JXB Advance Access originally published online on February 4, 2008
Journal of Experimental Botany 2008 59(3):575-583; doi:10.1093/jxb/erm336
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Evidence of phloem boron transport in response to interrupted boron supply in white lupin (Lupinus albus L. cv. Kiev Mutant) at the reproductive stage
1Centre for Mined Land Rehabilitation, The University of Queensland, St Lucia, Qld 4072, Australia
2School of Environmental Science
3School of Biological Sciences and Biotechnology, Murdoch University, Perth, WA 6150, Australia
* To whom correspondence should be addressed. E-mail: l.huang{at}uq.edu.au
Received 9 October 2007; Revised 28 November 2007 Accepted 30 November 2007
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Abstract |
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The present study investigates whether previously acquired boron (B) in mature leaves in white lupin can be retranslocated into the rapidly growing young reproductive organs, in response to short-term (3 d) interrupted B supply. In a preliminary experiment with white lupin in soil culture, B concentrations in phloem exudates remained at 300–500 µM, which were substantially higher than those in the xylem sap (10–30 µM). The high ratios of B concentrations in phloem exudates to those in the xylem sap were close to values published for potassium in lupin plants. To differentiate ‘old’ B in the shoot from ‘new’ B in the root, an experiment was carried out in which the plants were first supplied with 20 µM 11B (99.34% by weight) in nutrient solution for 48 d after germination (DAG) until early flowering and then transferred into either 0.2 µM or 20 µM 10B (99.47% by weight) for 3 d. Regardless of the 10B treatments, significant levels of 11B were found in the phloem exudates (200–300 µM in 20 µM 10B and 430 µM in 0.2 µM 10B treatment) and xylem sap over the three days even without 11B supply to the root. In response to the 0.2 µM 10B treatment, the translocation of previously acquired 11B in the young (the uppermost three leaves), matured, and old leaves was enhanced, coinciding with the rise of 11B in the xylem sap (to >15 µM) and phloem exudates (430 µM). The evidence supports the hypothesis that previously acquired B in the shoot was recirculated to the root via the phloem, transferred into the xylem in the root, and transported in the xylem to the shoot. In addition, some previously acquired 11B in the leaves may have been translocated into the rapidly growing inflorescence. Phloem B transport resulted in the continued net increment of 11B in the flowers over 3 d without 11B supply. However, it is still uncertain whether the amount of B available for recirculation is adequate to support reproductive growth until seed maturation.
Key words: 10B, 11B, B recirculation, Lupinus albus L., phloem exudate, xylem sap
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Introduction |
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Boron mobility in higher plants lacking polyol-assisted phloem transport (Brown and Shelp, 1997) has been reported to vary from very low to moderate, across a range of studies, for example, in wheat (Huang et al., 2001), cotton (Oertli, 1994), canola (Stangoulis et al., 2001), broccoli, and lupin (Marentes et al., 1997; Shelp et al., 1998), and various trees (Lehto et al., 2004). In many crop species, boron (B) deposited in older leaves is not remobilized into new growth, even upon the interruption of external B supply to roots (Oertli, 1994). A very limited retranslocation of B into the young ear was reported in wheat, where the B that did enter the ear was predominantly dependent on long-distance transport in the xylem and concurrent B uptake from the roots (Huang et al., 2001). In a B-efficient canola cultivar, B was retranslocated out of mature leaves into young growing leaves when external B supply to the roots was interrupted for 5 d (Stangoulis et al., 2001).
Observations of high B concentrations in phloem exudates of lupin and broccoli have been attributed to an indirect mobilization of B through xylem to phloem transfer of the B concurrently taken up by the roots supplied with B (Marentes et al., 1997; Shelp et al., 1998). However, this xylem to phloem transfer of B acquired from concurrent root B uptake is unlikely to function in plants where the B supply is interrupted for days, such as in the canola study (Stangoulis et al., 2001). To maintain high levels of B in the phloem, and in the new growth when the external B supply in the rooting medium is interrupted, the B previously acquired in mature leaves would have to be retranslocated into the actively growing inflorescence through direct phloem loading or recirculation into roots via the phloem and transport in the xylem up into the growing shoot organs. Preliminary experiments revealed a high B concentration in phloem exudates collected from vegetative stems of lupin. However, there has been no direct evidence of B retranslocation out of mature leaves when the external B supply is severely limited, nor on the likely B retranslocation from shoot to root in species without the B–polyol complexes in their phloem to facilitate B mobility.
The objectives of the present study are to investigate if there are significant levels of B translocation out of leaves into the rapidly growing reproductive organs of lupin plants at the early stages of reproduction. To differentiate clearly ‘old’ from ‘new’ B uptake in the plants, the present study used the sequential treatments of the plants with enriched stable B isotopes, firstly 11B (99.34% by weight) followed by 10B (99.47% by weight). Solution culture was used in the present study as the actual B concentrations in solid rooting media (such as soil/sand) may be higher due to concentration effects from water loss, such as in the case of Marentes et al. (1997).
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Materials and methods |
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General
White lupin (Lupinus albus L. cv. Kiev Mutant) was used because of the ease of phloem exudate collection (Pate et al., 1974). Plants were germinated on paper towels moistened with 2 mM CaSO4 at 25 °C in the dark, before being transplanted into soil or nutrient solution. The full-strength basal nutrient solution contained (µM): KNO3, 2800; NH4NO3, 2000; Ca(NO3)2, 1600; MgSO4, 1000; KH2PO4, 60; FeEDTA, 40; NaCl, 8; ZnSO4, 2; MnSO4, 2; CuSO4, 0.5; CoSO4, 0.2; and Na2MoO4, 0.08. Solution pH was maintained in the range of 5.0–6.0. Analytical grade chemicals were used to make up stock solutions of these nutrients, to which was added acid-cleaned B-specific resin (IRA-743, Sigma Chemical Co.) (5 g moist resin l–1) to remove residual B present in the solution. Water used for making up all chemical and nutrient solutions was purified by passing through a column packed with B-specific resin. Boron isotopes were supplied as boric acid enriched with 10B (99.47% weight) or 11B (99.34% weight) (Eagle Picher, USA).
Experiment 1: B levels in xylem and phloem
The purpose of this experiment was to assess the levels of B in phloem exudate and xylem sap in B-adequate plants at early podding. Germinated seeds were planted in potting mix (composted pine bark and sand—3:1) fertilized with the complete nutrient solution described above which contained 10 µM B (boric acid) throughout the experiment. The plants were maintained under glasshouse conditions similar to those in Huang et al. (2001).
Experiment 2: B isotope labelling
To investigate the movement of B into the inflorescence, the plants were cultured in nutrient solution in the same manner as described in Huang et al. (2001), under glasshouse conditions: air temperature 18–34 °C, relative humidity 35–76%, photosynthetically active radiation (PAR) 425–1135 µmol m–2 s–1 (natural sunlight between 10.00 h and 16.00 h in the glasshouse). Briefly, after being acclimatized for 4 d in the 1/3 strength complete nutrient solution containing 20 µM 11B, four plants were transplanted into each of the 5.0 l plastic pots lined with a polythene bag, containing 4.5 l of full-strength nutrient solutions. Plants were progressively thinned to one per pot by the date of commencement of the 10B treatments. The plants were supplied with 20 µM 11B for 48 d after germination (DAG)—until early flowering (14 d after budding) when the plants had 
16 leaflets on the main stem. During this period, aliquots of nutrients and 11B were added to the solution of each pot based on a programmed nutrient addition as used in a previous study (Huang et al., 2001).
On 48 DAG (between 15.00 h and 17.00 h), the plants were transferred into fresh nutrient solutions containing full-strength basal nutrients and subjected to two levels of 10B treatments: 0.2 µM or 20 µM 10B. Before transfer into the treatments, the roots were rinsed with double-deionized water and then three changes of triple-deionized water (purified with acid-cleaned B-specific resin) containing 2 mM CaSO4, to remove surface-adsorbed B. Each B treatment comprised 12 replicate pots, which allowed at least three consecutive harvests of three pots of plants at each time.
Phloem exudate and xylem sap collection and plant sampling
Experiment 1
At the early podding stage (61 DAG), phloem exudates were collected at shallow incisions according to Atkins (1999), from the young (1–2 cm long) pod tips, the base of the inflorescence axis, the upper stem (between the second and fourth leaves of the main stem from the top), the mid stem (between the fifth and seventh leaves of the main stem from the top), and the basal stem (between the ninth and 12th leaves of the main stem from the top). Phloem exudate was collected into fine plastic tips over a 1 h period between, 9.00 h and 10.00 h in the glasshouse. The phloem exudate samples at each sampling position from plants of the same pot were pooled to form one sample and stored in a plastic microcentrifuge tube at –20 °C before analysis.
After phloem exudate collection, the shoot was cut at the hypocotyl, and root bleeding (xylem) sap was collected from the root stump. The droplets emerging from the root stump within the first 5 min were discarded (Jeschke et al., 1985). The collection of xylem sap lasted 1 h between 11.00 h and 12.00 h in the shade. The measured pH values in the phloem exudate and xylem sap were consistent with published values for these respective solutions (Pate, 1975), indicating minimal mixing of xylem and phloem fluids during collection.
B concentrations in the phloem exudate and xylem sap samples were determined by inductively coupled atomic emission spectrometry (ICP-AES, Varian).
Experiment 2
The collection of phloem exudate and xylem sap was carried out in the same manner as in Experiment 1 on 1, 2, and 3 d after the commencement (48 DAG) of 10B treatments. Phloem exudates were collected from the base of the inflorescence axis and from the mid-stem position (between the fourth and sixth leaves from the top of the main stem). Xylem sap was collected from decapitated root stumps after phloem exudate collection (Jeschke et al., 1985).
All sap samples were stored at –20 °C for total B analysis by ICP-AES to determine initially the total B concentration. A known volume of each sap sample was then diluted with 4 ml of 1% nitric acid containing beryllium (Be) (2 µg l–1) as an internal standard before inductively coupled plasma-mass spectrometry (ICP-MS; Perkin Elmer, Elan 6000, USA) analysis.
After phloem exudate collection, the shoots were divided into five strata: the inflorescence; young open leaves (YOL; the uppermost three leaves); recently matured leaves (RML; leaves 4, 5, and 6 from the top); matured leaves (ML; leaves 7, 8, and 9); and the rest of leaves (RL; below the 10th leaf of the main stem). The petioles were separated from the leaflets for plant analysis. The inflorescence was separated into flower buds and inflorescence stems (termed flower stalks).
Boron isotope analysis
Plant samples were dried at 70 °C to constant weight, finely ground, and then digested in concentrated nitric acid by means of a microwave digestion system (CEM Mars5, USA) (Huang et al., 2004). Sample digests were dissolved in B-free water. A known level of Be was added as an internal standard to all sap and digest samples and standard solutions for the analysis of 10B and 11B concentrations by ICP-MS.
Data analysis
The contents and concentrations of 10/11B in plant parts, phloem exudate, and xylem sap were compared using the analysis of variance for treatment effects. Differences among mean values were determined by Duncan's multiple range test (P 
0.05) with the SPSS statistical package (SPSS, version 6.0, SPSS Inc., 1993).
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Results |
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Boron in phloem exudate and xylem sap
Boron concentrations in the phloem exudates were substantially higher than those in the xylem sap (Figs 1–3). In Experiment 1, using soil culture containing adequate B supply, B concentrations were the highest (400–500 µM) in the phloem exudates collected from the stem segment immediately adjacent to the inflorescence (the oldest first order branch), and 25% lower (300–420 µM) in the upper, mid, and basal stem segments. In contrast, B concentrations in the xylem sap were only 25–35 µM (Fig. 1).
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In Experiment 2, after the interruption of 11B supply in the nutrient solution, the level of 11B concentrations in the phloem exudates remained relatively stable at 200–300 µM over the first 2 d, but significantly increased up to 430 µM in the 0.2 µM 10B treatment at day 3 (Fig. 2). The 10B concentration in the phloem exudates from the terminal inflorescence and mid-stem linearly increased with time in the 20 µM 10B treatment, up to 120–130 µM 10B while, surprisingly, the concentration of previously acquired 11B remained at 200–300 µM in the phloem exudates, even 3 d after removal of 11B from the nutrient solution (Fig. 2).
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The level of 10B in the xylem sap rapidly increased up to
15 µM in the 20 µM 10B treatment at day 1 after commencing the treatment and remained relatively stable for the rest of the experimental period (Fig. 3). Regardless of the 10B treatments, significant levels of 11B in the xylem sap were detected over the 3 d (Fig. 3). However, the previously acquired 11B concentration in the xylem sap remained as high as 10–15 µM in the xylem sap for 2 d, and increased to >15 µM at day 3 in the 0.2 µM 10B treatment. In contrast, the 11B concentration in the xylem sap significantly declined to 7–8 µM at day 3 in the 20 µM 10B treatment (Fig. 3).
Boron partitioning in reproductive organs and leaves
Despite the biomass increment, 11B concentrations in the inflorescence components (flower buds and stalks) remained relatively stable at 37–50 mg kg–1 dry weight, in 0.2 µM 10B over the 3 d (Fig. 4). In contrast, there was a linear increase in 10B concentration in the flower buds and stalks over the 3 d, in response to 20 µM 10B supply. Coincidently, 11B concentrations declined significantly over the 3 d in both 10B treatments in the old leaves (ML), but decreased by day 2 and remained stable at day 3 in the oldest group of leaves (RL) in which early senescence was observed (Table 1). Comparatively, the decrease in 11B concentrations in the old leaves (ML and RL) was larger at 0.2 µM 10B than that at 20 µM 10B treatment.
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Even though the supply of 11B to the roots was interrupted, there was a continual increase in 11B content over the 3 d in both the flower buds and stalks at 0.2 µM 10B (Fig. 5). The net increment of 11B in the inflorescence at 20 µM 10B was smaller than that at 0.2 µM 10B (Fig. 5). The 10B contents in the flower buds and stalks of the inflorescence increased linearly with time in the 20 µM 10B plants, but only a small change occurred in the 0.2 µM 10B treatment (Fig. 5).
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The effects of the 10B treatments on leaf B content were observed mostly in the leaf blades (Fig. 6a, b). At both 0.2 µM and 20 µM 10B, the 11B content in YOL and RML blades rapidly declined over the 3 d. Regardless of the B treatments, the 11B content in the petiole of YOL remained relatively stable (Fig. 6a), but decreased in the RML petioles (Fig. 6b). The 10B content at 20 µM 10B increased linearly with time in the YOL and RML blades and petioles (Fig. 6a. b).
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Discussion |
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The present study is the first to demonstrate the retranslocation of B from the shoot to the root in crop species without a polyol transport mechanism, and B retranslocation out of mature leaves into actively growing reproductive organs via phloem and xylem. This is supported by several pieces of evidence, notably: (i) the presence of significant levels of 11B in the xylem sap 3 d after the removal of 11B supply in the nutrient solution. Indeed the 11B increased at day 3 in the 0.2 µM 10B treatment, but decreased in the 20 µM 10B treatment. (ii) The presence of high levels of 11B in the phloem exudates collected from the base of the terminal inflorescence and the mid-stem positions, which increased up to 430 µM in the 0.2 µM 10B treatment; and (iii) continued net transport of 11B into the flowers (particularly at 0.2 µM 10B) coinciding with the rapid loss of 11B out of the leaf blades of mature and old leaves, which resulted in stable 11B concentrations in the flowers despite their increase in dry matter and the termination of external 11B supply for 3 d.
It is highly likely that B in the shoot was recirculated into the root via the phloem, transferred into the xylem, and carried upwards in the xylem sap, because there were 7–17 µM 11B present in the xylem sap over the 3 d after withdrawing 11B supply in the external nutrient solution. The source of this 11B in the xylem sap cannot be quantitatively accounted for by the residual effects of water-soluble B stored in root cells (Dannel et al., 2000). A substantial proportion of B was detected in the symplastic pool of root cells of sunflower plants, when provided with 50 µM B, but not in those with 1 µM B (Dannel et al., 2000). In sunflower, the newly absorbed B accounted for >80% of the B in xylem sap when supplied with 10 µM B (Matoh and Ochiai, 2005). However, the ‘old’ B (11B) in the xylem sap still accounted for almost 40% of the xylem B in the present study. If only root uptake of external B occurred without recirculation of 11B from the shoot to the root, the high levels of 11B would not have been detected in the xylem sap of the lupin plants. The recycled 11B in the phloem may have been efficiently loaded into the xylem in the roots by the B transporters in the root pericycle cells (Takano et al., 2002).
In addition, the retranslocation of B from the shoot to the root seemed to be regulated by the concurrent B supply to the root, as the concentration of the previously absorbed 11B significantly increased in the xylem sap at day 3 in the 0.2 µM 10B treatment, while it declined in the xylem sap at 20 µM 10B. This suggests that increased recirculation of 11B from shoot to root, coinciding with increased translocation of 11B out of mature and old leaves into the phloem at 0.2 µM 10B, was a response to B demand in the inflorescence that was the most actively growing shoot organ at the time. In contrast, a decreased recirculation of 11B from shoot to root may have occurred when supplied with adequate B in the nutrient solution (20 µM 10B) due to the direct transport of concurrent 10B uptake into the inflorescence.
The possibility of B retranslocation in lupin plants is further supported by the substantial levels of 11B in the phloem exudates collected from both the mid-stem and the base of the terminal inflorescence. In particular, there was a trend for an increase in 11B concentration over the first 2 d (from 250 µM to 300 µM) and a significant increase at day 3 (430 µM) in the 0.2 µM 10B treatment. This coincided with the rise of 11B in the xylem sap at day 3 in the plants treated with 0.2 µM 10B. Increased 11B recirculation into the roots and transport upwards to the shoot would be needed to meet the demand of the rapidly growing sink—the inflorescence—when the roots were not supplied with adequate B. The circulation of K+ and Mg2+ has been reported previously in white lupin: 76% or 87% of the phloem-borne K+ and Mg2+ were circulated into the root and re-entered the xylem (Jeschke et al., 1985), and then were preferentially transported into the inflorescence (Jeschke et al., 1987). The preferential partitioning of recirculated B into growing tissues may have occurred as net 11B content actually declined in the top leaves (YOL group) while it increased in the rapidly growing inflorescence.
The 11B in the phloem exudates was most likely to have come from that translocated out of young, mature, and old leaves (YOL, RML, ML, and RL), but the proportion was not quantitatively determined. There may be some initial water-soluble 11B stored in stem tissues (Matoh and Ochiai, 2005), but this pool of B would have been depleted quickly without continual supply of 11B. Decreased B concentrations in mature/old leaves were observed in canola plants with interrupted B supply, particularly in B-efficient cultivars (Stangoulis et al., 2001). The retranslocation of 11B out of the mature leaves was enhanced when external 10B supply was very low (0.2 µM), coinciding with the rise of 11B concentration in the phloem sap at day 3. In contrast, the newly absorbed 10B in 20 µM 10B plants contributed <30% towards the total B in the phloem exudates.
B concentrations (300–500 µM) in the phloem exudates were substantially higher than those in the xylem sap (10–30 µM) within the 3 d period, regardless of B supply to the roots. By calculation, the ratios of total B concentrations in the phloem exudates to those in the xylem sap were 13 and 18 in 20 µM and 0.2 µM 10B treatments, respectively. In comparison, the ratios of K (phloem-mobile) and Ca (phloem-immobile) concentrations in the phloem exudates from lupin (L. albus) fruit tips to those in the xylem sap are 17 and 1.2, respectively (Pate, 1975). Marentes et al. (1997) found that the ratios of B concentrations in the phloem exudates (380–30 µM) to those in the xylem sap ranged from 4 to 23 in lupin plants.
Collectively, the evidence presented here strongly suggests that the previously acquired 11B in the shoot can be recirculated to the root via the phloem within the short term. This is supported by the significant levels of 11B in the xylem sap over the 3 d following interruption of 11B supply to roots, coinciding with the pattern and levels of 11B changes in the phloem exudates collected from the upper stem (the base of the terminal inflorescence) and the mid-stem. The results support the argument that previously acquired B can be translocated out of mature/old leaves of the lupin plant into the rapidly growing inflorescence via recirculation in the phloem to the root, followed by xylem transport to the shoot. Nevertheless, uncertainty remains regarding the amount of B available for recirculation and whether it would be adequate to support reproductive growth during a prolonged period of low B uptake by roots, since a significant proportion of B in vegetative tissues is bound in cell walls and not available for retranslocation, especially in low B plants (Matoh and Ochiai, 2005). Further investigation using techniques such as split-root studies with B isotopes are necessary to quantify the amount of B in the shoot that is recirculated to the root and from there to rapidly growing vegetative and reproductive tissues.
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Acknowledgements |
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We thank Dr Brenda Rohl and Mr Michael Smirk at the University of Western Australia for their invaluable help in the analysis of B isotopes by the ICP-MS (PE, Elan 6000). Ms Jinjuan Zhang provided assistance in the glasshouse experiments. The present study was funded by an Australian Research Council small grant at Murdoch University.
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