The effect of different oxygen availability on the nitrate reductase (NR, EC 1.6.6.1) activity in cucumber roots was studied. NR activity measured in the presence of Mg²⁺ (actual NR activity) as well as activity measured with EDTA (maximum NR activity) increased distinctly after 30 min of root incubation in a medium flushed with N₂ (anaerobic conditions). In contrast, aeration of roots (aerobic conditions) decreased both enzyme activities. Such inactivation of NR was rapidly reversed after transferring the roots to anaerobic conditions. An air-induced decrease of the actual enzyme activity was prevented by staurosporin, a protein kinase inhibitor; whereas microcistin LR, an inhibitor of protein phosphatases, completely eliminated the reactivation of NR actual activity under limited oxygen availability. An increase of the NR actual activity in roots incubated in a nitrogen-flushed buffer was correlated with a lower content of ATP in root tissues. These data suggest that reversible protein phosphorylation is involved in the regulation of NR activity under limited oxygen. On the other hand, feeding roots with inhibitors of protein kinases as well as phosphatases did not affect the maximal activity of NR indicating that other modification(s) of enzyme activity could also function in cucumber roots. Since the changes in the expression level of gene encoding nitrate reductase (CsNR) under different oxygen availability were not correlated with the enzyme activity, the transcription level of oxygen action was excluded. On the other hand, it was demonstrated that oxygen-induced alteration of NR was dependent on the ratio of oxidized/reduced pyridine nucleotides in tissues. In aerobic conditions, when maximal NR activity was inhibited, a drop of the NAD(P)H level was also observed. These data point to hysteretic modifications of NR protein induced by NAD(P)H as the target of reversible and rapid changes in maximal enzyme activity under different oxygen availability.

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5') and (3') KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and co-cultivation conditions were found to play a significant role in influencing tissue competence, Agrobacterium virulence and compatibility of both, for achieving the maximum transformation frequencies. The stable transformation with 4.31 % efficiency was achieved using the optimized conditions. The transformed green shoots that were selected and rooted on medium containing kanamycin and tested positive for nptII gene by polymerase chain reaction were established in soil to collect seeds. GUS activity was detected in leaves, roots, pollen grains and T₁ seedlings. Southern analysis of T₀ plants showed the integration of nptII into the plant genome.

Heat caused reduction in membrane protein thiol content, increased accumulation of thiobarbituric acid reactive substances and reduced germination rate and early growth in germinating Amaranthus lividus seeds. Imposition of heat stress during early germination also causes accumulation of reactive oxygen species like superoxide and hydrogen peroxide while activities of antioxidative enzymes catalase, ascorbate peroxidase, and glutathione reductase decreased. Calcium chelator (EGTA), calcium channel blocker (LaCl₃) and calmodulin inhibitor (trifluroperazine) aggravated these effects. Added calcium reversed the effect of heat, implying that protection against heat induced oxidative damage and improvement of germination requires calcium and calmodulin during the recovery phase of post-germination events in Amaranthus lividus.

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) in the three crassulacean acid metabolism (CAM) plants: Kalanchoë pinnata, K. daigremontiana and Ananas comosus (pineapple) undergoes regulatory phosphorylation during the dark period. We cloned PEPC kinase gene from two CAM Kalanchoë species using conventional RT-PCR approach. The PEPC kinase transcripts comprise only a protein kinase catalytic domain, encoding 272-276 amino acids with predicted M_r of 30.6-31.0 kDa. The expression of PEPC kinase gene in the Kalanchoë species was abundant at the beginning of dark phase, but that in pineapple cross-hybridized with Kalanchoë PEPC kinase probes was abundant at the end of dark phase. The PEPC kinase was encoded by a small gene family containing at least two members in each species. Treatment of detached leaves with the protein synthesis inhibitors cycloheximide and puromycin blocked the nocturnal appearance of PEPC kinase activity and maintained PEPC in the dephosphorylated state in the three CAM species. The calcium/calmodulin antagonist W7 blocked the apparent phosphorylation state of PEPC in pineapple, but not in Kalanchoë species. Furthermore, the transcript abundance of PEPC kinase matched the apparent in vivo phosphorylation state of PEPC in the Kalanchoë species, but unmatched that in the pineapple. These results implicated that the phosphorylation state of PEPC in Kalanchoë species is largely controlled by PEPC kinase transcript abundance, while that in pineapple may be controlled by both PEPC kinase transcript abundance and Ca²⁺-dependent protein kinase (CDPK).

Trigonelline (TRG) is known as a compatible solute in response to stress as well as a cell cycle regulator, and is more concentrated in legumes than other non-legume dicots. Four Glycine max L. genotypes (Essex, ExF 67, Forrest and Stressland) were used to examine TRG concentration in seeds and seedlings exposed to 30 or 100 mM NaCl, and to determine the association of TRG concentrations in seedlings with seedling growth. Seed germination across genotypes was inhibited by elevated salinity (71-91 %) in ExF 67 and Forrest and by accelerated aging (77-92 %) in Forrest. Length of seedlings in most genotypes stressed with NaCl apparently decreased. The TRG content in mature seeds of four genotypes was 44.4-74.6 µg g^-1(d.m.). TRG content significantly increased during early young seedling development, but remained or significantly reduced in some genotypes stressed with NaCl.

To investigate the molecular mechanisms of Al toxicity, cross-species cDNA array approach was employed to identify expressed sequence tags (ESTs) regulated by Al stress in root tips of Al-tolerant maize (Zea mays) genotype Cat100-6 and Al-sensitive genotype S1587-17. Due to the high degree of conservation observed between sugarcane and maize, we have analyzed the expression profiling of maize genes using 2 304 sugarcane (ESTs) obtained from different libraries. We have identified 85 ESTs in Al stressed maize root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding previously identified proteins induced by Al stress, such as phenyl ammonia-lyase, chitinase, Bowman-Birk proteinase inhibitor, and wali7. In addition, several novel genes up-and downregulated by Al stress were identified in both genotypes.

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.

Immunoblot analysis was used to assess the effects of light and redox-modifying chemicals on the 52 kDa protein disulfide isomerase (PDI) from chloroplasts of Arabidopsis thaliana. A monoclonal antiserum was used that preferentially cross-reacts with the 52 kDa relative to the 65 kDa isoform of PDI. The PDI-52 was most abundant in leaves, flowers, stems and seeds, but was undetected in roots. PDI-52 formed a ∼220 kDa protein complex on blue native gels, indicating that it associates with either itself or other proteins in chloroplasts. Light decreased the levels of PDI-52 by 80 %, relative to the control protein (the CF1 subunit of chloroplast ATP synthase). Treatment with dithiothreitol decreased the content of the 52 kDa protein by half. In contrast, when the reduction of plastoquinone is blocked by DCMU, or when the plants are treated with phosphate, PDI-52 contents increased by 1.5 to 2-fold relative to CF1. The effect of the chemical treatments coincided with the effect of the light/dark cycle and implied that light decreased PDI-52 protein content by way of the cellular redox environment.

Trichloroacetic acid (TCA), a secondary atmospheric pollutant, is also formed in forest soil and thus ranked among natural organohalogens. The observed biooxidation of atmospheric tetrachloroethene (PER) to TCA in chloroplasts has led to the investigation of the mode of action of TCA in spruce needles, since TCA is also accumulated in the needles after its rapid uptake from soil by roots. Being phytotoxic, TCA considerably influences conifers by affecting their photosynthetic apparatus. We examined the transport of TCA from soil into chloroplasts in order to compare the effects of TCA on conifers from both sources, i.e. endogenously produced within chloroplasts or taken up by roots. The influence of TCA formed in chloroplasts was found to be much more adverse than that of "soil" TCA.

We have determined a 1778 base sequence which includes the complete ndhCKJ operon of barley plastid DNA. This operon contains the ndhC, ndhK and ndhJ genes encoding the polypeptides NDH-C, NDH-K and NDH-J, respectively, of the thylakoid Ndh complex. Poly-and mono-cistronic transcripts were identified, with an increase in the latter under oxidative stress induced by herbicide Paraquat. Complete sequencing of transcript cDNAs and of the corresponding regions of five additional monocots revealed that the 13^th C (cytosine) base of ndhC is edited to U (uracil) converting the CAC codon (encoding histidine, H) to UAC (encoding tyrosine, Y). Dicots having the appropriate TAC codon at the genome sequence do not require editing. The new editing site can not be predicted by comparison with the Marchantia sequence (that has a C at the 13^th position) because, in contrast to Angiosperms, the amino-terminal sequence in lower plants is highly variable in NDH-C.

A highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47-100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm^-3 gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous.

An improved protocol for generation of viable cormlets from tissue culture derived shoots of saffron has been developed. Multiple shoots were generated from apical buds, small corms and in vitro developed single shoots. Bunches of two to three shoots when cultured on half strength Murashige and Skoog (MS) medium containing 3 mg dm^-3 benzyladenine (BA) and 80 g dm^-3 sucrose developed 1.89 cormlets per shoot bunch with an average fresh mass of 1.18 g. It took nine months from culture of apical buds to the harvest of cormlets but under field conditions 22 months. Sucrose appeared to be essential for cormlet induction as no cormlets were developed in the medium devoid of sucrose and only 0.29 per shoot in medium containing mannitol. In vitro derived cormlets sprouted from apical and axillary buds on MS medium containing 12 mg dm^-3 BA, 3 mg dm^-3 indolebutyric acid and 30 g dm^-3 sucrose. Daughter cormlet formation from in vitro derived cormlets was also observed.

Callus cultures initiated from shoot base explants of Curcuma aromatica Salisb. were maintained on Murashige and Skoog (MS) media supplemented with 2 mg dm^-3 2,4-dichlorophenoxyacetic acid alone or with 0.5 mg dm^-3 kinetin. Plantlets were regenerated from 60 and 180-d-old callus on MS media supplemented with 3 mg dm^-3 benzyladenine and 0.5 mg dm^-3 α-naphthalene acetic acid. Approximately 8-10 plantlets were produced after 30-40 d of culture per 50 mg of callus inoculated. Out of 113 regenerants analyzed 85 plants were exclusively diploid and 28 were predominantly diploid revealing presence of polyploid nuclei. Frequency of polyploid cells were more in regenerants obtained from 180-d-old callus then from 6-d-old callus which might be attributed to the ageing of callus.

The primary phytotoxic effect of aluminum (Al) is confined to the root apex. It is a matter of debate whether the primary injury of Al toxicity is apoplastic or symplastic. This review paper summarizes our current understanding of the spatial and metabolic sites of Al phytotoxicity. At tissue level, the meristematic, distal transition, and apical elongation zones of the root apex are most sensitive to Al. At cellular and molecular level, many cell components are implicated in Al toxicity including DNA in nucleus, numerous cytoplastic compounds, the plasma membrane, and the cell wall. Although it is difficult to distinguish the primary targets from the secondary effects so far, understanding of the target sites of Al toxicity is helpful for elucidating the mechanisms by which Al exerts its deleterious effects on root growth.

This paper describes a proteome analysis and changes in endogenous abscisic acid (ABA) contents during seed development of Araucaria angustifolia (Bert.) O. Ktze. Megagametophytes and embryonic axis tissues exhibited a similar ABA variation pattern during seed development, reaching maximum values at the pre-cotyledonary stage. The embryonic axis protein content increased until the cotyledonary stage with following stabilization at mature seed. The two-dimensional electrophoresis at the torpedo developmental stage showed approximately 230 polypeptides against 340 in the mature stage. Peptide mass fingerprinting analyses identified three polypeptides, corresponding to an AtSAC4, a late embryogenesis abundant (LEA) and a storage protein, respectively.

A greenhouse experiment was conducted to investigate the effects of silicon application on Phaseolus vulgaris L. under two levels of salt stress (30 and 60 mM NaCl in the irrigation water). Salinity significantly reduced growth, stomatal conductance and net photosynthetic rate, and increased Na⁺ and Cl^- content mainly in roots. Silicon application enhanced growth of salt stressed plants, significantly reduced Na⁺ content especially in leaves and counterbalanced the effects of NaCl on gas exchange; the effect was more evident at 30 mM NaCl. Cl^- content in shoots and roots was not significantly modified by silicon application; the drop in K⁺ content caused by salinity was partially counterbalanced by silicon, especially in roots.

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.

The activities and isoenzyme pattern of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) have been studied during germination of Chenopodium murale seeds. CAT and SOD activities were similar in dry seeds and during first 2 d of imbibition. CAT activity increased during radicle protrusion and early seedling development. The maximum SOD activity was found at final stages of germination and early seedling development. POD activity was not detected until the 6^th day of germination, indicating POD involvement not until early seedling development. Gibberellic acid (GA₃, 160 µM) delayed and synchronized C. murale germination.

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1-2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.

Changes in stomatal and chlorophyll fluorescence characteristics were analyzed in the course of leaf expansion in European beech (Fagus sylvatica L.) cultivars Aurea Pendula, Cristata, Rohanii, Rotundifolia and Viridivariegata. Stomatal length increased gradually from the second to the fifth phenological stage. Rotundifolia reached the highest mean stomatal length whereas Aurea Pendula and Cristata had the lowest values. Stomatal density for all cultivars decreased from the second to the fifth stage. Aurea Pendula reached the highest stomatal density in all phenological stages. The highest values of variable to maximum fluorescence ratio (F_v/F_m) were recorded in Rotundifolia, Rohanii, and the wild type, whereas Viridivariegata showed the lowest F_v/_Fm. Similar trend was found in maximum to initial fluorescence ratio (F_m/F₀), but extremely low F_m/F₀ values were recorded in Viridivariegata in the last phenological stage. The highest potential electron capacity was found in Rohanii, Viridivariegata and the wild type and lowest in Cristata. This parameter increased in the course of early leaf development.

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.

Agrobacterium tumefaciens mediated vacuum infiltration transformation in planta has been established in pakchoi, a kind of Chinese cabbage, but the transformation frequency in harvested seeds has varied in the range of 0.5 to 3.0 × 10^-4 over several years and is much lower than the transformation frequency in Arabidopsis thaliana. To understand that, the distribution and vitality changes of A. tumefaciens in plant tissues were examined. Results revealed that there was a majority of A. tumefaciens in the flower compared with that in the stem and in the leaf at all times after infiltration. As fact of transformants in the upper part of the treated plant (T₀) stalk and fact of the survival of A. tumefaciens in the plant were proved, possibilities of optimizing the transformation conditions to increase the transformation frequency in pakchoi was discussed.

We aimed to assess the potential effects of fumigation by methyl salicylate (MeSA) on plant monoterpene production and emissions. We evaluated monoterpene production and emissions both by chromatographic and proton transfer reaction mass spectrometry at the whole plant-and leaf-scales, in MeSa-fumigated (ca. 60 mm³ m^-3 in air) and control (without MeSa fumigation) holm oak (Quercus ilex L.) plants exposed to temperatures ranging from 25 to 50 °C. The MeSa-fumigated plants showed ca. 3-4-fold greater leaf monoterpene concentrations and emission rates than the control plants between the temperatures of 25 to 45 °C.

In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing 8.8 µM 6-benzylaminopurine (BAP) with 11.4 µM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different explants was achieved on medium containing 2.8 µM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7 µM IAA and 2.4 or 4.9 µM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the soil. The plantlets showed 90 % survival during transplantation.

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides Pall. was here developed. The protoplasts were isolated directly from the leaves of the hairy root-induced plants. The highest yield of protoplasts was obtained from fully expanded leaves of young plants. Their viability was up to 72 ± 2.3 %. The highest division frequency (32.4 ± 0.13 %) and sustained divisions were obtained in Durand, Potrykus and Donn (DPD) medium supplemented with 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid, 0.2 mg dm^-3 6-benzylaminopurine, 0.3 M mannitol, 2 % sucrose and 500 mg dm^-3 casein hydrolysate at the plating density of 3.0 × 10⁵ cm^-3. The frequency of shoot differentiation from protocalli reached to 91.75 ± 3.1 %. Opine synthesis and polymerase chain reaction analysis confirmed that T-DNA still existed in the protoplast regenerated plants.