bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of cell wall proteins in relation to the developmental anatomy of the carrot root apex
Authors: Smallwood M. , Beven A. , Donovan N. , Neill S.J. , Peart J. , Roberts K. , Knox J.P.
Published: March 2003

A panel of monoclonal antibodies that recognize a class of cell wall proteins, related to the hydroxyproline‐rich glycoproteins, has been assembled and characterized in relation to their restricted patterns of binding amongst the cells comprising the carrot root apex. The occurrence of the epitopes at the surface of cells and intercellular spaces in the region of the apex between the meristematic initials and the region of cell expansion indicates dynamic patterns that reflect aspects of the development of the anatomical pattern. The monoclonal antibody JIM11 reacts with the surface of cells in the central root cap and the region of the meristem. As the cortex/stele boundary becomes established the reactivity is seen in the inner cortical layers and finally in the whole cortex. Later in development the JIM11 epitope is also expressed by two pairs of pericycle cell files adjacent to the phloem region and also by the epidermis. The JIM12 monoclonal antibody is unreactive with cells in the region of the root cap and the meristem but is reactive with intercellular spaces formed at the junction of the oblique and radial walls in the double‐layered sectors of the pericycle opposite the xylem poles. This epitope is also transiently expressed by the two phloem sieve tube element mother cells. Later in development JIM12 recognizes the future metaxylem cells. The antibody JIM20 recognizes all the cells and intercellular spaces recognized by JIM11 and JIM12. Immuno‐chemical analyses indicate cross‐reactivity with carrot taproot extensin and Solanaceous lectins.
A light‐ and temperature‐entrained circadian clock controls expression of transcripts encoding nuclear proteins with homology to RNA‐binding proteins in meristematic tissue
Authors: Heintzen Christian , Melzer Slegbert , Fischer Ruth , Kappeler Stefan , Apel Klaus , Staiger Dorothee
Published: March 2003

To investigate physiological processes generated by endogenous circadian rhythms on the molecular level, we have identified clock‐controlled genes In the long‐day plant Sinapis aiba L. A cDNA library was differentially screened using cDNA probes representing transcripts expressed at either the middle of the light period or the middle of the dark period. Two closely related groups of transcripts, Sagrp1 and Sagrp2, controlled by a circadian rhythm have been isolated. RNA blot analysis verified that transcript levels oscillate in plants grown in light/dark cycles with maxima between ‘Zeitgober’ time (zt)8 and zt12 (8–12 h after onset of illumination) and minima around zt20. Steady‐state mRNA levels continue to oscillate in plants shifted from light/dark cycles to constant light. No synchronous mRNA oscillations are found in plants grown from seed in constant light at constant temperature, suggesting that the clock has to be entrained initially. In contrast, when plants grown in constant light are exposed to rhythmic temperature shifts oscillations of steady‐state Sagrp mRNA levels are induced, indicating that temperature acts as an alternative external stimulus (zeitgeber) other than light to entrain the oscillator. In situ hybridization reveals that both transcript groups are expressed predominantly in meristematic and growing tissue. Strong expression is observed in the leaf primordia of the shoot apex, the procambial strands, cambium and in all cell layers of young leaves around zt12. In contrast, little or no signal is found on tissue sections isolated at zt20. This indicates that the oscillator(s) regulating Sagrp transcript fluctuations operate(s) synchronously in different organs. For both transcript groups cDNAs were isolated corresponding to unspliced pre‐mRNAs or to transcripts generated by the use of a second 5′ splice site. The cDNAs corresponding to the fully spliced transcripts contain open reading frames for polypeptides of 16 kDa, each containing a putative N‐terminal RNA recognition motif and a C‐terminal region rich in glycine. The predicted proteins show strong homology to an ABA‐inducible glycine‐rich protein from maize embryos and to the mammalian RNA‐binding protein A1 of the heterogeneous nuclear ribonucleoprotein complex involved in pre‐mRNA splicing. The SaGRP protein fluctuates with a very low amplitude over light/dark cycles. Immunogold labeling demonstrates the presence of the SaGRP protein within the nucleus of the investigated meristematic cells of young leaves.
Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene
Authors: Grierson Claire , Du Jian‐Sheng , De Torres Zabala Marta , Beggs Kyle , Smith Caroline , Holdsworth Michael , Bevan Michael
Published: March 2003

The expression of genes encoding patatin, a major tuber protein, is highly tissue‐specific but is also modulated by exogenous sucrose. The patterns of transcription observed in potato plants could be due to mechanisms conferring tuber‐specificity or they could reflect the concentrations of sucrose found in different tissues. To distinguish between these possibilities, a detailed examination was made of the function of a region of the promoter previously implicated in conferring tissue‐specific and sucrose‐inducible expression. Internal deletions of this region revealed three separate functional domains regulating expression. The B repeat region acted as a positive activator of transcription in the tuber and was also responsible for a degree of sucrose‐inducibility. The distal region of the A repeat repressed transcription in leaf and tuber tissue, while the proximal region of the A repeat was able to confer sucrose‐responsiveness. Each of these regions specifically bound nuclear proteins which may be putative transcription factors involved in conferring these responses. The region found to confer sucrose‐inducible expression was conserved among some other genes that are also regulated by exogenous sucrose.
Complex, localized changes in CO2 assimilation and starch content associated with the susceptible interaction between cucumber mosaic virus and a cucurbit host
Authors: Técsi László I. , Maule Andy J. , Smith Alison M. , Leegood Richard C.
Published: March 2003

Infection of the cotyledons of Cucurbita pepo L. with cucumber mosaic virus (CMV) results in the formation of chlorotic, starch‐containing lesions. To characterize the physiological changes occurring within lesions, the distribution of the virus was examined by immunolocalization and correlated with starch accumulation, 14CO2 assimilation and chlorophyll a fluorescence quenching. These techniques resolved the lesion into a complex and reproducible arrangement of cell types of diverse physiology. The region of infected cells extended beyond specific circular zones of cells which variously showed enhanced rates of CO2 assimilation, enhanced chlorophyll a fluorescence quenching, starch accumulation, starch degradation and chlorosis. This indicates a series of physiological changes occurring over several days following viral replication within a cell. Starch accumulation in the lesion was shown to result from photosynthetic activity of cells within the lesion and not from the import of photosynthate from surrounding uninfected areas of the cotyledon.
Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping
Authors: Topping Jennifer F. , Agyeman Francesca , Henricot Beatrice , Lindsey Keith
Published: March 2003

The technique of promoter trapping has been exploited to identify markers of embryogenesis in Arabidopsis thaliana. A population of transgenic A. thaliana was generated containing the promoter trap vector pΔgusBin19, following Agrobacterium tumefaciens‐mediated transformation. This vector contains, at the T‐DNA left border, a promoterless gusA gene, which is activated following integration downstream of a native gene promoter that directs transgene transcription. Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimetric assay. Histochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to contain single T‐DNA inserts were analysed in detail. Each line showed a distinct pattern of GUS fusion activity. Fusion transcripts were identified, demonstrating that transcription was initiated in the genomic DNA flanking the T‐DNA left border. Inverse PCR was used to clone the T‐DNA flanking sequences, and for one line a corresponding cDNA was identified, demonstrating that the tagged sequences are transcribed. The markers represent the earliest embryonic genes known to be expressed in plants.
SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine‐tagged protein
Authors: Sauer Norbert , Stolz Jürgen
Published: March 2003

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy‐dependent, uncoupler‐sensitive loading into phloem cells via a sucrose‐H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy‐dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy‐dependent manner. The KM‐values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m‐chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH‐group inhibitors. The VMAX but not the KM‐values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1‐His6 cDNA encoding a histidine tag at the SUC1 C‐terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.
Expression of α‐amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient
Authors: Chen Min‐Huei , Liu Li‐Fei , Chen Yung‐Reui , Wu Hsin‐Kan , Yu Su‐May
Published: March 2003

A rice suspension cell culture system has been established to study how sugar depletion regulates α‐amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa α‐amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of α‐amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that α‐amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N‐termini of nine rice α‐amylases suggests hitherto unidentified pathways for import of α‐amylases into amyloplasts. The studies show that differential α‐amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar‐regulated characteristics of α‐amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of α‐amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.
Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development
Authors: Burton Rachel A. , Bewley J. Derek , Smith Alison M. , Bhattacharyya Madan K. , Tatge Helma , Ring Steve , Bull Vicky , Hamilton William D.O. , Martin Cathie
Published: March 2003

cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced. The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species. In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N‐terminus of the mature protein. This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule‐bound starch synthase II (GBSSII). However, in each case it is missing from the other isoform of SBE from the same species. On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families. There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different. Pea SBEI and SBEII are differentially expressed during embryo development. SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos. The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development. Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.
Arabinogalactan proteins in maize coleoptiles: developmental relationship to cell death during xylem differentiation but not to extension growth†
Authors: Schindler T. , Bergfeld R. , Schopfer P.
Published: March 2003

The spatial and temporal expression of arabinogalactan proteins (AGPs) in the coleoptile of maize seedlings was investigated with monoclonal antibodies (MAC207, JIM13, JIM14) raised against particular AGP epitopes in carrot. MAC207 binds to a buffer‐soluble AGP fraction of 90–210 kDa that also reacts with β‐glucosyl Yariv reagent and the lectin RCA120. Immunogold‐labelling showed that the MAC207 epitope is exclusively localized in the plasma membrane. JIM13 binds to a 120 kDa component of the buffer‐soluble AGP fraction localized in the plasma membrane of future sclerenchyma cells and secondary‐wall thickenings of future tracheids of vascular bundles. JIM14 binds to a 50 kDa component of the salt‐extractable fraction from cell walls localized in the innermost wall layer of sclerenchyma cells. These AGP epitopes demonstrate different temporal expression patterns which do not correlate with extension growth. Auxin had no effect on the amount of soluble AGP from coleoptile sections, containing the growth‐controlling epidermis but no vascular bundles, as measured by crossed electrophoresis. Moreover, incorporation of radioactive arabinose, galactose or proline into this fraction was not stimulated by auxin. These results contradict the hypothesis that AGPs function as wall‐loosening agents in auxin‐mediated extension growth. The results are compatible with the notion that AGPs serve as developmental markers defining particular features of future cell differentiation. The specific association of the epitopes recognized by JIM13 and JIM14 with disintegrating cells suggests that the related AGPs identify those cells of the coleoptile which are committed to programmed cell death.
Correlation between the induction of a gene for Δ1‐pyrroline‐5‐carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress
Authors: Yoshiba Yoshu , Kiyosue Tomohiro , Katagiri Takeshi , Ueda Hiroko , Mizoguchi Tsuyoshi , Yamaguchi‐Shinozaki Kazuko , Wada Keishiro , Harada Yoshinori , Shinozaki Kazuo
Published: March 2003

The isolation and characterization is reported of a cDNA for Δ1‐pyrroline‐5‐carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana. Southern blot analysis suggested that only one copy of the corresponding gene (AtP5CS) is present in A. thaliana. The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia. Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana. A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high‐salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.
Efficient isolation and mapping of Arabidopsis thaliana T‐DNA insert junctions by thermal asymmetric interlaced PCR
Authors: Liu Yao‐Guang , Mitsukawa Norihiro , Oosumi Teruko , Whittier Robert F.
Published: March 2003

Thermal asymmetric interlaced (TAIL‐) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T‐DNA insertions in Arabidopsis thaliana is described. Insertion‐specific products were amplified from 183 of 190 tested T‐DNA insertion lines. Reconstruction experiments indicate that the technique can recover single‐copy sequences from genomes as complex as common wheat (1.5 × 1010 bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T‐DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T‐DNA insertions confer hygromycin resistance, can be used for fine‐scale mapping of linked phenotypic loci.
Molybdenum co‐factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two‐domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins
Authors: Stallmeyer B. , Nerlich A. , Schiemann J. , Brinkmann H. , Mendel R.R.
Published: March 2003

The molybdenum co‐factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1, a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA. The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6. As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two‐domain structure: the N‐terminal domain is homologous to the E. coli Moco protein MoeA, the C‐terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli, is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two‐domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.
Molecular characterization of proteins in protein‐body membrane that disappear most rapidly during transformation of protein bodies into vacuoles
Authors: Inoue Kaori , Motozaki Asako , Takeuchi Yuka , Nishimura Mikio , Hara‐Nishimura Ikuko
Published: March 2003

During the post‐germination growth of seeds, protein bodies fuse with one another and are converted to a central vacuole. To investigate this transition, protein‐body membranes from dry seeds of pumpkin (Cucurbita sp.) were prepared and their protein components characterized. Five major proteins (designated MP23, MP27, MP28, MP32 and MP73) were detected in the protein‐body membranes. A cDNA clone encoding both MP27 and MP32 has been isolated. The deduced precursor polypeptide was composed of a hydrophobic signal sequence, MP27 and MP32, in that order. A putative site of cleavage between MP27 and MP32 was located on the COOH‐terminal side of asparagine 278, an indication that the post‐translational cleavage may occur by the action of a vacuolar processing enzyme that converts proprotein precursors of seed proteins into the mature forms. Immunoelectron microscopic analysis showed that MP27 and MP32 were associated with protein‐body membrane of dry pumpkin seeds. Among the five membrane proteins, MP27 and MP32 disappeared most rapidly during seedling growth. The degradation of MP27 and MP32 starts just after seed germination and proceeds in parallel with the transformation of the protein bodies into a vacuole.
Involvement of Ca2+ signalling in the sugar‐inducible expression of genes coding for sporamin and β‐amylase of sweet potato
Authors: Ohto Masa‐aki , Hayashi Koji , Isobe Minoru , Nakamura Kenzo
Published: March 2003

Genes coding for sporamin and β‐amylase of sweet potato are inducible not only by high levels of metabolizable sugars, such as sucrose, but also by a low concentration of polygalacturonic acid (PGA). Calmodulin inhibitors and EGTA inhibited both the PGA‐inducible and the sucrose‐inducible accumulation of mRNAs for sporamin and β‐amylase in sweet potato. Calmodulin inhibitors, EGTA and La3+, also inhibited the sucrose‐inducible expression, in leaves of transgenic tobacco, of a fusion gene, β‐Amy:GUS, which consists of the promoter of the β‐amylase gene and the coding sequence for β‐glucuronidase. The sucrose‐inducible expression of the β‐Amy:GUS fusion gene was also inhibited by two inhibitors of Ca2+ channels, diltiazem and nicardipine. These results suggest that the sugar‐inducible expression of genes for sporamin and β‐amylase involves, at least in part, Ca2+‐mediated signalling, and that the cytosolic free Ca2+ may mediate cross‐talk between signals related to carbohydrate metabolism and other stimuli. Treatment of coelenterazine‐loaded leaf discs of tobacco expressing a Ca2+‐binding photoprotein, aequorin, with 0.2 M sucrose for 24 h significantly reduced the level of luminescence that could be induced by cold shock, as compared to cold shock‐induced luminescence in coelenterazine‐loaded leaf discs treated with water. Repression of cold shock‐induced luminescence was due to the conversion of holoaequorin to apoaequorin during the treatment with sucrose. Treatment of coelenterazine‐loaded leaf discs with a 0.2 M solution of glucose or fructose, but not of mannitol or sorbitol, also reduced the cold shock‐induced luminescence. It is suggested that non‐synchronous increases in cytosolic level of free Ca2+ occur in leaf discs during treatment with high levels of metabolizable sugars.
Delayed leaf senescence in ethylene‐deficient ACC‐oxidase antisense tomato plants: molecular and physiological analysis
Authors: John Isaac , Drake Rachel , Farrell Aldo , Cooper Wendy , Lee Pam , Horton Peter , Grierson Don
Published: March 2003

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild‐type plants indicated that ACC oxidase mRNA accumulation normally begins in pre‐senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild‐type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild‐type plants. Northern analysis of the photosynthesis‐associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild‐type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening‐related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild‐type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild‐type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild‐type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay‐green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.
A new elicitor of the hypersensitive response in tobacco: a fungal glycoprotein elicits cell death, expression of defence genes, production of salicylic acid, and induction of systemic acquired resistance
Authors: Baillieul Fabienne , Genetet Isabelle , Kopp Marguerite , Saindrenan Patrick , Fritig Bernard , Kauffmann Serge
Published: March 2003

A 32 kDa glycoprotein whose effects in tobacco and other Nicotianae mimic a typical hypersensitive response, was isolated from Phytophthora megasperma. Infiltration of a few nanograms of the protein into leaves caused the formation of lesions that closely resemble hypersensitive response lesions. Transcripts of genes encoding enzymes of the phenylpropanoid and sesquiterpenoid pathways accumulated rapidly after elicitor application followed by salicylic acid production. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of PR protein genes was activated. After several days systemic acquired resistance was also induced. Thus, tobacco plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in a Nicotianae and possibly into crop plant species.
A galactose‐rich, cell‐wall glycoprotein from styles of Nicotiana alata
Authors: Sommer‐Knudsen Jens , Clarke Adrienne E. , Bacic Antony
Published: March 2003

A basic, galactose‐rich style glycoprotein (GaRSGP) encoded by a previously characterized style‐specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45–120 kDa on SDS—PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline‐rich glycoproteins (P/HRGPs) to be characterized both as a cDNA‐clone and protein. Glycans are attached to the protein backbone through both O‐ and N‐glycosidic linkages with the majority of the carbohydrate being O‐linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style‐specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style‐specific carbohydrate epitope(s) which may play a role in style function.
Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues
Authors: An Yong‐Qiang , McDowell John M. , Huang Shurong , McKinney Elizabeth C. , Chambliss Sharon , Meagher Richard B.
Published: March 2003

Arabidopsis
has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by
ACT2
and
ACT8
, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene‐specific RNA gel blot hybridization and reverse transcriptase‐mediated polymerase chain reaction (RT‐PCR) assays showed that
ACT2
and/or
ACT8
mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most
Arabidopsis
organs. The 5′ flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a β‐glucuronidase (GUS) reporter gene and transformed into
Arabidopsis
. The
ACT2/GUS
construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the
ACT8/GUS
construct was weaker. It was observed only in a subset of the organs and tissues expressing
ACT2/GUS
and was not significantly expressed in the flower.
ACT2, ACT8
, and
ACT8/GUS
mRNAs were present at moderate to high levels in pollen, and yet neither
ACT2/GUS
nor
ACT8/GUS
enzyme expression could be detected in pollen. This suggested a mechanism of translational control affecting
ACT2
and
ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.
Vectors carrying two separate T‐DNAs for co‐transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers
Authors: Komari Toshihiko , Hiei Yukoh , Saito Yasuhito , Murai Nobuhiko , Kumashiro Takashi
Published: March 2003

Novel ‘super‐binary’ vectors that carried two separate T‐DNAs were constructed. One T‐DNA contained a drug‐resistance, selection‐marker gene and the other contained a gene for β‐glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tume‐faciens LBA4404 that carried the vectors. Frequency of co‐transformation with the two T‐DNAs was greater than 47%. GUS‐positive, drug‐sensitive progeny were obtained from more than half of the co‐transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T‐DNAs. Thus, the non‐selectable T‐DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug‐resistant transformants. Since various DNA fragments may be inserted into the non‐selectable T‐DNA by a simple procedure, these vectors will likely be very useful for the production of marker‐free transformants of diverse plant species. Delivery of two T‐DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co‐transformation was relatively low.
A phosphonate monoester hydrolase from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants
Authors: Dotson Stanton B. , Lanahan Michael B. , Smith Alan G. , Kishore Ganesh M.
Published: March 2003

A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50‐fold less active. A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development. However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate. The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis. As an example of tissue‐specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal‐specific promoter and treating the mature plants with glyceryl glyphosate. A chromogenic phosponate ester substrate, 5‐bromo‐4‐chloro‐indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene. The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed.
The CER3 gene of Arabidopsis thaliana is expressed in leaves, stems, roots, flowers and apical meristems
Authors: Hannoufa Abdelali , Negruk Valentin , Eisner Galina , Lemieux Bertrand
Published: March 2003

The eceriferum3 (cer3) locus encodes one of 21 gene products known to be involved in wax biosynthesis in Arabidopsis thaliana. Mutations at these loci are readily identified by their bright, dark‐green stems when compared with the more glaucous wild‐type plant. Clones of a gene which encodes a 795 amino acid open reading frame have been isolated by using plant DNA flanking the site of a T‐DNA insertion in line BRL1. Molecular complementation of the cer3 mutant phenotype by clones of this gene establish that it corresponds to the CER3 gene. Although the 90 kDa predicted amino acid sequence of this gene shows no homology to any other known protein, the second exon of CER3 encodes a RRX12KK nuclear localization sequence (NLS). Southern blot and DNA sequence analysis revealed that the T‐DNA is inserted 89 bp downstream of the translation termination codon of this gene. Northern blot hybridization of RNA isolated from the BRL1 mutant with the CER3cDNA probe indicated that the transcript is absent in this mutant line. Unlike other CER genes that have been cloned to date, high levels of the CER3 transcript were found in all tissues from wild‐type plants, that is, leaves, stems, roots, flowers, and apical meristems.
Isolation and characterization of abscisic acid‐deficient Arabidopsis mutants at two new loci
Authors: Léon‐Kloosterziel Karen M. , Gil Marta Alvarez , Ruijs Gerda J. , Jacobsen Steven E. , Olszewski Neil E. , Schwartz Steven H. , Zeevaart Jan A.D. , Koornneef Maarten
Published: March 2003

Novel Arabidopsis mutants with lowered levels of endogenous abscisic acid (ABA) were isolated. These were selected in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol. Another mutant was isolated in a screen for NaCl tolerance. The ABA‐deficiency was caused by two monogenic, recessive mutations, aba2 and aba3, that were both located on chromosome 1. The mutants showed a phenotype that is known to be characteristic for ABA‐deficiency: a reduced seed dormancy and excessive water loss, leading to a wilty phenotype. Double mutant analysis, combining different aba mutations, indicated the leaky nature of the mutations.
The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering
Authors: Zagotta Michelle T. , Hicks Karen A. , Jacobs Carolyn I. , Young Jeff C. , Hangarter Roger P. , Meeks‐Wagner D. Ry
Published: March 2003

Flowering in Arabidopsis thaliana is promoted by long‐day (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short‐day (SD) photoperiods. The early‐flowering 3 (elf3) mutant of Arabidopsis, which is insensitive to photoperiod with regard to floral initiation has been characterized. elf3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf3 mutant seedlings were less responsive than wild‐type to all wavelengths of light, and most notably defective in blue and green light‐mediated inhibition. When analyzed for the flowering‐time phenotype, elf3 was epistatic to mutant alleles of the blue‐light receptor encoding gene, HY4. However, when elf3 mutants were made deficient for functional phytochrome by the introduction of hy2 mutant alleles, the elf3 hy2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome‐mediated pathway regulating floral initiation remains functional in elf3 single mutants. In addition, the inflorescences of one allelic combination of elf3 hy2 double mutants form a terminal flower similar to the structure produced by tfl1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.
Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium
Authors: Allen Gethyn J. , Sanders Dale
Published: March 2003

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole‐vacuole mode of patch‐clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At ‘zero’ to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage‐independent (non‐rectifying) and were typical of Vacuolar K,+‐selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time‐dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl− permeability was assumed then the apparent PCa was lower. However, substitution of Cl− by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl− permeability of the SV channel is invalid. Single‐channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi‐ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg2+‐activated and were demonstrated to be Mg2+‐permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions.

The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.
Accumulation of nitrate in the shoot acts as a signal to regulate shoot‐root allocation in tobacco†
Authors: Scheible Wolf‐Rüdiger , Lauerer Marianne , Schulze Ernst‐Detlef , Caboche Michel , Stitt Mark
Published: March 2003

Mutants and transformants of tobacco (Nicotiania tabacum L. cv Gatersleben 1) with decreased expression of nitrate reductase have been used to investigate whether nitrate accumulation in the shoot acts as a signal to alter allocation between shoot and root growth. (a) Transformants with very low (1–3% of wild‐type levels) nitrate reductase activity had growth rates, and protein, amino acid and glutamine levels similar to or slightly lower than a nitrate‐limited wild‐type, but accumulated large amounts of nitrate. These plants should resemble a nitrate‐limited wild‐type, except in responses where nitrate acts as a signal. (b) Whereas the shoot:root ratio decreases from about 3.5 in a well‐fertilized wild‐type to about 2 in a nitrate‐limited wild‐type, the transformants had a very high shoot:root ratio (8–10) when they were grown on high nitrate. When they were grown on lower nitrate concentrations their shoot:root ratio declined progressively to a value similar to that in nitrate‐limited wild‐types. Mutants with a moderate (30–50%) decrease of nitrate reductase also had a small but highly significant increase of their shoot:root ratio, compared to the wild‐type. The increased shoot:root ratio in the mutants and transformants was due to a stimulation of shoot growth and an inhibition of root growth. (c) There was a highly significant correlation between leaf nitrate content and the shoot:root ratio for eight genotypes growing at a wide range of nitrate supply. (d) A similar increase of the shoot:root ratio in nitrate reductase‐deficient plants, and correlation between leaf nitrate content and the shoot:root ratio, was found in plants growing on ammonium nitrate. (f) Split‐root experiments, in which the transformants were grown with part of their root system in high nitrate and the other part in low nitrate, showed that root growth is inhibited by the accumulation of nitrate in the shoot. High concentrations of nitrate in the rooting medium actually stimulate local root growth. (g) The inhibition of root growth in the transformants was relieved when the transformants were grown on limiting phosphate, even though the nitrate content of the root remained high. This shows that the nitrate‐dependent changes in allocation can be overridden by other signals that increase allocation to root growth. (h) The reasons for the changed allocation were investigated in transformants growing normally, and in split‐root culture. Accumulation of nitrate in the shoot did not lead to decreased levels of amino acids or protein in the roots. However, it did lead to a strong inhibition of starch synthesis and turnover in the leaves, and to decreased levels of sugars in the root. The rate of root growth was correlated with the root sugar content. It is concluded that these changes of carbon allocation could contribute to the changes in shoot and root growth.
Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes
Authors: Sun Chih‐Wen , Callis Judy
Published: March 2003

The highly conserved protein ubiquitin is encoded by five polyubiquitin genes in Arabidopsis thaliana ecotype Columbia that have been divided into two subtypes, the UBQ3/UBQ4 subtype and the UBQ10/UBQ11/UBQ14 subtype. Northern analysis using gene‐specific oligonucleotides as hybridization probes and enzyme activity measurements from transgenic plants expressing β‐glucuronidase (GUS) under the control of individual polyubiquitin 5′ flanking regions were used to determine the developmental and environmental regulation of polyubiquitin transcription and mRNA accumulation. Polyubiquitin mRNA levels within and between subtypes were independently modulated. UBQ3 mRNA levels were three‐fold higher than UBQ4 mRNA levels in vegetative organs, but only two‐thirds of the UBQ4 mRNA levels in flowers. UBQ3 mRNA was modulated by dark/light treatments, while mRNAs from UBQ4 and all members of the other subtype were unaffected. Similarly, within the UBQ10/UBQ11/UBQ14 subtype, UBQ11/UBQ14 mRNAs were modulated differently in seedlings after a two‐hour heatshock treatment. Among all the polyubiquitin genes, UBQ10 mRNA level was the most constant in all organs and environmental conditions examined. Transgenic plants transformed with a UBQ10 5′ flanking region::GUS gene contained higher levels of GUS activity than transgenic plants expressing GUS under the control of UBQ3 5′ flanking regions. In conclusion, the relative abundance of different Arabidopsis polyubiquitin mRNAs, even those produced from highly similar genes within a subtype, appears to be modulated independently in response to developmental and environmental cues.
Starch synthesis in amyloplasts purified from developing potato tubers
Authors: Naeem M. , Tetlow I.J. , Emes M.J.
Published: March 2003

Amyloplasts have been purified from potato tubers by mechanical homogenization and gravity sedimentation through Nycodenz. Based on the recovery and latency of organelle‐specific marker enzymes, the recovery of amyloplasts is approximately 13%, exhibiting 65% intactness, with less than 1% contamination by other subcellular fractions. These preparations were able to synthesize starch from glucose‐1‐phosphate plus ATP, or ADP‐glucose but not from glucose‐6‐phosphate. Rates of starch synthesis from glucose‐1‐phosphate plus ATP were linear for up to 1 h and sensitive to the inhibitor 4,4‐diisothiocyanato‐stilbene 2,2‐disulphonic acid (DIDS). Starch synthesis was optimal at pH 7.0 and was saturated by 5–10 mM glucose‐1‐phosphate and by 1 mM ADP‐glucose. The results are discussed in the context of the pathway of starch synthesis and the transport of metabolites across the amyloplast envelope.