Osmotic stress-induced alterations in rice (Oryza sativa L.) and recovery on stress release

In laboratory experiments, rice plants cv Kranti were stressed osmotically using polyethylene glycol 6000 and mannitol, whereas, in pots, plants were drought stressed by withholding water supply. Both osmotic and drought stress influenced different aspects of nitrogen metabolism, resulting in a decline in the activities of aspartate aminotransferase, alanine amino-transferase and an increase in protease activity accompanied by increased free proline and alterations in other amino acid content. An influence on -amylase activity, total free sugars and starch contents was also observed, reflecting the impact of water stress on interconversion between starch and simpler sugars. Effects of polyethylene glycol 6000 as an osmotic agent were more consistent than those of mannitol during short-term (18 h) stress imposition, probably because of slight absorption of mannitol. Since the recovery for most of the parameters was substantial on release of stress, one can infer that the duration and magnitude of stress applied in the present experiments did not cause major permanent alterations in the rice cultivar Kranti. The significant basic information gathered in such experiments, particularly on recovery potential, can be utilised for varietal screening.     

Does abscisic acid influence proline accumulation in stressed leaves?

Root treatments of barley (Hordeum distichum L.) plants with 10^-7 to 10^-4 M abscisic acid (ABA) caused an increase in proline content, especially at higher concentrations, within 2–3 h. Even 3 h after the removal of ABA from the medium the plants continued to accumulate proline. The higher the concentration of the ABA, the higher was the proline level at 6 h. When the highest ABA concentration, 10^-4 M, was tested with polyethylene glycol (PEG) (-5.0 bars) in the medium, the ABA treatment resulted in a higher proline content than in control plants. The treatments PEG alone and PEG + ABA resulted in heavy accumulation of proline, especially, 3 h after releasing the plants from the stress. The proline content in PEG+ABA-treated plants was always higher than plants treated with PGE or ABA alone. In peas (Pisum sativum L. cv Alaska) the same trend occurred although to a lesser degree. These findings indicate an influence of ABA on proline accumulation in water-stressed plants.Abbreviations ABA abscisic acid - PEG polyethylene glycol - RWC relative water content     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Effects of root herbivory by an insect on a foliar-feeding species,mediated through changes in the host plant

Summary The effects of root herbivory by larvae of the scarabaeid, Phyllopertha horticola, on the growth of Capsella bursa-pastoris were examined. Individuals of Aphis fabae were reared on the leaves to determine what effect, if any, root feeding has on the performance of this insect. The experiment was conducted under two watering regimes (low and high). Low watering and root feeding caused water stress in the plants and this was reflected in a reduction in vegetative biomass and an increase in the proportion of material allocated to reproduction. Supplying plants with ample water in the high treatment enabled the water stress caused by root herbivory to be offset, but not completely overcome. Low watering and root feeding caused an increase in aphid weight and growth rate, while root feeding also increased fecundity and adult longevity. These effects are attributed to an improvement in food quality, measured by total soluble nitrogen, and caused by amino acid mobilization due to the water stress. The implications of these results in agricultural and ecological situations are discussed.     

Overexpression of ornithine-δ-aminotransferase increases proline biosynthesis and confers osmotolerance in transgenic plants

Accumulationof proline is a way to increase tolerance to water stress in plants. Therefore,considerable attention has been devoted to optimise proline biosynthesis intransgenic plants. Glutamate and ornithine are both precursors of proline butwhile genes of the glutamate pathway were overexpressed in transgenic plants,no gene encoding an enzyme of the ornithine pathway was considered until now. Thepresent study aims to establish if the overexpression ofornithine--aminotransferase (-OAT) represents an additional wayto increase proline content. To achieve this goal, anArabidopsis -OAT cDNA was fused to the CaMV35Spromoter and introduced via Agrobacterium transformationinto Nicotiana plumbaginifolia. Overexpression of the-OAT cDNA in the analysed transgenic lines was linked to an increase in-OAT enzyme activity. The transgenic lines presenting high enzymaticactivity synthesized more proline than the control plants and showed a higherbiomass and a higher germination rate under osmotic stress conditions. Thesestudies reveal a new and efficient way to increase proline content in plantsand to enhance crop tolerance.     

Leaf anatomy and water loss of in vitro PEG-treated ‘Valiant’ grape

Polyethylene glycol was used to induce water stress of micropropagated Valiant grape. Reduced growth and slow rooting were observed in treated plantlets with 2, 4 and 6% polyethylene glycol as compared to control plantlets with no polyethylene glycol in the rooting medium. At high concentrations of 4 and 6%, leaves exhibited wilting and necrosis. At the 2% level, plantlets recovered and grew satisfactorily. Detached leaves of treated plantlets with 2% polyethylene glycol lost less water than controls when exposed to low humidity for 4 hours. Leaf anatomy of plantlets treated with 2% polyethylene glycol, control (in vitro plantlets) and greenhouse-grown plants were compared under light microscopy. Leaves from control plantlets contained larger mesophyll cells, lacked normal palisade layer formation, had greater intercellular pore spaces and fewer chloroplasts. Leaves from polyethylene glycol-treated plantlets, however, had smaller mesophyll cells, a more defined palisade layer, reduced intercellular pore space and the greatest number of chloroplasts. These results suggest that an osmoticum such as polyethylene glycol may be used to induce more normal leaf anatomy and reduced water loss in micropropagated Valiant grapes.Abbreviations BA 6-benzylaminopurine - FAA formalin-acetol-alcohol - MS Murashige & Skoog (1962) medium - MW molecular weight - NAA napthaleneacetic acid - PEG polyethylene glycol - TBA tertiary butyl alcohol     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

Water flux through a semi-deciduous forest grove of the Orinoco savannas

Water relations were analysed in a semi-deciduous forest grove occurring in the oxisols of the Orinoco savannas. This grove has a shallow unconsolidated ironstone cuirass, which is overlaid by a sandy loam layer (0.0–0.5 m) that contains more than 90% of the grove forest root phytomass. Evapotranspiration and through drainage were calculated by using data from the soil profile as related to physical characteristics of the site root zone, hydraulic conductivity, volumetric water content and potential hydraulic gradient. Mean annual evapotranspiration was 783 mm year^–1 and annual through drainage below the root zone was 14% (162 mm year^–1) of the gross rainfall. This drainage recharged the 42% of the annual saturation deficit of the water table. Similar mean annual evapotranspiration (770 mm year^–1) was also calculated by using the water balance components. The mean daily coupling omega factor () between the grove canopy and the surrounding atmosphere indicated that a high degree of coupling (=0.14±0.16) occurs in the grove and evapotranspiration was mainly controlled by surface conductance. As the dry season proceeded, the soil saturation deficit () increased rapidly resulting in a threshold surface conductance (0.030–0.005 m s^–1) for ranging from 0.05 to 0.10. Hypotheses to explain the omnipresence of perennial species in the wide range of physical conditions in neotropical savannas are discussed.     

The Effect of Polyethylene Glycol on Proline Accumulation in Rice Leaves

The regulation of proline accumulation in polyethylene glycol (PEG, –1.5 MPa) treated rice leaves was investigated. PEG treatment resulted in a decrease in relative water content, indicating that PEG treatment caused water stress in rice leaves. Proline accumulation caused by PEG was related to protein hydrolysis, an increase in ornithine--amino- transferase activity, an increase in the content of ammonia, and an increase in the contents of the precursors of proline biosynthesis, glutamic acid, ornithine, and arginine. Results also show that abscisic acid accumulation is not required for proline accumulation in PEG-treated rice leaves.     

Variation in Osmoregulation in Differentially Drought-Sensitive Wheat Genotypes Involves Calcium

Two wheat (Triticum aestivum L.) genotypes differing in their sensitivity to water deficit (stress tolerant - C306 and stress susceptible - HD2329) were subjected to osmotic stress for 7 d using polyethylene glycol (PEG-6000; osmotic potential –1.0 MPa), at initial vegetative growth. The plants were either supplemented with 1 mM CaCl₂ (Ca²⁺) alone or along with verapamil (VP; calcium channel blocker) to investigate the involvement of calcium in governing osmoregulation. Relative elongation rate (RER), dry matter (DM) production, water potential (_w), electrolyte leakage (EL), contents of proline (Pro) and glycine betaine (GB) and activities of -glutamyl kinase (GK) and proline oxidase (PO) in shoots and roots were examined during stress period. C306 showed relatively higher accumulation of Pro while HD2329 accumulated more GB under stress. RER, DM and _w were relatively higher in C306 than HD2329. Roots compared to shoots showed lower content of osmolytes but had faster rate of their accumulation. Presence of Ca²⁺ in the medium increased the activity of GK and decreased that of PO while in the presence of its inhibitor, decrease in activity of both the enzymes was observed. Ca²⁺ appeared to reduce the damaging effect of stress by elevating the content of Pro and GB, improving the water status and growth of seedlings and minimizing the injury to membranes. The protective effect of Ca²⁺ was observed to be more in HD2329 than C306.     

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

In vitro acclimatization of date palm (Phoenix dactylifera L.) plantlets: A quantitative comparison of epicuticular leaf wax as a function of polyethylene glycol treatment

Wax deposits on leaf surfaces ofin vitro-grown plantlets,in vitro plantlets treated with polyethylene glycol and greenhouse-grown seedlings from five cultivars of date palm (Phoenix dactylifera L.) were extracted and quantified. Significant variations among treatments and cultivars were obtained. Greenhouse-grown plants had the greatest wax deposits followed by the acclimatized plantlets.In vitro plantlets had an average of 15% of the wax of greenhouse plants. Cultivar and plant age differences had a significant effect on the quantity of wax deposits. Greenhouse seedlings of Majhool, Deglet Noor and Khadraoui (cultivars grown under irrigation) had less wax accumulation than Zahidi and Sayer, dryland cultivars.The increase in wax deposition as a result of polyethylene glycol treatment, explains in part, the decreased water loss observed in acclimatized plantlets when transferredex vitro. Abbreviations EW epicuticular wax - NAA naphthalene acetic acid - PEG polyethylene glycol     

Seasonal Changes in Photosynthesis and Stomatal Conductance of Five Plant Species from a Semiarid Ecosystem

In order to determine whether stomatal closure alone regulates photosynthesis during drought under natural conditions, seasonal changes in leaf gas exchange were studied in plants of five species differing in life form and carbon fixation pathway growing in a thorn scrub in Venezuela. The species were: Ipomoea carnea, Jatropha gossypifolia, (C3 deciduous shrubs), Alternanthera crucis (C4 deciduous herb), and Prosopis juliflora and Capparis odoratissima (evergreen phreatophytic trees). Xylem water potential () of all species followed very roughly the precipitation pattern, being more closely governed by soil water content in I. carnea and A. crucis. Maximum rate of photosynthesis, Pmax, decreased with in I. carnea, J. gossypifolia, and A. crucis. In I. carnea and J. gossypifolia stomatal closure was responsible for a 90 % decline in net photosynthetic rate (PN) as decreased from -0.3 to -2.0 MPa, since stomatal conductance (gs) was sensitive to water stress, and stomatal limitation on PN increased with drought. In A. crucis, PN decreased by 90 % at a much lower (-9.3 MPa), and gs was relatively less sensitive to . In P. juliflora and C. odoratissima, Pmax, gs, and intercellular CO2 concentration (Ci) were independent of soil water content. In the C3 shrubs stomatal closure was apparently the main constraint on photosynthesis during drought, Ci declining with in I. carnea. In the C4 herb, Ci was constant along the range of values, which suggested a coordinated decrease in both gs and mesophyll capacity. In P. juliflora Ci showed a slow decrease with which may have been due to seasonal leaf developmental changes, rather than to soil water availability.     

Further evidence of a cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion

Summary Somatic hybridization experiments in Citrus that involve the fusion of protoplasts of one parent isolated from either nucellus-derived embryogenic callus or suspension cultures with leaf-derived protoplasts of a second parent, often result in the regeneration of diploid plants that phenotypically resemble the leaf parent. In this study, plants of this type regenerated following somatic fusions of the following three parental combinations were analyzed to determine their genetic origin (nuclear and organelle): (embryogenic parent listed first, leaf parent second) (1) calamondin (C. microcarpa Bunge) + Keen sour orange (C. aurantium L.), (2) Cleopatra mandarin (C. reticulata Blanco) + sour orange, and (3) Valencia sweet orange (C. sinensis (L.) Osbeck) + Femminello lemon (C. limon (L.) Burm. f.). Isozyme analyses of PGI, PGM, GOT, and IDH zymograms of putative cybrid plants, along with RFLP analyses using a nuclear genome-specific probe showed that these plants contained the nucleus of the leaf parent. RFLP analyses using mtDNA-specific probes showed that these plants contained the mitochondrial genome of the embryogenic callus donor, thereby confirming cybridization. RFLP analyses using cpDNA-specific probes revealed that the cybrid plants contained the chloroplast genome of either one or the other parent. These results support previous reports indicating that acquisition of the mitochondria of embryogenic protoplasts by leaf protoplasts is a prerequisite for recovering plants with the leaf parent phenotype via somatic embryogenesis following somatic fusion.Abbreviations cp chloroplast - GOT glutamateoxaloacetate transaminase - IDH isocitrate dehydrogenase - mt mitochondria - PEG polyethylene glycol - PGI phosphoglucose isomerase - PGM phosphoglucomutase - RFLP restriction fragment length polymorphism Florida Agricultural Experiment Station Journal Series No. R-04631.     

Somatic embryogenesis and plant regeneration in callus from inflorescences of Hordeum vulgare X Triticum aestivum hybrids

Summary Embryogenic callus cultures were obtained by culturing young inflorescence tissues of Hordeum vulgare cv. PF51811 (2x)XTriticum aestivum cv. Chinese Spring (6x) hybrids on 2,4-D-containing N₆ medium. After subculture for about 10 months the calli retained a high potentiality for somatic embryogenesis and plant regeneration. Of about 300 regenerated plants, approximately 100 were transplanted to potting soil. Eight embryoids and three regenerated plants examined had 28 chromosomes identical to the original hybrid plants, while one regenerated plant was found to be a mixploid composed of cells with 28 and 56 chromosomes. The possibility for obtaining amphiploid hybrids through tissue culture is discussed.     

Irrigation effects on European corn borer — maize water relations

This study examined the impact of irrigation water on certain aspects of an insect-plant relationship in the field including the assessment of plant-mediated water effects on an herbivore's development, survival, and behavior, and plant damage parameters and host tissue water status. Maize (Zea mays L.) plants were arranged in a randomized complete block design in the field over two years in North Carolina (NC). Four blocks were subjected to three different irrigation treatments initiated ca. one week before anthesis: optimal, intermediate, deficit water supply. Each plant was infested with one (1986) or two (1987) black head stage, E-race European corn borer [Ostrinia nubilalis (Hübn.)] (ECB) egg masses at tasselling. ECB development, tunnelling site, and survival as well as plant tissue water status (tissue % water contents [] & leaf water potentials []) were recorded through July. The irrigation effect on ECB parameters was slight and variable. Internal stalk temperatures of optimal plants were consistently cooler than their deficit counterparts (1 day-degree/day). With degree-days included as an explanatory variable in the analyses, there were no significant irrigation effects on the ECB parameters, except for total proportion of ECB's bored into maize plant parts. More ECB's bored into drier plants than in optimal plants; however, this trend was not significant in 1987.Plant water indices showed that though responded to irrigation, there were only minor changes in tissue , particularly in view of the larger diurnal tissue changes observed and the relatively high, sustained stalk levels seen over all treatments. Examination of ECB pupal confirmed that dietary water changes were minor or non-limiting to the insects' developmental physiology, because pupal was not sensitive to the irrigation treatments.Though water supply changes have drastic developmental and agronomic consequences for the maize plant, little or no changes were seen in the ECB feeding environment. Furthermore, a plant damage model was developed whereby the total % of ECB's tunnelled into maize was related to the mean larval age. The implications of this model on the understanding of ECB tunnelling behavior, damage potential, and pest management is noted.     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Effect of restricted watering and its combination with root pruning on root growth capacity,water status and food reserves ofPinus caribaea var.hondurensis seedlings

Summary Six-month oldPinus caribaea var.hondurensis seedling were subjected to various regimes of restricted watering and their combinations with root pruning to determine a suitable technique of producing seedlings with optimum physiological condition for successful bare-root planting.From the range of treatments tested, plants subjected to watering once every 9 days (D9) were found to be the hardest in terms of highest root growth capacity, leaf water content, root starch content and in having a moderately low transpiration rate.P. caribaea seedlings subjected to this preconditioning treatment may be more able to survive bare-root planting.