Ascophyllum nodosum extract act as a biostimulants in tomato plants -

Ascophyllum nodosum extract act as a biostimulants in tomato plants

Biostimulants are an emerging category of crop management products which can enhance crop productivity under abiotic stress conditions. The ability of some biostimulant products such as Ascophyllum nodosum extracts (ANE) to enhance the tolerance of crops to drought stress has been observed by growers. The objective of this study was to investigate if different commercial ANE biostimulants provided the same tolerance to tomato plants (cv. Moneymaker) subjected to a defined drought period. A compositional characterisation of the key macromolecules of ANEs was performed. In addition, the role of ANE biostimulants in inducing changes of chlorophyll and osmolytes levels, MDA production, dehydrin isoform pattern and dehydrin gene expression levels was assessed. The three ANE biostimulants evaluated were found to provide different levels of tolerance to drought stressed tomato plants.

Plant material and growth condition:

Tomato seeds (Lycopersicon esculentum, cv. Moneymaker) were surface sterilized with sodium hypochlorite for 1 min before being thoroughly rinsed with distilled water. Seeds were set in plug trays using growth medium of compost: vermiculite: perlite (5: 1: 1). On day 21, seedlings were then transferred to 2 litre pots. The resultant plants were raised in a growth room at a temperature of 27/22 ± 2 °C (day/night; 16/8 h) and 70 ± 5% relative humidity (RH). Plants were irrigated with 125 mL water every other day in order to create equal soil moisture conditions in all the pots.

Treatment application and drought stress conditions

Three commercially available liquid seaweed extracts of A. nodosum (ANE A, ANE B and ANE C) manufactured using different methods were applied to plants as biostimulant treatments. ANE A was manufactured using a proprietary process at high temperatures and neutral pH. ANE B and ANE C were manufactured using a proprietary process at high temperatures and alkaline pH. Prior to imposition of severe drought, ANE biostimulants and control treatments were applied by foliar spray at a dilution of 0.33% (v/v) on 35-day-old tomato plants. Distilled water was applied as a control. After 24 h, drought stress was induced by withholding water for 7 days. To minimize the influence of any positional effect on drought stress responses, the relative position of the pots in the growth room was changed every other day. After the drought treatment, plants were re-watered, and 24 h later ANE treatments were applied again as foliar spray at 0.33% (v/v). Control plants were sprayed with equal volume of distilled water. Leaf tissue was sampled before first ANE biostimulant application (T0), at 7 days after subjecting plants to drought stress (T1), at 48 h after the second ANE treatment in the 3rd day of the recovery stage (T2) and at the end of the recovery stage (T3). The samples were snap-frozen in liquid nitrogen, ground and kept in −80 °C until further analysis. Similar tomato plants were selected and grown under unstressed conditions for 56 days. ANE biostimulants and control treatments were applied by foliar spray as described above to evaluate growth promoting effects on non-drought stressed tomato plants. Sampling points for unstressed plants corresponded to 42-day-old (T1), 45-day old (T2) and 56-day old tomato plants (T3).

Chemical compositional analysis of ANEs

Total solids from ANE liquid formulations were determined after drying in a convection oven for 18 h at 105 °C. These same samples were then used to determine ash by placing in a furnace for 6 h at 550 °C. Sulphate content linked to carbohydrate molecules was determined quantitatively after hydrolysing the samples with 2 M trifluoroacetic acid (TFA) for 5 h at 100 °C. Sulphate ion was precipitated in a strongly acid medium with barium chloride-gelatine. The resulting turbidity was measured spectrophotometrically at 420 nm. L-fucose, total uronic acids, laminarin and total polyphenol content were also determined spectrophotometrically. A quantitative analysis of soluble mannitol from ANEs was carried out by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

Growth parameters, relative water content and chlorophyll determination

Tomato plants were harvested at the end of the recovery stage and measurement of fresh weight (FW) of the plants (leaf + stem) was obtained. Plant dry weight (DW) was determined by drying frozen ground samples in a convection oven for 18 h at 105 °C. DW was measured and recorded before every subsequent metabolite analysis at all stated sampling times. The chlorophyll content was determined using an extraction method with an ammonia/acetone mixture (1/9, v/ v) and were expressed on a dry weight basis (mg·g⁻¹ DW).

Measurements of relative water content (RWC) were performed on leaves collected at three sampling times (T0, T1 and T2). FW of the leaves selected was immediately measured after cutting. In order to obtain the turgid weight (TW), the leaves were immersed in distilled water in a closed petri dish and incubated under normal room temperature and dim light for 18 h. At the end of the imbibition period, the leaves were taken out, properly wiped to remove the water on the surface and weighed. Afterwards, the leaves were put in a convection oven for 24 h at 80 °C to obtain DW.

RWC was calculated according the equation: (RWC in %) = [(FW-DW)/(TW-DW)]*100.

The content of malondialdehyde (MDA) was measured by thiobarbituric acid reactive substance (TBARS) assay. Proline content measured by reaction mixture of 1% (w/v) ninhydrin in acetic acid/water/ethanol (60/20/20, v/v/v)] and incubated for 20 min at 95 °C. After cooling at room temperature, the absorbance was measured at 520 nm and were expressed on a dry weight basis (mg/g DW).

Chromatographic determination of soluble sugars

Soluble sugars were extracted from 15 mg of frozen ground leaf material from three sampling times (T0, T1 and T2) with 0.5 mL of aqueous 2% (w/v) PVPP and incubated for 25 min at 90 °C. Then, leaf extracts were sonicated for 5 min and centrifuged at 20,000 x g for 20 min at 4 °C. The levels of glucose, fructose and sucrose were determined by HPAEC-PAD using a Carbopac PA-100 column. An isocratic gradient of 50 mM NaOH (degassed by bubbling with helium) at 1 mL/min was applied. Compounds were identified by comparison of retention time to that of commercial standards (Sigma Aldrich) and sugars were quantified by peak integration. Tomato leaf heat-stable protein fraction was determined by the Bradford method using the Bio-Rad protein assay as described by the manufacturer. 250 μg of protein per sample were trichloroacetic acid (TCA) precipitated (20%, v/v), washed three times with cold acetone and dried. Protein pellets were resuspended in 1 × Laemmli sample buffer with and without 5% (v/v) 2-mercapthoethanol and heated for 20 min at 80 °C for further SDS-PAGE and immuno-analysis under reducing and non-reducing conditions.

SDS-PAGE and immuno-analysis (western blot) of dehydrin isoforms 4 μg of heat-stable protein extracts were resolved on a 14% SDSPAGE using a Mini-Protean II Cell (Bio-Rad). Duplicate gels were prepared, one to transfer onto 0.2 μm nitrocellulose membrane (Whatman) with a Mini Trans-Blot Cell (Bio-Rad) and the other for staining with Coomassie Brilliant Blue R-250. Electrotransferred nitrocellulose membranes were blocked with phosphate-buffer saline containing 0.1%. The membranes were probed with polyclonal anti-dehydrin affinity purified serum (dilution 1/2000) raised against the conserved K-segment for dehydrin C-terminal (AS07-206A, Agrisera), which were detected with rabbit antiserum against IgG horseradish peroxidase conjugate diluted 5000-fold (NA934VS, GE-Healthcare). The immuno-complexes were visualized using the Pierce ECL chemiluminescence detection system (Thermo-Scientific). Immunoreactive bands were quantified by densitometry of scanned autoradiographs using the software ImageJ (NIH) and the results were expressed as the relative fold-change with respect to the levels of accumulation of control plants. The molecular mass of the separated polypeptides was estimated in comparison to the mobility of pre-stained electrophoresis marker (ColorBurst, Sigma-Aldrich).

Results

The liquid ANE formulations used in this study, ANE B showed the highest concentration of solids while ANE C displayed the lowest value, 1.5 and 2-fold more dilute than ANE A and ANE B formulations, respectively. On the whole, ANE A and ANE B formulations were primarily composed of uronic acids (representing mainly alginate), fucose, mannitol, laminarin, polyphenols and ash. The level of change of some of these components was not always proportional with the changes in total solids. For example, the concentration of mannitol, uronic acids or fucose in ANE A was found to be 15 to 86% higher than ANE B. On the other hand, this analysis showed that the amount of laminarin in ANE A was 1.5 fold lower than ANE B. The product composition pattern of ANE C showed similarities with ANE A and ANE B. However, this ANE contained lower amounts of carbohydrates such as fucose, uronic acids or mannitol while the presence of laminarin was not detected. All ANE biostimulants contained a high proportion of sulphate from the total ash content. Fucoidan is a sulphated, fucose rich, heteropolysaccharide that may be extracted from the cell wall of A. nodosum. Interestingly, the highest sulphate to fucose ratio was observed for ANE B (1.27) while ANE A showed the lowest value (1.06). The analysis of polyphenols, determined as phloroglucinol equivalents, indicated that ANE C and ANE B contained 2 to 3.4-fold higher amounts of this component on a dry weight basis than ANE A. The amount of unknown compounds for ANE A and ANE B was lower than 20% (w/w) while ANE C had the highest percentage of unidentified organic components.

Effects of ANEs on tomato growth and chlorophyll content under drought stress

To evaluate the capacity for drought stress tolerance induced by ANE biostimulants, 35-old day tomato plants (cv. Moneymaker) were treated with three different commercial ANEs while control plants were sprayed with distilled water. After 7 days without watering, severe drought stress was evident in untreated plants compared to nondrought control plants. Compared to unstressed control, the RWC (%) of drought untreated plants was decreased by 14.49%. The difference in drought tolerance between the untreated and ANE A-treated drought plants was also remarkable. While control plants showed severe wilting of all the leaves and plant growth inhibition, we found that ANE A-treated plants had less noticeable visual stress symptoms on leaves and significant higher plant height. On the contrary, plants treated with ANE B and ANE C displayed generalized wilting and a similar plant growth pattern compared to control. The RWC of ANE A-treated plants was also significantly higher under drought stress in comparison with control. However, tomato plants treated with ANE B and ANE C were only able to maintain hydration level around 70% of RWC throughout the dehydration period. These phenotypical and physiological differences clearly illustrate the higher tolerance to severe water deficit induced by ANE A application compared to the other two commercial ANEs. At the end of the recovery stage, after re-watering and applying the second ANE foliar application, an enhanced plant growth and greater foliar density was observed in both ANE A and ANE C-treated plants compared to untreated plants. Both growth parameters, above ground plant FW and DW, were significantly increased over control by between 25 and 30%. On the other hand, growth and biomass of ANE B-treated plants was almost identical to untreated drought plants and approximately 50% lower than unstressed control plants. The effects of ANE treatments on unstressed tomato plants over the same growth period were also tested.

The application of this biostimulant resulted in a significant chlorophyll content increase, exhibiting values that were between 10% and 14% higher with respect to tomato plants treated with ANE A and ANE C. Only the total chlorophyll content of ANE A-treated plants was significantly higher than in untreated drought plants by 9%.

Lipid peroxidation was measured in terms of MDA content. Accumulation of MDA is a typical symptom of membrane lipid damage under drought stress conditions and a noticeable increase of this parameter (up to 35%) was confirmed in untreated drought plants with respect to well-watered control. MDA accumulation in ANEtreated plants under water stress was significantly decreased in all the treatments tested compared to control and lowest values were found under the effect of ANE B and ANE C, representing a decrease of 30%. MDA accumulation of plants treated with ANE C was significantly decreased by 15% compared to ANE A-treated plants.

The osmoprotectant accumulation achieved under drought stress was measured by determining the variations in endogenous concentrations of proline and soluble sugars. After withholding water for 7 days, the leaf proline content in untreated plants accumulated 6.3-fold compared to control plants grown under unstressed conditions. Each ANE treatment stimulated a significant increase of proline levels with respect to control under drought stress conditions. The soluble sugar content of tomato leaves was quantified by HPAEC-PAD after detecting glucose, fructose and sucrose as the main chromatographic peaks of plant extracts. The results revealed that the total soluble sugar content, calculated as the sum of glucose, fructose and sucrose, ranged between 4.97 and 7.93 mg g−1 DW in plants growing under unstressed conditions before applying the first ANE treatment. In response to drought stress, these treated plants increased foliar sucrose, glucose and fructose concentrations by 40%, 28% and 15% with respect to untreated plants, respectively. In line with the observed variations in endogenous proline levels, plant re-watering resulted in a pronounced decrease in the concentration of soluble sugars. This effect was more pronounced in plants treated with ANE B, showing 22–25% lower glucose, fructose and sucrose values than untreated plants. On the other hand, total soluble sugars or sucrose content of ANE A-treated plants was 12 and 18% higher compared to control after 2 days of the second ANE application and 3 days of re-watering, respectively. This effect was more pronounced in plants treated with ANE B, showing 22–25% lower glucose, fructose and sucrose values than untreated plants. On the other hand, total soluble sugars or sucrose content of ANE A-treated plants was 12 and 18% higher compared to control after 2 days of the second ANE application and 3 days of re-watering, respectively.

Eight polypeptide bands were recognized in heat-stable protein extracts from leaves of tomato plants (cv. Moneymaker). These bands showed a molecular mass range determined by SDS-PAGE from 15 to 38 kDa. The analysis of the thermostable fractions revealed that 3 low molecular weight polypeptides (15, 18, 25 kDa) and one 32 kDa molecular specie slightly accumulated in untreated plants during drought stress. The presence of post-translational phosphorylation was estimated by evaluating the gel mobility shift of immunoreactive bands after alkaline phosphatase treatment of these heat stable fractions. The immunoblot performed on SDS-PAGE from samples of ANE Atreated plants under drought stress showed that most of the detected dehydrin-like protein appears to be phosphorylated. For example, the bands detected around 18 kDa ran at slightly lower mass when the protein extract was treated with alkaline phosphatase. This difference of molecular mass can be due to the phosphorylation of these dehydrin like proteins. On the other hand, the relative amount of the other detected proteins between 34 and 25 kDa decreased significantly while a new polypeptide was detected at 55 kDa.

At the start of the experiment, the relative gene expression of tas14 with respect to the reference gene actin was found at low constitutive levels and no statistically significant differences were observed between plants. Even though tas14 gene expression was clearly induced in all plants subjected to drought stress, ANEs induced a significant up-regulation compared to untreated plants.

Conclusions

Clear phenotypic differences were observed between ANE formulations at the end of the drought period with ANE A maintaining better plant growth without symptoms of drought stress. Physiological measurement of osmolytes supports a metabolic/physiological basis to the effect. Gene transcription and proteomic analysis of stress protective proteins support a potential mode of action in providing this tolerance. Although there are similarities between 2 of the ANEs in terms of their impact on the measured markers, the intensity of the tolerance appears to be different with ANE A providing stronger tolerance than ANE C. Taken together, this study results highlight that despite the ANE biostimulants being manufactured from the same raw material, their ability to maintain crop productivity during drought stress was not the same.

Reference:

Goñi, O., Quille, P. and O’Connell, S., 2018. Ascophyllum nodosum extract biostimulants and their role in enhancing tolerance to drought stress in tomato plants. Plant Physiology and Biochemistry, 126, pp.63-73