bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of enhanced nitrogen deposition and phosphorus limitation on nitrogen budgets of semi‐natural grasslands
Authors: Phoenix Gareth K. , Booth Rosemary E. , Leake Jonathan R. , Read David J. , Grime J. Philip , Lee John A.
Published: August 2003

Increased reactive atmospheric N deposition has been implicated in floristic changes in species‐rich acidic and calcareous grasslands, but the fate of this pollutant N in these ecosystems is unknown. This paper reports the first analysis of N budgets and N fluxes for two grasslands in the White Peak area of Derbyshire, one of the most heavily N‐polluted locations in the UK. N fluxes were monitored in lysimeter cores (retaining the original turfs) taken from field plots of unimproved acidic and calcareous grasslands that had received (in addition to ambient N deposition) simulated enhanced N deposition treatments of 3.5 and 14 g N m−2 yr−1 for 6 years. The influence of reducing phosphorus limitation was assessed by factorial additions of P. Seasonal leached losses of nitrate, ammonia and organic N were monitored in detail along with estimates of N removal through simulated grazing and gaseous losses through denitrification and volatilization. The rates of N fluxes by these pathways were used to create N budgets for the grasslands. Both grasslands were found to be accumulating much of the simulated additional N deposition: up to 89% accumulated in the calcareous grassland and up to 38% accumulated in the acidic grassland. The major fluxes of N loss from these grasslands were by simulated grazing and leaching of soluble organic N (constituting 90% of leached N under ambient conditions). Leached inorganic N (mainly nitrate) contributed significantly to the output flux of N under the highest N treatment only. Loss of N through ammonia volatilization accounted for less than 6% of the N added as simulated deposition, while denitrification contributed significantly to output fluxes only in the acidic grassland during winter. The implications of the results for ecosystem N balances and the likely consequences of N accumulation on these grasslands are discussed.
Controls on microbial CO2 production: a comparison of surface and subsurface soil horizons
Authors: Fierer Noah , Allen Andrew S. , Schimel Joshua P. , Holden Patricia A.
Published: August 2003

Although a significant amount of the organic C stored in soil resides in subsurface horizons, the dynamics of subsurface C stores are not well understood. The objective of this study was to determine if changes in soil moisture, temperature, and nutrient levels have similar effects on the mineralization of surface (0–25 cm) and subsurface (below 25 cm) C stores. Samples were collected from a 2 m deep unsaturated mollisol profile located near Santa Barbara, CA, USA. In a series of experiments, we measured the influence of nutrient additions (N and P), soil temperature (10–35°C), and soil water potential (−0.5 to −10 MPa) on the microbial mineralization of native soil organic C. Surface and subsurface soils were slightly different with respect to the effects of water potential on microbial CO2 production; C mineralization rates in surface soils were more affected by conditions of moderate drought than rates in subsurface soils. With respect to the effects of soil temperature and nutrient levels on C mineralization rates, subsurface horizons were significantly more sensitive to increases in temperature or nutrient availability than surface horizons. The mean Q10 value for C mineralization rates was 3.0 in surface horizons and 3.9 in subsurface horizons. The addition of either N or P had negligible effects on microbial CO2 production in surface soil layers; in the subsurface horizons, the addition of either N or P increased CO2 production by up to 450% relative to the control. The results of these experiments suggest that alterations of the soil environment may have different effects on CO2 production through the profile and that the mineralization of subsurface C stores may be particularly susceptible to increases in temperature or nutrient inputs to soil.
Regional‐scale measurements of CH4 exchange from a tall tower over a mixed temperate/boreal lowland and wetland forest
Authors: Werner Cindy , Davis Kenneth , Bakwin Peter , Yi Chuixiang , Hurst Dale , Lock Loreen
Published: August 2003

The biosphere–atmosphere exchange of methane (CH4) was estimated for a temperate/boreal lowland and wetland forest ecosystem in northern Wisconsin for 1997–1999 using the modified Bowen ratio (MBR) method. Gradients of CH4 and CO2 and CO2 flux were measured on the 447‐m WLEF‐TV tower as part of the Chequamegon Ecosystem–Atmosphere Study (ChEAS). No systematic diurnal variability was observed in regional CH4 fluxes measured using the MBR method. In all 3 years, regional CH4 emissions reached maximum values during June–August (24±14.4 mg m−2 day−1), coinciding with periods of maximum soil temperatures. In 1997 and 1998, the onset in CH4 emission was coincident with increases in ground temperatures following the melting of the snow cover. The onset of emission in 1999 lagged 100 days behind the 1997 and 1998 onsets, and was likely related to postdrought recovery of the regional water table to typical levels. The net regional emissions were 3.0, 3.1, and 2.1 g CH4 m−2 for 1997, 1998, and 1999, respectively. Annual emissions for wetland regions within the source area (28% of the land area) were 13.2, 13.8, and 10.3 g CH4 m−2 assuming moderate rates of oxidation of CH4 in upland regions in 1997, 1998, and 1999, respectively. Scaling these measurements to the Chequamegon Ecosystem (CNNF) and comparing with average wetland emissions between 40°N and 50°N suggests that wetlands in the CNNF emit approximately 40% less than average wetlands at this latitude. Differences in mean monthly air temperatures did not affect the magnitude of CH4 emissions; however, reduced precipitation and water table levels suppressed CH4 emission during 1999, suggesting that long‐term climatic changes that reduce the water table will likely transform this landscape to a reduced source or possibly a sink for atmospheric CH4.
The annual cycles of CO2 and H2O exchange over a northern mixed forest as observed from a very tall tower
Authors: Davis Kenneth J. , Bakwin Peter S. , Yi Chuixiang , Berger Bradford W. , Zhao Conglong , Teclaw Ronald M. , Isebrands J. G.
Published: August 2003

We present the annual patterns of net ecosystem‐atmosphere exchange (NEE) of CO2 and H2O observed from a 447 m tall tower sited within a mixed forest in northern Wisconsin, USA. The methodology for determining NEE from eddy‐covariance flux measurements at 30, 122 and 396 m above the ground, and from CO2 mixing ratio measurements at 11, 30, 76, 122, 244 and 396 m is described. The annual cycle of CO2 mixing ratio in the atmospheric boundary layer (ABL) is also discussed, and the influences of local NEE and large‐scale advection are estimated. During 1997 gross ecosystem productivity (947−18 g C m−2 yr−1), approximately balanced total ecosystem respiration (963±19 g C m−2 yr−1), and NEE of CO2 was close to zero (16±19 g C m−2 yr−1 emitted into the atmosphere). The error bars represent the standard error of the cumulative daily NEE values. Systematic errors are also assessed. The identified systematic uncertainties in NEE of CO2 are less than 60 g C m−2 yr−1. The seasonal pattern of NEE of CO2 was highly correlated with leaf‐out and leaf‐fall, and soil thaw and freeze, and was similar to purely deciduous forest sites. The mean daily NEE of CO2 during the growing season (June through August) was −1.3 g C m−2 day−1, smaller than has been reported for other deciduous forest sites. NEE of water vapor largely followed the seasonal pattern of NEE of CO2, with a lag in the spring when water vapor fluxes increased before CO2 uptake. In general, the Bowen ratios were high during the dormant seasons and low during the growing season. Evapotranspiration normalized by potential evapotranspiration showed the opposite pattern. The seasonal course of the CO2 mixing ratio in the ABL at the tower led the seasonal pattern of NEE of CO2 in time: in spring, CO2 mixing ratios began to decrease prior to the onset of daily net uptake of CO2 by the forest, and in fall mixing ratios began to increase before the forest became a net source for CO2 to the atmosphere. Transport as well as local NEE of CO2 are shown to be important components of the ABL CO2 budget at all times of the year.
Stress influence on genesis, onset and maintenance of cardiovascular diseases: literature review
Authors: Franco Gianfabio P. , de Barros Alba L.B.L. , Nogueira‐Martins Luiz A. , Michel Jeanne L.M.
Published: August 2003

Background. Recent mortality profiles in Brazil show that circulatory diseases are the leading cause of death in Brazil. These disorders contribute to 34% of deaths, with 50% of those deaths occurring in people under 50 years of age, that is, in people who are still active in the workforce. We assume that the growing incidence of cardiovascular diseases has occurred as the globalization of Brazil continues and brings with it the associated health risk factors of modern lifestyles, including stress.

Aim. This paper reports the evidence on the influence of stress in the development, onset and progress of cardiovascular diseases. We aim to define the concept of stress and to point systematically to the interrelationships between its emotional and bodily manifestations through a discussion of the history and study of stress. We then suggest that factors leading to the experience of stress in Brazil are no different than in any other modern nation. We further offer a perspective on nursing interventions currently undertaken in Brazil in both hospital and community settings, with their more recent emphasis on health promotion and prevention.

Methods. An extensive literature review was undertaken. The data presented here were selected from reviews and clinical studies described in MEDLINE, LILACS, SciELO databases, as well as in the classical literature. We also refer to the current Brazilian literature on the prevalence of cardiovascular diseases and their associated risk factors.

Conclusions. The incidence of cardiovascular diseases in Brazil is rising. Because of the globalization of Brazilian society, with its consequent competitiveness and accelerated modern lifestyles, Brazilians are no less immune to the usual health risk factors associated with cardiovascular diseases. Stresses associated with a modern lifestyle, however, are emerging as a new and major risk for developing cardiovascular diseases in Brazil.
The relationship of critical thinking skills and critical thinking dispositions of baccalaureate nursing students
Authors: Profetto‐McGrath Joanne
Published: August 2003

Background. Critical thinking is essential to nursing practice; therefore, knowledge and understanding of nursing students’ critical thinking skills (CTS) and related dispositions are important to nurse educators. This paper presents the results of a non‐experimental study conducted in spring 1998, identifies implications for nurse educators, and offers recommendations for future research.

Aim. The aim of the study was to investigate the CTS and critical thinking dispositions (CTD) of students enrolled in a 4‐year baccalaureate programme at a university in Western Canada.

Methods. The study used a cross‐sectional design. Data collection occurred during regularly scheduled classes. A volunteer sample of 228 students from all 4 years of the baccalaureate programme completed a background/demographic questionnaire, the California Critical Thinking Skills Test, and the California Critical Thinking Disposition Inventory. The reliability of the test and inventory were established using the Kuder Richardson 20 and Cronbach Alpha respectively. Both descriptive and inferential statistical analyses were carried out.

Findings. Critical thinking mean scores increased from years 1 to 4 with the exception of year 3. However, there was no statistically significant difference among the four student groups. Although differences in critical thinking disposition scores were not statistically significant, students’ scores differed significantly on the systematicity subscale. There was a significant relationship between students’ overall CTS and CTD scores.

Conclusions. Approximately 38% of the students in the current study had adequate levels of CTS and 85·5% of the students had adequate levels of CTD. Results indicate a need for students’ continued development in these areas. Dispositions are crucial to critical thinking; without them critical thinking does not happen or may be substandard.
Critical care nursing research priorities in Hong Kong
Authors: Lopez Violeta
Published: August 2003

Background. Research is vital to nursing practice especially in an area such as critical care, where nursing practice continues to increase in complexity and nurses assume greater responsibility and accountability for patient care. However, without knowledge of what are the most significant problems or questions affecting the welfare of critically ill patients in Hong Kong, nurses’ research efforts may be directed to areas that are not of highest priority in today's climate of decreasing health care resources and changing health care provision.

Aim. To identify and prioritize research questions of importance to Hong Kong critical care nurses.

Method. A three‐round Delphi technique was used to solicit, identify and prioritize problems for critical care nursing research.

Results. Respondents who completed the third round had a mean of 6·25 years of critical care nursing experience. Ninety‐one research topics were ranked important or extremely important. Twenty‐seven research priorities ranked as extremely important included topics relating to patient care, family care, nursing, technology, alternative interventions, and illness prevention/health promotion research domains. Thirteen research topics were ranked as least important. None of the participants ranked research topics as not important.

Study limitations. Only full‐time active members of the Hong Kong Association of Critical Care Nurses participated in this study and they may not necessarily represent the views of all critical care nurses in Hong Kong. In addition, no attempts were made to compare research priorities between specialty groups of nurses.

Conclusion. With the re‐structuring of Hong Kong's health care system, critical care nurses are expected to take an active role in advancing the profession, and to be accountable for improving patient outcomes by use of effective, evidence‐based interventions. The research priorities identified in this study may provide impetus and direction for critical care nursing research initiatives.
Agoraphobia: nurse therapist‐facilitated self‐help manual
Authors: Lovell Karina , Cox Debbie , Garvey Rachel , Raines David , Richards David , Conroy Patrick , Repper Dean
Published: August 2003

Background. Agoraphobia is a common and disabling mental health disorder. Substantial evidence supports the use of cognitive behaviour therapy (CBT), in particular the intervention termed exposure therapy, as the treatment of choice. However, although the evidence base for cognitive‐behaviour therapy is extensive, the service delivery evidence base is poor, and alternative ways of delivering therapy are required if mental health services are to achieve standards set out by the National Service Framework in the United Kingdom.

Aim. The study had two aims: (1) to develop a self‐help manual, which could be facilitated by a nurse trained in CBT, for clients suffering from agoraphobia and (2) to pilot the self‐help manual and evaluate its effectiveness.

Method. The self‐help manual was piloted with experienced nurses trained in CBT on three clinical sites for 10 weekly sessions of 30 minutes duration. A range of clinical outcome measures was administered by an independent assessor before and after treatment and at 1‐month follow‐up.

Results. A total of 18 clients completed treatment and results showed improvement on all clinical measures; improvement was maintained at 1‐month follow‐up. Importantly, 89% of clients were clinically significantly improved at post‐treatment assessment. Clients were satisfied with their treatment and the self‐help manual, and therapists found facilitated self‐help an acceptable way to deliver treatment.

Conclusion. Nurses can deliver effective support to patients using a self‐help manual for agoraphobia. Although the results are promising, further work is required with larger numbers, longer follow‐up and economic evaluation under controlled conditions. The work could also be adapted to different psychological conditions. Variation in the amount of specialist educational training is necessary to determine how many nurses are needed to support patients using self‐help.
Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana
Authors: Aeong Oh Sung , Park Joon‐Hyun , In Lee Gyu , Hee Paek Kyung , Ki Park Soon , Gil Nam Hong
Published: August 2003

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana. Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis. Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age‐dependent in planta senescence and dark‐induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis. The results further suggest that the three genes function at a common step of age‐dependent and dark‐induced senescence processes. It is further shown that one of the mutations is allelic to ein2‐1, an ethylene‐insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis.
Light‐regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene
Authors: Helliwell Chris A. , Webster Carl I. , Gray John C.
Published: August 2003

Expression of the pea plastocyanin gene (PetE) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5′ untranslated leader region does not direct light‐regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light‐regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5′ untranslated leader region, the coding region and the 3′ region of the PetE gene to light‐regulated expression. Both the coding region and the 5′ untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5′ leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5′ end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.
Ca2+‐independent phosphorylation of a 68 kDa pollen protein is stimulated by the self‐incompatibility response in Papaver rhoeas
Authors: Rudd Jason J. , Franklin F. Christopher H. , Franklin‐Tong Vernonica E.
Published: August 2003

The self‐incompatibility (SI) response in Papaver rhoeas involves a Ca2+‐based signalling pathway, which mediates the SI‐specific inhibition of incompatible pollen. We have previously reported the identification of p26.1, a pollen protein whose phosphorylation was increased specifically as a consequence of the SI response. We have investigated whether further specific protein phosphorylation events are induced in P. rhoeas pollen. Here we report the identification of an additional pollen protein, p68, which also responds to S proteins by an increase in its phosphorylation state. This phosphorylation event occurs in living pollen tubes grown in vitro, and can be observed specifically when pollen is challenged with biologically active S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible S proteins. The timing of the increase in phosphorylation of p68 is temporally later than that of p26.1, occurring between 240 sec and 400 sec after challenge. This suggests that its phosphorylation is downstream of p26.1 in the SI signalling pathway(s). Surprisingly, the kinases responsible for the phosphorylation of p68 are not Ca2+‐dependent. This, and the later timing of the p68 response, suggests that a ‘second wave’ of Ca2+‐independent signalling may follow the initial Ca2+‐dependent SI signalling. This indicates that the SI signalling pathway(s) in pollen may be quite complex.
Conserved expression of a TASSELSEED2 homolog in the tapetum of the dioecious Silene latifolia and Arabidopsis thaliana
Authors: Lebel‐Hardenack Sabine , Ye De , Koutnikova Hana , Saedler Heinz , Grant Sarah R.
Published: August 2003

To investigate the genetics of male sex determination and stamen development in the dioecious plant Silene latifolia (white campion), male‐specific transcripts were isolated from developing flowers by cDNA subtraction. One of the cDNAs identified, STA1, had high DNA and amino acid sequence homology to the male sex determining gene of Zea mays (maize), TASSELSEED2. Both genes are expressed in male and not in female flowers, However, they do not share the same expression pattern. The TASSELSEED2 gene product is expressed in the gynoecium primordia of male maize flowers where it is necessary for pistil abortion. STA1 is not expressed in the gynoecium primordia of male white campion and therefore its gene product cannot perform the same function in sex determination that TASSELSEED2 performs in maize. STA1 is expressed in tapetal cells of white campion male flowers and of white campion hermaphroditic mutants. A homologous gene is also expressed in the tapetum of hermaphroditic Silene species. Tapetal expression of a homologous gene (named ATA1) was also found in Arabidopsis thaliana. The similarity in primary sequence and expression pattern of STA1 and ATA1 indicate that these genes have a conserved role in tapetum development.
Movement and subcellular localization of a tobamovirus in Arabidopsis
Authors: Lartey Robert , Ghoshroy Soumitra , Ho Joe , Citovsky Vitaly
Published: August 2003

Tobamoviruses represent a well‐characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus–plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis. Here, we describe infection of Arabidopsis by a recently discovered crucifer‐infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis, causing mild and fast‐developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus–plant interaction.
Differential expression of two P5CS genes controlling proline accumulation during salt‐stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis
Authors: Strizhov Nicolai , Ábrahám Edit , Ökrész László , Blickling Stefan , Zilberstein Aviah , Schell Jeff , Koncz Csaba , Szabados László
Published: August 2003

Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of Δ1‐pyrroline‐5‐carboxylate synthase (P5CS) controlling the rate‐limiting step of glutamate‐derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1AtP5CS1 mapped to chromosome 2‐78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3–101.3 contributes 20–40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt‐treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt‐induction. Mutations abi1 and axr2, affecting ABA‐perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt‐stress, whereas ABA‐signalling functions defined by the abi2 and abi3 mutations have no effect on salt‐induction of the AtP5CS genes.
Systemic elevation of phosphatidic acid and lysophospholipid levels in wounded plants
Authors: Lee Sumin , Suh Sujeoung , Kim Seju , Crain Richard C. , Kwak June Myoung , Nam Hong‐Gil , Lee Youngsook
Published: August 2003

Plants develop systemic defense responses upon exposure to pathogens or wounding by herbivores. Lipids and lipid metabolites have previously been implicated in induction of defense molecules during plant responses to physical wounding. Possible involvement of changes in lipid composition in systemic wound signal transduction was examined in leaves of seedlings of several different plant species. In the wounded tomato leaf, phosphatidic acid increased approximately fourfold within 5 min whereas lysophosphatidylcholine and lysophosphatidylethanolamine increased over twofold within 15 min of wounding. Similar changes in these lipids were observed in the neighboring non‐wounded leaf. In broad bean, soybean, sunflower and pepper seedlings phosphatidic acid levels increased rapidly and systemically upon wounding. The results suggest that the role of phospholipid hydrolysis and accumulation of lipid metabolites in the early events are responsible for systemic wound signal transduction in plants. Furthermore, they indicate that the wound signal propagates outside the wounded leaf within 5 min in these plants.
PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana
Authors: Fujita Hironori , Syōno Kunihiko
Published: August 2003

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N‐1‐naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5‐triiodobenzoic acid (TIBA) and 9‐hydroxyfluorene‐9‐carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin‐efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.
Development of Arabidopsis thaliana leaves at low temperatures releases the suppression of photosynthesis and photosynthetic gene expression despite the accumulation of soluble carbohydrates
Authors: Strand Åsa , Hurry Vaughan , Gustafsson Petter , Gardeström Per
Published: August 2003

Arabidopsis thaliana plants were grown at 23°C and changes in carbohydrate metabolism, photosynthesis and photosynthetic gene expression were studied after the plants were shifted to 5°C. The responses of leaves shifted to 5°C after development at 23°C are compared to leaves that developed at 5°C. Shifting warm developed leaves to 5°C lead to a severe suppression of photosynthesis that correlated with a rapid and sustained accumulation of hexose phosphates and soluble sugars. Associated with the suppression of photosynthesis and the accumulation of soluble sugars was a reduction in the amount of transcript for genes encoding photosynthetic proteins (cab and rbcS). In contrast, leaves that developed at 5°C showed an increase in photosynthesis and control levels of photosynthetic gene expression. This recovery occurred even though leaves that developed at 5°C maintained large pools of soluble sugars. Leaves that developed at 5°C also showed a strong upregulation of the cytosolic pathway for soluble sugar synthesis but not of the chloroplastic pathway for starch synthesis. This was shown at the level of both enzyme activity and the amount of transcript. Thus, development of Arabidopsis leaves at 5°C resulted in metabolic changes that enabled them to produce and accumulate large soluble sugar pools without any associated suppression of photosynthesis or photosynthetic gene expression. These changes were also associated with enhanced freezing tolerance. We suggest that this reprogramming of carbohydrate metabolism associated with development at low temperature is essential to the development of full freezing tolerance and for winter survival of over‐wintering herbaceous annuals.
Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2
Authors: Molina A. , García‐Olmedo F.
Published: August 2003

Purified lipid transfer protein LTP2 from barley applied on tobacco leaves eliminated symptoms caused by infiltration of Pseudomonas syringae pv. tabaci 153. Growth of the pathogen in leaves of transgenic tobacco plants was retarded when compared with non‐transformed controls. The percentage of inoculation points that showed necrotic lesions was greatly reduced in transgenic tobacco 17–38% versus 78%) and the average size of these lesions was 61–81% that of control. The average total lesion area (necrosis and chlorosis) in the transgenic plants was also reduced (38% of control). Arabidopsis thaliana transgenic plants inoculated with P. syringae pv. tomato DC3000 also had lower percentages of necrotic lesions (22–38% versus 76%), a reduced average area for each lesion (53–67% of control), and a smaller total lesion area per inoculation (43% of control). These results further support the assignment of a defense role for LTPs and highlight their biotechnological potential.
The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes
Authors: Aarts Mark G.M. , Hodge Rachel , Kalantidis Kriton , Florack Dion , Wilson Zoe A. , Mulligan Bernard J. , Stiekema Willem J. , Scott Rod , Pereira Andy
Published: August 2003

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild‐type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter‐GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.
Light‐dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings
Authors: von Lintig Johannes , Welsch Ralf , Bonk Michael , Giuliano Giovanni , Batschauer Alfred , Kleinig Hans
Published: August 2003

In chloroplasts, carotenoids are essential pigments involved in photosynthesis. During photomorphogenesis, a coordinated increase in the amounts of chlorophylls and carotenoids, in conjugation with other components, leads to the formation of a functional photosynthetic apparatus. To investigate the regulation of carotenoid biosynthesis during this process at the molecular level, GGPS, PSY and PDS cDNAs have been cloned from white mustard (Sinapis alba L.). GGPS encodes a key enzyme in plastid isoprenoid metabolism, while the products of PSY and PDS catalyse the subsequent steps in carotenoid biosynthesis. Due to the low mRNA levels of the genes involved, the use of a RT‐PCR protocol was necessary to measure gene expression during photomorphogenesis. With light, there is an up‐regulation of PSY expression, the first gene within the carotenoid biosynthetic pathway, while PDS and GGPS expression levels remain constant. Treatment with different light qualities reveals a phytochrome‐mediated regulation of PSY expression in developing white mustard seedlings. To obtain more detailed information on the light‐regulation, Arabidopsis thaliana wild‐type and phytochrome mutants were utilized. Continuous far‐red and red light both increase the expression of PSY in wild‐type seedlings, demonstrating that both light‐labile and light‐stable phytochromes are involved in PSY regulation. The response to far‐red light is completely abolished in the phyA mutant, showing that PHYA mediates the increase in PSY transcript levels under these light conditions. In the phyB mutant, the red light response is normal, indicating that PSY expression is not controlled by PHYB but by other light‐stable phytochromes. Measurement of chlorophylls and carotenoids under the same light regimes shows that the up‐regulation of PSY expression does not necessarily result in an increase of the carotenoid content. Only those light conditions which allow chlorophyll biosynthesis lead to a significant increase of the carotenoid content. Therefore, it is proposed that up‐regulation of PSY mRNA levels leads to an increased capacity for the formation of carotenoids. However, this only takes place under light conditions leading to protochlorophyllide photoconversion.
Developmental and auxin‐induced expression of the Arabidopsis prha homeobox gene
Authors: Plesch Gunnar , Störmann Katja , Torres Jorge Tovar , Walden Richard , Somssich Imre E.
Published: August 2003

A single‐copy Arabidopsis homeobox gene, prha, which encodes a homeodomain protein with a molecular weight of 90 500 has been characterized. The position of the gene was mapped to the distal part of chromosome 4. Expression of the gene differs in various vegetative and floral plant tissues and is positively influenced by the phytohormone auxin. In Arabidopsis plants, a complex pattern of prha promoter‐driven GUS expression is observed, often associated with regions of developing vascular tissue. Exogenously applied auxin strongly increased endogenous prha transcript levels. In addition, the prha promoter is highly responsive to the synthetic auxin, naphthalene acetic acid, in transient assays using tobacco protoplasts. The PRHA protein has the capacity to bind to TAATTG core sequence elements but requires additional adjacent bases for high‐affinity binding. These findings are discussed in relation to studies of other plant homeobox genes as well as possible in vivo target genes for PRHA.
Overexpression of light‐dependent PORA or PORB in plants depleted of endogenous POR by far‐red light enhances seedling survival in white light and protects against photooxidative damage
Authors: Sperling Ulrich , van Cleve Barbara , Frick Geneviève , Apel Klaus , Armstrong Gregory A.
Published: August 2003

The structurally related light‐dependent protochlorophyllide (Pchlide) oxidoreductases PORA and PORB mediate the only light‐requiring step in chlorophyll (Chl) biosynthesis in higher plants. Correlative evidence suggests that some in vivo functions of PORA and PORB may be unique, including a postulated photoprotective role for PORA. For example, wild‐type Arabidopsis thaliana seedlings grown in non‐photooxidative far‐red light (cFR) resemble those grown in white light (WL), but they are yellow and do not green normally thereafter in WL. This defect is accompanied by the absence of detectable PORA and reduced levels of PORB expression. Here, direct evidence is provided that the presence of POR, either as PORA or PORB, can confer photoprotection in plants. In contrast to the wild‐type, the plastids of transgenic PORA‐ or PORB‐overexpressing Arabidopsis seedlings grown in cFR possess extensive prolamellar bodies. Upon a subsequent shift to WL, POR‐overexpressing seedlings develop thylakoid membranes, accumulate large amounts of Chl and are viable at fluence rates lethal to the wild‐type. Intriguingly, the plastid membrane architectures of greening transgenic seedlings seem to depend on whether PORA or PORB has been over‐produced. POR‐overexpressing seedlings shifted from cFR to WL of fluence rates from 20 to 500 μE m−2 sec−1 accumulate substantially higher amounts of Chl than does the wild‐type. Furthermore, the WL fluence rate that permits maximal Chl accumulation increases from 8 μE m−2 sec−1 in the wild‐type to 125 μE m−2 sec−1 in transgenic seedlings. POR Overexpression during growth in cFR also correlates with a fourfold decrease in the steady‐state content of Pchlide, a potentially lethal photosensitizer.
The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very‐low‐fluence and high‐irradiance responses
Authors: Yanovsky Marcelo J. , Casal Jorge J. , Luppi Juan P.
Published: August 2003

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very‐low‐fluence responses (VLFR), low‐fluence responses (LFR) and high‐irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a poly‐morphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far‐red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by mapmaker/QTL. Two quantitative trait loci, one on chromosome 2 (VLF1) and another on chromosome 5 (VLF2), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.
Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2
Authors: Molina A. , García‐Olmedo F.
Published: August 2003

Purified lipid transfer protein LTP2 from barley applied on tobacco leaves eliminated symptoms caused by infiltration of Pseudomonas syringae pv. tabaci 153. Growth of the pathogen in leaves of transgenic tobacco plants was retarded when compared with non‐transformed controls. The percentage of inoculation points that showed necrotic lesions was greatly reduced in transgenic tobacco (17–38% versus 78%) and the average size of these lesions was 61–81% that of control. The average total lesion area (necrosis and chlorosis) in the transgenic plants was also reduced (38% of control). Arabidopsis thaliana transgenic plants inoculated with P. syringae pv. tomato DC3000 also had lower percentages of necrotic lesions (22–38% versus 76%), a reduced average area for each lesion (53–67% of control), and a smaller total lesion area per inoculation (43% of control). These results further support the assignment of a defense role for LTPs and highlight their biotechnological potential.
Cloning and characterization of LRG5, a gene involved in blue light signaling in Chlamydomonas gametogenesis
Authors: Glöckner Gernot , Beck Christoph F.
Published: August 2003

The Chlamydomonas reinhardtii mutant lsrg2 exhibits a partial defect in the blue‐light activated signal transduction chain that controls gametogenesis. By genomic complementation, a cosmid clone that corrects the Irg2 phenotype was isolated. The smallest fragment of this cosmid that gave complementation was used in RFLP analysis, which revealed that the cloned gene was not linked to the LRG2 locus. The sequence of the genomic clone and a cDNA of this gene, which we named LRG5, was determined. Although no significant homology with sequences in the databases was found, the nuclear localization signal and the presence of negatively and positively charged domains suggests a function for the LRG5 protein in the regulation of transcription. The correction of the Irg2 phenotype is probably caused by overexpression of the LRG5 gene in the transformants, as these expressed a level of LRG5 approximately twofold higher than wild‐type or mutant strains. Southern blot analysis provided evidence for the existence of homologous sequences in other green algae as well as in higher plants, suggesting a conserved function for the LRG5 protein.
Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants
Authors: Grebenok Robert J. , Lambert Georgina M. , Galbraith David W.
Published: August 2003

The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post‐translationally targeted to the nucleus via energy‐dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying ‘classical’ NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications.

The green‐fluorescent protein (GFP) of Aequorea victoria has recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described. This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.
A negative selection scheme based on the expression of cytosine deaminase in plastids
Authors: Serino German , Maliga Pal
Published: August 2003

The enzyme cytosine deaminase (CD) encoded by codA catalyzes deamination of cytosine to uracil. CD is present in prokaryotes and in many eukaryotic micro‐organisms, but is absent in higher plants. 5‐fluorocytosine (5FC) is metabolized in CD‐expressing cells, causing cellular death. A chimeric codA has been introduced into the tobacco plastid genome and 5FC was used to select against tissue culture cells and seedlings expressing CD. This negative selection scheme will be useful in identifying nuclear genes which control plastid gene expression in higher plants.
Construction of an ∼2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2
Authors: Wang Ming Li , Huang Lu , Bongard‐Pierce Deverie K. , Belmonte Stephen , Zachgo Eve Ann , Morris John W. , Dolan Maureen , Goodman Howard M.
Published: August 2003

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.
Heat sensitivity of human parvovirus B19
Authors: Yunoki M. , Tsujikawa M. , Urayama T. , Sasaki Y. , Morita M. , Tanaka H. , Hattori S. , Takechi K. , Ikuta K.
Published: August 2003

Background and Objectives To date there has been no published report on a systematic evaluation of the heat sensitivity of human parvovirus B19 (B19) and the related safety of the plasma‐derived fractionated products. In this study, we examined the heat sensitivity of B19 by using the infectivity assay with cultured cells.

Materials and Methods The heat sensitivity of B19 was examined by measuring the reduction in viral infectivity titres after heating liquid containing B19 at 60 °C. Viral infectivity was assayed by detection of viral antigens or viral mRNA in infected cells. As a control, canine parvovirus (CPV) was also heat‐treated.

Results B19 displayed quite different inactivation kinetics to CPV when both were heated in liquid at 60 °C. In sharp contrast to the latter, B19 was rapidly inactivated within 1 h when the virus was suspended in 5% or 25% human serum albumin solution, phosphate‐buffered saline, or complete medium. However, B19 appeared to be resistant to heat inactivation in liquid containing 60% sucrose.

Conclusions The heat sensitivity of B19 in liquid was clearly different from that of CPV. Significantly, the efficiency to inactivate B19 and reduce its infectivity following heating in liquid was mainly affected by the composition of the solutions used for virus suspension.