Polyamines (PAs) are a group of low molecular weight polycationic compounds that are ubiquitous in nature and important for many physiological, biochemical and developmental processes (Smith 1985). Current views about the plasticity of the PA pathway in plants are conflicting, with one school of thought supporting the notion that the pathway is rigidly controlled and as a result it is unlikely that any changes in end product accumulation can be achieved by modulating levels of enzyme activity in the pathway (Burtin and Michael 1997). An alternative view which is increasingly supported by more in depth molecular and biochemical studies using transgenic plants suggests that the pathway can indeed exhibit plasticity, however this depends on spacial control of expression consistent with a threshold model in terms of levels of accumulation of the first PA, Put, in specific tissues (Bassie et al. 2000). Most profound changes appear to take place in storage tissues such as seeds, followed by roots with the least effect seen in vegetative tissues (Thrun- Nghia et al. unpublished). The simplest PA, putrescine (Put), is derived from ornithine by ornithine decarboxylase (ODC). Putrescine may then be converted into the longer aliphatic PAs spermidine (Spd) and spermine (Spm) by spermidine and spermine synthases respectively. Plants provide an interesting eukaryotic system for investigating the physiological and biochemical role of PAs because an alternative route to Put via arginine decarboxylase (ADC) is present. Ornithine decarboxylase converts ornithine to Put directly whereas ADC uses arginine as substrate to also generate Put via agmatine and Ncarbamoylputrescine (Smith 1985).

We had previously shown that manipulation of ADC in transgenic rice by either over-expression (Fig. 1A) or down-regulation using an antisense construct (Fig. 1B) resulted only in Put concentration increases in one clone (Noury et al. 2000) or decreases (Capell et al. 2000) in seeds (Fig. 2A and B). In contrast, by generating transgenic plants expressing odc either in a constitutive or a seed-specific manner, we were able to detect changes in all three PAs in seeds (Fig. 3) in a large number of independent transgenic plants. Results from a comparative analysis of enzyme activities and PA levels in different tissues or organs in transgenic rice plants expressing heterologous adc (sense or antisense) or odc also support our hypothesis that fluctuations in PA levels as a result of over-expression or down-regulation of the two early enzymes in the pathway are regulated at the tissue or organ level. Furthermore, heterologous odc expression in rice tissues resulted in an increase in Put concentration in leaves in two specific lineages (clones 2 and 48, Fig. 4A) a behaviour we rarely observed in any plants engineered with adc driven by a strong constitutive promoter (Noury et al. 2000, Fig. 4B). Line 2 also had a small but significant

increase in Spd and Spm accumulation in leaves (Fig. 4A). We postulate that the increases in Spd and Spm are consistent with the notion that the transgenic Put pool in leaf tissue needs to reach a minimal level before excess Put may be channeled into the biosynthesis of the higher PAs. However, no linear correlation could be demonstrated between increases in ODC activity in leaf or root tissue and end product accumulation in seeds.

Our data suggest strongly that the spacial control of the PA pathway in plants has been largely ignored by investigators in the field. This is a significant deficiency which is largely responsible for what we believe to be an erroneous view about the plasticity of the PA pathway in plants. By comparing the expression profile of ADC and ODC enzymes and PA levels in transgenic rice populations engineered with adc (either in sense or antisense

orientation) and odc, we conclude that ODC is the main enzyme responsible for Put biosynthesis in plants.

In summary, our results indicate that: (a) whereas previous results showed that the expression of a heterologous arginine decarboxylase (adc) in rice only resulted in increases in Put but no Spd and Spm levels in seeds in one clone, plants engineered with odc exhibited changes in all polyamines in seeds; this was independent of the promoter controlling expression of the transgene(s); (b) there was no linear correlation between either odc/adc mRNA levels, enzyme activity and PA levels; (c) PA levels in seeds were not dependent on odc or adc expression levels; (d) PA levels and enzyme activity varied in different tissues, indicating that the pathway is plastic in a spacial manner. The redundancy of the alternative ADC pathway may be attributed to evolutionary reasons, as among living organisms only plants and bacteria are known to contain this part of the pathway. Having established the key role of ODC in PA biosynthesis in plants we are now in a position to design more rational strategies to determine how flux through the subsequent steps in the pathway influences end product accumulation and also how changes in PA levels may influence related pathways such as ethylene and tropane alkaloid biosynthesis.

References

Bassie L, M. Noury, O. Lepri, T. Lahaye, P. Christou and T. Capell, 2000. Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat arginine decarboxylase cDNA constitutively. Transgenic Res. 9: 33-42.

Burtin D and T. Michael, 1997. Over-expression of arginine decarboxylase in transgenic plants. Biochem. J. 325: 331-337.

Capell T, L. Bassie, L. Topsom, E. Hitchin and P. Christou, 2000. Simultaneous down-regulation of two related enzymes in early steps of the polyamine biosynthetic pathway in transgenic rice by a single antisense mRNA species. Mol. Gen. Genetics (in press).

Noury M, L. Bassie, O. Lepri, I. Kurek, P. Christou and T. Capell, 2000. A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues. Plant Mol. Biol. 43: 537-544.

Smith A, 1985. Polyamines. Ann. Rev. Plant Physiol. 36: 117-143.