New disease resistance genes in soybean against Pseudomonas syringae pv glycinea: evidence that one of them interacts with a bacterial elicitor

Summary Soybean [Glycine max (L.) Merr.] cultivars Flambeau and Merit differed in their resistance to Pseudomonas syringae pv glycinea (Psg) race 4, carrying each of four different avirulence (avr) genes cloned from Psg or the related bacterium, Pseudomonas syringae pv tomato. Segregation data for F₂ and F₃ progeny of Flambeau x Merit crosses indicated that single dominant and nonallelic genes account for resistance to Psg race 4, carrying avirulence genes avrA, avrB, avrC, or avrD. Segregants were also recovered that carried all four or none of the disease resistance genes. One of the disease resistance genes (Rpg1, complementing bacterial avirulence gene B) had been described previously, but the other three genes — designated Rpg2, Rpg3, and Rpg4 — had not here to fore been defined. Rpg3 and Rpg4 are linked (40.5 ± 3.2 recombination units). Rpg4 complements avrD, cloned from Pseudomonas syringae pv tomato, but a functional copy of this avirulence gene has not thus far been observed in Pseudomonas syringae pv glycinea. Resistance gene Rpg4 therefore may account in part for the resistance of soybean to Pseudomonas syringae pv tomato and other pathogens harboring avrD.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Responses of stomata of senescing and nonsenescing leaves of Nicotians glauca to changes in intercellular concentrations of leaf carbon dioxide

Abstract. Gas exchange measurements were performed to test the hypothesis that failure of stomata to open in senescing leaves of Nicotiana glauca is caused by elevated concentrations of carbon dioxide in the intercellular spaces of leaf mesophyll tissue (c_i). Senescing leaves selected for experiments were completely chlorotic and lacked positive rates of photosynthesis. When stomata in detached epidermis from senescing leaves were illuminated in CO2-free air, they opened to similar apertures as those in detached epidermis from nonsenescing leaves. To compare the effects of changes in c_i on stomatal responses of the two leaf types, leaf 'flags' of either nonsenescing or senescing leaves were illuminated at a photosynthetic photon flux density of 500 μmol m⁻² s⁻¹ in a gas exchange cuvette. Leaf temperatures were maintained at 23.5 ± 0.5°C, and vapour pressure differences between leaves and the air were maintained between 0.70 and 0.75kPa. C_i was adjusted by changing external concentrations of carbon dioxide in air circulating through the cuvette. Conductances and photosynthetic rates of nonsenescing leaves changed in response to changes in c_i, but neither the conductances nor the photosynthetic rates of senescing leaves were affected significantly by changes in q. We conclude that guard cells of senescing leaves of Nicotiana glauca do not lose the capacity to respond to changes in carbon dioxide concentration and that increases in c_i resulting from declining rates of mesophyll photosynthesis are not the sole cause of maintenance of stomatal closure during leaf senescence. The data suggest that factors external to guard cells may prevent them from responding to changes in carbon dioxide concentrations in intact senescing leaves.     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell