Maine Potato Board Project
Report 2005
Screening the exotic Solanum germplasm for tolerance to
abiotic stresses
Benildo G. de
los Reyes
Dept. of Biological Sciences,
University of Maine-Orono
Executive Summary
Abiotic stresses such as low temperature, drought and salinity
are agronomic problems that limit the productivity of most food crops including
potato. Breeding to enhance stress
tolerance of commercial cultivars has not been very successful due the narrow
genetic base of cultivated germplasm. In this study, we used a combination of
physiological and molecular strategies to assess the extent of natural
variation for abiotic stress tolerance between the cultivated and wild species of
Solanum, with the goal of identifying
potential donors for future use in cultivar improvement.
Species of the genus Solanum exhibit wide variation
with respect to tolerance to chilling and freezing temperatures. Results of physiological
studies that measured the differences in low temperature-induced damage to the
cell membrane showed that wild species are generally hardier than the
cultivated potatoes. Species can be ranked according to increasing sensitivity
to chilling as follows: S. commersonii < S. polytrichon = S.
stoloniferum < S. boliviense = S. tuberosum < S. trifidum.
Furthermore, the South Argentinian
species S. commersonii also exhibited
the highest degree of tolerance to freezing (< -10oC). Freezing
tolerance of this species is a consequence of its ability to cold acclimate
during extended exposure to chilling (2oC to 4oC). S. polytrichon was also able to cold
acclimate but exhibited moderate tolerance to freezing compared to S. commersonii. The other species
including S. trifidum and the
cultivated S. tuberosum are not
capable of cold acclimation and thus are very sensitive to freezing. Analysis
of the differential responses of the species to salinity and dehydration/osmotic
stress induced artificially in hydroponics medium showed that the cold
acclimating species exhibited moderate tolerance to salinity and osmotic/dehydration
stresses. The current results indicate that the physiological and biochemical
basis of tolerance to each of the three stress regimes are very complex and not
completely identical. Comparative molecular studies showed that cold-acclimating
species can be distinguished from non-acclimating species by the expression
patterns of homologous genes associated with cold and drought tolerance in
other plant species. Further genetic tests are currently in progress to
determine if these genes can be used to develop markers for large-scale
germplasm screening.
Project Accomplishments
Funds obtained in 2005 from the Maine Potato Board were used
to establish procedures for screening the Solanum
germplasm for abiotic stress tolerance. Experiments conducted were focused on
the development of simple physiological and molecular techniques that allowed
preliminary assessment of the extent of genetic variation for stress tolerance
within the genus. The major aspects of the project results are summarized as follows:
Variation in low temperature
stress tolerance.
Five wild species accessions (S.
commersonii, S. boliviense, S. polyctrichon, S. trifidum, S. stoloniferum)
and four cultivated genotypes (Red Pontiac, Superior, Kennebec, Russett
Burbank) were compared with respect to their responses to extended exposure to
chilling temperature (2oC). Chilling injury was assessed by the
leakage of cellular electrolytes resulting from physical damage to the cell
membrane (Membrane Injury Index, MII). The results study showed a continuous
variation for tolerance to chilling among the wild and cultivated species. The relative ranking of species and
cultivars according to increasing sensitivity to are follows: S. commersonii
< S. polytrichon = S. stoloniferum < S. boliviense =
S. tuberosum < S. trifidum (Fig. 1). S. commersonii, a
species that originated from South Argentina can also acclimate to low
temperature, which leads to further hardening. Two weeks (11 to 14 days)
exposure to 2oC can efficiently induce the cold acclimation response
(Fig. 2). S. polytrichon can also cold acclimate but to a relatively lesser
degree compared to S. commersonii. Exposure to 2oC does not have
significant effect on the freezing hardiness of S. boliviense, S. polytrichon, S.stoloniferum, S. trifidum and the
cultivated S. tuberosum.
Figure
1. Variation in
chilling-induced membrane injury among Solanum species. Plants at
mid-vegetative stage were incubated for a maximum of 11 days at 2oC, the
optimum temperature previously reported to induce cold acclimation in some Solanum
species (Chen and Li, 1980). Membrane Injury Index (MII) is equal to the ratio
of the percentage electrolyte leakage (%EL) between the plants grown at
chilling (2oC) and control (28oC) temperatures. MII = 1
indicates no significant leakage of electrolytes due to chilling, while MII
> 1 indicates significant leakage due to chilling. 1 S. boliviense (PI 265860); 2 S. commersonii (Oka 5040); 3 S. trifidum (PI
255541); 4 S. polytrichon (PI 184773); 5 S. stoloniferum (PI
283109); 6 S. commersonii (PI 472833); 7 S. tuberosum (Red Pontiac); 8 S. tuberosum
(Superior); 9 S. tuberosum (Russet Burbank); 10 S. tuberosum
(Kennebec). The relative ranking of Solanum species according to
increasing levels of chilling sensitivity are as follows: S. commersonii
< S. polytrichon = S. stoloniferum < S. boliviense =
S. tuberosum < S. trifidum. ** Means were significantly
greater than 1 at P<0.005; * Means were significantly greater than 1 at
P<0.01; Error bars are shown as standard error of the mean, n = 4.
Figure
2. Differential
responses of Solanum species to cold-acclimation. Statistically significant differences in the
mean MII were detected only in S. commersonii and S. polytrichon,
indicating that CA in Solanum is species-dependent. 1 S. boliviense (PI 265860); 2 S. commersonii (Oka 5040); 3 S.
trifidum (PI 255541); 4 S. polytrichon (PI 184773); 5 S.
stoloniferum (PI 283109); 6 S. commersonii (PI 472833); 7 S. tuberosum (Red Pontiac); 8 S.
tuberosum (Superior); 9 S. tuberosum (Russet Burbank); 10 S.
tuberosum (Kennebec). ** Means were significantly different between
control and CA plants at P<0.005; * Means were significantly different
between control and CA plants at P<0.025; Error bars are shown as standard
error of the mean, n = 4.
Variation in salinity
and dehydration stress tolerance. We developed a quick and simple method for physiological
screening for salinity and dehydration (physiologically induced by osmotic
stress) tolerance. This methodology involves exposure of plants during their
mid-vegetative growth phase to high NaCl (300 mM) and high mannitol (200mM)
concentrations in hydroponics medium (0.25x Hoaglands). Like in the low temperature experiments,
species and cultivars responded differently to high salt and osmotic stress
treatments (Fig. 3). The species that was able to cold acclimate (S. commersonii) exhibited moderate
tolerance to salinity and osmotic stresses. The relative cultivar ranking based
on the level of salt and dehydration sensitivity was not completely identical to
the ranking based on low temperature sensitivity. These results indicate that
the physiological and biochemical basis of tolerance to each of the three
stress regimes are very complex and not completely identical. Further
physiological experiments are required to assess the extent of inter-specific
variation.
Figure 3. The same set of species differentials used in the
cold acclimation experiments were studied for their responses to 3 day-exposure
to salinity (300mM NaCl) and osmotic/dehydration (200mM mannitol) stresses. In
general, the relative cultivar ranking based on cold hardiness was much similar
to the NaCl than the mannitol stress ranking. 1 S. boliviense (PI 265860);
2 S. commersonii (Oka 5040); 3 S. trifidum (PI 255541);
4 S. polytrichon (PI 184773);
5 S. stoloniferum (PI 283109); 6 S. commersonii (PI
472833); 7 S. tuberosum (Red Pontiac); 8 S.
tuberosum (Superior); 9 S. tuberosum (Russet Burbank); 10 S.
tuberosum (Kennebec). ** Means were significantly greater than 1 at
P< 0.005; * Means were significantly greater than 1 at P<0.01; Error bars
are shown as standard error of the mean, n = 5.
Differential
expression of cold-acclimation associated genes. The cold acclimation and drought
stress response genetic network has been elucidated in plant genetic models.
The network is comprised of hundreds of genes that function either as a
regulator or effector of cellular defense mechanisms. We sequenced the
cDNAs corresponding to some of the well characterized regulators (CBF1, ZAT12 and RAV1 transcription
factors) and effectors of physiological defense mechanisms (COR78, GolS-2, GolS-3). Analysis of the
stress-induced activities of these genes by gene-specific semi-quantitative
polymerase chain reaction indicated that the cold-acclimating species (stress
tolerant) can be distinguished from the non-acclimating species (stress
intolerant) by genotype-specific expression signatures. The stress tolerant
species generally exhibited more robust induction of the expression of both the
regulator and effector genes under cold stress conditions (Fig. 4).
Tools for further
genomics studies.
The current findings will be used to further establish a robust molecular
method for screening the Solanum
germplasm for tolerance to abiotic stresses and to elucidate the molecular
basis of tolerance mechanisms by semi-global (large-scale) analysis of
stress-induced changes on gene expression. Hence, we have used a portion of the
grant from the Maine Potato Board to purchase a set of gene chips (containing
>30,000 potato genes) from The Institute of Genomics Research (TIGR). The
gene chips are currently being used for more in-depth analysis of the variation
revealed by the current data.
Figure
4. Solanum homologs of genes associated with
the CA response in Arabidopsis were investigated by semi-quantitative
RT-PCR. Cold-acclimating and non-acclimating species can be distinguished by
the expression patterns of cold acclimation-associated genes. The transcription factor CBF1
is a major regulator of the cold acclimation gene regulon (Stockinger et al.,
1997). RAV1 and ZAT12 are newly identified transcription factors
with possible roles in cold acclimation (Fowler and Thomashow, 2002). COR78
is one of the known targets of CBF1 via the C-repeat/DRE cis-elements. GolS-2
and GolS-3 encode galactinol synthases involved in the synthesis of
osmoprotectants (raffinose-family of oligosaccharides) during cold stress (Taji
et al., 2002). These genes are also potentially regulated through the CBF1/RAV1/ZAT12
pathway. In general, the transcription factors and their putative targets
exhibited distinct expression profiles in each of the Solanum species.
For instance, cold-induced expression occurred earlier in the cold-acclimating
species (S. commersonii, Oka 5040) than in the non-acclimating species (S.
boliviense, S. trifidum and S. tuberosum cvs. Red Pontiac,
Superior). ZAT12 transcripts were not detected in S. trifidum. GolS-3
transcripts were not detected in S. trifidum and S. tuberosum.
Experimental Strategy
Cold-Acclimation. Plants were established to
five-leaf stage at 28oC, 12 hr photoperiod (control condition). For
cold-acclimation (CA), plants were incubated for a maximum of 11 days at 2oC,
10 hr photoperiod. To assess the differences in chilling-induced membrane
injury during CA, electrical conductivity (EC) was measured on leaf discs
collected from control and cold-treated plants at 4, 7 and 11 days. Percent electrolyte
leakage (%EL) in the control and cold-treated plants was determined by the
following equation: EC-Tr/EC-Total
x 100, where EC-Tr is the amount of electrolytes that leaked
from the tissue and EC-Total is the total tissue electrolytes measured
after boiling the leaf discs for 30 min in water bath. Membrane Injury Index
(MII) was calculated by the following equation: %EL-Treatment/%EL-control.
Freezing
Tolerance.
Plants that have been cold acclimated at 2oC for 11 days were transferred to a
freezing chamber (-10oC) for 1 hr. Leaf discs were collected and membrane
injury was assessed with the same procedures used in the cold acclimation
experiment.
Salinity
and Osmotic Stress Tolerance. Plants grown under control condition (28oC, 12 hr
photoperiod) in the soil were transferred and acclimated in hydroponics medium
(0.25X Hoaglands) for one week. At the beginning of the second week, the
hydroponics medium was supplemented with high NaCl and high mannitol at a final
concentration of 300mM and 200mM, respectively. EC was measured in each sample
after 3 days of stress.
Semi-quantitative
RT-PCR. Total
RNA was isolated from leaf tissues with the Trizol reagent (Invitrogen). The
cDNA templates were synthesized from 0.5 ug total RNA with the ImProm-II
Reverse Transcription System (Promega). Semi-quantitative RT-PCR was performed
on 0.05 ug of cDNA using the PCR Master Mix (Promega) for 22 to 24 cycles in
the iCycler thermal cycler (Biorad). Gene-specific primers were designed from the potato cDNA sequences and ESTs available
in the GenBank. The expression of the actin
gene was used to normalize transcript abundance between the treatments and
control.
Conclusion
Significant genetic genetic variation for abiotic stress
tolerance was detected among cultivated and exotic species of Solanum using both physiological and
molecular parameters. Correlation between low temperature stress tolerance with
salinity and dehydration stress tolerance was moderately significant. S. commersonii, a wild species from
Southern Argentina is a potential source of genes or alleles associated with
stress tolerance. Further physiological and molecular characterization is
currently underway to understand the genetic and biochemical basis of the
stress tolerance exhibited by this species.
References
Chen HH, Li
PH (1980) Plant Physiol 65:1146-1148.
Fowler S,
Thomashow MF (2002) Plant Cell 14:1675-1690.
Stockinger
EJ, Gilmour SJ, Thomashow MF (1997) Proc Natl Acad Sci USA 94:1035-1040.
Taji T,
Ohsumi C, Iuchi S, et al. (2002) Plant J 29:417-426.