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Plant Physiol, December 2001, Vol. 127, pp. 1836-1844
Overexpression of Malate Dehydrogenase in Transgenic Alfalfa
Enhances Organic Acid Synthesis and Confers Tolerance to
Aluminum1
Mesfin
Tesfaye,
Stephen J.
Temple,2
Deborah L.
Allan,
Carroll P.
Vance, and
Deborah A.
Samac*
Departments of Plant Pathology (M.T., D.A.S.), Agronomy and Plant
Genetics (S.J.T., C.P.V.), and Soil, Water, and Climate (D.L.A.), and
U.S. Department of Agriculture-Agricultural Research Service-Plant
Science Research Unit (C.P.V., D.A.S.), University of Minnesota, St.
Paul, Minnesota 55108
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ABSTRACT |
Al toxicity is a severe impediment to production of many
crops in acid soil. Toxicity can be reduced through lime application to
raise soil pH, however this amendment does not remedy subsoil acidity,
and liming may not always be practical or cost-effective. Addition of
organic acids to plant nutrient solutions alleviates phytotoxic Al
effects, presumably by chelating Al and rendering it less toxic. In an
effort to increase organic acid secretion and thereby enhance Al
tolerance in alfalfa (Medicago sativa), we produced
transgenic plants using nodule-enhanced forms of malate dehydrogenase
and phosphoenolpyruvate carboxylase cDNAs under the
control of the constitutive cauliflower mosaic virus 35S promoter. We
report that a 1.6-fold increase in malate dehydrogenase enzyme specific
activity in root tips of selected transgenic alfalfa led to a 4.2-fold
increase in root concentration as well as a 7.1-fold increase in root
exudation of citrate, oxalate, malate, succinate, and acetate compared
with untransformed control alfalfa plants. Overexpression of
phosphoenolpyruvate carboxylase enzyme specific activity
in transgenic alfalfa did not result in increased root exudation of
organic acids. The degree of Al tolerance by transformed plants in
hydroponic solutions and in naturally acid soil corresponded with their
patterns of organic acid exudation and supports the concept that
enhancing organic acid synthesis in plants may be an effective strategy
to cope with soil acidity and Al toxicity.
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INTRODUCTION |
Al makes up about 7% of the
Earth's crust (Delhazie and Ryan, 1995 ). The solubility of Al in
neutral and alkaline soils is too low to be toxic to plants, but in
acid soils, which make up about 40% of the world's arable land, Al
becomes soluble and readily enters roots where it inhibits root growth
and development (Delhazie and Ryan, 1995; Kochian, 1995). As a result,
the root system becomes inefficient in water and nutrient uptake,
leading to lower crop yields (Delhazie and Ryan, 1995; Kochian, 1995).
A common agricultural practice for acid soils is to apply lime to raise
soil pH. However, farmers in developing countries frequently have
neither access to the infrastructure required nor capital available to
remedy acid soil and Al toxicity via lime application (Devine et al., 1990). In addition, liming acid soils does not remedy acidity in the
subsoil layer. An alternative is to tailor plants to suit acid soils by
identifying and/or developing plants with improved tolerance to Al in
acid soils.
Al-tolerant maize and snapbean genotypes release more citrate into the
rhizosphere in response to Al, whereas Al-tolerant wheat genotypes
release more malate and succinate in response to Al (Miyasaka et al.,
1991; Delhazie and Ryan, 1995; Pellet et al., 1996). The amount of
organic acid exudation depends on both the amount of Al in the growth
medium and the duration of exposure (Delhazie and Ryan, 1995). The main
organic acids known to effectively detoxify Al are citrate, oxalate,
and tartarate followed by malate, malonate, succinate, and acetate (Hue
et al., 1986; Ginting et al., 1998). In research to increase organic
acid production in plants, a citrate synthase gene from
Pseudomonas aeruginosa controlled by the cauliflower mosaic
virus (CaMV) 35S promoter was used to produce transgenic tobacco
(Nicotiana tabacum) and papaya (Carica
papaya) (de la Fuente et al., 1997). These plants were reported to
have an increased internal citrate concentration, secrete up to 4-fold
more citrate than control plants, and have enhanced Al tolerance.
However, a recent study has shown that the increased citrate synthase
activity in those same transgenic tobacco plants and in additional
transgenic tobacco and alfalfa (Medicago sativa) did not
correlate with increased citrate production nor was Al tolerance
enhanced in transgenic plants (Delhazie et al., 2001). In the present
study we explored the possibility that modification of expression of
specific plant genes controlling other steps in organic acid
biosynthesis might increase organic acid synthesis and Al tolerance in alfalfa.
Malate is a key metabolite in plants. It is involved in numerous
processes, including C4 and Crassulacean acid metabolism photosynthesis, stomatal and pulvinual movement, nutrient uptake, respiration, nitrogen assimilation, fatty acid oxidation, and providing
energy to bacteroids in root nodules (Vance and Heichel, 1991; Vance,
1997; Miller et al., 1998). Malate is synthesized via the concerted
action of phosphoenolpyruvate carboxylase (PEPC; EC
4.1.1.31), which catalyzes conversion of phosphoenolpyruvate to oxaloacetate, and by malate dehydrogenase (MDH; EC 1.1.1.37), which
causes oxidation of oxaloacetate to form malate (Vance, 1997). Plants
contain several isoforms of MDH (Miller et al., 1998) and PEPC (Chollet
et al., 1996), and their expression is dependent upon function and
tissue (Vance et al., 1994; Vance, 1997).
Legume root nodules contain specific forms of MDH and PEPC genes that
are expressed 5- to 15-fold higher than in other tissues (Vance et al.,
1994; Miller et al., 1998). These non-photosynthetic isoforms play a
crucial role in providing the large amount of malate required by
bacteroids to fix N2 and by the plant for
assimilation of N2 (Vance, 1997). A particularly
novel form of MDH (noduleenhanced; neMDH) has been isolated from
alfalfa nodules (Miller et al., 1998). This isoform has unusual kinetic
properties that drive the reaction toward the production of large
quantities of malate in nodules (Miller et al., 1998). We hypothesized
that over-expression of neMDH and PEPC would result in increased
organic acid biosynthesis and Al tolerance. Thus, our objectives were
to constitutively over-express neMDH and PEPC enzymes in alfalfa using
the CaMV 35S as a promoter, to test whether such transgenic plants had enhanced malate synthesis and excretion, and to evaluate whether selected transgenic plants had increased Al tolerance.
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RESULTS |
Transgenic Alfalfa with Increased Expression of neMDH or
PEPC
We first analyzed enzyme specific activities in root tips of
transgenic plants in comparison with the untransformed parent (M. sativa cv Regen-SY). As shown in Figure
1, nine transgenic lines with the
35S::neMDH transgene showed up to a 1.6-fold increase in MDH
specific activity, ranging from 23.4 to 35.0 µmol of NADH min 1 mg1 protein (Fig.
1A). Six neMDH transgenic lines (M20-10, M16-27, M2-14, M16-26, M10-7,
and M16-28) had significantly higher enzyme activity levels than the
untransformed control (P < 0.05). By contrast, two
transgenic lines with the 35S::PEPC transgene showed an
increase in PEPC-specific activity (Fig. 1B), and only PEPC-3 had
significantly higher enzyme activity than untransformed alfalfa cv
Regen-SY (P < 0.05).
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Figure 1.
Enzyme specific activities from root-tips of
transgenic and untransformed alfalfa lines. A, MDH-specific activity;
B, PEPC-specific activity. Bars = means + SE
(n = 5).
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Transcript levels of neMDH and PEPC mRNA were analyzed in selected
transgenic and alfalfa cv Regen-SY plants by quantitative reverse
transcription (RT)-PCR as well as RNA blotting using neMDH cDNA as a
probe. The neMDH cDNA probe specifically hybridizes to neMDH mRNA
(S. Miller, personal communication). In addition, plants of an
alfalfa germplasm, GA-AT, previously selected for acid soil tolerance
(Bouton, 1996), were analyzed for comparison. The RT-PCR results were
similar to northern-blot results (data not shown). Root tips of
transgenic plants transformed with the 35S::neMDH transgene
showed up to a 4.1-fold increase in neMDH transcript accumulation
compared with root-tips of untransformed alfalfa cv Regen-SY plants
(Fig. 2). Leaf samples of transgenic plants showed up to 60% reduction in the amount of neMDH transcripts compared with untransformed alfalfa cv Regen-SY plants. GA-AT plants
also showed a 1.8-fold greater neMDH transcript accumulation than
alfalfa cv Regen-SY in root-tips but not in leaf samples. In contrast,
there was no detectable change in PEPC transcript levels from root-tip
samples between PEPC transformants and untransformed plants (data not
shown). Variation in the amounts of neMDH and PEPC transcripts was
observed between different transformants, and this variation appeared
to reflect differences in enzyme specific activities.
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Figure 2.
Relative RT-PCR analysis of neMDH mRNA expression
in selected transgenic and untransformed alfalfa lines. Relative
expression of neMDH mRNA (upper band) was estimated after normalization
with signals from the 18S transcript levels (lower band) used as an
endogenous control in a multiplex RT-PCR reaction. Hybridization
signals were analyzed using a Storm 840 PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
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Monospecific antisera against alfalfa neMDH and PEPC were used to
analyze expression of these proteins in transgenic, alfalfa cv
Regen-SY, and GA-AT plants by western blotting. Using 10% (w/v) SDS- polyacrylamide gels, antisera to neMDH recognizes two bands of
approximately 34 kD (S. Miller and C.P. Vance, unpublished data). Amounts of neMDH polypeptides in root-tip samples of selected neMDH transgenic lines were 4.2- to 4.7-fold greater than in root tips
of the untransformed lines (alfalfa cv Regen-SY and GA-AT; Fig.
3A). At comparable total soluble protein
loadings on SDS-PAGE, neMDH proteins were 6.7-fold higher in root tips
of transgenic plants than in leaf samples (Fig. 3A). Consistent with
the mRNA expression pattern, neMDH proteins in leaf samples of
transgenic plants were reduced to about 50% of the amount in alfalfa
cv Regen-SY. Compared with the amount of alfalfa cv Regen-SY protein,
neMDH protein expression in GA-AT plants was reduced in root-tip
samples but greater by 2.7-fold in leaf samples. The amount of PEPC
protein in root-tip samples of PEPC-3 plants was 1.5-fold greater than amounts in control root tips (Fig. 3B). Also, the amount of PEPC polypeptide was elevated in root tips of PEPC-3 when compared with leaf
samples (Fig. 3B).
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Figure 3.
Immunoblot analysis of selected transgenic and
untransformed alfalfa lines. Total soluble protein (10 µg/lane) from
root-tips and leaf samples was separated by SDS-PAGE, blotted, and
probed with neMDH (A) and PEPC (B) monospecific antibodies.
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Over-Expression of neMDH or PEPC in Alfalfa Leads to Altered
Organic Acid Production
Organic acid content in roots and leaves and exudation of organic
acids from roots into the rhizosphere by selected transgenic plants
were compared with that of alfalfa cv Regen-SY and GA-AT plants using
HPLC. Low amounts of malate were detected in root samples of M16-27 and
alfalfa cv Regen-SY plants, although root tissues from line M20-10
showed considerably higher concentration of malate (Fig.
4A). Both transgenic lines evaluated
(M16-27 and M20-10) containing the 35S::neMDH transgene,
showed significantly elevated levels of citrate, oxalate, succinate,
and acetate in root tissues compared with alfalfa cv Regen-SY plants
(Fig. 4, A and B). Leaves from plants containing the
35S::neMDH transgene also showed increased organic acid
concentrations compared with leaves from alfalfa cv Regen-SY and GA-AT
plants (Fig. 4, C and D). Malate accumulation in leaves of M16-27 and
M20-10 was 4- to 10-fold greater compared with alfalfa cv Regen-SY and
GA-AT plants (Fig. 4C). Citrate and oxalate concentrations in root and leaf tissues of PEPC-3 plants were not significantly different from
root and leaf concentrations in alfalfa cv Regen-SY and GA-AT plants
(Fig. 4, A and C). Succinate was depressed in root samples of PEPC-3
when compared with alfalfa cv Regen-SY and GA-AT (Fig. 4B). Amounts of
citrate, oxalate, succinate, and acetate were similar between root
tissues of GA-AT and alfalfa cv Regen-SY (Fig. 4, A and B).
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Figure 4.
Oraganic acid content and exudation in transgenic
and untransformed alfalfa lines under neutral pH growth conditions. A
and B, Root organic acid content (n = 7); C and D, leaf
organic acid content (n = 2); and E and F, organic acid
exudation into the rhizosphere (n = 4). Bars = means + SE. Note that the figures are drawn to different
log scales.
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We investigated whether overexpression of neMDH or PEPC would increase
the amounts of organic acids exuded from roots under neutral pH
conditions. Over a 24-h period, M16-27 plants exuded significantly more
citrate, oxalate, malate, succinate, and acetate into the rhizosphere
than did alfalfa cv Regen-SY plants (Fig. 4, E and F). Similar amounts
of citrate and malate were measured in root exudates of PEPC-3 and
alfalfa cv Regen-SY plants (Fig. 4E). There was no detectable amount of
oxalate in root exudates of M20-10, alfalfa cv Regen-SY, GA-AT, or
PEPC-3 plants. PEPC-3 plants did not show detectable amounts of
succinate or acetate in their root exudates. Amounts of citrate and
malate released by roots of the conventionally bred acid soil tolerant
alfalfa, GA-AT, were significantly higher than that of alfalfa cv
Regen-SY (Fig. 4E). In contrast, exudation of succinate by roots of
GA-AT plants was reduced from alfalfa cv Regen-SY levels (Fig.
4F).
Transgenic Plants Show Increased Al Tolerance in Hydroponic
Solution and Acid Soil
Plants of the transgenic lines and untransformed controls had
similar leaf morphology and flowering patterns. After 30 d of growth in neutral pH (soil:sand, 1:1, v/v) under greenhouse conditions, however, alfalfa cv Regen-SY plants had accumulated more dry matter than did transgenic plants (data not shown). A reduced biomass accumulation by transgenic plants was observed during the 1st year of
growth in field plots with soil pH 7.25 (Fig.
5). However, there was no significant
difference in shoot dry matter accumulation in the following year (Fig.
5).
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Figure 5.
Shoot biomass accumulation in transgenic and
untransformed alfalfa lines grown in field plots with soil pH 7.25. Plants were transplanted on June 2, 1999, and aerial biomass was cut by
hand on the dates shown. Bars = means + SE
(n = 3 with up to 10 plants in each replicate
plot).
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We evaluated root elongation of transgenic and untransformed control
plants under acidic conditions minus Al in hydroponic culture. Root
elongation of all plants occurred at pH 4.3, but root elongation of the
untransformed alfalfa cv Regen-SY plants was significantly faster than
that of any of the transgenic lines tested (Fig.
6). A slower rate of root growth by
transgenic lines is consistent with the trends seen in shoot biomass
accumulation of field-grown alfalfa plants (pH 7.25) during the 1st
year of growth (Fig. 5). PEPC-3 plants showed the slowest root growth rate in acidic nutrient solutions without the addition of Al.
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Figure 6.
Influence of Al on root elongation of transgenic
and untransformed alfalfa lines. Relative root growth = (root
elongation with Al/root elongation without Al) * 100. Plants were
preconditioned in hydroponic solution containing 0.5 mM
CaCl2 in deionized water, pH 4.3. After 3 d,
Al was added to fresh preconditioning medium, and root length was
measured before and after 24 h of growth. Bars = means + SE (n = 10). Root elongation in the
preconditioning medium without the addition of Al during the same
24 h period was: M16-27 = 14.9 mm, M20-10 = 11.0 mm,
alfalfa cv Regen-SY = 23.6 mm, GA-AT = 16.5 mm, and
PEPC-3 = 8.0 mm.
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The primary response of plants to Al toxicity is the inhibition of root
elongation, which is observed as early as 1 h after Al exposure
(Pellet et al., 1996). Relative root growth, expressed as a percentage
of the root elongation in an Al treatment compared with root elongation
without Al treatment, is considered to be a reliable screening measure
for Al tolerance in plants (Howeler, 1991). The root elongation rates
of transgenic, untransformed alfalfa cv Regen-SY and GA-AT plants were
evaluated in pH 4.3 hydroponic solutions containing 20, 50, or 100 µM Al. Compared with elongation in the absence of Al, the
addition of 20, 50, or 100 µM Al inhibited root
elongation of all plant lines (Fig. 6). Addition of Al at 20 µM resulted in a 40% to 80% root growth inhibition in
transgenic and untransformed GA-AT and alfalfa cv Regen-SY plants (Fig.
6). The transgenic lines and GA-AT plants showed enhanced tolerance at
the higher Al concentrations. The relative root growth at 100 µM Al was on the order of M16-27  GA-AT > M20-10 PEPC-3 > alfalfa cv Regen-SY plants (Fig.
6).
We evaluated the dry matter yield of alfalfa plants grown on naturally
acidic, high-Al-containing soil. Biomass accumulation (shoot and root)
of 35S::neMDH transgenic plants (lines M16-27 and M20-10) was
significantly greater than that of GA-AT, alfalfa cv Regen-SY, and
PEPC-3 plants (P < 0.01; Fig.
7). After 15 d of growth, neMDH
transgenic lines produced 1.6-fold more biomass than alfalfa cv
Regen-SY plants, although Al stress-like symptoms occurred in all
plants.
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Figure 7.
Biomass accumulation of transgenic and
untransformed alfalfa lines. Plants were grown for 15 d in acid
soil (pHH2O = 4.0, AlKCl = 71 µg mL1). Bars = means + SE
(n = 10).
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The amount of Al and P accumulated by alfalfa plants grown on naturally
acid soil was evaluated. Consistent with increased biomass
accumulation, M16-27 and M20-10 showed significantly higher Al content
in roots and shoots compared with untransformed plants grown in the
same soil (Table I). The content of Al in
shoot tissue was significantly lower than the content in root tissue. P
content in shoot and root tissues of M16-27 and M20-10 plants was up to
2.5-fold greater than P content in alfalfa cv Regen-SY and GA-AT plants
(Table I). Root and shoot P content in PEPC-3 plants did not differ
significantly from untransformed alfalfa cv Regen-SY and GA-AT levels
(Table I). In contrast to the pattern of Al accumulation, P content was
considerably higher in shoots than in roots.
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Table I.
Shoot and root Al and P content of transgenic and
untransformed alfalfa lines as determined by inductively coupled
plasma-atomic emission spectroscopy analysis (n = 10)
Al or P contents (µg plant1) were derived from dry
matter yield (mg dry tissue plant1) × Al or P
concentrations (µg mg dry tissue1). Means with
different superscript letters in columns are significantly different at
P < 0.05.
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DISCUSSION |
The present study showed that overexpression of neMDH cDNA in
alfalfa resulted in modest but significant increases in malate concentration in tissues of some transgenic lines. Plants with overexpression of neMDH cDNA also had enhanced concentration of other
organic acids in roots and leaves, increased organic acid exudation
from roots, and a concomitant increase in tolerance to Al. By contrast,
over-expression of the PEPC cDNA in alfalfa resulted in a reduction of
organic acid content in roots and root exudates, although PEPC-3 plants
displayed an increased malate concentration in leaf tissues. Increased
root concentration of organic acids evidently does not necessarily
correspond with increased exudation by roots.
The relative reduction in biomass accumulation of neMDH transgenic
lines compared with untransformed alfalfa cv Regen-SY plants grown in
soil of neutral pH during the early stages of plant growth may be due
to the increased exudation of citrate and malate by these plants. Root
exudation represents an appreciable drain on plant resources, thereby
imposing an energy cost to plants (Smucker, 1984). When plants were
grown in Al-containing acid culture, however, transgenic plants,
especially those with the 35S::neMDH transgene, showed
greater root elongation rates and higher biomass accumulation than did
untransformed alfalfa cv Regen-SY plants, indicating that transgenic
plants are much more tolerant to Al. The amounts of organic acids in
roots and leaves and the exudation profiles for M20-10 plants were
consistently different from that of M16-27 plants. The reason for this
is unclear, because MDH-specific activity of roots in M20-10 plants was
higher than in M16-27 plants. Although we have no direct evidence
regarding the mechanism of Al tolerance in the transgenic alfalfa, the
enhanced Al tolerance by 35S::neMDH transgenic lines and the
poor growth of untransformed alfalfa cv Regen-SY plants in
Al-containing hydroponic solution or soil culture corresponded with the
organic acid synthesis and exudation patterns of these plants. For
instance, M16-27 plants showed 4.2-fold more citrate, oxalate, malate,
succinate, and acetate in roots and exuded 7.1-fold greater amounts of
these organic acids into the rhizosphere compared with alfalfa cv
Regen-SY plants. From the enhanced exudation of organic acids by roots
of the transgenic M16-27 line, for example, it is appealing to
speculate that Al-tolerance by this transgenic line may be in part due
to chelation of Al in the rhizosphere. The observation that transgenic
alfalfa with overexpression of neMDH also had increased P uptake when
grown in high-Al-containing acid soil supports the idea that Al is
complexed by organic acids released by the plant roots. Release of
organic acids has been shown to improve P availability in acid soils by chelating Al from Al-P complexes (Fox et al., 1990).
Considering the constitutive nature of the 35S promoter, the decreased
amount of neMDH transcript or protein in leaf samples of M16-27 and
M20-10 plants was unexpected, and the reasons are not clear.
Nevertheless, the 35S promoter in transgenic alfalfa leaves has been
found to be affected by environmental conditions (D.A. Samac,
unpublished data), which may be reflected in mRNA and protein
accumulation of cloned genes driven by this promoter. Because the root
apex is the site of Al toxicity (Kochian, 1995), targeting increased
synthesis of organic acids to root tips may significantly improve Al
tolerance and/or P uptake. To test this, additional transgenic alfalfa
plants are being produced with the neMDH cDNA driven by promoters that
are active in root meristematic tissues. This strategy may minimize the
metabolic drain that appears to be associated with overexpression of
organic acids in neMDH transgenic alfalfa.
Results from a number of laboratories have provided strong evidence for
the role of organic acids in Al tolerance. Addition of organic acids to
nutrient solutions alleviates phytotoxic Al effects, presumably by
chelating Al and lowering the activity of free Al in the rhizosphere
(Hue et al., 1986). In hydroponic systems an equimolar amount of
citrate is needed to significantly reduce Al toxicity, whereas a 3-fold
excess of malate and a 2-fold excess of oxalate relieves Al toxicity
(Ryan et al., 1995; Ginting et al., 1998; Zheng et al., 1998). Al in
the growing medium induces exudation of malate, citrate, succinate, or
oxalate in many Al-tolerant plants (Miyasaka et al., 1991; Delhazie and
Ryan, 1995; Pellet et al., 1996; Ma et al., 1997).
The transgenic plants over-expressing neMDH in the present study showed
increased Al content in roots and shoots compared with untransformed
plants grown in high-Al-containing acid soil. The increase in Al
content was largely a reflection of a significantly greater biomass
accumulation of M16-27 and M20-10 plants compared with dry matter yield
of untransformed plants in the same soil. Increased Al uptake by
tolerant plants was reported previously for Al-tolerant buckwheat (Ma
et al., 1997) and alfalfa (Campbell, 1999). Recent studies show that a
large fraction of the Al in hydroponic culture enters the root system
fairly rapidly (Lazof et al., 1994; Vázquex et al., 1999).
Therefore, an additional Al tolerance mechanism(s) appears to be active
in tolerant varieties in which Al accumulates in the symplasm. An
internal detoxification of Al by forming an Al-oxalate complex was used
to explain Al tolerance of buckwheat that had accumulated high levels
of Al in root tissues (Ma et al., 1997). Magnetic resonance imaging of
roots of resistant and sensitive clones of alfalfa indicated that Al is
complexed within roots of resistant clones and remains free in
Al-sensitive clones (Campbell, 1999). Although it is unclear where
complexing might have occurred, it is possible that increased organic
acids in root and shoot tissue of M16-27 and M20-10 plants might have
resulted in internal Al-complexing, rendering Al less toxic. Although
there is strong evidence for a central role of organic acids in Al
tolerance by crops, other suggested mechanisms of Al tolerance include
(a) H+ influx at root tips resulting in a net
increase in the root surface pH (Degenhardt et al., 1998), (b) binding
of Al by excreted proteins (Basu et al., 1999), (c) localized excretion
of phosphate at the root apex to precipitate Al (Pellet et al., 1996),
(d) sequestration within the vacuole, and (e) presence of metal-binding
and/or Al-tolerant enzymes (Taylor, 1991; Vázquex et al., 1999).
The alfalfa germplasm GA-AT was developed through recurrent selection
in acid soil for Al tolerance (Bouton, 1996). In hydroponic
experiments, GA-AT plants demonstrated a level of Al tolerance similar
to M16-27. The root organic acid synthesis and exudation pattern of
GA-AT plants were different from M16-27, suggesting that an additional Al tolerance mechanism may be operating in GA-AT plants. Because organic acid exudation was measured in alfalfa plants under neutral conditions in the absence of Al, the profile of organic acids exuded by
GA-AT plants may be altered by exposure to Al. We have made crosses of
M16-27 with GA-AT plants and have found cosegregation of the transgene
and enhanced organic acid synthesis. These crosses will allow us to
test whether Al tolerance is subject to additive or synergetic effects
related to germplasm source.
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MATERIALS AND METHODS |
Alfalfa Transformation and Plant Culture
The neMDH cDNA and PEPC cDNA isolated from alfalfa
(Medicago sativa) nodule libraries were characterized
previously (Pathirana et al., 1992; Miller et al., 1998). Full-length
cDNAs were cloned separately in a polylinker between the CaMV 35S
promoter and nopaline synthase 3' terminator. Standard molecular
techniques were used for DNA manipulation (Maniatis et al., 1992). The
chimeric genes were transferred into Agrobacterium
tumefaciens LBA 4404 by triparental mating (Horsch and Klee,
1986). Leaf pieces from a highly regenerable clone of M.
sativa cv Regen-SY (Bingham, 1991) were transformed with
A. tumefaciens as previously described (Austin et al.,
1995). Transgenic plants were grown in sand:soil (1:1, v/v) in a
greenhouse. Plants were watered daily with tap water and fertilized
with water soluble fertilizer (20:10:20, v/v; N:P:K) every 2 weeks.
Nine alfalfa lines with the neMDH transgene and eight alfalfa lines
containing the PEPC construct were confirmed by PCR to carry the NPTII
selectable marker gene (data not shown). Transgenic plants were also
verified by PCR analyses using primer sequences specific for the 35S
promoter and the transgene(s) (data not shown). Because of the
out-crossing nature of alfalfa, all experiments were performed with
primary transformants (T0) propagated from stem cuttings in
vermiculite in a greenhouse. All experiments using alfalfa cv Regen-SY
and GA-AT plants were also from propagated cuttings. Unless otherwise
stated, experiments were conducted in a growth chamber set at
21°C/19°C, day/night temperature and 16-h light at 500 µmol
m2 s1.
Enzyme-Specific Activities and Immunoblotting
Root tip samples (approximately the distal 20 mm of root) and
leaf samples (150 mg) were ground in liquid nitrogen and total soluble
proteins were extracted as described (Gantt et al., 1992). Protein in
the tissue supernatant was estimated using the Bio-Rad protein reagent
(Bio-Rad, Hercules, CA; Bradford, 1976).
In vitro MDH or PEPC activity was measured spectrophotometerically as
described previously (Egli et al., 1989). For western blotting, soluble
proteins were separated on 10% (w/v) SDS-polyacrylamide gels,
electroblotted to nitrocellulose membranes (Amersham, Buckinghamshire, UK), probed with rabbit anti-neMDH (Miller et al., 1998) or anti-PEPC (Pathirana et al., 1992), and visualized with a perioxidase-coupled secondary antibody and enhanced chemiluminescence western-blot detection reagent (Amersham). Cross-reacting protein levels were quantitated by densitometry using an AMBIS imaging system
(Scananalytics, Billerica, MA).
RT-PCR Analysis
Root tips (400 mg) and leaf samples (200 mg) were ground in
liquid nitrogen, and total RNA was extracted using the Trizol reagent
(Life Technologies, Cleveland) according to the manufacturer's instructions. To remove contaminating DNA, 15 µg of RNA was incubated with 1 unit of RQ1 RNase-free DNase (Promega, Madison, WI) for 1 h
at 37°C. RNA samples were purified with the RNeasy Plant Mini Kit
(Qiagen USA, Madison, WI) and used as a template in a multiplex RT-PCR
reaction using the access RT-PCR system (Promega). Primer sequences
were M11F (5'-GACCTGCATCTCTATGATATCG-3'), MRTR
(5'-CAACAACTGGAACATCCACATC-3'), and the universal 18S primer pairs and
competimers (Ambion, Austin, TX) for amplifying an internal control.
The M11F and MDHR primers correspond to positions 384-405 and 801-822
of the M. sativa neMDH cDNA sequence (GenBank accession
no. AF020273). The multiplex RT-PCR amplification reactions (50 µL)
consisted of 5 units of AMV Reverse Transcriptase, 5 units of
Tfl DNA Polymerase, 1× AMV/Tfl reaction
buffer, 1 mM MgSO4, 250 µM dNTPs,
20 pmol each of M11F and MRTR primers, 15 nM universal 18S
primers, 35 nM competimers, and 250 ng of total RNA.
Amplification conditions were one cycle of 48°C for 45 min, one cycle
of 94°C for 2 min, 40 cycles of 94°C for 30 s, 53°C for 1 min, and 68°C for 2 min followed by a final extension at 68°C for 7 min. The PCR products were electrophoresed on 1% (w/v) agarose gel,
transferred to Zetaprobe membrane (Bio-Rad), prehybridized, hybridized,
and washed under highly stringent conditions. Probes were a
32P-labeled 1.65-kb neMDH cDNA fragment obtained by
BamHI-NotI digestion of the pMDH-11 cDNA
clone (Miller et al., 1998) and a 0.3-kb 18S cDNA obtained by RT-PCR
amplification of total RNA from an alfalfa root tissue. Probes were
labeled using a random priming kit (Amersham). Hybridization signals
were analyzed using a storm 840 PhosphorImager and ImageQuant software
(Molecular Dynamics). Relative transcript levels of neMDH were
estimated after normalization with signals from the 18S transcript levels.
Organic Acid Extraction
Roots were washed gently to remove vermiculite and blotted dry
with paper towels. About 0.3 to 0.5 g of root tips and leaves was
collected, frozen in liquid nitrogen, and stored at  80°C until
extracted. Organic acid extraction methods were essentially as
described previously (Johnson et al., 1996). Acidic fractions were
concentrated to dryness in a speed-vac (Savant Instruments, Holbrook,
NY), redissolved in 4 mM sulfuric acid, filtered through a
0.45-µm syringe filter, and stored at 20°C until analysis. A
20-µL sample was analyzed by HPLC (Spectra Physics, San Jose, CA) equipped with an Aminex HPX-87H 300- × 7.8-mm column
(Bio-Rad) and an organic acid guard column (Bio-Rad). The mobile phase
was 4 mM sulfuric acid with a flow rate of 0.6 mL/min at
25°C. Individual components were detected at 210 nm on a UV/VIS
detector (Spectroflow 757, ABI Analytical, Sunnyvale, CA).
To evaluate organic acid exudation, 3-week-old alfalfa cuttings were
kept for 24 h in plastic pots filled with industrial quartz
(Unimin Corporation, New Cannan, CT) containing 10 mL of deionized
water. Plants were placed in a growth chamber and exudates were
collected after rinsing roots and quartz with approximately 25 mL of
sterile deionized water. Washes were filtered through Whatman paper
(Clifton, NJ) and mixed with 1 volume of ethanol. Organic acids were
extracted and analyzed as described above.
Alumimum Tolerance Experiments
Two-week-old cuttings were placed in a hydroponic chamber filled
with a preconditioning solution consisting of 12 L of 0.5 mM CaCl2 in deionized water, pH 4.3. pH was
adjusted by adding HCl. After 3 d of growth in the preconditioning
solution, root length was measured, and the solution was replaced with
12 L of preconditioning solution containing 0, 20, 50, or 100 µM AlCl3 at pH 4.3. pH was adjusted by adding
NaOH or HCl. Root length was measured before and after 24 h of
growth in Al treatments.
To evaluate Al tolerance in soil, cuttings trimmed to 1 shoot node were
planted in plastic pots filled with 300 g (dry soil weight) of a
highly weathered unlimed soil: pHH2O = 4.0, AlKCl = 71 µg g1,
PBray = 40 µg g1, Total
n = 0.1%, NNO3 = 60.7 µg
g1, NNH4 = 8.2 µg g1,
K = 332 µg g1, OM = 2.3%. The soil was
obtained from Garden Plain, Sedgwick County in central Kansas. Growth
experiments were conducted in a growth chamber set as described above.
The soil in each pot was watered daily with deionized water. At
harvest, plants were excavated from soil, roots were washed, plants
were partitioned into shoots and roots, and dry matter yield was
determined. Al and P concentrations in plant tissues were measured
using ARL 3560 inductively coupled plasma-atomic emission
spectroscopy by the Research Analytical Laboratory (University of Minnesota).
| |
ACKNOWLEDGMENTS |
We thank Susan Miller for assistance in protein work and Drs.
Howard Rines and Richard Zeyen for critical reading of the manuscript. We thank Drs. Paul St. Amand and Daniel Skinner for supplying soil.
| |
FOOTNOTES |
Received April 19, 2001; returned for revision July 10, 2001; accepted September 14, 2001.
1
This work was funded by the North Central
Biotechnological Initiative (Purdue grant no. 593-0244-05/U.S.
Department of Agriculture [USDA] grant no. 97-34340-3987). This
paper is a joint contribution from the Plant Science Research Unit,
USDA, Agricultural Research Service, and the Minnesota Agricultural
Experiment Station. Mention of a trademark, proprietary product, or
vendor does not constitute a guarantee or warranty of the product by
the USDA and does not imply its approval to the exclusion of other
products and vendors that might also be suitable.
2
Present address: Forage Genetics International, N5292
Gills Coulee Road, West Salem, WI 54669.
*
Corresponding author; e-mail debbys{at}puccini.cdl.umn.edu; fax
651-649-5058.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.010376.
| |
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TOP
ABSTRACT
INTRODUCTION