bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐angiosperm phytochromes and the evolution of vascular plants
Authors: Schneider‐Poetsch Hansjörg A. W. , Kolukisaoglu Üner , Clapham David H. , Hughes Jon , Lamparter Tilman
Published: March 2003

The phytochromes, a class of plant light‐sensing pigments, are a gene family with a long, complex evolutionary history. Angiosperms each have five or more phytochromes (designated A to E in Arabidopsis) with distinct functions as light receptors and only moderate sequence identities for different types within a species. The long‐term challenge taken up here is to trace the origin and function of the various motifs within the angiosperm phytochromes through gymnosperm phytochromes (types N, O and P) and lower plant phytochromes, sometimes reaching even to bacterial progenitor molecules. Particularly intriguing are the findings of homology of a C‐terminal region of phytochromes with bacterial transmitter modules and of a large N‐terminal region with a protein encoded by a gene from the cyanobacterum Synechocystis. Phylogenetic analysis helps to answer general questions such as the times of divergence of mono‐ and dicotyledons, of groups of gymnosperms or of ferns. Phytochrome sequences suggest (1) that mono‐ and dicotyledons became separated 150‐200 million years earlier than indicated by the fossil record and (2) that Ginkgo and Cycas have been separated unexpectedly late from the lineage giving rise to the Pinidae. (3) The status of Psilotum as a close relative of the primeval vascular plants is not supported. Phytochrome gene sequences additionally reveal that (4) moss and fern phytochromes have erratically acquired C‐termini which, though kinase‐like, are different from the common ones and that (5) introns have been lost, gained or shifted in position from algae to angiosperms. Phytochromes promise to be a rich source of phylogenetic information into the future as more sequences and functional data emerge, not least from studies of lower plants.
Carbohydrate levels among winter wheat cultivars varying in freezing tolerance and snow mold resistance during autumn and winter
Authors: Yoshida Midori , Abe Jiro , Moriyama Masahisa , Kuwabara Tatsuo
Published: March 2003

The LT50 values and soluble carbohydrate levels in wheat (Triticum aestivum L.) crowns and leaves were monitored throughout autumn and winter in cultivars varying in freezing tolerance and snow mold resistance during 1993/1994 and 1994/1995 in the field at Sapporo, Japan. During the first stage of hardening, from sowing to mid‐November, the pattern of accumulation of mono‐ and disaccharides was similar for all cultivars. During the second stage of cold hardening, from mid‐November to mid‐December, the greatest accumulation of mono‐ and disaccharides, without a corresponding increase in fructan, was observed among the freezing‐tolerant cultivars; and levels of simple saccharides rapidly decreased under snow cover. Conversely, levels of mono‐ and disaccharides in snow mold‐resistant cultivars were less than 70% of those in freezing‐tolerant cultivars before snow cover and maintained low levels throughout winter, while polysaccharide levels in snow mold‐resistant cultivars were about 120% of those in freezing‐tolerant cultivars in December. Sugar metabolism during the winter was examined using 18 cultivars in 1994/1995. LT50 values were correlated to the greatest extent with total mono‐ and disaccharide and fructan content among wheat cultivars excluding snow mold‐resistant cultivars in December. Snow mold‐resistant cultivars tended to metabolize carbohydrates more slowly until the end of the snow cover period. This result suggested that the enzymatic metabolism of the synthesis of sugars and the conversion of fructan to cryoprotective sugars in response to low temperatures, especially subzero ones, might be different between the two contrasting types in resistance to winter stress.
Storage behavior of Typha latifolia pollen at low water contents: Interpretation on the basis of water activity and glass concepts
Authors: Buitink Julia , Walters Christina , Hoekstra Folkert A. , Crane Jennifer
Published: March 2003

Germplasm must be stored under optimal conditions to maximize longevity and efficiently maintain genetic resources. In order to identify optimal storage conditions, we investigated the effects of temperature (−5 to 45°C) and water content (<0.17 g H2O g−1 dry weight) on longevity of Typha latifolia L. pollen. Longevity was highest at water contents corresponding to storage relative humidity (RH) of 11‐15% which corresponded to the shoulder of water sorption isotherms. Also coinciding with this shoulder were abrupt changes in heat capacity of water present in the pollen. Consistent with changes in isotherms with temperature and the concept of critical RH for storage, optimum water contents increased with decreasing temperature. An attempt was made to explain the aging behavior according to the glass concept. The water content‐temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. We also found a change in activation energy of aging in Arrhenius plots around Tg, demonstrating a change in aging kinetics when the glassy state is lost. We concluL that Tg curves cannot be used solely to predict precise conditions of optimum storage, but might be useful for predictions of storage longevity above optimum water contents. The data imply that too much drying reduces longevity and should be avoided, particularly when cryogenic storage is considered.
A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942
Authors: Papageorgiou George C. , Alygizaki‐Zorba Aikaterini , Ladas Nectarios , Murata Norio
Published: March 2003

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na+ and K+ salts caused rapid (t1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.
Respiratory metabolism during cold storage of apple fruit. I. Sucrose metabolism and glycolysis
Authors: Duque Paula , Barreiro Maria G. , Arrabaça João D.
Published: March 2003

This study provides the first report on the occurrence of the respiratory climacteric during cold storage of apple fruit (Malus domestica Borkh. cv. Reinette du Canada). The respiratory pattern at 4°C was very similar to that observed during postharvest ripening at room temperature, except that shelf life was considerably extended and the onset of the climacteric delayed. Increasing the calcium content of the apple fruit significantly reduced loss of firmness during cold storage, but showed no effect on respiration or on the other parameters determined. A gradual accumulation of soluble sugars occurred during the first 60 days after harvest and was effectively completed before the climacteric peak was reached. This increase in sugars correlated with an increase in the activity of sucrose‐phosphate synthase (EC 2.4.1.14), and a marked change in the kinetic properties of the enzyme was observed after sucrose accumulation ceased. Changes in the hexose‐phosphate pool and in glycolytic and gluconeogenic activities indicated an initial increase in the gluconeogenic flow at early stages of the climacteric, followed by activation of glycolysis, with the carbon flow being most likely regulated at the reversible phosphorylation of fructose‐6‐phosphate to fructose‐1,6‐bisphosphate (mostly via pyrophosphate:fructose‐6‐phosphate phosphotransferase, EC 2.7.1.90) and at the pyruvate kinase (EC 2.7.1.40) steps. The results presented indicate that the respiratory climacteric does not occur to accommodate extra ATP requirements during sucrose synthesis nor can it be a consequence of an increased supply of respiratory substrate.
Complementary DNA cloning, sequencing and expression of an unusual dehydrin from blueberry floral buds
Authors: Levi Amnon , Panta Ganesh R. , Parmentier Cecile M. , Muthalif Mubarack M. , Arora Rajeev , Shanker Savita , Rowland Lisa J.
Published: March 2003

Levels of three major dehydrins of 65, 60, and 14 kDa have been observed to increase in blueberry (Vaccinium smpp.) floral buds during chill unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. Indeed, levels of the 65‐, 60‐, and 14‐kDa dehydrins increase such that they become the most predominant proteins visible on sodium dodecyl sulfate (SDS)‐polyacrylamide gels. The peptide sequence information from the 65‐ and 60‐kDa dehydrins was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding the dehydrins. One pair of primers amplified a 174‐bp fragment. The 174‐bp fragment was used to screen a cDNA library (prepared from RNA from cold‐acclimated blueberry floral buds) and resulted in the isolation of a clone with a 2.0‐kb insert. The cDNA was sequenced and found to be a full‐length clone encoding a K5‐type dehydrin (5 K boxes). Five high‐confidence peptide sequences, ranging from 9 to 25 amino acids long, obtained from the 60‐kDa dehydrin exactly matched sequences encoded within the cDNA clone. Furthermore, amino acid composition of the 60‐kDa dehydrin agreed well with the expected amino acid composition based on the cDNA sequence. However, the DNA sequence and coupled in vitro transcription/translation reactions of the cDNA clone indicated that it encodes a dehydrin with a native molecular mass of ∼40 kDa instead of 60 kDa. Experiments to determine if the dehydrins undergo post‐translational modifications revealed that the 65‐ and 60‐kDa dehydrins are glycosylated. Thus, our results indicate that the 2.0‐kb dehydrin cDNA encodes the native version of the 60‐kDa dehydrin. The dehydrin cDNA hybridized on RNA blots to two chilling/cold‐responsive messages of 2.0 and 0.5 kb. Both the 2.0‐ and 0.5‐kb messages increased to higher levels more quickly in the cold‐hardy cultivar Bluecrop than in the less hardy cultivar Tifblue. In addition, the 0.5‐kb message remained at a higher level longer in Bluecrop than in Tifblue.
Effects of ozone on apoplast/cytoplasm partitioning of ascorbic acid in snap bean
Authors: Burkey Kent O.
Published: March 2003

Apoplast/cytoplasm partitioning of ascorbic acid (AA) was examined in four genotypes of snap bean (Phaseolus vulgaris L.) known to differ in ozone sensitivity. Plants were grown in pots under field conditions using open‐top chambers to establish charcoal‐filtered (CF) air (36 nmol mol−1 ozone) or elevated ozone (77 nmol mol−1 ozone) treatments. AA in fully expanded leaves of 36‐day‐old plants was separated into apoplast and cytoplasm fractions by vacuum infiltration methods using glucose 6‐phosphate as a marker for cytoplasm contamination. Apoplast ascorbate levels ranged from 30 to 150 nmol g−1 fresh weight. Ozone‐sensitive genotypes partitioned 1–2% of total AA into the apoplast under CF conditions and up to 7% following a 7‐day ozone exposure. In contrast, an ozone‐tolerant genotype partitioned 3–4% of total leaf AA into the leaf apoplast in both CF and ozone‐treated plants. The results suggest that genetic background and ozone stress are factors that affect AA levels in the extracellular space. For all genotypes, the fraction of AA in the oxidized form was higher in the apoplast compared to the cytoplasm, indicative of a more oxidizing environment within the cell wall.
Dynamic metabolism of photosystem II reaction center proteins and pigments
Authors: Mattoo Autar K. , Giardi Maria‐Teresa , Raskind Alexander , Edelman Marvin
Published: March 2003

Photosystem II (PSII) reaction center is an intrinsic membrane‐protein complex in the chloroplast that catalyzes primary charge separation between P680, a chlorophyll a dimer, and the primary quinone acceptor QA. This supramolecular protein complex consists of D1, D2, α and β subunits of cytochrome b559, the psbI gene product, and a few low molecular mass proteins. Ligated to this complex are pigments: chlorophyll a, pheophytin a, β‐carotenes, and non‐heme iron. One of the major outcomes of light‐mediated photochemistry is the fact that in the light, D1 protein is rapidly turned over compared to the other proteins of the reaction center; the relative lability of proteins being: D1≫D2>Cyt b559. D1 degradation in visible light exhibits complex, multiphasic kinetics. D1 degradation can be uncoupled from photosynthetic electron transport, which suggests that degradation may perform some separate function(s) beyond maintaining photosynthetic activity. The presence of a physiologically relevant level of ultraviolet‐B (UV‐B) radiation in a background of photosynthetically active radiation stimulates D1/D2 heterodimer degradation in a synergistic manner. D1 undergoes several post‐translational modifications including N‐acetylation, phosphorylation, and palmitoylation. Light‐dependent phosphorylation of D1 occurs in all flowering plants but not in the green alga Chlamydomonas or in cyanobacteria, and the same may be true for D2. The roles of these modifications in D1/D2 assembly, turnover, or function are still a matter of conjecture. Nor do we yet know about the fate of the liganded pigments, such as the chlorophyll and carotenoids bound to the reaction center proteins. Environmental extremes that negatively impact photosynthesis seem to involve D1 metabolism. Thus, D1 protein is a major factor of PSII instability, and its replacement after its degradation is a primary component of the PSII repair cycle.
Calcium is involved in regulation of the synthesis of HSPs in suspension‐cultured sugar beet cells under hyperthermia
Authors: Trofimova Marina S. , Andreev Igor M. , Kuznetsov Vladimir V.
Published: March 2003

Exposure of plants to elevated temperatures induces a complex set of changes that enable plants to adapt following heat stress. In order to test the effect of Ca2+ on heat shock‐induced changes in cell protein synthesis the incorporation of [

S]methionine into protein was studied in cultured sugar beet (Beta vulgaris L.) cells incubated in media containing different calcium concentrations. Heat shock inhibited the synthesis of non‐heat shock proteins (non‐HSPs) and promoted the synthesis of a set of HSPs, typical of plants. The synthesis of non‐HSPs was greatly inhibited by external Ca2+ removal by treatment of the cells with ethylene glycol‐bis(β‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid. In contrast, extracellular Ca2+ appeared not to be strictly required for the de novo production of HSPs, but this cation exerted different effects on the synthesis of individual HSPs. Cell injury increased if the cells were exposed simultaneously to high temperature and Ca2+‐deficient medium. Recovery of HSP synthesis and reduced cell injury were observed after addition of exogenous calcium to Ca2+‐depleted cells. These findings are consistent with a Ca2+ requirement for the survival of the cells under heat shock, and likely for the development of cell thermotolerance.
The impact of elevated carbon dioxide on the growth and gas exchange of three C4 species differing in CO2 leak rates
Authors: Ziska Lewis H. , Sicher Richard C. , Bunce James A.
Published: March 2003

Recent work has suggested that the photosynthetic rate of certain C4 species can be stimulated by increasing CO2 concentration, [CO2], even under optimal water and nutrients. To determine the basis for the observed photosynthetic stimulation, we tested the hypothesis that the CO2 leak rate from the bundle sheath would be directly related to any observed stimulation in single leaf photosynthesis at double the current [CO2]. Three C4 species that differed in the reported degree of bundle sheath leakiness to CO2, Flaveria trinervia, Panicum miliaceum, and Panicum maximum, were grown for 31–48 days after sowing at a [CO2] of 350 μl l−1 (ambient) or 700 μl l−1 (elevated). Assimilation as a function of increasing [CO2] at high photosynthetic photon flux density (PPFD, 1 600 μmol m−2 s−1) indicated that leaf photosynthesis was not saturated under current ambient [CO2] for any of the three C4 species. Assimilation as a function of increasing PPFD also indicated that the response of leaf photosynthesis to elevated [CO2] was light dependent for all three C4 species. The stimulation of leaf photosynthesis at elevated [CO2] was not associated with previously published values of CO2 leak rates from the bundle sheath, changes in the ratio of activities of PEP‐carboxylase to RuBP carboxylase/oxgenase, or any improvement in daytime leaf water potential for the species tested in this experiment. In spite of the simulation of leaf photosynthesis, a significant increase in growth at elevated [CO2] was only observed for one species, F. trinervia. Results from this study indicate that leaf photosynthetic rates of certain C4 species can respond directly to increased [CO2] under optimal growth conditions, but that the stimulation of whole plant growth at elevated carbon dioxide cannot be predicted solely on the response of individual leaves.
Characterization of two cDNAs encoding glutathione S‐transferases in rice and induction of their transcripts by the herbicide safener fenclorim
Authors: Wu Jingrui , Cramer Carole L. , Hatzios Kriton K.
Published: March 2003

The safener fenclorim (4,6‐dichloro‐2‐phenyl‐pyrimidine) induces glutathione S‐transferases (GSTs; EC 2.5.18) in rice (Oryza sativa L. cv. Lemont), including those with activity toward pretilachlor [2‐chloro‐2′,6′‐diethyl‐N‐(2‐propoxyethyl)acetamide] and this is associated with increased tolerance to this herbicide. In this study, we report on the isolation and characterization of two partial cDNA clones encoding GSTs in rice. The two cDNA clones were isolated by using GST‐specific degenerate primers and the RT‐PCR (reverse transcriptase‐polymerase chain reaction) technique from control (unsafened) and fenclorim‐treated (safened) rice roots, respectively. Sequence analyses demonstrated that the two cDNAs share 60% amino acid sequence identity and 62% nucleotide sequence identity, appearing to represent two different rice GSTs (RGST I and II). Southern blot analysis with the two clones confirmed the presence of at least three GST genes in the rice genome. Northern blotting with the two clones as probes showed that the GST gene(s) are constitutively expressed in roots of unsafened rice seedlings. Treatment with the safener fenclorim induced the expression of the rice GST gene(s). The fenclorim‐mediated induction of mRNA expression coincided with a concomitant induction of total GST activity toward pretilachlor in roots of fenclorim‐safened rice. Phylogenetic analysis based on the amino acid sequence alignment of the two rice cDNAs with sequences of other plant GSTs showed that RGST I seems to be more closely related to wheat GSTs, while RGST II is more closely related to the maize GST I and IV isozymes.
Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate N‐(indol‐3‐ylacetyl)‐glutamine and initiation of a mutant screen
Authors: Barratt Natalie M. , Dong Weiqin , Gage Douglas A. , Magnus Volker , Town Christopher D.
Published: March 2003

The accumulation of conjugates of indole‐3‐acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole‐3‐acetyl‐aspartate, indole‐3‐acetyl‐glutamate, and indole‐3‐acetyl‐glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μM IAA. In addition, a new metabolite was detected and positively identified as indole‐3‐acetyl‐glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole‐3‐acetyl‐aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole‐3‐acetyl‐glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole‐3‐acetyl‐glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA‐inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole‐3‐acetyl‐glutamine and less indole‐3‐acetyl‐aspartate than wild‐type, and is allelic to an existing class of photorespiration mutants (gluS) deficient in chloroplastic glutamate synthase.
Light modulation and in vitro effects of adenine nucleotides on leaf nitrate reductase activity in cucumber (Cucumis sativus)
Authors: Agüera Eloísa , Poblete Lourdes , De La Haba Purificación , Maldonado José M.
Published: March 2003

Nitrate reductase (NR, EC 1.6.6.1) activity in attached cucumber (Cucumis sativus L. cv. Ashley) leaves changed rapidly and reversibly during light/dark transitions, especially when assayed in the presence of free Mg2+. Light decreased and darkness increased the sensitivity of the enzyme to inhibition by Mg2+. The NR activation state, i.e. activity in the presence of Mg2+ relative to activity in the absence of Mg2+, increased with light intensity up to 400 μmol m−2 s−1 PAR (photosynthetically active radiation). When a desalted crude extract from illuminated leaves was preincubated with ATP, NR was gradually inactivated. Inactivation was only observed when activity was assayed in the presence of Mg2+. The ATP‐inactivated NR remained inactive after removing the excess of ATP by gel filtration and it did not occur in partially purified NR preparations. NR extracted from darkened attached leaves was markedly activated when preincubated with 5′‐AMP. These results support the view that inactivation/activation of cucumber‐leaf NR in response to light/dark signals most likely involves phosphorylation/dephosphorylation of the enzyme catalysed by endogenous proteins. A substantial activation of NR by preincubation with 5′‐AMP was also observed when activity was assayed in the absence of Mg2+, thus indicating that 5′‐AMP can directly activate NR. Irradiation of an extract from darkened leaves containing FAD promoted a partial activation of NR. This effect was observed both in the +Mg2+ and in the −Mg2+ assay, indicating that activation was caused by photoexcited flavin and did not involve dephosphorylation of the enzyme.
The role of ethylene and cell wall modifying enzymes in raspberry (Rubus idaeus) fruit ripening
Authors: Iannetta Pietro P. M. , Van Den Berg Jan , Wheatley Ronald E. , McNicol Ronald J. , Davies Howard V.
Published: March 2003

This study focuses on four raspberry (Rubus idaeus) genotypes from two different genetic backgrounds: cvs Glen Prosen and Glen Clova, bred at the Scottish Crop Research Institute (SCRI) and genotypes bred at Horticulture Research International (HRI), East Malling (EM), EM 4997 and EM 5007. The ripe fruit of each genotype pair were characterised subjectively by raspberry breeders as relatively firm or soft, respectively. Different stages of fruit development from each genotype were used to quantify fruit firmness, rates of ethylene evolution and ripening rate. Penetrometry data confirmed suspected firmness differences. Firmness correlated with rates of ethylene evolution. Rates of ethylene production also correlated with receptacle size. Storage of green fruits in 20 μl l−1 ethylene reduced fruit firmness, enhanced respiration rate and colour (anthocyanin) development and stimulated the development of cell wall hydrolase activities. However, during natural ripening in the field, fruit respiration rate declined, which indicates a non‐climacteric ripening pattern. In drupelets, the activities of polygalacturonase (PG), pectin methylesterase (PME), Cx‐cellulase (Cx) and β‐galactosidase (β‐gal.) increased substantially as ripening progressed. More detailed studies with ripe fruit of cv. Glen Clova indicated major isoforms of PG at pIs 3.3, 8.6 and 10.1; of PME at pIs 7.2, 8.5, 8.7, 8.8; of Cx at pI 2.4; and of β‐gal. at pIs 6.3 and 6.7.
Purification and characterization of nitrate reductase from nodule cytosol of soybean plants
Authors: Kanayama Y. , Kimura K. , Nakamura Y. , Ike T.
Published: March 2003

Nodulated soybean (Glycine max [L.] Merr.) plants were grown in a nitrogen‐free liquid culture medium prepared with distilled water. The cytosol fraction from root nodules showed a significant level of NADH‐dependent nitrate reductase activity, even when the root did not show activity. This nitrate reductase was purified by column chromatography and native polyacrylamide gel electrophoresis (PAGE). The purified protein showed a main band at 100 kDa on sodium dodecyl sulfate (SDS)‐PAGE. The Km value for nitrate was 0.16 mM, and the highest activity was obtained at around pH 7.5. These characteristics are very similar to the inducible type of nitrate reductase, previously purified from soybean leaves. The developmental change in activity of this enzyme corresponded to that in nitrogenase activity.
Protective effect of glutathione on the cytotoxicity caused by a combination of aluminum and iron in suspension‐cultured tobacco cells
Authors: Yamaguchi Yukiko , Yamamoto Yoko , Ikegawa Hiroshi , Matsumoto Hideaki
Published: March 2003

The role of endogenous glutathione (GSH) in the protection of suspension‐cultured tobacco cells from aluminum (Al) toxicity was examined. Cells at the logarithmic phase of growth were treated with or without Al in nutrient medium prepared without Pi and EDTA. In the absence of Al, total GSH content (including oxidized glutathione [GSSG]) increased gradually. In the presence of Al, the increase of GSH was repressed. This effect was observed before the loss of plasma membrane integrity and the loss of cell viability. In contrast, GSSG content in cells increased in the presence of Al. GSH‐deprived cells were prepared by culturing cells with buthionine sulfoximine (an inhibitor of Γ‐glutamylcysteine synthetase) for 24 h. Total GSH content in GSH‐deprived cells was 6% of that in normal cells. The GSH‐deprived cells exhibited a higher degree of lipid peroxidation, increased accumulation of Al, and greater loss of viability than normal cells. These results suggest that GSH protects cells from the oxidative membrane damage caused by a combination of Al and Fe(II) possibly by both direct consumption of GSH and oxidation of GSH.
Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)
Authors: Padmasree K. , Raghavendra A. S.
Published: March 2003

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea (Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O2 evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO2 (1.0 mM NaHCO3) than that at limiting CO2 (0.1 mM NaHCO3). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO2. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO2 than at optimal CO2. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO2 (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.
Change in plasma membrane ATPase activity during dormancy release of vegetative peach‐tree buds
Authors: Aue Henri‐Luc , Lecomte Isabelle , Gendraud Michel , Pétel Gilles
Published: March 2003

Plant dormancy and dormancy breaking depend, at least partially, on peculiar short distance relationships between buds and tissues underlying buds (bud stands). In peach‐tree, it was previously observed that dormancy was related to a high nutrient absorption capacity in tissues underlying buds. This situation could be linked to higher plasma membrane ATPase activity (EC 3.6.1.3), inducing a higher nutrient absorption, in bud stands. This work consists of characterization of the plasma membrane ATPase activity in vegetative buds and bud stands during the rest period and dormancy release. During the dormant period (October and November), plasma membrane ATPase activity was found to be higher in bud stands than in buds. This was correlated with a lower amount of plasma membrane ATPase in buds compared to bud stands during this period. Moreover, plasma membrane ATPase activation by trypsin treatment was not the same in both tissues and different levels of ATPase activation could be noted within the same tissue during the different stages of dormancy release. According to these results, it can be postulated that dormancy release in peach‐tree, is related to modifications of plasma membrane ATPase properties in buds and bud stands during winter time.
Regulation of Γ‐aminobutyric acid synthesis in situ by glutamate availability
Authors: Scott‐Taggart Catherine P. , Van Cauwenberghe Owen R. , McLean Michael D. , Shelp Barry J.
Published: March 2003

The aim of this study was to evaluate the hypothesis that Γ‐aminobutyric acid (Gaba) synthesis in situ is regulated by Glu availability. The Gly decarboxylase (EC 2.1.2.10) inhibitor, aminoacetonitrile (AAN), was investigated as a means of preventing the recycling of photorespiratory ammonia, thereby causing the accumulation of Glu. In vitro measurements of crude tobacco (Nicotiana tabacum L. cv. Samsun NN) leaf extracts demonstrated that AAN effectively inhibited Gaba:pyruvate transaminase (EC 2.6.1.19) activity, but not Glu decarboxylase (EC 4.1.1.15), Glu:oxaloacetate transaminase (EC 2.6.1.1) or Glu:pyruvate transaminase (EC 2.6.1.1) activities. Gly decarboxylase activity was inhibited preferentially over Gaba transaminase, and secondary effects that can lead to an inhibition of photosynthesis were minimized by the use of a short‐time course of only 90 min to investigate the impact of 25 mM AAN on the steady state metabolism of [U‐14C]Glu by young transpiring shoots of tobacco seedlings. With time, AAN treatment caused the accumulation of Gly/Asn, but not Gaba. Furthermore, AAN increased the C‐radioactivity in Glu, Pro and Krebs cycle organic acids, and reduced the C‐radioactivity in Gln and Gaba. The data provide evidence for increased flux of carbon through the Gaba shunt when the availability of Glu as a substrate for Glu decarboxylase was enhanced.
Plastid photodamage and Cab gene expression in barley leaves
Authors: La Rocca Nicoletta , Dalla Vecchia Francesca , Barbato Roberto , Bonora Angelo , Bergantino Elisabetta , Rascio Nicoletta
Published: March 2003

The effects of norflurazon (NF) and amitrole (AM), two bleaching herbicides which inhibit carotenogenesis, were compared in leaves of 7‐day‐old barley (Hordeum vulgare L. cv Express) plants grown in damaging light. The herbicide effects were analysed with respect to chloroplast organization, photosynthetic functionality and nuclear photodependent expression of the Lhcb1 gene, which codes for the Lhcb1 light‐harvesting chlorophyll a/b binding protein of photosystem II. Both herbicides caused dramatic photooxidation of organelles, which were photosynthetically unfunctional. Plastids of NF‐treated plants lacked thylakoids and pigments. Plastids of AM‐treated plants had some strikingly altered membranes and contained only very small quantities of chlorophylls. Despite the presence of severely photodamaged plastids, cells of AM‐treated leaves contained high levels of Lhcb1 transcript. This transcript, on the contrary, was completely absent in the cells of NF‐treated plants. These findings suggest that in order to block expression of nuclear genes coding for plastid‐resident proteins, photodamage leading to the complete dismantling of thylakoids and to the total absence of any form of photosynthetic pigment is required.
Regulation of pollination‐induced ethylene and its role in petal abscission of Pelargonium×hortorum
Authors: Hilioti Zoe , Richards Christina , Brown Kathleen M.
Published: March 2003

Pollination in geranium (Pelargonium×hortorum) results in rapid non‐autocatalytic ethylene production followed by abscission of turgid petals. In the present work, the regulation of post‐pollination ethylene was investigated in various floral organs. Ethylene production and petal abscission increased as the number of stigmatic lobes pollinated increased. Direct application of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) to the stigma induced ethylene production and petal abscission only at high concentrations, which are not naturally present in the pollen. Application of aminoethoxyvinylglycine (AVG), an inhibitor of ACC synthase activity, to the stigma effectively prevented pollination‐induced ethylene production and petal abscission. Pollination increased the levels of ACC 46‐fold in stigma plus style tissue and 6‐fold in the basal portion of petals. Enhanced accumulation of N‐malonyl‐ACC (MACC), a conjugated form of ACC, was also observed in stigma and style tissues after pollination. Pollination increased the activity of ACC synthase 2.5‐fold in stigma and style tissues and 3‐fold in the basal portion of petals. At 2 h after pollination, a 4‐fold increase in the expression of the ACC synthase gene GAC‐1 was detected specifically in the stigma and style. In vivo activity of ACC oxidase was 1.5‐fold higher in pollinated than in unpollinated or wounded stigma and style. In the lower part of the pistil, the activity of ACC oxidase was lower than in the upper pistil, regardless of treatment. The activity of ACC oxidase was significantly higher in the basal portion of petals than the upper portion of petals. Pollination had no effect on the activity of ACC oxidase in petals. Treatment of florets with cycloheximide effectively prevented pollination‐induced petal abscission, but ethylene production was completely unaffected, providing evidence for the post‐transcriptional regulation of ethylene production. Stigma and style tissues accounted for 57% of the total ethylene production in the flower following pollination. When the stigma and style were removed and the top of the sterile ovary pollinated, there was no pollination‐induced ethylene production or petal abscission. We propose that rapid petal abscission in response to pollination results from increased ethylene production, which occurs via activation of ACC synthase primarily in the stigma and style. This contrasts with post‐pollination ethylene production in some wilting‐type flowers in timing, lack of autocatalytic ethylene and predominantly post‐transcriptional regulation.
Interaction between metabolism of atmospheric H2S in the shoot and sulfate uptake by the roots of curly kale (Brassica oleracea)
Authors: Westerman Sue , De Kok Luit J. , Stuiver C. Elisabeth E. , Stulen Ineke
Published: March 2003

Exposure of curly kale (Brassica oleracea L.) to gaseous H2S resulted in a decreased sulfate uptake by the roots. At 0.2 μl l−1 H2S, a level sufficient to meet the sulfur need of plants for growth, the sulfate uptake was maximally decreased by 50% after 3 or 4 days of exposure. Higher levels up to 0.8 μl l−1 H2S did not further affect the sulfate uptake. The nitrate uptake was not affected upon exposure to 0.2–0.8 μl l−1 H2S. H2S exposure did not affect the sulfate content of the plants, but it resulted in an increased content of thiols and cysteine in the shoots, whereas that in the roots was hardly affected. Plants grown under sulfate‐deprived conditions had a decreased biomass production, very low content of sulfate and decreased content of thiols in both shoot and roots. Sulfate‐deprived plants had a two‐fold higher sulfate uptake after transfer to a sulfate‐containing solution, while nitrate uptake was decreased by 50%. When sulfate‐deprived plants were exposed to 0.25 μl l−1 H2S, plant biomass production and nitrate uptake were restored but the sulfate uptake after transfer to a sulfate‐containing solution remained high. Also here, H2S exposure resulted in an increase in the thiol and cysteine content of both shoot and roots, whereas the content of sulfate remained low. The presented results clearly demonstrate a direct interaction between the regulation of sulfate uptake by the roots and the metabolism of gaseous H2S by the shoot. Curly kale is able to use both sulfate and H2S as a sulfur source for growth, and matching the supply of sulfur in the form of pedospheric or atmospheric sulfur to the sulfur needed for growth appears to be regulated nicely. However, the significance of thiols as signal in the shoot/root coordination of sulfate uptake appears to be limited. From the data it is evident that there is no direct mutual regulation between the uptake of sulfate and nitrate by the roots.
Biolistic transformation of chincherinchee (Ornithogalum) and regeneration of transgenic plants
Authors: De Villiers Santie M. , Kamo Kathryn , Thomson Jennifer A. , Bornman Chris H. , Berger David K.
Published: March 2003

Ornithogalum (chincherinchee) is a genus in the Hyacinthaceae. It is popular as a cut flower or pot plant. However, susceptibility to disease, especially ornithogalum mosaic virus, prevents commercial exploitation of micropropagated hybrids. Provided that it is possible to transform Ornithogalum, this problem might be alleviated by the transfer of genes that code for resistance to the virus. The purpose of this study was to develop a transformation protocol using the pat gene as a selectable marker. Callus, induced on leaf segments of an Ornithogalum thyrsoides×O. dubium hybrid cultured in vitro, was bombarded with a particle gun 4–6 weeks after initiation. We first used β‐glucuronidase transient expression to optimise the bombardment parameters and then for stable transformation used both a conventional microprojectile‐mediated method as well as a modification that entailed complexing single‐stranded p35SAC DNA, containing the pat gene, with histone H1 prior to bombardment. Transgenic plants were regenerated from the bombarded tissues and cultured on a medium containing 15 μM phosphinothricin as selective agent. Rooted plants were tested for the presence of the pat gene by polymerase chain reaction. Integration of the gene into the genomic DNA was verified by Southern blotting. Northern blots, enzyme‐linked immunosorbent assay, and leaf paint assays with the herbicide Ignite® (glufosinate ammonium) confirmed expression of phosphinothricin acetyl transferase, the enzyme that detoxifies the herbicide.
Tuber morphology and starch accumulation are independent phenomena: Evidence from ipt‐transgenic potato lines
Authors: Sergeeva Lidiya I. , De Bruijn Steef M. , Koot‐Gronsveld Elly A. M. , Navratil Oldrich , Vreugdenhil Dick
Published: March 2003

Tuber formation and carbohydrate metabolism in potatoes were studied using transgenic potato plants carrying the Agrobacterium tumefaciensipt gene, involved in cytokinin biosynthesis. Three independent transformants, viz. clones 1, 11 and 13, whose cytokinin and auxin content had previously been shown to be different from each other and from the wild‐type, were analysed in vitro. Clones 11 and 13 showed a higher ability to form stolons and tubers, as evident from: (1) stolon development in whole plants grown under non‐inductive conditions, (2) total number and weight of tubers formed by cuttings of this clone in darkness, (3) tubers appeared earlier than tubers of wild‐type plants and at a lower sucrose concentration in the medium. Clone 1 did not form stolons or tubers under any conditions tested, but rather formed short shoots. A series of metabolic changes, known to be characteristic for tubers, were analysed in leaves, stems and developing buds. It was found that the short type of shoots, formed by clone 1, had metabolic characteristics very similar to tubers formed in wild‐type or clones 11 and 13, including glucose, fructose, sucrose, and starch levels, and activities of invertase, sucrose synthase and fructokinase. It is concluded that the regulation of the stolon swelling and of carbohydrate metabolism, normally occurring simultaneously, can be uncoupled, and are thus, at least partly independent phenomena. The present data obtained with a high‐cytokinin line indicate that cytokinins (probably in concert with auxins) might be mainly involved in the regulation of tuber morphology.
Heterogeneity of photosystem II reaction centers as influenced by heat treatment of barley leaves
Authors: Bukhov Nikolai G. , Carpentier Robert
Published: March 2003

Three functionally distinct populations of PSII reaction centers differing in the ability to keep the primary acceptors in a reduced state and to transfer electrons to PSI were estimated using chlorophyll fluorescence measurements in primary barley leaves exposed to elevated temperatures in the range of 37–51°C. The capacity of the PSII reaction centers to perform at least one light‐induced charge separation was not affected by a 5‐min heat treatment at temperatures up to 51°C. The first population containing QB‐non‐reducing centers corresponded to 15–20% of the total PSII activity up to 45°C. In a second population, PSII reaction centers maintained QA reduction under light in the presence of oxygen, but not in the presence of a strong artificial PSI electron acceptor, methyl viologen. In a third population that gradually increases from zero at 37°C to about 60% at 45°C, the PSII centers were not able to keep QA in the reduced state even in the presence of oxygen as the sole electron acceptor. Three electron transport pathways, the pseudocyclic one involving both PSII and PSI, the NAD(P)H‐dependent pathway mediated by PSI alone after the loss of activity in some PSII centers, and the PSI‐driven ferredoxin‐dependent route enhanced by weakly efficient PSII centers that are able to provide only catalytic amounts of electrons, are suggested to create a proton gradient in chloroplasts of heat‐stressed leaves thus protecting PSII reaction centers from photodamage.
Gene expression in flavonoid biosynthesis: Correlation with flavonoid accumulation in developing citrus fruit
Authors: Moriguchi Takaya , Kita Masayuki , Tomono Yasuhiko , Endo‐Inagaki Tomoko , Omura Mitsuo
Published: March 2003

Two genes active in the early steps of flavonoid biosynthesis were isolated from a citrus cDNA library, and their expression patterns were analyzed during citrus (Citrus unshiu Marc.) fruit development in relation to flavonoid contents. The respective cDNA clones from chalcone isomerase (CHI, CitCHI) and flavanone 3‐hydroxylase (F3H, CitF3H) are 931 and 1360 bp long, encoding 222 and 362 amino acid residues. CitCHI seems to be a single‐copy gene, whereas a few CitF3H‐related sequences are present in the citrus genome. Messenger RNA levels of the flavonoid biosynthetic genes CitCHI and CitF3H and two previously isolated chalcone synthases (CHS, CitCHS1, and CitCHS2) were analyzed in various tissues during fruit development. Transcript levels for CitCHS1, CitCHS2, CitCHI, and CitF3H were generally high in young tissues and low in senescent tissues. Flavonoid changes were analyzed in relation to gene expression. High flavonoid contents were found in young tissues. The high expression of flavonoid biosynthetic genes and high accumulation of flavonoids in young fruits suggest that flavonoids are synthesized in the early developmental stage.
Does the chloroplast small heat shock protein protect photosystem II during heat stress in vitro?
Authors: Härndahl Ulrika , Sundby Cecilia
Published: March 2003

It has been suggested that the function of the chloroplast‐localized small heat shock protein (sHsp) is to protect photosystem II (PSII) from heat inactivation. This paper reports that addition of purified sHsp protein to isolated thylakoid membranes gave no protection of PSII and questions that there is any direct effect of the sHsp on PSII. The opinion is forwarded that the primary role for the chloroplast‐localized sHsp may not even be protection of PSII.
Purification of a novel lipoxygenase from eggplant (Solanum melongena) fruit chloroplasts
Authors: Pérez‐Gilabert Manuela , López‐Nicolás José Manuel , García Carmona Francisco
Published: March 2003

A novel membrane lipoxygenase (LOX; EC 1.13.11.12) from eggplant (Solanum melongena L. cv. Belleza negra) fruit chloroplasts has been purified 20‐fold to a specific activity of 207 enzymatic units per mg of protein with a yield of 72%. The purification was carried out by sonicating the chloroplastic membranes in the presence of Triton X‐114 followed by phase partitioning and anion exchange chromatography. The purified membrane LOX preparation consisted of a single major band with an apparent molecular mass of 97 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results obtained using intact chloroplasts indicate that the enzyme is not localized in the stroma. When the enzyme reacts with linoleic acid, it produces a single peak, which comigrates with standard 9‐hydroperoxy‐octadecadienoic acid. A physiological role for this chloroplastic LOX is proposed.