Photoperiod Extension Enhances Sexual Megaspore Formation and Triggers Metabolic Reprogramming in Facultative Apomictic Ranunculus auricomus

Abstract

Meiosis, the key step of sexual reproduction, persists in facultative apomictic plants functional to some extent. However, it still remains unclear how and why proportions of reproductive pathways vary under different environmental stress conditions. We hypothesized that oxidative stress mediates alterations of developmental pathways. In apomictic plants we expected that megasporogenesis, the stage directly after meiosis, would be more affected than later stages of seed development. To simulate moderate stress conditions we subjected clone-mates of facultative apomictic Ranunculus auricomus to 10 h photoperiods, reflecting natural conditions, and extended ones (16.5 h). Reproduction mode was screened directly after megasporogenesis (microscope) and at seed stage (flow cytometric seed screening). Targeted metabolite profiles were performed with HPLC-DAD to explore if and which metabolic reprogramming was caused by the extended photoperiod. Prolonged photoperiods resulted in increased frequencies of sexual vs. aposporous initials directly after meiosis, but did not affect frequencies of sexual vs. asexual seed formation. Changes in secondary metabolite profiles under extended photoperiods affected all classes of compounds, and c. 20% of these changes separated the two treatments. Unexpectedly, the renowned antioxidant phenylpropanoids and flavonoids added more to clone-mate variation than to treatment differentiation. Among others, chlorophyll degradation products, non-assigned phenolic compounds and more lipophilic metabolites also contributed to the dissimilarity of the metabolic profiles of plants that had been exposed to the two different photoperiods. The hypothesis of moderate light stress effects was supported by increased proportions of sexual megaspore development at the expense of aposporous initial formation. The lack of effects at the seed stage confirms the basic assumption that only meiosis and sporogenesis would be sensitive to light stress. The concomitant change of secondary metabolite profiles, as a systemic response at this early developmental stage, supports the notion that oxidative stress could have affected megasporogenesis by causing the observed metabolic reprogramming. Hypotheses of genotype-specific responses to prolonged photoperiods are rejected.

Keywords: FCSS; Ranunculus; apomixis; light stress; oxidative stress; reproduction mode; secondary metabolites; seed formation.

FIGURE 1

Development of sexual ovules (%) in Ranunculus carpaticola × cassubicifolius plants grown in climate chambers under prolonged photoperiod (16.5 h, light stress) and shorter photoperiod (10 h, control plants). N = 146 sexual ovules from 20 plants in the control and n = 181 sexual ovules from 18 plants in the stress treatment.

FIGURE 2

Mean percentage of sexual ovules for three hexaploid Ranunculus carpaticola × cassubicifolius clones (T, I, and V) grown in climate chambers under enhanced photoperiod (16.5 h) and shorter photoperiod (10 h). Error bars show standard deviation. Differences between treatments are significant for clone T (P < 0.01) but not for the other two clones (P > 0.20). N = number of sexual ovules.

FIGURE 3

Influence of the treatment on the production of sexual seeds (%) in Ranunculus carpaticola × cassubicifolius plants grown in climate chambers under prolonged photoperiod (16.5 h, N = 224) and shorter photoperiod (10 h, N = 257).

FIGURE 4

Similarity analysis of HPLC-DAD detectable secondary metabolites in closed buds of three hexaploid Ranunculus carpaticola × cassubicifolius clones (I, V, T) exposed to a 10 and 16.5 h photoperiod. (A) NMDS of Bray–Curtis dissimilarity of standardized peak area patterns of 66 metabolites; bracket numbers are percentages of sexual ovules formed in one flower; P-values (ANOSIM) inform about similarity and dissimilarity of the indicated factors photoperiod (N = 12) and clone (N = 8); (B–D) variable contributions of different metabolite classes illustrated by 3–4 representative UV spectra (220–450 nm) of (B) chlorophyll degradation products (Chl) and carotenoids (Ca), (C) cinnamic acids (C), flavonoids (F) and other polar phenols or alkaloids (P), (D) lipohilic phenols, alkaloids or other more unsaturated metabolites (LP), and (E) aliphatic and more saturated metabolites (A). To correlate to mode of reproduction in the early developmental stage, percentage of sexual ovules of each individual was included as one variable in the NMDS ordination.