Introduction

According to the Food and Agriculture Organisation of the United Nations (FAO), the world population will reach 9.7 billion by 2050, leading to a significant increase in global food demand (United Nations, Department of Economic and Social Affairs, 2019). Therefore, modern agriculture faces the critical challenge of ensuring higher crop yields while also meeting consumer demands for healthy, safe, and sustainably produced foods. In addition, lifestyle changes over the past decade have driven consumer demand for fresh, ready-to-eat products that are convenient and quick to consume. In this context the food industry has increasingly promoted minimally processed vegetables on the market. Leafy vegetables commonly known as “baby leaf” are generally used by the industry for minimally processed and ready-to-eat vegetable products. These fresh vegetables are selected, washed, cut, packaged and prepared for immediate consumption. Among the main leafy vegetables, there is lettuce (Lactuca sativa L.), a species belonging to the Asteraceae family (Compositae). It is one of the most widely consumed vegetables in the world due to its exceptional nutritional properties: it is low in calories, fat and sodium, and is a good source of fibre, iron, folic acid and vitamin C1. The cultivation of this species is highly significant for both the Italian and global agricultural economies. Baby leaf vegetables are typically cultivated in protected environments and are characterized by short growth cycles, lasting from a few weeks to a few months. This agronomic approach involves intensive soil use, requiring a continuous supply of nutrients. Hence, agronomists and farmers are looking for innovative techniques and products to enhance the agronomic performance, economic viability, and quality sustainability of baby leaf vegetable production. For ready-to-use products, it is important to implement effective agronomic strategies that enhance yield, quality attributes, and shelf life to ensure high-quality plant material2. One promising solution is the application of plant biostimulants, which are defined as fertilizing products aimed at improving tolerance to abiotic stress, enhancing nutrient uptake and fruit quality, and generally stimulating plant physiological processes. Biostimulants promote nitrogen metabolism and may reduce nitrate accumulation in plant tissues. Indeed, positive results have been tested on wild rocket3, lettuce and spinach4,5. Among the various categories of plant biostimulants, microalgae are gaining increasing attention from both researchers and farmers6. Microalgae, also called phytoplankton, are photosynthetic prokaryotic or eukaryotic organisms (1–50 µm in size) currently employed in biodiesel production7 or as dietary supplements8. According to Siringi et al.9, treatment with Spirulina (Arthrospira platensis) in an aquaponic system used both as a foliar spray on lettuce and as fish feed improved plant height, number of leaves per plant and chlorophyll content, resulting in a 25.5% increase in dry weight compared to untreated controls. Other studies on the use of plant biostimulants in lettuce have reported the use of protein hydrolysates and algae extracts with auxin-like activity10, which led to an increase in secondary root development and increased nutrient utilisation efficiency. In terms of quality, biostimulants have also been associated with higher chlorophyll content, producing greener leaves that are more visually appealing to consumers11. The use of plant biostimulants can also induce the synthesis of multiple antioxidant compounds by stimulating the biosynthetic pathway of polyphenols via the enzyme phenylalanine ammonia-lyase (PAL)12. These compounds, which are rich in double bonds, help dissipate excess energy under light stress and water deficit by binding reactive molecules, thereby protecting the cell wall and enabling plants to tolerate stress. From a genetic point of view, it is known that plant biostimulants, depending on their origin, composition, and route of administration, can induce extensive reprogramming of gene expression in plants. For example, seaweed extracts activate protection against drought stress through modulation of specific signalling networks in Arabidopsis thaliana L. and Zea mays L.13,14. Plant biostimulants based on algae extracts have also been reported to influence the expression of genes involved in carbon fixation, secondary metabolism, MAPK signalling, plant hormone signal transduction and plant-pathogen interactions in Solanum lycopersicum L.15. More recently, Gitau and collaborators16 demonstrated that treatment with Chlorella microalgae led to an empirical upregulation of genes involved in abiotic stress, especially cold and water stress in tomato. Considering the importance of the baby leaf vegetables and lettuce and the interest of farmers and agronomists in plant biostimulants, our hypothesis was to assess the agronomic effects of marine microalgae extracts used as foliar spray biostimulant in baby leaf lettuce production. In addition, based on phenotypic and physiological data collected under our experimental conditions, we studied the expression of a group of genes mainly involved in plant growth and nitrogen utilization efficiency. Due to the increased interest in the use of plant biostimulants and their importance in lettuce and small leafy vegetables, this study evaluated the agronomic effects of extracts derived from marine microalgae such as Nannochloropsis gaditana and Porphyridium sp. which can be applied as foliar treatment in baby leaf lettuce production. Based on the phenotypic and physiological data collected during the experimental phase, the expression of a group of genes primarily involved in plant development was studied.

Materials and methods

Chemical characterisation of lyophilised powder marine microalgae

Two marine microalgae, Nannochloropsis gaditana (N. gaditana) and Porphyridium sp. were purchased from “NeoAlgae” (Spain) as lyophilized powder and used in this investigation. The carbon and nitrogen contents were determined according to the Dumas method17 using a CHN elemental analyzer (Perkin Elmer, Milan, Italy) and crude protein content was calculated using nitrogen-to-protein conversion factor (6.25) and expressed as percent of dry matter (% DM). The total lipids were determined by solvent extraction in a Soxhlet apparatus with hexane for 8 h according to an official method. Total and soluble carbohydrates were first extracted from the microalgae as reported in Nogami et al.18 and then quantified using an anthrone colorimetric method19. Total soluble proteins were evaluated according to Bradford20, while pigments (chlorophyll a and b and carotenoids) were quantified according to Porra et al.21. Total polyphenol content was determined using the Folin–Ciocalteu reagents according to Julkunen–Titto22 and expressed as gallic acid equivalents. The content of proline was performed according to Bates et al.23, while free amino acids were determined by the method reported in Rosen24 and expressed as leucine equivalents.

Microalgae culture and extract preparation

To prepare the aqueous extracts, 2.5 mg of lyophilized powder was taken from each microalgae sample and dissolved in 50 mL of distilled, sterilized water. The resulting solution was sonicated for 30 min using a GRANT ULTRASONIC BATH XUBA3. After sonication, the solution was centrifuged at 2000 rpm for 5 min using a THERMO IEC CL30R. The supernatant was then collected, and from each microalgae extract, dilution series were prepared at ratios of 1:10, 1:100, and 1:1000 (v/v) in water. These dilutions were subsequently applied as foliar spray biostimulants on the experimental crops.

Seed germination and phytotoxicity test

Prior to the application of the prepared plant biostimulants, pH and electrical conductivity were measured. The phytotoxicity test is widely recognized as an effective method to determine whether the extracts have phytotoxic effects or promote biostimulation in plants. The tests were carried out on garden cress, Lepidium sativum L., an annual Brassicaceae which is very commonly used for such assays due to its rapid germination within 24 h at a temperature of 27 °C and its sensitivity to substances in the substrate25. Specifically, 15 seeds were placed in a Petri dishes lined with filter paper discs sprayed with 4 mL of each microalgae solution. For each marine microalga concentration, as well as for the control (distilled water only), three replicates were performed. The plates were incubated in a dark growth chamber at a constant temperature of 27 °C for 36 h. After incubation, germinated seeds were counted, and root lengths were measured using a digital caliper. The germination index was subsequently calculated using the following formula:

$$GI\% \, = \,100\, \times \,\left( {G1/G2} \right)\, \times \,(R1/R2).$$

where G1 and G2 are germinated seeds in the sample and control and R1 and R2 are the average root length for the sample and control, respectively.

Greenhouse experiment

The baby leaf lettuce (Lactuca sativa L.) genotype “Oxana” was purchased from Maraldi S.r.l. and used in the experiment. To test the effect of the obtained plant biostimulants, the cultivation was carried out in a greenhouse located on the campus of the University of Salerno. The selected marine microalgae at the best dilution obtained from the GI test were evaluated as a foliar spray treatment on the crop and compared with two controls (one treated only with water, and one treated with the commercial microalgal biostimulant Agrialgae® using the dose reported on the label). The temperature and humidity inside the greenhouse were measured with reference to a mobile meteorological station (PCE instruments PCE-FWS 20N) placed inside the structure at a height of 130 cm above the ground. The temperature was kept constant within a range of 25–28 °C and with a relative humidity (RH) ranging from 37 to 50%. Illumination was provided by lamps (indium art 1158 Sap-T 400 Cnr-L Graphite Disano, 250 W, 230 V, 50 Hz) with a photoperiod of 16 h of light and 8 h of darkness. The treatments were applied weekly as foliar spray applications. A mixture of peat and composted soil amendment “Triplo bio” from the company TerComposti S.p.a. with the following chemical and physical characteristics was used as a substrate for growing the cultures: pH of 7, electrical conductivity of 3.0 dS m−1, bulk density of 175 kg m−3 and porosity of 85% v/v. The potting soil was placed inside plastic pots (10 cm diameter and a volume of 500 mL). Fifteen lettuce seeds were sown in each pot. Irrigation was carried out every two days throughout the cultivation cycle by distributing 60 mL of water per pot. For fertilization, only the nitrogen requirement was considered, the administration of which was divided into two applications. The first application was carried out 15 days after sowing by distributing 0.45 g N, as calcium nitrate, per pot. The second one was carried out 10 days after the first by distributing again 0.45 g of N, as calcium nitrate, per pot. Treatments with biostimulants were carried out every seven days starting on the 18th day after sowing for a total of four treatments until the end of the crop cycle. Application was carried out by foliar spray treatment by applying 30 mL of solution. The harvest was carried out 35 days after sowing, cutting the stem at the height of the collar. The experiment was carried out according to the following treatment: (T1) control-1 (CTRL), (T2) control-2 (commercial biostimulant “Agrialgae®”), (T3) Nannochloropsis gaditana 1:100, (T4) Porphyridium sp. 1:100.

Agronomic surveys

For each pot, the number of leaves was counted, and plant height was measured using a ruler. The above- and belowground fresh weight (g pot−1) were recorded with an electronic balance. Samples were then placed in an oven at 65 °C for 48 h to determine the corresponding dry weights. The harvest index (HI) was calculated as the ratio of aboveground dry weight to the total above- and belowground dry weights indicating the allocation of photosynthates between productive and vegetative tissues. Additionally, crop water productivity (CWP) and agronomic nitrogen efficiency (NAE) were calculated according the methods described by Ronga et al.26.

Physiological surveys

Canopy temperature was measured using a “FLIR EXSERIES” thermal imaging camera by pointing at the leaves at 1 cm, taking a single measurement per pot at the time of harvest. In addition to the leaf temperature, the temperature of the growth medium was recorded using a ‘HANNA INSTRUMENTS HI 145’ digital thermometer, with one measurement per pot. Stomatal conductance at the leaf level was measured using a DELTA-T SERVICE AP4 porometer. Finally, at harvest, the chlorophyll index was determined instantaneously using a SPAD meter (Konica Minolta).

Qualitative surveys

Qualitative surveys were also carried out which involved the leaf area using the portable leaf area meter Li-cor model Li-3000. In addition, was calculated the specific leaf area (SLA) which is the ratio between the leaf area and the aboveground fresh weight area expressed in cm2 g−1 and the values of mass leaf area (MLA), that is the inverse of SLA (g cm−2). Leaf colour was assessed using a CR-210 Chroma Meter (Minolta Corp., Osaka, Japan) equipped with a standard D65 illuminant. The resulting colorimetric data were expressed in the CIELAB colour space as L*, a*, and b* values. Chroma (C*) and hue angle (h°) were subsequently calculated using the following equations:

$$c = \, [a2 \, + b2]^{1/2}$$
$$h = \tan - 1\;b/a$$

RNA extraction and qRT-PCR analysis of gene expression

RNA was extracted by Trizol Thermo Fisher Scientific (Wilmington, DE, United States) according to the manufacturer’s instructions. RNA quality and concentration were evaluated by agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States), respectively. Reverse transcribed DNase-treated total RNA (1 μg) was obtained using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, United States) and random hexamers. qRT-PCR was performed by QuantStudio™ 5 Real-Time PCR Instrument. Each PCR reaction consisted of 1 μl of 1: 20 diluted cDNA, 5 μl of 2X PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, CA, United States), and 0.4 μM of each gene-specific primer. The thermal cycling conditions were 50 °C for 2 min (one step), one cycle at 95 °C for 2 min, followed by 40 cycles of two steps at 95 °C for 15 s and 56 °C for 15 s. Oligonucleotides, listed in the Table 1, were designed by using Primer3 (https://primer3.ut.ee). Three independent experiments were carried out and three technical replicates were tested for each sample. To normalize gene expression values Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene was used as internal control. Expression was calculated by the 2−ΔΔCT method27,28. Expression levels of analysed transcripts were compared to the untreated control leaves used as calibrator. qRT-PCR data were subjected to one-way ANOVA by GraphPad Prism version 8.0 (GraphPad Software, Inc.; San Diego, CA, USA).

Table 1 List of primers used for RNA extraction and qRT-PCR analysis of gene expression.
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Statistical analysis

All the collected data were subjected to analysis of variance (ANOVA) and the averages were separated by Duncan’s p < 0.005 test using the GenStat 12th programme.

Results

Chemical characterisation of lyophilised powder marine microalgae

The chemical composition of the microalgae used in this study is crucial for evaluating their effects as biostimulants. The main constituents of the two marine microalgae are reported in Table 2, while other minor components, such as phenolics, free aminoacids and pigments are listed in Tables 3 and 4. As shown, the two microalgae differ in their chemical composition. Total carbon, total protein and total lipid content are higher in N. gaditana than in Porphyridium, whereas total and soluble sugars are found in greater amounts in Porphyridium than in N. gaditana (Table 2). Total phenolics and free aminoacids are higher in Porphyridium, while soluble proteins and proline levels are higher in N. gaditana (Table 3). The two microalgae also differ in their photosynthetic pigments composition. Chlorophylls and carotenes are predominantly detected in N. gaditana, whereas phycobiliproteins, characteristic of blue and red microalgae, are exclusively present in Porphyridium and completely absent in N. gaditana (Table 4).

Table 2 Composition of marine microalgae. Principal constituents (% DM).
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Table 3 Composition of marine microalgae. Total phenolics, soluble proteins and amino acids (mg/g DM).
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Table 4 Composition of marine microalgae. Photosynthetic pigments (mg/g DM).
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Phytotoxicity test on lettuce seeds

For each aqueous extract, regardless of concentration, an average pH of 8.2 and an average conductivity of 330 μS cm−1 was measured. Following these measurements, the phytotoxicity test was performed. The Phytotoxicity tests (GI%, Germination Index) conducted on garden cress seeds yielded notable results, which are summarized in Table 5. According to this test (also referred to as Zucconi’s test), GI% values below 60% indicate phytotoxicity of the tested product. All treatments in this study showed GI% values > 60%. In particular, the most significant biostimulant effects were observed with N. gaditana at a concentration of 1:10 and 1:100 with GI% of 146.40% and 128.20%, respectively, and Porphyridium sp. at a concentration of 1:10 and 1:100 with GI% of 154.50% and 118.90%, respectively.

Table 5 Result of phytotoxicity test.
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Greenhouse treatment

Greenhouse treatments were selected based on the germination test results and the highest concentrations that reported biostimulant effects. the most effective in vivo treatments on lettuce were N. gaditana 1:100 and Porphyridium sp. 1:100 compared to the control sprayed with water only and the commercial biostimulant ‘Agrialgae®’.

Results of agronomic surveys

Table 6 shows the results of the agronomic surveys carried out at harvest. Both marine microalgae promoted greater plant height compared to the untreated control and compared to the commercial biostimulant, which reported lower values than the other treatments tested. In particular, T3 and T4 increased plant height by 40% and 17% respectively compared to the untreated control. No statistical differences were reported between the treatments for soil temperature. The number of leaves did not show statistically significant results, and no significant differences were observed among the treatments.

Table 6 Results of agronomic surveys at harvest.
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Table 7 presents the results of the production parameters performed at harvest. In addition to increasing plant height, both marine microalgae treatments increased aboveground fresh weight. Specifically, T3 increased the aboveground fresh weight by about 44% compared to the untreated control followed by T4, which increased it by about 41.5% compared to the control. The commercial biostimulant, in contrasts, reported the lowest value of this parameter. No statistically significant differences were observed among treatments for aboveground dry weight and belowground fresh weight, although T3 showed higher values than the other treatments. Furthermore, T3 increased belowground dry weight by approximately 30% compared to untreated lettuce plants.

Table 7 Results of agronomic surveys at harvest.
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Table 8 shows the latest agronomic findings at harvest. Both marine microalgae increased the above- and belowground fresh weights. Treatment of the lettuce plants with T3 favoured an increase in above- and belowground fresh weights of about 40% compared to the untreated control while T4 favoured an increase in above- and belowground fresh weights of about 37% compared to the untreated control. Once again, the commercial biostimulant reported the lowest value. Above- and belowground dry weight reported no statistically significant difference between treatments although both marine microalgae promoted a longer shelf-life. T3 reported statistically significant higher values of the harvest index. Both marine microalgae promoted crop water productivity (CWP) and improved nitrogen agronomic efficiency (NAE). Specifically, T3 and T4increased CWP by 58% and 34%, respectively, and improved NAE by 66% and 42%, respectively, compared to the untreated control.

Table 8 Results of agronomic surveys at harvest.
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Results of physiological surveys

Table 9 reports the mean values of SPAD (chlorophyll index), canopy temperature, and stomatal conductance for the different applied treatments. Regarding the SPAD index, significant differences were observed among treatments: treatment T2 showed the highest value, significantly greater than T3, which exhibited the lowest value. T1 and T4 showed intermediate values that were not significantly different from either T2 or T3. Canopy temperature varied significantly among treatments: T3 recorded the lowest temperature, significantly lower than T1 and T2. T4 exhibited an intermediate value. Regarding stomatal conductance, T3 and T4 showed significantly higher values compared to T1 and T2. This suggests an increase in stomatal activity in microalgae treatments compared to the control and the commercial biostimulant.

Table 9 Results of physiological surveys at harvest.
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Results of qualitative surveys

Table 10 shows the results of the qualitative surveys carried out at harvest. Both marine microalgae treatments increased the leaf area of lettuce. T4 increased the leaf area by 52% compared to the untreated control, followed by T3, which increased the leaf area of the lettuce by approximately 14% compared to the untreated control. Additionally, Table 10 shows that the commercial biostimulant treatment resulted in statistically significantly lower leaf area values. Specific leaf area (SLA) and mean leaf area (MLA) did not show statistically significant differences among the treatments.

Table 10 Results of qualitative surveys at harvest.
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To assess the qualitative effects of the different treatments, leaf colour was measured at harvest. The results presented in Table 11, show that the treatments did not affect the brightness index (L) but reported statistically significant differences in the red index (a) and the yellow index (b). In particular, the commercial biostimulant reported statistically significant higher values of the green index than the other treatments, while T4 and T1reported higher values of the yellow index and chroma. Finally, T4 also showed statistically significantly higher values of the hue angle, followed by the untreated control.

Table 11 Results of qualitative surveys at harvest.
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RNA extraction and qRT-PCR analysis of gene expression

In order to obtain molecular information on the effects of microalgae extracts in lettuce leaves, the expression levels of selected genes involved in major plant growth processes were assessed by qRT-PCR. The genes of interest included Cytosolic glutamine synthetase 1 (LsGS1), Chloroplast glutamine synthetase 2 (LsGS2) and Nitrate transporter 2.1 (LsNRT2.1), which are involved in nitrogen transport and assimilation. Additionally, genes involved in hormone metabolism and signalling, including Gibberellin 20 oxidase (LsGA20OX1) and Ethylene receptor 1 (LsETR1), were analysed. Lastly, genes associated with plant cell growth, such as Growth regulatory factor 5 (LsGRF5) and Expansin-A1 (LsEXPA1), were also assessed to evaluate their role in cellular expansion and growth regulation in response to microalgae treatments. Interestingly, we found that biostimulation with T3 and T4 significantly increased the expression levels of LsNRT2.1 and LsETR1 compared to the control, whereas Agrialgae® slightly reduced the transcript levels of LsNRT2.1. A significant increase in LsGS1 expression levels (2.95- fold-change) was also detected in plants treated with T3, while the expression of the chloroplast isoform LsGS2 did not change significantly between treatments. In contrast, treatments with T3 and T4 induced downregulation of LsEXPA1. Finally, biostimulant treatments did not significantly influence the expression level of LsGRF5 (Fig. 1).

Fig. 1
figure 1

Relative expression of LsETR1, LsGRF5, LsGS1, LsGS2, LsNRT2.1, LsGA20OX1 and LsEXPA1 genes in control and Agrialgae®, N. gaditana, and Porphyridium sp. treated leaves. Lettuce GAPDH served as endogenous control. Error bars represent the standard deviation of the mean of three biological replicates. Statistical significance for each gene among control and treated samples was determined by using one-way ANOVA with Duncan’s multiple comparisons test. Different letters indicate significant differences (p < 0.05). T1: control; T2: commercial biostimulant “Agrialgae®”; T3: N. gaditana 1:100; T4: Porphyridium sp. 1:100.

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Discussion

The present study demonstrates that treatments with marine microalgae extracts reported promising results in the production of baby leaf lettuce, improving several agronomic traits, including plant fresh weight and leaf number, as well as plant height and physiological parameters. While numerous studies have explored the benefits of marine macroalgae and microalgae on lettuce growth and yield, there is a lack of research specifically addressing the agronomic efficiency of marine microalgae. For instance, previous work on microalgae as biofertilizers in hydroponic systems reported that treatments with Chlorella vulgaris enhanced yield by promoting leaf expansion and biomass accumulation compared to untreated controls29. These findings are consistent with our results, which suggest that marine microalgae can reduce reliance on mineral fertilizers while maintaining or even improving plant growth. In this study, aboveground fresh weight increased in both treated groups relative to the control, resulting in higher gross commercial yield, a critical factor for growers. Moreover, treatments with T3 and T4 led to increases in leaf area of 14% and 52%, respectively, thereby maximizing photosynthetic capacity, biomass production, and overall plant development. The aboveground dry weight observed in the two treatments with marine microalgae suggests a reduced water content, which may hypothetically indicate an improvement in the product’s shelf life, an important factor for crops intended for minimally processed vegetables. Furthermore, the biostimulant treatments led to an increase in belowground dry weight, indicating the development of a more robust root system capable of more efficient water and nutrient uptake, potentially contributing to enhanced overall plant growth. These results align with those of Santoro et al.30, who reported that applications of Chlorella vulgaris and Scenedesmus quadricauda significantly improved both root and shoot biomass. According to Parmar et al.31, the use of microalgae as biostimulants has demonstrated potential in enhancing various aspects of plant health and productivity, including nutrient use efficiency and stress tolerance. In the present study, treatments with T3 and T4 increased agronomic nitrogen use efficiency (NAE) by 58% and 34%, respectively, and crop water productivity (CWP) by 66% and 42%, respectively. Additionally, baby leaf lettuce treated with T3 and T4 exhibited higher stomatal conductance values, 45% and 35.5% greater than the control, respectively, along with a reduction in canopy temperature. These physiological responses may be linked to the presence of proline in both marine microalgae. This amino acid is known for its protective roles as an osmoprotectant and reactive oxygen species (ROS) scavenger, and it may also function as a metabolic signal involved in plant development and the activation of cellular defence mechanisms32. The genetic mechanism governing all these observed phenomena is dictated by complex regulatory mechanisms. Here, we evaluated the impact of biostimulants on a panel of selected genes associated with critical plant growth processes such as cell proliferation and expansion, nitrogen transport and metabolism, and plant cell division and expansion. Interestingly, we found that extracts of T3 and T4 modulate the expression of genes involved in nitrogen uptake and assimilation, such as LsNRT2.1 and LsGS1. These changes in gene expression may contribute to the observed improvement in NAE after treatment with marine microalgae in lettuce. Indeed, both genes are crucial in enhancing plant nitrogen use efficiency33. In particular, LsNRT2.1 expression was increased by foliar spray treatment of T3 and T4, whereas LsGS1 was up-regulated exclusively by biostimulation with T3. Nitrogen transporters (NRT) are responsible for the uptake, transport, assimilation and remobilisation of nitrogen in plants. High-affinity NRT2 transporters belong to the Porter Nitrate- Nitrite family and play a key role in NO3− uptake, especially under nitrogen-limiting conditions34. In such environments, they also regulate auxin transport activity, thereby influencing root growth35. Glutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. Different cytosolic (GS1) or plastidic (GS2) isoforms have been reported in many plant species36. For example, it has been shown that overexpression of GS1 can improve NAE and yield in many crops37,38,39,40. The increase in glutamine synthase activity and nitrogen transporters after treatment with different seaweed extracts has already been associated with a positive effect on nitrogen assimilation and yield parameters in lettuce, bean, winter rapeseed and many other crops41,42. In this scenario, our data suggests that activation of these two genes contributed to triggering an increase in NAE after foliar spray treatment of algal extracts in lettuce. Based on these results, it will be useful to determine in future work whether overexpression of these genes also contributes to increased uptake of N derived from seaweed extracts by lettuce aerial parts. Noteworthy, we also observed a significant upregulation of the ethylene receptor LsETR1 in lettuce treated with extracts ofT3 and T4. compared to control and Agrialgae®. It has been reported that ETR1 is the strongest negative regulator of ethylene responses43,44. Ethylene is considered a plant hormone that normally inhibits plant growth by affecting cell elongation and accelerating senescence processes45. Considering this, upregulation of LsETR1 may promote ethylene insensitivity and contribute to the increase in lettuce biomass after biostimulation with T3 and T4. As a final remark, we anticipate that transcriptomic analyses may help to elucidate the complex mechanisms through which T3 and T4 trigger positive effects on plant growth and NAE, thereby providing valuable insights to optimize the application of these algae extracts in lettuce cultivation.

Conclusion

The use of marine microalgae on baby leaf vegetable intended for minimal processing has proven to be an excellent eco-sustainable solution, firstly because microalgae cultivation has a sustainable impact on the environment and, secondly, because their use on the crop is able to provide several benefits, in this case an improvement in the efficiency of nutrient assimilation and management of the crop’s water supply as well as an increase in the final yield. In this regard, marine microalgae, as biostimulants, seem to be interesting products to address the correct management of nitrogen fertilisation, improving nitrogen assimilation and avoiding its loss by leaching or volatilisation, and reducing water loss and thus making the plant less susceptible to environmental stress, while stimulating growth and increasing the harvested biomass. At the end of growth cycle, the measured parameters demonstrated a biostimulant effect of the marine microalgae on the tested crop, which showed greater efficiency in nitrogen assimilation and water uptake, consequently leading to increased biomass production. Furthermore, improvements were observed in qualitative traits, including enhanced productivity in terms of weight (+ 31%), number of leaves, increased plant height, and nutrient assimilation. Gene expression analysis revealed that the investigated extracts influenced the expression of genes involved in nitrogen uptake and assimilation, contributing to enhance nitrogen agronomic efficiency in lettuce. Consequently, this research demonstrates the possibility of carrying out foliar treatments with T3 and T4 as an effective and safe biostimulant for sustainable agriculture and opens new prospects for experiments on the use of marine microalgae as a foliar spray treatment for other crops.