Profiles of Semi-Polar Metabolites from Leaves of In Vitro- Derived Plants of Indonesian Pepper Varieties (Capsicum annuum) after Gamma Irradiation Treatments

Gamma irradiation has been widely utilized for mutation breeding as it induces random mutations in plant cells. Due to limitations on other breeding approaches, including cross-breeding and transgenic plants, gamma irradiation-induced mutation breeding has regained its popularity among breeders and scientists. Untargeted metabolomics analysis can be used to profile any perturbation between untreated samples and treated sample groups without having prior knowledge of particular metabolites, including semi-polar metabolites, which cover phenolic acids, flavonoids, glycosylated steroids, alkaloids, and other glycosylated species. This study aimed to profile semi-polar metabolite modification in leaves of plants derived from in vitro cultures after gamma irradiation treatments in Indonesian chili pepper varieties. Seeds of Laris and Kopay were exposed to gamma-ray doses (0, 100, 200, 300, 400, and 500 Gy), after which they were germinated and cultivated in vitro. Plantlets were acclimated in the greenhouse and leaves were collected for metabolite analysis at 80 days after the germination of irradiated seeds. Semi-polar metabolites from leaves were extracted using methanol and the extracts were subjected to LC-MS analysis. Results showed that the composition and levels of semi-polar metabolites of Laris 100, 200, 300, 400, and 500 Gy were similar to Laris 0 Gy (control; unirradiated seeds), although several abundancies of that of 200 Gy gamma irradiation dose were different than control. Except at dose 400 Gy, Kopay at 0 Gy, 200 Gy, and 300 Gy also showed similar metabolite profiles, indicating that gamma-ray doses did not induce mutation at genes regulating metabolite biosynthetic pathways. In contrast, Kopay 400 Gy showed low levels of terpenoids and flavonoids, indicating that a 400 Gy dose of gamma ray may affect the upstream part of the shikimate biosynthetic pathway, resulting in low levels of precursors at the upstream biosynthetic pathway of terpenoids and flavonoids. Hence, the accumulation of terpenoids and flavonoids was very low. These findings provide insights into the effect of gamma irradiation for mutation breeding that may be important for future pepper breeding programs.


Introduction
Peppers, Capsicum, are one of the important horticultural commodities in Indonesia used as a condiment in cooked foods, spices, and herbal supplements for health problem treatments.Of five Capsicum species, Capsicum annuum is widely cultivated in Indonesia, consisting of numerous varieties developed from local varieties and breeding programs.The varieties are popular sources of sweet and hot peppers; thus, they are economically essential in the market and for consumers.Total production of fresh peppers in Indonesia reached 2,7 million tonnes in 2021, comprised of bird peppers and red peppers [1,2].This 1255 (2023) 012057 IOP Publishing doi:10.1088/1755-1315/1255/1/012057 2 production value was categorized as the third major production in the world.Kopay and Laris are two Indonesian long-red-fruited pepper varieties.Kopay is one of the valuable pepper varieties in West Sumatra.It has long fruits and reaches up to 33 cm, which becomes its superior quality trait [19].Laris is a commercial pepper variety with some important quality traits, such as high yields (7.5 tons/ha) and anthracnose resistance [20,21].
While the total production of fresh peppers has already been extensive, market demands are always growing as the population increases yearly.Thus, the total production of fresh peppers should be escalated to cope with the market demands.Agricultural intensification by applying fertilizers, pesticides, and technologies could be an option to increase the level of plant yields [3].However, these applications work only under optimized conditions.Global climate change increases the tension in the environment, including extreme temperatures and altered rainfall patterns.This leads to drought, floods, and increased crop pathogens and pests.Plant breeding is driven by the selection of crops with improved yields while adapting to climate change [4].In addition, plant breeding aims to alter crop traits through traditional breeding (crossing) and modern breeding (mutation using mutagen or genetic engineering).
Gamma-ray irradiation has been widely used as a physical mutagen to induce crop mutation.The gamma-ray ions will interact with plant cell molecules causing substantial damage to DNA or chromosomes, called genetic mutation [5].The alteration in irradiated plants could be seen in plants' morphology, anatomy, biochemistry, and physiology, which is dependable on the irradiation doses.Also, it has been shown to enhance the production of scavenging damaging reactive oxygen species (ROS) inside plant cells and in plant defence mechanisms against numerous stresses, such as UV radiation [6,7].In C. annuum, gamma-ray irradiation improved the adaptability against whitefly transmitted begomo virus [8].Gamma-ray irradiation has been reported to increase the accumulation of phenolics and flavonoids in leaves and fruits of pomegranates [5] and in in vitro callus culture of rosemary [6].Gammaray irradiation could be combined with in vitro culture, defined as in vitro mutagenesis.It allows efficiencies in handling large mutant populations, mutant selection, mutant recoveries, and mutant propagation [9].
Metabolomics, targeted and untargeted approaches, are approaches to detect primary and secondary metabolites in biological samples [22].Metabolites are sensitive to perturbations, including diseases, environmental changes, and mutational genetics.The targeted metabolomics approach focuses on specific metabolite groups related to particular biosynthetic pathways.The untargeted metabolomics approach covers detections of a wide range of metabolite groups in biological samples.This approach which can be used to profile changes in metabolite composition and levels due to perturbations of the environment and biotic stresses has an advantage that no prior knowledge of particular metabolites, including semi-polar metabolites, which cover phenolic acids, flavonoids, glycosylated steroids, alkaloids, and other glycosylated species is required.Liquid chromatography coupled with mass spectrometry (LC-MS) is an analytical technique for profiling semi-polar metabolites in biological samples.
This study aimed to investigate the effects of gamma-ray irradiation on semi-polar metabolite profiles in leaves of in vitro-derived plants of Indonesian pepper varieties (C.annuum).The gamma-ray doses tried were 0, 100, 200, 300, 400 to 500 Gy.The ability to detect the alteration as early as possible such as during the early growth phase would be beneficial for selecting superior plants with specific healthrelated accumulated metabolites for further crop improvement.

Plant materials
Seeds of C. annuum varieties, Laris and Kopay, were obtained commercially and exposed to gamma ray doses (0, 100, 200, 300, 400, and 500 Gy).Under a laminar airflow cabinet, all seeds were rinsed with sterile distilled water thrice before surface sterilization.Seeds were surface-sterilized by soaking the seeds in ethanol solution 70% (v/v) for 5 min and rinsed with sterile distilled water thrice.Seeds were soaked in hypochlorite solution 30% (v/v) for 5 min and rinsed with sterile distilled water three times.

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Seeds were dried on sterile tissue papers, after which they were germinated and cultivated in vitro on Murashige and Skoog (MS) without any supplements (MS 0).Seed viabilities of unirradiated and irradiated seeds were recorded by counting the total numbers of germinating seeds at six weeks after sowing.After 60 days in in vitro culture, plantlets were acclimated in the greenhouse, and their leaves were collected for metabolite analysis at 80 days after the seed germination.Collected leaves were ground under liquid nitrogen and kept at -80 o C for further analysis.

Metabolite extraction
Semi-polar metabolites from 100 mg of freeze-powdered leaves were extracted with 1 mL methanol.The sample mixtures were vortexed for 10 sec, sonicated for 15 min, and centrifuged for 10 min at 2500 rpm.The mixtures were filtered using a 0.2 µM PVDF filter and air-dried under nitrogen gas in a fume hood.Prior to liquid chromatography coupled with mass spectrometry (LC-MS) analysis, the dried extracts were resuspended with 300 µL of methanol acidified with 0.125% formic acid.The extracts were subjected to LC-MS analysis.

Liquid chromatography-mass spectrometry (LC-MS) analysis
LC-MS analysis was performed using an ultra-high pressure liquid chromatography (UPLC), as described in Skubel et al. 2020 [25].The UPLC system was Dionex UltiMate 3000 Rapid Separation LC system equipped with a photodiode array (PDA) detector DAD-3000RS.For high mass accuracy mass spectrometry, the eluent flow was directed to a Q Exactive Plus Orbitrap high-resolution highmass-accuracy mass spectrometer (HR-MS) (Thermo Scientific, Waltham, MA).A complete MS scan from 100 to 1000 m/z in a negative ionization mode using an electrospray interface was used to detect metabolite masses in the samples.The auxiliary gas flow rate was 7, the sweep gas flow rate was 1, and the sheath gas flow rate was 30 arbitrary units.The ESI voltage was 3500 volts (−3500 for negative ESI) with a capillary temperature of 275 °C and the mass resolution was 140,000 m/Δm FWHM.Semi-polar metabolites were separated on a reverse phase C8 column (Phenomenex Kinetex) with the length size 100, column diameter 2.1 mm, particle size 2.6 μm, and pore size 100 Å.Two mobile phase components, i.e., A: 0.5% ACS grade acetic acid in water pH 3-3.5 (LCMS grade) and B: 100% Acetonitrile (LCMS grade).The mobile phase flow was 0.2 ml/min, and a gradient mode was used for all analyses with the initial gradient conditions were 95% A and 5% B for 30 min, then the gradient changed to 5% A and 95% B, which was kept for the next 8 minutes.During the following 4 min, the mobile phase ratio was brought to initial gradient conditions.Between subsequent injections, an 8 min equilibration interval was included.The average pump pressure was around 3900 psi for the initial conditions.

Data analysis
Data acquisition and analysis were performed using the Xcalibur software (Thermo Fisher Scientific).Targeted metabolite analysis was performed using MAVEN [23,24].Data were pre-treated by log2 transformation and mean centering (subtracting its mean and dividing by its standard deviation).Pretreated data were subjected to analysis of variance (ANOVA) and post hoc test (Bonferroni test) to determine the least significant variances among samples.Metabolites with significant differences between accessions, determined by p-values lower than 0.001, were subjected to hierarchical cluster analysis (HCA).HCA was performed by using complete linkage and Euclidian method in http://www.heatmapper.ca/expression/.

Seed viability
Seed viabilities after gamma-ray irradiation were observed and compared with unirradiated seeds by counting the total number of germinated seeds in vitro.This experiment was set in a controlled in vitro environment to reduce external factors that may disturb plant growth, such as high temperature and disease, and to inspect internal factors that may reduce seed viability due to gamma-ray irradiation treatment.Data on seed viabilities are shown in Table 1.Of all treatments, unirradiated seeds (0 Gy) and irradiated seeds (100 -500 Gy) showed that all seeds were viable to germinate.However, their viabilities varied among all treatments and varieties.Unirradiated seeds showed at least 50% viability in C. annuum var Kopay and 70% in C. annuum Laris.Meanwhile, C. annuum var Kopay seeds, after gamma-ray irradiation at 100 Gy to 500 Gy doses, were still viable.The highest seed viability in Kopay occurred in seeds treated with 200 Gy of gamma-ray, and the lowest seed viability was observed in seeds after 100 Gy and 400 Gy in Kopay (Table 1).The fact that only 55% of control seeds of Kopay were germinated might be caused by the quality of original seeds.Irradiated seeds of Kopay with doses of 100 Gy and 400 Gy showed that 30% of seeds were germinated.However, the plantlets could not continue growing due to microbial contamination.Rescuing the plantlets with subculturing in a new culture media had been conducted.However, the contamination still occurred and caused the mortality of plantlets.The contamination might be due to factors such as endophyte microbes-infected seeds.It has been shown that endophytes survived and continued to grow in culture media during germination on gamma-ray irradiated seeds of tall fescue grass [13].After gamma-ray irradiation at 100 to 500 Gy doses, Laris seeds were still viable to germinate.The 500 Gy seeds of Laris had 100% viabilities, while seeds of Kopay treated with an equivalent gamma-ray dose showed 45% seed viabilities.Higher seed viabilities at highdose gamma-ray irradiation showed that Laris seeds were more resistant to higher irradiation than Kopay and that irradiation might have led to breaking of seed dormancy.However, seeds exposed to higher doses of gamma-ray showed a disturbance in seed tissues' physiological and biochemical activities, resulting in a decrease in seed germination percentages [10,11,12].

Semi-polar metabolite profiles
To investigate any biochemical process alteration after gamma-ray irradiation on pepper, we analysed untargeted semi-polar metabolites, such as flavonoids, terpenoids, phenolics, and alkaloids, of leaves from 80-day seedlings using LC-MS.Semi-polar metabolite profiles of unirradiated and irradiated seeds of C. annuum var Kopay and Laris were depicted by heatmap in Figure 1 and Figure 2. We retrieved, in total, 387 semi-polar metabolites, including 15 metabolites with putative identification, from C. annuum var Kopay and Laris.These metabolites were subjected to Hierarchal Cluster Analysis (HCA).HCA of all samples based on variations of 387 metabolites showed three sample clusters: Clusters A1, A2, and A3 (Figure 1).Cluster A1 consisted of only Kopay 400 Gy and displayed a distinguished separation between Kopay 400 Gy with most samples.Cluster A2 distinguished most Laris samples, including unirradiated Laris (0 Gy) and irradiated Laris (100, 200, 300, and 500 Gy).Cluster A3 was differentiated Kopay 0, 200 and 300 Gy, as well as Laris 400 Gy.
HCA of semi-polar metabolites revealed three metabolite clusters (A-C).These clusters displayed variations in the accumulation of metabolites of all samples (Figure 1).Almost semi-polar metabolites of cluster A expressed higher accumulation in all samples, suggesting that gamma-ray irradiation was not impacted to biosynthetic pathway of metabolites in cluster A. Metabolites in cluster B displayed variations of metabolite expressions across all samples.Based on metabolite cluster B, some metabolites' abundancies were dependent on gamma-ray irradiation doses.Several metabolite abundancies were higher in Laris 200 Gy than in Laris 0 Gy, indicating a biosynthesis flux perturbation due to irradiation treatment.In addition, all metabolites in cluster B were at similar abundancies in Kopay 400 Gy, which are clearly distinguished from those in other samples.In contrast to clusters A and B, most metabolites in cluster C were expressed at lower levels.Pepper samples clustered in A3 had the lowest levels of metabolites in cluster C, suggesting that abundancies of metabolite cluster C differentiate cluster A3 from the rest of the samples.
Fifteen metabolites were putatively identified as phenylpropanoid biosynthetic pathway derivatives, including flavonoids and terpenoids (Figure 2A).Eight metabolites were identified as flavonoids, including rutin, kaempferol, isoquercetin, phloretin, and apigenin.Seven metabolites were identified putatively as terpenoids, including capsianosides, diterpene glycosides, and lyoniresinol.Flavonoids and terpenoids metabolites were derivatives of the shikimate biosynthetic pathway.These metabolites are resulted from different intermediates in the biosynthetic pathway from phosphoenolpyruvates to phenylalanines, precursors of phenylpropanoid pathway, and pyruvates, precursors of methyl-Derythritol phosphate (MEP) together with 3-phosphoglycerates pathway (Figure 2B).Flavonols (quercetin, rutin, and kaempferol) and flavones (apigenin) are all the major flavonoids in peppers [14].In addition, capsianosides and diterpene glycosides have been reported in pepper leaves to correlate with the least susceptibility of pepper plants against thrips-borne disease [15,16].An approximately 5-to 20fold increase in secondary metabolites in certain plants is noted with as low as 20 Gy irradiation dose [17].On the contrary, paprika required as high as 10 kGy to have a significant increase (around 10%) of all components of capsaicinoids.The level of different compounds such as isodihydrocapsaicin was not affected by all irradiation doses, while doses up to 5 kGy led to higher increases in capsaicin and dihydrocapsaicin levels [18].
Based on the putative metabolites, Kopay 400 Gy was clearly distinguished from other samples due to the lowest levels of all flavonoids and terpenoids (Figure 2A).Low levels of both metabolites may indicate that gamma-ray irradiation at a dose of 400 Gy in Kopay seeds affected the shikimate biosynthesis, which is in the upstream part of the flavonoids and terpenoids biosynthetic pathway (Figure 2B).Probably, the biosynthesis flux in the shikimate pathway was interrupted.Hence, downstream precursors for terpenoids and flavonoids are produced very low.

Conclusion
Our results showed that gamma-ray irradiation doses did not influence seed viabilities but more to genotypic dependent factors, although Laris seemed more resistant to irradiation than Kopay.At 200 Gy gamma irradiation dose, several semi-polar metabolite abundancies were different from those in unirradiated plants, showing an effect of induced mutation due to gamma-ray irradiation on the metabolite biosynthesis in Laris.Kopay 0 Gy, 200 Gy, and 300 Gy also showed similar metabolite profiles, indicating that gamma-ray doses did not induce any clear mutation at genes regulating metabolite biosynthetic pathways.At dose 400 Gy, Kopay showed low levels of terpenoids and flavonoids, indicating that gamma-ray irradiation may affect the upstream part of the shikimate biosynthetic pathway, resulting in low levels of precursors at the upstream biosynthetic pathway of terpenoids and flavonoids.Hence, the accumulation of terpenoids and flavonoids was very low.Our findings showed secondary metabolite alteration, which may provide insights into the effect of gamma irradiation for mutation breeding that may be important for future pepper breeding programs.

Figure 1 .
Figure 1.Semi-polar metabolite profiles from leaves of seedlings originated from seeds irradiated with 0, 100, 200, 300, 400, and 500 Gy of Capsicum annuum var Kopay and Laris.The coloured matrix represents the metabolite intensity in samples.The metabolite intensity value has been transformed by log2 calculation and normalized by mean-centered.Metabolite clusters were represented by the alphabets A to C.

Table 1 .
Effect of gamma-ray irradiation on seed viability of Capsicum annuum var Kopay and Laris.