Methylotroph bacteria and cellular metabolite carotenoid alleviate ultraviolet radiation-driven abiotic stress in plants

Abstract

Increasing UV radiation in the atmosphere due to the depletion of ozone layer is emerging abiotic stress for agriculture. Although plants have evolved to adapt to UV radiation through different mechanisms, but the role of phyllosphere microorganisms in counteracting UV radiation is not well studied. The current experiment was undertaken to evaluate the role of phyllosphere Methylobacteria and its metabolite in the alleviation of abiotic stress rendered by ultraviolet (UV) radiation. A potential pink pigmenting methylotroph bacterium was isolated from the phylloplane of the rice plant (oryzae sativa). The 16S rRNA gene sequence of the bacterium was homologous to the Methylobacter sp. The isolate referred to as Methylobacter sp N39, produced beta-carotene at a rate (μg ml^-1 d^-1) of 0.45-3.09. Biosynthesis of beta-carotene was stimulated by brief exposure to UV for 10 min per 2 days. Carotenoid biosynthesis was predicted as y = 3.09 × incubation period + 22.151 (r ² = 0.90). The carotenoid extract of N39 protected E. coli from UV radiation by declining its death rate from 14.67% min^-1 to 4.30% min^-1 under UV radiation. Application of N39 cells and carotenoid extract also protected rhizobium (Bradyrhizobium japonicum) cells from UV radiation. Scanning electron microscopy indicated that the carotenoid extracts protected E. coli cells from UV radiation. Foliar application of either N39 cells or carotenoid extract enhanced the plant's (Pigeon pea) resistance to UV irradiation. This study highlight that Methylobacter sp N39 and its carotenoid extract can be explored to manage UV radiation stress in agriculture.

Keywords: UV irradiation; beta carotene; carotenoid; methylotroph; plant-microbe interaction.

FIGURE 1

Experimental set up to evaluate the response of methylotroph to UV irradiation. (A) Isolation of UV-resistant methylotrophs from the phylloplane of plant leaf biomass. Methylotrophs isolated by enriching leaf surface bacteria in ammonium mineral salt (AMS) agar. Colonies were UV irradiated for 15 min, and the most resistant one was selected for further study. The 16S rRNA gene of the isolate was homologous (>98%) to Methylobacter sp. (B) Experimental layout for evaluating the effect of UV irradiation on growth of methylotrophs, carotenoid biosynthesis, and Crt1 gene abundance. (C) Sequence of steps followed to study the effect of UV irradiation on methylotrophs growth and carotenoid biosynthesis. Ino – Inoculation of isolate; Est – Estimation of cell numbers, carotenoid concentration, and abundance of Crt1 gene copies (only Crt1 was estimated at 0d and 10d); Inc – Incubation of vials; UV – UV irradiation. Broth without UV irradiation served as control, where the petriplates were covered with polypropylene lid to restrict UV radiation. Experiments were conducted in three replicates.

FIGURE 2

Carotenoid extraction from the methylotroph bacterial isolate (Methylobacter sp N39) and qualitative analysis of metabolites or extracts to identify key molecules. (A) Extraction of bacterial metabolites. (B) Thin layer chromatography (TLC) of the extract to identify the key metabolite. (C) High-pressure liquid chromatography (HPLC) analysis of bacterial extract to determine its composition. HPLC chromatograms of standard beta-carotene (top) and metabolites or extracts (bottom). Chromatogram of bacterial metabolite was dominated by a peak representing standard beta-carotene. X-axis represents retention time (RT) in minute and Y-axis represents volt. (D) Relative area of HPLC peaks of sample (methylotroph bacterial extract). X-axis represents retention time in minutes and Y-axis represents relative peak area (%). Values are arithmetic mean and error bar as standard deviation of three replicated observations.

FIGURE 3

Effect of UV irradiation on cellular activities of Methylobacter sp N39. The cellular activities were rates of carotenoid biosynthesis, growth rate, and carotenoid biosynthesis gene (CrtI) copy number. Methylobacter sp N39 was grown in ammonium mineral salt broth. Broth was exposed to UV irradiation for 0, 10, 20, and 30 min every 2 days. After exposure broth was re-incubated allowing cells to grow and biosynthesize carotenoid. The process of exposure was repeated at 2 days interval for 10 days. Cell numbers (top left) and carotenoid (top right) concentration were estimated at 2 days interval. Rates were estimated as slope of values over incubation period. After completion of incubation period (10 days), DNA was extracted from broth to quantify carotenoid biosynthesis gene, CrtI by real-time PCR using SYBR green chemistry (bottom left). X-axis represents UV irradiation (min in 2 days). Y-axis represents different parameters. Each data point represents arithmetic mean and standard deviation as error bar of three replicated observations.

FIGURE 4

Growth and carotenoid production by a pink pigmenting Methylobacter sp N39 (Genbank Acc no MW276130.1) in response to UV radiation. The bacterium was cultured in a synthetic medium. Broth was exposed to UV radiation for different timings (0, 10, 20, and 30 min). For each data point, a portion of broth was sampled at 2 days interval and used for estimating cell abundance and carotenoid concentration. Rest of the broth was exposed to UV radiation and incubated till next sampling. X-axis represents the incubation period, while Y-axis represents cell abundance (left panel) and carotenoid concentration (right panel). Values are presented in 4 replicates.

FIGURE 5

Effect of Methylobacter sp N39 on extending UV resistance to E. coli bacteria. Cells of E. coli were multiplied in LB media and prepared for different treatments and UV irradiation. The treatments were (1) E. coli suspension (control), (2) E. coli + commercial beta-carotene at 10 μg ml^–1, (3) E. coli + extracts of Methylobacter sp N39 extract containing 10 μg beta-carotene ml^–1, and (4) E. coli + Methylobacter sp N39. Cell suspensions were spread plated on LB plates and UV irradiated for 0, 5, 10, 15, 20, 25, and 30 min. Colonies were counted after incubation. Left panel (A) represents survival fraction (%) of E. coli in response to UV irradiation. X-axis represents irradiation time in minutes, and Y-axis represents the survival fraction. Right panel (B) represents the inhibition rate of E. coli in response to UV irradiation. Rate of inhibition is determined from the slope of survival fraction vs irradiation time. Each value represents arithmetic mean ± standard deviation as error bar of three replicated observations.

FIGURE 6

Scanning electron micrograph of bacterial cells under UV radiation and the effect of carotenoid extract on protecting cells from radiation. Methylobacter sp N39 cells without UV exposure (A); Methylobacter sp N39 cells after 15 min of UV exposure. Cells survived UV radiation without any apparent disruption of cellular structure (B). E. coli cells exposed to UV C radiation for 5 min (C). The cellular structure damaged exhibiting deformed cells due to UV radiation; (D) E. coli cells treated with crude carotenoid after exposure to UV C radiations for 5 min. E. coli cells were protected from UV radiation by the carotenoid extract of Methylobacter sp N39 cells.

FIGURE 7

Effect of Methylobacter sp N39 and carotenoid extract on plant–rhizobium interaction in response to UV irradiation. Seeds of pigeon pea and soybean were prepared with the following treatments. 1 - control (no rhizobium no UV), 2 - rhizobium no UV, 3 - rhizobium with UV, 4 - rhizobium + Methylobacter sp N39 with UV, and 5 - rhizobium + carotenoid extract with UV. After treatments seeds were air dried and UV irradiated for 15 min. Seeds were planted and grown under controlled condition. Plants were harvested after 30 days of sowing and parameters (shoot dry weight, number of nodules, and acetylene reduction assay) were estimated. Y-axis represents parameters, and X-axis represents treatments. Values are presented as arithmetic mean ± standard deviation of three replicated observations.

FIGURE 8

Effect of foliar application of cells of Methylobacter sp N39 and carotenoid on plant’s UV resistance. The test plant Pigeon pea (Cajanus cajan var Asha) was grown under controlled condition. On 30th day of sowing, plants were subjected to foliar application of Methylobacter sp N39 (10⁶ cells/ml), carotenoids extracted (10 μg/ml) from Methylobacter sp N39, and water as control. The treated plants were UV irradiated for 0.5, 1, and 1.5 h per day for 3 consecutive days. On 40th day of sowing, plants were analyzed for (A) chlorophyll A content and (B) shoot dry weight. X-axis represents treatments and Y-axis represents parameters. Values are presented as arithmetic mean with error bar as standard deviation of three replicated observations.