Rod Scott

Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene β-glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and α-amylase inhibitors.

Characterisation of a wound-induced transcript from the monocot asparagus that shares similarity with a class of intracellular pathogenesis-related (PR) proteins

We report the isolation and characterisation of a wound-induced cDNA designated AoPR1 from a suspension of mesophyll cells that had been mechanically isolated from cladodes of light-grown Asparagus officinalis seedlings by grinding in a mortar and pestle. The transcript abundance is up-regulated following cell separation and in chopped mesocotyl tissue from dark-grown seedlings. The expression of AoPR1 was shown by northern analysis to be located around the site of damage. Sequence analysis revealed similarity between the predicted AoPR1 polypeptide and bean PvPR1 and PvPR2 proteins, the potato pSTH2 protein, the pea PI49 protein, the parsley PcPR1-1 protein and a major pollen allergen from birch (BetvI). These transcripts have been shown to be induced in response to microbial attack or fungal elicitation. To our knowledge, this is the first example of a monocot cDNA belonging to this class of intracellular pathogenesis-related proteins (IPRs).

Patterns of gene expression in developing anthers of Brassica napus

The relationship between bud length, anther length and stage of anther development has been investigated in Brassica napus using a series of cytological markers that define steps in the process of male gametogenesis. It was determined that bud length is directly related to anther length and that anther or bud length is tightly linked to the stage of male gametogenesis within the anther. This simple correlation has enabled the construction of cDNA libraries representing transcripts expressed in defined stages of anther development, and the detailed examination of the developmental pattern of expression of anther RNAs.Two anther cDNA libraries were constructed, one from anthers of 1.2–1.8 mm long buds (sporogenesis library) and one from anthers of 1.8–4.0 mm long buds (microspore development library). A total of 19 independent cDNAs have been isolated by differential screening whose temporal expression patterns overlap and which together cover the stages of anther development from pre-meiotic microsporocytes to tri-nucleate pollen grains. The pattern of expression of each of these clones is unique and indicates that stages of anther development which cannot be easily distinguished by light microscopy can be recognised by virtue of the absence or presence of certain RNAs. Three cDNAs isolated from the sporogenesis library have been shown by in situ hybridisation to be tapetum-specific. In contrast, five clones isolated from the microspore development library are microspore-specific. These clones exhibit a pattern of expression different to those previously described in that their transcripts are absent in mature pollen grains. Thus these RNAs are probably required in microspore development rather than for the growth of the germinating pollen grain.

A Brassica napus mRNA encoding a protein homologous to phospholipid transfer proteins, is expressed specifically in the tapetum and developing microspores

The E2 cDNA was isolated by differential screening of a Brassica napus library made from anthers of 1.8–4 mm in length. Northern gel and RNA dot blot analysis revealed that the E2 cDNA hybridised to a 0.7-kb mRNA transcript in RNA extracted from anthers and extruded immature microspores. High expression is limited to the microspore development phase. In situ hybridisation indicated that E2 transcript is also expressed within the tapetal cells. The sequence of 658 nucleotides was determined from clone E2B containing one major open reading frame, encoding a putative protein of Mr 12.5 kDa. This protein is cysteine rich, encodes a potential signal sequence and shows high homology with plant phospholipid transfer proteins.

A Brassica napus mRNA expressed specifically in developing microspores

The I3 cDNA isolated from a library made from 2–4 mm (immature) anthers of Brassica napus shows microspore-specific expression. Homologous transcripts are detected in buds and anthers of male-fertile plants, but not in green tissues, roots, or in cytoplasmic male-sterile buds. High expression of the transcript is limited to microspores entering and undergoing mitosis. The predicted peptide sequence of the cDNA shows an unusual repeated alanine/proline motif at the C-terminus, which may be of importance in the native protein structure.

Correct processing of the kiwifruit protease actinidin in transgenic tobacco requires the presence of the C-terminal propeptide.

A 35S cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-level accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cleave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing.

Activation and developmental regulation of an Arabidopsis anther-specific promoter in microspores and pollen of Nicotiana tabacum

SummarySeven cytologically distinct stages during micro spore development were identified and used to define the activation of an Arabidopsis anther-specific gene (apg) in transgenic tobacco plants containing an apg promoter-gus fusion. Histochemical analysis of GUS activity showed that the apg promoter was activated in miduninucleate microspores prior to equatorial and polar nuclear migration. Quantitative analysis in isolated spores showed that GUS activity per milligram of protein decreased progressively during pollen development, but on a per spore basis showed a second peak of activity in mid-bicellular pollen. Activation of the apg promoter in isolated gametophytic cells during development was also investigated following DNA delivery by microprojectile bombardment. Levels of transient expression were detectable in uninucleate microspores, but peaked in mid-bicellular pollen, in contrast to the progressive increase in activity of the promoter of the ‘late’ pollen gene lat52. These data show that the apg promoter is activated in a biphasic pattern, and indicate that the activity of transcription factors which mediate apg promoter activity persist through microspore mitosis, but decrease during pollen maturation.

Genomic male sterility in lettuce, a baseline for the production of F1 hybrids

Abstract Male sterile plants of lettuce ( Lactuca sativa L.) cv. ‘Lake Nyah’ were produced using a genotype-independent transformation method employing Agrobacterium tumefaciens with a binary vector. The latter carried the neomycin phosphotransferase II gene controlled by the nopaline synthase promoter and terminator sequences, enabling transformed shoots to be selected on medium containing kanamycin sulphate. In addition, expression of a pathogenesis-related glucanase gene linked to a tapetum-specific promoter, caused the dissolution of the callose wall of developing microspores in transgenic plants. All transformed plants were male sterile. Plants regenerated from uninoculated, excised cotyledons of ‘Lake Nyah’ exhibited normal microspore development and were fertile. These results suggest that callose degradation may cause male sterility and provides the opportunity for producing F1 hybrid seed in lettuce.

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.