Species-Specific Variation in Abscisic Acid Homeostasis and Responses Impacts Important Traits in Crassocephalum Orphan Crops

Abstract

Crassocephalum rubens and Crassocephalum crepidioides are plant species native to Africa, but grow in most tropical and subtropical regions of the world. They are rich in vitamins, minerals, and essential oils and are traditional leafy vegetables and medicinal plants in Sub-Saharan Africa. The plants are still mainly collected from the wild but shall be taken into cultivation and an important aim in the domestication of these species is to improve traits that are relevant for crop production. Here, seed formation and germination capacities in C. crepidioides and C. rubens were investigated, and it was found that C. crepidioides exhibits a higher level of seed dormancy, which could be broken with light, and was correlated with higher amounts of abscisic acid (ABA), a plant hormone that promotes seed dormancy. ABA is also very well-known for its role in abiotic stress tolerance, and it is shown that tetraploid C. crepidioides exhibits a higher level of resistance against drought and heat stress than diploid C. rubens, traits that will benefit the cultivation of these plants, particularly in rain-fed cropping systems. The potential of Crassocephalum to improve nutrition and increase the resilience of marginal cropping systems in Africa is discussed.

Keywords: ABA; dormancy; drought stress; ebolo; neglected crop; redflower ragleaf.

FIGURE 1

Crassocephalum rubens and C. crepidioides form composite flowers, whose disk florets differ in corolla color. (A) Stages of flower development of C. crepidioides Ile-Ife. (B–E) Flower details of C. rubens Mali and C. crepidioides Ile-Ife (F). Nectarous secretions on the tip of a capitulum prior to anthesis at stage 9 (B). Side view of a capitulum after anthesis showing the outer (a) and inner (b) involucral bracts (C). Side view of a capitulum with open florets and emerged stigmas (D). Top view of a capitulum of C. rubens at stage 12 (E). (F) Top view of a capitulum of C. crepidioides at stage 11. (G–I) Scanning electronic microscopic pictures of flower details of C. crepidioides Ile-Ife. Side view of a closed capitulum (G). Top view of a capitulum with different floret stages being visible (H). Open anthers containing pollen (I). The stigma of C. crepidioides with pollen attached (J).

FIGURE 2

Crassocephalum crepidioides forms more seeds than C. rubens, which exhibit a higher level of seed dormancy. (A) Seed of C. crepidioides with hairy pappus. (B) Seed development in capitula of C. rubens and C. crepidioides. Sections of representative capitula are shown. (C) The number of seeds/capitulum of different accessions of C. rubens and C. crepidioides, grown in the greenhouse at 16 h light/8-h dark cycles. Twenty capitula were evaluated and the mean and SD are shown. Different letters over the error bars indicate significant differences (p < 0.05; ANOVA, post hoc Tukey). (D) One thousand-seed-weight of plants grown as in panel (B) (means ± SD; n = 4). Different letters over the error bars indicate significant differences (p < 0.05; ANOVA, post hoc Tukey). (E,F) Seed germination of C. rubens and C. crepidioides in the dark (E) or in the light (F). Fifty seeds of C. rubens Mali (top) and C. crepidioides Ile-Ife (bottom) were plated on 1/2 MS and incubated in either the dark or in 16-h 30 μmol m^–2s^–1 white light/8-h dark cycles at a temperature of 21 or 25°C, respectively. Seed germination, defined as radicle emergence from the seeds coat, was evaluated at the indicated time points. Data show the mean ± SD with n = 50.

FIGURE 3

Crassocephalum crepidioides is hypersensitive to ABA and Fluridone treatment during germination. (A) Response of C. rubens and C. crepidioides to externally applied ABA in terms of germination capacities and cotyledon greening. 50 seeds of each accession were plated on 1/2 MS medium without (control) or with 0. 1-, 0. 3-, or 0.5-μM ABA and incubated in 16-h 30 μmol m^–2s^–1 white light/8-h dark cycles at a temperature of 25°C. Seed germination (left graph), was defined as radicle emergence from the seeds coat. Cotyledon greening (right graph) was defined as fully developed, green cotyledons. The means and SDs are shown. Photos of representative plates are shown below the graphs. (B) Response of C. rubens and C. crepidioides seeds to externally applied Flu during germination in the light and in the dark. 50 seeds of each accession were plated on 1/2 MS medium without (control) or with 1-μM Flu, 5-μM Flu, or 10-μM Flu and incubated in 16-h 30 μmol m^–2s^–1 white light/8-h dark cycles (left graph) or in the dark (right graph) at a temperature of 25°C. (C) The ABA levels in seeds and seedlings of C. crepidioides and C. rubens. The ABA levels were determined using LC/MS-MS in fresh dry seeds, fresh seeds imbibed for 24 h in water and 3-day-old seedlings; n = 4–5. In all charts, the means and SDs are shown. In all graphs, the asterisks indicate significant differences compared to the wild-type by Student’s t-test (***p ≤ 0.001).

FIGURE 4

Crassocephalum crepidioides shows increased resistance to Mannitol. Response of C. rubens Mali and C. crepidioides Ile-Ife to externally applied Mannitol in terms of germination capacities and cotyledon greening. Fifty seeds of each accession were plated on 1/2 MS medium without (control) or with 0, 75, 150, and 200 mM of Mannitol and incubated in 16-h 30 μmol m^–2s^–1 white light/8-h dark cycles at a temperature of 25°C. Seed germination (left graphs) was defined as radicle emergence from the seeds coat. Cotyledon greening (right graphs) was defined as fully developed, green cotyledons. The means and SDs are shown.

FIGURE 5

Crassocephalum crepidioides shows reduced leaf water loss and delayed accumulation of ABA and its catabolites as compared to C. rubens. (A) Detached leaf water loss assays with leaves of 4-week-old C. rubens Mali and 5-week-old C. crepidioides Ile-Ife plants, grown in 16 h 80 μmol m^–2s^–1 white light/8-h dark cycles at a temperature of 25°C; n = 10. (B,C) The ABA measurements in drought-stressed plants. (B) Seven-week-old C. rubens Mali and 8-week-old C. crepidioides Ile-Ife plants, grown as in A, were subjected to progressive drought by withholding water for 3 days. Photos of representative plants are shown. (C) Levels of ABA, ABA-GE, PA, and DPA were then measured in these plants with LC-MS/MS; n = 4–5. In all graphs, the data show the mean ± SD; the asterisks indicate significant differences between genotypes by Student’s t-test (*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001).

FIGURE 6

Crassocephalum crepidioides shows a higher recovery potential following drought than C. rubens. Seven-week-old plants of C. rubens Mali and 8-week-old plants of C. crepidioides Ile-Ife, grown in 16-h white light/8-h dark cycles at 25°C, were subjected to progressive drought by withholding water for 7 days before they were re-watered. Left: Photos of representative plants, taken at the indicated time-points. Right: Survival rates, which are defined as the ability to form new leaves. The mean and SD of three biological replicates, with at least 12 individual plants each, are shown. The asterisk indicates significant differences between genotypes by Student’s t-test (***p ≤ 0.001); n.d.: not detected.