bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of embryo growth in mature fruits of celery (Apium graveolens)
Authors: van der Toorn Peter , Karssen Cees M.
Published: May 2006

A celery ‘seed’ is a schizocarpic fruit and consists largely of endosperm surrounded by a thin testa and a thick pericarp. Embryo growth in fruits of celery (Apium graveolens L. var. rapaceum [Miller] D. C.) cv. Monarch with low and high densities was studied during incubation in water and in polyethylene glycol (PEG). Embryo growth in water was characterized by both cell division and an increase in cell size, while embryo growth in PEG solutions solely occurred through cell division. Low density fruits differed from high density fruits in a higher cell division rate during incubation both in water and PEG; in water also the cell size of the embryo was larger. As a consequence, the low density fruits reached the embryo length of 1.5 mm, which was critical for radicle protrusion, already after 6.1 days in water and 1.7 days before the high density fruits. In ‐1.2 MPa PEG the embryos in both fractions reached their maximum length of 0.9 and 0.7 mm in low and high density fruits, respectively, after 13 days of incubation. When incubation for 17 days in PEG was followed by incubation in water, the embryo growth in both fractions was faster than without a pre‐incubation in PEG and also the cell size increased. The differences between the fractions persisted, however. It was concluded that embryo cell wall extensibility increased during PEG‐incubation. During incubation in water, low density fruits had a higher water content than high density fruits. It was concluded that this difference was caused by a lower pericarp pressure potential (Ψp, pericarp). When the pericarp was removed, the differences in water content disappeared, the cell size in both fractions became similar, and the difference in mean germination time was reduced. The difference in embryo growth rate of the two fruits during incubation both in water and in PEG could be eliminated by lowering the osmotic potential (Ψπ) of the incubation medium of low density fruits. It was concluded that the pericarp of celery fruits is an important factor in regulation of embryo growth.
The CO2concentrating mechanism in cyanobactiria and microalgae
Authors: Badger Murray R. , Price G. Dean
Published: May 2006

Over the past 10 years it has become clear that cyanobacteria and microalgae possess mechanisms for actively acquiring inorganic carbon from the external medium and are able to use this to elevate the CO2 concentration around the active site of the primary photosynthetic carboxylating enzyme, ribulose bisphosphate carboxylase‐oxygenase (Rubisco). This results in a vastly enhanced photosynthetic affinity for inorganic carbon (Ci) and improved photosynthetic efficiency. The CO2 concentrating mechanism is dependent on the existence of membrane bound Ci transport systems, and a microenvironment within the cell where the accumulated Ci can be used to elevate CO2 at the site of Rubisco. Evidence presented in this review suggests that in cyanobacteria this is achieved by the packaging of Rubisco and carbonic anhydrase (CA) into discrete structures, which are termed carboxysomes. Analogous structures in microalgae, termed pyrenoids, may perform a similar function. The recovery and analysis of high‐CO2‐requiring mutants has greatly advanced our understanding of the mechanisms and genes underlying these systems, especially in cyanobacteria, and this review places particular emphasis on the contribution made by molecular genetic approaches.
Structure, function and regulation of nitrate reductase in higher plants
Authors: Hoff Tine , Stummann Bjarne M. , Henningsen Knud W.
Published: May 2006

Two types of nitrate reductase (NR) have been recognized in higher plants. Most plants contain a NADH‐specific NR, a few plants contain only a NAD(P)H‐bispecific NR and some plants contain both NR types. Most NRs are nitrate inducible. Many NRs are expressed in both roots and leaves, but in some species a root‐specific NR has been identified. All NRs from higher plants are dimers of identical subunits with 881 (bean) to 926 (spinach) amino acid residues. The complete nucleotide sequence of NR genes or cDNAs from several higher plants have been obtained. The amino acid identity between the species analyzed ranges from 60 to 90%. NR is organized into 3 domains containing FAD, heme and a molybdenum cofactor. Electrons are transferred from NAD(P)H to nitrate via these cofactors. Nitrate triggers transcription of inducible genes for NR. NR activity and mRNA accumulation can be induced to a low level in etiolated and green dark‐adapted plants by addition of nitrate. The accumulation of NR mRNA and activity is strongly stimulated by subsequent exposure to white light. Light also stimulates NR mRNA translation and/or the stability of the NR protein. In light/dark grow plants the mRNA level generally peaks at the end of the dark periods and the NR activity peaks a few hours later. These diurnal oscillations are controlled via transcription initiation.
Fatty acid composition and the hydrophilic properties of phospholipids in seedlings of spring and winter wheat growing at 20°C and 2°C
Authors: Filek Maria , Dubert Franciszek , Skoczowski Andrzej
Published: May 2006

The work of adhesion between water and a heptane solution of phospholipids extracted from spring (cv. Jara) and winter (cv. Grana) wheat (Triticum aestivum L.) grown at 2°C and 20°C was measured in order to determine the effect of changes in the composition of fatty acids on the hydrophilic properties of the lipid bilayer of membranes. The work of adhesion water/phospholipids was positively correlated with the contents of the oleic and linoleic acids (18:1 and 18:2, respectively) and negatively correlated with the content of linolenic acid (18:3) in the phospholipids. The sensitivity of the work of adhesion to changes in the particular fatty acids decreased with increasing number of double bonds. In addition, it depended on the genotype and on the temperature of growth of the seedlings.
Role of roots and shoots in the regulation of the Fe efficiency responses in sunflower and cucumber
Authors: Romera Francisco J. , Alcántara Esteban , De La Guardia Manuel Díaz
Published: May 2006

Several experimental approaches were used to study the role of roots and shoots in the regulation of the Fe efficiency responses in the roots of Fe deficient plants of sunflower (Helianthus annuus L., inbred line RHA 274) and cucumber (Cucumis sativus L., cv. Burpee pickler). The presence of the shoot apex and the youngest leaves in sunflower and cucumber plants was not indispensable to the development of the responses. In split‐root experiments with sunflower plants, with either one or two shoots obtained by grafting, different parts of the root system could receive different Fe additions. Roots growing in a medium without Fe developed Fe efficiency re‐sponses even when the other part of the root system was growing with Fe. Also, the part of the root system that was growing in a medium with Fe, developed some Fe efficiency responses, although delayed and at a lower level as compared with the root without external Fe. The results are discussed in relation to the idea that the development of the responses in the roots is controlled not only by the Fe content in the root, but also by some information transmitted between parts of a root system that differ in their Fe nutritional status.
Effects of turgor potential on 1‐aminocyclopropane‐1‐carboxylic acid uptake into the vacuolar compartment and ethylene production in tomato pericarp slices
Authors: Saftner Robert A.
Published: May 2006

While solute transport and ethylene production by plant tissue are sensitive to the osmotic concentration of the solution bathing the tissue, the influence of tissue water relations and specifically tissue turgor potential on the kinetics of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) uptake into the vacuolar compartment and ethylene production have not been examined. 1‐Aminocyclopropane‐1‐carboxylic acid transport and ethylene production were examined in tomato (Lycopersicon esculentum Mill. cv. Liberty) pericarp slices incubated in solutions having a range of mannitol, polyethylene glycol 3350 and ethylene glycol concentrations known to affect tissue water relations. Tissue osmotic and turgor potentials were derived from osmolality measurements of cell saps recovered by freeze‐thawing and corrected for the contribution of the free‐space solution. When relatively nonpermeable (mannitol or polyethylene glycol 3350) osmotica were used, both ACC uptake and ethylene production were greatest at a solution osmolality of 230 milliosmolal where tissue turgor potential ranged between 120 and 140 kPa. At higher and lower turgor potentials, the high‐affinity saturating component of ACC uptake and ethylene production were inhibited, and ACC efflux from the vacuolar compartment was increased. The inhibition of ACC uptake was evident as a decrease in Vmax with no effect on Km. Turgor potential changes caused by adjusting solution osmolality with mannitol or polyethylene glycol 3350 were accompanied by changes in the osmotic potential and water potential of the tissue. The effects of turgor potential vs the osmotic and water potentials of tomato pericarp slices were differentiated by comparing responses to nonpermeable osmotica and mixtures of nonpermeable and permeable osmotica. Ethylene glycol‐mannitol mixtures had effects on the osmotic potential and water potential of the tissue similar to those of nonpermeable osmotica but had less effect on tissue turgor, ACC transport and ethylene production. Incubating tissue in solutions without nonpermeable osmotica osmotically shocked the tissue. Increasing solution osmolality with ethylene glycol in the absence of nonpermeable osmotica increased tissue turgor and ethylene production. The present study indicates that tissue turgor is an important factor affecting the kinetics of ACC uptake into the vacuolar compartment and ethylene production in tomato pericarp slices.
Galactomannan hydrolyzing activity develops during priming in the micropylar endosperm tip of tomato seeds
Authors: Nonogaki Hiroyuki , Matsushima Hisashi , Morohashi Yukio
Published: May 2006

Development of galactomannan hydrolyzing activity was followed in seeds of tomato [Lycopersicon esculentum (L.) Mill. cv. Toyonishiki] during priming and germination. The activity developed in seeds that were being primed in polyethylene glycol (‐0.8 MPa). The activity was detected exclusively in the endosperm portion just adjacent to the radicle tip. Part of the activity remained active after desiccation of the primed seeds. After transfer to water, the activity in the primed seeds immediately began to increase, while in unprimed seeds the beginning of the increase in activity was delayed by about 1 day. In scanning electron microscopy, the inner surface of the cell walls of the micropylar endosperm portion appeared eroded in primed seeds that had been imbibed in water for 16 h (before germination), but not in unprimed seeds imbibed for the same period. These results support the hypothesis that galactomannan hydrolyzing enzyme, which is believed to be responsible for breakdown of tomato endosperm cell walls and hence for the weakening of mechanical restraint against radicle growth, may be involved in the improved germination of primed tomato seeds.
Phenylalanine ammonia‐lyase activity and capsaicin‐precursor compounds in p‐fluorophenylalanine‐resistant and ‐sensitive variant cells of chili pepper (Capsicum annuum)
Authors: Ochoa‐Alejo Neftalí , Salgado‐Garciglia Rafael
Published: May 2006

Cell suspension cultures of chili pepper (Capsicum annuum L. cv. Tampiqueño 74) displaying differences in their resistance to p‐fluorophenylalanine (PFP) and in their contents of capsaicin (the compound which is responsible for the hot taste of chili pepper fruits) were characterized in relation to the activity of phenylalanine ammonia‐lyase (PAL; EC 4.3.1.5), the levels of free l‐phenylalanine, phenolics and the phenylpropanoid acids involved in capsaicin biosynthesis. A nonselected cell line, a sensitive line (CA‐02), a moderately resistant cell line (CA‐29) and two resistant cell lines (CA‐04 and CA‐16) were studied. Higher PAL activities and higher levels of phenylalanine and phenolics were found in the PFP‐resistant cells even after a minimum of 9 subcultures (15 days each) in the absence of the analog, indicating that the selected trait was stable. PFP‐resistant chili pepper cells accumulated higher amounts of capsaicin precursors (cinnamic, caffeic and ferulic acids) than either the nonselected cells or the sensitive cell line. p‐Coumaric acid was not detected at significant levels in any of the cell cultures. Overall, accumulation of free phenyl‐alanine correlated well with PAL activity, phenolics, phenylpropanoids and capsaicin levels, suggesting an active flow through the phenylpropanoid pathway in PFP‐resistant cells of chili pepper.
Culture of cotyledons of Douglas‐fir on a medium for the induction of adventitious shoots induces rapid changes in polypeptide profiles and mRNA populations
Authors: Campbell Michael A. , Gaynor John J. , Kirby Edward G.
Published: May 2006

Cotyledons of 3‐ to 4‐week‐old seedlings of Douglas‐fir [Pseudotsuga menziesii (Mirb). Franco] were treated with shoot induction medium (SIM) containing 5 μM 6N‐benzylaminopurine (BAP) and 5 nM naphthaleneacetic acid (NAA). Fresh weight, dry weight and soluble protein levels were not altered within the first 48 h of SIM treatment. SIM‐treated cotyledons were labelled in vivo with 35SO42‐, and TCA‐insoluble proteins were analyzed electrophoretically by a 2‐dimensional system consisting of non‐equilibrium electrophoresis (NEPHGE) followed by sodium dode‐cylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). A basic polypeptide with a relative molecular weight of 14.5 kDa was detected 32 h after induction and after 48 h a number of polypeptides in the 14 to 35 kDa range were induced. Translation products of poly‐A+ RNA isolated from cotyledons treated with SIM for 4, 16, 32 and 48 h were analyzed by using 2‐dimensional NEPHGE‐SDS‐PAGE. Both qualitative and quantitative differences in the translation products were observed at all time points investigated. Expression of a specific RNA coding for a 30 kDa polypeptide was demonstrated as early as 4 h after culture on SIM. This RNA was also present at 16 h, but decreased with longer SIM treatments. Thus, culture of excised cotyledons on a medium inducing the formation of adventitious shoots results in rapid quantitative and qualitative changes in the polypeptide composition and translatable RNA population prior to morphological evidence of shoot induction.
Immunogold localization of photosynthetic enzymes in leaves of Aristida latifolia, a unique C4 grass with a double chlorenchymatous bundle sheath
Authors: Ueno Osamu
Published: May 2006

C4 species of the genus Aristida (Poaceae) have 3 distinct types of photosynthetic cells in their leaves, the mesophyll (M) cells, the outer bundle sheath (BS) cells, and the inner BS cells, and exhibit a unique Kranz‐type leaf anatomy. The cellular localization of C3 and C4 photosynthetic enzymes was investigated in leaves of Aristida latifolia Domin by the protein A‐gold immunocytochemical technique. The outer BS cells contained centripetally located small chloroplasts, which were structurally similar to those of the M cells. The inner BS cells contained centrifugally located large chloroplasts, which lacked well‐developed grana and exhibited rudimentary grana for the most part. The leaves contained high levels of NADP‐malic enzyme (EC 1.1.1.40) activity, and the plant was classified as being of the NADP‐malic enzyme type. The immunocytochemical study revealed that labeling of ribulose 1,5‐bisphos‐phate carboxylase/oxygenase (EC 4.1.1.39) was present in the chloroplasts of the outer and inner BS cells, but was undetectable in the M cells. Labeling of phoshoen‐olpyruvate carboxylase (EC 4.1.1.31) was observed in the cytosol of M cells, but not in that of BS cells. By contrast, labeling of pyruvate, Pi dikinase (PPDK, EC 2.7.9.1) was evident not only in the chloroplasts of M cells but also in those of outer BS cells, but was absent from the inner BS cells. The density of labeling in the chloroplasts of M cells was higher than that in chloroplasts of outer BS cells. These results indicate that the two carboxylating enzymes are differentially distributed between the M cells and the two types of BS cells, whereas PPDK shows a more complex distribution pattern. The locations of these enzymes are discussed in relation to C4 photosynthesis.
Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA
Authors: Simmonds John , Stewart Pamela , Simmonds Daina
Published: May 2006

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.
Partial purification of plant plasma membrane K+, Mg2+‐ATPase by ion exchange chromatography
Authors: Bérczi Alajos , Morré D. James
Published: May 2006

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H+‐pumping ATPase (EC 3.6.1.35) from various plants. Soybean (Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two‐phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non‐ionic detergent (either Triton X‐100 or C12E8). The Mg‐ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl‐eluted fractions contained a Mg‐ATPase which showed the characteristics of Mg‐ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg−1 min−1 when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.
Conifer decline in the north‐east of France: Characteristic changes in chloroplast protein pattern and absence of anti‐oxidative defense capability point to an involvement of ozone
Authors: Guillemaut Pierre , Weber‐Lotfi Frédérique , Blache Denis , Prost Michel , Rether Benoit , Dietrich André
Published: May 2006

A method yielding intact and biologically active chloroplasts from needles of mature spruce (Picea abies L. cv. Karsten) or fir (Abies alba Mill.) growing in the field is presented. Using this method, chloroplasts were prepared from yellow and green needles of declining spruce and fir from two forest areas of the Vosges mountains (north‐east of France). Both quantitative and qualitative changes could be shown in the two‐dimensional chloroplast protein pattern of yellowing needles, as compared to that of apparently healthy needles. We observed that an accumulation of photosystem II D1‐protein in the thylakoids was associated with needle decline. Furthermore, a lack of resistance capability against free radical attack was observed in yellow needles. We have attempted to correlate our observations with pollutant levels and we propose that, in the forest areas studied, O3 is one of the main factors involved in conifer decline. It also appears from our results that decline and senescence are distinct phenomena.
Effect of nitrate limitation on the photosynthetically active pools of aspartate and malate in maize, a NADP malic enzyme C4 plant
Authors: Khamis Saadi , Lamaze Thierry , Farineau Jack
Published: May 2006

After two weeks of moderate N restriction, growth of 3‐week‐old Zea mays L. plants was less than half that of the control and aspartate and malate levels in the leaves were severely suppressed (45 and 65% decrease, respectively). Since in NADP malic enzyme type C4 plants, such as maize, malate and aspartate are intermediates in the C4 photosynthetic pathway, the operation of the latter was investigated. Moderate nitrogen deficiency had only a small effect on the rate of photosynthesis (20% decrease) measured under 1000 umol m−2 s−1 irradiance. 14CO2 pulse‐12CO2 chase experiments combined with measurements of in vitro photosynthetic enzyme activities demonstrated the operation of a typical C4 photosynthetic pathway in N‐restricted plants. The turnover rates of malate and aspartate molecules involved in the C4 cycle were determined by the loss of label in the carbon 4 moiety of these molecules during the chase period. It is shown that N restriction did not alter the turnover of malate but greatly accelerated that of aspartate. The amounts of malate and aspartate moving through photosynthetically active pools were estimated using a kinetic model. For malate, the size of this pool appeared to be only slightly diminished whereas for aspartate the size of the corresponding pool decreased by a factor of 3. It is proposed that under moderate NO3− deficiency, despite deviations in malate metabolism leading to a pronounced decrease in the size of its cellular pool, a large amount of malate remained in the operation of the C4 pathway. By contrast, the participation of aspartate in the operation of the C4 pathway was greatly reduced.
Sonication: A new method for gene transfer to plants
Authors: Joersbo Morten , Brunstedt Janne
Published: May 2006

Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects are reviewed. The state of the art of acoustic transformation is presented and possible mechanisms are discussed.
Genetics and molecular biology of nitrate metabolism in higher plants
Authors: Warner Robert L. , Kleinhofs Andris
Published: May 2006

Nitrate uptake and reduction are highly regulated processes. In many plant species, nitrate uptake is induced by nitrate, Little, however, is known about the genetic and molecular aspects of nitrate transport. Reduction of nitrate to ammonia is carried out by nitrate and nitrite reductases. Nitrate and light enhance expression of the nitrate and nitrite reductase genes in most species. Mutants have been selected and characterized to identify genes controlling nitrate reductase in several higher plant species. Six loci are known to control the synthesis or assembly of the molybdenum cofactor of nitrate reductase, xanthine dehydrogenase and aldehyde oxidase. The nitrate reductase apoenzyme is encoded by a single gene, except in allopolyploid species and in those species possessing both NADH‐specific and NAD(P)H‐bispecific nitrate reductases. Comparison of NADH‐specific nitrate reductase amino acid sequences deduced from cloned genes reveals considerable sequence conservation in regions believed to encode the functional domains of nitrate reductase, but less conservation in the N‐terminal and hinge regions of the enzyme. For both nitrate and nitrite reductases, sequence identity is greater among species of the same subclass than between Monocotyledoneae and Dicotyledoneae subclass species.
Control of nitrogen and carbon metabolism in root nodules
Authors: Vance Carroll P. , Gantt J. Stephen
Published: May 2006

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O2 limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O2‐limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO2 fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O2 regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH‐glutamate synthase (NADH‐GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.
Advances in cereal protoplast research
Authors: Vasil Indra K. , Vasil Vimla
Published: May 2006

Beginning in 1986, plants have been regenerated from protoplasts of all of the important cereal species, including wheat, rice, maize, and barley, and grasses such as sugarcane. In addition, somatic hybrids/cybrids as well as transgenic plants with introduced useful agronomic traits have been obtained in several instances. This rapid and impressive progress in the genetic manipulation of cereals has been made possible by two critical technical advances during the past decade: the establishment of embryogenic suspension cultures as a source of totipotent protoplasts and the direct delivery of DNA into protoplasts for genetic transformation.
Plant development from protoplasts of members of Bryophyta, Pteridophyta and Spermatophyta under identical conditions
Authors: Binding Horst , Görschen Eckart , Hassanein Ahmed M. , Qing Ling Hong , Mordhorst Gudrun , Puck Gabriele , Rudnick Joachim , Rong Wang Gui , Truberg Bernd
Published: May 2006

Organized development from isolated protoplasts using a single protocol has been obtained with two bryophyte species, one fern species, and 34 dicotyledonous species. Regeneration is described here for the first time with the dicotyledonous species Cyphomandra betacea, Euphorbia pulcherrima, Linum catharticum, and Senecio fuchsii. In co‐cultures with a Solanum hybrid, interfamilial and even interdivisional support was observed acting at different developmental steps. The results are discussed with respect to standardization of culture conditions, variety of morphogenic pathways, and interspecific correlations such as tolerance, allelopathy and nursery/feeding in unrelated systems.
Molecular analysis of nuclear genes in somatic bybrids
Authors: Bates George W.
Published: May 2006

This paper reviews recent developments in the use of molecular probes for analyzing the genetic makeup of somatic hybrids. Successful application of somatic hybridization to the interspecific transfer of traits encoded in the nucleus is still having limited success. A major difficulty is hybrid infertility, particularly in hybrids between sexually incompatible species. The formation of asymmetric hybrids is being explored as an approach for improving hybrid fertility. Evaluation of the degree of chromosome elimination and chromosome stability and instability in asymmetric hybrids is difficult when the traditional approaches of chromosome counting and isozyme analysis are used. Two new approaches are resolving this difficulty. The use of species‐specific repetitive DNA probes in dot blotting and in situ hybridization to chromosomes is providing quantitative data on chromosome elimination and allows detection of translocations. Use of restriction fragment length polymorphism (RFLP) probes for analysis of hybrids between genetically mapped species makes it possible to account for the presence or absence of individual chromosomes and chromosomes arms. Wider use of such molecular probes should greatly improve our understanding of the genetics of both symmetric and asymmetric somatic hybrids and may lead to new strategies for the effective interspecific transfer of nucleus‐encoded traits by protoplast fusion.
Microprotoplast isolation, enrichment and fusion for partial genome transfer in plants
Authors: Ramulu K. S. , Dijkhuis P. , Verhoeven H. A. , Famelaer I. , Blaas J.
Published: May 2006

Somatic hybridization using protoplasts with whole genomes has often resulted in complex hybrids with many unwanted chromosomes or genes. Several researchers have attempted to reduce the number of undesired chromosomes through irradiation of the donor protoplasts, but so far without much success. Alternatively, micropro‐toplasts containing one or a few chromosomes can be used for partial genome transfer, as has been demonstrated in human and other mammalian cell systems using microcells. Recently, we have optimized the ‘microprotoplast system’ for several donor cell lines (potato, Nicotiana, sugar beet) carrying various genetic markers, such as kanamycin resistance, β‐glucuronidase, nitrate reductase deficiency, hormone autotrophy, opines, etc. Protocols were developed to obtain higher yields of micro‐protoplasts as well as to enrich sub‐diploid microprotoplasts containing one or a few chromosomes. These microprotoplasts were fused with whole mesophyll protoplasts of recipient lines using polyethylene glycol. Various requirements for lines used as donor and recipient partners in microprotoplast‐protoplast fusions are described. The results are discussed in the context of partial genome transfer.
DNA radiation damage and asymmetric somatic hybridization: Is UV a potential substitute or supplement to ionising radiation in fusion experiments?
Authors: Hall Robert D. , Krens Frans A. , Rouwendal Gerard J. A.
Published: May 2006

Recent reports have revealed that the asymmetric nature of the nuclear genome of somatic hybrids, produced following the irradiation of one of the parents with X‐ or gamma rays, is generally much less than had been anticipated. As a consequence, we have begun to investigate whether UV radiation might be used as an alternative or indeed a supplement to the presently‐used ionising radiation techniques in such experiments. Cell culture studies have revealed that UV radiation induces the desired physiological effects in sugar beet (Beta vulgaris) protoplasts, namely, a prevention of cell division without immediate cytotoxicity. Preliminary studies using denaturing and pulsed field gel electrophoresis have shown that UV can also induce substantial physical fragmentation of DNA. When using the same techniques, less breakdown was observed following gamma radiation. All results were highly reproducible. Such results augur well for the potential use of UV in asymmetric somatic cell fusion experiments.
Organelle assortment and mitochondrial DNA rearrangements in Brassica somatic hybrids and cybrids
Authors: Earle E. D. , Temple M. , Walters T. W.
Published: May 2006

Protoplast fusion permits manipulations of organelle genomes not readily achieved by other in vitro procedures or sexual crosses. Although considerable information is now available about the fate of chloroplasts and mitochondria in fusion products of various genera, many additional questions about factors affecting organelles after fusion remain to be answered. Brassica species are particularly favorable materials for such studies. Organelle assortment, mitochondrial DNA (mtDNA) recombination, and plant phenotypes observed after fusion of protoplasts from cytoplasmic male sterile B. oleracea with protoplasts from B. campestris, B. oleracea or B. napus are described. The somatic hybrids and cybrids obtained at Cornell have been used for detailed studies of recombinant mtDNA, including correlation of a specific mtDNA region with the ogura type of cytoplasmic male sterility, and have provided plant materials for possible use in hybrid breeding programs.
Protoplasts in organelle research: Transfer and transformation of plastids
Authors: Koop Hans‐Ulrich , Eigel Lutz , Spörlein Brigitte
Published: May 2006

Protoplasts, because they lack the wall of a typical higher plant cell, offer unique opportunities for experimental manipulation of their organellar constituents. Here, we report on modification of the organellar content of Nicotiana tabacum protoplasts by microfusion‐induced transfer of defined numbers of chloroplasts into albino recipient cells. A single chloroplast is found to be sufficient for establishing a new plastid population in the progeny of the recipient cell. The frequency of green or variegated regenerants is shown to be genotype dependent. It can be drastically increased by using selection pressure for the transferred organelle. We also report on transient expression of plastid specific reporter gene constructs in plastids after PEG‐mediated direct gene transfer into Nicotiana plumbaginifolia protoplasts. The expression is shown to be localized in the plastids by determining gene expression in isolated chloroplasts under conditins which completely remove cytoplasmic enzyme activity derived from a nuclear reporter gene construct. These data demonstrate for the first time that functional DNA, introduced into the cytoplasm by direct gene transfer, enters the organellar compartment and is expressed.
Polyethylene glycol‐mediated direct gene transfer in Nicotiana spp.
Authors: Gharti‐Chhetri Gokarna B. , Cherdshewasart Wichai , Dewulf Jocelyne , Jacobs Michel , Negrutiu Ioan
Published: May 2006

Direct gene transfer to protoplasts is a widespread experimental procedure. In this review, it is discussed in relation to the fate and expression of transforming DNA and its genetic transmission upon integration, and expression (in)stability during successive meiotic generations. The results suggest that the major differences between Nicotiana plumbaginifolia and N. tabacum were not in DNA uptake but rather in the specific fate of the transforming DNA. Differences between integrative transformation versus transient gene expression are discussed. Transient gene expression experiments with cotransformed independent constructs enable accurate assays of gene activity modulation, including both activation and repression. The study of the time of integration of foreign DNA using haploid mesophyll protoplasts showed that foreign DNA integration took place during the first two division cycles of the protoplasts. Most of the transfomants inherited the foreign genes as a monogenic trait: N. plumbaginifolia exhibited a higher proportion of multiple insertions compared with N. tabacum. Furthermore, gene integration occurs as a random process. Compared to ‘simple’ transformation, cotransformation with independent constructs showed a higher level of rearrangement of the inserted DNA. Foreign gene transmission, followed through different generations, indicated that inactivation of the inserted gene could happen through a variety of processes. These results open up a series of questions concerning the applied aspects of direct gene transfer to plants.
Protoplasts as tools for plant genome modifications
Authors: Paszkowski Jerzy , Peterhans Alex , Schlüpmann Henriette , Basse Christoph , Lebel Edouard G. , Masson Jean
Published: May 2006

We present here the application of protoplast technology in the selection and recovery of rare, spontaneous plant genome alterations. Using protoplasts as a cell cloning system allowed the detection and molecular characterization of intrachromosomal recombination events between genomic repeats. The mechanism, frequencies and the induction of intrachromosomal recombination are discussed as well as its application for genome mutagenesis.
Flow cytometry and plant protoplast cell biology
Authors: Bergounioux Catherine , Brown Spencer C. , Petit Patrice X.
Published: May 2006

Flow cytometry, which requires individualized or single cell preparations, is particularly adapted to studies of protoplast biology. It has been developed at three levels: descriptions of populations, measurement of cell function, and positive or negative selection of rare protoplast types. It enables measurement of several parameters in single protoplasts at a high rate of analysis. Without protoplast purification, excellent resolution of subpopulations is still possible. The quantitative data obtained on each protoplast normally have relative units but absolute calibration of intensity scale is available. Specific probes allow one to correlate multiparameter data on each cell. DNA, RNA, proteins, metabolites, organelles, virus and plasmid content of protoplasts have been described.

The essential conceptual advantage of flow cytometry is the processing of heterogeneous material, typical of biological samples, but inadequately handled through overall mean values. Biological properties of protoplast subpopulations are followed using fluorescence intensity as a measure of DNA versus RNA or protein synthesis, cytoplasmic and nuclear antigens, membrane potentials, membrane viscosity, cell wall regeneration and pH.
Plant cell wall removal: Cause for microtubule instability and division abnormalities in protoplast cultures?
Authors: Simmonds Daina H.
Published: May 2006

Cell wall removal from plant cells can destabilize the cortical microtubules (MTs) in isolated protoplasts. The degree of destabilization depends on the origin and physiological condition of the cells, enzyme purity and digestion protocol, and the presence, in the digestion medium, of stabilizing factors such as Ca2+ or taxol. Disorientation of MTs in protoplasts and the absence of a “normal’ cell wall during early culture periods results in abnormalities in mitotic spindles, phragmoplasts, new cross‐walls and chromosome segregation. These abnormalities are greatly reduced in older protoplast cultures, where a substantial cell wall had regenerated. It is suggested that the cell wall may serve to stabilize MTs through transmembrane proteins and may play a role in the spatial organization of MT nucleating sites.
Protoplasts as tools in cell biology
Authors: Fowke L. C. , Wang H.
Published: May 2006

Studies of plant protoplasts using both fluorescent dyes and electron dense probes have demonstrated endocytosis in plants. Ultrastructural work with soybean protoplasts using cationized ferritin (CF) revealed an endocytotic pathway from coated pits at the plasma membrane to coated vesicles, the partially coated reticulum, Golgi bodies, multivesicular bodies and finally the vacuole. Endocytosis may be responsible for membrane retrieval from the cell surface or degradation of elicitors or toxins during host‐pathogen interactions. Immunofluorescence studies of dividing plant protoplasts have provided new information about the preprophase band (PPB) of microtubules and the shape of spindles. Studies of PPBs in soybean protoplast cultures permitted detailed examination of PPB development and an assessment of the usefulness of the PPB index for identifying morphogenic cultures. In multinucleate protoplasts the size and number of PPBs were apparently not controlled by nuclear number. Research with conifer protoplasts resulted in the discovery of new features of gymnosperm spindles.
The isolation and purification of surface specific proteins of somatic and reproductive protoplasts of lily and rapeseed
Authors: Blomstedt Cecilia K. , Xu Huiling , Singh Mohan B. , Knox R. Bruce
Published: May 2006

The plasma membrane surface proteins of intact somatic (leaf) and reproductive (pollen, generative cell or sperm cell) protoplasts of lily (Lilium longiflorum) and rapeseed (Brassica napus cv. Midas) were compared after probing with N‐hydroxysuccinimido‐ (NHS) or sulfo‐NHS‐biotin. The plasma membranes of intact protoplasts are impermeable to these biotin probes, which bind covalently to the free amino groups of surface proteins. Enzyme‐labelled streptavidin was used to detect membrane proteins after separation by SDS‐PAGE and western blotting. In lily, six proteins specific to the surface membrane of leaf protoplasts were identified varying from 25–64 kDa, three proteins to pollen protoplasts in the range 35–64 kDa and two proteins to generative cell protoplasts, 63 and 67 kDa. In rapeseed leaf protoplasts, seven proteins in the range 22–69 kDa were detected, while in the sperm enriched fraction five proteins were present in the same kDa range. The proteins identified as membrane specific for generative cell protoplasts of lily have been isolated and were used as antigens for monoclonal antibody production. Preliminary results indicate the successful production of antibodies to surface antigens. These antibodies will be used to localise surface specific epitopes which are likely to be involved in cell‐cell recognition at fertilization.
Regulation of maize root nitrate reductase mRNA levels
Authors: Long Deborah M. , Oaks Ann , Rothstein Steven J.
Published: May 2006

The regulation of maize (Zea mays L. cv. W64A x W182E) root nitrate reductase (NR) transcript levels has been investigated. To facilitate this process a partial nitrate reductase cDNA clone has been isolated from a maize root cDNA library. Its derived amino acid sequence was found to be more similar to that of the barley nicotinamide adenine dinucleotide (phosphate)‐reduced from [NAD(P)H] bispecific isoform of NR than to that of the maize leaf NADH:NR. The amount of NR mRNA in maize roots was found to increase very rapidly upon the addition of nitrate, reaching a maximum approximately 30 min after induction in the root tip. The level of NR mRNA was analysed in leaf and root tissue. There was very little cross‐hybridizing mRNA in leaf tissue when the root cDNA clone was used as a probe. Conversely, the leaf NADH:NR cDNA clone hybridized to an mRNA that was present at considerably higher levels in leaf tissue than in root tissue, although there was a clearly detectable level of this transcript present in the roots. Finally, the level of root NR mRNA was analyzed by using the tissue print technique to study its localization at the cellular level. The greatest amount of NR transcript was found in the region just above the root tip. At the cellular level, NR mRNA was found in all root cell types.
Temporary storage compounds and sucrose‐starch metabolism in seed coats during pea seed development (Pisum sativum)
Authors: Rochat Christine , Boutin Jean‐Pierre
Published: May 2006

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea (Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem‐unloaded sucrose and high activities of some enzymes of sucrose‐starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.
Protein synthesis at low temperatures in two soybean cultivars differing by their cold sensitivity
Authors: Cabané M. , Vincens P. , Boudet A. M.
Published: May 2006

The effect of low temperatures (14°C/8°C, day/night) on polypeptide synthesis in leaves of two soybean (Glycine max [L.] Merr.) cvs (Verdon and Maple Arrow) differing in cold sensitivity was investigated. The two cultivars were initially characterized in terms of cold tolerance according to their growth at the young plant stage at 14°C/8°C. Verdon was found to be more tolerant than Maple Arrow. In vivo [35S]‐methionine labeled polypeptides were resolved by two‐dimensional electrophoresis. Autoradiograms were computer analyzed to evidence and quantify significative changes occurring after 5 days at 14°C/8°C, and to compare the response of the two cultivars. Most of the observed changes were quantitative. The two cultivars essentially exhibited a common modified polypeptide pattern in response to cold temperatures, but the changes were quantitatively more pronounced in the most tolerant cultivar. Computer analysis of two‐dimensional electrophoresis gels allowed, for the first time, characterization of cultivar differences in terms of protein pattern under cold conditions.
Respiratory pathways in germinating maize radicles correlated with desiccation tolerance and soluble sugars
Authors: Leprince Olivier , van der Werf Adrie , Deltour Roger , Lambers Hans
Published: May 2006

We have assessed the activities of the cytochrome and alternative pathways in total respiration and their role in each stage of germination of Zea mays L. radicles. Throughout imbibition, the salicylhydroxamic acid (SHAM) concentration needed to inhibit the cyanide‐resistant pathway, without any side effects, decreased from 15 mM in quiescent embryos to 5 mM at 72 h after imbibition. Electrons predominantly flowed through the cytochrome pathway although the alternative pathway was already present at early imbibition. The capacity of the alternative path was about 70% of the control rate of respiration. Its engagement progressively increased from 18% after 10 min of imbibition to 70% at the radicle emergence and then decreased to 50% at 96 h after imbibition, concomitant with the onset of radicle growth. The alternative pathway was, however, not essential for germination. The observed activity of the alternative path correlated with the monosaccharide (glucose + fructose) content, suggesting that the alternative pathway could be acting according to the ‘energy overflow model’. On the other hand, up to 24 h after imbibition at 16°C, maize radicles tolerate a severe desiccation, becoming intolerant at 72 h. On reimbibition of tolerant radicles, respiration increased immediately and the alternative pathway was rapidly engaged. At 72 h, no respiration was measured, indicating a total loss of the respiratory systems. The possible correlation between carbohydrate content, loss of desiccation tolerance and activity of the two respiratory pathways is discussed.
Effect of light and riboflavin on indolebutyric acid‐induced root formation on apple in vitro
Authors: van der Krieken Wim M. , Breteler Hans , Visser Marcel H. M. , Jordi Wilco
Published: May 2006

Maximum root formation on apple (Malus) shoots cultured in vitro occurred after an incubation in the dark on medium containing 3.2 or 10 μM indolebutyric acid (IBA) plus riboflavin. Omission of riboflavin or culture in light resulted in a significant decrease in the number of roots formed. About 95% of the absorbed IBA was inactivated by conjugation, ca 4% was extracted as the free IBA acid (IBAH) and only 1% as IAAH. It was investigated whether the decrease in root formation caused by exposure to light or omission of riboflavin during culture was parallelled by a shift in the concentrations of the physiologically active auxin compounds (IBAH and IAAH) in the stem base, i.e. the location where the roots emerge. At least 90% of the absorbed 3H‐IBA was located in the stem base. Omission of riboflavin, either in the dark or in the light, had no effect on the IBAH and IAAH concentrations, whereas root formation decreased significantly. Incubation in the light on medium containing 10 μM IBA with or without riboflavin and culture in the dark on medium containing 3.2μM IBA plus riboflavin resulted in similar IBAH and IAAH concentrations. However, the number of roots was significantly lower after culture in the light. Therefore, we conclude that the synergistic effect of riboflavin and the antagonistic effect of light on IBA‐induced root formation are not solely based on changes in the concentrations of the active auxin components resulting from IBA uptake.