An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.     

Characterization and regional mapping of new anonymous chromosome 20-specific DNA markers isolated from a flow-sorted DNA library

Employing the flow-sorted chromosome 20-specific DNA library LL20NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2-p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12-q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.     

Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy.

The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455).     

Light-induced division and genomic synchrony in phototrophically growing cultures of Rhodopseudomonas sphaeroides.

An experimental procedure for rapidly obtaining cell populations of phototrophically growing Rhodopseudomonas sphaeroides which display division and genomic synchrony has been developed. The basis of the procedure resides with the normal physiological response displayed by cells of R. sphaeroides that have been subjected to an immediate decrease in incident light intensity. After an abrupt high- to low-light transition of an asynchronously dividing cell population, an immediate cessation of increases in culture turbidity, total cell number, and net accumulations of culture deoxyribonucleic acid and phospholipid occurs. Total cell number remains constant for 2.5 h after the transition to low light, after which time, it undergoes a sharp increase. Reinitiation of high-light conditions of growth 1 h subsequent to this increase in total cell number results in a cell population possessing a high degree of division and genomic synchrony. A characterization of this procedure, together with a demonstration of its utility for studies on intracytoplasmic membrane assembly, is presented.     

Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.     

Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin

A simple adaptation of a commercial spectrofluorometer allows selective excitation of fluorescent biomolecules adsorbed to a solid surface while they are in equilibrium with a bulk solution. As a demonstration of this technique, we have detected a change in the effective singlet-singlet energy transfer in fluorescence-labeled bovine serum albumin (BSA) upon adsorption to a fused silica surface. The technique combines total internal reflection fluorescence excitation of surface-adsorbed BSA with a fluorescence spectroscopic examination of energy transfer between two different fluorophores that are covalently bound to amino groups in each BSA molecule. Two donor--acceptor pairs were used, 4-chloro-7-nitro-2,1,3-benzoxadiazole-rhodamine and dansyl-eosin. For studies of surface-adsorbed BSA, we constructed a device in which the excitation light of a standard fluorescence spectrometer totally internally reflects from a surface at which adsorbed BSA is in equilibrium with the bulk solution. A shallow evanescent wave is created, which excites fluorescence from only those BSA molecules in close proximity to the surface. Spectral examination shows significantly less effective singlet-singlet energy transfer from the donor to the acceptor in surface-adsorbed BSA relative to that in native bulk-dissolved BSA. Under appropriate and reasonable assumptions, the energy transfer change between native and adsorbed states of fluorescent BSA can be interpreted as a conformational change of BSA upon adsorption.     

Variation in the phenology of photosynthesis among eastern white pine provenances in response to warming

In higher‐latitude trees, temperature and photoperiod control the beginning and end of the photosynthetically active season. Elevated temperature (ET) has advanced spring warming and delayed autumn cooling while photoperiod remains unchanged. We assessed the effects of warming on the length of the photosynthetically active season of three provenances of Pinus strobus L. seedlings from different latitudes, and evaluated the accuracy of the photochemical reflectance index (PRI) and the chlorophyll/carotenoid index (CCI) for tracking the predicted variation in spring and autumn phenology of photosynthesis among provenances. Seedlings from northern, local and southern P. strobus provenances were planted in a temperature‐free‐air‐controlled enhancement (T‐FACE) experiment and exposed to ET (+1.5/3°C; day/night). Over 18 months, we assessed photosynthetic phenology by measuring chlorophyll fluorescence, gas exchange, leaf spectral reflectance and pigment content. During autumn, all seedlings regardless of provenance followed the same sequence of phenological events with the initial downregulation of photosynthesis, followed by the modulation of non‐photochemical quenching and associated adjustments of zeaxanthin pool sizes. However, the timing of autumn downregulation differed between provenances, with delayed onset in the southern provenance (SP) and earlier onset in the northern relative to the local provenance, indicating that photoperiod at the provenance origin is a dominant factor controlling autumn phenology. Experimental warming further delayed the downregulation of photosynthesis during autumn in the SP. A provenance effect during spring was also observed but was generally not significant. The vegetation indices PRI and CCI were both effective at tracking the seasonal variations of energy partitioning in needles and the differences of carotenoid pigments indicative of the stress status of needles. These results demonstrate that PRI and CCI can be useful tools for monitoring conifer phenology and for the remote monitoring of the length of the photosynthetically active season of conifers in a changing climate.     

Soil and root nutrient chemistry structure root-associated fungal assemblages in temperate forests

Root-associated fungi (RAF) link nutrient fluxes between soil and roots and thus play important roles in ecosystem functioning. To enhance our understanding of the factors that control RAF, we fitted statistical models to explain variation in RAF community structure using data from 150 temperate forest sites covering a broad range of environmental conditions and chemical root traits. We found that variation in RAF communities was related to both root traits (e.g., cations, carbohydrates, NO₃⁻) and soil properties (pH, cations, moisture, C/N). The identified drivers were the combined result of distinct response patterns of fungal taxa (determined at the rank of orders) to biotic and abiotic factors. Our results support that RAF community variation is related to evolutionary adaptedness of fungal lineages and consequently, drivers of RAF communities are context-dependent.     

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.