Effects of Phloretin on Seedling Growth and Histochemical Distribution of Phenols, Polysaccharides and Lipids in Capsella bursa-pastoris (L.) Medik

Abstract

The present study evaluates the phytotoxic effects of phloretin, a prevalent secondary metabolite of apple trees, on the broadleaf weed Capsella bursa-pastoris (L.) Medik. known for its resistant myxospermous seeds that form a long-lasting soil bank. The results indicate a significant, dose-dependent inhibitory effect of phloretin on the growth and morphological parameters of weed seedlings grown in vitro. Although the applied phloretin concentrations (250-1000 µM) were not lethal to the C. bursa-pastoris seedlings after two weeks, the metabolism of the seedlings was impaired, resulting in an accumulation of lipid droplets in the root tips and root hairs. Histochemical analysis shows deposits of phenols in the root epidermal cells, which are probably aggregates of phloretin or its metabolic derivatives. The accumulation of pectin in the cell walls of root border cells in phloretin-treated seedlings indicates an attempt to reduce the uptake of phloretin and reduce its concentration in the cells. Inhibition of shoot growth associated with chlorosis and reduced photosynthetic pigment content is a consequence of seedling exposure to phloretin. This study provides a basis for further evaluation of phloretin as a new bioherbicidal compound and for elucidating the mechanism underlying its phytotoxic activity.

Keywords: Malus; allelopathy; apple; chlorophyll; dihydrochalcones; pectin; phytotoxic effect; starch.

Figure 1

Germination of C. bursa-pastoris seeds on ½MS medium with different concentrations of phloretin (0, 250, 500 and 1000 μM) in in vitro culture. (A) Percentage of germination during 14-day period; (B) germination index at 14th day of seedling growth. Values represent means ± SE (standard error) of 30 seeds per each treatment repeated three times (n = 90). Values denoted by the same letter of the same color are not significantly different at p ≤ 0.05 per Fisher’s least significant difference (LSD) test.

Figure 2

C. bursa-pastoris seedlings after 14 days of vertical growth in in vitro culture on ½MS nutrient medium with different concentrations of phloretin. Seedlings on the medium (A) without phloretin—control; (B) with 250 μM phloretin; (C) 500 μM phloretin; and (D) 1000 μM phloretin. Bar = 10 mm.

Figure 3

Phloretin effects on C. bursa-pastoris seedlings during 14 days of vertical growth in in vitro culture: (A) root length (mm); (B) number of lateral roots per seedling; (C) shoot length (mm); and (D) number of leaves per seedling. Values represent means ± SE of 30 seedlings per each treatment repeated three times (n = 90). Values denoted by the same letter of the same color are not significantly different at p ≤ 0.05 per Fisher’s least significant difference (LSD) test.

Figure 4

Dose-dependent curves of phloretin-induced root growth inhibition of C. bursa-pastoris seedlings during 14 days of vertical growth in in vitro culture.

Figure 5

Phloretin effects on C. bursa-pastoris seedlings. (A) Fresh weight and (B) vigor index during 14 days of seedling growth in in vitro culture. Values represent means ± SE of 30 seedlings per each treatment repeated three times (n = 90). Values denoted by the same letter of the same color are not significantly different at p ≤ 0.05 per Fisher’s least significant difference (LSD) test.

Figure 6

Phloretin effects on (A) chlorophyll a and b content; (B) chlorophyll a and b ratio; (C) total chlorophyll and carotenoid contents; and (D) chlorophylls and carotenoids ratio in C. bursa-pastoris seedling shoots after 14 days of seedling growth in in vitro culture. The results represent mean of three biological replicates. Different letters indicate statistically significant differences based on Fisher’s LSD test (p ≤ 0.05).

Figure 7

Toluidine Blue O staining of phenols in the roots of C. bursa-pastoris seedlings grown on the ½MS nutrient medium without (left) or with 500 µM phloretin (right) in culture in vitro for 14 days. Mature zone (A) and root apex (B) of control root; mature zone (C) and root apex (D) of phloretin-treated root. Bar = 50 µm (A,C) and 20 µm (B,D). rp—lateral root primordium.

Figure 8

The root of C. bursa-pastoris seedlings grown on ½MS nutrient medium without (left) and with 500 µM phloretin (right) in culture in vitro for 14 days. Iodine–potassium iodide (IKI) staining of starch in the root tips of control (A) and phloretin-treated root (B). Bar = 20 µm (A,B). Rhutenium red staining of pectin in the root tips of control (C) and phloretin-treated root (D). Bar = 50 µm (C,D). Sudan Black B staining of lipids in the root tips of control (E) and phloretin-treated seedling (F). Bar = 20 µm (E,F).