bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryTo 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

SummaryThe 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

SummaryInfection 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

SummaryThe 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

SummaryAn 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

SummaryA 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.
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

SummaryDuring 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

SummaryGenes 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

SummaryTo 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

SummaryA 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.
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

Summary 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

SummaryNovel ‘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.