Agrobacterium-mediated genetic transformation of plants: The role of host

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

MPEC: an important gene in the chlorophyll biosynthesis pathway in photosynthetic organisms

One of the least understood enzymatic steps in chlorophyll biosynthesis is the formation of isocyclic ring, which is catalyzed by the Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase that is involved in the conversion of MgPME to protochlorophyllide. Several genes encoding part of this enzyme have been identified and functional analysis of them has been performed. The enzyme plays important roles in higher plants and photosynthetic bacteria. The review focuses on the current knowledge of MgPME cyclase coding genes, with emphasis on their organization, expression pattern, and functional analysis obtained from mutants.     

Polyamine metabolism in hants : Arginine and omithine decarboxylase activity in ripeningTrlticum durum seeds

The pattern of the activity of arginine decarboxylase (ADC) and omithine decarboxylase (ODC) involved in polyamine synthesis in ripening wheat seeds was examined. The aim was to study the polyamines and the activity of the two enzymes in correlation with the growth processes occurring in the developing wheat seeds. The results obtained showed a very different pattern of polyamine content in the two organs of caryopsis, and that the two enzymes in the embryos have a higher activity than in the endosperms. Moreover, while in the embryos the ADC exhibits higher activity than the ODC, in the endosperms the activity of ODC is about similar to that of ADC. This pattern is discussed in relation to the different histological characteristics of embryo and endosperm tissues during seed development.     

Irreversible changes in barley leaf chlorophyll fluorescence detected by the fluorescence temperature curve in a linear heating/cooling regime

The chlorophyll fluorescence (F) temperature curves in a linear time-temperature heating/cooling regime were used to study heat-induced irreversible F changes in primary green leaves of spring barley (Hordeum vulgare L. cv. Akcent). The leaf segments were heated in a stirred water bath at heating rates of 0.0083, 0.0166, 0.0333, and 0.0500 °C s⁻¹ from room temperature up to maximal temperature T _m and then linearly cooled to 35 °C at the same rate. The F intensity was measured by a pulse-modulated technique. The results support the existence of the two critical temperatures of irreversible F changes postulated earlier, at 45–48 and 53–55 °C. The critical temperatures are slightly dependent on the heating rate. Two types of parameters were used to characterize the irreversibility of the F changes: the coefficient of irreversibility μ defined as the ratio of F intensity at 35 °C at the starting/ending parts of the cycle and the slopes of tangents of linear parts of the F temperature curve. The dependence of μ on T _m revealed a maximum, which moved from 54 to 61 °C with the increasing heating/cooling rate v from 0.0083 to 0.0500 °C s⁻¹, showing two basic phases of the irreversible changes. The Arrhenius and Eyring approaches were applied to calculate the activation energies of the initial increase in μ. The values varied between 30 and 50 kJ mol⁻¹ and decreased slightly with the increasing heating rate.     

Induction of <Emphasis Type="Italic">in vitro</Emphasis> flowering in the orchid <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20 μM N ⁶-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed.     

Ozone sensitivity and ethylenediurea protection in ash trees assessed by JIP chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence transient analysis

The effect of ethylenediurea (EDU) was tested using the chlorophyll (Chl) a fluorescence transient analysis, performed with JIP-test, to assess ambient ozone (O₃) effects on photosynthesis of adult trees under natural conditions. Twelve adult European ash (Fraxinus excelsior L.) trees, known to be sensitive or tolerant to O₃, determined by presence symptomatic (S) or absence asymptomatic (AS) trees of foliar symptoms in previous years, were treated either with distilled water containing 450 g m⁻³ EDU or with distilled water. Once a month across the growing season [the accumulated exposure over a threshold of 40 nmol(O₃) mol⁻¹ was 32.49 μmol mol⁻¹ h⁻¹], Chl a fluorescence transients were measured in vivo on dark-adapted leaves of 1-year-old labeled shoots, from the lower crown part. Twenty-five parameters were calculated. The maximum quantum yield of primary photochemistry (ϕ_Po or F_v/F_m) did not differentiate between S-and AS-trees, while increased Chl content and de-excitation rates suggested compensation of O₃ injury in S-trees. Seasonal reductions in absorbing fluxes and increase in heat and fluorescence dissipation processes was due to leaf ageing and drought, the latter suggesting water deficit influenced Chl a fluorescence stronger than ambient O₃ exposure. AS-trees showed elevated probability of connectivity among photosystem 2 units, a mechanism to stimulate energy dissipation and reduce photo-oxidative injury. EDU prevented the inactivation of reaction centers. This slight effect does not warrant EDU as a tool to assess O₃ effects on photosynthesis, while the JIP-test is suggested for a quantitative assessment in adult trees.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Modelling the dynamics of the electron transport rate measured by PAM fluorimetry during Rapid Light Curve experiments

We propose a dynamic model specifically designed to simulate changes in the photosynthetic electron transport rate, which is calculated from fluorescence measurements when plants are exposed, for a short time, to a series of increasing photon flux densities. This model simulates the dynamics of the effective yield of photochemical energy conversion from the maximum and natural chlorophyll fluorescence yields, taking into account a cumulative effect of successive irradiations on photosystems. To estimate a characteristic time of this effect on photosystems, two series of experiments were performed on two benthic diatom culture concentrations. For each concentration, two different series of irradiations were applied. Simplified formulations of the model were established based on the observed fluorescence curves. The simplified versions of the model streamlined the parameters estimation procedure. For the most simplified version of the model (only 4 parameters) the order of magnitude of the characteristic time of the residual effect of irradiation was about 38 s (within a confidence interval between 20 and 252 s). The model and an appropriate calibration procedure may be used to assess the physiological condition of plants experiencing short time-scale irradiance changes in experimental or field conditions.     

Elevated CO2 concentration enhances the role of the ear to the flag leaf in determining grain yield of wheat

Net photosynthetic rate (P _N) of ear and flag leaf during grain filling stage and grain yield of plants with non-darkened or darkened flag leaf or darkened ear were examined in two different CO₂ concentrations: ambient (AC) and AC+200 μmol mol⁻¹ (EC). Ear showed much higher enhancement (56 %) of P _N than flag leaf (23 %) under EC. Moreover, CO₂ enrichment shortened the photosynthetic duration of flag leaf relative to ear. In this way the ratio of ear to flag leaf contribution to grain yield increased from 1.18 (AC) to 1.39 (EC).     

Increased cold tolerance in Arabidopsis thaliana transformed with Choristoneura fumiferana glutathione S-transferase gene

A glutathione S-transferase (GST) gene cloned from the lepidopteran spruce budworm, Choristoneura fumiferana Clem. was transformed into the model plant Arabidopsis thaliana. The CfGST-transgenic and wild-type A. thaliana were subjected to 4 and 10 °C for 48 h and their cold resistance was studied. The GST activity of the transgenic plants was 46.6 and 35.7 % higher than that of the wild-type plants after 48 h under 4 and 10 °C, respectively. Relative membrane permeability and malondialdehyde content in the transgenic plants were lower while contents of the chlorophyll and proline were higher than those in the wild-type plants under 4 and 10 °C. The survival rate of the transgenic plants was 43.7 % for 24 h under 0 °C, while survival rate of wild-type plants was 28.3 %. The results indicated that the insect GST could enhance cold resistance in the transgenic A. thaliana.     

Genetic relationships among <Emphasis Type="Italic">Hystrix patula,H. duthiei</Emphasis> and <Emphasis Type="Italic">H. longearistata</Emphasis> according to meiotic studies and genome-specific RAPD assay

Hybrids including Hystrix patula, H. duthiei and H. longearistata were obtained and genetic relationships among them were studied. Meiotic pairing in hybrids of H. duthiei × Psathyrostachys juncea (Ns), H. longearistata × Psa. juncea (Ns), Leymus multicaulis (NsXm) × H. duthiei, L. multicaulis (NsXm) × H. longearistata, Elymus sibiricus (StH) × H. patula, Roegneria ciliaris (StY) × H. patula, R. ciliaris (StY) × H. duthiei and R. ciliaris (StY) × H. longearistata averaged 5.76, 5.44, 11.94, 10.88, 10.08, 3.57, 0.46 and 0.90 bivalents per cell, respectively. The results indicated that H. duthiei and H. longearistata had the NsXm genomes of Leymus, while H. patula contained the StH genomes and had a low genome affinity with the StY genomes of Roegneria. Results of genome-specific RAPD assay were comparable with the chromosome pairing data. According to the genomic system of classification in Triticeae, H. patula should be considered as Elymus hystrix L., while H. duthiei and H. longearistata as Leymus duthiei and Leymus duthiei ssp. longearistata, respectively.     

Inhibition of germination and α-amylase induction by 6-methoxy-2-benzoxazolinone in twelve plant species

6-Methoxy-2-benzoxazolinone (MBOA) inhibited germination of rice (Oryza sativa L.), wheat (Triticum aethiopicum Jakubz), rye (Secale cereale L.), onion (Allium cepa L.), wild oat (Avena fatua L.), barnyard grass [Echinochloa crus-galli (L.) Beauv.], ryegrass (Lolium rigidum Gaudin), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), tomato (Lycopersicum esculentum Mill.), carrot (Daucus carota L.) and amaranth (Amaranthus retroflexus L) and the inhibition increased with increasing MBOA concentrations. MBOA also inhibited the induction of α-amylase in these plant seeds and the inhibition increased with increasing MBOA concentrations. There were variations in sensitivity of these plant species to MBOA, and species of family Poaceae (barnyard grass, wild oat, rice, rye, ryegrass, and wheat) were less sensitive to MBOA than the other plant species.     

In vivo activity of recombinant human lewis fucosyltransferase III in leaves of Nicotiana tabacum L.

Fucosylation in plants occurs in glycoproteins and polysaccharides but the function of fucosylation is largely unknown. We aimed to analyze the effects of over-expression of human fucosyltransferase III (hFucT III) on in vivo N-glycan accumulation in tobacco plant leaves and focused on comparing the amount of Lewis a (Le^a)-epitope accumulation in transgenic and in wild-type plants. Fucosyltransferase assays, Western blot and mass spectrometry were used to identify, quantify and analyse Le^a N-glycans. We found that constitutive overexpression of hFucT III activity had no effect on Le^a complex type N-glycans accumulation. Our results suggest that tobacco recombinant hFucT III acts more as a hydrolase than as a transferase.     

Determination of oxalate in plant tissues with oxalate oxidase prepared from wheat

A method for determination of oxalate with oxalate oxidase (OxO, EC 1.2.3.4) prepared from wheat bran, is based on specific oxidation of oxalate to produce H₂O₂. The H₂O₂ formed was colorimetrically determined using horseradish peroxidase-catalyzed oxidation of 4-aminoantipyrine and N,N-dimethylaniline by H₂O₂. The new method was tested on rice, buckwheat, soybean and oxalis leaves, showing it is precise, sensitive, inexpensive, highly reproducible and simple to perform. Good agreement could be obtained between this method and the HPLC.     

Carbon isotope ratios and the variation in the diurnal pattern of malate accumulation in aerial roots of CAM species of <Emphasis Type="Italic">Phalaenopsis</Emphasis> (Orchidaceae)

We investigated the carbon isotope ratios and the diurnal pattern of malate accumulation in leaves and aerial roots of eight species of Phalaenopsis grown in greenhouses. The leaves of all the species showed carbon isotope ratios and the diurnal patterns of malate content typical of CAM plants. However, the aerial roots exhibited a large variation in the diurnal pattern of malate content among species and even among plants within the same species, although carbon isotope ratios were always CAM-like values. Some aerial roots showed the typical diurnal pattern of CAM, but others maintained high or low malate contents during a day without fluctuation. In order to characterize more strictly the nature of the malate variation in the aerial roots, we further investigated a possible variation of the diurnal pattern of malate among different aerial roots within an individual for Phalaenopsis amabilis and P. cornu-cervi. The diurnal pattern of malate content was varied even among different aerial roots within the same plant. Thus the photosynthetic carbon metabolism in aerial roots of orchids is fairly complex.     

Different levels of inbreeding depression between outcrossing and selfing Serapias species

We quantified inbreeding depression for fruit production, embryo vitality and seed germination in three deceptive orchids, Serapias vomeracea, S. cordigera and S. parviflora, which do not provide any reward to their pollinators, and are predicted to experience high outcrossing. Of the three species examined only S. parviflora was autonomously selfing. Both S. vomeracea and S. cordigera showed highly significant differences in fitness between selfed and outcrossed progenies, resulting in high levels of inbreeding depression, which increased in magnitude from seed set to seed germination. Inbreeding depression may promote outcrossing in Serapias by acting as a post-pollination barrier to selfing. Cumulative inbreeding depression across three stages in S. parviflora was lower that in both outcrossing species. The large difference in germination between selfed and outcrossed seeds is an important issue in conservation biology.