Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions

Abstract

Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological "tools" to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.

Keywords: Brassica napus; boron; boron deficiency tolerance; boron efficiency; phenotyping; rapeseed.

FIGURE 1

(A) Boxplots showing the distribution of boron (B) efficiency parameters recorded for 234 spring-type Brassica napus accessions. Left panel: length of leaf 1 ( = L1) in millimeters. Middle panel: hypocotyl length in millimeters. Right panel: shoot dry weight (DW) in milligrams. Crosses inside boxes show average values, horizontal lines within boxes mark the median value. Each boxplot consists of 234 parameter average values. Each parameter average value derives from n ≥ 10 plants. Significance between treatments (B 0/B 1) was calculated using t-test: ^∗∗∗p < 0.001. (B) Exemplary pictures of plants of B-inefficient B. napus cultivars grown on B-deficient zerosoil-substrate and displaying B deficiency phenotypes such as arrested shoot apical meristem growth and leaf- and vasculature deformations. (C) Origin-dependent distribution of B efficiency indices (BEIs), calculated from the B 0/B 1 ratios of the B-related parameters L1 length and DW. Crosses inside boxes show average values, horizontal lines inside boxes mark the median value. Each boxplot consists of the BEI average values from n ≥ 10 plants. (D) Phenotypes (top panel), growth parameters (lower panel), and B concentrations (lower panel) in leaves of 25-day-old plants of B-inefficient (CR2262 = IE) and B-efficient (CR2267 = E) B. napus cultivars growing on zerosoil-substrate containing sufficient (B 1) or deficient (B 0) B levels. Growth parameters and B concentrations are means ± SD from n = 12 plants. Significance was calculated using t-test: ^∗∗∗p < 0.001.

FIGURE 2

B-dependent growth behavior and analyses of B. napus cultivars grown in a LemnaTec cultivation system. (A) Left panel: Exemplary pictures of selected cultivars [IE (CR2262) = B deficiency-sensitive cultivar; E (CR2267) = B deficiency-tolerant cultivar] growing in B-deficient (-boron) or B-sufficient (+boron) zerosoil-substrate taken at days 5, 9, or 12 after sowing (DAS). Right panel: segmented plant images which are used for image analysis. (B) Principal component analysis (PCA) for 13 rapeseed cultivars based on recorded imaging-based growth parameters. Symbols indicate different B soil substrate conditions [ = B-deficient (-), = B-sufficient (+) or = B-surplus (++) conditions].

FIGURE 3

(A) B-dependent relative growth rates (RGR) based on the measurement of the leaf surface area (border length) of different B-efficient (E = CR2267, E2 = CR2280), moderate (Darmor; D), or inefficient (IE = CR2262, IE2 = CR3153) B. napus cultivars recorded in the LemnaTec facility at early (4–7 DAS) (A), or later (10–13 DAS) (B) growth stages. Different letters indicate significantly different RGRs. Red and green crosses mark outliers outside the 5 and 2.5% range of the standard deviation of the value distribution, respectively. (C) Measurements of aboveground physical growth parameters of B. napus plants grown in the automated phenotyping facility. DW per plant part [cotyledons, leaf 1 (L1), leaf 3 (L3), remaining leaves (RL), stems] of indicated B. napus cultivars grown under B-deficient (–), B-sufficient (+), or B-surplus (++) conditions. DW values are means ± SD from n = 8 plants.

FIGURE 4

Macro- and microelement concentrations of different B. napus plant parts. HR–ICP–MS-based element analysis of different aerial B. napus plant parts of two B deficiency tolerant (E = CR2267, E2 = CR2280) and two B deficiency sensitive (IE = CR2262, IE2 = CR3153) cultivars grown in an automated phenotyping facility under B-deficient (–), B-sufficient (+), or B-surplus (++) conditions. Bars represent concentrations of specific plant parts or organs (cot, cotyledon; L1, first vegetative leaf; L3, third vegetative leaf; RL, remaining leaves; stem, remaining stem tissue without leaves). Values show mean element concentrations in ng or μg per mg DW from n = 3 biological replicates ± SE.

FIGURE 5

Soluble sugar concentrations [Glc, glucose (A); Frc, fructose (B); and Suc, sucrose (C)] in cotyledons from B. napus plants of B-deficiency tolerant (E = CR2267) and B-deficiency sensitive (IE = CR2262) cultivars grown in an automated phenotyping facility under B-deficient (–), B-sufficient (+), or B-surplus (++) conditions. Values are means ± SD from n = 4 plants. Significance was calculated using t-test: ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05.

FIGURE 6

Analysis of B-dependent Brassica napus root growth. (A) Root cessation assays of the identified B-efficient (E = CR2267) and B-inefficient (IE = CR2262) B. napus cultivars. Plants were transferred from B-sufficient (= control = 5 μM B) to control or B-deficient ( = 0.01 μM B) conditions. IE-, but not E-plants showed growth arrest of root and shoot tissues after transfer to B-deficient conditions. (B) Primary root (PR) growth depth per day was recorded for all cultivars and acceleration curves plotted. Acceleration curves for the IE (CR2262) and the E (CR2267) cultivar after transfer to control or B-deficient conditions are shown. Values represent the average ± SD of three replicates with five plants each (n = 15). (C) Slope coefficients calculated from the acceleration curves of 229 spring-type B. napus accessions plotted against their BEIs. CR2267 (E) and CR2280 (E2) exhibited both BEIs greater 0.7 and a positive slope coefficient of PR growth gain. Cultivar CR2285 with the highest BEI of all tested accessions exhibited a negative slope coefficient. Slope coefficient values of all spring-type rapeseed cultivars are listed in Supplementary Data Sheet S1.

FIGURE 7

B seed concentrations and contents. B seed concentration (A), 1000-kernel weights (B), and B seed contents (C) of selected B-efficient (E = CR2267, plain blue; E2 = CR2280, vertically dashed blue; E3 = CR2285, yellow) and inefficient (IE = CR2262, plain gray; IE2 = CR3153, diagonally dashed gray; IE3 = CR3035, horizontally dashed green) cultivars. (D) B efficiency does not correlate with B seed contents. B seed contents of 20 cultivars plotted against their BEIs. Values in A and (C) represent means from n = 3 measurements ± SE.

FIGURE 8

Root system architecture parameters and shoot and root fresh weights (FW) from B-efficient (E = CR2267) and B-inefficient (IE = CR2262) Brassica napus cultivars grown in hydroponic culture medium with different nutrient deficiencies. (A) Representative pictures of seedlings grown under indicated nutrient deficiencies for 7–8 days. (B) Shoot (left panel) and root (right panel) FW from E and IE plants grown under above conditions. Values represent averages ± SD from n ≥ 6 plants. Asterisks indicate significant differences (^∗p < 0.05, t-test) of the recorded parameter in comparison to the control (“none”) condition. Bars = 1 cm.

FIGURE 9

Boron toxicity growth assays of B-efficient (E = CR2267, blue chart bars) and B-inefficient (IE = CR2262, gray chart bars) B. napus cultivars on zerosoil-substrate with toxic B levels. Values represent averages ± SE from shoot material (n ≥ 17). Asterisks indicate significant differences (^∗∗p < 0.01, ^∗∗∗p < 0.001, t-test) of the recorded parameter between the two cultivars in the same growth condition (Ns = not significant). (A) Shoot FW in grams, (B) shoot DW in grams, and (C) water content in % are displayed.

FIGURE 10

Boron (B)- and cultivar-dependent B accumulation and growth-reflecting parameters during early vegetative development (until 25 days after germination; DAG) of B. napus plants from B-inefficient (IE = CR2262, gray dots) and B-efficient (E = CR2267, blue squares) cultivars growing on B-deficient (B 0), B-adequate (B 1), or B-surplus (B 2) zerosoil-substrate conditions. (A) Shoot FW in mg per plant, (B) shoot DW in mg per plant, (C) water content in %, and shoot B concentrations are displayed. Measurements are means ± SD from n = 4 biological replicates containing each six to eight plants.

FIGURE 11

Growth of B deficiency tolerant and sensitive B. napus cultivars under near-field conditions with sufficient B supply (0.39 mg B/kg soil; hot water extract). Exemplary pictures of flowering plants of the IE (CR2262) (A) or E (CR2267) (B) cultivars grown under near-field conditions. (C) B concentrations of indicated plant tissues from IE (gray chart bars) and E (blue chart bars) cultivars grown under near-field conditions.