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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

Relative expression of CmBOR1 and BOR4 genes measured by real-time RT-PCR analysis, 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 95% confidence level, was performed. Different letters in the same organ indicate significant differences between treatments (P <0.05).
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pone.0134372.g004: Relative expression of CmBOR1 and BOR4 genes measured by real-time RT-PCR analysis, 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 95% confidence level, was performed. Different letters in the same organ indicate significant differences between treatments (P <0.05).

Mentions: We also monitored the expression level of CmBOR1 gene (Fig 4), which has been reported to modulate B transport in plants due to its role in loading B into the xylem, and therefore to translocate B from roots to shoots [7,12]. However, no differences in the CmBOR1 expression level in the leaves and roots of Ct and +B seedlings were found. This is in accordance with the high capacity of Citrus to accumulate B in their leaves at high external B levels, which suggests that citrus plants have an effective system for B transport from roots to leaves, even under B-excess conditions. Therefore, the presented data indicate that B toxicity tolerance is not linked to a repressible B transport mechanism. To support this, it has been reported that the CmBOR1 transcript level is not affected by high B supply and the activity of this gene does not prevent high B accumulation in citrus leaves [11]. In Oryza sativa, OsBOR1 loads B into the xylem and also participates in the absorption of this element in roots [42]. In Brassica napus, BnBOR1;1c and BnBOR1;2a are up-regulated in roots under low B stress, but no differences in their expression have been found between B-efficient and B-inefficient cultivars in low or normal B environments [13]. However, some evidence has indicated that the removal of the BOR1 protein from membranes and its rapid degradation through the endocytic pathway may occur in response to high B levels in A. thaliana [43]. This suggests that this mechanism in other species could be involved in the regulation of B transport from roots to shoots at high B levels.


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)

Relative expression of CmBOR1 and BOR4 genes measured by real-time RT-PCR analysis, 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 95% confidence level, was performed. Different letters in the same organ indicate significant differences between treatments (P <0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134372.g004: Relative expression of CmBOR1 and BOR4 genes measured by real-time RT-PCR analysis, 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 95% confidence level, was performed. Different letters in the same organ indicate significant differences between treatments (P <0.05).
Mentions: We also monitored the expression level of CmBOR1 gene (Fig 4), which has been reported to modulate B transport in plants due to its role in loading B into the xylem, and therefore to translocate B from roots to shoots [7,12]. However, no differences in the CmBOR1 expression level in the leaves and roots of Ct and +B seedlings were found. This is in accordance with the high capacity of Citrus to accumulate B in their leaves at high external B levels, which suggests that citrus plants have an effective system for B transport from roots to leaves, even under B-excess conditions. Therefore, the presented data indicate that B toxicity tolerance is not linked to a repressible B transport mechanism. To support this, it has been reported that the CmBOR1 transcript level is not affected by high B supply and the activity of this gene does not prevent high B accumulation in citrus leaves [11]. In Oryza sativa, OsBOR1 loads B into the xylem and also participates in the absorption of this element in roots [42]. In Brassica napus, BnBOR1;1c and BnBOR1;2a are up-regulated in roots under low B stress, but no differences in their expression have been found between B-efficient and B-inefficient cultivars in low or normal B environments [13]. However, some evidence has indicated that the removal of the BOR1 protein from membranes and its rapid degradation through the endocytic pathway may occur in response to high B levels in A. thaliana [43]. This suggests that this mechanism in other species could be involved in the regulation of B transport from roots to shoots at high B levels.

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