Download Enhancing Alkali Stress Tolerance in Tomato Plants Using SAMS

ISB NEWS REPORT
MAY 2014
Enhancing Alkali Stress Tolerance in Tomato Plants Using SAMS Genes:
Outcomes, Problems and Perspectives
Biao Gong and Qinghua Shi
Background
Abiotic stresses such as salinity, drought, and alkalinity
limit crop productivity worldwide. There are 831 million
hectares of soil affected by excessive salinity and
alkalinity in the world. Of these, 434 million hectares are
sodic soils (alkaline) and 397 million hectares are saline
soils1. Soil salinity is mainly due to the accumulation
of neutral salts (NaCl and Na2SO4), and sodic soils
are mainly due to the accumulation of alkaline salts
(NaHCO3 and Na2CO3). Salt stress generally involves
osmotic and ionic stresses. Comparison of alkali with
salt stress reveals an added high-pH effect of alkali
stress. High pH can damage the normal physiological
functions of roots and destroy the root cell structure;
this damage inhibits absorption of inorganic anions such
as Cl–, NO3–, and H2PO4–, greatly affects the selective
absorption of K+/Na+, and disrupts ionic balance and
pH homeostasis in tissue. Thus, plants in alkaline soil
must cope with physiological drought, high pH, and ion
toxicity, and also maintain intracellular ionic balance.
Plants’ adaptation to environmental stresses
usually depends on the activation cascades of
molecular networks involving stress perception, signal
transduction, and the expression of specific stress-related
genes and metabolites. Consequently, engineering genes
that protect and maintain the function and structure of
cellular components can enhance tolerance to stress.
Our limited knowledge of stress-associated metabolism
remains a major gap in our understanding; therefore,
comprehensive profiling of stress-associated metabolites
is most relevant to the successful molecular breeding of
stress-tolerant crop plants. Unraveling additional stressassociated gene resources will enable future molecular
dissection of salinity–alkalinity tolerance mechanisms
in important crop plants.
Function of SAM-S gene
S-adenosyl-L-methionine (SAM), synthesized by
S-adenosyl-L-methionine synthetase (SAM-S) from
methionine and ATP, is a common substrate for many
biochemical reactions in plants. The highly reactive
methylated sulphur of SAM is used by a broad range
of methyltransferases and can methylate a myriad of
substrates, including DNA, RNA, and some proteins as
well as sterols, pectin, lignin, and flavonoids. It also plays
an important role in regulating plant development, abiotic
or biotic stress response, and metabolism. Synthesis of
polyamine is also dependent on SAM levels in plants.
In the process of PA synthesis, SAM is first catalyzed
to decarboxylated S-adenosyl-L-methionine (dcSAM)
by S-adenosyl-L-methionine decarboxylase (SAMDC).
dcSAM provides an aminopropyl group donor for the
formation of polyamine, which is well known to regulate
plant tolerance to both abiotic and biotic stresses. The
introduction of the SAMDC gene confers plant tolerance
to abiotic and biotic stresses, which is highly correlated
with higher polyamine accumulation in the transgenic
plants. SAM also serves as a precursor for the synthesis
of ethylene, which is a gaseous hormone involved in
a large variety of physiological processes of plants.
Because both polyamine and ethylene synthesis depend
on SAM as a precursor, SAMS may play a critical role in
improving plant tolerance to various stressors.
SAMS are multiple stress responsive family
genes
Four SAMS isogenes have been identified in tomato
(Solanum lycopersicum); among them, SAMS1 and
SAMS3 are significantly up regulated by NaCl, abscisic
acid (ABA), and mannitol in tomato roots2. Significantly,
up-regulation of SAMS has also been observed in coldstressed alfalfa3, iron-deficient Arabidopsis thaliana4,
alkali-stressed tomato1, and so on. Meanwhile,
overexpression of SAMS has been shown to improve
salt stress tolerance in tobacco (Nicotiana tobacum)5
and alfalfa (Medicago sativa)6, alkali stress tolerance
in tomato1, and cold stress tolerance in alfalfa3. It has
been reported that salt stress induces expression of
tomato SAMS and subsequent SAM accumulation,
which occurs predominantly in lignified tissues. This
results in greater selectivity and reduced Na+ uptake
and compensates for diminished bulk flow of water and
solutes along the apoplectic pathway by enhancing the
cell-to-cell pathway for water transport7. In addition,
high levels of SAM concentration and SAMS activity
have been correlated with lignification during plant
defense responses against pathogen stress.
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SAMS activates multiple genes/enzymes involving ‘stress response’
Research shows that raising the levels of SAMS
expression by plant transformation enhances the
expression of downstream target genes encoding
arginine decarboxylase (ADC), SAMDC, spermidine
synthase (SPDS), and polyamine oxidase (PAO),
also known as superoxide dismutase (SOD), catalase
(CAT), and ascorbate peroxidase (APX)1,3. In wild-type
plants, ADC, SAMDC, SPDS, and PAO genes are active
in plant tissues exposed to salt, cold, or heavy metals,
and are normally induced by a range of abiotic stresses
through the regulation of polyamine metabolism.
Expression of genes in reactive oxygen species (ROS)
scavenging is usually higher in stress tolerant varieties
than in stress sensitive varieties. During oxidative-stress
acclimation, some SOD, CAT, and APX genes express
to very high levels3. Products of these genes are often
quite benefitial for ROS scavenging and are involved
in the direct protection of cell components such
as proteins, membranes, and organelles from damage
caused by ROS.
The SAMS regulatory pathway in higher plants
appears to be complex, despite having only two
downstream metabolism pathways (polyamine and
ethylene signal pathways). Ethylene is a gaseous
phytohormone that can act synergistically with either
abscisic acid (ABA), salicylic acid (SA), or other
phytohormones to play key roles in physiological
processes throughout the life cycle of plants as well as
induce stress tolerance. In addition, polyamines are low
molecular weight aliphatic polycations that are quite
common in living organisms. Being positively charged,
polyamines can interact with negatively charged
molecules such as proteins, nucleic acids, membrane
phospholipids, and cell wall constituents, thereby
activating or stabilizing these molecules to alleviate
cell injury under stress conditions.
The strong antioxidant nature of polyamines results
in the neutralization or scavenging of ROS under
normal and oxidative stress conditions. Additionally,
there is also evidence that interplay occurs among
ethylene, polyamines, and ROS signaling in response
to abiotic stress. Increasingly, evidence shows that
H2O2 generated through the PAOs pathway plays an
important role in plant development and plant response
to environment stresses1,3. For example, polyamines
and ethylene can, along with PAO activity, increase
H2O2 and Ca2+ concentrations in guard cells to induce
MAY 2014
stomatal closure under osmotic stress. The production
of H2O2 through PAO has been correlated with the
growth of soybean lateral roots, and PAO activity
contributes to cantharidin-induced H2O2 synthesis in
the apoplastic milieu of maize mesocotyl. Additionally,
H2O2 generated from the PAO signaling pathway could
reduce canker susceptibility in sweet orange (Citrus
sinensis Osbeck), and it is involved in methyl jasmonate
mediated wound signaling transduction. In maize,
H2O2 derived from polyamine catabolism behaves as a
signal for secondary wall deposition and for induction
of developmental programmed cell death. These
observations indicate that SAMS genes have multiple
biological functions and should be considered a prime
target gene for improving stress tolerance in plants.
Current outcomes
Our recent study demonstrates that overexpression of
SAMS dramatically confers alkali stress tolerance in
tomato plants. Based on the analysis of physiological
indices, it may be concluded that the function of SAMS is
mainly due to an increased accumulation of spermidine
(Spd) and spermine (Spm), which affect nutrient
balance, ROS scavenging, and higher photosynthetic
capacity under stress conditions. Furthermore, the
transgenic plants, when used as rootstock, significantly
increase the growth capacity of scions, promote fruit
sets, and enhance fruit yield under alkali stress.
Plant survival may require sacrificing production under normal conditions
One way plants react to abiotic stress is to slow plant
growth, delay development, and subsequently reduce
flowering and fruit yields. In general, wild species
show more tolerance to abiotic stress but have less crop
production compared with cultivated species. It appears
that the stress response process is antagonistic with
growth and development processes in plants. Transgenic
plants with constitutive overexpression of SAMS under
normal conditions can regulate the expression of some
other stress response genes that indirectly regulate the
defense process and disturb normal plant growth and
development. It has been observed that overexpression
of SAMS usually contributes to lower fruit yields
if grown in normal conditions. Transgenic tomato
plants usually produce fewer inflorescences and show
significantly lower fruit and seed yields. However,
no significant difference in plant growth rate can be
observed in transgenic plants compared with wild type
under normal conditions.
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Reducing the negative consequences of SAMS
overexpression via grafting
As mentioned above, our study indicated that
overexpression of SAMS gene can significantly improve
the alkali stress tolerance of tomato plants. Nevertheless,
fruit yield in transgenic lines was remarkably decreased
from the T1 generation. However, no significant
difference in growth potential was observed between
wild-type and transgenic plants; instead, the numbers of
flowers and seeds were significantly reduced in transgenic
plants. Thus, we speculated that overexpression of the
SAMS gene influences fruit yield independently of the
energetic metabolism pathway. Perhaps overexpression
of the SAMS gene reduces fertility or the ability to flower
by regulating the polyamines and ethylene metabolism
pathway. However, the specific reasons remain to be
determined in further studies. To widen the application
of genetic engineering, we used grafting to reduce
the negative consequences of overexpressing SAMS.
However, we can’t provide the mechanism of how
grafting reduces the negative consequences of fruit yield
while preserving the stress tolerance ability. We provide
the novel hypothesis that grafting may be a savior for
some transgenic plants with undesirable-agronomic
traits based on our incomplete finding.
Reviews and perspectives
There are many alternative strategies for engineering
salinity and alkalinity tolerance, including improving
the ability of Na+ and ROS detoxification, promoting the
root system, and so on. However, SAMS may be more
effective when combined with these strategies for alkali
tolerance. Most published evaluations of transgenic
plants for abiotic stress tolerance are based on survival
rather than final productivity or fruit yield. The alkali
stress protection offered by overexpression of SAMS
is largely related to survival and recovery from severe
alkali stress. This pattern of response may be of limited
value under slight and long term alkali stress.
As is well known, transgenic crops, especially fresh
vegetables and fruits, have raised security concerns
worldwide. So breeders who create germplasm via
genetic engineering should not only consider the
question of gene function or its advantages, but also
should pay increased attention to the combination of
MAY 2014
research, production, and sales when genes are used
in horticultural crops. In many studies, large numbers
of genes have demonstrated the ability to control yield
and quality, improve biotic or abiotic stress tolerance,
etcetera. However, the number of target genes used for
testing and production are less than one in ten thousand,
most likely because of the difference in focus of basic
scientific research and commercial interests. On the one
hand, we attempted to reveal the theoretical significance
of gene transfer by investigating the function of SAMS
gene; on the other hand, we used transgenic plants as
rootstock to improve saline-alkaline stress tolerance of
wild-type scions, which is a benefit for both experimental
and production purposes.
Grafting is widely used in horticultural crops,
especially in Cucurbitaceae, Solanaceae and fruit trees,
to solve the above-mentioned problems. We did not
find related genes of the plant binary expression vector
(PROKII) in both scion and tomato fruits when we
assayed for the labeled genes (NPTII and 35S promoter)
using PCR analysis (Fig. 1). We therefore believe
that more attention should be paid to using transgenic
rootstock in commercial crops, not only for improving
crop production characteristics and economic viability,
but also because use of transgenic rootstock has strategic
significance for food safely. And lastly, although grafting
could solve some problems of food safety, more attention
should be paid in future studies to the environmental
concerns, especially for soil ecology, caused by genetic
engineering.
Figure 1 PCR analysis of labeled genes (NPTII and 35S promoter) in wild-type scions and tomato fruits that are grafted
into transgenic rootstock. “M” = marker; “+” = positive control (left for
NPTII and right for 35S).
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MAY 2014
References
1. Gong B, Li X, VandenLangenberg KM, Wen D, Sun S, Wei M, Li Y, Yang F, Shi Q, Wang X. (2014) Overexpression of S-adenosyl-L-methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism. Plant biotechnology journal, doi: 10.1111/pbi.12173.
2. Espartero J, Pintor-Toro JA, Pardo JM. (1994) Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress. Plant
Molecular Biology, 25(2): 217-227.
3. Guo Z, Tan J, Zhuo C, Wang C, Xiang B, Wang Z. (2014) Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine
synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation. Plant biotechnology journal, doi:
10.1111/pbi.12166.
4. Lan P, Li W, Wen TN, Shiau JY, Wu YC, Lin W, Schmidt W. (2011) iTRAQ protein profile analysis of Arabidopsis roots reveals new aspects critical for
iron homeostasis. Plant physiology, 155(2): 821-834.
5. Qi YC, Wang FF, Zhang H, Liu WQ. (2010) Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco. Acta physiologiae plantarum, 32(2): 263-269.
6. Hua Y, Zhang BX, Cai H, Li Y, Bai X, Ji W, Wang ZY, Zhu YM. (2012) Stress-inducible expression of GsSAMS2 enhances salt tolerance in transgenic
Medicago sativa. African Journal of Biotechnology, 11(17): 4030-4038.
7. Sánchez-Aguayo I, Rodríguez-Galán J M, García R, Torreblanca J, Pardo JM. (2004) Salt stress enhances xylem development and expression of Sadenosyl-L-methionine synthase in lignifying tissues of tomato plants. Planta, 220(2): 278-285.
Biao Gong and Qinghua Shi
State Key Laboratory of Crop Biology, PR China
Scientific Observing and Experimental Station of Environment Controlled Agricultural Engineering in Huang-Huai-Hai Region,
Ministry of Agriculture, PR China
College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, PR China.
[email protected] (Qinghua Shi)