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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 TIP5, V-PPiase and V-ATPase A 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.g005: Relative expression of TIP5, V-PPiase and V-ATPase A 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: In this study, boron excess promoted the expression level of putative gene TIP5, and the number of transcripts increased in the leaves and roots of +B seedlings by 3.3- and 2.4-fold, respectively, in comparison with Ct ones (Fig 5). Aquaporin TIP5 has been related to a decreasing B concentration in the cytoplasm through its transport across the tonoplast membrane and to compartmenting it into the vacuole complexed with B-polyols [6]. Thus at high B levels in culture media, the up-regulation of putative gene TIP5 might be responsible for the intracellular B allocation, which could contribute to B tolerance in Citrus. Pang et al. [16] pointed out this capacity in A. thaliana, and provided evidence to support that this gene may be involved in B transport to vacuoles. Vacuolar sequestration has also been widely assumed to play a fundamental role in plant tolerance to excess micronutrients and heavy metals [46].


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 TIP5, V-PPiase and V-ATPase A 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.g005: Relative expression of TIP5, V-PPiase and V-ATPase A 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: In this study, boron excess promoted the expression level of putative gene TIP5, and the number of transcripts increased in the leaves and roots of +B seedlings by 3.3- and 2.4-fold, respectively, in comparison with Ct ones (Fig 5). Aquaporin TIP5 has been related to a decreasing B concentration in the cytoplasm through its transport across the tonoplast membrane and to compartmenting it into the vacuole complexed with B-polyols [6]. Thus at high B levels in culture media, the up-regulation of putative gene TIP5 might be responsible for the intracellular B allocation, which could contribute to B tolerance in Citrus. Pang et al. [16] pointed out this capacity in A. thaliana, and provided evidence to support that this gene may be involved in B transport to vacuoles. Vacuolar sequestration has also been widely assumed to play a fundamental role in plant tolerance to excess micronutrients and heavy metals [46].

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