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We used a novel system of branch enclosures to study the impact of elevated CO2 (900 ppm) on the gas-exchange characteristics of developed and developing leaves of white poplar (Populus alba L.), as well as of leaves subsequently developing at ambient CO2, outside the enclosures in which the CO2 concentration was raised. We found no significant effect of elevated CO2 on photosynthesis, respiration and isoprene emission, as the rates of developed and developing leaves inside the enclosures, and of leaves developing outside the enclosures, were similar to those recorded using enclosures maintained at ambient CO2. The enclosure system, however, largely influenced the rates of gas-exchange. In fact, leaves already developed inside the enclosures showed rates of photosynthesis, stomatal conductance, and isoprene emission higher than leaves developing inside the enclosures, and also higher than leaves developing outside the enclosure. These differences were caused by a higher efficiency in the light use and by a higher Ribulose 1.5 bisphosphate carboxylase (Rubisco) activity in leaves fully developed inside enclosures than in the other leaf classes. The experiment overall suggests that branch enclosures may alter the physiology of the plants, reducing or counteracting the impact of elevated CO2, which we predicted to stimulate photosynthesis and uncouple isoprene emission from photosynthesis. This may be an important bias against the use of enclosure systems for studies of the impact of environmental constraints and global change factors on physiological features.

The innovative Web-O3-Fumigation (WOF) is a suitable technique to study O3 effects in plants. Through WOF, a very high dose of O3 fumigation may be obtained at the level of selected leaves, while allowing a gradient of ozone stress in branches at different distance from branch directly fumigated with O3 diffusing from perforated Teflon tubes. We used WOF to study the impact of O3 on the emission of isoprene, a putatively powerful antioxidant, in leaves of Quercus pubescens. In our experiment we produced with WOF: i) approximately 300 ppb O3 on the leaves of the branch directly exposed to the pollutant; ii) approximately 190 ppb on the leaves of the branches distant less than 30 cm from the treated branch; iii) a summer ambient O3 concentration (100 ppb) on the leaves of the branches distant more than 30 cm from the branch directly fumigated. High O3 concentrations induced a long-lasting inhibition of photosynthesis and isoprene emission in leaves which were directly fumigated. However, isoprene emission was stimulated by intermediate O3 concentration 288 hours after the end of the treatment. Isoprene stimulation, and the activation of the related antioxidant mechanism, may therefore be dependent on the O3 concentration and may be different in plant canopies depending on their exposure to the pollutant.

RuBPcarboxylase activity was measured in extracts of barley ( Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per CO fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO fixed. It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed. [ABSTRACT FROM AUTHOR]

The carbon isotope ratio (δ13C%) in leaves and berries of Vitis vinifera L. was determined during the fruiting season. The δ13C was from -24.6 to -26.3 in total leaf tissue, while it is from -22.7 to -24.3 in the aqueous extract of leaves. The δ13C in the berries was more stable, around -24, with no change during ripening. Malic and tartaric acid, isolated from the berries at the onset of ripening, showed a δ13C of -23.5 and -23.0 respectively. It was concluded that malic acid accumulated in the berries at the onset of ripening is not formed directly through β-carboxylation. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a large T 0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium-delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain-regulatory genes, TaCKX2-1, TaGLW7, TaGW2, and TaGW8, in T 0 , T 1 , and T 2 generation plants at an average edit rate of 10% without detecting off-target mutations in the most Cas9-active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160-bp deletion in TaCKX2-D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium-delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

The expression of SPANX (sperm protein associated with the nucleus in the X chromosome) gene family has been reported in many tumors, such as melanoma, myeloma, glioblastoma, breast carcinoma, ovarian cancer, testicular germ cell tumors, and hematological malignancies. However, no systematic approach has so far been devised to estimate the percentage of cancer cells expressing SPANX. This study was undertaken to quantify the expression of SPANX proteins in melanomas. The expression of SPANX proteins was evaluated by immunohistochemistry in normal skin (n = 12), melanomas (n = 21), and benign nevi (n = 10), using a polyclonal antibody raised in our laboratory. Seventeen of the 21 melanomas (80.9%) examined expressed SPANX proteins. A high percentage of their cells (49.0% +/- 5.5%) stained positively for SPANX proteins compared with no expression found in normal skin cells. Benign nevi had an intermediate number of cells expressing SPANX proteins (25% +/- 8.5%), which resulted significantly higher than normal skin cells and significantly lower than skin melanoma cells. In melanoma cells, the labeling was mostly nuclear, sometimes incomplete or limited to the perinuclear wall, even if cytoplasmic staining was also seen in SPANX-positive tumor cells. In contrast, the 5 of 10 SPANX-positive nevi had a clear nuclear localization of the signal. These data suggest that the SPANX protein family is expressed in the vast majority of the melanomas tested. The mechanism(s), which brings up SPANX gene expression and the role of these proteins are not known.

Parthenocarpy is potentially a desirable trait for many commercially grown fruits if undesirable changes to structure, flavour, or nutrition can be avoided. Parthenocarpic transgenic tomato plants (cv MicroTom) were obtained by the regulation of genes for auxin synthesis (iaaM) or responsiveness (rolB) driven by DefH9 or the INNER NO OUTER (INO) promoter from Arabidopsis thaliana. Fruits at a breaker stage were analysed at a transcriptomic and metabolomic level using microarrays, real-time reverse transcription-polymerase chain reaction (RT-PCR) and a Pegasus III TOF (time of flight) mass spectrometer. Although differences were observed in the shape of fully ripe fruits, no clear correlation could be made between the number of seeds, transgene, and fruit size. Expression of auxin synthesis or responsiveness genes by both of these promoters produced seedless parthenocarpic fruits. Eighty-three percent of the genes measured showed no significant differences in expression due to parthenocarpy. The remaining 17% with significant variation (P <0.05) (1748 genes) were studied by assigning a predicted function (when known) based on BLAST to the TAIR database. Among them several genes belong to cell wall, hormone metabolism and response (auxin in particular), and metabolism of sugars and lipids. Up-regulation of lipid transfer proteins and differential expression of several indole-3-acetic acid (IAA)- and ethylene-associated genes were observed in transgenic parthenocarpic fruits. Despite differences in several fatty acids, amino acids, and other metabolites, the fundamental metabolic profile remains unchanged. This work showed that parthenocarpy with ovule-specific alteration of auxin synthesis or response driven by the INO promoter could be effectively applied where such changes are commercially desirable.

We investigated the expression of the SpanX protein family in cells of normal testes and in testicular germ cell tumours, mainly seminomas and embryonal carcinomas, using an immunohistochemical approach. Most of the normal germ cells, belonging to spermatogonial and primary spermatocytic classes, showed a strong nuclear positivity. In contrast, post-meiotic germ cells showed diffused cytoplasmic and sometimes also perinuclear localization of the signal. The vast majority of cells were also positive in eight seminomas, six embryonal cell carcinomas and one teratocarcinoma. In all seminomas, nuclei were either exclusively or preferentially labelled; whereas, the nuclear signal intensity decreased in parallel with the appearance of some cytoplasmic staining in embryonal carcinomas. In conclusion, these data suggest that the SpanX protein family is not exclusively expressed post-meiotically and that seminomas and embryonal carcinomas may originate from SpanX-positive carcinoma-in-situ cell.

Apple (Malus x domestica) is one of the most consumed fruit crops in the world. The major production areas are the temperate regions, however, because of its excellent storage capacity it is transported to distant markets covering the four corners of the earth. Transformation is a key to sustaining this demand - permitting the potential enhancement of existing cultivars as well as to investigate the development of new cultivars resistant to pest, disease, and storage problems that occur in the major production areas. In this paper we describe an efficient Agrobacterium tumefaciens-mediated transformation protocol that utilizes leaf tissues from in vitro grown plants. Shoot regeneration is selected with kanamycin using the selectable kanamycin phosphotransferase (APH(3)II) gene and the resulting transformants confirmed using the scorable uidA gene encoding the bacterial beta-glucuronidase (GUS) enzyme via histochemical staining. Transformed shoots are propagated, rooted to create transgenic plants that are then introduced into soil, acclimatized and transferred to the greenhouse from where they are taken out into the orchard for field-testing.

The carbon of the four main monoterpenes emitted by Quercus ilex L. leaves was completely labeled with 13C after a 20-min feeding with 99% 13CO2. This labeling time course is comparable with the labeling time course of isoprene, the terpenoid emitted by other Quercus species and synthesized in leaf chloroplasts. It is also comparable with that of phosphoglyceric acid. Our experiment therefore provides evidence that monoterpenes emitted by Q. ilex are formed from photosynthesis intermediates and may share the same synthetic pathway with isoprene. By analyzing the rate and the distribution of labeling in the different fragments, we looked for evidence of differential carbon labeling in the [alpha]-pinene emitted. However, the labeling pattern was quite uniform in the different fragments, suggesting that the carbon skeleton of the emitted monoterpenes comes from a unique carbon source.

We studied the emission of [alpha]-pinene from Quercus ilex leaves. Only the abaxial side of the hypostomatous Q. ilex leaf emits [alpha]-pinene. Light induced photosynthesis and [alpha]-pinene emission. However, the response of photosynthesis to dark-to-light transitions was faster than that of [alpha]-pinene, suggesting that ATP controls the emission. The emission was higher at 30 than at 20[deg]C, whereas photosynthesis did not change. Therefore, the relationship between photosynthesis and [alpha]-pinene emission does not always hold. When CO2 was removed from the air, transpiration was stimulated but photosynthesis and [alpha]-pinene emission were inhibited. [alpha]-Pinene inhibition was more rapid under low O2. When CO2 in the air was increased, photosynthesis was stimulated and transpiration was reduced, but [alpha]-pinene emission was unaffected. Therefore, the emission depends on the availability of photosynthetic carbon, is not saturated at ambient CO2, and is not dependent on stomatal opening. The pattern of [alpha]-pinene emission from Q. ilex is different from that of plants having specialized structures for storage and emission of terpenes. We suggest that [alpha]-pinene emitted by Q. ilex leaves is synthesized in the chloroplasts and shares the same biochemical pathway with isoprene emitted by isoprene-emitting oak species.

Photosynthetic electron transport drives the carbon reduction cycle, the carbon oxidation cycle, and any alternative electron sinks such as nitrogen reduction. A chlorophyll fluorescence- based method allows estimation of the total electron transport rate while a gas-exchange-based method can provide estimates of the electron transport needed for the carbon reduction cycle and, if the CO2 partial pressure inside the chloroplast is accurately known, for the carbon oxidation cycle. The gas-exchange method cannot provide estimates of alternative electron sinks. Photosynthetic electron transport in flag leaves of wheat was estimated by the fluorescence method and gasexchange method to determine the possible magnitude of alternative electron sinks. Under non-photorespiratory conditions the two measures of electron transport were the same, ruling out substantial alternative electron sinks. Under photorespiratory conditions the fluorescence-based electron transport rate could be accounted for by the carbon reduction and carbon oxidation cycle only if we assumed the CO2 partial pressure inside the chloroplasts to be lower than that in the intercellular spaces of the leaves. To further test for the presence of alternative electron sinks, carbon metabolism was inhibited by feeding glyceraldehyde. As carbon metabolism was inhibited, the electron transport was inhibited to the same degree. A small residual rate of electron transport was measured when carbon metabolism was completely inhibited which we take to be the maximum capacity of alternative electron sinks. Since the alternative sinks were small enough to ignore, the comparison of fluorescence and gas-exchange based methods for measuring the rate of electron transport could be used to estimate the mesophyll conductance to CO2 diffusion. The mesophyll conductance estimated this way fell as wheat flag leaves senesced. The age-related decline in photosynthesis may be attributed in part to the reduction of mesophyll conductance to CO2 diffusion and in part to the estimated decline of ribulose 1,5-bisphosphate carboxylase amount.

A study was undertaken to interpret the nature and function of the "plug like formation" observed inside the spermathecae of many phlebotomine sandflies dissected during field surveys. Trials were carried out on two laboratory reared species, Phlebotomus perniciosus and P. papatasi. The results showed that the "plug like formation" is a true mating plug (MP) containing immotile sperms. Our studies showed also that the MP evolves differently according to the physiological state of the mated female. In blood-fed females, it persists 22-25 hrs post mating. Follows a period of 3-4 days in which the spermathecae acquire different form from that of the virgin spermathecae, but sperms are still undistinguishable. Active sperms appear only a few hours before oviposition. After oviposition, however, spermathecae are void as in the virgin female. Thus, during the complete gonotrophic cycle the spermathecae display five different forms: a) normal and void; b) showing MP; c) distended and yellowish, MP absent; d) active sperms present; e) normal and void. In unfed females, MP was observed to last for 6 days post copula. The results show also that MP is interfering with reinsemination.

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O2 evolution of the leaf tissue in saturating light and CO2 was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO2 assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.

Mesophyll protoplasts were isolated from leaves of 10 day old aseptically grown soybean seedlings, or from surface disinfested leaves of 3 week old plants grown in environmental chambers. The protoplasts were encapsulated in 2mm diameter Ca alginate beads. Immobilized protoplasts were induced to divide by culturing in shaker flasks containing an actively growing soybean cell suspension. The feeder cell suspension supported the division of protoplasts independent of the protoplast density in the Ca alginate beads. At day 18 after encapsulation, the alginate matrix was dissolved, releasing viable callus colonies. The feeder cell suspension obviated plating of protoplasts at high density which is usually required for subsequent cell division and colony development. Since the protoplasts were embedded at low density, the cell colonies were derived from single cells.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14-17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.