Evaluation of Borage Extracts As Potential Biostimulant Using a Phenomic, Agronomic, Physiological, and Biochemical Approach

Abstract

Biostimulants are substances able to improve water and nutrient use efficiency and counteract stress factors by enhancing primary and secondary metabolism. Premise of the work was to exploit raw extracts from leaves (LE) or flowers (FE) of Borago officinalis L., to enhance yield and quality of Lactuca sativa 'Longifolia,' and to set up a protocol to assess their effects. To this aim, an integrated study on agronomic, physiological and biochemical aspects, including also a phenomic approach, has been adopted. Extracts were diluted to 1 or 10 mL L^-1, sprayed onto lettuce plants at the middle of the growing cycle and 1 day before harvest. Control plants were treated with water. Non-destructive analyses were conducted to assess the effect of extracts on biomass with an innovative imaging technique, and on leaf photosynthetic efficiency (chlorophyll a fluorescence and leaf gas exchanges). At harvest, the levels of ethylene, photosynthetic pigments, nitrate, and primary (sucrose and total sugars) and secondary (total phenols and flavonoids) metabolites, including the activity and levels of phenylalanine ammonia lyase (PAL) were assessed. Moreover, a preliminary study of the effects during postharvest was performed. Borage extracts enhanced the primary metabolism by increasing leaf pigments and photosynthetic activity. Plant fresh weight increased upon treatments with 10 mL L^-1 doses, as correctly estimated by multi-view angles images. Chlorophyll a fluorescence data showed that FEs were able to increase the number of active reaction centers per cross section; a similar trend was observed for the performance index. Ethylene was three-fold lower in FEs treatments. Nitrate and sugar levels did not change in response to the different treatments. Total flavonoids and phenols, as well as the total protein levels, the in vitro PAL specific activity, and the levels of PAL-like polypeptides were increased by all borage extracts, with particular regard to FEs. FEs also proved efficient in preventing degradation and inducing an increase in photosynthetic pigments during storage. In conclusion, borage extracts, with particular regard to the flower ones, appear to indeed exert biostimulant effects on lettuce; future work will be required to further investigate on their efficacy in different conditions and/or species.

Keywords: Borago officinalis L.; Lactuca sativa L.; image analysis; non-destructive measurements; phenols; photosynthesis.

FIGURE 1

Estimated fresh weight of Romaine lettuce plants treated with water (control), 1 or 10 mL L^–1 borage leaf (LE) or flower extract (FE). Data were obtained by processing of multi-view angles images from undisturbed potted lettuce plants at three time points of growth (days after transplanting). Values are means ± SE (n = 9). Data were subjected to one-way ANOVA. Different letters, where present, indicate significant differences among treatments.

FIGURE 2

Chlorophyll a fluorescence parameters measured in Romaine lettuce plants treated with water (control), 1 or 10 mL L^–1 borage LE or FE. (A) Maximum quantum efficiency of PSII, (B) performance index, (C) number of reaction centers per cross section, (D) energy dissipated per reaction center. Values are means ± SE (n = 3). Data were subjected to two-way ANOVA. Different letters, where present, indicate significant differences among treatments or times.

FIGURE 3

Leaf gas exchanges in Romaine lettuce plants treated with water (control), 1 or 10 mL L^–1 borage LE or FE. (A) Net photosynthesis, (B) transpiration, (C) stomata conductance, (D) photosynthetic water use efficiency. Values are means ± SE (n = 3). Data were subjected to one-way ANOVA. Different letters, where present, indicate significant differences among treatments.

FIGURE 4

Ethylene emission in Romaine lettuce heads treated with water (control), 1 or 10 mL L^–1 borage LE or FE. Values are means ± SE (n = 3). Data were subjected to one-way ANOVA.

FIGURE 5

Chlorophyll a+b (A) and carotenoids (B) concentrations in Romaine lettuce leaf tissue treated with water (control), 1 or 10 mL L^–1 borage LE or FE. Values are means ± SE (n = 3). Data were subjected to one-way ANOVA.

FIGURE 6

Phenolics (A) and total flavonoids (B) concentrations, and antioxidant capacity (C) in Romaine lettuce leaf tissue treated with water (control), 1 or 10 mL L^–1 borage LE or FE. Values are means ± SE (n = 8). Data were subjected to one-way ANOVA. Different letters, where present, represent significant differences among treatments.

FIGURE 7

Total soluble proteins in Romaine lettuce leaf tissue treated with water (control), 1 or 10 mL L^–1 borage LE or FE. Values are means ± SE (n = 8). Data were subjected to one-way ANOVA. Different letters represent significant differences among treatments.

FIGURE 8

In vitro PAL specific activity (A) and levels of PAL-like polypeptides (B) in Romaine lettuce leaf tissue treated with water (control), 1 or 10 mL L^–1 borage LE or FE. In vitro PAL activity data are means ± SE (n = 8). For immunoblotting, polyclonal antibodies raised against a PAL protein of Petroselinum crispum, (kind gift of Dr. Imre E. Somssich) were used. Loading was 10 μg protein per lane. The results of one experiment, representative of three, are shown.