www.czu.cz ; www.raupp.info

Engineering the plant polyamine biosynthetic pathway.
Manipulation of metabolic pathways in plants through molecular genetics is
now possible because of a significant increase in our knowledge base of how
such, often complex, networks are controlled and regulated. In our ongoing
efforts to implement rational molecular approaches to modulate plant
metabolism, we chose the polyamine pathway as a model to unravel key factors
that still present bottlenecks in engineering plant biosynthetic pathways,
August 2004 by Teresa Capell .



The polyamine pathway is ubiquitous in living organisms.
Polyamines are low molecular weight polycationic molecules, which are
thought to play important roles in a number of physiological and
developmental processes (1). In animals and fungi, the diamine putrescine
(the precursor of the higher polyamines spermidine and spermine) is
synthesized directly from ornithine by the enzyme ornithine decarboxylase
(ODC). Plants have an alternative route to the production of putrescine that
is catalyzed by arginine decarboxylase (ADC). Additional reactions convert
putrescine into spermidine and spermine. These steps are catalyzed by
spermidine and spermine synthases, which add propylamino groups generated
from S-adenosylmeth-ionine by S-adenosylmethionine decarboxylase (SAMDC).

Engineering of the plant polyamine biosynthetic pathway has concentrated
mostly on two species, tobacco and rice (2,3). We have generated a diverse
rice germplasm with altered polyamine content. Transgenic rice plants
expressing the Samdc cDNA accumulated spermidine and spermine in seeds at
two to three-fold higher levels compared to wild type. In a different set of
experiments, we were able to measure a ten-fold putrescine accumulation in
transgenic rice plants harboring oat adc cDNA compared to wild type.
Reduction in endogenous adc transcript levels in rice resulted in depletion
of putrescine and spermidine pools, with no concomitant changes in
expression of downstream genes in the pathway.

In general, studies focusing on spatial expression of these transgenes
demonstrate that more dramatic changes in polyamine content occur in storage
compared to vegetative tissues, such as leaves and roots. Therefore, we
believe that the polyamine biosynthetic pathway in plants is regulated
strongly in a spatial manner (4). In tomato, enhanced fruit juice quality
and prolonged vine life of fresh fruits with increased lycopene was achieved
by expression of yeast Samdc driven by the ripening-inducible E8 promoter
(5).

Role of polyamines in stress response In plants, polyamines accumulate under
several abiotic stress stimuli, including salt and drought. It has been
suggested that this increase in polyamine concentration could be considered
as an indicator of plant stress. The first demonstration of involvement of
polyamines in stress responses in plants was documented by accumulation of
putrescine in response to sub-optimal K+ levels (6). Since then, the link
between increased putrescine levels and several abiotic stresses was
established. For example, Krishnamurthy and Bhagwat (7) reported
accumulation of spermidine and spermine in salt tolerant rice cultivars and
accumulation of putrescine in rice sensitive cultivars in response to
salinity stress.

The physiological role of an increase in putrescine accumulation following
abiotic stresses is still unclear and is a matter of considerable debate. It
has been very difficult to establish directly a cause and effect
relationship between increased polyamine levels in plants and abiotic
stress. The increase in putrescine levels in plants under stress might be
the cause of stress-induced injury or alternatively a means of protection
against stress. Earlier experiments by Roy and Wu (8,9) expressing oat adc
cDNA in rice under control of an ABA-inducible promoter resulted in
transgenic rice plants with increased biomass when grown under salt stress.
The same authors expressed Tritordeum Samdc cDNA in rice, under control of
the same promoter. Under salt stress these plants showed increased seedling
growth compared to wild type. Results from a number of studies suggest that
polyamines, particularly spermidine and spermine, are involved in regulation
of gene expression by enhancing DNA-binding activities to particular
transcription factors. Polyamines are believed to have an osmoprotectant
function in plan cells under water deficit.

A threshold model linking polyamines and abiotic stress response in plants
Osmoregulatory processes are important to all organisms for stabilization of
the intracellular milieu against environmental fluctuations of water and
ions. Despite divergence of biochemical pathways, both prokaryotic and
eukaryotic organism share several physiological responses to osmotic stress.

We put forward a threshold model that is consistent with high levels of adc
gene expression leading to production of putrescine. The production of
putrescine needs to exceed basal levels in order to generate a large enough
metabolic pool to trigger polyamine flux through the pathway leading to
increases in the levels of spermidine and spermine. Transgenic rice plants
expressing the Datura adc gene accumulated up to two-fold putrescine in leaf
tissues compared to wild type. Such plants, when subjected to drought stress
induced by 20% polyethylene glycol, exhibit a very significant divergent
behavior compared to wild type under the same conditions. Following 3 and 6
days of drought stress, all wild type plants wilt and show drought-induced
rolling of leaves. Such symptoms are completely absent from Dadc -transgenic
plants, which exhibit significant putrescine accumulation during the same
period. After the 6-day drought stress period, the phenotype of transgenic
plants is indistinguishable from non-stressed wild type. Transgenic plants
with 2- to 4-fold higher levels of putrescine develop and set seed normally.

Based on these observations, we put forward a model that is consistent with
a mechanism linking polyamine metabolism to drought tolerance. Expression of
the Dadc transgene driven by the strong maize Ubi-1 promoter would augment
the putrescine pool to levels that extend beyond the critical threshold
required to initiate the conversion of excess putrescine to spermidine and
spermine (10). Spermidine and spermine de novo synthesis in transgenic
plants under drought stress is corroborated by the activation of the rice
samdc gene. Transcript levels for rice samdc reach their maximum levels at 6
d after stress induction. Such increases in the endogenous spermidine and
spermine pools of transgenic plants not only regulate the putrescine
response, but also exert an anti-senescence effect at the whole plant level,
resulting in phenotypically normal plants. Wild type plants, however, are
not able to raise their spermidine and spermine levels after 6 d of drought
stress and consequently exhibit the classical drought-stress response (11).

Transgenic germplasm we have generated exhibiting increased tolerance to
drought stress is currently being evaluated in field trials. We are very
excited about the prospects of this germplasm to make a positive
contribution towards sustainable rice production under stress conditions.

References

(1) Malmberg RL et al. (1998) Critical Rev Plant Sci 17, 199-224.

(2) Kumar A, Minocha SC (1998) Transgenic manipulation of polyamine
metabolism. In: Lindsey K (ed) Transgenic research in plants. Harwood
Academic Publishers UK, pp 187-199.

(3) Capell T, Christou P (2004) Current Opinion in Biotechnology 15,
148-154.

(4) Trung-Nghia P et al. (2003) Planta 218, 125-134.

(5) Mehta RA et al. (2002) Nature Biotech 20, 613-618.

(6) Richards FJ, Coleman RG (1952) Nature 170, 460.

(7) Krishnamurthy R, Bhagwat KA (1989) Plant Physiol. 91, 500-504.

(8) Roy M, Wu R (2001) Plant Science 160, 869-875.

(9) Roy M, Wu R (2002) Plant Science 163, 987-992.

(10) Bassie L et al. (2000) Trans. Res. 9, 33-42.

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

Teresa Capell
Department of Crop Genetics and Biotechnology Schmallenberg, Germany

[www.isb.vt.edu]

------------------------------------------
Posted to Phorum via PhorumMail