Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate

Abstract

Background and aims: Cress seeds release allelochemicals that over-stimulate the elongation of hypocotyls of neighbouring (potentially competing) seedlings and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here.

Methods: Low-molecular-weight cress-seed exudate (LCSE) from imbibed Lepidium sativum seeds was fractionated by phase partitioning, paper chromatography, high-voltage electrophoresis and gel-permeation chromatography (on Bio-Gel P-2). Fractions, compared with pure potassium salts, were bioassayed for effects on Amaranthus caudatus seedling growth in the dark for 4 days.

Key results: The LCSE robustly promoted amaranth hypocotyl elongation and inhibited root growth. The hypocotyl inhibitor was non-volatile, hot acid stable, hydrophilic and resistant to incineration, as expected for K+. The root inhibitor(s) had similar properties but were organic (activity lost on incineration). The root inhibitor(s) remained in the aqueous phase (at pH 2.0, 6.5 and 9.0) when partitioned against butan-1-ol or toluene, and were thus hydrophilic. Activity was diminished after electrophoresis, but the remaining root inhibitors were neutral. They became undetectable after paper chromatography; therefore, they probably comprised multiple compounds, which separated from each other, in part, during fractionation. On gel-permeation chromatography, the root inhibitor co-eluted with hexoses.

Conclusions: Cress-seed allelochemicals inhibiting root growth are different from the agent (K+) that over-stimulates hypocotyl elongation and the former probably comprise a mixture of small, non-volatile, hydrophilic, organic substances. Abundant components identified chromatographically and by electrophoresis in cress-seed exudate fitting this description include glucose, fructose, sucrose and galacturonic acid. However, none of these sugars co-chromatographed and co-electrophoresed with the root-inhibitory principle of LCSE, and none of them (in pure form at naturally occurring concentrations) inhibited root growth. We conclude that the root-inhibiting allelochemicals of cress-seed exudate remain unidentified.

Keywords: Allelochemicals; amaranth (Amaranthus caudatus); bioassay; chromatography; cress (Lepidium sativum); electrophoresis; hypocotyl elongation; potassium salts; root growth; seed exudate.

Fig. 1.

Effect of cress seed(ling)s on germination and seedling growth of amaranth. Amaranth seeds (ten per dish) were incubated in the dark on damp filter paper in 5-cm Petri dishes in the presence of 0–12 cress seeds. (A, B) After 4 days, the amaranth seedlings were measured (A, hypocotyls; B, roots). (C) The number of amaranth seeds that germinated is also recorded. n = 61–67 dishes for each number of cress seeds tested; asterisks indicate a significant effect of cress seeds, **P < 0.002.

Fig. 2.

Low-molecular-weight cress-seed exudate (LCSE) promotes amaranth hypocotyl growth and inhibits amaranth root growth through two different agents. Amaranth seeds were incubated on damp filter paper for 4 days in the presence of LCSE (total solute concentration ~1.6 mg mL⁻¹) that had been stored frozen, compared with LCSE treated by: freeze-drying; ashing in a Bunsen flame; incubation with 0.25 m formic acid for 1 h at 20 or 120 °C; or partitioning against ethyl acetate (acidified to pH 2.7 with 20 mm formic acid; the upper hydrophobic organic phase and lower hydrophilic aqueous phase were bioassayed separately). Any formic acid or ethyl acetate was dried off before the bioassays, and all LCSE specimens were reconstituted in deionized water to the original volume. The right-hand three bars represent controls with no LCSE present: ‘acid-only hydrophobic’ and ‘acid-only hydrophilic’ were the organic and aqueous phase, respectively, after 20 mm formic acid was shaken with ethyl acetate. n = 15 Petri dishes for each treatment; **P < 0.002 compared with frozen/thawed LCSE. Dashed line, frozen/thawed LCSE; solid line, water-only control.

Fig. 3.

Effect of salts on amaranth seedling growth. Ten amaranth seeds per dish were incubated for 4 days in the dark in the presence of various salts at 5 mm (A) or 10 mm (B–D), then the hypocotyl and root lengths were measured. Each treatment was applied in three Petri dishes; the histograms show the mean ± s.e.m. organ lengths. In each case, the germination was 70–80 %. In C, the salts were prepared in house by adjustment of 10 mm KOH to pH 6.0 with the appropriate acid. In D, the 10 mm potassium acetate was adjusted to pH 3.0 with a small excess of acetic acid, mimicking a potential ‘K⁺-trapped’ anion present in low-molecular-weight cress-seed exudate, then dried from a 100 mm solution of the acid (or water) named on the x-axis. The dashed line indicates the water-only control. *P < 0.01, **P < 0.001 (in each histogram compared with the water-only control).

Fig. 4.

Behaviour of the active principles of low-molecular-weight cress-seed exudate (LCSE) on electrophoresis. Replicate samples (n = 48) of LCSE were fractionated by paper electrophoresis at pH 6.5 for 13 min at 2.5 kV. Each of the 48 electrophoretograms carried a marker mixture containing K⁺, Na⁺, Mg²⁺, Cu²⁺, XXXGol-sulphorhodamine (oligosacch–SR; a neutral marker, fluorescent), lepidimoic acid (Lep), galacturonic acid (GalA), Orange G, phosphate (P_i) and sulphate (SO₄²⁻). (A) The marker mixture was cut off each of the 48 electrophoretograms, together with a fringe of the neighbouring LCSE loading, and stained with Bromophenol Blue; 16 representative runs are illustrated. The unstained majority of each electrophoretogram was then cut into ten zones, each of which was eluted with water and the eluate bioassayed on hypocotyl growth (B) and root growth (C) of amaranth seedlings. The histograms show the mean seedling lengths (±s.e.m.; n ≈ 48). In B, the dashed line indicates the mean of the shortest four; in C, it is the mean of the longest four. Asterisks indicate that the specific zone differed significantly from the mean (n ≈ 192) of the relevant four zones: *P < 0.01, **P < 0.001.

Fig. 5.

Paper chromatography of active principle(s) present in low-molecular-weight cress-seed exudate (LCSE). (A, B) Paper chromatography (in butan-1-ol/acetic acid/water) of five examples out of 15 independent LCSE samples: (A) stained with Bromophenol Blue, revealing ionic constituents; and (B) same chromatograms stained with silver nitrate, revealing sugars. Abbreviation: ‘LCSE’, a 100-µL streak loading (~4 cm × 1 cm) of 20-fold concentrated LCSE; MM, marker mixture [10 µL, containing 30 mm of each of lepidimoic acid, galacturonic acid, potassium sulphate and a trace of Orange G]. Spots labelled in yellow represent components of marker mixture. Spots labelled in white represent components of LCSE (Fru, fructose; GalA, galacturonic acid; Glc, glucose; Sucr, sucrose; Unk, unidentified anion). [Spots are labelled only on the fourth chromatogram.] Chromatography was on acid-washed Whatman No. 3 paper, developed in butan-1-ol/acetic acid/water (12:3:5) for 20 h. After thorough drying, the paper was dipped rapidly through methanol/acetone (1:2) and re-dried, repeated several times, and finally re-dried in a draught of air overnight, helping to remove the last traces of acetic acid. (C, D) Strips corresponding to zones 1–10 were excised from replicate 100-µL streak-loaded chromatograms (identical to A and B but not stained; not shown) of the 15 concentrated LCSE samples; each strip was eluted into 1 mL water, and the eluates were bioassayed for effects on the growth of amaranth hypocotyls (C) and roots (D). The approximate migration positions of various markers (with some variation between the 15 chromatograms) are indicated above histogram C.

Fig. 6.

Behaviour of the active principles of low-molecular-weight cress-seed exudate (LCSE) on gel-permeation chromatography. The LCSE was run through a Bio-Gel P-2 column, and selected even-numbered fractions were tested for thymol-reactive sugars (A) and for the ability to promote amaranth hypocotyl growth (B) and to inhibit root growth (C). Abbreviations in A are as follows: B1, unidentified sugar as named by Iqbal et al. (2016); DP3, probable neutral trisaccharide; Fru, fructose; Glc, glucose; Mlt2, maltose; Mlt3, maltotriose; PS, polysaccharides; Sucr, sucrose. Asterisks indicate a significant effect of the fraction (mean ± s.e.m.; n ≈ 20) compared with the mean of all fractions; *P < 0.01, **P < 0.001. The most effective fraction for root inhibition is marked with a vertical arrow.