Tetraploidy Enhances Boron-Excess Tolerance in Carrizo Citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.)

Abstract

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport.

Highlights: Tetraploidy enhances B excess tolerance in citrange CarrizoExpression of NIP5 and BOR1 transporters and cell wall-bounded B are similar between ploidiesB tolerance is attributed to root anatomical modifications induced by genome duplicationThe rootstock 4x citrange carrizo may prevent citrus trees from B excess.

Keywords: 4x; BOR1; Casparian strip; NIP5; citrus; poliploidy; rootstock.

FIGURE 1

Boron (B) concentration (μg g^-1 DW) measured in the 2x and 4x CC seedlings grown in vitro (Experiment 1) for 45 days in nutrient media supplemented with 50 μM (Ct) or 400 μM (+B) H₃BO₃. Values are the means ± SE of six plants (n = 6). Ploidy (P) and treatment (T) effects tested by two-way ANOVA are indicated as follows: ^∗P < 0.05; ^∗∗∗P < 0.001; ns, not significant; nc, not considered. Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.

FIGURE 2

Boron (B) uptake rate (μmol g^-1 root DW day^-1) and B transport rate (μmol g^-1 root DW day^-1) measured in the 2x and 4x CC seedlings grown in vitro (Experiment 1) for 45 days in nutrient media supplemented with 50 μM (Ct) or 400 μM (+B) H₃BO₃. Values are the means ± SE of six plants (n = 6). Ploidy (P) and treatment (T) effects tested by two-way ANOVA are indicated as follows: ^∗∗∗P < 0.001. Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.

FIGURE 3

Relative gene expression level of (A)NIP5 and (B) BOR1 genes measured by qRT-PCR analysis in roots of the 2x and 4x CC seedlings grown in vitro (Experiment 1) for 45 days in nutrient media supplemented with 50 μM (Ct) or 400 μM (+B) H₃BO₃. Values are the means ± SE of nine plants (n = 9). Ploidy (P) and treatment (T) effects tested by two-way ANOVA are indicated as follows: ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns, not significant; nc, not considered. Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.

FIGURE 4

Free-hand cross-sections of the roots of (A) the 2x and (B) 4x CC seedlings grown in vitro, taken at 9–12 mm from the root apex, cleared in chloral hydrate, stained with berberine hemisulfate, post-stained with aniline blue and visualized under the UV light of a fluorescence microscope. Casparian strip is clearly differentiated in the 4x root, while is slightly visible in the 2x root. PXy, Protoxylem; CS, Casparian strip; Ex, Exodermis.

FIGURE 5

Number of root tips in 6-month-old 2x and 4x CC seedlings grown in greenhouse conditions. Values are the means ± SE of three plants (n = 3). Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.

FIGURE 6

(A) Boron concentration (μg g^-1 DW) in fully expanded leaves from the 6-month-old 2x and 4x CC seedlings grown in greenhouse conditions (Experiment 2) for 5 weeks in nutrient solutions to which 50, 200, 400, or 800 μM H₃BO₃ were added. (B) Boron concentration (μg g^-1 DW) in fully expanded leaves from 6-month-old 2x and 4x CC seedlings grown in glasshouse for 5 weeks in nutrient solutions to which 50 μM (Ct) or 800 μM (+B) H₃BO₃ were added. Values are the means ± SE of six plants (n = 6). Ploidy (P) and treatment (T) effects tested by two-way ANOVA are indicated as follows ^∗∗∗P < 0.001. Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.

FIGURE 7

Visual symptoms of B toxicity in leaves from the 6-month-old 2x and 4x CC seedlings grown in greenhouse conditions (Experiment 2) for 10 weeks in nutrient solutions added with 50 μM (Ct) and 800 μM H₃BO₃ (+B).

FIGURE 8

(A) Leaf damage (%) and (B) leaf abscission (%) in the 6-month-old 2x and 4x CC seedlings grown in greenhouse conditions (Experiment 2) for 10 weeks in nutrient solutions to which 800 μM (+B) H₃BO₃ was added. Values are the means ± SE of six plants (n = 6). Ploidy (P) and treatment (T) effects tested by two-way ANOVA are indicated as follows: ^∗P < 0.05; ^∗∗∗P < 0.001. Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.

FIGURE 9

Boron (B) concentration (μg g^-1 DW) in 9-month-old spring flush leaves of 5-year-old VL orange trees grafted onto the 2x and 4x CC rootstocks grown under field conditions (Experiment 3). Ploidy (P) and year (Y) effects tested by two-way ANOVA are indicated as ^∗∗∗P < 0.001. Values are the means ± SE of six leaf samples pooled from the four trees of each block. Different letters indicate significant differences at P < 0.05 using Duncan’s multiple range test.