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Keywords:

  • osmotic potential;
  • soluble carbohydrate

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Cultivar differences in root elongation under B toxic conditions were observed in barley (Hordeum vulgare L.). A significant increase in the length and width of the root meristematic zone (RMZ) was observed in Sahara 3771 (B tolerant) when it was grown under excessive B concentration, compared to when grown at adequate B supply. This coincided with an increase in cell width and cell numbers in the meristematic zone (MZ), whereas a significant decrease in the length and no significant effect on the width of the MZ was observed in Clipper (B intolerant) when it was grown under excessive B supply. This was accompanied by a decrease in cell numbers, but an increase in the length and width of individual cells present along the MZ. Excessive B concentrations led to a significantly lower osmotic potential within the cell sap of the root tip in SloopVic (B tolerant) and Sahara 3771, while the opposite was observed in Clipper. Enhanced sugar levels in the root tips of SloopVic were observed between 48 and 96 h after excess B was applied. This coincided with an increase in the root elongation rate and with a 2.7-fold increase in sucrose level within mature leaf tissue. A significant decrease in reducing sugar levels was observed in the root tips of Clipper under excessive B concentrations. This coincided with significantly lower root elongation rates and lower sucrose levels in leaf tissues. Results indicate a B tolerance mechanism associated with a complex control of sucrose levels between leaf and root tip that assist in maintaining root growth under B toxicity.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Boron is an essential trace element for higher plants; however, when present at toxic concentrations it becomes a limitation for plant growth, leading to a subsequent reduction in crop yield (Cartwright, Zarcinas & Spouncer 1986; Nable, Banuelos & Paull 1997). Although the mechanism of B toxicity is still not clear, a primary phenotypic effect of B toxicity is a poorer root growth compared to that of plants grown at optimal levels of B supply (Lovatt & Bates 1984; Nable 1988; Holloway & Alston 1992; Chantachume et al. 1995; Stangoulis & Reid 2002 and references therein; Reid et al. 2004; Choi et al. 2006). Genotypic variation in root elongation has been effectively used as an indicator of B tolerance (Chantachume et al. 1995; Jefferies et al. 2000). Recent research has identified a range of genotypic variation in response to B toxicity with mechanisms including B exclusion (Nable, Cartwright & Lance 1990; Jefferies et al. 1999) and an inherent ability to tolerate excessive B concentration in plant tissues (Torun et al. 2006). It was observed that the B-tolerant barley cultivar Sahara 3771 has the capacity to maintain much lower B concentrations in roots as well as in xylem and leaves (Hayes & Reid 2004), for which the authors propose a mechanism of active efflux of the borate anion. A lower B concentration in the root of B-tolerant Sahara 3771 than that in the root of B-intolerant cultivars may prevent the toxicity effect on root growth that is generally accompanied by shorter root axes and fewer lateral roots (Huang & Graham 1990).

Reid et al. (2004) identified the root tip as a site of B toxicity because an inhibition of root growth occurred if excess B was applied to the root tip region, but not if excess B was applied to mature sections of the root. At the cellular level, B toxicity is associated with reduced mitotic index in the root meristem, and this is coupled with an increase in chromosome fragments, chromosome stickiness and micronuclei development (Liu et al. 2000). Mitotic activity in the root tip of pea (Pisum sativum L. cv. ‘Alaska’) was also reported to decline within 24 h after exposure to toxic B concentrations (Klein & Brown 1981). These observations provide an insight into how B toxic concentrations impede root growth through increased incidence of abnormal mitosis that in turn impacts on cell division, and ultimately decrease the rate of root elongation.

In general, root growth utilizes sucrose as the major carbon and energy source (Gasparikova 1992). Cellular growth in the root elongation zone occurs with a physical stretching of the cell wall through the accumulation of intracellular solutes (including soluble sugars) to generate an osmotic potential difference between the cell and cell wall that allows water uptake through the cell membrane (Cosgrove 1999; Pritchard, Winch & Gould 2000). There are a few reports demonstrating that carbon deficiency causes a reduction in the rate of cell division, cell density and subsequently root elongation (Van't Hoff 1968; Muller, Stosser & Tardieu 1998). With this in mind, the site of B toxicity in the root tip may indicate an effect of B toxicity on sugar metabolism. There is evidence for a specific effect of B on carbohydrate metabolism (Bowen 1972; Bonilla et al. 1980). A lower content of protein was observed in the root tips of sugarcane grown at high B supply, which resulted in a decrease in the activity of a number of enzymes, specifically aldolase and glyceraldehyde-3-phosphate dehydrogenase, involved in carbohydrate metabolism (Bowen 1972). A decline in glucose of both the leaf and root sap of sugar beet was found under B toxicity, and also nitrate nitrogen accumulated in the leaf sap commensurate with a reduction of nitrate reductase activity (Bonilla et al. 1980). However, the relationship between B toxicity and sugar transport, or metabolism associated with root elongation, remains unclear. The aims of this study were therefore also to investigate the reasons for the apparent variability in root growth between the barley cultivars, B-tolerant Sahara 3771, B-tolerant SloopVic and B-intolerant Clipper under B toxicity. In particular, we investigated whether reduced or enhanced root elongation in these cultivars results from changes in sugar status in both source and sink organs.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Experiment 1: selection of an adequate B concentration for growth studies and genotypic variation in B tolerance

Seeds of Sahara 3771 and Clipper were germinated on moist filter paper in glass Petri dishes with 4 mL of distilled water at 20 °C for 2 d in a dark growth cabinet. Germinated seeds were selected for uniformity and transplanted into seedling trays with aerated nutrient solution for a further 2 d, and then transferred to PVC black-coloured containers filled with 2 L of aerated quarter-strength Hoagland's solution (Hoagland & Arnon 1950) containing the following nutrients: Ca(NO3)2, 1.25 mm; KNO3, 1.25 mm; MgSO4, 0.5 mm; KH2PO4, 0.25 mm; MnSO4, 5.9 µm; ZnSO4, 8.0 µm; CuSO4, 1 µm; NaMoO4, 0.01 µm; KCl, 50 mm; Fe ethylenediamine tetraacetic acid (EDTA), 20 µm; and B(OH)3 at designated levels. The solution was adjusted to pH 6.0 with KOH or HCl. Plants were grown in a growth chamber at 20/10 °C day/night temperature and 14/10 h day/night photoperiod. The first experiment was a 2 × 7 factorial of two genotypes (Sahara 3771 and Clipper) and seven B levels [0, 0.5, 1, 2, 5, 10 and 15 µm of B(OH)3] in a completely randomized design with eight replicates. Analysis of variance (anova) was performed with computer software GenStat (Lane et al. 1988), version 6.0.

Relative shoot growth was calculated as the shoot dry matter (DM) at each B level expressed as a percentage of shoot DM at an adequate B level, where maximum shoot growth was achieved. The growth curves were established using the modified Mitscherlich model.

  • image

where y is the plant yield at B concentration x in the nutrient solution (external B) or the youngest emerged blade (YEB) (internal B); α, β and γ are parameters established based on the observed data. The critical concentrations, which are associated with 90% maximum shoot yield, were calculated by letting y = 90.

The second part of this study aimed to look at the variation in B tolerance between three cultivars: Sahara 3771, Clipper and SloopVic. Plants were grown in a growth chamber at 20/10 °C day/night temperature and 14/10 h day/night photoperiod for 2 weeks in 15 µm B (B-adequate) solution and then transferred to their treatments (either 15 or 5000 µm B) for a further week before harvesting to record variation in shoot and root dry weight (DW), and B concentration of those plant parts.

Experiment 2: effects of high B concentration in culture solution on root tips: meristematic morphology and growth

Sahara 3771 and Clipper were grown in nutrient solution under the same conditions as described in experiment 1. The experiment was a 2 × 2 factorial of two genotypes and two external B levels (15 and 5000 µm B) in a completely randomized design with four replicates.

To investigate cell structure in the root meristematic zone (RMZ) at 4 d after B treatments were applied, the apical 10 mm of the root from four plants of each treatment was taken. The segments were stained with aniline blue using a method modified from Kaneko et al. (1999). Root tips were washed in deionized water, boiled in 95% (v/v) ethanol for 1 h, soaked in water for a further 5 min and then stained with 0.05% (w/v) aniline blue (water soluble; BDH Ltd, Poole, England) solution in 0.15 m K2HPO4 (pH 11.0) for 2 h at room temperature and overnight at 5 °C. The stained samples were washed in deionized water for 5 min before observation. Stained root tips were observed with a confocal microscope (MRC 1000 UV; Bio-Rad, Hercules, CA, USA) using a ×10 lens and an argon laser with an excitation filter of 488/10 nm and an emission filter of 527/32 nm. The same settings for confocal aperture, laser power, camera gain and camera black level were used for examination of all roots. The length and width of the RMZ were measured using confocal images 1733 × 1155 µm in size. To calculate the total area of the meristematic zone (MZ), image pixel number in the aniline blue-labelled zone was measured, and then the value was multiplied by pixel area (2.26 × 2.26 µm = 5.1076 µm2). The distal end of the MZ was estimated to occur at a cell length of 2.5 times the length of the shortest cells (Lenoble et al. 1996). Electronically zoomed-in images were taken to measure the cells in the MZ using image analysis software ImageJ (version 1.28, NIH). Measurements were made for 10 adjacent cells for each MZ and repeated for 10 roots per treatment. Cell number along the MZ was calculated using the length of the MZ divided by the average length of a single cell. Cell number across the MZ was calculated from the width of the MZ divided by the average width of a single cell.

Root imaging was carried out using a flatbed scanning technique (Richner et al. 2000). Individual primary roots of each plant were placed in a shallow transparent tray (20 × 30 cm) filled with 2–3 mL of nutrient solution in which they had been grown to facilitate separation of roots and to enable correct imaging. Images were analysed for the diameter of the apical 10 mm segments in individual primary roots, using a commercially available image analysis software package (WinRhizo; Regent Instrument, Quebec, Canada).

Experiment 3: effects of high B concentration in culture solution on root tips: reducing sugar concentrations, osmotic potential and root elongation rate

SloopVic and Clipper were grown in nutrient solution with the same conditions as described in experiment 2. Roots were harvested at different times (i.e. 0, 24, 48 and 96 h after B treatment). The experiment was a 2 × 4 factorial of two genotypes and four harvests in a completely randomized design with four replicates. Harvested plants were quickly rinsed in high-purity water (18 MΩ cm−1 resistivity) for 10 s, quickly blotted dry with tissue paper and measured for root length. Root elongation rate (mm d−1) was calculated as root length (mm) during B treatment divided by the time of treatment (4 d). Root tips of 10 plants were collected from the region, 2–10 mm from the root apex and kept in Eppendorf tubes (Eppendorf, Hamburg, Germany) stored in liquid N during the harvesting, and then dried in a VirTis automatic freeze-drier (VirTris Inc, Gardiner, NY, USA) for 3 d. Shoot and the remaining root of each plant were also collected separately and oven dried at 80 °C for 24 h for DW measurements. Freeze-dried samples were weighed and then diluted in a fresh Eppendorf tube with 750 µL nano-pure water (>18 MΩ resistivity), shaken on an agitator platform for 10 min and centrifuged at 4293 g for 10 min. Supernatant (500 µL) was collected, placed in a fresh 1.5 mL Eppendorf tube and centrifuged again at 4293 g for 10 min, then stored at 4 °C until further use. Supernatant (200 µL) was diluted to 300 µL with nano-pure water with the addition of 0.5 mL 2% 3,5-dinitrosalicylic acid (DNSA) reagent in a 0.7 M NaOH solution. This procedure was repeated for duplicate samples. All samples were then mixed by vortexing for 5 s and then kept on ice for a further 10 min prior to transferring to a boiling water bath for 5 min, after which samples were immediately transferred to an iced water bath for a further 10 min. Reducing sugars were spectrophotometrically measured at 590 nm with comparison to glucose standards.

To measure osmotic potentials in root tips, harvested roots were quickly rinsed in high-purity water (>18 MΩ cm−1 resistivity) for 10 s and quickly blotted dry with tissue paper. Several apical 5 mm root segments were placed in a 5-mm-diameter filter paper disc laid between two slides of microscope glass. Glass slides were pressed together, and the cell sap was collected onto the paper disc that was then quickly placed in a vapour pressure osmometer (model 5100; Wescor, Logan, UT, USA) to avoid evaporation. The osmotic potential was calculated according to the Van't Hoff equation (Henton et al. 2002), π = −nRT, where π is the osmotic potential, R is the gas constant, T is the absolute temperature and n is osmolity.

Experiment 4: effects of high B concentration in culture solution on root tips and leaf tissue: soluble carbohydrate concentration and invertase activity

SloopVic and Clipper were grown in a growth chamber at 20/10 °C day/night temperature and 14/10 h day/night photoperiod for 1 week in 15 µm B (B-adequate) solution and then transferred to their respective treatments (either 15 or 5000 µm B) for a further week before measurements were taken.

For sugar measurements, leaf tissue (the second youngest mature leaf blade) and root tips (0–10 mm from the root apex) were extracted for soluble carbohydrates using a modified method of Madore, Michell & Boyd (1988). The root tips and the leaf tissues were cut off and placed in 1.5 mL Eppendorf tubes. The tubes were immediately plunged into liquid nitrogen (within 20 s of excising) to minimize enzymatic activity. The samples were dried for 48 h in a VirTis automatic freeze-drier, and then weighed. Soluble carbohydrates were extracted by heating for 20 min in 1 mL of hot 80% EtOH [EtOH: water (80:20 v/v) at 80 °C]. The procedure was repeated three times to ensure complete extraction. The supernatant of the ethanolic extracts was collected and dried in a Speed-Vac vacuum centrifuge (Savant Instruments Inc., Farmingdale, NY, USA). Immediately prior to analysis, samples were resuspended in 900 µL of high-purity water, and filtered through a 0.2 µm syringe filter. Sugars were separated by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) using a Dionex GP40 gradient pump, a Dionex ED 40 electrochemical detector (Dionex, Sunnyvale, CA, USA) and a Shimadzu SIL-10AD autoinjector (Shimadzu, Chiyoda-Ku, Tokyo, Japan). Sample (10 µL) was injected into a CarboPac PA-1 column (Dionex) and eluted using a linear gradient of sodium hydroxide (100 mm, made from low CO2, 50% w/w liquid NaOH) over 13 min with a flow rate of 1 mL min−1. Sugar concentrations were estimated by comparing the peak areas of standards to the peak areas of each sample using Dionex Peaknet software.

Acid and neutral invertase (NI) activity measurement

Leaf tissue (the second youngest mature leaf blade) and root tips [0–10 mm from apex and around 30 mg fresh weight (FW)] were harvested and immediately frozen in liquid nitrogen and stored at −80 °C. For extraction, the tissues were ground into fine power in liquid nitrogen. For the assay of NI activity, extraction buffer [N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (Hepes)–NaOH (pH 7.0), 50 mm; MgCl2, 10 mm; Na2 EDTA, 1 mm; dithiothreitol (DTT), 2.6 mm; ethylene glycol, 10%; and Triton 0.02% in water] (Pelleschi, Rocher & Prioul 1997) was added to ground tissues to a final volume of 60 µL. For the assay of acidic invertase (AI) activity, extraction buffer [50 mm NaH2PO4 buffer (pH 6.0), 1 mm DTT and 1 mm EDTA, 10% ethylene glycol in water) (Shaikh, Quick & Rolfe 2000) was added into ground tissues to a final volume of 60 µL. The extracts were centrifuged for 15 min at 12 225 g and 4 °C. Aliquots of the supernatant collected were desalted by a G25 Sephadex column (Pharmacia Fine Chemicals, Upsala, Sweden). To measure NI activity, 50 µL of 0.9 m sucrose was added to the desalted samples. The samples were incubated for 25 min at 37 °C and then boiled for a further 5 min to stop invertase activity. The glucose was assayed by the method of Moreno et al. (1981). Briefly, the reaction mixture (500 µL) contained 50 µL 0.1 m NaH2PO4 (pH 7.0) buffer, 10 units of glucose oxidase, 2 units of peroxidase, 150 µg of o-dianisidine and 325 µL of high-purity water. After 30 min incubation, the reaction was stopped with the addition of 500 µL of 3 m HCl. The color produced was measured spectrophotometrically at 540 nm. The glucose amount was calculated after referring to a glucose standard curve. The glucose present in tissue was subtracted after a control reaction was performed with a 25 µL tissue extract, which had been boiled for 5 min before adding sucrose to inactivate invertase activity. The enzyme activity was expressed as nmol glucose g−1 FW h−1. To measure AI activity, 30 µL of desalted extract was boiled for 5 min and the other 30 µL of desalted extract was not boiled. Leaf extract (30 µL) was incubated in 120 µL 200 mm sodium acetate buffer (pH 5.0) and 150 µL of 200 mm sucrose for 30 min at 37 °C. The reaction was stopped with the addition of 500 µL DNSA reagent [1% (w/v) DNSA in 0.7 N NaOH solutions] and cooled on ice for a further 5 min. The colour was developed by boiling for 10 min and then cooling on ice for further 5 min. The presence of reducing sugars resulted in the development of a deep orange colour, which was assayed spectrophotometrically at 560 nm.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Selection of an adequate B concentration for growth studies and genotypic variation in B tolerance

Two barley cultivars, Clipper and Sahara 3771, differed in B requirement. Clipper had generally higher internal B concentrations when compared to Sahara 3771. In the B0 treatment, shoot B concentrations were very low, but differences between the two genotypes were observed (0.1 ± 0.07 and 1.1 ± 0.09 mg kg−1 for Sahara and Clipper, respectively). This caused a reduction in shoot yield by 30% for Sahara 3771 and 38% for Clipper (Fig. 1). The B-tolerant Sahara 3771 had a lower external and internal B requirement than the B-intolerant Clipper to achieve 90% of the maximal shoot yield. The critical B deficiency concentration was estimated by the Mitscherlich growth model and was calculated at 0.5 ± 0.06 and 10 ± 0.14 mg B kg−1 DW for Sahara 3771 and Clipper, respectively, corresponding to external B supply of 0.5 and 5.0 µm, respectively. From these results, 15 µm B was chosen as an adequate B level (control) for further experiments, while the B toxicity level was chosen from previously published work (Nable 1988).

image

Figure 1. Relationship between B concentration in the youngest emerged blade (YEB) (the youngest expanded leaf) (a), growth medium (b) and yield [based on shoot dry matter (DM) production] in barley genotypes, Sahara 3771 (○) and Clipper (▿) grown for 20 d in solution culture. The curves were fitted according to the Mitscherlich growth model. Data points represent the mean values of eight plants in each treatment ± SE.

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Cultivar difference in B tolerance

Cultivar difference in sensitivity to B toxicity was observed. In the B-sensitive Clipper, shoot and root growths were significantly decreased by treatment with B toxic concentration, while no reduction was observed in Sahara 3771 and SloopVic (Table 1). Measured in DW, root growth was more sensitive than shoot growth. The B concentration in shoots grown at 15 µm B was higher in Clipper (24 mg kg−1) than in Sahara 3771 (15 mg kg−1) and SloopVic (16 mg kg−1) (Table 1). At excess B supply, the B concentration in the shoot increased in all three barley cultivars compared to their control supplied with 15 µm B; however, this increased B concentration varied between cultivars, in the order Clipper > SloopVic > Sahara 3771. The B concentration in the shoot of Clipper was around sixfold and twofold higher than those of Sahara 3771 and SloopVic, respectively. The B concentration in the root of Clipper at high B supply was 2.6-fold and 1.4-fold greater than those of Sahara 3771 and SloopVic, respectively. Thus, in general, B accumulation in the shoot and root tissue was highest in Clipper followed by SloopVic and Sahara 3771.

Table 1.  Effect of B treatment on shoot and root dry weight (DW) (mg per plant), and B concentration (mg kg−1 DM) in the shoots and roots of three barley cultivars Clipper, Sahara 3771 and SloopVic
MeasurementClipperSahara 3771SloopVic
155000155000155000
  • *

    Significant at P < 0.001.

  • Plants were grown in a growth chamber at 20/10 °C day/night temperature and 14/10 h day/night photoperiod for 2 weeks in 15 µm B (B-adequate) solution and then transferred to their treatments (either 15 or 5000 µm B) for a further week. Values represent means ± SE for three plants.

  • DM, dry matter.

DW
 Shoot275 ± 15199 ± 6*175 ± 6187 ± 11181 ± 8206 ± 10
 Root82 ± 434 ± 5*42 ± 940 ± 149 ± 346 ± 2
B concentration
 Shoot24 ± 0.73100 ± 58*15 ± 0.4517 ± 3.3*16 ± 1.11590 ± 12*
 Root22 ± 1.5887 ± 12*43 ± 2.8333 ± 8.8*42 ± 1.2620 ± 5.8*

Effects of high B concentration in culture solution on root tips: meristematic morphology and growth

At both adequate and excess B supply, the length and width of the RMZ differed between cultivars (Table 2). In plants grown at 15 µm B, the average length of the RMZ was greater in Clipper (636 ± 15 µm) than in Sahara 3771 (557 ± 3 µm), and a corresponding difference was also found for their total root length. However, at high B supply, the length of the RMZ was significantly (< 0.05) increased by 15% in Sahara 3771, but significantly (P < 0.05) decreased by 20% in Clipper. A slight increase (10%; P < 0.05) in the width of the RMZ of Sahara 3771 was also found at high B supply. In plants grown in adequate B supply for 4 d, individual cell length was similar between Sahara 3771 and Clipper (Table 2); however, B toxicity increased the cell length of Clipper by 11% (P < 0.05), while the cell length in Sahara 3771 remained unchanged. Cell width also increased with higher B supply, 8 and 17% for Sahara 3771 and Clipper, respectively. The increase of cell width in Sahara 3771 did not significantly change the final cell area. Overall, total cell number in the MZ was reduced in Clipper, but increased in Sahara 3771. Boron toxicity significantly (P < 0.001) reduced the root tip diameter of the two barley cultivars by around 20%.

Table 2.  Analyses of various parameters in the meristematic zone (MZ) of root tips by confocal microscopy and the diameter of root tips by flatbed scanning
Measurements (µm)ClipperSahara 3771
155000155000
  • *

    Significant at P < 0.005.

  • **

    Significant at P < 0.001.

  • Apical 10 mm segmented root tips were collected from the primary root grown for 4 d at 15 µm B (B adequate) or 5000 µm B (B toxic). Values represent means ± SE for replications.

Cell number along MZ66 ± 0.849 ± 0.9*59 ± 1.370 ± 0.7*
Cell number across MZ37 ± 0.533 ± 0.436 ± 1.337 ± 0.3
Individual cell length9.4 ± 0.210.5 ± 0.2*9.3 ± 0.29.2 ± 0.1
Individual cell width10.9 ± 0.212.8 ± 0.2**10.8 ± 0.411.7 ± 0.2*
Length of MZ636 ± 15506 ± 23*557 ± 3638 ± 3.9*
Width of MZ399 ± 8418 ± 8384 ± 8428 ± 3.0*
Root tip diameter (mm)0.34 ± 0.020.26 ± 0.01**0.34 ± 0.020.27 ± 0.01**

Effects of high B concentration in culture solution on root tips: reducing sugar concentrations, osmotic potential and root elongation rate

Osmotic potentials in the cell sap of the root tips (2–10 mm from apex) were compared. At 15 µm external B, the osmotic potential was higher in the B-tolerant Sahara 3771 (−0.60 MPa ± 0.02) and SloopVic (−0.60 MPa ± 0.03) than in the B-sensitive Clipper (−0.84 MPa ± 0.02) (Fig. 2a). However, at 5000 µm B, the osmotic potential significantly (P < 0.001) decreased in both Sahara 3771 (−0.78 MPa ± 0.04) and SloopVic (−0.70 MPa ± 0.01), whereas in the B-sensitive Clipper, the osmotic potential increased (−0.73 MPa ± 0.03). In SloopVic, the total contribution of the three soluble sugars to the osmotic potential was −0.1 MPa (1 bar). Boron toxicity significantly (P < 0.001) increased the reducing sugars in the root tips (2–10 mm from root apex) of Sahara 3771 and SloopVic by 31 and 119%, respectively, whereas a significant (P < 0.001) reduction by 85% was found in Clipper (Fig. 2b). These results were associated with a fivefold reduction in root elongation in Clipper under high B supply, whereas in Sahara 3771 and SloopVic, the root elongation rate increased twofold (Fig. 2c). These results suggest that the B-tolerant cultivars may require increased levels of osmoticum in the growing zone to maintain root growth under excess B supply.

image

Figure 2. Osmotic potential, reducing sugar concentrations and root elongation rates were measured in the root of Sahara 3771, SloopVic and Clipper barley. (a) Osmotic potential measured in the cell sap of the apical 2–10 mm segmented root. (b) Reducing sugar concentration standardized as glucose concentration in the apical 2–10 mm segmented root. (c) Root elongation rate calculated over the 7 d period of B treatment. Plants were first grown for 2 weeks at 15 µM B and then transferred to either 15 or 5000 µM B for a further week before measurements were taken. Values represent means ± SE for six plants. DW, dry weight.

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A time-course was developed to determine when the response in reducing sugars occurred in two barley cultivars, Clipper and SloopVic, under high concentration of B supply (Fig. 3). Reducing sugar concentrations in root tips of SloopVic remained relatively constant for the first 2 d after the addition of high B and then between 2 and 4 d dramatically increased (Fig. 3a). This remarkable increase in reducing sugars preceded a gradual increase in root elongation rate (Fig. 3b). In contrast, an increase in reducing sugars was observed in Clipper within the first 24 h after the addition of toxic B, a flattening off for the next 24 h and between 2 and 4 d, a gradual decline in reducing sugars was observed (Fig. 3a). With a decline in reducing sugars between 2 and 4 d in Clipper, we also saw a drastic decline in the rate of root elongation (Fig. 3b).

image

Figure 3. The effect of B toxicity on relative reducing sugar (RRS) in root tips (a) and relative root elongation (RRE) rate (b) of Clipper (○) and SloopVic (●) grown for 4 d in 5000 µm B. RRS was calculated as {100 × [reducing sugars (mg g−1) at day 1 (d1)/reducing sugars at day 0 (d0)]}. RRE was calculated as {100 × [root elongation rate (mm d−1) at day 1 (d1)/root elongation rate at day 0 (d0)]}. Bars represent the mean of 27 plants for the reducing sugar concentrations and 30 plants for the root elongation rate ± SE.

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Effects of high B concentration in culture solution on root tips and leaf tissue: soluble carbohydrates and invertase activities

Soluble carbohydrates (glucose, fructose and sucrose) were measured in apical 10 mm of the root and mature leaf tissue (the second youngest mature leaf blade). Sucrose was the main soluble sugar in the leaf tissue, whereas glucose concentrations were higher in the root tip tissue (Fig. 4a,b). At 15 µm B, sucrose concentrations in the leaf of Clipper were around threefold higher than in SloopVic. However, B toxicity significantly (P < 0.001) altered the sucrose concentration in both tolerant and sensitive cultivars. A 2.7-fold increase in sucrose concentration was observed in the leaf tissue of SloopVic, whereas a 1.4-fold reduction was observed in the leaf tissue of Clipper (Fig. 4b). The low sucrose concentration in the leaf tissue of Clipper under high B supply corresponded to a significant increase of glucose and fructose in the leaf. In the leaf tissue of Clipper, the increase of glucose concentration was greater than that of fructose concentration. Likewise, both glucose and fructose were also increased in the leaf tissue of SloopVic. However, the major difference between the two cultivars was that sucrose concentration was increased in SloopVic but decreased in Clipper. In the root tips of SloopVic, B toxicity resulted in a 1.4-fold increase in both glucose and fructose concentration (Fig. 4a), while the concentrations of glucose and fructose in the root tips of Clipper were reduced sevenfold and threefold, respectively.

image

Figure 4. Soluble carbohydrate concentrations in (a) the apical 0–10 mm segmented roots and (b) the second youngest matured leaf of SloopVic and Clipper. Plants were grown in a growth chamber at 20/10 °C day/night temperature and 14/10 h day/night photoperiod for 1 week in 15 µm B (B-adequate) solution and then transferred to their treatments (either 15 or 5000 µm B) for a further week before measurements were taken. Values represent mean ± SE for three plants. DW, dry weight.

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The involvement of invertase activity in the changes of carbohydrate status in response to high B supply was investigated. NI activity did not respond to high B supply in either roots or shoots of SloopVic, whereas in Clipper NI activity increased by 30% in the shoot and decreased it by up to 90% in the root (Fig. 5) compared to control B supply. The NI activity in Clipper in response to high B supply was accompanied by changed carbohydrate status in the root and shoot as shown previously. AI activity responded to B toxicity in roots and shoots of both Clipper and SloopVic (Fig. 5). In the shoot, AI activity was enhanced by 73% for Clipper and 68% for SloopVic in response to high B supply, and this was accompanied by an increase in glucose and fructose concentration in the shoots of both cultivars (Fig. 5). In contrast, the AI activity was decreased by high B supply in roots of both cultivars by 74 and 54% in Clipper and SloopVic, respectively, compared to control B supply, although the reduction in SloopVic's roots was not as great as in Clipper's roots. The decreased AI activity in the root tip of SloopVic under high B supply was not consistent with the enhanced reducing sugars in the region.

image

Figure 5. The effect of B on neutral invertase (NI) and acidic invertase (AI) activities in the root (a) and second youngest mature leaf (b) of two barley cultivars, SloopVic and Clipper. Plants were grown in a growth chamber at 20/10 °C day/night temperature and 14/10 h day/night photoperiod for 2 weeks in 15 µm B (B-adequate) solution and then transferred to their treatments (either 15 or 5000 µm B) for a further week before measurements were taken. Values represent mean ± SE for three plants of Clipper and six plants of SloopVic. FW, fresh weight.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The aim of this study was to investigate the mechanism of B tolerance in barley as expressed in the maintenance of root growth under high concentration of B in the root environment. A control level of B was first established for subsequent experiments, and a lower internal and external B requirement was observed in Sahara 3771 when compared to Clipper (Fig. 1). Lower external and internal requirements may indicate increased efficiency in B uptake and/or utilization within plant tissues. Given the structural role of B in higher plants (Kobayashi et al. 1999; Vidal et al. 2000), the lower B requirements in Sahara 3771 may also indicate lower cell wall B requirements. Recent evidence also suggests that in some species (e.g. Brassica napus L.), plants are able to remobilize B even though sucrose (and not polyol) is a primary photo-assimilate (Stangoulis et al. 2001). One might hypothesize that Sahara 3771 remobilizes more B leading to increased efficiency in utilization, and therefore, a lower internal B requirement. Further studies are required to test these theories.

Our results show an adverse effect of toxic B concentration on the root tip region where there was a decrease in the length of the MZ and in cell density associated with larger cells within it in the B-intolerant barley cultivar Clipper. This result confirmed previously reported accounts (Reid et al. 2004) about an adverse effect of toxic B concentration on the root tip region. In contrast, the B-tolerant barley cultivar Sahara 3771 showed an increase in the length of the MZ and cell density, which was associated with increased root elongation rate. Importantly, the contrary response of B-tolerant and -intolerant cultivars was also observed in the concentration of reducing sugars in the root tips, thus explaining the energy source for root elongation under stressful conditions in the tolerant cultivars.

At adequate B supply, Clipper has much higher levels of sugars in both the leaf and root tip with a twofold higher biomass than those of the B tolerant cultivars, indicating a higher demand for carbon in Clipper. However, at high B concentrations, there was a dramatic decrease of sucrose in leaf tissue, and soluble sugars also declined dramatically in the root tip of Clipper. Although similar osmotic potential values were observed in the three cultivars when grown at high B supply, enhanced soluble sugars in the root tip of SloopVic contributed around −0.1 MPa (1 bar) to the total osmotic potential which one may hypothesize to have an effect on osmoregulation.

A time-course investigation for root elongation and reducing sugars in root tips of SloopVic and Clipper demonstrated that a significant decline in the root elongation rate was found in Clipper between 48 and 96 h after toxic B was applied. This coincided with a reduction in sugars in Clipper, while there was no reduction of the root elongation rate with a significant increase in reducing sugars in SloopVic. The effect of B toxicity on sugar status is consistent with the results from a recent study (Roessner et al. 2006), where all phosphorylated sugars in the root of the B-intolerant Clipper decreased, while the B-tolerant Sahara roots exhibited the opposite pattern, with an increase in phosphorylated sugar levels within this area. It appears that when B concentrations are sufficiently high, sugar metabolism, transport or utilization is affected. However, further evidence is still required to link the altered sugar metabolism to changes in root elongation, thus elucidating the primary effect of high B concentration on root elongation. Cause and effect are therefore not necessarily proven by the association of high root elongation rates with high root tip reducing sugars under B toxic conditions. Rather, we may suggest a hypothesis, integrating all the observations in this study. Soluble sugars in the root tip contribute to the osmotic pressure required for cell elongation under toxic B conditions, but do not directly contribute to osmotic relations. To determine whether the relationship is causative or not, it is necessary to obtain more direct and detailed investigations on root cell turgor pressure linked to cell elongation, or physical/chemical changes of cell wall (e.g. wall extensibility) under toxic B conditions. Interestingly, a similar mechanism has been recently reported for Al tolerance in wheat (Tabuchi, Kikui & Matsumoto 2004) where an accumulation of soluble sugars occurred in the Al-tolerant wheat (Atlas 66) with a lower osmotic potential in the cell sap of the root tips. The authors postulate that the reduction of osmotic potential enables the cells to take up water for maintaining cell turgor and to elongate against the pressure produced by cell wall rigidity under Al stress. Notably, the present study has shown that a reduction in intracellular B concentrations is not always required for the B tolerance mechanism responsible for maintenance of root growth. While B concentrations in the shoot and root tissues of SloopVic were quite higher at excess B supply (threefold and twofold higher than those of Sahara 3771), this did not impact negatively on shoot growth, although it is noted that the experiment was run for a short time period, and with a longer growth period we may have seen some adverse effects of B toxicity. Even taking this into account, in the short time period witnessed, B tolerance was clearly evident, and given that the barley cultivar SloopVic does not have the 4H B-tolerance allele associated with the ability to maintain lower levels of B in plant tissue, its B tolerance mechanism is therefore not associated with active B efflux proposed for the B-tolerant Sahara 3771 (Hayes & Reid 2004).

From a molecular perspective, enhanced sucrose levels in the leaf tissue and enhanced glucose and fructose levels in the root tip of SloopVic appear to be associated with other important quantitative trait locus (QTL), perhaps the 2H and 3H QTLs from the donor parent Sahara 3771 (Jefferies et al. 1999, 2000). New research should investigate mapping of the high sugar trait in the root tips of a Clipper × Sahara 3771 double haploid population to see if this new B tolerance trait maps to the 2H and 3H loci previously reported, and whether other QTLs, such as for osmotic adjustment, are also important in the tolerance mechanism.

In summary, from this study, there appears to be growing evidence that B tolerance in barley is associated with root morphological changes under B toxicity, leading to an increase in root elongation as witnessed in Sahara 3771 and SloopVic. The maintenance of root growth in the B-tolerant barley could be a result of osmotic adjustment, underlying a role of enhanced reducing sugar levels to maintain root elongation, and hence, whole plant growth under B toxicity. Further investigation using the Clipper/Sahara 3771 mapping population (Islam & Shepherd 1981; Karakousis et al. 2003) is needed to identify the gene location for this new B tolerance trait.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This research was supported by an Australian Commonwealth Government-funded scholarship to the principal author. We thank Professor David Coventry and Dr. Ian Nuberg for their help to get the scholarship.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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