bluuebunny/crossref_metadata_2025 huggingface dataset:
The Middle Asian Element in the Southern Rocky Mountain Flora of the western United States: a critical biogeographical review
Authors: Weber William A.
Published: May 2003

Aim Presentation of an hypothesis suggesting that the extraordinarily similarity of the Russian Altai and the American Southern Rocky Mountain Flora represents an Oroboreal Flora; that had to have had an essential continuity across the northern part of the world in the Tertiary period, constituting a highland and steppe component of the better‐known Arcto‐Tertiary Flora of eastern and far‐western North America and eastern Asia.

Location North America and Middle (Altai) Asia.

Methods Summarization of the author's field and herbarium studies of whole floras over a period of over 60 years, consisting of successive specializations in vascular plants, lichens, and bryophytes.

Main conclusions (1) The modern alpine and associated marginal steppe and montane floras contain taxa of Tertiary age. (2) The floras of the southern mountains antedate those of the present‐day Arctic. (3) The Middle Asiatic and the North American floras once enjoyed a contiguous existence over a broad area involving connections between North America and Asia across the North Pole by way of Greenland. Their present disjunctions are products of extinction and attrition of ranges, not of long‐distance migration or dispersal mechanisms. (4) North‐eastern North American disjunctions of so‐called Cordilleran species (the Nunatak hypothesis) need not require explanations involving long‐distance dispersal or migration, but represent relictual populations of the once widely distributed Oroboreal flora.
Trophic diversity of the otter (Lutra lutraL.) in temperate and Mediterranean freshwater habitats
Authors: Clavero Miguel , Prenda José , Delibes Miguel
Published: May 2003

AimTo analyse the geographical patterns in the composition and diversity of otter's (Lutra lutraL.) diet and their relationship with climatic characteristics.

LocationEuropean freshwater habitats under Mediterranean and temperate climatic regimes.

MethodsThirty‐seven otter diet studies were reviewed, twenty‐one from temperate and sixteen from Mediterranean areas. All studies were based on spraint analysis and their results expressed as relative frequency of occurrence of seven main prey categories. Principal Component Analysis was performed to extract the main gradients of diet composition. Pearson's correlation andt‐tests were used to assess the relationship between diet characteristics (composition, diversity and taxonomic richness) and geographical and climatic variables.

ResultsA clear latitudinal gradient in diet composition was observed. Otter diet was more diverse and featured more prey classes in southern localities, while the species was more piscivorous towards the north, where it predated upon a higher number of fish families. This pattern was similar when temperate and Mediterranean localities of Europe were compared. Mediterranean otters behaved as more generalist predators than temperate ones, relying less on fish, and more on aquatic invertebrates and reptiles.

Main conclusionsGeographical differences in otter feeding ecology in Europe seem to be related with the two contrasted climatic conditions affecting prey populations. The otter can act as a highly specialized piscivorous predator in temperate freshwater ecosystems, which do not suffer a dry season and have a comparatively stable water regime compared to Mediterranean ones. However, the unpredictable prey availability in Mediterranean areas, affected by strong spatial and temporal water shortages, favours a diversification of the otter's diet.
Understanding Late Quaternary extinctions: the case of Myotragus balearicus (Bate, 1909)
Authors: Bover Pere , Antoni Alcover Josep
Published: May 2003

Aim  In this study we present a new view on the extinction of Myotragus balearicus, an extinct highly modified dwarf caprine from the Gymnesic Islands (or eastern Balearic Islands), as a methodological case study for interpretation of Late Quaternary extinctions (LQEs).

Methods  We analyse all available 14C ages obtained from M. balearicus bones from the uppermost part of the Pleistocene and the Holocene, together with the available chronological data of the putative causes of Myotragus extinction.

Results  It has been possible to define two critical dates that allow us to establish an ‘uncertainty period for the Myotragus extinction’ (UPME) in each analysed island (Mallorca, Menorca and Cabrera). For Mallorca, the UPME corresponds to the interval c. 3700 to 2030 calbc (i.e. c. 1670 years of uncertainty). In the case of Menorca, the UPME spans from 10,000 to 1930 calbc (8070 years of uncertainity). In Cabrera the UPME is placed between 3650 and 300 calbc (3350 years of uncertainty). These periods, together with a review of the available information on the chronology of human arrival and the chronology of Holocene climatic change, shed light on the possible causes of the extinction of this species.

Main conclusions  Extinction of Myotragus because of climatic change can be definitively rejected. The Myotragus extinction must be attributed to the rapid effects of the first human occupation. The use of uncertainity periods for the disappearance of species represents a useful tool for the analysis of LQEs.
Phylogeography of the giant harlequin beetle (Acrocinus longimanus)
Authors: Zeh David W. , Zeh Jeanne A. , Bonilla Melvin M.
Published: May 2003

Aim The extent to which cryptic species contribute to neotropical diversity remains inadequately investigated. Based on its highly distinctive morphology, the giant harlequin beetle, Acrocinus longimanus, is currently described as a single species, ranging from southern Mexico to northern Argentina. However, the discovery of cryptic species in Cordylochernes scorpioides, a pseudoscorpion with obligate dependence on the harlequin beetle for dispersal, strongly suggests the existence of barriers to gene flow in A. longimanus. The aim of this study was therefore to determine whether levels of DNA divergence between geographical populations provided evidence of genetically distinct lineages in the harlequin beetle.

Location Trinidad and Panamá.

Methods Sequencing of 1245 bp of the mitochondrial cytochrome oxidase I (COI) gene of A. longimanus from seven locations in Trinidad and Panamá.

Results Mitochondrial haplotype diversity in the harlequin beetle shows limited evidence of geographical structuring, with a maximum sequence divergence between populations of only 1.29%. This is an order of magnitude less than the level of COI divergence between harlequin beetle riding pseudoscorpions from the same geographical locations.

Main conclusions The molecular data on populations from northern South America and Panamá are consistent with the current, morphologically based classification of A. longimanus as a single, pan‐neotropical species. In addition, the relatively low level of population divergence detected in this study indicates that speciation in the hitchhiking pseudoscorpion has occurred in the absence of significant barriers to gene flow in its beetle host. It is proposed that, in the harlequin beetle, the phylogenetic signal of colonization and vicariance associated with the formation of the Isthmus of Panamá has been obscured, although not fully erased, by historical and contemporary gene flow.
Variable photosynthetic acclimation in consecutive cohorts of Scots pine needles during 3 years of growth at elevated CO2 and elevated temperature
Authors: LUOMALA E.‐M. , LAITINEN K. , KELLOMÄKI S. , VAPAAVUORI E.
Published: May 2003

In this experiment, the photosynthetic acclimation of successive needle cohorts of Scots pine were studied during 3 years of growth at elevated CO2 and temperature. Naturally regenerated Scots pine (Pinus sylvestris L.) trees were subjected to elevated CO2 concentration (+CO2, 700 p.p.m), elevated temperature (+T, ambient +2 to +6 °C) and to a combination of elevated CO2 and temperature (+CO2 + T) in closed‐top chambers, starting in August 1996. Trees growing in chambers with ambient CO2 and ambient temperature served as controls (AmbC). Elevated CO2 influenced the dark reactions more than the light reactions of photosynthesis, as in the 1996 and 1997 cohorts the carboxylation capacity of Rubisco was reduced in the first and second year of exposure, but there was no consistent change in chlorophyll fluorescence. Net photosynthesis measured at growth concentration of CO2 was higher at +CO2 than at AmbC on only one measuring occasion, was generally lower at +T and was not changed at +CO2 + T. However, trees grown at +T tended to invest more nitrogen (N) in Rubisco, as Rubisco/chlorophyll and the proportion of the total needle N bound to Rubisco occasionally increased. The interaction of +CO2 and +T on Rubisco was mostly negative; consequently, in the second and third year of the experiment the carboxylation capacity decreased at +CO2 + T. In the 1996, 1997 and 1998 cohorts, the structural N concentration of needles was lower at +CO2 than at AmbC. Elevated CO2 and elevated temperature generally had a positive interaction on N concentration; consequently, N concentration in needles decreased less at +CO2 + T than at +CO2. At +CO2 + T, the acclimation response of needles varied between years and was more pronounced in the 1‐year‐old needles of the 1997 cohort than in those of the 1998 cohort. Thus, acclimation was not always greater in 1‐year‐old needles than in current‐year needles. In the +CO2 + T treatment, elevated temperature had a greater effect on acclimation of needles than elevated CO2.
Calcium requirement for ethylene‐dependent responses involving 1‐aminocyclopropane‐1‐carboxylic acid oxidase in radicle tissues of germinated pea seeds*
Authors: PETRUZZELLI L. , STURARO M. , MAINIERI D. , LEUBNER‐METZGER G.
Published: May 2003

The Ca2+ requirements of ethylene‐dependent responses were investigated in germinating seeds of Pisum sativum L. using 1‐aminocyclopropane‐1‐carboxylic acid (ACC) oxidase (Ps‐ACO1), ACC synthase (Ps‐ACS2) and class I β‐1,3‐glucanase as molecular markers. Ethylene biosynthesis and responsiveness are localized to the elongation and differentiation zones of the pea radicle. Ethylene treatment induced ectopic root hair formation in the cell elongation zone and promoted root hair elongation growth in the radicles of germinated seeds. Characterized Ca2+ antagonists, including EGTA, lanthanum, verapamil, ruthenium red, W‐7, lithium and neomycin, were used to test for the involvement of the apoplastic and the intracellular Ca2+‐pool, the Ca2+/calmodulin complex and the phoshoinositide (PI) cycle in the ethylene responses. Ca2+ release from internal pools, but no appreciabe apoplastic Ca2+, is involved in the transcriptional induction by ethylene of Ps‐ACO1 and in ectopic root hair formation in the radicle elongation zone of germinated pea seeds. Furthermore, the Ca2+/calmodulin complex and the PI cycle seem to be involved in these ethylene responses. In contrast, both the intracellular and the apoplastic Ca2+‐pools are required for the negative and positive ethylene responses to the gene expression of PS‐ACS2 and class I β‐1,3‐glucanase, respectively; and, apoplastic Ca2+ also promotes root hair elongation growth. Tissues from adult plants and germinating seeds exhibit temporal and spatial differences in the signal/response coupling by Ca2+ of ethylene‐regulated processes.
Two tomato non‐symbiotic haemoglobin genes are differentially expressed in response to diverse changes in mineral nutrient status
Authors: WANG Y.‐H. , KOCHIAN L. V. , DOYLE J. J. , GARVIN D. F.
Published: May 2003

The role of plant non‐symbiotic haemoglobins remains undefined, but recent findings suggest a possible role in plant nitrate nutrition. This study sought to characterize patterns of gene expression for two tomato (Lycopersicon esculentum L.) non‐symbiotic haemoglobin genes, and to examine how diverse changes in nutrient status influences the expression of these two genes. The deduced amino acid sequences of the genes examined, SOLly GLB1 and SOLly GLB2, are 55% identical. SOLly GLB1 mRNA is present in highest abundance in roots and older stems, whereas SOLly GLB2 mRNA abundance is highest in leaves, but detectable in other tissues including roots. The SOLly GLB1 mRNA levels increased rapidly in roots in response to each nutrient treatment examined in hydroponic culture, including the individual removal of phosphate, potassium and iron from the culture medium, as well as by the addition of nitrate. In contrast, the levels of SOLly GLB2 mRNA were not significantly altered in response to these same treatments. These results are the first report that non‐symbiotic haemoglobin gene expression can be influenced by a broad range of changes both in mineral macronutrient and micronutrient status, suggesting a previously unrecognized generic role for non‐symbiotic haemoglobins in processes associated with mineral nutrient nutrition.
The auxin responsive gene Pp‐C61 is up‐regulated in Pinus pinaster roots following inoculation with ectomycorrhizal fungi
Authors: REDDY S. M. , PANDEY A. K. , MELAYAH D. , MARMEISSE R. , GAY G.
Published: May 2003

An indoleacetic acid (IAA) up‐regulated cDNA, referred to as Pp‐C61, was isolated by differential screening of a cDNA library constructed from auxin‐treated roots of Pinus pinaster. The Pp‐C61 cDNA contains a 1068 bp open reading frame which encodes a polypeptide sharing no homology with known proteins. The N‐terminal protein region contains a hydrophobic signal peptide suggesting that Pp‐C61 is an extracellular protein. Pp‐C61 is present as a single copy in the P. pinaster genome and homologous genes were detected in other gymnosperm and angiosperm trees. Pp‐C61 was transcriptionally regulated by auxin with a maximal mRNA accumulation 6 h after treatment. Pp‐C61 was up‐regulated by IAA concentrations as low as 10− 10m. Up‐regulation was found to be a primary (direct) response to auxin. Ethylene and abscisic acid, but not quercetin were able to promote Pp‐C61 mRNA accumulation. As a putative extracellular protein, Pp‐C61 was postulated to play a role in ectomycorrhiza formation. Pp‐C61 was up‐regulated in P. pinaster roots following inoculation with ectomycorrhizal fungi. Transcript accumulation was the highest (5.5‐fold over control) in the presence of an IAA‐overproducing mutant of Hebeloma cylindrosporum; it was less in the presence of wild‐type strains of H. cylindrosporum and Rhizopogon roseolus. Pp‐C61 transcript also accumulated upon inoculation with a non‐mycorrhizal mutant of H. cylindrosporum. These results suggest that Pp‐C61 up‐regulation corresponds to a defence reaction in response to fungal colonization.
Protein turnover in relation to maintenance metabolism at low photon flux in two marine microalgae
Authors: QUIGG A. , BEARDALL J.
Published: May 2003

Acclimation to very low photon fluxes involves adjusting a suite of physiological characteristics that collectively elicit a physiological response. Facilitating such changes is pro‐tein turnover. Dunaliella tertiolecta (Butcher) and Phaeodactylum tricornutum (Bohlin) were grown in turbidostats at a range of photon fluxes between 2 and 300 µmol photons m−2 s−1. The kinetics of pulse‐chase labelling of the protein with 3H showed that (1) two protein pools were present, one of which turned‐over rapidly (hours), and a second which turned over more slowly (days); and (2) protein turnover rates were slower in P. tricornutum than in D. tertiolecta. Phaeodactylum tricornutum had a lower maintenance coefficient for protein turnover than D. tertiolecta, and correspondingly a smaller proportion of its respiratory demands (30%) were associated with protein turnover than in D. tertiolecta (36%). There appears to be a correlation between lower metabolic activity, requiring lower protein concentrations, and an associated decreased cost of maintenance processes in P. tricornutum compared to D. tertiolecta. Differences between protein turnover rates and maintenance metabolic costs may be one of the photo‐acclimation strategies that determine which photon niches microalgae can successfully exploit.
Is stimulation of leaf photosynthesis by elevated carbon dioxide concentration maintained in the long term? A test with Lolium perenne grown for 10 years at two nitrogen fertilization levels under Free Air CO2Enrichment (FACE)
Authors: AINSWORTH E. A. , DAVEY P. A. , HYMUS G. J. , OSBORNE C. P. , ROGERS A. , BLUM H. , NÖSBERGER J. , LONG S. P.
Published: May 2003

Photosynthesis is commonly stimulated in grasslands with experimental increases in atmospheric CO2 concentration ([CO2]), a physiological response that could significantly alter the future carbon cycle if it persists in the long term. Yet an acclimation of photosynthetic capacity suggested by theoretical models and short‐term experiments could completely remove this effect of CO2. Perennial ryegrass (Lolium perenne L. cv. Bastion) was grown under an elevated [CO2] of 600 µmol mol−1 for 10 years using Free Air CO2Enrichment (FACE), with two contrasting nitrogen levels and abrupt changes in the source : sink ratio following periodic harvests. More than 3000 measurements characterized the response of leaf photosynthesis and stomatal conductance to elevated [CO2] across each growing season for the duration of the experiment. Over the 10 years as a whole, growth at elevated [CO2] resulted in a 43% higher rate of light‐saturated leaf photosynthesis and a 36% increase in daily integral of leaf CO2 uptake. Photosynthetic stimulation was maintained despite a 30% decrease in stomatal conductance and significant decreases in both the apparent, maximum carboxylation velocity (Vc,max) and the maximum rate of electron transport (Jmax). Immediately prior to the periodic (every 4–8 weeks) cuts of the L. perenne stands, Vc,max and Jmax, were significantly lower in elevated than in ambient [CO2] in the low‐nitrogen treatment. This difference was smaller after the cut, suggesting a dependence upon the balance between the sources and sinks for carbon. In contrast with theoretical expectations and the results of shorter duration experiments, the present results provide no significant change in photosynthetic stimulation across a 10‐year period, nor greater acclimation in Vc,max and Jmax in the later years in either nitrogen treatment.
Photoprotective role of rhodoxanthin during cold acclimation in Cryptomeria japonica
Authors: HAN Q. , SHINOHARA K. , KAKUBARI Y. , MUKAI Y.
Published: May 2003

To examine the role of rhodoxanthin in long‐term acclimation to low temperatures, we monitored seasonal changes in pigment composition, photosynthesis, chlorophyll fluorescence and the level of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in needles of wild‐type and mutant forms of Cryptomeria japonica. In winter, rhodoxanthin accumulated in sun‐exposed needles of wild‐type plants, but not in those of the mutant. The level of chlorophyll decreased in both types of plant in winter. In contrast, the level of the xanthophyll cycle pool increased in both cases. The level of the pool in the mutant was twice that in the wild type in winter, on a Chl basis, even though the levels in both were similar in summer. The synthesis of rhodoxanthin might be triggered by photo‐inhibitory conditions, as suggested by the sustained elevated levels of zeaxanthin (Z) and antheraxanthin (A). In the wild type and the mutant, the quantum yield of CO2 fixation (φ), the photosynthetic capacity, the photochemical efficiency of photosystem II (PSII), the photochemical quenching and the level of Rubisco in summer were similar. However, all these values for the wild type were higher than those for the mutant in winter. The non‐photochemical quenching (NPQ) in the mutant in winter increased rapidly even under low light conditions due to the high sustained levels of Z and A. In contrast, in the wild type, the conversion of Z via A to rhodoxanthin prevented the rapid increase in NPQ to maintain the relatively high level of φ. These findings suggest that rhodoxanthin might play an important photoprotective role in long‐term acclimation to cold. The dynamic regulation of the amount of rhodoxanthin relative to the level of the xanthophyll cycle pool might act to maintain an appropriate balance between light absorption, photosynthesis and the thermal dissipation of energy due to excess absorbed light in winter.
Growth and water relations of Tamarix ramosissima and Populus euphratica on Taklamakan desert dunes in relation to depth to a permanent water table
Authors: GRIES D. , ZENG F. , FOETZKI A. , ARNDT S. K. , BRUELHEIDE H. , THOMAS F. M. , ZHANG X. , RUNGE M.
Published: May 2003

The hypothesis that water relations and growth of phreatophytic Tamarix ramosissima Ledeb. and Populus euphratica Oliv. on dunes of varying height in an extremely arid Chinese desert depend on vertical distance to a permanent water table was tested. Shoot diameter growth of P. euphratica was inversely correlated with groundwater depth (GD) of 7 to 23 m (adj. R2 = 0.69, P = 0.025); growth of T. ramosissima varied independent of GD between 5 and 24 m (P = 0.385). Pre‐dawn (pd) and midday (md) water potentials were lower in T. ramosissima (minimum pd −1.25 MPa, md −3.6 MPa at 24 m GD) than in P. euphratica (minimum pd −0.9 MPa, md −3.05 MPa at 23 m GD) and did not indicate physiologically significant drought stress for either species. Midday water potentials of P. euphratica closely corresponded to GD throughout the growing season, but those of T. ramosissima did not. In both species, stomatal conductance was significantly correlated with leaf water potential (P. euphratica: adj. R2 = 0.84, P < 0.0001; T. ramosissima: adj. R2 = 0.64, P = 0.011) and with leaf‐specific hydraulic conductance (P. euphratica: adj. R2 = 0.79, P = 0.001; T. ramosissima: adj. R2 = 0.56, P = 0.019); the three variables decreased with increasing GD in P. euphratica. Stomatal conductance of P. euphratica was more strongly reduced (> 50% between −2 and −3 MPa) in response to decreasing leaf water potential than that of T. ramosissima (30% between −2 and −3 MPa). Tolerance of lower leaf water potentials due to higher concentrations of leaf osmotically active substances partially explains why leaf conductance, and probably leaf carbon gain and growth, of T. ramosissima was less severely affected by GD. Additionally, the complex below‐ground structure of large clonal T. ramosissima shrub systems probably introduces variability into the assumed relationship of xylem path length with GD.
Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms
Authors: WANG X.‐J. , LOH C.‐S. , YEOH H.‐H. , SUN W. Q.
Published: May 2003

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L−1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ∼25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.
Changes in mesophyll anatomy and sink–source relationships during leaf development in Quercus glauca, an evergreen tree showing delayed leaf greening
Authors: MIYAZAWA S.‐I. , MAKINO A. , TERASHIMA I.
Published: May 2003

Changes in mesophyll anatomy, gas exchange, and the amounts of nitrogen and cell wall constituents including cellulose, hemicellulose and lignin during leaf development were studied in an evergreen broad‐leaved tree, Quercus glauca, and in an annual herb, Phaseolus vulgaris. The number of chloroplasts per whole leaf in P. vulgaris increased and attained the maximal level around 10 d before full leaf area expansion (FLE), whereas it continued to increase even after FLE in Q. glauca. The increase in the number of palisade tissue cells per whole leaf continued until a few days before FLE in Q. glauca, but it had almost ceased by 10 d before FLE in P. vulgaris. The radius and height of palisade tissue cells in Q. glauca, attained their maximal levels at around FLE whereas the thickness of the mesophyll cell wall and concentrations of the cell wall constituents increased markedly after FLE. These results clearly indicated that, in Q. glauca, chloroplast development proceeded in parallel with the cell wall thickening well after completion of the mesophyll cell division and cell enlargement. The sink–source transition, defined to be the time when the increase in daily carbon exchange rate exceeds the daily increase in leaf carbon content, occurred before FLE in P. vulgaris but after FLE in Q. glauca. During leaf area expansion, the maximum daily increase in nitrogen content on a whole leaf basis (the maximum leaf areas were corrected to be identical for these species) in Q. glauca was similar to that in P. vulgaris. In Q. glauca, however, more than 70% of nitrogen in the mature leaf was invested during its sink phase, whereas in P. vulgaris it was 50%. These results suggest that Q. glauca invests nitrogen for cell division for a considerable period and for chloroplast development during the later stages. We conclude that the competition for nitrogen between cell division and chloroplast development in the area of expanding leaves can explain different greening patterns among plant species.
Gene expression associated with N‐induced shifts in resource allocation in poplar
Authors: COOKE J. E. K. , BROWN K. A. , WU R. , DAVIS J. M.
Published: May 2003

Surprisingly little is known about molecular mechanisms by which nitrogen (N) availability acts to modulate the growth of forest trees. To address this issue, differential display was used in conjunction with filter‐based arrays to identify 52 partial cDNA clones that were significantly regulated within days in response to limiting or luxuriant levels of NH4NO3 fertilization in Populus trichocarpa Torr. & Gray × deltoides Bartr. ex Marsh. A subset of these cDNAs also demonstrated shifts in expression patterns in stem‐girdled trees, a manipulative physiology technique that disrupts phloem transport. Stem girdling also induced changes in glutamine and asparagine pools which were correlated with the observed changes in expression profiles for these genes. The identity of these genes provides insight into biochemical processes that are altered by N availability in poplar. Carbon–nitrogen interactions appear to figure prominently in the N‐response. The gene expression data suggest that N availability modulates the partitioning of C and N resources into metabolic fates that have the potential to alter both wood quality and quantity, including synthesis of vegetative storage proteins, cell wall components, and terpenoids.
Growth and physiological responses of cotton (Gossypium hirsutumL.) to elevated carbon dioxide and ultraviolet‐B radiation under controlled environmental conditions
Authors: ZHAO D. , REDDY K. R. , KAKANI V. G. , READ J. J. , SULLIVAN J. H.
Published: May 2003

Better understanding of crop responses to projected changes in climate is an important requirement. An experiment was conducted in sunlit, controlled environment chambers known as soil–plant–atmosphere–research units to determine the interactive effects of atmospheric carbon dioxide concentration [CO2] and ultraviolet‐B (UV‐B) radiation on cotton (Gossypium hirsutumL.) growth, development and leaf photosynthetic characteristics. Six treatments were used, comprising two levels of [CO2] (360 and 720 µmol mol−1) and three levels of 0 (control), 7.7 and 15.1 kJ m−2 d−1biologically effective UV‐B radiations within each CO2level. Treatments were imposed for 66 d from emergence until 3 weeks after the first flower stage. Plants grown in elevated [CO2] had greater leaf area and higher leaf photosynthesis, non‐structural carbohydrates, and total biomass than plants in ambient [CO2]. Neither dry matter partitioning among plant organs nor pigment concentrations was affected by elevated [CO2]. On the other hand, high UV‐B (15.1 kJ m−2 d−1) radiation treatment altered growth resulting in shorter stem and branch lengths and smaller leaf area. Shorter plants at high UV‐B radiation were related to internode lengths rather than the number of mainstem nodes. Fruit dry matter accumulation was most sensitive to UV‐B radiation due to fruit abscission. Even under 7.7 kJ m−2 d−1of UV‐B radiation, fruit dry weight was significantly lower than the control although total biomass and leaf photosynthesis did not differ from the control. The UV‐B radiation of 15.1 kJ m−2 d−1reduced both total (43%) and fruit (88%) dry weights due to smaller leaf area and lower leaf net photosynthesis. Elevated [CO2] did not ameliorate the adverse effects of UV‐B radiation on cotton growth and physiology, particularly the boll retention under UV‐B stress.
Enhancement of virus‐induced gene silencing through viral‐based production of inverted‐repeats
Authors: Lacomme Christophe , Hrubikova Katarina , Ingo Hein
Published: May 2003

Plant virus‐based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology‐dependent RNA degradation mechanism. This phenomenon, called virus‐induced gene silencing (VIGS), has potential as a powerful reverse‐genetics tool in functional genomic programmes through transient, loss‐of‐function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40–60 base direct inverted‐repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)‐based vector carrying such inverted‐repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real‐time RT‐PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted‐repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted‐repeats in plant virus‐based vectors, generating a more robust loss‐of‐function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.
The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress
Authors: Boyce Joy M. , Knight Heather , Deyholos Michael , Openshaw Mark R. , Galbraith David W. , Warren Gareth , Knight Marc R.
Published: May 2003

The sfr6 mutant of Arabidopsis displays a deficit in freezing tolerance after cold acclimation. We previously observed that the transcripts of three cold‐, ABA‐ and drought‐inducible genes, each having a C‐repeat motif or the drought‐responsive element (CRT/DRE) in its promoter, failed to normally accumulate in this mutant. We now report that the effects of sfr6 upon transcript levels are reflected in the levels of the encoded proteins, confirming that the cold‐inducible protein expression is affected by the sfr6 mutation. Using microarray analysis, we found not only that this effect may be general to cold‐inducible genes with CRT/DRE promoter elements, but also that it extends to some other genes whose promoters lack a CRT/DRE element. The role of the CRT/DRE has been empirically tested by use of a synthetic promoter, confirming that the CRT/DRE is sufficient to confer the sfr6 effect upon expression. Tolerance of osmotic stress was also found to be reduced in sfr6, consistent with a role in osmotic stress tolerance for the cold‐, ABA‐ and drought‐inducible genes whose expression is affected by the sfr6 mutation.
Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male‐sterile Kosena radish
Authors: Koizuka Nobuya , Imai Ritsuko , Fujimoto Hideya , Hayakawa Takahiko , Kimura Yusuke , Kohno‐Murase Junko , Sakai Takako , Kawasaki Shinji , Imamura Jun
Published: May 2003

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7‐kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35‐amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS‐associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR‐containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.
Ku80‐ and DNA ligase IV‐deficient plants are sensitive to ionizing radiation and defective in T‐DNA integration
Authors: Friesner Joanna , Britt Anne B.
Published: May 2003

Double‐strand break (DSB) repair pathways catalyze the rejoining of broken chromosomes and the integration of transforming DNAs. These processes have been well characterized in bacteria, fungi, and animals. Plants are generally thought primarily to utilize a non‐homologous end joining (NHEJ) pathway to repair DSBs and integrate transgenes, as transforming DNAs with large tracts of homology to the chromosome are integrated at random. In order to test the hypothesis that NHEJ is an important pathway for the repair of DSBs in plants, we isolated T‐DNA insertion mutations in the Arabidopsis homologs of the Ku80 and DNA ligase IV genes, required for the initiation and completion, respectively, of NHEJ. Both mutants were hypersensitive to the cytostatic effects of gamma radiation, suggesting that NHEJ is indeed a critical pathway for the repair of DSBs. T‐DNA insertion rates were also decreased in the mutants, indicating that Ku80 and DNA ligase IV play an important role in either the mechanism or the regulation of T‐DNA integration in Arabidopsis.
The C‐terminal tail of Arabidopsis 14‐3‐3ω functions as an autoinhibitor and may contain a tenth α‐helix
Authors: Shen Wei , Clark A. Clay , Huber Steven C.
Published: May 2003

The eukaryotic regulatory protein 14‐3‐3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me2+) and some polyamines interact with the loop 8 region of the 14‐3‐3 proteins and allow them to bind and inhibit pNR in vitro. The role of the highly variant C‐terminal regions of the 14‐3‐3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C‐terminal tail of the Arabidopsis 14‐3‐3ω isoform and evaluated its contributions to the inhibition of pNR. Nested C‐terminal truncations of the recombinant 14‐3‐3ω protein revealed that the removal of the C‐terminal tail renders the protein partially Mg2+‐independent in both pNR binding and inhibition of activity, suggesting that the C‐terminus functions as an autoinhibitor. The C‐terminus of 14‐3‐3ω appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys‐247. C‐terminal truncation of 14‐3‐3ω at Thr‐255 increased its interaction with antibodies to the C‐terminus of 14‐3‐3ω in non‐denaturing conditions, but not in denaturing conditions, suggesting that the C‐terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C‐terminal peptide, from Trp‐234 to Lys‐249, revealed that the C‐terminal tail might contain a tenth α‐helix, in agreement with the in silico predictions. The function of the putative tenth α‐helix is not clear because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg2+. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14‐3‐3 protein is restricted by the C‐terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.
Identification and expression of a soybean nodule‐enhanced PEP‐carboxylase kinase gene (NE‐PpcK) that shows striking up‐/down‐regulation in vivo
Authors: Xu Wenxin , Zhou You , Chollet Raymond
Published: May 2003

Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post‐translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC‐kinase (PpcK), a novel, ‘minimal but functional’ Ser/Thr kinase. To date, this plant‐specific kinase has been investigated in molecular terms exclusively in non‐leguminous plants, such as Crassulacean‐acid‐metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up‐regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady‐state abundance of this former, nodule‐enhanced transcript (NE‐PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up‐/down‐regulation of NE‐PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation‐state of PEPC, and the abundance of a previously identified, nodule‐enhanced transcript (GmPEPC7) that encodes the target enzyme (NE‐Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK‐Ppc‘expression‐partners’ in plants, exemplified by NE‐PpcK and NE‐Ppc in the soybean nodule.
Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4‐dichloroaniline
Authors: Loutre Caroline , Dixon David P. , Brazier Melissa , Slater Malcolm , Cole David J. , Edwards Robert
Published: May 2003

The pollutant 3,4‐dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N‐β‐d‐glucopyranosyl‐DCA exported into the medium. The N‐glucosyltransferase (N‐GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5‐trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N‐GT and O‐GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4‐hydroxyphenylpyruvic acid. Both O‐GT and N‐GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.
The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation
Authors: Amor Besma Ben , Shaw Sidney L. , Oldroyd Giles E. D. , Maillet Fabienne , Penmetsa R. Varma , Cook Douglas , Long Sharon R. , Dénarié Jean , Gough Clare
Published: May 2003

Establishment of the Rhizobium–legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation‐defective (Nod−) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor‐activated signal transduction pathway. Characterisation of a new M. truncatula Nod− mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod− mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild‐type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor‐induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild‐type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.
Nanolitre‐scale assays to determine the activities of enzymes in individual plant cells
Authors: Roy Stuart J. , Cuin Tracey A. , Leigh Roger A.
Published: May 2003

There are a variety of methods for characterising gene expression at the level of individual cells and for demonstrating that the cells also contain the encoded proteins. However, measuring the activity of enzymes at the resolution of single cells in complex tissues, such as leaves, is problematic. We have addressed this by using single‐cell sampling to extract 10–100 pl droplets of sap from individual plant cells and then measuring enzyme activities in these droplets with nanolitre‐scale fluorescence‐based assays. We have optimised these assays and used them to measure and characterise the activities of acid phosphatase, cysteine protease and nitrate reductase in sap samples from epidermal and mesophyll cells of barley (Hordeum vulgare L.) and Arabidopsis thaliana leaves exposed to different developmental and environmental conditions. During leaf senescence in barley, we found that the dynamics with which acid phosphatase and protease activities changed were different in each cell type and did not mirror the changes occurring at the whole‐leaf level. Increases in nitrate reductase activities after exposure of barley plants to nitrate were large in mesophyll cells but small in epidermal cells. The technique was applied successfully to Arabidopsis and, as in barley, revealed cell‐specific differences in the activities of both acid phosphatase and nitrate reductase. The assays add to the spectrum of techniques available for characterising cells within complex plant tissues, thus extending the opportunity to relate gene expression to biochemical activities at the single‐cell level.
Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non‐dehiscent anthers
Authors: Steiner‐Lange Sabine , Unte Ulrike S. , Eckstein Luca , Yang Caiyun , Wilson Zoe A. , Schmelzer Elmon , Dekker Koen , Saedler Heinz
Published: May 2003

A male sterile mutant with a defect in anther dehiscence was identified in an Arabidopsis thaliana population mutagenized with the Zea mays transposon En‐1/Spm. Mutants produce viable pollen that can fertilize when released mechanically from the anthers. Mutant stamens are of normal size and shape, but lack cell wall fortifications in the endothecial cell layer of the anther, which are required for the dehiscence process. The mutant phenotype was shown to be caused by a transposon insertion in AtMYB26, disrupting the putative DNA‐binding domain of this R2R3‐type MYB transcription factor. RT‐PCR revealed that expression of AtMYB26 is restricted to inflorescences. Sterility was shown to be stable under several environmental conditions. The high stability of the sterile phenotype, together with the fact that pollen is functional, makes AtMYB26 and its orthologs a valuable tool for manipulating male fertility in higher plants.