Role of Bacteria-Derived Flavins in Plant Growth Promotion and Phytochemical Accumulation in Leafy Vegetables

Sinorhizobium meliloti 1021 bacteria secretes a considerable amount of flavins (FLs) and can form a nitrogen-fixing symbiosis with legumes. This strain is also associated with non-legume plants. However, its role in plant growth promotion (PGP) of non-legumes is not well understood. The present study evaluated the growth and development of lettuce (Lactuca sativa) and kale (Brassica oleracea var. acephala) plants inoculated with S. meliloti 1021 (FL+) and its mutant 1021ΔribBA, with a limited ability to secrete FLs (FL−). The results from this study indicated that inoculation with 1021 significantly (p < 0.05) increased the lengths and surface areas of the roots and hypocotyls of the seedlings compared to 1021ΔribBA. The kale and lettuce seedlings recorded 19% and 14% increases in total root length, respectively, following inoculation with 1021 compared to 1021ΔribBA. A greenhouse study showed that plant growth, photosynthetic rate, and yield were improved by 1021 inoculation. Moreover, chlorophylls a and b, and total carotenoids were more significantly (p < 0.05) increased in kale plants associated with 1021 than non-inoculated plants. In kale, total phenolics and flavonoids were significantly (p < 0.05) increased by 6% and 23%, respectively, and in lettuce, the increments were 102% and 57%, respectively, following 1021 inoculation. Overall, bacterial-derived FLs enhanced kale and lettuce plant growth, physiological indices, and yield. Future investigation will use proteomic approaches combined with plant physiological responses to better understand host-plant responses to bacteria-derived FLs.


Introduction
The challenges imposed by sustained climate change and global warming have had tremendous negative impacts on the global food supply chain and human health. Part of this challenge is caused by increased population growth and increased food and nutrition insecurity [1]. To feed this increased population, it is crucial that agricultural productivity must be remarkably increased within the next few years. Farmers worldwide are making efforts to increase food production either by increasing crop production acreages or by enhancing productivity on existing agricultural lands through inputs, such as fertilizer and irrigation, and adopting new methods, such as precision farming and artificial intelligence [2]. However, excessive usage of synthetic chemical fertilizers is harmful to the environment as it pollutes the soil, water, and air [3,4]. The main environmental impacts associated with fertilizers are nitrate leaching into ground water, emission of greenhouse gases, toxic heavy metal pollution in soils, and surface runoff of nitrogen (N) and phosphorus (P), leading to aquatic eutrophication [5][6][7][8].
Providing sufficient food for an increasing human population will require different strategies and approaches. The usage of plant growth-promoting (PGP) microbes as biofertilizers or  S. meliloti 1021 inoculation in lettuce seedlings exhibited significant (p < 0.05) creases in root and hypocotyl growth parameters, such as total length and total surf  S. meliloti 1021 inoculation in lettuce seedlings exhibited significant (p < 0.05) increases in root and hypocotyl growth parameters, such as total length and total surface area ( Table 2). The total root length and surface area were significantly (p < 0.05) increased by 13.7% and 14.3% in FL + lettuce seedlings, respectively, compared to FL − lettuce seedlings.

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Total hypocotyl length and surface area were higher (i.e., 7% and 4%, respectively) in FL + lettuce seedlings compared to FL − seedlings (Table 2).  Figure 2). Stem diameter was increased by 10% in FL + plants than in FL − plants (Table S1). The kale leaf elongation rate was also higher in FL + plants compared to the FL − and control plants ( Figure S1). Our results showed a significant (p < 0.05) difference in the stem diameter of kale between the FL + plants and no inoculation controls (Table S1). Chlorophyll fluorescence indices, Fv/Fo, and Fv/Fm were enhanced by bacterial-derived FLs (Table S1). The results showed Fv/Fo and Fv/Fm were slightly increased in FL + plants compared to the FL − plants, even though there was no significant difference (p > 0.05) between treatments (Table S1). Moreover, the fresh weight of kale was increased by 7% in FL + plants compared to FL − plants (Table S1).

Plant Growth, Physiology, and Yield
Kale plants were not significantly (p > 0.05) different in the number of leaves height, and stem diameter between FL + inoculated plants (FL + plants) and FL − inoc plants (FL − plants). However, the plant height and stem diameter were higher plants than in the FL − plants and control plants (Table S1; Figure 2). Stem diamet increased by 10% in FL + plants than in FL − plants (Table S1). The kale leaf elongati was also higher in FL + plants compared to the FL − and control plants ( Figure S results showed a significant (p < 0.05) difference in the stem diameter of kale betw FL + plants and no inoculation controls (Table S1). Chlorophyll fluorescence indices, and Fv/Fm were enhanced by bacterial-derived FLs (Table S1). The results showed and Fv/Fm were slightly increased in FL + plants compared to the FL − plants, even there was no significant difference (p > 0.05) between treatments (Table S1). Moreov fresh weight of kale was increased by 7% in FL + plants compared to FL − plants (Ta  Lettuce plants inoculated with FL + were shown to have increased plant height, stem diameter, chlorophyll indices, and fresh weight compared to the FL − plants, even though they were not significantly (p > 0.05) different between treatments (Table S2; Figure 2). The lettuce leaf elongation rate was also higher in FL + plants than in the FL − and control plants ( Figure S2). The number of leaves, plant height, and stem diameter were significantly (p < 0.05) increased in FL + plants compared to the control plants (Table S2). Moreover, the fresh weight of the lettuce plants was significantly (p < 0.05) increased in the FL + plants compared to the control plants (Table S2). The fresh weight of the plants was increased by 5% with bacterial FLs. Significant differences were not observed in chlorophyll fluorescence indices (i.e., the maximum quantum efficiency of PSII and potential photosynthetic capacity) between treatments. However, the chlorophyll fluorescence indices were slightly increased in the FL + plants compared to the other treatments (Table S2).

Leaf Tissue Phytochemical Analysis
In kale leaves, chlorophyll a and b and carotenoid content in the leaves were significantly (p < 0.05) increased in bacteria-inoculated plants compared to control plants, although they were not significantly (p > 0.05) different between FL + and FL − kale plants ( Figure 3A-C). FL + plants showed that chlorophylls a and b, and carotenoid contents were 35%, 35%, and 26% increased, respectively, compared to the control plants ( Figure 3A-C). Similarly, chlorophylls a and b were higher in FL + plants than FL − kale plants ( Figure 3A-C). The total flavonoid content in kale leaves was significantly (p < 0.05) higher in FL + plants by 28% and 12% more than in the FL − and control plants, respectively ( Figure 3D). The leaves of the FL + plants had significantly (p < 0.05) higher total phenolic content than the FL − and control plants ( Figure 3E). compared to the control plants (Table S2). The fresh weight of the plants was increased by 5% with bacterial FLs. Significant differences were not observed in chlorophyll fluorescence indices (i.e., the maximum quantum efficiency of PSII and potential photosynthetic capacity) between treatments. However, the chlorophyll fluorescence indices were slightly increased in the FL + plants compared to the other treatments (Table S2).

Leaf Tissue Phytochemical Analysis
In kale leaves, chlorophyll a and b and carotenoid content in the leaves were significantly (p < 0.05) increased in bacteria-inoculated plants compared to control plants, although they were not significantly (p > 0.05) different between FL + and FL − kale plants (Figure 3A-C). FL + plants showed that chlorophylls a and b, and carotenoid contents were 35%, 35%, and 26% increased, respectively, compared to the control plants ( Figure 3A-C). Similarly, chlorophylls a and b were higher in FL + plants than FL − kale plants ( Figure 3A-C). The total flavonoid content in kale leaves was significantly (p < 0.05) higher in FL + plants by 28% and 12% more than in the FL − and control plants, respectively ( Figure 3D). The leaves of the FL + plants had significantly (p < 0.05) higher total phenolic content than the FL − and control plants ( Figure 3E). In lettuce leaves, even though chlorophyll a, chlorophyll b, and carotenoid contents were not significantly (p > 0.05) different between FL + and FL − plants, chlorophyll a and chlorophyll b were higher in the FL + plants compared to the FL − plants ( Figure 4A-C). Total flavonoids and total phenolics content were significantly (p < 0.05) higher in the FL + plants compared to the FL − and control plants ( Figure 4D,E). We observed that in FL + plants, total flavonoids and total phenolics were 57% and 102% higher, respectively, than those found in the FL − plants ( Figure 4D,E). In lettuce leaves, even though chlorophyll a, chlorophyll b, and carotenoid contents were not significantly (p > 0.05) different between FL + and FL − plants, chlorophyll a and chlorophyll b were higher in the FL + plants compared to the FL − plants ( Figure 4A-C). Total flavonoids and total phenolics content were significantly (p < 0.05) higher in the FL + plants compared to the FL − and control plants ( Figure 4D,E). We observed that in FL + plants, total flavonoids and total phenolics were 57% and 102% higher, respectively, than those found in the FL − plants ( Figure 4D,E).

Leaf Micro-and Macronutrient Elemental Compositions
Plant leaf tissue elemental analysis showed that micro and macro elements found in lettuce leaf tissues were influenced by bacterial-derived FLs ( Table 3). The kale plants inoculated with FL + had the highest phosphorus, potassium, magnesium, boron, and iron contents compared to the FL − and control plants (Table 3). Nitrogen was higher in the FL − plants than in the FL + and control plants (Table 3). In the lettuce leaves, the N content was approximately the same in the FL + and control plants. However, N was increased by 9% in the FL + plants compared to the FL − plants (Table 3). Lettuce leaf tissue P was increased in the FL + plants compared to the control and FL − plants (Table 3). Leaf P content in the FL + plants was 14% higher than in the FL − plants. K was slightly reduced (4%) in the FL + plants compared to the FL − lettuce plants. Among the other nutritional, calcium, sodium, manganese, and zinc were increased by 2%, 24%, 18%, and 3% in the FL+ plants, respectively, compared to the FL − plants (Table 3).

Leaf Micro-and Macronutrient Elemental Compositions
Plant leaf tissue elemental analysis showed that micro and macro elements found in lettuce leaf tissues were influenced by bacterial-derived FLs ( Table 3). The kale plants inoculated with FL + had the highest phosphorus, potassium, magnesium, boron, and iron contents compared to the FL − and control plants (Table 3). Nitrogen was higher in the FL − plants than in the FL + and control plants (Table 3). In the lettuce leaves, the N content was approximately the same in the FL + and control plants. However, N was increased by 9% in the FL + plants compared to the FL − plants (Table 3). Lettuce leaf tissue P was increased in the FL + plants compared to the control and FL − plants (Table 3). Leaf P content in the FL + plants was 14% higher than in the FL − plants. K was slightly reduced (4%) in the FL + plants compared to the FL − lettuce plants. Among the other nutritional, calcium, sodium, manganese, and zinc were increased by 2%, 24%, 18%, and 3% in the FL+ plants, respectively, compared to the FL − plants (Table 3).

Discussion
Recent research showed that FLs play a key role in plant growth and promotion [20,24]. This is the first study to investigate the effect of bacterial-derived FLs in leafy vegetable plant growth, physiological functions, yield, and chemical composition. RF is an essential constituent in the coenzymes of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN and FAD play key roles in several flavoprotein-mediated redox reactions in different metabolic pathways [42]. They also act as essential cofactors for a multitude of mainstream metabolic enzymes that mediate hydride, oxygen, and electron transfers. RF is easily converted enzymatically or photochemically into lumichrome [21,24], which is considered a potent molecule that stimulates plant growth and development [43]. According to Dakora [20], lumichrome and RF can influence ecological cues for sensing environmental stress, plant growth factors, signals for stomatal functioning, and act as protectants or elicitors of plant defense. While the mechanisms behind these functions remain unknown, it is possible that lumichrome induced the biosynthesis of classical phytohormones that caused the observed developmental changes in the plants [20].
Many cellular processes in plants directly depend on FLs, as flavoenzymes catalyze a broad spectrum of different reactions [44][45][46]. The present study showed that FLs facilitated plant growth and promoted metabolic activities from the seedling to the harvesting stage. The S. meliloti 1021 (FL + strain) seedlings showed the highest root and hypocotyl growth compared to the S. meliloti 1021∆ribBA (FL − strain). Moreover, FL factors can both directly and indirectly enhance plant growth. For example, flavoproteins influenced Arabidopsis plant growth and development of hormonal levels, especially the metabolism of auxin and cytokinin [47].
A significant number of flavoproteins also play key roles in the primary and secondary metabolism of carbon, nitrogen, or sulfur assimilation, amine metabolism as well as the plant's response to abiotic stress [47][48][49][50]. Interestingly, the results in the present study also evidenced that inoculation of FLs secreting S. meliloti 1021 improved plant physiological processes, mainly photosynthesis, leading to the enhanced yield of the kale and lettuce plants. Chlorophyll fluorescence indices are broadly used to determine the photosynthesis performance of plants [51]. Therefore, this finding can be attributed to increased Fv/Fo and Fv/Fm following the FLs secreting S. meliloti 1021 inoculation, although they were not significantly different (p > 0.05) between treatments. Chlorophyll has a key role in photosynthesis and the overall productivity of crops [52][53][54] and its level in plant leaves reveals the potency of the photosynthetic process. From the present study, quantitative analysis of photosynthetic pigments clearly showed that chlorophylls a and b (i.e., primary photosynthetic pigments) were higher in FL + plants than in the FL − plants and control plants. Carotenoids are accessory photosynthetic pigments that cannot transfer sunlight energy directly to the photosynthetic pathway, however, must pass their absorbed energy to chlorophyll and function as a photoprotector under excessive light incidence [55,56]. They also supply substrates for the biosynthesis of key plant growth regulators, such as abscisic acid and strigolactones [57,58]. The increased carotenoid content in kale and lettuce leaves with FLs secreting bacterial inoculation might have contributed to increased plant growth, physiological activities, and yield. Other than these photosynthetic pigments, plants require a balance of micro-and macronutrients to effectively perform photosynthesis [59]. Nitrogen (N) is one of the most important elements that determines plant growth [60,61]. Furthermore, N is found in the composition of numerous compounds, including amino acids, which make up proteins, nucleic acids, and nitrates and nitrites [62]. Previous studies have shown that leaf N content positively affects photosynthesis [63] through its influence on photosynthetic enzymes, pigment content, and the size, number, and composition of chloroplasts [64]. The present results also indicated that in lettuce, the percentage N was more increased in FL + plants than in FL − plants. In addition to N, we found out that phosphorus (P) in lettuce was enhanced by FL + . In kale, potassium (K) was increased in FL + plants compared to the other treatments. Both P and K are essential elements that determine plant growth and productivity and are involved in several physiological processes, such as the activation of numerous enzymes, and the regulation of the cation-anion balance [65]. These physiological processes are essential for cell division and the development of the growing tip of plants [66].
Phenolic compounds are inherent in plants and are known as potential agents for preventing and mitigating many oxidative stress-related diseases [67]. Our results indicated that total phenolics and flavonoids in kale and lettuce were increased in FL + plants. We demonstrated that FL-secreting bacterial inoculation in plants can be associated with enhanced phytochemical content in both kale and lettuce plants, as previously reported for herbal tea by Fu [67]. In addition, FL molecules, such as RF, can act as antioxidant agents, which could facilitate more benefits to plants through the enhancement of their leaf nutritional quality [68]. These findings suggest that the FLs secreting bacterial application can be considered as a greener and eco-friendly plant growth-promoting technique to increase the productivity and nutritional qualities of leafy vegetables and promote their health benefits.

Materials and Methods
This study was conducted in the Compost and Biostimulant Laboratory, Department of Plant, Food and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Canada.

Inoculum Preparation and Seed Treatment
Kale (Brassica oleracea var. acephala 'Dwarf Green Curled') and lettuce (Lactuca sativa 'Simpson') seeds were purchased from Halifax Seeds (Halifax, NS, Canada). S. meliloti strains (FL + , FL − ) were grown for 5 days in MMNH 4 medium plates. Cultures were scrubbed and suspended in sterile water to prepare bacterial solutions with an OD 600 of 0.6. The lettuce and kale seeds were sterilized with 10% sodium hypochlorite for 10 min and washed thoroughly three times with sterile distilled water (ddH 2 O). The cleaned seeds were treated by soaking in solutions of FL + , FL − , and sterile ddH 2 O, which was the control treatment, for 90 min. The treated seeds were air-dried under a laminar flow for 30 min. Ten seeds per treatment were placed in a 16.5 × 18 cm CYG germination pouch (Mega International, Roseville, MN, USA) with six biological replicates, and saturated with 25 mL of the respective solution per seed treatment.

Seedling Growth Components
The pouches were incubated in the dark for two days at 22 • C, before being transferred into a growth chamber under a day/night temperature regime of 25 • C/20 • C, 16/8 h day/night illumination, light intensity of 300 µmol m −2 ·s −1 , and relative humidity of 70%. The experiment was arranged in a completely randomized design with six replications and repeated twice. Every day, 3 to 5 mL of sterile ddH 2 O was added to maintain regular moisture, for nine days.
Root and hypocotyl morphological parameters were analyzed for 5 seedlings per pouch on the 9th day using a root scanning apparatus STD4800 Scanner, Epson Perfection V850 Pro, equipped with WinRHIZO™2000 software (Regent Instruments, Quebec, QC, Canada). The WinRHIZO™2000 software recognized scanned digital root and hypocotyl images and analyzed morphological traits, such as total lengths and total surface areas of root and hypocotyl [71][72][73].

Statistical Analysis
All statistical analyses were performed using Minitab software version 20 (Minitab Inc., State College, PA, USA). Data obtained with different treatments were subjected to a one-way analysis of variance (ANOVA). Post hoc means were compared using Fisher's least significant difference (FSD) at α = 0.05.

Greenhouse Environment and Experimental Design
Pot experiments were carried out from March to June and September to December 2022 in the Department of Plant, Food, and Environmental Sciences greenhouse as two trials with seven biological replicates. Seeds selection and sterilization processes were the same as described in Section 4.1.2. The seedlings of each plant species were raised for 4 weeks in a growth chamber with a day/night temperature regime of 24 • C/22 • C, 16/8 h d −1 illumination at a light intensity of 300 µmol m −2 ·s −1 , and relative humidity of 70%. The seedlings were transplanted into 15 cm diameter plastic pots with a 10.5 cm depth. The mean day/night temperature cycle in the greenhouse during the experiment from transplanting to final harvest was 28 • C/20 • C with 71% relative humidity. Supplementary lighting was set to a 16 h day length cycle using a 600 W HS2000 high-pressure sodium lamp with NAH600.579 ballast (P.L. Light Systems, Beamsville, ON, Canada). The growing medium was a general-purpose Pro-mix BX potting mix and transplanted seedlings were acclimatized for a week under greenhouse conditions before treatment application. Treatments were inoculation with FL + strain, FL − strain, and a control (without inoculation). Inoculum was prepared as described in Section 4.1.2 and applied 5 mL per pot at 2-week intervals.

Plant Growth, Physiology, and Yield Parameters
The number of green leaves for each plant was counted at 4 weeks after transplanting (AT). Plant height was measured from the apical meristem to the collar of the stem. The fourth leaf of each plant was tagged to estimate the plant growth rate by measuring the leaf elongation rate. Chlorophyll fluorescence indices were determined from three leaves per plant at 30 days AT using a chlorophyll fluorometer (Optical Science, Hudson, NH, USA). Leaves were first dark adapted for 25 min before the initial fluorescence yield (Fo) was measured, after that the maximum chlorophyll fluorescence (Fm) emitted was measured during a saturating light pulse. Variable chlorophyll fluorescence (Fv) was calculated as Fv = Fm − Fo. Fluorometric parameters, including maximum quantum efficiency (Fv/Fm), and potential photosynthetic capacity (Fv/Fo), were determined at each saturating pulse. Fresh weights of leaf samples from each treatment were recorded at the final harvest, i.e., 35 days AT, using a portable tabletop balance (VWR-2002P2, VWR International, Mississauga, ON, Canada). Harvested leaves were composed of leaf blades attached to petiole that had ≤5% blemishes or damage on the leaf surfaces. Those with blemishes or damage > 5% of the total leaf surface area were culled and not considered edible or marketable and were not included in the yield data. The leaves were dried in a 52100-10 Cole-Parmer mechanical convection oven dryer (Cole-Parmer Instrumental Company, Vernon Hills, IL, USA) at 65 • C to constant weights for 72 h, to determine leaf tissue micro and macro elements at the Nova Scotia Department of Agriculture Laboratory, Truro, NS, using inductively coupled plasma mass spectrometry (PerkinElmer Perkin Elmer 2100DV, Waltham, MA, USA).

Chlorophylls a and b, and Carotenoid Determination
Chlorophylls a and b, and carotenoid contents in kale and lettuce leaves were estimated as described by [74,75], with some modification. Ground leaf tissues (200 mg) were separately homogenized in 2 mL of 80% acetone. The mixture was centrifuged at 13,000× g for 15 min and the absorbance of the supernatant was measured at 646.8, 663.2, and 470 nm against 80% acetone as a blank using a Bio-Tek Synergy H1 Hybrid Multi-Mode Reader and the Gen5 software application version 3.08 (BioTek ® Instruments, Inc., Winooski, VT, USA). Leaf chlorophylls a and b, and the total carotenoid contents of leaves were calculated by the following formulas and were expressed as a µg/g fresh weight (FW). Total phenolic content (TPC) was estimated using the Folin-Ciocalteu method, in terms of gallic acid equivalent (GAE) in mg/g of the extract, as reported by [76], with some modifications. A total of 200 mg of kale and lettuce ground leaf tissues were separately added to 2 mL of ice-cold 95% methanol and incubated at room temperature for 48 h under dark conditions. The mixture was centrifuged at 13,000× g for 5 min before 100 µL of the supernatant was added to 200 µL of 10% (v/v) Folin-Ciocalteau reagent. The mixture was incubated in the dark at 25 • C for 2 h, after the addition of 800 µL of 700 mM sodium carbonate (Na 2 CO 3 ). The absorbance of the supernatant was measured at 765 nm, using 95% (v/v) methanol as the blank. TPC was calculated using a gallic acid standard curve and expressed in terms of GAE (mg) per FW (g).

Total Flavonoid Determination
Total flavonoid content (TFC) was calculated using the aluminum chloride (AlCl 3 ) colorimetric method, as described by Chang [77] and Pourmorad [78], with some slight modifications. Ground kale and lettuce leaf samples (200 mg) were separately mixed with 2 mL of ice-cold 95% methanol and centrifuged at 13,000× g for 15 min. The reaction mixture containing 1.5 mL of 95% methanol, 0.1 mL of 10% AlCl 3 , 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water was added to 500 µL of the supernatant. The solution was incubated at room temperature for 30 min and the absorbance was measured at 415 nm, using 95% (v/v) methanol as the blank without AlCl 3 . For TFC determination, quercetin was used to make the standard calibration curve and it was expressed in µg quercetin/g FW.

Statistical Analysis
All statistical analyses were conducted using Minitab software version 20 (Minitab Inc., State College, PA, USA). Data obtained with different treatments were subjected to a one-way analysis of variance (ANOVA). Post hoc means were compared using Fisher's least significant difference (FSD) for plant growth, physiology, and yield parameter analysis and Tukey test for phytochemical analysis at α = 0.05. GraphPad Prism 9 software was used for visualization purposes.

Conclusions
This is the first study to examine the role of bacterial-derived FLs on plant growth and the development of green leafy vegetables. The results revealed that PGP from bacterialderived FLs can enhance kale and lettuce growth and the development from the seedling to the harvest stage. The present results indicated that inoculation with a FL + wildtype strain significantly increased the growth of kale and lettuce plants compared to inoculation with a FL − mutant strain, which secrete fewer FLs than S. meliloti 1021. The total phenolics and total flavonoid contents in the kale and lettuce leaves were significantly increased with bacterial FLs secretion. Two of the primary elements, nitrogen, and phosphorus, were increased by the bacterial-derived FLs in lettuce. Moreover, inoculation with FL + apparently improved plant growth better than FL − . This study has the great potential to aid the development of a new methodology for improving the health and productivity of agriculturally important crops through inoculation of bacterial-derived FLs. However, in future investigations, we will use proteomic approaches combined with plant physiological responses to better understand host-plant responses to bacterial-derived FLs. Moreover, it will be important to investigate how bacterial-derived FLs impact on central carbon metabolism in kale and lettuce plants.

Data Availability Statement:
The data for this study are available from the corresponding author upon reasonable request.