The effect of UV-B on Arabidopsis leaves depends on light conditions after treatment

Abstract

Background: Ultraviolet B (UV-B) irradiation can influence many cellular processes. Irradiation with high UV-B doses causes chlorophyll degradation, a decrease in the expression of genes associated with photosynthesis and its subsequent inhibition. On the other hand, sublethal doses of UV-B are used in post-harvest technology to prevent yellowing in storage. To address this inconsistency the effect of short, high-dose UV-B irradiation on detached Arabidopsis thaliana leaves was examined.

Results: Two different experimental models were used. After short treatment with a high dose of UV-B the Arabidopsis leaves were either put into darkness or exposed to constant light for up to 4 days. UV-B inhibited dark-induced chlorophyll degradation in Arabidopsis leaves in a dose-dependent manner. The expression of photosynthesis-related genes, chlorophyll content and photosynthetic efficiency were higher in UV-B -treated leaves left in darkness. UV-B treatment followed by constant light caused leaf yellowing and induced the expression of senescence-related genes. Irrespective of light treatment a high UV-B dose led to clearly visible cell death 3 days after irradiation.

Conclusions: High doses of UV-B have opposing effects on leaves depending on their light status after UV treatment. In darkened leaves short UV-B treatment delays the appearance of senescence symptoms. When followed by light treatment, the same doses of UV-B result in chlorophyll degradation. This restricts the potential usability of UV treatment in postharvest technology to crops which are stored in darkness.

Fig. 1

The overall scheme of the experiment. The leaves from 6 week old Arabidopsis thaliana were detached either from dark adapted overnight plants or directly from plants growing in the growth chamber during the light period (2 h after the photoperiodic light had been turn on). The leaves were put on petri dishes on water-soaked paper. Half of each leaf was covered with black paper (control) and the leaves were irradiated with UV-B (8 W·m⁻²) for 5 min. After irradiation the leaves were either left in darkness (leaves from dark-adapted plants) or under continuous illumination with white light (100 μmol ·m⁻²· s⁻¹) for up to 4 days. Thus, 4 different kinds of samples were analyzed, i) darkened control (CD), ii) control illuminated with continuous light (CL), iii) UV-B irradiated leaves left in darkness (UVD) and finally, iv) UV-B irradiated leaves kept under continuous illumination (UVL)

Fig. 2

Photographs of the detached leaves of 6-week old A. thaliana with one half covered with black paper, and another half irradiated with UV-B (8 W·m⁻²) for the indicated time and left in darkness for 4 days

Fig. 3

Photographs of detached A. thaliana leaves with one half covered with black paper, and another half irradiated with UV-B (8 W·m⁻²) for 5 min and left in darkness (a and b) or under constant illumination (100 μmol·m⁻² ·s⁻¹ of white light, c) for the indicated time. The leaves were taken from plants dark-adapted overnight (a), or from plants kept in a growth chamber for 2 h after the dawn (b and c)

Fig. 4

Changes in photosynthetic pigments (a chlorohyll a, b chlorophyll b, c chlorophyll a/b, d violaxanthin, e neoxanthin, f lutein, g β-carotene ) in detached Arabidopsis leaf halves either irradiated with UV-B (8 W·m⁻²) for 5 min or covered with black paper (control) and left either in darkness or under constant white light (100 μmol·m⁻²·s⁻¹) for the indicated time. Day 0 means 1 h after the treatment. Non-irradiated leaf halves served as a control. Pigments were separated by HPLC with detection by absorbance at 436 nm (chlorophylls) or 405 nm (carotenoids) and their content was determined from the area under the peak of the chromatogram using the extinction coefficients listed in Additional file 2: Table S2. Statistical significance of the differences between treatments was assessed with one-way ANOVA and the results of this analysis are listed in Additional file 3: Table S3

Fig. 5

Influence of 5 min UV-B (8 W·m⁻²) irradiation on photosynthesis in Arabidopsis leaves. After irradiation samples were kept either in darkness or under constant light (100 μmol·m⁻²·s⁻¹) for the given time. Day 0 means 1 h after the treatment. Non-irradiated leaf halves served as a control. a Changes in PSII maximal quantum yield (Fv/Fm) during experimental treatment, measured with an imaging fluorometer. The results are the means of measurements for at least 12 different leaves. Statistical significance of the differences between treatments was assessed with one-way ANOVA and the results of this analysis are listed in Additional file 3: Table S3. b Total proteins (upper- Coomassie stained SDS-PAGE) and D1 protein (lower- Western blot with anti-D1 antibodies) in examined leaves. Each well contains proteins extracted from 120 mg of tissue The degradation product of D1 protein is marked with an arrow. c and d Relative expression levels of photosynthesis-related genes (CAB, RBSC1) measured with real-time RT-PCR and normalized for the expression of four housekeeping genes (PDF2, UBC9, UBQ10, SAND). After the specified time period leaves were cut into halves and control and treated halves from 4 different leaves were pooled. Each measurement was repeated at least 3 times. A single dark-adapted overnight control sample from day 0 was used as a reference for calculating relative expression levels. Error bars indicate the standard error

Fig. 6

Influence of 5 min UV-B (8 W·m⁻²) irradiation on the senescence and cell death of Arabidopsis leaves. After irradiation samples were kept either in darkness or under constant light (100 μmol·m⁻²·s⁻¹) for given time. a-d Time-course of the relative expression of senescence associated genes: (a) SAG13, (b) SAG12, (c) SEN1 and (d) WRKY53 normalized for the expression of four housekeeping genes (PDF2, UBC9, UBQ10, SAND). Day 0 means 1 h after the treatment. Non-irradiated leaf halves served as a control. After the specified time period leaves were cut into halves and control and treated halves from 4 different leaves were pooled. Each measurement was repeated at least 3 times. A single dark-adapted overnight control sample from day 0 was used as a reference for calculating relative expression levels. Error bars indicate the standard error. e Trypan blue staining for cell death of leaves irradiated with UV-B with the middle part covered and transferred either to light or to dark conditions

Fig. 7

Influence of UV-B on anthocyanins in Arabidopsis leaves. After the specified time period leaves were cut into halves and control and treated halves from 4 different leaves were pooled. Each measurement was repeated at least 3 times. Error bars indicate the standard error. a Anthocyanin content was analyzed by measuring absorbance at 532 nm and normalized to fresh weight in examined leaves. b and c Time-course of the relative expression of genes involved in anthocyanin biosynthesis: (b) PAL1 and (c) CHS normalized for the expression of four housekeeping genes (PDF2, UBC9, UBQ10, SAND). A single dark-adapted overnight control sample from day 0 was used as a reference for calculating relative expression levels