bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinated accumulation of the chloroplastic and cytosolic pyruvate,Pi dikinases with enhanced expression of CAM in Kalanchoë blossfeldiana
Authors: Kondo Ayumu , Nose Akihiro , Ueno Osamu
Published: March 2003

Recent studies reveal that the intracellular localization of pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) in mesophyll cells of malic enzyme (ME)‐dependent Crassulacean acid metabolism (CAM) plants varies among species, occurring not only in the chloroplasts but also in the cytosol in some species. The facultative CAM plant Kalanchoë blossfeldiana accumulates PPDK in both compartments of the mesophyll cells. In this study, the patterns of accumulation of the chloroplastic and cytosolic PPDKs were investigated for K. blossfeldiana plants with different CAM activities by immunogold labeling and electron microscopy. Greater CAM activity was found in plants grown under drought conditions with short days than under well‐watered conditions with long days, and in lower leaves than in higher leaves. There was a trend that plants and leaves with greater CAM activity show denser labeling for PPDK in both the cytosol and chloroplasts. However, the ratio of the density of PPDK labeling in the cytosol to that in the chloroplasts was almost constant (2.4–3.0). Higher labeling for phosphoenolpyruvate carboxylase (EC 4.1.1.31) in the cytosol was also correlated with higher CAM activity but there was almost no difference in the density of labeling for ribulose‐1,5‐bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the chloroplasts. These results indicate that the increase in accumulation of cytosolic PPDK is closely associated with the increase of chloroplastic PPDK during enhanced CAM expression. This suggests that both PPDKs are involved in CAM function.
Advances in the selection of transgenic plants using non‐antibiotic marker genes
Authors: Joersbo Morten
Published: March 2003

Production of transgenic plants started more than a decade ago, but it is still a time‐consuming operation. One of the critical points is the selection procedure used for the recovery of transgenic shoots after transformation. Moreover, as more transgenic traits are to be incorporated into crops that already have been transformed, it is clear that there is a need for new methods with higher efficiencies. In this article, recently developed selection systems are reviewed. They differ from conventional selection techniques as they are based on supplementing the transgenic cells with a metabolic advantage rather than killing the non‐transgenic cells. In many cases, these new selection systems have been found to be superior to conventional methods.
Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules
Authors: McCloud Sally A. , Smith Ron G. , Schuller Kathryn A.
Published: March 2003

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N2‐fixing soybean (Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phosphoenolpyruvate (PEP)‐utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free‐living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross‐reacted with a 52‐kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 mM malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid‐containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N2 fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.
Growth temperature can alter the temperature dependent stimulation of photosynthesis by elevated carbon dioxide in Albutilon theophrasti
Authors: Ziska L. H.
Published: March 2003

Stimulation of photosynthesis in response to elevated carbon dioxide concentration [CO2] in the short‐term (min) should be highly temperature dependent at high photon flux. However, it is unclear if long‐term (days, weeks) adaptation to a given growth temperature alters the temperature‐dependent stimulation of photosynthesis to [CO2]. In velveltleaf (Albutilon theophrasti), the response of photosynthesis, determined as CO2 assimilation, was measured over a range of internal CO2 concentrations at 7 short‐term measurement (12, 16, 20, 24, 28, 32, 36°C) temperatures for each of 4 long‐term growth (16, 20, 28 and 32°C) temperatures. In vivo estimates of VCmax, the maximum RuBP saturated rate of carboxylation, and Jmax, the light‐saturated rate of potential electron transport, were determined from gas exchange measurements for each temperature combination. Overall, previous exposure to a given growth temperature adjusted the optimal temperatures of Jmax and VCmax with subsequently greater enhancement of photosynthesis at elevated [CO2] (i.e., a greater enhancement of photosynthesis at elevated [CO2] was observed at low measurement temperatures for A. theophrasti grown at low growth temperatures compared with higher growth temperatures, and vice versa for plants grown and measured at high temperatures). Previous biochemical based models used to predict the interaction between rising [CO2] and temperature on photosynthesis have generally assumed no growth temperature effect on carboxylation kinetics or no limitation by Jmax. In the current study, these models over predicted the temperature dependence of the photosynthetic response to elevated [CO2] at temperatures above 24°C. If these models are modified to include long‐term adjustments of Jmax and VCmax to growth temperature, then greater agreement between observed and predicted values was obtained.
Plasma membrane H+‐ATPase activity is involved in adaptation of tomato calli to NaCl
Authors: Kerkeb Loubna , Donaire Juan Pedro , Rodríguez‐Rosales María Pilar
Published: March 2003

A tomato (Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl‐sensitive calli in medium supplemented with 50 mM NaCl. NaCl‐tolerant calli grew better than NaCl‐sensitive calli in media supplemented with 50 and 100 mM NaCl. Analysis of callus ion content showed a strong increase in Na+ and Cl− both in NaCl‐tolerant and ‐sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl‐tolerant calli showed a higher H+ extrusion activity than those from NaCl‐sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H+ extrusion by NaCl‐sensitive cells was correlated with an inhibition of microsomal vanadate‐sensitive H+‐ATPase (EC 3.6.1.35) and ATP‐dependent H+ transport, while the stimulation of H+ extrusion by cells tolerant to 50 mM NaCl was correlated with an increase in plasma membrane ATP‐dependent H+ transport. The increase of ATP‐dependent H+ extrusion in plasma membranes isolated from 50 mM NaCl‐tolerant calli was not a result of stimulation of a vanadate‐sensitive ATP hydrolytic activity or an increase in passive permeability to H+. Relative to NaCl‐sensitive calli, plasma membrane H+‐ATPase from calli tolerant to 50 mM NaCl showed a lower Km for Mg2+‐ATP. Our results indicate that tolerance of tomato calli to 50 mM NaCl increases the affinity of plasma membrane H+‐ATPase for the substrate ATP and stimulates the H+‐pumping activity of this enzyme without modifying its phosphohydrolytic activity.
The characterization of LeNUC1, a nuclease associated with leaf senescence of tomato
Authors: Lers Amnon , Lomaniec Ella , Burd Shaul , Khalchitski Andrei
Published: March 2003

Induction of nuclease and RNase activities, together with decreases in nucleic acid content are considered to be characteristics of senescence in higher plants. However, little is known about the specific identities or functions of the enzymes involved or the mechanisms controlling their activation. Here we report the identification of a 41‐kDa‐tomato nuclease, LeNUC1, which is specifically induced during tomato leaf senescence but not in ripening fruits. LeNUC1 is a glycoprotein, which can degrade both RNA and DNA and has optimal activity at pH 7.5–8. EDTA inhibits the activity of LeNUC1, while the addition of Co2+ or Mn2+ can restore its activity in the presence of the chelating agent. Interestingly, the activity of LeNUC1 is also induced in young leaves upon treatment with ethylene, which is known to be a senescence‐promoting hormone in tomato. Constitutive activity of a 39‐kDa nuclease, LeNUC2, similar in its biochemical requirements to LeNUC1, was also detected. LeNUC2 is not induced by ethylene and does not seem to be glycosylated. Based on their characteristics, LeNUC1 and LeNUC2 can be classified as Nuclease I enzymes. LeNUC1 may be involved in nucleic acid metabolism during tomato leaf senescence.
Purification and characterization of an aspartic protease from potato leaves
Authors: Guevara María G. , Daleo Gustavo R. , Oliva Claudia R.
Published: March 2003

A protease was isolated from potato (Solanum tuberosum L. cv. Pampeana) leaves 48 h after detaching, when aspartic protease (AP) activity is markedly increased. Purification was performed by ammonium sulfate precipitation, ion exchange chromatography and affinity chromatography. A size of 40 kDa was estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; it is monomeric and its properties are consistent with those of aspartic proteinases (EC 3.4.23): it has a pH optimum of 3 and it is inhibited by pepstatin. Like other plant APs, leaf AP appears to be glycosylated with a complex‐type N‐glycan. The enzyme has properties different from those of a tuber AP previously described, indicating that they may have different physiological roles.
A calcium‐binding protein with similarity to serum albumin localized to the ER‐Golgi network and cell walls of spinach (Spinacia oleracea)
Authors: Lait Cameron G. , Zwiazek Janusz J.
Published: March 2003

Using polyclonal antibodies raised against human serum albumin (HSA), a 70‐kDa microsomal protein with an isoelectric point of approximately 6.5 was detected in spinach (Spinacia oleracea L.). The protein was purified by selective ammonium sulfate precipitation and anion exchange HPLC. The protein shared 100% identity with the first 15 amino acids at the NH2 terminus of HSA, including the X‐X‐H amino acid region, which was identified in HSA as being responsible for binding of copper, zinc, indole derivatives and calcium. Blue staining of the protein with the cationic carbocyanine dye ‘Stains‐all’ and 45Ca overlay following SDS‐PAGE also suggest that the 70‐kDa plant protein binds calcium. The protein reacted positively with carbohydrate specific thymol stain, and the carbohydrates associated with the protein were identified by gas chromatography‐mass spectrometry (GC‐MS) as galactose and galacturonic acid. The 70‐kDa plant protein was present in the detergent‐poor phase following Triton X‐114 extraction of the microsomal proteins. Cell fractionation using continuous sucrose gradients showed that the protein is present in membrane fractions with high activity of endoplasmic reticulum (ER) and Golgi marker enzymes. Using nitrocellulose tissue prints probed with anti‐HSA antibodies, we demonstrated that the protein is present in the apoplastic space of petioles, suggesting that the protein is secreted to the apoplast of cortex cells in plants. Localization and binding properties suggest that the plant protein identified in the present study may participate in secretion processes, possibly involved with the transport of precursors required for cell‐wall synthesis.
Enhanced ATP‐dependent copper efflux across the root cell plasma membrane in copper‐tolerant Silene vulgaris
Authors: Van Hoof Nathalie A. L. M. , Koevoets Paul L. M. , Hakvoort Henk W. J. , Ten Bookum Wilma M. , Schat Henk , Verkleij Jos A. C. , Ernst Wilfried H. O.
Published: March 2003

We studied copper uptake in inside‐out plasma membrane vesicles derived from roots of copper‐sensitive, moderately copper‐tolerant and highly copper‐tolerant populations of Silene vulgaris (Amsterdam, Marsberg and Imsbach, respectively). Plasma membrane vesicles were isolated using the two‐phase partitioning method and copper efflux was measured using direct filtration experiments. Vesicles derived from Imsbach plants accumulated two and three times more copper than those derived from Marsberg and Amsterdam plants, respectively. This accumulation was ATP‐dependent. Also, 9‐amino‐6‐chloro‐2‐methoxyacridine fluorescence quenching rates upon copper addition decreased in the order Imsbach>Marsberg>Amsterdam. Our results support the hypothesis that efflux of copper across the root plasma membrane plays a role in the copper tolerance mechanism in S. vulgaris.
Cloning and characterization of cDNA of the GPI‐anchored purple acid phosphatase and its root tissue distribution in Spirodela oligorrhiza
Authors: Nishikoori Miwa , Washio Kenji , Hase Akira , Morita Naoki , Okuyama Hidetoshi
Published: March 2003

A cDNA clone of the glycosylphosphatidylinositol (GPI)‐anchored purple acid phosphatase (PAP) has been obtained by a combination of cDNA library screening and 5′ rapid amplification of cDNA ends from Spirodela oligorrhiza plants grown under phosphate‐deficient (−P) conditions. The open reading frame of the S. oligorrhiza PAP cDNA consists of 1 365 bp encoding a 455 amino acid protein. Its deduced amino acid sequence shows 82 and 80% similarity to Arabidopsis thaliana and Phaseolus vulgaris PAP, respectively. The amino acid residue, Ala439, followed by two more small amino acid residues, Asp and Ser, is predicted to be the GPI‐anchoring (ω) site. The absence of a dibasic motif upstream of the putative ω site suggests that the PAP is a cell wall protein. This presumption is supported by the finding that PAP was released by digestion of the cell wall fraction with cellulase. The GPI anchor is speculated to be a signal for transporting PAP to the cell wall. Immunohistochemical results using −P plant roots demonstrate that PAP is preferentially distributed in the outermost cortical cells of roots but not in the epidermis, suggesting its role in acquiring inorganic phosphate under phosphate‐deficient conditions. Northern blot analysis using the S. oligorrhiza PAP cDNA as a probe demonstrates that expression of the PAP gene increased during growth of −P plants and this time‐dependent occurrence in mRNA levels of the PAP in −P plants was also observed in their protein and activity levels.
Activation of pyrophosphate:fructose‐6‐phosphate 1‐phosphotransferase by fructose 2,6‐bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate‐deficient tobacco cells
Authors: Fernie Alisdair R. , Roscher Albrecht , Ratcliffe R. George , Kruger Nicholas J.
Published: March 2003

The aim of this work was to investigate the contribution of fructose 2,6‐bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6‐phosphofructo‐2‐kinase/fructose 2,6‐bisphosphatase that increased the fructose 2,6‐bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2‐deoxyglucose, a phosphate‐sequestering agent, and by growth of callus on phosphate‐depleted media. 31P‐NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6‐bisphosphate content of each line was unaffected by 2‐deoxyglucose and increased during growth on phosphate‐limited media. Short‐term treatment of callus with 2‐deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long‐term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U‐14C]glucose in response to incubation with 2‐deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate‐depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6‐bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate‐deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose‐6‐phosphate 1‐phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate‐depleted media, it is concluded that an increase in flux through pyrophosphate:fructose‐6‐phosphate 1‐phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.
Retardation and inhibition of the cation‐induced superoxide generation in BY‐2 tobacco cell suspension culture by Zn2+ and Mn2+
Authors: Kawano Tomonori , Kawano Nakako , Muto Shoshi , Lapeyrie Frédéric
Published: March 2003

Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt‐stress response. Recently, we have demonstrated that treatment of tobacco (Nicotiana tabacum) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li+, Na+, K+), alkali earth metals (Mg2+, Ca2+) or lanthanides (La3+, Gd3+). In this study, we tested the effect of extracellular supplementation of Zn2+ and Mn2+ on the cation‐induced oxidative burst in tobacco cell suspension culture, measured with a superoxide‐specific Cypridina luciferin‐derived chemiluminescent reagent. Extracellular supplementation of Zn2+ and Mn2+ inhibited the generation of superoxide in response to addition of salts. Although both Zn2+ and Mn2+ inhibited the salt‐induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn2+ simply inhibited total production of superoxide while Zn2+ inhibited the early phase of superoxide production and induced the slow release of superoxide. Roles of Mn2+ and Zn2+ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane‐bound NADPH oxidase, respectively, are discussed.
Peach (Prunus persica) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness
Authors: Bregoli Anna M. , Scaramagli Sonia , Costa Guglielmo , Sabatini Emidio , Ziosi Vanina , Biondi Stefania , Torrigiani Patrizia
Published: March 2003

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 mM), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 mM), spermidine (0.1, 1 and 5 mM) and spermine (2 mM) on peach (Prunus persica L. Batsch cv. Redhaven) fruit ripening was evaluated under field conditions. Treatments were performed 19 (polyamines) and 8 (AVG) days before harvest. Fruit growth (diameter, fresh and dry weight), flesh firmness, soluble solids content and ethylene emission were determined on treated and untreated (controls) fruits. Moreover, endogenous polyamine content and S‐adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity were determined to check for a possible competition between polyamines and ethylene for their common precursor S‐adenosylmethionine (SAM). Both treatments strongly inhibited ethylene emission and delayed flesh softening. On a biochemical level, AVG and exogenous polyamines both reduced the free‐to‐conjugate ratio of endogenous polyamines, and transiently altered SAMDC activity. The possible use of these compounds to control fruit ripening is discussed also in the light of their rejuvenating effect on peach fruits.
Pyrophosphate import and synthesis by plant mitochondria
Authors: Casolo Valentino , Micolini Stefano , Macrì Francesco , Vianello Angelo
Published: March 2003

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea (Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 mM. Pyrophosphate could enter from the cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F– and Ca2+ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (ΔΨ) in succinate‐energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP‐stimulated oxygen consumption that was inhibited by F– or Ca2+. In a similar experiment, ADP induced the dissipation of ΔΨ when it was added after the succinate‐generated ΔΨ had reached a steady state and, again, F– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H+‐ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H+‐PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F– or Ca2+. The physiological role of the mitochondrial H+‐PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction.
Secondary xylem development in Arabidopsis: a model for wood formation
Authors: Chaffey Nigel , Cholewa Ewa , Regan Sharon , Sundberg Björn
Published: March 2003

Our understanding of the molecular controls regulating the identity of the vascular cambium and the development of secondary xylem and phloem have not yet benefited much from the use of Arabidopsis as a genetic system. Under appropriate growth conditions Arabidopsis undergoes extensive secondary growth in the hypocotyl, with the development of both a vascular and a cork cambium. The secondary xylem of the hypocotyl develops in two phases, an early phase in which only vessel elements mature and a later stage in which both vessel elements and fibres are found. During this second phase the secondary xylem of Arabidopsis closely resembles the anatomy of the wood of an angiosperm tree, and can be used to address basic questions about wood formation. The development of the vascular cambium and secondary growth in Arabidopsis hypocotyl is described and its utility as a model for wood formation in trees is considered.
The third myrosinase gene TGG3 in Arabidopsis thaliana is a pseudogene specifically expressed in stamen and petal
Authors: Zhang Jiaming , Pontoppidan Bo , Xue Jiaping , Rask Lars , Meijer Johan
Published: March 2003

Genomic clones and full‐length cDNA for the myrosinase gene TGG3 from Arabidopsis thaliana ecotype Columbia were sequenced. The TGG3 gene was similar with the earlier described myrosinase genes and shared the conserved intron/exon splice sites but had an insertion of one nucleotide in exon 5, a deletion of two nucleotides in exon 6 and a deletion of approximately 210 nucleotides in exon 12. These mutations shifted the open reading frame in exon 5 and resulted in a truncated protein. Analysis of the TGG3 DNA sequence from five other Arabidopsis ecotypes showed polymorphisms, but in no case did a functional TGG3 gene appear to be present. Although TGG3 apparently is a pseudogene, it was expressed specifically in stamen and petal according to RT‐PCR analysis, while TGG1 and TGG2 transcripts were present in most of the tested tissues. Western blot analysis showed only one myrosinase band of 68 kDa corresponding to TGG1 and TGG2 in flower samples, while no band corresponding to TGG3 was encountered. Apparently only two functional myrosinases are present in this gene family in Arabidopsis.
Cloning and functional characterization of a homoglutathione synthetase from pea nodules
Authors: Iturbe‐Ormaetxe Iñaki , Heras Begoña , Matamoros Manuel A. , Ramos Javier , Moran Jose F. , Becana Manuel
Published: March 2003

The thiol tripeptide glutathione (GSH; γGlu‐Cys‐Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; γGlu‐Cys‐βAla), in addition to or instead of GSH. We have isolated from a pea (Pisum sativum L.) nodule library a cDNA, GSHS2, that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min−1 mg−1 protein with a standard substrate concentration (5 mM β‐alanine) and Km values of 1.9 mM for β‐alanine and 104 mM for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3‐fold more specific for β‐alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.
Plant aquaporins
Authors: Baiges Isabel , Schäffner Anthony R. , Affenzeller Matthias J. , Mas Alberto
Published: March 2003

Aquaporins are ubiquitous membrane channel proteins that facilitate and regulate the permeation of water across biological membranes. Aquaporins are members of the MIP family and some of them seem to be also able to transport other molecules such as urea or glycerol. In the plant kingdom, a single plant expresses a considerably large number of MIP homologues. These homologues can be subdivided into four groups (PIP, TIP, NIP, SIP) with highly conserved amino acid sequences and intron positions in each group. Since their discovery, advancing knowledge of their structure led to an understanding of the basic features of the water transport mechanism. An optimal water balance is essential to the homeostasis of most organisms, and aquaporins may be one of the mechanisms involved under changing environmental and developmental conditions. In fact, this may be one reason for the abundance and diversity of aquaporins, in particular in plants. In addition, exposure to different types of stress alters water relations and thus, aquaporins may be involved in stress responses as well. The transcriptional and/or post‐translational regulation of aquaporins would determine changes in membrane water permeability. Both phosphorylation and translocation to/from vesicles have been reported as post‐translational mechanisms. However, translocation in plants has not yet been shown. Although significant advances have been achieved, complete understanding of aquaporin function and regulation remains elusive.
Salicylic acid modulates ozone‐induced hypersensitive cell death in tobacco plants
Authors: Pasqualini Stefania , Della Torre Guido , Ferranti Francesco , Ederli Luisa , Piccioni Claudia , Reale Lara , Antonielli Marisa
Published: March 2003

Ozone‐tolerant Bel B and ozone‐sensitive Bel W3 tobacco cultivars were subjected to acute ozone fumigation (200 p.p.b. for 3 h) and the subcellular localization of H2O2 was then studied. H2O2 accumulated on the cell walls and plasma membrane of both cultivars but the accumulation pattern differed greatly. H2O2 production was high in both cultivars immediately after fumigation, but, in the tolerant Bel B cultivar, after 7 h was only detected in some spongy cells adjacent to epidermal cells. Instead, in the sensitive Bel W3 cultivar, accumulation was still abundant in the cell walls of palisade, spongy and epidermal cells at this time. Significant changes in apoplastic ascorbate pool were noted in both cultivars in the first hours after fumigation. As the reduced ascorbate content remained unchanged, the marked increase in total ascorbate must have originated from the striking increase in dehydroascorbate, particularly in the ozone‐sensitive Bel W3. Exposure of plants to ozone resulted in a marked transient increase in both free and conjugated salicylic acid (SA) as well as an increase in the activity of benzoic acid 2‐hydroxylase which catalyses SA biosynthesis. SA induction differed greatly in the two cultivars, in that: (1) SA accumulation was far greater in the ozone‐sensitive Bel W3 cv. and (2) the maximum SA peak was delayed in Bel W3 and observed only 7 h after fumigation ended. These results suggest that a high SA content, as documented in the ozone‐sensitive Bel W3 cultivar, could trigger the production of ROS with subsequent SA‐mediated cell‐death.
Scopoletin expression in elicitor‐treated and tobacco mosaic virus‐infected tobacco plants
Authors: Costet Laurent , Fritig Bernard , Kauffmann Serge
Published: March 2003

Localized acquired resistance (LAR) characterizes a narrow zone of living cells expressing strong defense responses and surrounding cells undergoing a hypersensitive response (HR). In Samsun NN tobacco plants, tissues undergoing tobacco mosaic virus‐induced or elicitor‐induced LAR exhibit a strong blue fluorescence under UV light. We have shown that scopoletin and its glucoside, scopolin, accounted for the fluorescence: (1) both compounds were identified after extraction and purification by thin layer and high performance liquid chromatography; (2) there was a strict correlation between the occurrence of fluorescence and accumulation of high amounts of scopoletin; and (3) infiltration of commercial scopoletin caused a similar fluorescence to that occurring in LAR tissues. There was a 20‐fold increase in scopoletin levels in LAR tissues compared to tissues treated with a non‐HR dose of elicitor, while PR1 protein accumulated in similar amounts in both types of tissues. Scopoletin was able to suppress the elicitor‐induced HR only when co‐infiltrated with very low HR‐dose of elicitor. These two observations suggested that, although scopoletin alone would not be able to control the development of the HR through its known antioxidant activity, it may nevertheless participate to such function of LAR tissues in combination with other antioxidant molecules.
Photoinhibition and loss of photosystem II reaction centre proteins during senescence of soybean leaves. Enhancement of photoinhibition by the ‘stay‐green’ mutation cytG
Authors: Guiamét Juan J. , Tyystjärvi Esa , Tyystjärvi Taina , John Isaac , Kairavuo Marja , Pichersky Eran , Noodén Larry D.
Published: March 2003

The ‘stay‐green’ mutation cytG in soybean (Glycine max) partially inhibits the degradation of the light‐harvesting complex II (LHCII) and the associated chlorophyll during monocarpic senescence. cytG did not alter the breakdown of the cytochrome b6/f complex, thylakoid ATP synthase or components of Photosystem I. In contrast, cytG accelerated the loss of oxygen evolution activity and PSII reaction‐centre proteins. These data suggest that LHCII and other thylakoid components are degraded by separate pathways. In leaves induced to senesce by darkness, cytG inhibited the breakdown of LHCII and chlorophyll, but it did not enhance the loss of PSII‐core components, indicating that the accelerated degradation of PSII reaction centre proteins in cytG was light dependent. Illumination of mature and senescent leaves of wild‐type soybean in the presence of an inhibitor (lincomycin) of chloroplast protein synthesis revealed that senescence per se did not affect the rate of photoinhibition in leaves. Likewise, mature leaves of the cytG mutant did not show more photoinhibition than wild‐type leaves. However, in senescent cytG leaves, photoinhibition proceeded more rapidly than in the wild‐type. We conclude that the cytG mutation enhances photoinhibition in senescing leaves, and photoinhibition causes the rapid loss of PSII reaction‐centre proteins during senescence in cytG.
Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases
Authors: Rubio Maria C. , González Esther M. , Minchin Frank R. , Webb K. Judith , Arrese‐Igor Cesar , Ramos Javier , Becana Manuel
Published: March 2003

The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants (Medicago sativa L. × Sinorhizobium meliloti 102F78) of the elite genotype N4 and three derived transgenic lines have been studied in detail. These transgenic lines overproduced, respectively, Mn‐containing superoxide dismutase (SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all lines, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves and nodules, but had distinct tissue‐dependent effects on the activities of the peroxide‐scavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and catalase activities increased moderately in leaves but decreased in nodules. At mild water stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the parental line, which suggests a superior tolerance of transgenic plants under these conditions. However, the untransformed and the transgenic plants performed similarly during moderate and severe water stress and recovery with respect to important markers of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the base genotype used for transformation and the background SOD isozymic composition may have a profound effect on the relative tolerance of the transgenic lines to abiotic stress.
Identification of a grapefruit cDNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions
Authors: Porat Ron , Pavoncello David , Lurie Susan , Mccollum T. Gregory
Published: March 2003

Citrus fruits are sensitive to low temperatures and this often results in the development of chilling injuries during postharvest storage. In order to gain more insight into the molecular mechanisms involved in the acquisition of fruit chilling tolerance, we initiated a grapefruit (Citrus paradisi, cv. Marsh Seedless) flavedo cDNA sequencing project and used it to identify a cDNA similar to other Poncirus trifoliata and Citrus unshiu dehydrin genes reported to be responsive to low temperatures. The grapefruit dehydrin cDNA, designated cor15, encodes a predicted polypeptide of 15.1 kDa, that is almost completely identical with other reported citrus dehydrin proteins, except that it contains two large amino acid repeats, whereas P. trifoliata COR11 has only one such repeat and P. trifoliata COR19 and C. unshiu COR19 have three repeats. Together, the various grapefruit, P. trifoliata and C. unshiu dehydrins form a closely related and unique dehydrin gene family that differs from most other plant dehydrins in having an unusual K‐segment similar to that of gymnosperms and in having a serine cluster (S‐segment) at an unusual position at the carboxy‐terminus. The grapefruit cor15 gene is consistently expressed in the fruit peel tissue at harvest, but its message levels dramatically decrease during storage at 2°C. However, a pre‐storage hot water treatment, which enhances fruit chilling tolerance, elicited retention of the constant level of cor15 gene expression during cold storage and eliminated its decline. The hot water treatment had no inductive effect on cor15 gene expression when the fruit were held at non‐chilling temperatures. The effects of other stresses, such as exposure to ethylene, UV irradiation and wounding, on cor15 gene expression, were temporary and persisted for 1‐2 days after the treatments.
Enhanced H+/ATP coupling ratio of H+‐ATPase and increased 14‐3‐3 protein content in plasma membrane of tomato cells upon osmotic shock
Authors: Kerkeb Loubna , Venema Kees , Donaire Juan Pedro , Rodríguez‐Rosales María Pilar
Published: March 2003

Modulation of proton extrusion and ATP‐dependent H+ transport through the plasma membrane in relation to the presence of 14‐3‐3 proteins in this membrane in response to osmotic shock was studied in tomato (Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H+ extrusion by cells was activated rapidly and significantly after adding 100 mM NaCl, 100 mM KCl, 50 mM Na2SO4, 1.6% sorbitol or 2 µM fusicoccin to the medium. The increase in H+ extrusion by cells treated with 100 mM NaCl was correlated with an increase of H+ transport by the plasma membrane H+‐ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14‐3‐3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 mM NaCl as compared to control cells without changing the amount of plasma membrane H+‐ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14‐3‐3/H+‐ATPase complex.
Phenylalanine ammonia lyase gene expression during abscission in citrus
Authors: Kostenyuk Igor A. , Zoń Jerzy , Burns Jacqueline K.
Published: March 2003

Phenylalanine ammonia lyase (PAL) gene expression was examined in fruit and leaf abscission zones of Valencia orange for periods up to 72 h after induction of abscission with Ethrel® (CEPA) or the citrus mature fruit‐specific abscission material 5‐chloro‐3‐methyl‐4‐nitro‐1H‐pyrazole (CMN‐P). PAL gene expression was detected in mature fruit abscission zones beginning 6 and 24 h after CEPA and CMN‐P application, respectively, and continued up to 72 h. PAL gene expression was detected in leaf abscission zones 6 h after CEPA application and continued throughout the remainder of the study. In contrast, PAL gene expression was not detected in leaf abscission zones treated with CMN‐P. 2‐aminoindan‐2‐phosphonic acid (AIP), a specific inhibitor of PAL enzyme activity, decreased CMN‐P‐induced PAL expression in fruit abscission zones, and this was accompanied by a lack of fruit drop. PAL transcripts were low in abscission zones of immature fruit; however, the transcript was induced by CMN‐P but less by CEPA application. The data suggest that downstream products of PAL activity may be important not only for wound healing and defense reactions that occur at the abscission zone fracture plane, but also for regulation of PAL gene expression during abscission.
A unique pantoyllactone glycoside system is activated in rice seedlings developing aerobically in the dark
Authors: Menegus Faustino , Lilliu Ilaria , Scaglioni Leonardo
Published: March 2003

Rice (Oryza sativa L.) seedlings developing aerobically in the dark accumulated in the expanding coleoptile millimolar amounts of pantoyllactone glucoside (PLG) together with sugars and amino acids. Following leaf emergence and seedling development, the transformation of PLG into pantoyllactone primeveroside (PLP) was initiated inside the coleoptile, whose senescence was suspended in spite of the exhaustion of seed reserves and cessation of shoot growth. PLG, PLP and most of the sugars and amino acids remained inside the coleoptile. Light administration triggered shoot greening and coleoptile senescence that was accompanied by the conversion of PLG into the shoot‐translocatable species PLP and by the utilization of the coleoptile‐stored metabolites. Light‐induced retrieval of amino acids and sugars from the coleoptile was 90% complete within 24 h while translocation of pantoyllactone glycosides started only 24–48 h after light administration. Conversely, no accumulation of PLG and PLP was detected in seedlings germinated and grown either aerobically in the light or in the dark under hypoxic conditions. The possible significance of the activation of the PLG‐PLP system under specific environmental conditions is discussed.
Light‐dependent photosynthetic characteristics indicated by chlorophyll fluorescence in five mangrove species native to Pohnpei Island, Micronesia
Authors: Kitao Mitsutoshi , Utsugi Hajime , Kuramoto Shigeo , Tabuchi Ryuichi , Fujimoto Kiyoshi , Lihpai Saimon
Published: March 2003

To investigate the adaptive capacity to strong light among mangrove species, we examined light‐dependent properties of photosynthesis in relation to photoinhibition using chlorophyll fluorescence for sun‐leaves of five mangrove tree species; Sonneratia alba, Rhizophora stylosa, Rhizophora apiculata, Bruguiera gymnorrhiza and Xylocarpus granatum. These species were classified into three groups; pioneer —S. alba and R. stylosa, intermediate —R. apiculata and B. gymnorrhiza and climax species —X. granatum. A further distinction can be made between the two intermediate species, the less shade‐tolerant R. apiculata and the shade‐tolerant B. gymnorrhiza. There was a clear association between successional status and light‐saturated electron transport rate (ETR) where ETR was higher in S. alba and R. stylosa > R. apiculata and B. gymnorrhiza > X. granatum. Based on its lower initial slope of light‐dependent ETR and lower ratio of variable to maximum fluorescence (Fv/Fm) after a dark‐adaptation, X. granatum appeared to suffer greater photoinhibition than other species. The response of qP, which represents PSII openness, to incident light indicates a ranking of tolerant capacity to photoinhibition of S. alba and R. stylosa > R. apiculata > B. gymnorrhiza and X. granatum. The difference in qP response between R. apiculata and B. gymnorrhiza might be attributed to the higher leaf absorptance and the lower degree of thermal dissipation indicated by the higher F′v/F′m in B. gymnorrhiza. It appears that the co‐ordination of leaf absorptance, thermal dissipation and consumption of absorbed light energy through electron transport is important in the acclimation of mangrove species to exposed habitats.
Phytochrome controlled signalling cascades in higher plants
Authors: Kevei Éva , Nagy Ferenc
Published: March 2003

Plants can sense the changes in the environmental light conditions with highly specialized photoreceptors. Phytochromes are sensitive to red and far‐red light and have a dual role in the life of plants. These photoreceptors play an important role in plant growth and development from germination to seed maturation and they are also involved in synchronizing the circadian clock with light/dark cycles. Biochemical, cell biological and genetic studies have been carried out to elucidate the molecular mechanism by which phytochromes transduce light signals. A major step in this process seems to be the light‐dependent nuclear import of phytochromes. In the nuclei phytochromes interact with transcription factors and regulate the expression of numerous genes, resulting in complex physiological and developmental responses to light. This review focuses on the recently obtained results leading to the identification of some factors and processes involved in phytochrome signalling.
Infective endocarditis in renal transplant recipients
Authors: Bishara J. , Robenshtok E. , Weinberger M. , Yeshurun M. , Sagie A. , Pitlik S.
Published: March 2003

Because of the increasing number of renal transplantations performed and the rarity of reported cases of infective endocarditis in these patients, we studied the clinical characteristics of this infection in this population. We report on two cases from our experience and review reported cases of infective endocarditis in renal transplant recipients retrieved from the MEDLINE system. In addition, we reviewed a large series of infective endocarditis looking for patients with renal transplants. In addition to our 2 cases, 12 previously reported cases were found. The mean time from transplantation to diagnosis of infective endocarditis was 3.5 years (range 2 months to 15 years). Causative organisms included fungi, Staphylococcus aureus (3 cases each), Corynebacterium sp. (2 cases), Streptococcus viridans, VRE, Brucella sp., Clostridium sp., Nocardia sp. and Erysipelothrix sp. (one case each). Skin manifestations of endocarditis and/or splenomegaly were not reported in these patients. Septic emboli and mycotic aneurysms were relatively common. The overall mortality rate was 50% (7 of 14 patients died). Infective endocarditis seems to be rare in renal transplant recipients. The few reported cases are characterized by unusual causative micro‐organisms and atypical clinical presentation. Further studies are needed to delineate the magnitude and scope of this association.
Infection in solid organ transplantation
Authors: Snydman D.R.
Published: March 2003

Infection in the organ transplant recipient is a major determinant of the outcome of transplantation. The attributable mortality from invasive fungal disease and cytomegalovirus disease has been 50 and 30%, respectively. Determinants of infection include epidemiologic exposures to environmental pathogens or exposure to latent infection in the donated organ or recipient, and changes in the net state of immunosuppression. For the past two decades significant improvements have been made in the recognition, prevention and treatment of infections, such as cytomegalovirus and P. carinii. Future research will need to focus on treatment of invasive mold infections, and the management of hepatitis B and C in liver transplantation. In addition, improvement in the prevention of the post‐transplant lymphoproliferative disease syndrome will be necessary (
Note).
Historical overview of the use of cytomegalovirus hyperimmune globulin in organ transplantation
Authors: Snydman D.R.
Published: March 2003

A historical review of the development of cytomegalovirus hyperimmune globulin (CMV‐IG, CytoGam®) shows its increasing use in solid organ transplant (SOT) recipients, either alone or in combination with ganciclovir. A review of clinical trials of CytoGam in renal transplant recipients shows reductions in CMV‐associated syndromes and fungal and parasitic superinfections, and increases in graft survival, while CytoGam prophylaxis trials in orthotopic liver transplant (OLT) recipients have produced reductions in severe CMV‐associated disease and invasive fungal disease. A combination of CytoGam plus ganciclovir in OLT recipients has resulted in reductions in CMV hepatitis and infection, and CMV disease and viremia, plus a trend in improved 1‐ and 2‐year survival rates.
Antibody inhibition of cytomegalovirus: the role of natural killer and macrophage effector cells
Authors: Forthal D.N. , Phan T. , Landucci G.
Published: March 2003

To explore mechanisms by which antibody might inhibit cytomegalovirus (CMV), we measured the ability of intravenous CMV‐IgG (CytoGam® to reduce viral yield in the presence of effector cells. Foreskin fibroblasts were infected with a clinical strain of CMV, and CytoGam® was added along with effector cells consisting of either unfractionated peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, or macrophages. The combination of CytoGam® and either of the effector cell types markedly inhibited established CMV infection in vitro. In addition, CytoGam® combined with effector cells protected the monolayer from CMV‐induced cytopathic effects. Antibody‐dependent, effector cell‐mediated functions may underlie the ability of CytoGam® to prevent or modulate CMV infection in vivo.