Inge Czaja

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.

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.

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.

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts.

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

T‐DNA tagging reveals a novel cDNA triggering cytokinin‐ and auxin‐independent protoplast division

Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.

retraction: Identification and role of adenylyl cyclase in auxin signalling in higher plants

This corrects the article DOI: 37810