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Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

Martínez-Cuenca MR, Martínez-Alcántara B, Quiñones A, Ruiz M, Iglesias DJ, Primo-Millo E, Forner-Giner MÁ - PLoS ONE (2015)

Bottom Line: Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones.Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%.In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Citriculture and Vegetal Production, Valencian Institute of Agrarian Research, Moncada, Valencia, Spain.

ABSTRACT
This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

No MeSH data available.


Related in: MedlinePlus

Boron concentration ([Bf], μg g-1 DW) and boron content (Bf, μg) in (A) soluble in water, (B) soluble in organic solvents and (C) insoluble fractions measured in roots and leaves of Citrus macrophylla seedlings grown for 25 days in B-normal (50 μM, Ct) and B-toxic (400 μM, +B) nutrient solutions.Values are the means ± SE of three independent experiments (n = 3). For a comparison of means, an ANOVA followed by the LSD test, calculated at the 95% confidence level, was performed. Different letters indicate significant differences for each parameter and within each plant organ (P <0.05).
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pone.0134372.g006: Boron concentration ([Bf], μg g-1 DW) and boron content (Bf, μg) in (A) soluble in water, (B) soluble in organic solvents and (C) insoluble fractions measured in roots and leaves of Citrus macrophylla seedlings grown for 25 days in B-normal (50 μM, Ct) and B-toxic (400 μM, +B) nutrient solutions.Values are the means ± SE of three independent experiments (n = 3). For a comparison of means, an ANOVA followed by the LSD test, calculated at the 95% confidence level, was performed. Different letters indicate significant differences for each parameter and within each plant organ (P <0.05).

Mentions: Plants contain boron in both soluble and insoluble forms [4]. According to Liu et al. [4], B extracted in water is likely to be localized in the plant free space, or apoplast, while the remaining soluble B, extracted using organic solvents, belongs to B located inside cells or the protoplast and linked to sugars, alcohols and polyhydroxycarbolates [49]. Both fractions represent not only mobile B, but also the only form in plant tissues that can be re-translocated in the phloem [5]. Finally, the B-insoluble fraction represents the B bound to cell walls linked to peptic polysaccharides [25]. Fig 6 shows the partitioning of B in soluble (water and organic solvents) and insoluble forms in roots and leaves. Insoluble B (Fig 6C) was mostly the main fraction of B in both Ct and +B seedlings (more than 65.8% of total B), followed by the water-soluble fraction (ranging from 31.8% to 9% of total B; Fig 6A), and lastly by soluble B in the organic solvents fraction (below 7.6%; Fig 6B). However, the seedlings grown under B-excess conditions increased the concentration and total amount of B located in cell walls and in the soluble fraction from the apoplast when compared with Ct plants, which is likely in equilibrium with the external medium. Nevertheless, the concentration and the amount of B extracted with organic solvents, which is attributed to cytoplasmic B, was similar in the organs from both +B and Ct seedlings. As boron exerts its toxicity inside rather than outside cells, the capability to retain increased B amounts linked to cell walls under B-excess conditions enables this genotype to block excess B in an insoluble form, thus preventing its entry to the cytoplasm to protect cells from B toxicity. Accordingly, Nozawa et al. [50] found a negative correlation between the B concentration inside yeast cells and the degree of tolerance to a high external B level, and concluded that cells with lower protoplasmic B were more tolerant to high B levels. Other authors have also observed a lower B concentration in leaf protoplasts of B-toxicity-tolerant cultivars of barley and wheat, which suggests different B partitioning within the shoot as a tolerance mechanism [42]. Hence it is likely that insolubilisation of B outside the cytoplasm might constitute another mechanism of B toxicity tolerance.


Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

Martínez-Cuenca MR, Martínez-Alcántara B, Quiñones A, Ruiz M, Iglesias DJ, Primo-Millo E, Forner-Giner MÁ - PLoS ONE (2015)

Boron concentration ([Bf], μg g-1 DW) and boron content (Bf, μg) in (A) soluble in water, (B) soluble in organic solvents and (C) insoluble fractions measured in roots and leaves of Citrus macrophylla seedlings grown for 25 days in B-normal (50 μM, Ct) and B-toxic (400 μM, +B) nutrient solutions.Values are the means ± SE of three independent experiments (n = 3). For a comparison of means, an ANOVA followed by the LSD test, calculated at the 95% confidence level, was performed. Different letters indicate significant differences for each parameter and within each plant organ (P <0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520451&req=5

pone.0134372.g006: Boron concentration ([Bf], μg g-1 DW) and boron content (Bf, μg) in (A) soluble in water, (B) soluble in organic solvents and (C) insoluble fractions measured in roots and leaves of Citrus macrophylla seedlings grown for 25 days in B-normal (50 μM, Ct) and B-toxic (400 μM, +B) nutrient solutions.Values are the means ± SE of three independent experiments (n = 3). For a comparison of means, an ANOVA followed by the LSD test, calculated at the 95% confidence level, was performed. Different letters indicate significant differences for each parameter and within each plant organ (P <0.05).
Mentions: Plants contain boron in both soluble and insoluble forms [4]. According to Liu et al. [4], B extracted in water is likely to be localized in the plant free space, or apoplast, while the remaining soluble B, extracted using organic solvents, belongs to B located inside cells or the protoplast and linked to sugars, alcohols and polyhydroxycarbolates [49]. Both fractions represent not only mobile B, but also the only form in plant tissues that can be re-translocated in the phloem [5]. Finally, the B-insoluble fraction represents the B bound to cell walls linked to peptic polysaccharides [25]. Fig 6 shows the partitioning of B in soluble (water and organic solvents) and insoluble forms in roots and leaves. Insoluble B (Fig 6C) was mostly the main fraction of B in both Ct and +B seedlings (more than 65.8% of total B), followed by the water-soluble fraction (ranging from 31.8% to 9% of total B; Fig 6A), and lastly by soluble B in the organic solvents fraction (below 7.6%; Fig 6B). However, the seedlings grown under B-excess conditions increased the concentration and total amount of B located in cell walls and in the soluble fraction from the apoplast when compared with Ct plants, which is likely in equilibrium with the external medium. Nevertheless, the concentration and the amount of B extracted with organic solvents, which is attributed to cytoplasmic B, was similar in the organs from both +B and Ct seedlings. As boron exerts its toxicity inside rather than outside cells, the capability to retain increased B amounts linked to cell walls under B-excess conditions enables this genotype to block excess B in an insoluble form, thus preventing its entry to the cytoplasm to protect cells from B toxicity. Accordingly, Nozawa et al. [50] found a negative correlation between the B concentration inside yeast cells and the degree of tolerance to a high external B level, and concluded that cells with lower protoplasmic B were more tolerant to high B levels. Other authors have also observed a lower B concentration in leaf protoplasts of B-toxicity-tolerant cultivars of barley and wheat, which suggests different B partitioning within the shoot as a tolerance mechanism [42]. Hence it is likely that insolubilisation of B outside the cytoplasm might constitute another mechanism of B toxicity tolerance.

Bottom Line: Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones.Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%.In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Citriculture and Vegetal Production, Valencian Institute of Agrarian Research, Moncada, Valencia, Spain.

ABSTRACT
This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

No MeSH data available.


Related in: MedlinePlus