Richard Walden

Modification of Phytohormone Response by a Peptide Encoded by ENOD40 of Legumes and a Nonlegume

The gene ENOD40 is expressed during early stages of legume nodule development. A homolog was isolated from tobacco, which, as does ENOD40 from legumes, encodes an oligopeptide of about 10 amino acids. In tobacco protoplasts, these peptides change the response to auxin at concentrations as low as 10−12 to 10−16 M. The peptides encoded by ENOD40 appear to act as plant growth regulators. Sequence alignment of full ENDO40 gene sequences from soybean, pea, alfalfa, and tocacco plants.

Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

Cell signalling by oligosaccharides

Only recently has it begun to be understood how plants use the structural diversity of oligosaccharides to regulate important cellular processes such as growth, development and defence. A characteristic feature of these regulatory molecules is that they are biologically active at extremely low concentrations. A central remaining question is how cells perceive and transduce oligosaccharide signals, and current research is aimed at providing the answer.

Gene-transfer and plant-regeneration (techniques)

The production of transgenic plants depends on the stable introduction of foreign DNA into the plant genome, followed by regeneration to produce intact plants, and the subsequent expression of the introduced gene(s). A variety of transformation methods have been developed, and this has allowed many of the worlds important crop plants to be transformed. Transformation mediated by Agrobacterium has provided a reliable means of creating transgenics in a wide variety of species that are amenable to tissue culture and regeneration. Vectorless gene-transfer systems include particle bombardment (biolistics), electroporation and membrane permeabilization using chemicals. Of these, biolistics has proved to be particularly successful with plants that are less amenable to tissue culture, such as cereals and legumes.

Growth of Tobacco Protoplasts Stimulated by Synthetic Lipo-Chitooligosaccharides

Nodulation (Nod) factors are lipo-chitooligosaccharides (LCOs) secreted by rhizobia to trigger the early steps of nodule organogenesis in leguminous plants. A method to synthesize LCOs in vitro was developed. Synthetic LCOs alleviated the requirement for auxin and cytokinin to sustain growth of cultured tobacco protoplasts. LCOs containing C18:1 trans—fatty acyl substituents were more effective than those containing cis—fatty acids in promoting cell division as well as in activating an auxin-responsive promoter and the expression of a gene implicated in auxin action. These data indicate that LCOs redirect plant growth also in nonlegumes by activating developmental pathways also targeted by phytohormones.

Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants

SummaryTransgenic tobacco plants (Nicotiana tabacum L.) expressing the rolC gene of Agrobacterium rhizogenes under the transcriptional control of the 35S RNA promoter are male sterile. When these plants are genetically crossed with others containing the rolC gene linked in antisense orientation to the 35S RNA promoter, hybrid progeny display restoration of male fertility. Moreover, hybrid progeny are revertant for other features of the rolC phenotype, such as restoration of plant height, leaf pigment content and female fertility. The level of restoration of the characteristics of untransformed tobacco appeared to be independent of the steady-state level of antisense RNA. Addition of six transcriptional enhancer sequences upstream of the 35S transcriptional start region in the antisense construct led to a higher steady-state level of antisense RNA than that produced using a promoter linked to a single enhancer sequence. However no significant difference was observed in the level of attenuation of the rolC phenotype in the progeny of crosses with either one or six transcriptional enhancers linked to the antisense rolC gene. Antisense constructs comprising only 189 by of the rolC 5′ coding region appeared less efficient in attenuating the rolC phenotype than those including the whole rolC coding region as well as its 3′ untranslated region. Furthermore, results from experiments on light-controlled rolC gene expression indicate that microsporogenesis is sensitive to rolC gene action during the early stages of flower development.

Identification and role of adenylyl cyclase in auxin signalling in higher plants.

Cyclic AMP is an important signalling molecule in prokaryotes and eukaryotes, but its significance in higher plants has been generally doubted because they have low adenylyl cyclase activity and barely detectable amounts of cAMP. Here we used activation T-DNA tagging to create tobacco cell lines that can proliferate in the absence of the phytohormone auxin in the culture media,. The sequence tagged in one line, axi 141, was used to isolate a complementary DNA encoding adenylyl cyclase, the first from a higher plant. Sequence analysis reveals that the tobacco adenylyl cyclase is probably soluble, contains characteristic leucine-rich repeats, and bears similarity with adenylyl cyclase from the yeast Schizosaccharomyces pombe. Expression of the cDNA in Escherichia coli results in an increase in endogenous cAMP levels, and in yeast its expression functionally complements the cry1 mutation. Tobacco protoplasts treated with cAMP, or the adenylyl cyclase activator forskolin, no longer require auxin to divide. This finding, together with the observation that the adenylyl cyclase inhibitor dideoxyadenosine inhibits cell proliferation in the presence of auxin, suggests that cAMP is involved in auxin-triggered cell division in higher plants.

Developmental and UV Light Regulation of the Snapdragon Chalcone Synthase Promoter.

Expression directed by the 1.1-kb snapdragon chalcone synthase (CHS) promoter linked to the [beta]-glucuronidase reporter gene has been studied in transgenic tobacco. The pattern of expression of the chimeric gene was compared with the expression of the endogenous CHS genes in tobacco and snapdragon. We demonstrate that expression of the CHS promoter is controlled in both an organ-specific and tissue-specific manner. The highest level of expression was observed in immature seeds. Deletions were used to define regions of the promoter required for expression in roots, stems, leaves, seeds, and flower petals of transgenic plants. We have defined the minimal sequences required for expression in different organs and mapped regions of the promoter that influence expression in either a positive or negative manner. A promoter fragment truncated to -39 activates transcription in roots of 4-week-old seedlings, whereas a fragment extending to -197 bp directs expression in petals and seeds. A positive regulatory element located between -661 and -566 and comprising a 47-bp direct repeat is active in all tissues investigated except petals. UV light-regulated expression in leaves of transgenic tobacco seedlings is dependent on the presence of sequences also required for leaf-specific expression. Within the intact promoter, sequences that individually confer different patterns of expression interact to produce the highly regulated expression pattern of CHS.

Rol genes alter hormonal requirements for protoplast growth and modify the expression of an auxin responsive promoter

SummaryGrowth characteristics of tobacco protoplasts containing rolA linked to its own promoter, or the rolB, or rolC genes of Agrobacterium rhizogenes linked to the Cauliflower Mosaic Virus 35S RNA promoter were compared with those from untransformed plants. RolA protoplasts require auxin and cytokinin for callus formation. Protoplasts overexpressing rolB and C form callus in the absence of exogenously applied auxin and cytokinin, respectively. Long term callus growth requires auxin, but the requirement for cytokinin is not critical. Optimal transient expression of an auxin responsive promoter element occurred at lower external levels of auxin in rolB and rolC protoplasts compared with untransformed protoplasts. Addition of putrescine was required for auxin responsive transient gene expression in rolA protoplasts suggesting that polyamines, or their products affect gene expression in rolA plants.

A plant cation–chloride co-transporter promoting auxin-independent tobacco protoplast division

Although auxin plays a central role in plant development, little is known about the signal transduction pathways triggered by auxin regulating cell elongation, division and differentiation. We describe the molecular analysis of the mutant tobacco line axi 4/1, which was regenerated from an auxin‐independent callus created by activation T‐DNA tagging. Transcriptional enhancer‐mediated deregulated expression of the tagged plant gene axi 4 uncouples division of axi 4/1 protoplasts from external auxin stimuli, whereas in untransformed protoplasts expression of axi 4 and cell division are auxin dependent. axi 4 encodes a 109 kDa protein with significant homology to a family of electroneutral cation–chloride co‐transporters (CCCs). We show that overexpression of AXI 4 or another member of the CCC family, the Na+/K+/2Cl−‐co‐transporter from shark, triggers auxin‐independent growth of tobacco protoplasts. We suggest that Na+/K+/2Cl−‐co‐transporters may play a role in signalling cell division and that this function is highly conserved between AXI 4 and the shark Na+/K+/2Cl−‐co‐transporter. We also demonstrate that a C‐terminal fragment of AXI 4 is sufficient to promote auxin‐independent cell division, showing that the C‐termini of CCCs are functional subunits triggering cell division. This may allow a molecular dissection of this process not only in plants but also in animal cells.