John Simmonds

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.

A constitutive gene expression system derived from the tCUP cryptic promoter elements

Abstract.A limited number of constitutive promoters have been used to direct transgene expression in plants and they are often derived from non-plant sources. Here, we describe novel gene-regulatory elements which are associated with a cryptic constitutive promoter from tobacco, tCUP, and modifications that were made to create a strong gene-expression system that is effective across all living cell types from a wide range of plant species, including several important crops (Arabidopsis, canola, flax, alfalfa, tobacco). The tCUP 5′ untranslated region was mutated to eliminate translational interference by upstream ATGs, and the influence of the Kozak consensus sequence on the levels of a β-glucuronidase (GUS) reporter gene activity was demonstrated. These modifications resulted in expression that was greatly enhanced in all organs. A TATA consensus sequence was added to the core promoter to complement an existing Initiator (Inr) sequence. Although this addition was known to elevate core promoter activity by 3-fold the additive effect on the overall gene-expression system was marginal in all of the transgenic plants tested. Two transcriptional enhancers were identified and the region containing them were oligomerized, yielding a significant increase in marker gene-expression in some but not all plant species. In general, the enhanced tCUP gene-expression system generated levels of GUS activity which exceeded that of the 35S promoter in most plant species and the elevation in activity occurred uniformly among the various plant organs. The potential benefit of cryptic elements for the construction of gene-expression systems for crop species is discussed

Reduced herbivory of the European corn borer (Ostrinia nubilalis) on corn transformed with germin, a wheat oxalate oxidase gene

Abstract The strategy of targeting expression of a constitutively regulated gene to generate H 2 O 2 in the extracellular matrix, to reduce herbivory of the European corn borer (ECB) [ Ostrinia nubilalis (Hubner)] was tested by transforming corn with germin, a wheat oxalate oxidase (OXO) gene, regulated by the rice actin promoter elements (pAct-OXO). With two independent transformation events, enzyme activity was stable over seven generations of backcrossing into three maize inbred lines. Enzyme activity remained associated with the cell wall debris fraction of water extracted tissues. Leaf tissue of the germin transgenics had elevated levels of H 2 O 2 . In vitro leaf feeding bioassays demonstrated that ECB larvae feeding was significantly reduced and larval growth and development were delayed on all ECB infested germin transgenic lines. This reduced ECB feeding was confirmed under field conditions. Most significantly, stalk tunneling damage, measured at plant harvest, was substantially reduced in all germin transgenic lines. The reduction of tunneling by 50% in the transgenic lines is indicative of lower levels of ECB survival which should be significant in ECB epidemiology. Possible mechanisms of resistance include modifications in plant cell wall chemistry, activation of pathogen resistance genes and effects of H 2 O 2 and germin on insect physiology are discussed.

Developmental and environmental regulation of anthocyanin pigmentation in wheat tissues transformed with anthocyanin regulatory genes

SummaryCell autonomous anthocyanin pigmentation, produced by the anthocyanin regulatory genes B and C1 controlled by the constitutive CaMV35s promoter (pBC1-7), was used to optimize biolistic gene delivery into embryogenic wheat (Triticum aestivum L. cv ‘Chris’) scutellum cultures. Intensely pigmented callus cells were observed 24 h postbombardment but these cells did not continue to divide and were developmentally terminal. A population of nonexpressing cells generated transgenic sectors which showed light-dependent anthocyanin pigmentation. Anthocyanin pigmentation was suppressed in regenerating shoot cultures but reverted to light-dependent production in the pericarp of developing seeds. Similarly, following microtargeted gene delivery into apical meristems, anthocyanin production was developmentally suppressed in leaf base meristems but prominent anthocyanin sectors developed in mature tissues beyond this region and persisted throughout leaf growth. In three developmental situations, callus proliferation, plant regeneration, and leaf growth, perpetuation of cells with anthocyanin regulator genes under the control of constitutive promoters was dependent on a higher level of regulation to suppress pigmentation at developmentally sensitive stages of meristematic activity. These findings provide additional evidence that the anthocyanin regulatory genes may be responsive to a variety of developmental and environmental stimuli.

Effects of low temperature stress on excitation energy partitioning and photoprotection in Zea mays

Analysis of the partitioning of absorbed light energy within PSII into fractions utilised by PSII photochemistry (ΦPSII), thermally dissipated via ΔpH- and zeaxanthin-dependent energy quenching (ΦNPQ) and constitutive non-photochemical energy losses (Φf,D) was performed in control and cold-stressed maize (Zea mays L.) leaves. The estimated energy partitioning of absorbed light to various pathways indicated that the fraction of ΦPSII was twofold lower, whereas the proportion of thermally dissipated energy through ΦNPQ was only 30% higher, in cold-stressed plants compared with control plants. In contrast, Φf,D, the fraction of absorbed light energy dissipated by additional quenching mechanism(s), was twofold higher in cold-stressed leaves. Thermoluminescence measurements revealed that the changes in energy partitioning were accompanied by narrowing of the temperature gap (ΔTM) between S2/3QB- and S2QA- charge recombinations in cold-stressed leaves to 8°C compared with 14.4°C in control maize plants. These observations suggest an increased probability for an alternative non-radiative P680+QA- radical pair recombination pathway for energy dissipation within the reaction centre of PSII in cold-stressed maize plants. This additional quenching mechanism might play an important role in thermal energy dissipation and photoprotection when the capacity for the primary, photochemical (ΦPSII) and zeaxanthin-dependent non-photochemical quenching (ΦNPQ) pathways are thermodynamically restricted in maize leaves exposed to cold temperatures.

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H2O2 generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations.

Molecular analysis of two Brassica napus genes expressed in the stigma.

A partial cDNA clone, Pis 63, corresponding to a mRNA highly expressed in Brassica napus pistils, was isolated by differential screening. PCR was used to complete the Pis 63 sequence (Pis 63-1) and to obtain the sequence of another related cDNA (Pis 63-2). Northern blot and in situ analyses demonstrated that these transcripts are expressed in the stigma throughout flower development. Pis 63-1 and Pis 63-2 display similarity to a cotton fibre cDNA clone.

Mitotic activity in wheat shoot apical meristems: effect of dissection to expose the apical dome

An efficient method, less laborious than histological procedures, is described to screen relatively large numbers of shoot apices for mitotic activity. Mitotic activity of shoot apices of Triticum aestivum L. was observed by differential interference contrast (DIC) microscopy of apices infiltrated with a clearing fluid (chloral hydrate/phenol/lactic acid/dibutylphthalate/benzyl benzoate). Serial optical sections were viewed through entire vegetative apical domes and floral primordia. In vegetative shoots, mitotic cells were observed throughout the light and dark cycles of plants maintained in either 8 or 16 h photoperiods. Mitotic activity was lower in the dark phase and increased through the light cycle in both photoperiods. Cells in L1 and L2 layers at the summit of the apex were mitotically active and contributed to the developing shoot and floral structures. Thus, cells in L2 at the summit of vegetative apices are valid targets for transformation leading subsequently to modified germ line cells. Dissections to expose apices for DNA delivery inhibited mitotic activity; recovery periods greater than 48 h would be needed for restoration of normal activity. This suggests that a period of recovery from dissection would be beneficial for attempts at integrative transformation of apical cells.

Inhibitory effects of filter-sterilized media on microspore development and embryogenesis in Brassica napus and Triticum aestivum

SummaryPlant tissue culture media, sterilized through commercial filter membrane units, inhibited plant cell growth and development. Embryogenesis was suppressed in microspore cultures of Brassica napus and anther cultures of Triticum aestivum, and in vitro pollen development was suppressed in B. napus. Inhibition of growth and development occurred on media that had been filter-sterilized in low volumes through non-washed filter membrane units of all brands tested, including Nalgene, Corning, Millipore, and Sartorius membrane products. Similar results were obtained with Costar Transwells and Millipore inserts (Millicell-CM.-HA, and-PCF). These deleterious effects were eliminated by washing the membranes with water followed by a small volume of the medium. Media sterilized by filtration through washed filters produced significant improvements of growth and development of embryos and pollen.