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.

Highlights

  • Boron (B) is an essential micronutrient required in major physiological functions for the normal growth and development of higher plants [1]

  • As boron accumulates in leaves as they age, B toxicity symptoms usually appear on older leaves, first as leaf tip and margin yellowing or mottling, with a brownish burnt appearance, which ends in premature fall at high concentration levels [3]

  • Seeds of Citrus macrophylla W. were sterilised for 5 min in a 2% v/v commercial bleach (0.5 M NaClO) solution prior to seed coat removal, rinsed 3 times with sterilized deionized water and transferred to a media containing distilled water with 0.4% agar added (Difco Bacto)

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Summary

Introduction

Boron (B) is an essential micronutrient required in major physiological functions for the normal growth and development of higher plants [1] This element participates in cell wall structure formation through the borate-diol bonding of two rhamnogalacturonan II molecules. As boron accumulates in leaves as they age, B toxicity symptoms usually appear on older leaves, first as leaf tip and margin yellowing or mottling, with a brownish burnt appearance, which ends in premature fall at high concentration levels [3]. It can occasionally appear resinous gum spots on the undersides of leaves and it is as well associated with a shortened distance between leaf nodes. Severe B toxicity may induce loss of vigour, shorter branches, and even twig dieback

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