He–Ne laser accelerates seed germination by modulating growth hormones and reprogramming metabolism in brinjal

A plant’s ability to maximize seed germination, growth, and photosynthetic productivity depends on its aptitude to sense, evaluate, and respond to the quality, quantity, and direction of the light. Among diverse colors of light possessing different wavelengths and red light shown to have a high impact on the photosynthetic and growth responses of the plants. The use of artificial light sources where the quality, intensity, and duration of exposure can be controlled would be an efficient method to increase the efficiency of the crop plants. The coherent, collimated, and monochromatic properties of laser light sources enabled as biostimulator compared to the normal light. The present study was attempted to use the potential role of the He–Ne laser as a bio-stimulator device to improve the germination and growth of brinjal and to investigate the possible interactions of plant and laser photons. A substantial enhancement was observed in germination index, germination time and seed vigor index of laser-irradiated than control groups. The enhanced germination rate was correlated with higher GA content and its biosynthetic genes whereas decreased ABA content and its catabolic genes and GA/ABA ratio were noted in laser-irradiated groups during seed germination than control groups. Further the expression of phytochrome gene transcripts, PhyA and PhyB1 were upregulated in laser-irradiated seedlings which correlate with enhanced seed germination than control. Elevated levels of primary metabolites were noted in the early stages of germination whereas modulation of secondary metabolites was observed in later growth. Consequently, significantly increased photosynthetic rate, stomatal conductance, and transpiration rate was perceived in laser-irradiated seedlings compare with control. The current study showed hormone and phytochrome-mediated mechanisms of seed germination in laser-irradiated groups along with the enhanced photosynthetic rate, primary and secondary metabolites.


RNA extraction and quantitative real-time PCR analysis.
The RNA was quantified using nanodrop spectrophotometry (Thermo Fisher Scientific, USA) and stored at -80 °C till further use. Prior to the cDNA conversion, 2 µg of RNA was treated with 2 U DNase I (RNase-free) (Invitrogen) as per the manufacturer's protocol. The treated RNA samples were reverse transcribed to cDNA using high capacity cDNA transcription kit (Applied Biosystems Inc., Foster City, CA) and stored at -80 ºC until further use. Quantitative Real-time PCR was performed using the KAPA SYBR FAST qPCR kit on the 7500 Fast real-time PCR system (Applied Biosystems). The relative transcript level of GA (GA3ox1 and GA3ox2), ABA (CYP707A1 and CYP707A2) and phytochrome (PhyA, PhyB1 and PhyB2) was calculated by the comparative ΔCt and normalized to 18s rRNA transcript levels. The primers were designed by Primer 3 Input (version 0.4.0) and the primer sequences are listed in Supplementary Table 1.
Electrospray ionization (ESI) was used as ion source with the following parameters: Capillary voltage 3000 V, dry gas temperature 350 °C and dry gas flow rate 8 L/min. The profiling was performed in both positive and negative ESI mode in duplicates. The ion range was given within a range of 60 to 1500 mass to charge ratio (m/z) by the Mass Hunter Workstation Software version B.04.00 (Agilent Technologies, Santa Clara, California, United States).

The metabolic pathway analysis
The pathway analysis was carried out using Metaboanalyst (MetPA) with the identified metabolites from each developmental stage to identify the most relevant metabolic pathways involved in response to laser irradiation. The compound name was provided for the analysis with Arabidopsis thaliana as a pathway library. Overrepresentation analysis was performed using the hypergeometric test to avoid the repetition of compounds provided. The pathway topology analysis was performed to estimate the node importance using Relativebetweenness centrality.

Data Processing and Analysis
The parameters used were as follows: minimum absolute abundance as 5000 counts, a minimum number of ions was 2, retention time window as 0.5 min and the mass window was 2 mDa. The normalized data were subjected to Principal Component Analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) using Metaboanalyst software. Further, the OPLS-DA tool used for dimension reduction and identification of spectral features for group separation was applied. The metabolites present at least in 50% of the samples from each experimental group were subjected to metabolite identification via plant metabolic network database (PMN 12.0) in addition to pre-published data, and other databases (METLIN). The abundance values of metabolites from control and irradiated groups were transformed into log10 values and subjected to statistical analysis an unpaired t-test (Graphpad Prism 5.0) and represented. Pathway enrichment analysis and correlation analysis of the identified metabolites was performed using Metabolomics Pathway Analysis (MetPA) using MetaboAnalyst software 3.0.

Altered germination traits in response to laser irradiation
The germination curve, index, mean germination time and seed vigour index were noted from the control and laser-irradiated groups.
A significant elevation was observed in the germination curve and index of the laser-irradiated group over control (**p<0.01) ( Figure S1a,b).
Mean germination time was significantly reduced in the laser-irradiated groups (**p<0.01) ( Figure S1c). The lower germination time indicates the rapid seed germination and seed vigour index, showed the superior seed germination, as well as rapid seedling growth, was highest for laser-irradiated groups as compared to un-irradiated control (***p<0.001) ( Figure S1d).

Identification and quantification of primary metabolites
The organic acids and the intermediates of TCA cycles such as succinate, fumarate, citrate/isocitrate, and malate were exhibited a significant low abundance in the laser-irradiated seeds after 24 h incubation ( Figure S2a), whereas the abundance level of these metabolites elevated significantly during the later stage of seedling development ( Figure S2b-e). The most predominant fatty acids in the brinjal seed, linoleic acid was reported in the study throughout the developmental stages. During the initial days of germination (1 DAI), the level was lower in the laser-irradiated seeds, which was gradually increased by 7 DAI ( Figure S2b), and linoleic acid was not detected in the leaves of the seedlings. Besides, the essential fatty acids such as stearic acid and arachidic acids showed an elevated level from the initial stages to later stages of seedling development. The stearic acid was detected in 7 and 28 DAI, and shown an up-regulation. A higher abundance of arachidic acid was detected in the 14 and 21 DAI laser-irradiated group ( Figure S2c and d).

The metabolic pathway analysis
In contrast, the intermediates of TCA cycles were down-regulated in the laser-irradiated seeds ( Figure S3). The pathway analysis of identified metabolites from 7 DAI has also altered 21 pathways, which is similar to 1 DAI. In contrast to the previous phase of development, the intermediates of the TCA cycle were highly up-regulated at 7 DAI groups and the intermediates in starch and sucrose metabolism were constantly up-regulated.
The flavonoid biosynthesis pathway has also been found to be up-regulated due to laser irradiation ( Figure S3b Figure   S3e). The flavonoid biosynthesis intermediates were found to be altered with higher accumulation in the 28 DAI groups. Besides, the stilbene, diarylheptanoid and gingerol biosynthesis, and carbon fixation in the photosynthetic biosynthesis pathway were also highly up-regulated in the laser-irradiated seedlings. Further, the comprehensive metabolic map was constructed and it is showing the identified primary and its associated secondary metabolites with the abundance value of laser-irradiated seeds and seedlings from 1-28 DAI ( Figure   S4a-e).     Gulla in response to He-Ne laser and un-irradiated control (n=3). Data are expressed as mean ± SD and significant at ***p < 0.001, **p< 0.01, and *p< 0.05 compared with un-irradiated control. Figure S6. Diagrammatic representation of He-Ne laser source for seed irradiation.