Plant Physiol. (1999) 119: 735-742
Manipulation of in Vivo Sorbitol Production Alters
Boron Uptake and Transport in Tobacco1
Nacer Bellaloui,
Patrick H. Brown*, and
Abahaya M. Dandekar
Department of Pomology, University of California, Davis, California
95616
 |
ABSTRACT |
Recent evidence that some species can
retranslocate boron as complexes with sugar alcohols in the phloem
suggests a possible mechanism for enhancing boron efficiency. We
investigated the relationship between sugar alcohol (sorbitol) content,
boron uptake and distribution, and translocation of
foliar-applied, isotopically enriched 10B in three lines of
tobacco (Nicotiana tabacum) plants differing in sorbitol
production. In tobacco line S11, transformed with sorbitol-6-phosphate
dehydrogenase, the production of sorbitol was accompanied by an
increase in the concentration of boron in plant tissues and an
increased uptake of boron compared with either tobacco line A4,
transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated
to the meristematic tissues only in line S11. These results demonstrate
that the concentration of the boron-complexing sugar alcohol in the
plant tissue has a significant effect on boron uptake and distribution
in plants, whereas the translocation of the foliar-applied
10B from the mature leaves to the meristematic tissues
verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can
result in increased boron uptake, with no apparent negative effects on
short-term growth.
| |
INTRODUCTION |
Current information implies that boron uptake is a passive process
and that its rate is determined by the boron concentration in the
medium and the formation of boron complexes in plants (Brown and Hu,
1994
). Species and cultivars, however, differ significantly in boron
uptake even when grown under identical environmental conditions that
are characteristic of selective ion uptake. For example, Brown and
Jones (1971) reported that the boron-efficient tomato cv Rutgers was 15 times more efficient in using boron from the medium than the
boron-inefficient cv T3238. They concluded that cv T3238 lacks the
ability to transport boron to the shoot. Nable et al. (1990) found that
barley and wheat cultivars tolerant to boron-toxic soils accumulated
less boron in the youngest expanded leaf than the less-tolerant
cultivars. In both of these examples, species were grown under
identical conditions and uptake kinetics were consistent with a passive
process. The mechanisms underlying species differences in boron uptake
are unknown, but may result from differences in membrane permeability
(Nable and Paull, 1991), the ability of a species to translocate
boron (Bellaloui and Brown, 1998) and mobilize it within the plant
(Brown and Shelp, 1997), the formation of boron complexes in the cell
(Hu and Brown, 1997), or other, as-yet-unidentified mechanisms.
Boron is generally considered an immobile element in most species,
because it accumulates in leaves and does not retranslocate to other
plant organs. The immobility of boron, however, is not a general
phenomenon in all species. For example, Shelp et al. (1995) found that
the concentration of boron in the floral parts of broccoli was higher
than in the leaves, and that foliar-applied B
was translocated to a small extent to florets mainly via the phloem.
Further, it was reported that boron was highly mobile in apple (van
Goor and van Lune, 1980), and that foliar-applied boron could be
retranslocated from mature leaves of Prunus,
Pyrus, and Malus species (Hanson, 1991; Picchioni
et al., 1995). Recently, Brown and Hu (1996) observed that
foliar-applied 10B was phloem mobile in
sorbitol-rich species within Prunus, Pyrus, and
Malus, but there was no significant translocation of the
foliar-applied B in nonsorbitol species
included in the study. The mobility of boron in sorbitol-rich species
was subsequently verified by Hu et al. (1997), who isolated and
characterized soluble boron complexes from the extrafloral nectar of
peach and the phloem sap of celery. In peach and celery boron is
translocated in the phloem as sorbitol-boron-sorbitol and
mannitol-boron-mannitol, respectively (Hu et al.,
1997).
The presence of high concentrations of sorbitol (Moing et al., 1992),
the free phloem mobility of boron in sorbitol-rich species (Brown and
Hu, 1996), and the finding that boron is present in the phloem as a
sorbitol-boron-sorbitol complex (Hu et al., 1997) strongly suggest that
polyols (sorbitol) play a significant role in boron transport in
Prunus, Pyrus, and Malus species.
Whether this phenomenon also influences boron uptake is unknown.
However, the observation that boron uptake is partially determined by
the underlying rate of boron-complex formation suggests that sugar alcohols may also affect boron uptake.
The aim of the present study was to determine if differences in
sorbitol content affect boron uptake and translocation. To achieve this
goal we determined boron uptake and translocation in tobacco
(Nicotiana tabacum) genetically manipulated to produce sorbitol (S11) and contrasted this with the antisense strain, A4, and wild-type tobacco (strain SR1), in which sorbitol is absent.
| |
MATERIALS AND METHODS |
Plant Material
Three tobacco (Nicotiana tabacum) lines were selected
for this study. The first line, S11, was genetically engineered to
produce sorbitol using cDNA encoding NADP-dependent S6PDH (Tao et al., 1995). In the experiment of Tao et al. (1995), an apple cDNA encoding S6PDH was stably integrated and expressed in transgenic tobacco (line
S11). They demonstrated that S6PDH was expressed in sufficient quantity
for the synthesis of sorbitol in tobacco and suggested that S6PDH is a
key enzyme for sorbitol synthesis in apple. The second line (positive
control), A4, was genetically engineered to contain the antisense of
the cDNA encoding S6PDH (Tao et al., 1995). The third line (negative
control), SR1, is the wild type.
Seeds of tobacco lines S11, A4, and SR1 were germinated in Petri
dishes. We treated seeds of strains S11 and A4 with kanamycin solution
(100 mg L
) for 30 min, then placed them on
moistened filter paper. Seeds of tobacco line SR1 were soaked in water
for 30 min. We subsequently chilled all seeds at 5°C for 3 d,
after which the seeds were transferred to a controlled-environment room
for germination. After 7 d of germination, the seedlings were
transplanted to a clean sand medium supplied with one-quarter-strength
Hoagland solution (Hoagland and Arnon, 1950) under greenhouse
conditions with natural lighting at a temperature of 30°C/15°C
(day/night). The pH of the nutrient solution was adjusted to 5.5 to 6.5 and plants were irrigated twice a week. The nutrient irrigation was
applied after the growth medium was rinsed with double-distilled water
to avoid salt and boron accumulation. Isotopic boron (99.43%
10B:0.57% 11B) (Eagle
Picher, Quapaw, OK) as boric acid was used as a tracer for uptake and
translocation of boron.
We screened all of the transgenic plants for gene expression using the
GUS assay according to the method of Jefferson (1987), and tested a
number of nontransgenic tobacco (SR1, wild-type) plants as a negative
control. For each experiment there were four replicates of each
treatment and line combination. Replicates within treatments were
assigned based on the activity of the GUS, such that the average GUS
activity within a given treatment did not differ significantly from any
other treatment.
Experiment 1: Uptake and Distribution of Boron
This experiment was designed to investigate whether sorbitol
production and distribution is influenced by boron supply (0.1, 1.0, and 10 mg L1), and whether soribitol
affects the uptake and distribution of boron.
Two weeks after germination, plants were supplied with a concentration
of 0.04 mg L1 natural abundance boron as boric
acid in a nutrient solution background (described above), for 2 weeks.
After an additional 2 weeks, the boron source was changed to 99.43%
10B-enriched boric acid supplied at either
0.1, 1, or 10 mg L1 with three-quarter-strength
Hoagland solution (Hoagland and Arnon, 1950). Plants were harvested at
0 (before 10B treatment), 4, 12, 24, and 504 h after 10B-enriched boric acid application.
We determined the 10B uptake and distribution in
plant tissues (mature leaves, meristematic tissues, stems, and roots)
as described below. S11, was contrasted with the positive (A4) and
negative (SR1) controls. Sorbitol concentrations were determined in all lines, as described below by Tao et al. (1995) and Greve and Labavitch (1991), and compared to boron uptake and distribution.
Experiment 2: Translocation and Distribution of Boron
The aim of this experiment was to verify the role of sorbitol in
boron translocation from the mature leaves to the meristematic tissues
after foliar application of 10B.
Tobacco seeds were germinated and transplanted to sand culture, and
were then supplied with a concentration of 0.1 mg
L1 boron as natural abundance boric acid. After
2 weeks of growth, the plants were supplied with a concentration of
1.0 mg L1 boron in three-quarter-strength
Hoagland solution. After 1 week of growth in 1 mg L1 boron,
the mature leaves (three leaves from each plant) of S11, A4, and SR1
plants were treated (foliar application) with
10B-enriched (99.43 atom %) boric acid at a
concentration of 700 mg L1. The mature leaves
were immersed in 700 mg L1
10B with 0.05% (v/v) L-77 surfactant (Loveland
Industries, Greeley, CO) for 15 s. Mature leaves and meristematic
tissues of all lines were harvested at 0, 1, 12, 24, 48, and 240 h
after foliar-applied B. Untreated plants of
each line were used as controls.
Sampling and Plant Analysis
At each harvest, four individual plants (replicates) were selected
from each line, and each plant was divided into mature leaves,
meristematic tissues, stems, and roots. Roots were removed from the
sand, cleaned of adhering substrate, and then collected without being
washed. Preliminary experiments demonstrated that washing removes from
6% to 12% of root boron. This amount was highly variable between
replicates. Replicate plants were more uniform in boron content when
washing was omitted. We analyzed recently fully mature leaves. At each
harvest mature leaves and meristematic tissues were divided into two
groups: one for isotopic boron (10B)
determination and the other for cell wall boron determination. We
determined the isotopic boron content in mature leaves, meristematic tissues, stems, and roots of each plant, as described below.
Boron Analysis
Plant tissues were dry ashed at 500°C and analyzed for boron
content using an inductively coupled plasma MS (model Elan 500, Perkin-Elmer, Norwalk, CT), as described by Brown and Hu (1994).
IM
IM of boron was calculated according
to the equation of Williams (1948):
where R1 and
R2 are the initial and final root dry
weights at t1 and
t2, respectively, and
M1 and M2 are
the initial (t1) and final
(t2) boron contents.
Sorbitol Analysis
One gram of fresh tissue was homogenized with an ice-cold mortar
and pestle in 10 mL of 80% ethanol. The extract was centrifuged and
the supernatant dried by a stream of air. An internal standard, 250 µL of 50 µg of inositol, was added to the samples. The samples were
dried again by a stream of air. Four-hundred microliters of acetic
anhydride and 60 µL of 1-methyl imidazol were added to acetylate the
sorbitol. After 10 min, the reaction was stopped by adding 2 mL of
water. The acetylated sugars were partitioned in 2 mL of
dichloromethane and dried. Acetylated samples were then dissolved in
100 µL of acetone and analyzed using a Perkin-Elmer gas chromatograph
(model 8320). MS was carried out using a mass-selective detector (model
5970, Hewlett-Packard) to confirm the retention time (Greve and
Labavitch, 1991; Tao et al., 1995).
Experimental Design
We used a randomized complete block design in this experiment. We
repeated the experiment without the use of the antisense line. All
values shown in tables and graphs represent the means of four
replicates. Error bars indicate SEs. We performed the statistical analysis with the General Linear Models procedure (SAS,
1982).
| |
RESULTS |
The plants were healthy throughout the experiment and did not show
any symptoms of boron deficiency or toxicity.
Experiment 1: Uptake and Distribution of Boron
At 504 h the concentrations of 10B in
all tissues of S11 plants were greater than in tissues of the A4 or SR1
plants at each solution concentration of boron (Fig.
1). This difference was particularly
marked when plants were grown at 10 mg 10B
L1. In this treatment the highest
concentrations of 10B were present in the
meristematic tissue of S11, and were 300% higher than the
10B in the stems of S11 and approximately 300%
higher than in any tissues of A4 or SR1. The distribution of boron in
all plants changed with time and with the concentration of boron
in the medium. With increasing boron concentrations and at
later harvest dates, the proportion of boron found in aboveground
plant parts (meristems and mature leaves) increased; this was
particularly pronounced in S11 (Fig. 1).
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| Figure 1.
10B concentration in mature leaves,
meristematic tissues, stems, and roots of tobacco plants. Transgenic
S11, sense orientation (A-D); transgenic A4, antisense orientation
(E-H); and wild-type SR1 (I-L) plants were grown in 0.1 ( ), 1.0 ( ), and 10 mg L1 ( ) 10B for a period of
3 weeks. Bars represent means ± SE of four
replicates.
|
|
At all concentrations (0.1, 1.0, and 10 mg L1)
of 10B, the total net uptake of
10B (Fig. 2, A, B,
and C) in S11 was higher than in A4 and SR1. In S11 the major site of
10B accumulation was in the meristematic tissues,
whereas in A4 and SR1 the major site of 10B
accumulation was in the mature leaves and roots.
IM was also significantly higher in S11
than in A4 and SR1 (Fig. 2D). IM in S11
grown with 10 mg L1 boron was 174% and 197%
compared with IM in A4 and SR1. There was
no difference in plant dry weight among the lines (Fig.
3). This indicates that the increase in
10B uptake in line S11 was due mainly to the
ability of S11 to acquire more 10B from the
medium, and was not a result of differences in plant growth.
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| Figure 2.
Net uptake of 10B (after
10B treatment) per organ in S11 (A), A4 (B), SR1 (C), and
IM (D) of tobacco plants grown in 0.1, 1.0, and 10 mg L1 10B for a period of 3 weeks.
Bars represent means of four replicates ± SE.
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|
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| Figure 3.
Total dry weight in S11, A4, and SR1. Plants were
grown in 0.1, 1.0, and 10 mg L1 10B for a
period of 3 weeks. Bars represent means ± SE of four
replicates.
|
|
Sorbitol in Plant Tissues
No sorbitol was detected in either A4 or SR1. The concentration
and content of sorbitol plants in S11 were higher in meristematic tissues compared with mature leaves, stems, or roots, and this pattern
was observed when the plants were grown with 0.1, 1.0, or 10 mg
L1 10B in the medium
(Table I). Total sorbitol (micromoles per
plant) increased as 10B increased in the medium
(Table I).
View this table:
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|
Table I.
Sorbitol in mature leaves, meristem tissues, stems,
and roots of transgenic (sense) plants
Plants were grown in 0.1, 1.0, and 10 mg/L-1
10B for 3 weeks. Values are means of four replicates ± SE.
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Correlation between Sorbitol and Boron Uptake
The 10B IM and
the content of 10B in plant tissues were closely
correlated with the content of sorbitol in the plant, and plants receiving the highest 10B treatment had both the
highest IM of 10B and
the highest tissue sorbitol content (Fig.
4).
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| Figure 4.
Correlation between sorbitol content and
IM of 10B (A) and
10B content (B) in S11. Plants were grown in 0.1 (), 1.0 ( ), and 10 mg L1 ( ) 10B for 3 weeks.
**, P < 0.01.
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Experiment 2: Translocation and Distribution of Boron
In lines A4 and SR1, foliar boron application increased boron
concentration in treated leaves and had no effect on meristematic tissue leaves (Fig. 5, B and D). In line
S11 foliar 10B application initially caused an
increase in mature leaf 10B (24 h) followed by a
decline in leaf 10B to control values by 48 h
(Fig. 5B). Simultaneously, there was an increase in
10B in meristematic tissues of S11 (Fig. 5D).
10B in meristematic tissues of S11 increased from
19 µg g1 dry weight at zero time to 46.6 µg
g1 dry weight (245%) by 48 h. This
increase of 10B observed in the S11 meristematic
tissues was not observed in the meristematic tissues of untreated S11,
A4, or SR1 (Fig. 5, A and C) or treated A4 or SR1.
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| Figure 5.
Changes in 10B concentration in mature
leaves in untreated (A) and treated (B) plants, and meristematic
tissues in untreated (C) and treated (D) plants over a period of
250 h. 10B was applied to the mature leaves of S11
(), A4 (), and SR1 () at a concentration of 700 mg
L1.
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| |
DISCUSSION |
Mature leaves, meristematic tissues, stems, and roots of
transgenic (S11) tobacco plants had higher concentrations, greater net
uptake, and IM of 10B
than the antisense transformed or wild-type plants. There was a close,
positive correlation between sorbitol production and total
10B content in the plant and
IM of 10B. Foliar
10B labeling further demonstrated that the
synthesis of sorbitol enhanced the transport of boron in S11.
These results suggest that the presence of sorbitol increased both
boron uptake and transport, and support the results of Brown and Hu
(1996), who demonstrated that the mobility of boron was mediated by the
presence of sorbitol and the formation and transport of sorbitol-boron
complexes in sorbitol-producing species.
The results presented here also suggest that the presence of
boron-binding compounds in the cell may increase boron uptake. This
conclusion is consistent with the current understanding of the
mechanism of boron uptake, which is thought to be a nonmetabolic process determined in part by the formation of nonexchangeable boron
complexes within the cytoplasm and cell wall (Brown and Hu, 1994).
Although an increase in boron-binding compounds (e.g. sorbitol) in
transgenic tobacco (S11) might be expected to increase boron uptake by
maintaining a favorable gradient for boron diffusion into the plant, it
should be noted that boron uptake occurs in tobacco plants containing
no sorbitol. Clearly, sorbitol is not required for boron uptake,
although its presence apparently enhances both uptake and translocation
to the shoot. The influence of sorbitol production on boron uptake
cannot be attributed to changes in cellular osmotic status, as the
concentrations of sorbitol present are not osmotically significant.
Both of the experiments that we conducted here and reported previously
(Brown et al., 1999) demonstrate that sorbitol synthesis influences
boron mobility. When foliar 10B was applied to
mature leaves of S11, A4, and SR1 plants, meristematic tissues in S11
had the highest concentrations of 10B, whereas
mature leaves of A4 and SR1 had the highest 10B
concentrations. An increase in the proportion of boron allocated to
meristematic tissues is typical of species in which sorbitol is
abundant and boron is phloem mobile (Brown and Hu, 1996). Higher apical
than basal concentrations of a nutrient are considered to be indicative
of phloem mobility (Van Goor and Van Lune, 1980). Foliar
10B applications further verified the phloem
mobility of 10B in strain S11. The rate at which
10B disappeared from leaves receiving
10B, and the appearance of
10B in nontreated meristematic tissues of S11,
but not A4 or SR1, clearly confirm that the synthesis of sorbitol
facilitated boron mobility.
The lack of mobility of foliar-applied 10B to the
meristematic tissues in A4 and SR1 could be due to the high
boron-fixing capacity of these lines, which would make the
foliar-applied 10B and naturally root-absorbed
boron unavailable for retranslocation (Brown and Hu, 1994). An
alternative explanation is that the translocation of free boron as
H3BO3 cannot take place
because of the inherently high membrane permeability of
H3BO3, which may lead to a leakage of free boron from
the phloem vessels to the adjacent xylem vessel (Oertli and Richardson,
1970). The boron-sorbitol complex may facilitate boron translocation by
preventing its complexation to insoluble compounds within the leaves,
or the formation of sorbitol-boron complexes may alter the membrane
permeability of boron, thereby overcoming the theoretical constraints
to boron mobility proposed by Oertli and Richardson (1970).
The results of the current experiments suggest that the enhanced boron
uptake in S11 tobacco plants resulted from increased uptake of boron by
roots and by enhanced translocation of boron from roots to shoots.
Together, these processes would reduce the concentration of free
H3BO3 in the root-cell
symplasm and hence favor increased boron uptake.
The production of sorbitol in line S11 was influenced by the presence
of boron in the medium. The mechanism by which this occurred is unknown
but may suggest that the formation of the sorbitol-boron complex favors
further sorbitol synthesis by removing end-product inhibition. The
concentration of sorbitol in S11 was approximately 0.3 to 1.0 µmol
g1 fresh weight, whereas boron was present at
0.1 to 0.5 µmol g1 fresh weight. A
preliminary experiment showed that with a high boron concentration in
the medium (10 mg L-1), a majority of the cellular
boron was soluble. If we presume that sorbitol and boron were localized
together (the formation of the boron-sorbitol complex would favor
this), then adequate boron was present to effectively complex most
available sorbitol and favor more sorbitol production.
The genetic manipulation of tobacco to produce sorbitol clearly
increased uptake and enhanced boron mobility. These two factors would
be expected to result in overall improvement of the plants' ability to
tolerate low-boron soils and to withstand brief periods of boron
deficiency. Evidence suggests that boron plays a critical role in
flowering and seed yield, and that short-term deficiencies of boron (as
a result of drought, low transpiration, or rapid plant growth) can
result in substantial yield reductions (Dell and Huang, 1997; Brown et
al., 1999). Enhanced boron uptake and the ability to remobilize boron
to supply reproductive boron requirements would clearly be a
significant adaptive advantage with important agricultural
implications. These advantages occurred with no apparent decrease in
plant growth over the period used here (not shown). Long-term trials
should be conducted to verify the utility of this approach in improving
plant tolerance to a low-boron environment.
| |
FOOTNOTES |
1
This work was supported by the U.S. Department
of Agriculture (grant no. 9801010).
*
Corresponding author; e-mail phbrown{at}ucdavis.edu; fax
1-530-752-8502.
Received June 25, 1998;
accepted October 30, 1998.
| |
ABBREVIATIONS |
Abbreviations:
IM, specific uptake
rate.
S6PDH, sorbitol-6-P dehydrogenase.
| |
ACKNOWLEDGMENTS |
We wish to thank Henry Fisk, Sandy Uratsu, David Zeng, and Carl
Greve for technical assistance. We are grateful to Richard Bell,
Murdoch University, and Hening Hu for their critical reading of the
manuscript.
| |
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Top
Abstract
Introduction
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