3.1. Effects of different LED lights on root growth and morphology in AMHRCs
Generally, the long-term exposure of plant in vitro cultures to LED lights with different spectral qualities may cause diverse influences on growth and morphology of cells/tissues/organs (Landi et al. 2020). Therefore, the root growth and morphology in AMHRCs under different colors of LED lights (red, green, blue, RGB, and white) and dark were investigated in the present study. In contrast to the typical characteristics of root growth (lack of geotropism) in dark cultures, LED light treatment regardless of colors resulted in the light-avoidance growth of hairy roots, i.e., root growth toward the medium being evident (Fig. 1). As shown in Fig. 2A and B, all light treatment significantly promoted root growth in terms of fresh/dry weights. However, the effects of different colors of LED lights on root growth varied significantly, with the magnitude of biomass productivity during the plateau period (48 to 62 days) being in the order of red > RGB > green > blue > white > dark (Fig. 2B). The observation of micromorphology of hairy roots was beneficial for understanding the effects of LED lights on their growth. Interestingly, all light treatment was found to induce the formation of more root hairs in comparison with dark (Fig. 3), which was similar to the previous report that the root hair formation in Arabidopsis thaliana was strongly stimulated by light illumination (Simone et al. 2000). Accordingly, the enhanced density of root hairs upon LED light treatment might contribute to the uptake of nutrients in the medium thus promoting the root growth in AMHRCs.
3.2. Effects of different LED lights on phytochemical accumulation in AMHRCs
Apart from the influence on plant growth and morphology, the long-term light exposure can trigger plant defense response to cope with light stress by enhancing the synthesis of photoprotective compounds that are generally possess biological/pharmacological activities, such as flavonoids, carotenoids, and terpenoids (Alrifai et al. 2019; Landi et al. 2020). It is well documented that the supplementation of different colors of LED lights could induce the production of bioactive secondary metabolites in various plant in vitro cultures (Hashim et al. 2021). Nevertheless, the effects of LED lights on phytochemical production are species-specific (Landi et al. 2020). Accordingly, the effects of different colors of LED lights (red, green, blue, RGB, and white) on the production of high-value isoflavonoids (CA and FO) and astragalosides (AG IV and AG I) in AMHRCs need to study in the present study.
It is clearly observed from Fig. 4 that blue and red LED lights exhibited superiority in promoting the accumulation of four target compounds in comparison with dark, which was consistent with many findings that blue and red lights were effective in stimulating the production of secondary metabolites in plants (Alrifai et al. 2019; Landi et al. 2020; Zhang et al. 2020). Moreover, blue LED light was found most effective in enhancing the accumulation of isoflavonoids and astragalosides, which might be ascribed to that blue light could cause more severe light stress in plant cells due to its higher photon energy (Alrifai et al. 2019). Additionally, contents of all target compounds in blue-grown AMHRCs were noticed to increase significantly after 41 days (Fig. 4). This supported that light, acting as a unique environmental factor that can interfere with plant metabolism, required the long-term exposure to stimulate the biosynthesis of secondary metabolites (Landi et al. 2020). Overall, the results obtained here, together with the findings of “3.1” section, demonstrated the promising potential of blue light that could achieve the purpose of both improving the productivity of root biomass and enhancing the accumulation of medicinally important compounds in AMHRCs.
3.3. Effects of different initial inoculum sizes on root growth and phytochemical accumulation in AMHRCs under blue LED light
The initial inoculum size of cells/tissues/organs is a crucial factor that determines the productivity of biomass and secondary metabolites in plant in vitro cultures due to the limited nutrients in culture medium (Isah et al. 2018). In this regard, it is necessary to study the effects of different initial inoculum sizes (0.2%, 0.4%, 0.6%, and 0.8%) on root growth and phytochemical accumulation in AMHRCs under the selected blue LED light. As exhibited in Fig. 5A and B, different inoculum sizes indeed had a significant effect on fresh/dry weights of hairy roots. The magnitude of biomass productivity during the plateau period (55 to 76 days) was in the order of 0.6% > 0.8% > 0.4% > 0.2% (Fig. 5B). As shown in Fig. 6, the accumulation profiles of four target compounds were also significantly influenced by different inoculum sizes. For the two isoflavonoids (CA and FO), both yields were highest during the period of 48 to 62 days for inoculum size of 0.6% (Fig. 6). For the two astragalosides (AG IV and AG I), inoculum size of 0.8% was found more favorable for their accumulation, but both yields were also at higher levels from 48 to 55 days for inoculum size of 0.6% (Fig. 6). Overall, the initial inoculum size of 0.6% with exposure time of 55 days under blue LED light was determined as the appropriate conditions for obtaining the optimal productivity of root biomass and high-value phytochemicals in AMHRCs, which provided a valuable reference for the possible industrial application in the future.
Generally, hairy roots are cultured under dark conditions for growth and phytochemical production (Chandra and Chandra 2018). In this study, AMHRCs cultured in darkness under the aforementioned conditions (inoculum size of 0.6% and 55 days) were used as control to know the augmentation in phytochemical yields and biomass productivity in blue-light grown AMHRCs. Four target compounds in extracts of control and blue-light grown AMHRCs were determined by a developed UPLC-MS/MS method via screening of the specific precursor ion-to-product ion transitions, including [M-H]−283.1 → 268.1 for CA (Fig. 7A), [M-H]− 267.1 → 252.1 for FO (Fig. 7B), [M + Na]+ 807.5 → 627.4 for AG IV (Fig. 7C), and [M + Na]+ 891.5 → 711.5 for AG I (Fig. 7D). After calculation, yields of CA (153.97 ± 8.72 µg/g DW), FO (106.59 ± 4.61 µg/g DW), AG IV (206.49 ± 16.24 µg/g DW), and AG I (1431.26 ± 76.83 µg/g DW) increased by 3.17-fold, 2.66-fold, 1.78-fold, and 1.52-fold in blue-light grown AMHRCs as compared with control (48.52 ± 3.86 µg/g DW of CA, 40.11 ± 3.10 µg/g DW of FO, 115.96 ± 7.21 µg/g DW of AG IV, and 940.47 ± 55.56 µg/g DW of AG I), respectively. Moreover, the productivity of root biomass (12.68 ± 1.57 g/L) in blue-light grown AMHRCs was 1.40-fold higher than control (9.03 ± 0.49 g/L). The obtained findings here indicated that the simple supplementation of blue LED light could make AMHRCs industrially attractive as plant factory in controlled growing systems for obtaining higher productivity of root biomass and valuable isoflavonoids and astragalosides.
3.4. Contents of FAA and RS in AMHRCs under blue LED light
It is known that secondary metabolites such as flavonoids and terpenoids are derived from glycolysis, TCA cycle, pentose phosphate pathway, aliphatic amino acids, and aromatic amino acids (Aharoni and Galili 2011). Additionally, RS can provide energy after glycolysis, which is favorable for biosynthesis of secondary metabolites to counteract environmental stresses (Saddhe et al. 2020). As shown in Fig. 8A and B, contents of both FAA and RS in blue-light grown AMHRCs decreased significantly after 34 days. Meanwhile, yields of four target compounds in blue-light grown AMHRCs were found to increase obviously after 34 days (Fig. 6), which suggested that the enhanced accumulation of isoflavonoids and astragalosides might require more consumption of primary metabolites such as FAA and RS. In other words, the light stress in AMHRCs caused by long-term exposure of blue LED light could direct the primary metabolic flow toward the biosynthesis of photoprotective secondary metabolites such as isoflavonoids and astragalosides.
3.5. Antioxidant response in AMHRCs under blue LED light
Blue LED light was reported to have higher photon energy that likely induced the photooxidative stress in plant cells (Alrifai et al. 2019). It is known that plant can cope with oxidative stress mainly by modulating antioxidant enzyme system (Gill and Tuteja 2010). H2O2 is considered as the most stable reactive oxygen species (ROS), and CAT is able to catalyze the conversion of H2O2 to oxygen and water (Gill and Tuteja 2010). As shown in Fig. 8C and E, the change in H2O2 content was noticed to be negatively correlated with that in CAT activity, which indicated that antioxidant enzyme system was indeed activated as a positive-feedback response to scavenge the excessive ROS in AMHRCs caused by blue LED light. In addition, there was no significant change in MDA content along with POD activity (except 62 days) during the entire period (Fig. 8D and E), which suggested that the accumulation of intracellular ROS in blue-light grown AMHRCs did not reach the level that could trigger the lipid oxidation on cell/organelle membranes. Moreover, it is worth mentioning that H2O2 is an important signal molecule in plants that can be involved in the acclimatory signal transduction responsive to environmental stresses (Gill and Tuteja 2010). It was reasonable to speculate that the high level of H2O2 in blue-light grown AMHRCs at 48 days could act as the secondary messenger that possibly activated light signal transduction.
3.6. Expression of biosynthesis genes in AMHRCs under blue LED light
Upon the illumination of blue light, the photoreceptors i.e., cryptochromes in plants that can transduce light information to downstream signaling, thereby activating the expression of genes involved in regulating plant growth, development, and secondary metabolism (Alrifai et al. 2019; Landi et al. 2020; Xu 2020). Thus, the investigation of transcriptional profiles of enzyme genes involved in biosynthesis pathways of isoflavonoids (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, and I3′H) and astragalosides (AACT, HMGR, MK, PMK, MVD, IDI, FPS, SS, SE, and CAS) would contribute to shedding light on molecular events associated with the enhanced accumulation of isoflavonoids and astragalosides in blue-light grown AMHRCs.
As shown in Fig. 9A, the expression of all tested genes in isoflavonoid biosynthesis pathway was gradually activated from 20 to 48 days in AMHRCs upon blue LED light treatment, which should be responsible for the increasing accumulation of CA and FO during this period (Fig. 6). Factually, the photoactivated cryptochromes can enhance the production of a master regulator of light signaling i.e., HY5 (ELONGATED HYPOCOTYL5), which is able to bind to cis-acting elements such as G-box and ACE-box in promoters of structural genes in flavonoid biosynthesis pathway thus promoting their transcription (Xu 2020; Zhao et al. 2022). The superior expression of CHI (10.40-fold increase) and CHR (9.17-fold increase) at 48 days might be attributed to the mechanism stated above. Notably, HMGR exhibited the tremendous transcriptional abundance (96.44-fold increase at 48 days) among all investigated genes in astragaloside biosynthesis pathway (Fig. 9B), which suggested that this gene might play a key role in the induction of astragaloside biosynthesis in AMHRCs under blue LED light. It was reported that phytochrome-interacting factor 3 (PIF3, a basic helix-loop-helix transcription factor) could bind to the G-box in the promoter of HMGR (Kim et al. 2013). As inferred, blue LED light might activate the light-responsive PIF3 thus remarkably up-regulating HMGR expression for promoting AG IV and AG I biosynthesis in AMHRCs. Overall, the results obtained here demonstrated that blue LED light could significantly activated the transcription of biosynthesis genes (such as CHI, CHR, and HMGR) via cryptochrome-mediated light signal transduction for the enhanced accumulation of isoflavonoids and astragalosides in AMHRCs.