Physiological and biochemical responses of sugar beet (Beta vulgaris L) to ultraviolet-B radiation

Growth condition and UV-B treatment

The seeds of BR1 cultivar of sugar beet (Beta vulgaris L.) (Obtained from Agriculture Organization of West Azerbaijan Province, Urmia, Iran) were sterilized with 10% sodium hypochlorite for 10 min and then soaked in distilled water. After 4 days germination the seedlings were transferred to pots filled with soil. All plants were grown in a growth chamber (Jeiotech GC-3007 LH, Korea) at 25/20 °C (day/night), with a 16 h light/8 h dark photoperiod for 30 days under light intensity of 500 µmol m⁻²s⁻¹in PAR region. Afterwards, the plants were divided into 4 groups including control and three experimental groups exposed to 3.042, 6.084 and 9.126 kJm⁻²d⁻¹UV-B radiation for one week. UV-B radiation was provided by TL20W/12 RS SLV/25 lamp (Philips, Germany) positioned 20 cm above the plants. The lamp was wrapped in a 0.13 mm cellulose diacetate film and polyethylene foil to absorb radiation below 290 nm (UV-C) and above 320 nm (UV-A), respectively. Intensity of radiation was measured with an ASD Handheld 2 spectro radiometer.

Growth and leaf relative water content measurement

Plant height, fresh weight and leaf area were measured immediately after removal from the pots. Dry weight was determined after drying at 75 °C for 48 h. The leaf relative water content was determined in the fully expanded youngest leaf of the main shoot. The fresh weight (FW) of the sample leaves was recorded and the leaves were put in dark room in distilled water for 24 h in order to obtain the turgid weight (TW). Then, the samples were dried in an oven at 75 °C to obtain dry weight (DW). Leaf relative water content was calculated using the following formula (Sade, Galkin & Moshelion, 2015): ${\displaystyle LRWC\left(\text{\%}\right)=\left[\left(FW-DW\right)∕\left(TW-DW\right)\right]\times 100.}$

Photosynthetic pigments measurement

In order to measure chlorophyll and carotenoid content, 0.1 g of complete leaves were ground in 1 ml of 80% acetone and centrifuged at 2,700 rpm for 10 min. The supernatant was directly used for spectrophotometric measurements of absorbance at 647, 663 and 470 nm. To estimate chlorophyll a, chlorophyll b, total chlorophyll and carotenoids contents by spectrophotometer, following equations were used (Gross, 1991): ${\displaystyle \mathrm{Total}\mathrm{chlorophyll}\left(\mathrm{\mu }\mathrm{g∕ml}\right)=\left(20.21\times A_{645}\right)+\left(8.02\times A_{663}\right)}$ ${\displaystyle \mathrm{Chlorophyll}\mathrm{a}\left(\mathrm{\mu }\mathrm{g∕ml}\right)=\left(12.25\times A_{663}-2.79\times A_{647}\right)}$ ${\displaystyle \mathrm{Chlorophyll}\mathrm{b}\left(\mathrm{\mu }\mathrm{g∕ml}\right)=\left(21.50\times A_{647}-5.10\times A_{663}\right)}$ ${\displaystyle \mathrm{Carotenoid}\left(\mathrm{\mu }\mathrm{g∕ml}\right)=\left(1000\times A_{470}-1.82\mathrm{chlorophyll}\mathrm{a}-85.02\mathrm{chlorophyll}\mathrm{b}\right)∕198.}$

Photosynthetic O₂ evolution measurement

The Qubit Systems’ S104 Differential O₂ Analyzer (DOX) was used to measure the photosynthetic O₂ evolution. The chamber was calibrated using nitrogen gas and then oxygen (O₂) was added to 21%. Leaf samples were placed into the chamber and ambient air was injected. The released oxygen from leaves was measured by the device sensor for 1000 s. The light was set at 66 µmol m⁻²s⁻¹.

Maximum quantum yield of PSII and relative chlorophyll level determination

Before removing plants from pots, chlorophyll concentration in the4thleavesof all plants was measured by a chlorophyll content meter (Hansatech Instruments, CL-01, UK). Maximum quantum yield of PSII (F_v∕F_m) (F_v: variable fluorescence; F_m: maximum fluorescence) measurement was determined before plants removal from pots by a plant efficiency analysis meter (Hansatech Instruments, Handy PEA, UK).

Free amino acids, proline and glycine betaine content measurements

In order to evaluate the concentration of free amino acids, fresh sample was homogenized in 50 mM phosphate buffer (pH 6.8). The extracts were centrifuged for 20 min at 3,000 rpm then ninhydrin reagent was added to supernatant and heated for 7 min in water bath at 100 °C. Absorbance at 570 nm was determined immediately after cooling in the cold water bath. Different concentrations of glycine were used to make standard curve (Razavi, Nasrollahi & Ghasemian, 2017). To estimate proline content, 0.5 g of fresh leaf tissue was grounded in 10 ml sulfosalicylic acid. After addition of 2 ml ninhydrin reagent, 2 ml acetic acid and 2 ml toluene, two different phases appeared. Absorption of colored supernatant containing toluene and proline was measured at 520 nm. Standard curve was made using different concentrations of proline (Mahajan et al., 2018). For measuring glycine betaine concentration, 0.5 g of dried leaf tissues were grounded, homogenized with 20 ml deionized water and shaken for 24 h at 25 °C. After filtering the samples, they were diluted 1:1with 2N sulfuric acid, cooled for 1 h and 10 ml Lugol’s iodine was added. After vortexing, all samples were centrifuged and stored at 4 °C for 16 h. Residual crystals were dissolved in 1, 2-dichloroethane and the absorbance was measured at 365 nm. Standard curve was prepared by different concentrations of glycine betaine in 1N H₂SO₄ (Al Hassan et al., 2016).

Hydrogen peroxide (H₂O₂) level measurement

For measurement of H₂O₂ content, fresh leaf tissues were ground on ice in a mortar with 1% trichloro acetic acid. Samples were centrifuged at 4 °C for 15 min at 10,000 g. 0.5 ml of supernatant was mixed with 0.5 ml of 10 mM phosphate buffer (pH 7) and 1 ml of 1 M potassium iodide. Content of H₂O₂ was measured spectrophotometrically at 390 nm. Different concentrations of H₂O₂ were used to make standard curve (Velikova, Yordanov & Edreva, 2000).

Protein extraction

0.05 g of fresh leaf tissue was homogenized with 2 ml of 0.1 M phosphate buffer (pH 6.8). All extracts were centrifuged at 13,000 g for 15 min at 4 °C and used to measure the enzyme activities [19] and protein content. Protein concentration was estimated with Bradford reagent using bovine serum albumin as standard (Bradford, 1976).

Catalase (CAT) activity measurement

In order to examine CAT activity, a reaction mixture including 2.5 ml of 0.05 M phosphate buffer (pH 7), 0.3 ml of 3% H₂O₂ and 0.2 ml protein extract was prepared. Enzyme activity was examined using Beer–Lambert’s method as H₂O₂ removal measured through decrease in absorbance at 240 nm (Aebi, 1984).

Polyphenol oxidase (PPO) activity assessment

For PPO activity, the reaction mixture including 2.5 ml 0.2 M phosphate buffer (pH 6.8) and 0.2 ml of 0.02 M pyrogallol was incubated at 40 °C water bath. After adding 0.2 ml of the enzyme extract, absorbance changes were measured at 430 nm (Patra & Mishra, 1979). Enzyme activity was estimated by Beer-Lambert’s method.

Ascorbate peroxidase (APX) activity measurement

In order to evaluate APX activity, the reaction mixture included 2.5 ml 0.2 M phosphate buffer (pH6.5), 0.2 ml of 3% H₂O₂, 5 mM ascorbic acid and 0.2 ml enzyme extract (Nakano & Asada, 1981). The APX activity was estimated by Beer-Lambert’s method at 290 nm.

Protease activity determination

To estimate protease activity, 0.5 ml of 1% casein (provided in 0.5 M phosphate buffer, pH 6) with 0.2 ml enzyme extract were incubated at 45 °C for 1 h. The reaction was stopped by adding 0.1 ml of 40% trichloroacetic acid (TCA). Protease activity was calculated based on an extinction coefficient of 21.5 mM⁻¹cm⁻¹ andabsorbance measurement at 280 nm (Drapeau, 1974).

Malondialdehyde (MDA) measurement

For MDA determination, 0.2 g fresh leaf tissues were ground in 5 ml of 0.1% TCA. After centrifuging at 4,000 rpm for 20 min, 2.5 ml 0.5% thiobarbituric acid (TBA) in 20% TCA were added to the supernatants. Extracts were incubated in water bath at 95 °C for 30 min and immediately cooled on ice. Afterwards, all samples were centrifuged at 4,000 rpm for 30 min and absorbance was measured at 532 nm and 600 nm. The MDA concentration was calculated by subtracting the non-specific absorption at 600 nm from the absorption at 532 nm using absorbance extinction coefficient of 155 mM⁻¹cm¹ (Sadak, Abdelhamid & El-Saady, 2010).

Total betalain content

Betalain extraction was carried out by a modified method of Francis (2000). 5 g fresh leaves were ground in 25 ml of 20% ethanol and 0.5% citric acid solution and left in darkness for 24 h. After filtering, the supernatant was used for betalain purification by normal phase column chromatography. 10 g of silica gel 60packed with the binary solvent mixture of methanol/water 8:2 v/v with 1% v/v glacial acetic acid were used as stationary phase. The same binary solvent was applied as an elution at flow rate of 0.7 ml/min. The elution was used for spectrophotometric measurement of absorbance at 535 nm.

Total betalain content was estimated using following equation: ${\displaystyle \mathrm{Betalain}\mathrm{content}\left(\mathrm{mg}∕100\mathrm{gFW}\right)=\frac{A.F.M}{\epsilon }\cdot \frac{V}{1000\mathrm{m}}}$

A: sample absorption

M: average molecular mass

ε: molar extinction coefficient

V: extract volume

m: mass of vegetable solid used for extraction

DF: dilution factor.

Statistical analysis

Statistical analysis was carried out by one-way ANOVA (Version 18; SPSS Inc., Chicago, USA). Means compression were performed by Duncan Test at P < 0.05. Graphs were drawn in Excel Software (Microsoft Office, 2007).

Growth Parameters and Relative Water Content

The UV-B treated plants showed leaf chlorosis and a significant decrease in the plant’s height irrespective of the UV-B dose (Fig. 1A). Furthermore, UV-B irradiance caused reduction by 10–20% in both fresh and dry weight of leaves (Fig. 1B). The fresh weight of plants treated with the highest dose of UV-B showed the greatest decrease in fresh weight, whereas the weights of plants treated with the lowest and middle doses were identical. On the other hand, no dose-dependent difference in the dry weight of the three UV-B treated samples was observed. However, compared with the control, leaf relative water content was not affected in the UV treated plants (Fig. 1C). Thus, while UV-B treatment affected the growth of the sugar beet, a three-fold difference in dose of UV-B irradiation had only a minor influence on the BR1 cultivar (Dataset S1, Dataset S2, Dataset S3).

Photosynthetic performance

We then tested whether the reduction in growth is caused by decreased photosynthetic performance. The contents of photosynthetic pigments, chlorophyll a and b, as well as total chlorophyll, declined in all experimental groups to a similar degree (Fig. 2A). The same was true for carotenoid content which was reduced by 40% in all UV-B treated plants, compared to the control (Fig. 2B). However, compared with the control, photochemical efficiency of photosystem II (F_V/ F_M index) declined only in the plants exposed to 6.084 and 9.126 kJm⁻²d⁻¹ UV-B, by 28%, despite no difference in the content of pigments(Fig. 3A). On the other hand, the reduction in relative chlorophyll content (SPAD) in UV-B exposed plants was significant compared to control plants but not between the different UV-B doses (Fig. 3B).

Significant reduction in photosynthetic O₂ evolution was observed in all plants exposed to UV-B compared with the control, with the plants treated with 9.126 kJm⁻²d⁻¹UV-B releasing only 15% O₂ compared to controls (Fig. 3C). Clearly, photosynthesis performance of sugar beet is greatly affected by UV-B, but the irradiation dose seems to have only a minor effect (Dataset S1, Dataset S2, Dataset S3).

Metabolite Contents

There was maximum 22% increase in total free amino acids level in plants exposed to 9.126 kJm⁻²d⁻¹UV-B. However, plants exposed to 3.042 and 6.084 kJm⁻²d ⁻¹UV-B didn’t exhibit significant changes in free amino acids accumulation (Fig. 4A). Compared with the control, 3.042 kJm⁻²d⁻¹ UV-B caused no significant change in proline content. However, 6.084 and 9.126 kJm⁻²d ⁻¹ UV-B doses increased proline content by 24% and 29% respectively (Fig. 4B). The glycine betaine content was increased by 9.126 kJm⁻²d⁻¹ UV-B, while 3.042 and 6.084 kJm⁻²d⁻¹ UV-B did not cause significant changes in glycine betaine content in comparison with the control (Fig. 4C). As indicated in Fig. 4D, total betalain concentration increased in a dose dependent manner by 8%, 28% and 34% respectively, compared to the control (Dataset S1, Dataset S2, Dataset S3).

Protein content and enzyme activities

Exposure to 3.042 kJm⁻²d ⁻¹UV-B did not affect protein content, however, plants treated with 6.084 and 9.126 kJm⁻²d ⁻¹UV-B, showed 12% increase in protein concentration (Fig. 5A). Different UV-B doses caused dose dependent significant increase in CTA activity by 41%, 50%, and 56%, respectively (Fig. 5B). Similarly, a gradual increase in APX activity was observed (Fig. 5B). The activity of PPO was raised significantly in all UV-B exposed plants to the same extent, irrespective of the UV-B dose. Protease activity was increased significantly only in 9.126 kJm⁻²d⁻¹ UV-B treatment, while plants under 3.042 and 6.084 kJm⁻²d ⁻¹ UV-B did not show any differences in protease activity compared with the control (Fig. 5B). Thus, the increase in antioxidant enzyme activity in response to UV stress seems to contribute to the relatively similar damage of the plants at different UV-B doses (Dataset S1, Dataset S2, Dataset S3).

MDA and H₂O₂ content

The highest UV-B dose caused a 75% increase in MDA content, however, lower doses (3.042 and 6.084 kJm⁻²d⁻¹ UV-B) did not cause significant alterations (Fig. 6A). Interestingly, the H₂O₂ analyses demonstrated that UV-B did not cause any significant change in H₂O₂ levels compared with the control (Fig. 6B). Thus, the UV-B clearly led to oxidative stress, but not to accumulation of H₂O₂ (Dataset S1, Dataset S2, Dataset S3).