Crop productivity relies heavily on abundant supplies of water for
irrigation. Although drought and salt tolerance genes are present in wheat,
breeding for stress tolerance is time- and labour-intensive and complicated
by the multigenic nature of stress tolerance and the complexity of wheat
genetics. The polyamine biosynthetic pathway in higher plants is a useful
model in which to examine the components that affect the levels of
intermediates and end products in the pathway.

By introducing appropriate transgenes into plants and measuring the effects
of transgene products on end product accumulation, we may begin to
understand how individual components of the pathway contribute towards their
concerted regulation.

The polyamines spermidine and spermine, and their precursor putrescine, are
ubiquitous in all living organisms and are involved in many diverse
physiological, developmental, and biochemical processes. Pyridoxal phosphate
(PLP)-dependent ornithine decarboxylase (ODC) is the initial enzyme in the
pathway committed to polyamine synthesis. In plants and some bacteria,
putrescine can also be synthesized from arginine via arginine decarboxylase
(ADC) through the intermediate agmatine. Putrescine is further converted
into spermidine and spermine by spermidine synthase (SPDS) and spermine
synthase (SPMS), respectively. These enzymes add aminopropyl groups
generated from S-adenosylmethionine (SAM) by SAM decarboxylase (SAMDC). In
plants, the two alternative pathways appear to have specific roles in growth
and development. While ODC appears to be implicated in the regulation of the
cell cycle in actively dividing cells and meristematic zones, ADC is the
primary enzyme for putrescine synthesis in non-dividing elongating cells,
secondary metabolic processes, and in cells under various stresses2.

Arginine decarboxylase is a low copy number nuclear gene that lacks introns
in the sequences described to date. Several ADC cDNA clones have been
isolated and characterized from various species. Whereas in some plants a
single gene encodes ADC, in the Brassicaceae family at least two paralogues
exist in all members studied to date except for the basal genus Aethionema.
In Arabidopsis, protein sequences derived from ADC1 (U58851) and ADC2
(AF009647) genes show 80% homology; however the activities of these enzymes
differ. Much of the difference between ADC1 and ADC2 protein amino acid
sequences is at the N-terminus, suggesting that the subcellular location of
the two proteins might be different. Polyclonal antibodies raised against
tobacco ADC (AF321137; 99% homology to the tobacco ADC2 AF127241) detect ADC
protein in all plant organs analyzed: flowers, seeds, stems, leaves, and
roots; however, depending on the tissue, the protein is localized in two
different subcellular compartments, the nucleus and the chloroplasts. These
results suggest that the intracellular location of ADC in plants might
account for different roles for the enzyme in different locations. Thus the
notion was advanced that chloroplastic ADC might be involved in
photosynthesis, whereas the nuclear form may play a role in cellular
signalling3.

Engineering the polyamine biosynthetic pathway in wheat
Wheat (Triticum aestivum L.) is a staple crop for about 35% of the human
population. Its great adaptability to varied climatic conditions makes it
one of the most widely cultivated crops, with a short growing season and a
good yield per unit area. Breeders have produced disease-resistant,
drought-tolerant (to a certain degree) and high-yielding varieties using
conventional methodology. However, wheat productivity has declined as a
result of deteriorating soil conditions, the quality and quantity of
available water for irrigation, and general environmental degradation.
Consequently, increases in productivity have fallen below the rate of
population growth.

One of the major targets for wheat improvement is drought tolerance. Our
group is interested specifically in elucidating the role of polyamines in
abiotic stress tolerance in cereal crops. We generated transgenic wheat
plants expressing an oat (Avena sativa L.) ADC cDNA driven by the maize 1
ubiquitin (Ubi) promoter and first intron4. These plants accumulate up to
2-fold putrescine, spermidine, and spermine in leaves. The two-fold increase
in the three polyamines measured in leaves of primary transformants is
maintained in the T1 generation, thus confirming the heritable nature of
polyamine accumulation in transgenic wheat plants expressing the transgene.

Whereas an increase in polyamine content is rare in rice leaves, such
increases are more common in seeds. For example spermidine and spermine
levels are significantly increased in seeds of transgenic rice plants
expressing Ubi:DsSAMDC5. Multiple independent transgenic wheat lines
expressing Ubi:AsADC accumulate up to 7-fold putrescine, 1.5-fold
spermidine, and two-fold spermine in seeds, compared to wild type plants.
These levels are maintained and even enhanced in progeny.

Results from our earlier studies in rice and also our current investigation
in wheat demonstrate that less metabolically active tissue, such as seeds,
accumulate higher levels of polyamines. Our results are in line with
experiments in which metabolites such as pre-vitamin A and pharmaceutical
antibodies accumulate at high levels in seeds of rice, wheat, and pea6. It
is not surprising that higher levels of accumulation occur in seeds, because
these storage organs are dormant or certainly less metabolically active
compared to vegetative tissue. The above examples show that this behavior is
not limited to small molecular weight metabolites. Rather it is more
general, extending to the accumulation of recombinant proteins, which in
extreme cases can form paracrystalline structures in the endosperm6.

The wheat genome contains at least two arginine decarboxylase paralogs
Genomic characterization of the transgenic wheat plants indicated high
homology between the oat and wheat ADC. Digestion with several enzymes and
hybridization with a cloned partial wheat ADC probe confirmed the presence
of at least two ADC genes in the wheat genome. Only two ADC genes have been
cloned from monocotyledonous plants to date?the first from oat (X56802) and
more recently a second gene from rice (BAA84799). Chattopadhyah et al.7,
upon digestion of rice and oat genomic DNA with a specific enzyme and
subsequent probing with a 498-OsADC probe, observed two distinct molecular
species in both rice and oat genomic DNA blots. An ancestral ADC gene
appears to have been duplicated early in the origin of the Brassicae family,
thus yielding two paralogs. These two different ADC1 and ADC2 genes have
been described in Arabidopsis. Although the two genes share 80% homology in
their amino acid sequence, they exhibit different expression patterns: ADC1
is expressed constitutively, whereas ADC2 is mainly expressed in cauline
leaves and siliques and is induced by different abiotic stresses. Two ADC
paralogous genes have been characterized also in Pringlea antiscorbutica,
Nicotiana tabacum, and Malus sylvestris L (crabapple).

Using the gene-specific probe for TaADC, we detect two transcripts of ca.
2.6 and 3.1 kb in wheat. The transcripts are expressed in leaves and roots,
with the 2.6 kb species exhibiting higher steady-state accumulation. No
significant changes are measured in levels of steady-state TaADC (long or
short mRNA species) or in TaSAMDC levels in transgenic plants expressing the
AsADC gene. It is therefore apparent that the heterologous transgene
operates independently of its wheat homologs, and consequently any increase
in polyamine content is due to expression of the transgene alone. Some
evidence exists for post-transcriptional and post-translational regulation
of ADC in plants. TaADC might generate two different forms of the transcript
through alternative splicing. Alternative processing of a transcript is
common in animal cells, where expression from a single gene may produce
protein variants that differ in function, tissue specificity, or
sub-cellular localization. There are also reports of alternative splicing in
plants. For example Zhang et al.8 described the cloning of three cDNAs from
apple (MdSPDS-1, -2a and -2b) encoding SPDS. MdSPDS-2a and MdSPDS-2b
originate from SPDS2 by alternative splicing and are differentially
regulated in a tissue- and developmentally-specific manner.

In conclusion, we generated and characterized a transgenic wheat population
expressing AsADC. In the course of this characterization, we detected the
presence of multiple ADC homologs in wheat, coinciding with gene duplication
in other plant species with complex genomes. This transgenic wheat
population will be useful in studies to elucidate further the role of
polyamines in drought tolerance and also to ascertain differences and
similarities between rice and wheat in their general response to abiotic
stresses. Such studies are important because they will allow us to determine
whether polyamines are general response mediators that enhance tolerance of
plants to abiotic stress.

References
1. Capell T, Christou P (2004) Current Opinion in Biotech. 15, 148-154

2. Capell T, Bassie L, Christou P (2004) Proc. Natl. Acad. Sci. of USA 101,
9909-9914

3. Hanfrey C, Sommer S, Mayer MJ, Burtin D, Michael AJ (2001) Plant J. 27,
551-560

4. Bassie L, Zhu C, Romagosa I, Christou P, Capell T (2008) Mol. Breeding
DOI 10.1007/s11032-007-9154-2

5. Thu-Hang P, Bassie L, Safwat G, Trung-Nghia P, Capell T (2002) Plant
Physiol. 129, 1744-1754

6. Stoger E, Williams S, Keen D, Christou P (1999) Trans. Res. 8, 73-82

7. Chattopadhyay MK, Gupta S, Sengupta DN, Ghosh B (1997) Plant Mol. Biol.
34, 477-483

8. Zhang Z, Honda C, Kita M, Hu C, Nakayama M, Moriguchi T (2003) Mol.
Genet. Genomics 268, 799-807

www.checkbiotech.org