Hormopriming to Mitigate Abiotic Stress Effects: A Case Study of N 9-Substituted Cytokinin Derivatives With a Fluorinated Carbohydrate Moiety

Abstract

Drought and salinity reduce seed germination, seedling emergence, and early seedling establishment, affect plant metabolism, and hence, reduce crop yield. Development of technologies that can increase plant tolerance of these challenging growth conditions is a major current interest among plant scientists and breeders. Seed priming has become established as one of the practical approaches that can alleviate the negative impact of many environmental stresses and improve the germination and overall performance of crops. Hormopriming using different plant growth regulators has been widely demonstrated as effective, but information about using cytokinins (CKs) as priming agents is limited to only a few studies using kinetin or 6-benzylaminopurine (BAP). Moreover, the mode of action of these compounds in improving seed and plant fitness through priming has not yet been studied. For many years, BAP has been one of the CKs most commonly applied exogenously to plants to delay senescence and reduce the impact of stress. However, rapid endogenous N ⁹-glucosylation of BAP can result in negative effects. This can be suppressed by hydroxylation of the benzyl ring or by appropriate N ⁹ purine substitution. Replacement of the 2' or 3' hydroxyl groups of a nucleoside with a fluorine atom has shown promising results in drug research and biochemistry as a means of enhancing biological activity and increasing chemical or metabolic stability. Here, we show that the application of this chemical modification in four new N ⁹-substituted CK derivatives with a fluorinated carbohydrate moiety improved the antisenescence properties of CKs. Besides, detailed phenotypical analysis of the growth and development of Arabidopsis plants primed with the new CK analogs over a broad concentration range and under various environmental conditions revealed that they improve growth regulation and antistress activity. Seed priming with, for example, 6-(3-hydroxybenzylamino)-2'-deoxy-2'-fluoro-9-(β)-D-arabinofuranosylpurine promoted plant growth under control conditions and alleviated the negative effects of the salt and osmotic stress. The mode of action of this hormopriming and its effect on plant metabolism were further analyzed through quantification of the endogenous levels of phytohormones such as CKs, auxins and abscisic acid, and the results are discussed.

Keywords: Arabidopsis; abiotic stress; antisenescence; cytokinin analogs; hormopriming; plant biostimulant characterization index.

FIGURE 1

Scheme for the preparation of substituted 6-benzylamino-2′-deoxy-2-′fluoro-9-(β)-D-arabinofuranosylpurine derivatives by a method using Castro’s reagent and Hunig base.

FIGURE 2

Structures of the newly synthesized 6-benzylamino-2′-deoxy-2-′fluoro-9-(β)-D-arabinofuranosylpurine derivatives.

FIGURE 3

Parallel coordinate plot of the traits (Early seedling establishment, slope of the growing curve, RGR, AGR and the final rosette area) obtained from multi-trait high-throughput screening of Arabidopsis seedlings non-primed (MOCK) or primed with four different N⁹-substituted CK derivatives with a fluorinated carbohydrate moiety at four concentrations (10^–7, 10^–6, 10^–5, and 10^–4 M) and grown under optimal (upper panels), or salt (100 mM NaCl, middle panels) or osmotic (100 mM mannitol, bottom panels) stress conditions (N = 48). BAP at 10^–8 M was used as positive control.

FIGURE 4

RGB images of the non-primed Arabidopsis seedlings (MOCK) and those primed with the best performing concentration of compound 2 (according to the PBC index, Table 3) grown under optimal growth condition (left images), salt (100 mM NaCl, middle images) or osmotic (100 mM mannitol, right images) stress.

FIGURE 5

Color indices [NGRDI (A), VARI (B), or GLI (C)] used as greenness parameters for Arabidopsis seedlings from non-primed (MOCK) seeds or seeds primed with compound 2 at four concentrations (10^–7, 10^–6, 10^–5, and 10^–4 M) grown under optimal (upper panel), or salt (100 mM NaCl, middle panel), or osmotic (100 mM mannitol, bottom panel) stress conditions for 7 days (N = 48). Different letters mean significant differences among treatments (priming effect) for each growth conditions according to Dunn’s test after Kruskal–Wallis’ test.

FIGURE 6

Heatmap (A) representing the changes in CKs, auxins, and ABA for Arabidopsis seedlings from non-primed (MOCK) seeds or seeds primed with compound 2 at four concentrations (10^–7, 10^–6, 10^–5, and 10^–4 M) grown under optimal (black line), or salt (100 mM NaCl, red line) or osmotic (100 mM mannitol, blue line) stress conditions for 7 days. Principal component analysis (Dimension, Dim) (B) of the same Arabidopsis seedlings.