji , Sun Tai‐ping
Published: March 2003

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type‐specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3‐oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent‐kaurene oxidase, exhibited a similar cell‐specific expression pattern, we predicted that the synthesis of bioactive GAs from ent‐kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non‐germinating ga1–3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.
Gene targeting in Arabidopsis
Authors: Hanin Moez , Volrath Sandra , Bogucki Augustyn , Briker Markus , Ward Eric , Paszkowski Jerzy
Published: March 2003

Precise modification by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for flowering plants. These were at low frequencies precluding reliable estimation of targeting efficiency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 × 10−3. Approximately one‐third of events were true GT (TGT) leading to the anticipated modification of the chromosomal PPO copy. The remaining events could be classified as ectopic GT (EGT) arising by modification of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 × 10−3. In view of the high efficiency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modification of non‐selectable targets. Moreover, the system presented here should contribute significantly to future improvement of GT technology in plants.
APETALA1 and SEPALLATA3 interact to promote flower development
Authors: Pelaz Soraya , Gustafson‐Brown Cindy , Kohalmi Susanne E. , Crosby William L. , Yanofsky Martin F.
Published: March 2003

In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS‐box genes share overlapping roles in promoting flower meristem identity. Later in flower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS‐domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during flower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two‐hybrid assay. Among the five MADS‐box genes identified in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early flowering phenotype of plants constitutively expressing AP1 is significantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal flower development.
TUMOROUS SHOOT DEVELOPMENT (TSD) genes are required for co‐ordinated plant shoot development
Authors: Frank Markus , Guivarc'h Anne , Krupková Eva , Lorenz‐Meyer Irina , Chriqui Dominique , Schmülling Thomas
Published: March 2003

This report describes the identification of novel plant genes that are required to ensure co‐ordinated post‐embryonic development. After germination the tumorous shoot development mutants of Arabidopsis thaliana develop disorganized tumorous tissue instead of organized leaves and stems. This results in green callus‐like structures, which are capable of unlimited growth in vitro on hormone‐free medium. The tsd mutants are recessive and belong to three complementation groups (tsd1, tsd2, tsd3). The genes were mapped to the bottom of chromosomes 5 and 1, and the top of chromosome 3, respectively. Histological analyses showed that the tsd mutants have different developmental defects. The shoot apical meristem of tsd1 formed only rudimentary leaves and was characterized by a degenerating L1 cell layer. tsd2 mutants had reduced cell adhesion and altered cell division planes in the L2 and L3 cell layers. The tumorous tissue of tsd3 mutants originated from the base of the leaf. Cytokinin levels that are inhibitory to the growth of wild‐type seedlings bring about an enhanced growth response in all the tsd mutants. The steady state transcript levels of the histidine kinase CKI1 gene and the KNAT1 and STM homeobox genes were increased in tsd mutants, while mRNA levels of cell cycle genes were not altered. We hypothesize that the TSD gene products negatively regulate cytokinin‐dependent meristematic activity during vegetative development of Arabidopsis.
Trehalose‐6‐phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation
Authors: Eastmond Peter J. , Van Dijken Anja J. H. , Spielman Melissa , Kerr Aimie , Tissier Alain F. , Dickinson Hugh G. , Jones Jonathan D. G. , Smeekens Sjef C. , Graham Ian A.
Published: March 2003

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose‐6‐phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up‐regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose‐6‐phosphate (T‐6‐P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1–1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T‐6‐P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1–1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.
Geminivirus‐based vectors for gene silencing in Arabidopsis
Authors: Turnage Michael A. , Muangsan Nooduan , Peele Charles G. , Robertson Dominique
Published: March 2003

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene‐silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene‐replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.
Extended leaf longevity in the ore4‐1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene
Authors: Woo Hye Ryun , Goh Chang‐Hyo , Park Joon‐Hyun , de la Serve Bernard Teyssendier , Kim Jin‐Hee , Park Youn‐Il , Nam Hong Gil
Published: March 2003

The longevity of plant leaf organs is genetically determined. However, the molecular mechanisms underlying the control of longevity are still largely unknown. Here, we describe a T‐DNA‐insertional mutation of Arabidopsis thaliana that confers extended leaf longevity. The mutation, termed ore4‐1, delays a broad spectrum of age‐dependent leaf senescence, but has little effect on leaf senescence artificially induced by darkness, abscisic acid (ABA), methyl jasmonate (MeJA), or ethylene. The T‐DNA was inserted within the promoter region of the plastid ribosomal small subunit protein 17 (PRPS17) gene, and this insertion dramatically reduced PRPS17 mRNA expression. In the ore4‐1 mutant, the leaf growth rate is decreased, while the maturation timing is similar to that of wild‐type. In addition, the activity of the photosystem I (PSI) is significantly reduced in the ore4‐1 mutant, as compared to wild‐type. Thus, the ore4‐1 mutation results in a deficiency in various chloroplast functions, including photosynthesis, which may decrease leaf growth. Our results suggest a possible link between reduced metabolism and extended longevity of the leaf organs in the ore4‐1 mutation.
pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves
Authors: Goodin Michael M. , Dietzgen Ralf G. , Schichnes Denise , Ruzin Steven , Jackson Andrew O.
Published: March 2003

We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co‐localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co‐localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co‐expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co‐localization and interactions of proteins in a variety of experimental dicotyledonous hosts.
Mutations at CRE1 impair cytokinin‐induced repression of phosphate starvation responses in Arabidopsis
Authors: Franco‐Zorrilla José M. , Martin Ana C. , Solano Roberto , Rubio Vicente , Leyva Antonio , Paz‐Ares Javier
Published: March 2003

Plants display a number of responses to low phosphate availability, involving biochemical and developmental changes. Recently we have shown that many of these responses can be repressed in roots by exogenous addition of cytokinins. In order to understand the genetic basis to this effect of cytokinins, and its relation with the better known roles of cytokinins in the control of cell‐cycle and differentiation, we have undertaken mutant screening and characterization using a transgenic line of Arabidopsis thaliana harbouring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS). One type of mutant identified displayed reduced sensitivity of AtIPS1::GUS to cytokinin repression. Several other Pi starvation response genes showed reduced cytokinin sensitivity in these lines. These mutants also showed reduced cytokinin repression of the anthocyanin accumulation induced by Pi starvation in the aerial part of the plants. Mapping and molecular characterization of these mutants showed that they were allelic of CRE1/WOL, a locus known to encode a cytokinin receptor. CRE1 is downregulated by Pi starvation and induced by cytokinins, both in the wild‐type and in the cre1 mutants, in which cre1 mRNA levels are higher. These results reveal the existence of a positive feed‐back loop, in addition to the already established negative feedback loop, in cytokinin signalling and indicate that the negative regulation of Pi starvation responses by cytokinins involves a two‐component signalling circuitry, as it is the case of other types of cytokinin response.
Heavy metal toxicity: cadmium permeates through calcium channels and disturbs the plant water status
Authors: Perfus‐Barbeoch Laetitia , Leonhardt Nathalie , Vavasseur Alain , Forestier Cyrille
Published: March 2003

Because plant wilting has been described as a consequence of cadmium (Cd2+) toxicity, we investigate Cd2+ effects on plant water losses, gas exchanges and stomatal behaviour in Arabidopsis thaliana L. Effects of 1‐week Cd2+ application in hydroponic condition (CdCl2 10–100 µm) were analyzed. A 10‐µm Cd2+ concentration had no significant effect on the plant–water relationship and carbon assimilation. At higher Cd2+ concentrations, a Cd2+ ‐dependent decrease in leaf conductance and CO2 uptake was observed despite the photosynthetic apparatus appeared not to be affected as probed by fluorescence measurements. In epidermal strip bioassays, nanomolar Cd2+ concentrations reduced stomatal opening under light in A. thaliana, Vicia faba and Commelina communis. Application of 5 µm ABA limited the root‐to‐shoot translocation of cadmium. However, the Cd2+‐induced stomatal closure was likely ABA‐independent, since a 5‐day treatment with 50 µm Cd2+ did not affect the plant relative water content. Additionally, a similar Cd2+‐induced stomatal closure was observed in the ABA insensitive mutant abi1‐1. Interestingly, this mutant displayed a higher transpiration rate than the wild type but did not accumulate more Cd2+, arguing that Cd2+ uptake is not dependent only on the transpiration flow. Application of putative calcium channels inhibitors suppressed the inhibitory effect of Cd2+ in epidermal strip experiments, suggesting that Cd2+ could enter the guard cell through calcium channels. Patch‐clamp studies with V. faba guard cell protoplasts showed that plasma membrane K+ channels were insensitive to external Cd2+ application whereas Ca2+ channels were found permeable to Cd2+. In conclusion, we propose that Cd2+ affects guard cell regulation in an ABA‐independent manner by entering the cytosol via Ca2+ channels.
The stability of cortical microtubules depends on their orientation
Authors: Wiesler Bodo , Wang Qi‐Yan , Nick Peter
Published: March 2003

Auxin controls the orientation of cortical microtubules in maize coleoptile segments. We used tyrosinylated α‐tubulin as a marker to assess auxin‐dependent changes in microtubule turnover. Auxin‐induced tyrosinylated α‐tubulin, correlated with an elevated sensitivity of growth to antimicrotubular compounds such as ethyl‐N‐phenylcarbamate (EPC). We determined the affinity of α‐tubulin to EPC and found that it was dramatically increased when the tubulin was de‐tyrosinylated. By proteolytic cleavage of the carboxy terminal tyrosine, such an increased affinity could be induced in vitro. Thus, the auxin‐induced sensitivity of growth to EPC is not caused by an increased affinity for this inhibitor, but caused by a reduced microtubule turnover. Double visualization assays revealed that the transverse microtubules induced by auxin consist predominantly of tyrosinylated α‐tubulin, whereas the longitudinal microtubules induced by auxin depletion contain de‐tyrosinylated α‐tubulin. The results are discussed in terms of direction‐dependent differences in the lifetime of microtubules.
A role for HvGAMYB in anther development
Authors: Murray Fiona , Kalla Roger , Jacobsen John , Gubler Frank
Published: March 2003

HvGAMYB is a transcription factor that was first identified in barley aleurone cells and shown to be upregulated by gibberellin (GA). Using RNA and immunoblot analysis we have shown HvGAMYB is also strongly expressed in barley anthers. Transgenic barley expressing a HvGAMYB:GFP fusion gene have been created and GAMYB expression in anthers analysed. GFP expression was clearly visible during early anther development in the nuclei of the epidermis, endothecium, middle layer and tapetum. Expression in the epidermis and endothecium persists until just prior to anther dehiscence, expression in the other two cell layers is visible until they are compressed and broken down as the microspores develop. Further evidence of a role for HvGAMYB in anther development was provided by the creation of transgenic barley over‐expressing the HvGAMYB gene. Associated with the increase in HvGAMYB levels was a progressive decrease in anther size, particularly a decrease in anther length. Anthers also became increasingly lighter in colour. Anthers with fourfold more HvGAMYB protein than non‐transgenic controls failed to dehisce and were male sterile, anthers with approximately three to fourfold endogenous GAMYB protein levels were smaller and paler but still shed normally. To investigate the hormonal regulation of HvGAMYB expression in anthers, HvGAMYB and SLN1 protein levels in anthers were analysed following application of GA3. As in cereal aleurone, HvGAMYB levels were found to increase and SLN1 levels decrease following GA3 application suggesting a similar GA‐signalling pathway to that in aleurone exists in anthers.