Evidence of physiological phototropin1 (phot1) action in response to UV-C illumination

Abstract

Stem growth kinetics were measured in cucumber (Cucumis sativus) and Arabidopsis thaliana using highly-sensitive monitoring with 5-minute resolution, in darkness and in response to a short, single pulse of UV-C illumination. The results show that UV-C, like blue light, induces a rapid decrease in seedling growth rate. The fluence-response kinetics and time course were similar to the phototropin1-mediated response observed following a blue pulse. Arabidopsis seedlings were used to assess the genetic mechanism of this response. The phot1 mutant exhibited defects in stem growth rate inhibition, with sustained growth inhibition completely absent following specific treatments. The cryptochrome and phytochrome mutants exhibited responses comparable to wild type, suggesting that these receptor classes do not contribute to this response. The work demonstrates in two species that UV-C has an effect on a rapid plant photomorphogenic response and that the response is partially mediated by the phot1 photoreceptor.

The plant photosensory receptor series contains a set of light sensitive pigments that initiate signaling processes leading to changes in growth and development in response to various wavelengths of light. The phytochromes act in response to red, far-red and blue light.¹ The cryptochromes initiate responses following illumination with UVA or blue light.² The phototropins, and other LOV domain proteins such as ZEITLUPE, LKP2 and FKF, bind flavin and act as UV/blue light activated switches that govern discrete processes related to optimizing photosynthesis, circadian rhythmicity and flowering time respectively.³ Together this set of light-sensitive pigments promotes the integration of environmental information, guiding appropriate plant responses.

Some of the most rapid and conspicuous responses to light are observed in the developing stem. The stem is a dynamic plant organ, continually adjusting its growth rate and direction in response to prevailing light conditions.⁴ The measurement of changes in stem growth in response to varying light environments has historically been used to explore the nature of plant photoreceptors, at least as early as the initial descriptions of phytochrome.⁵ Through analysis of stem growth kinetics Meijer⁶ subsequently demonstrated the existence of a separate blue-light mediated photosensory process by determining that the time course for the response to blue light was different from the response to red light. Rich et al.⁷ later used the same approach to address questions central to models of differential growth in phototropism. Koornneef et al.⁸ returned to the simpler method of end point growth measurements in a pioneering study uncovering a range of mutants in light-induced responses in Arabidopsis. More recently Parks and Spalding⁹ utilized a computer-based imaging method that provided the resolution necessary to perform time course studies on Arabidopsis hypocotyl elongation despite the small size of the seedlings. This method successfully dissected the contributions of multiple photoreceptors to responses developing over widely varying time scales, and the hardware and computational tools supporting these methods continue to improve.¹⁰

Shinkle et al.¹¹ applied the kinetic approach to responses of etiolated cucumbers to light from several regions in the UV waveband. This study identified unique characteristics for growth responses to UV-A (320–380 nm) conventional UV-B (significant energy at wavelengths between 300–320 nm, which was identified as long-wavelength UV-B, LW-UVB) and UV-B extending into the UV-C (conventional UV-B plus significant energy at wavelengths between 290–300 nm, which was identified as full-spectrum UV-B, FS-UVB). The time courses for the inhibition in hypocotyl elongation caused by each type of light showed one important common feature: a decline in elongation rate occurring within four minutes after the onset of irradiation. As noted in the previous report, this rapid decline in elongation rate occurs in the same time frame as the response to blue light described for this plant and conditions¹² known to be regulated by phototropins.¹³ These physiological studies indicate that phototropins may be responsive to a wide range of wavelengths extending deep into the UV region, a finding consistent with protein phosphorylation studies.¹⁴

A subsequent report characterizing the response to UV-B extending into the UV-C described end point growth measurements of plant responses to narrow band width (254 nm) UV-C alone.¹⁵ This report approached the question of whether the time course of this response would show features different from those reported for other wavebands. We here report that the time course for the inhibition of hypocotyl elongation in cucumbers caused by UV-C is essentially the same as for the response to FS-UVB, including the most rapid phase of growth suppression tentatively ascribed to regulation by phototropins. Further, we provide evidence from Arabidopsis seedlings deficient in phototropin1 that indicates that it at least partly responsible for the most rapid UV-C induced inhibition of hypocotyl elongation in this species as well.

Results

Figure 1 shows the effect of a 10 min pulse of UV-C radiation on hypocotyl elongation rates in 4–5-day-old DRL-grown cucumber seedlings. There is a transient decline in elongation rate detectable at the first 3 min time point after the onset of irradiation, with the local minimum rate occurring at 12 min. The elongation rate then rises from 12–21 min. A second decline in elongation rate begins at 30 min and the rate drops to near zero by 60 min.

Figure 1.

Growth inhibition in response to UV-C pulse in dim-red-light-grown Cucumis sativus seedlings. Hypocotyl elongation rate was measured by radial displacement transducer. At time 0 plants were irradiated with 600 mW m⁻² UVC (peak wavelength 254 nm) for 10 min. Error bars indicate SE of the mean. N = 8 seedlings.

The time course of the growth kinetics between 0–12 min in cucumber resembles that mediated by phototropins in blue light responses. This possibility could be formally tested by examining the same response in Arabidopsis seedlings so that the rich genetic tool set could be exploited. Before mutants were tested, 2-day-old, etiolated wild-type Arabidopsis seedlings were analyzed under various filters to isolate the UV-C effect from effects caused by the visible fluors in the germicidal bulb. Figure 2 shows that the addition of a clear plexiglass mitigates several effects of the treatment. Neither the initial growth inhibition response (within minutes of treatment) nor the sustained growth inhibition response was observed. The sharp peak at 20 min was still observed and rapid growth continued after treatment. This pattern is reminiscent of seedling growth patterns in response to green light treatment. To test this possibility the same light source was filtered with green plexiglass. The seedling response pattern paralleled that of the clear plexiglass, with a rapid growth rate upon illumination with this narrower and lower fluence rate visible treatment.

Figure 2.

Hypocotyl growth rate in response to a filtered UV-C pulse. The hypocotyl growth rate of Arabidopsis thaliana seedlings (Col-0) was monitored for 1 h in darkness. A 10 min pulse of UV-C at 750 mW m⁻² either unfiltered (solid line), filtered by plexiglass (filled circles) or green plexiglass (open circles) was delivered to seedlings at 0 min. Growth rates were measured for 120 min. Growth rate is measured as the change in height over 5 min, and individual seedling growth rates were normalized to the mean growth rate for the 30 min prior to treatment. Error bars represent SE of the mean. N = 10 (green plexiglass), 16 (clear plexiglass) and 24 for wild type.

Fluence response experiments were performed in Arabidopsis seedlings. Dark-grown seedlings were treated with a 10 minute pulse of UV-C irradiation consisting of 100 mW m⁻², 300 mW m⁻² or 750 mW m⁻². The results are presented in Figure 3, and show that there is a slight dose-response effect in Arabidopsis seedlings. While the 100 mW m⁻² and 300 mW m⁻² rates cannot be differentiated, a general lower growth rate trend is observed for the 750 mW m⁻² treatment, with some data points suggesting significant differences.

Figure 3.

Fluence-rate response kinetics of seedling growth inhibition in response to a 10 min UV-C pulse. The growth of dark grown Col-0 Arabidopsis seedlings was monitored for 1 h in complete darkness. A 10 min pulse of UV-C was delivered and hypocotyl growth kinetics were measured for 120 min. UV-C was provided at three treatments, 100 mW m-2 (triangles), 300 mW m-2 (filled circles) or 750 mW m-2 (open circles, also shown in Fig. 2). Growth rate is calculated from the change in hypocotyl length over 5 min. Each seedling's growth rate is normalized to the average growth rate over the 30 min prior treatment. Error bars represent standard error of the mean. N = 21–24 seedlings.

The strong primary growth inhibition of hypocotyl elongation rate suggested a likely role for phototopins. This hypothesis was tested using Arabidopsis phototropin mutants in the conditions defined in Figure 2. Dark grown phot1phot2 Arabidopsis seedlings were given the same UV-C treatments as the wild type plants in Figure 4. The results are presented in panels A–D. Following a 100 mW m⁻² pulse the phot1phot2 mutants respond essentially as wild type, showing a general trend of growth inhibition of 59% of the dark growth rate 45 min after treatment. This response was comparable to wildtype, as well as cry1cry2 mutants. Most notably the seedlings exhibit the normal initial growth inhibition between 0–10 min, the response normally mediated by phototropin 1.

Figure 4.

Analysis of photomorphogenic mutants. UV-C induced hypocotyl growth inhibition was measured in phyAphyB (triangles), cry1cry2 (open circles) and phot1phot2 (closed circles) double mutants in response to a 10 min pulse of light at 100, 300 mW m⁻² or 750 mW m⁻² (A–C, respectively). The wild-type response is depicted as a dashed line for clarity. (D) presents the response in phot1 (closed circles), phot2 (open circles) or phot1phot2 double mutants (open triangles) exposed to 300 mW m⁻² UV-C. For all trails the dark rate of seedling growth was monitored for one hour and then plants were treated with a UV-C pulse for 10 min. The subsequent changes in growth rate were monitored for 120 min. Individual seedling growth rates were normalized to the mean growth rate 30 min before treatment. Error bars show standard error of the mean. N = 13 to 32 seedlings.

At higher fluence rates the effect of photoreceptor mutation becomes apparent. While phytochrome and cryptochrome mutants respond similarly to wild type seedlings, the phot double mutants show immediate growth inhibition that differ from inhibition in the wild type seedlings in several ways. Such differences were observed in response to both 300 mW m⁻² (Fig. 4B) and 750 mW m⁻² (Fig. 4C). Seedlings carrying the phot1phot2 mutations recover from initial inhibition caused by the 300 mW m⁻² irradiation. Other major differences were observed during sustained growth inhibition. After 40 min the phot mutant seedlings treated with 300 mW m⁻² exhibit no mean growth inhibition, whereas the 750 mW m⁻² treatment results in inhibition that is not as complete as wild type, cry1cry2 or phyAphyB seedlings.

The lack of growth inhibition in the phot1phot2 double mutant was analyzed further using the single mutants, phot1 (nph1-5) and phot2 (cav1-1). The results are presented in Figure 4D. The phot1 mutants fail to show sustained growth inhibition, almost identically to the phot1phot2 double mutant. On the other hand, the phot2 mutant exhibits inhibition patterns reminiscent of those observed in wild-type plants.

Discussion

Although the biosphere is shielded from UV-C irradiation by the Earth's atmosphere, fundamental study of photobiological responses to these wavebands is informative and relevant. Indeed Steinmetz and Wellmann¹⁶ reported an action spectrum for growth inhibition in cress hypocotyl that exhibited maximal quantum efficiency at 260 nm. Hence these studies have application in understanding how plant growth and development may be affected by UV-C in the event of sustained changes in the ozone layer. In terms of basic science the study of photoreceptor-mediated responses to these extreme wavelengths helps to expand the understanding of receptor photochemistry and the fundamental roles of plant light sensors.

A significant finding of this study is that the phot1 receptor at least partially mediates the response to UV-C. This is especially noteworthy because the known absorption spectra for phototropins would not predict any biological activity of the receptor in response to UV-C. The broad range of fluence rates and wavelengths that can be detected by phototropin1 and rapid and dynamic response that it mediates, point to this receptor in regulating early development processes in response to UV-C irradiation. While the phot1phot2 double mutant was utilized across all fluences to analyze the genetics of the response (Fig. 4A–C), the receptors mediating the response could be more finely determined by testing the phot1 and phot2 mutants. Individual phot mutants were then examined at the 300 mW m⁻² level. Tests at this fluence might best differentiate phot1 from phot2 influence because it represents the maximum difference observed between the phot1phot2 double mutant and wild type. When tested individually the phot1 mutant response tracked closely the patterns observed in phot1phot2 double mutants whereas the phot2 single mutant closely paralleled the wild type response (Fig. 4D). These results indicate that phot1 is the primary photosensor mediating the response to UV-C, and at least at this fluence rate can fully account for the response observed.

The participation of phototropin1 is plausible because flavin mononucleotide, the phototropin chromophore,¹⁸ undergoes conformational changes induced by 260–300 nm light when bound to the phototropin LOV2 domain.^19,20 Consistent with these findings UV-C (280 nm) was shown to induce autophosphorylation of phot1, presumably due to stimulation of electron transfer between aromatic amino acids.¹⁴ The patterns of phosphorylation are significantly different than those observed by normal phot activation. In the current report we demonstrate that this state is biologically active and modulates the amplitude of hypocotyl growth rate, especially in response to specific fluence rates.

Figure 1 illustrates the growth kinetics for etiolated cucumber seedlings in response to a 10 min pulse of UV-C. The geometry of the curve during this time course indicates three distinct phases that match well with published data from tests with other light qualities. First, a rapid inhibition of growth rate occurs within minutes, followed by a transient increase between 20–40 min, and ending with decreasing growth rates that ultimately reach strong suppression of growth by 60 min after initiation of the treatment. The various kinetics of this response could be analyzed genetically by studying the same response in Arabidopsis seedlings. Figure 3 shows that generally similar trends can be observed in Arabidopsis seedlings. The initial decrease in growth, a rapid and transient increase and long-term sustained inhibition represent three comparable phases that can be studied genetically using the resources of the species.

The transient increase in growth occurs in parallel with a described response to pulsed or constant low-fluence rate green light.²¹ Germicidal bulbs contain low-pressure mercury vapour that emits visible light. It stands to reason that these wavelengths could potentially contribute to the observed kinetics, particularly the transient increase in growth rate. The results in Figure 2 show that removal of UV-C completely eliminated the immediate and sustained phases of growth inhibition. These results provide evidence that UV-C is causing the inhibition and that green wavebands likely account for the transient increase observed in these experiments. The results of fluence-response experiments performed in various mutant backgrounds are presented in the three panels of Figure 4. Figure 4A presents the response after treatment with 100 mW m⁻² of UV-C. All mutants tested respond similar to wild-type seedlings. However, upon higher fluence rates the phot1phot2 double mutant shows incomplete immediate growth inhibition and decreased sustained inhibition. The phot1phot2 seedlings treated with 300 mW m⁻² fail to exhibit any appreciable decrease in growth inhibition where a pronounced but incomplete inhibition is observed in the 750 mW m⁻² treatments of this genotype. This feature plus the larger magnitude inhibition seen in the phyAphyB mutants only at the 300 mW m⁻² irradiation suggests that there are multiple signaling systems interacting to produce the final response. Three photosensory systems appear to be involved: (1) an unknown UV-C sensor; (2) a phytochrome and (3) phot1. An ad hoc model that would explain our results is as follows: At 100 mW m⁻² only the unknown sensor is active and induces inhibition. At 300 mW m⁻² all three are active with the effect of the unknown sensor counterbalancing the stimulation of elongation caused by the phytochrome. At 750 mW m⁻² the unknown sensor mediated response is increased relative to the lower fluence rates while the magnitude of the other two responses remain constant. If the unknown sensor is the same as reported in Shinkle et al.¹⁵ it is not unreasonable that this response is more fluence dependent than the other two responses because it has been shown to not obey Bunsen-Roscoe reciprocity, and hence would exhibit the combined effect of increasing fluence and increased fluence rate. One unexpected outcome is that phot1 alone is required for the maintenance of growth rate inhibition occurring 40–120 min after illumination. The phot1 receptor is a reasonable candidate because the phot1 receptor is necessary and sufficient to drive the first phase of growth inhibition in blue light (ref. ¹³, Folta, unpublished). In studies with blue light, phototropin1 mediated only primary growth inhibition, the phase occurring between 0 and 30–40 min after illumination begins.¹³ Phototropin activity could be detected in response to constant or pulsed blue light, as discerned from genetic and pharmacological analyses.^13,22 In this case the role for phototropins is not as apparent in primary growth inhibition, as the phot1phot2 mutants exhibit at least some degree of growth inhibition. It is clear that the phototropins do play a role in the maintenance of growth inhibition after the growth rates decrease in the final phase of inhibition, as the mutants grow at dark rates under certain fluence conditions relatively long after the pulse has been delivered. Following a blue light treatment the seedlings would trend toward dark growth rates at this time, and even cry-mediated growth inhibition would be only evident under full illumination. The finding that UV-C activation of phototropins leads to sustained growth inhibition defines a new mechanism for photo signaling, a process that usually is rapid and complete soon after photoreceptor light sensor or excited signaling pathway that maintains growth inhibition even in sustained instances of darkness following UV-C activation.

However, phototropin1 is not the sole receptor important for early photomorphogenic responses. As we have shown here, absence of the phototropin1 receptor only partially alleviates primary growth inhibition in response to UV-C irradiation. Another UV-B receptor has been postulated based on UV-B-mediated stomatal opening responses,³¹ but the action spectrum indicated a steep decline in quantum efficiency below 270 nm. Another possible explanation is that oxidative and/or DNA damage act as the “receptor” that mediates this response. Although these signals may have some role in hypocotyl growth inhibition, the full responsibility for this response cannot be attributed to this pathway. Shinkle et al. demonstrated that activation of photolyase with white light could partially restore hypocotyl growth rate in cucumber seedlings that had been exposed to UV-C.¹⁵ The instantaneous hypocotyl elongation inhibition with UV-C exposure and that lack of complete growth rate recovery with DNA repair indicate that this response is mediated by more than just cellular damage. Further, Shinkle et al.³² have demonstrated that UV-C radiation initiates a rapid accumulation of UV-absorbing pigments and lateral expansion of hypocotyls. It seems unlikely that the biosynthesis and developmental regulation involved in these two responses is consistent with extensive cellular damage.

There are many potential candidate sensors for the UV-C mediated response we describe, as many biomolecules absorb in these wavebands. Nucleic acids are often proposed as targets.²⁴ Tryptophan has an absorption maximum of approximately 280 nm²⁴ and in animals, it has been shown to react with UV-B to form 6-formylindolo[3,2-b]carbazole (FICZ). FICZ is recognized by an arylhydrocarbon receptor that travels to the nucleus to regulate the expression of genes associated with UVB radiation toxicity responses.²⁵ Other candidates UV-B and UV-C sensors include flavins,²⁶ indole acetic acid (IAA),²⁷ provitamin D²⁸ and pterins.²⁹ Recent studies of responses to UV-C utilize light in this waveband as a surrogate for oxidative stress, and document effects related to apoptosis.³⁰ These studies used tissue culture cells and UV-C treatments incorporating energy levels at least two orders of magnitude higher than those used here, leaving the relationship of these responses to the ones reported here unclear and likely remote.

There has been much progress in identifying downstream responses initiated by UV-B and their proximal regulators. These include the UIL3 gene,³³ the UVR-8 gene³⁴ the contribution of the Hy5 gene³⁵ the RUS1 gene³⁶ and the role of chromatin structure.³⁷ It should be noted that these advances have principally studied responses to UV-B radiation in the 300–320 nm range, while the few studies of responses to shorter wavelengths have indicated that the responses to radiation at wavelengths <290 nm are mediated by signal response processes separate from those induced by longer wavelength radiation.^15,38 Hence our study here has added one more response to the set of UV-B/C regulated phenomena whose signal transduction processes are the least well characterized.

Materials and Methods

Plant material and growth conditions.

Cucumber (Cucumis sativus L., cv. Burpee's Pickler) seeds were germinated and grown in 50 ml beakers containing moist vermiculite. Beakers were placed in 42 × 27 × 16 cm clear plastic storage boxes which had a 2 to 4 mm layer of distilled water on the bottom and which were loosely-fitted with lids to reduce evaporation. Storage boxes were placed in a growth room maintained at 24 ± 1°C, under dim red light (DRL, 0.3–0.5 µmol m⁻² s⁻¹) as described previously.¹¹ Hypocotyl elongation rate was measured by radial position transducer as described in Shinkle et al.¹¹

Arabidopsis thaliana (Col-0) wild-type and mutant seeds were surface sterilized with ethanol and sown on a minimal medium of 1 mm KCl and 1 mm CaCl₂ solidified with 1% Difco agar (Beckton, Dickinson and Company; Sparks, MD). The genotypes of mutant seeds are phot1phot2 (nph1-5 cav1), phot1-5, phot2 (cav1) cry1cry2 (cry1-304cry2-1) and phyAphyB [a novel allele of phyA from the SALK collection (col-0) crossed with phyB-5 (ler)]. Seeds were stratified for 1–4 d at 4°C in darkness and then were given a pulse of white light (15 min, 100 µmol m⁻² s⁻¹) to synchronize germination. Seedlings were grown vertically in darkness at 23°C for 38–50 h until seedlings fully emerge and hypocotyls reached 1–2 mm in height, corresponding to their most rapid hypocotyl elongation rate.

Irradiation treatments.

The UV-C light source was as described in Shinkle et al. 2005. Output spectra were measured with a double-monochromator spectroradiometer (Model 752, Optronic Laboratories, Orlando, FL) that was calibrated for wavelength accuracy (emission line at 312.9 nm from a 4 W fluorescent lamp) and absolute responsiveness (200 W tungsten-halogen lamp traceable to NIST standard) prior to measurement. For individual experiments, irradiances were verified using a UVX radiometer/UVX-31 sensor (UVP Inc., San Gabriel CA) that was calibrated against the spectroradiometer. Overall irradiance (and fluence rate) was 600 mW m⁻² (1.3 µmol m⁻² s⁻¹).

High-resolution image capture and analysis were performed as described,^9,13 except that the seedlings were placed onto vertical agar in a square Petri dish featuring a a UV-C transmissible polycarbonate window above the seedlings. UV-C was provided from a 15 W germicidal lamp (FG15T8, Bulbrite, Moonachie, NJ). Seedlings were grown for 1 h in darkness and then given a 10 min pulse of UV-C, followed by growth in complete darkness. Seedlings were imaged every five minutes for the duration of the experiments. UV-C pulses were administered at the following fluence rates and conditions: 100 mW m⁻², 300 mW m⁻², 750 mW m⁻² or 300 mW m⁻² filtered with clear plexiglass, or filtered with green plexiglass. Fluence rates were determined using UVX radiometer/UVX-31 sensor (UVP Inc., San Gabriel, CA). It is important to note that the UV light source was activated for 10 min prior to seedling treatment, as the bulb generates significant infrared energy on start-up that interferes with image acquisition.

Image analysis.

Growth rates were determined by analyzing images using custom software written in the National Instruments LabView environment.¹³ The growth rates of individual seedlings were calculated as the change in hypocotyl length over five minutes. Individual seedling growth rates were made relative to each other by normalizing their growth rate relative to the dark rate, obtained by averaging the growth rate 30 min prior to treatment.