Phototropin is partly involved in blue-light-mediated stem elongation, flower initiation, and leaf expansion: A comparison of phenotypic responses between wild Arabidopsis and its phototropin mutants

Journal Pre-proof Phototropin is partly involved in blue-light-mediated stem elongation, flower initiation, and leaf expansion: A comparison of phenotypic responses between wild Arabidopsis and its phototropin mutants Yun Kong, Youbin Zheng

PII:

S0098-8472(19)31565-5

DOI:

https://doi.org/10.1016/j.envexpbot.2019.103967

Reference:

EEB 103967

To appear in:

Environmental and Experimental Botany

Received Date:

19 June 2019

Revised Date:

28 November 2019

Accepted Date:

16 December 2019

Please cite this article as: Kong Y, Zheng Y, Phototropin is partly involved in blue-light-mediated stem elongation, flower initiation, and leaf expansion: A comparison of phenotypic responses between wild Arabidopsis and its phototropin mutants, Environmental and Experimental Botany (2019), doi: https://doi.org/10.1016/j.envexpbot.2019.103967

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Phototropin is partly involved in blue-light-mediated stem elongation, flower initiation, and leaf expansion: A comparison of phenotypic responses between wild Arabidopsis and its phototropin mutants Yun Kong and Youbin Zheng* School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada * Corresponding author. E-mail address: [email protected] (Y. Zheng)

ro of

Highlights  Phtotropins are partly involved in blue-light-promoted stem elongation  Phtotropins contribute to flowering promotion by blue light  Phototropins play a role in blue-light-mediated leaf expansion

Jo

ur

na

lP

re

-p

Abstract Our previous study on bedding plants indicated that blue light in association with low phytochrome activity, relative to red light, can promote elongation growth as one of the shadeavoidance responses. We hypothesized that phototropin is involved in the blue light promotion effects. To test the hypothesis, plant growth and morphology were examined in wild Arabidopsis (Col-0), and its three phototropin-deficient mutants (phot1, phot2, and phot1phot2) under four light quality treatments: (1) R, pure red light (660 nm); (2) B, pure blue light (455 nm); (3) BR, unpure blue light created by mixing B with low-level (6%) R; and (4) BRF, unpure blue light created by adding low-level far-red light (735 nm) to BR with red/far-red ≈ 1. Continuous (24-h) light-emitting diode lighting with 100 μmol·m−2·s−1 photosynthetic photon flux density, and an air temperature of ≈ 23 ℃ were used with the above treatments. The calculated phytochrome photoequilibrium, an indicator of phytochrome activity, was 0.89, 0.69, 0.50, and 0.60 for R, BR, B, and BRF, respectively. After 20 days of light treatments, for the wild-type plants, B or BRF, compared to R or BR, promoted stem elongation, plant flowering, and leaf expansion, showing typical shade-avoidance responses. However, for the phototropin mutants, the promotion effects of B or BRF, relative to R or BR, on the above plant traits were reduced. In the absence of the phototropin(s), plants had reduced stem elongation and delayed flower initiation under B or BRF rather than R or BR, or reduced leaf expansion to a larger degree under B or BRF than R or BR. Our results suggest that phototropin is partly involved in the promotion of stem elongation, plant flowering, and leaf expansion mediated by blue light in association with low phytochrome activity. Keywords: Arabidopsis, phototropin mutants, plant morphology, flowering time, leaf size, phytochrome photoequilibrium, 1. Introduction Our recent study using light-emitting diodes (LED) indicated that the blue-light-mediated elongation growth is affected by phytochrome activity (Kong et al., 2018). In this study, pure blue light (B) increased elongation growth relative to red light (R). However, unpure blue light (BR), created by adding a low level (10%) of R to B, reversed the promotion effect induced by B, and

Jo

ur

na

lP

re

-p

ro of

showed a similar or greater inhibitory effect compared to R. When a low level of far-red light (FR) was further added to BR (with a ratio of R/FR ≈ 1), the resulting unpure blue light (BRF) recovered the elongation growth promotion effect induced by B, and showed a similar promotion effect as B, compared to R. The phytochrome photostationary state (PPS) value, an indicator of phytochrome activity, was greater for R (0.89) and BR (0.74) than BRF (0.63) and B (0.49). It appeared that plant elongation growth was promoted by blue light in association with lower phytochrome activity (i.e., B or BRF), but inhibited by blue light in association with higher phytochrome activity (i.e., BR). However, the inhibition of elongation growth by BR cannot be explained only by higher phytochrome activity, since BR, despite having a lower PPS, showed a greater inhibitory effect on elongation than R for some species. This suggests that the elongation inhibition from the blue light photoreceptor, cryptochrome, may also be playing a role. In this case, blue-light-mediated cryptochrome activity could be modified by phytochrome activity (Liu et al., 2016), which can be affected by background light conditions. The co-action of blue light and other light signals can be easily found in natural conditions. For example, under canopy shade, depletion of blue light does not occur alone, but is accompanied by low R/FR ratios (Ballaré and Pierik, 2017). Possibly, plants under B or BRF had reduced cryptochrome activity with decreased phytochrome activity. In other words, there is a cross talk between cryptochrome and phytochrome (Liu et al., 2016). However, another question raised is whether the elongation promoted by B or BRF resulted only from lower activity of phytochrome and cryptochrome? Phototropin, in addition to cryptochrome, is another blue light receptor system, and phototropin controls a range of responses which include phototropism, chloroplast movements, stomatal opening, and leaf expansion (Christie, 2007). Due to phototropism, phototropins change the growth direction of stems and leaves towards blue light under canopy shade, where phytochrome activity is weak because of a lower R/FR ratio (Fraser et al., 2016). Also, for sesame seedlings at de-etiolation stage, the phototropism triggered by a blue-light gradient is enhanced by simultaneously decreased phytochrome activity (Woitzik and Mohr, 1988; Goyal et al., 2016). Shoot phototropism has two growth phases: the first-phase growth occurs in response to short light pulses, while the secondphase growth is induced by prolonged irradiation (Christie and Murphy, 2013; Sullivan et al., 2016). It is possible that through second-phase shoot phototropism, phototropin participated in elongation promoted by B and BRF (i.e., blue lights in association with low phytochrome activity). Arabidopsis has two phototropins, including phot1 and phot2, which show overlapping functions besides their own unique roles (Christie, 2007). In hypocotyls of Arabidopsis, phot1 operates in a broaden range of light intensity (0.01–100 μmol m−2 s−1), but phot2 functions only under higher light conditions. i.e., > 1 μmol m−2 s−1 (Matsuoka et al., 2007). For shoot elongation, further study is needed to determine whether one phototropin (i.e., either phot1 or phot2) or both phototropins contribute to elongation promoted by B and BRF under ≈ 100-µmol m−2 s−1 photosynthetic photon flux density (PPFD). Our recent study on bedding plants indicated that the elongation growth promoted by blue light in association with low phytochrome activity was a shade-avoidance response (Kong et al., 2018). In addition to promoting elongation growth, B or BRF vs. R or BR initiated earlier flowering, and changed leaf morphology (e.g., increased leaf size, and reduced greenness), with varied sensitivity among species. The typical blue-light-controlled shade-avoidance response is phototropism and this process is mediated by phototropins, including phot1 and phot2 found in Arabidopsis (Briggs and Christie, 2002; Ballaré and Pierik, 2017). In Arabidopsis seedlings, blue-light-controlled

phototropism is enhanced by light signaling of low R/FR ratios which are normally found under vegetated shade and result in low phytochrome activity(Goyal et al., 2016; Ballaré and Pierik, 2017). Also, it has been shown that phytochromes can interact with phototropins to affect plant growth under vegetated shade (Goyal et al., 2016). It may be possible that phototropins also contribute some other shade-avoidance responses (e.g., changes in flower initiation and leaf morphology) that are mediated by blue light in association with low phytochrome activity. The objective of this study was to examine whether phototropins contribute to blue-lightmediated stem elongation, flower initiation and leaf expansion by comparing the phenotypic responses between wild type and phototropin-deficient mutants of Arabidopsis under four light treatments: R, B, BR, and BRF.

Jo

ur

na

lP

re

-p

ro of

2. Materials and methods The experiment was conducted in a walk-in growth chamber at the University of Guelph, Guelph, ON, Canada. Four genotypes of Arabidopsis, one wild type (Col-0) and three phototropindeficient mutants (phot1, phot2, and phot1phot2) (Sullivan et al., 2016) were used as plant materials for the experiment. Seeds were stratified for 3 days at 4 °C and then sown (one seed per cell) in 50cell (5×10) containing rockwool cubes (Starter Plugs, Grodan Inc., Ontario, Canada). The four genotypes were randomly distributed in the middle eight rows of a tray, and wild-type seeds were sown in the remaining two rows as border plants. The sown trays were placed inside the growth chamber for the light treatments. Plants were sub-irrigated with nutrient solution made from 20–8– 20 water soluble fertilizer (Master Plant-Prod Inc., Brampton, Ontario, Canada). The nutrient solution had a fertilizer:water mass ratio of 1:1600. The irrigation frequency depended on plant growth stage. The set points for air temperature and relative humidity in the growth chamber were around 23 °C and 65%, respectively. The light quality treatments were: (1) R, pure red light with a peak wavelength at 660 nm; (2) B, pure blue light with a peak wavelength at 455 nm; (3) BR, unpure blue light created by mixing B with low-level (6%) R; and (4) BRF, unpure blue light created by further adding low-level far-red light (peak wavelength at 735 nm) to BR with red/far-red ≈ 1 (Fig. 1). The PPS values for the four light quality treatments were 0.89, 0.69, 0.60 and 0.50 for R, BR, BRF and B, respectively, which were calculated according to Sager et al. (1988). Light spectrum treatments were provided by spectrum-tunable LED fixtures LX602C (Heliospectra AB, Gothenburg, Sweden). The four light quality treatments were randomly allocated to four compartments in the growth chamber. The compartments were separated by opaque curtains to prevent neighboring effects. For each treatment, a PPFD of around 100 µmol m−2 s−1 at the plant canopy level was achieved by adjusting the light intensity output of the LED fixtures. Light spectra and intensities were set up and verified using a radiometrically-calibrated spectrometer (XR-Flame-S; Ocean Optics, Inc., Dunedin, FL, USA). Once over 50% of the seeds had germinated under all the treatments, the investigation of germination percentages was carried out. At 20 d after light treatments began, five plants were randomly selected from each species under each light treatment to observe main stem length, rosette canopy height, total leaf number, rosette leaf number, flowering index, and leaf morphologies. The flowering index was defined as follows: 0, flower stalk invisible; 1, flower stalk visible; 2, flower petal visible; 3, flower(s) opened. For leaf morphology observations, two fully expanded leaves, with petioles, were sampled from the top rosette canopy of each plant. The detailed observation

method of leaf morphology can be found in the Kong et al (2019). Briefly, the leaves and a standard scale were scanned using a digital scanner (CanoScan LiDE 25; Canon Canada Inc., Brampton, ON, Canada). Petiole length, leaf area, maximum blade length, and leaf color (RGB values) were determined from the scanned images using ImageJ 1.42 software (National Institutes of Health, USA). Hue angle was calculated from the above RGB values using the formulas provided by Karcher and Richardson (2003). Analysis of variance was performed using the Data Processing System software (DPS, version 7.05; Refine Information Tech. Co., Hangzhou, China), and data were presented as means ± SE (standard error). The means were separated using Duncan's new multiple range test at the P ≤ 0.05 level.

ro of

3. Results There were no differences in germination among the treatments (data not shown). At harvest, plant morphology response differed among light quality treatments, as well as between wild types and phototropin mutants (supplemental Fig. S1).

Jo

ur

na

lP

re

-p

For main stem length, the patterns of response to light treatments were different between phototropin mutants and wild type (Fig. 2A). For wild type, B and BRF increased main stem length compared to R and BR, and BR showed a similar inhibitory effect as R, but BRF showed a greater promotion effect than B. For phototropin mutants, P1P2 plants showed a similarly shorter main stem length under B, R and BR, than under BRF due to the reduced promotion effect of BRF, and the eliminated promotion effect of B. Despite similar light response patterns to wild-type plants, P1 and P2 plants reduced the promotion effect of BRF, and P2 plants reduced the promotion effect of B. The decreased promotion effects of B or BRF resulted from shorter main stem length under BRF for P1 plants and under both B and BRF for P2 and P1P2 plants vs. wild-type plants, despite similar main stem lengths under R and BR. This suggests that B or BRF vs. R or BR promoted stem elongation, but the promotion effect was reduced under B in the absence of phot2, and under BRF in the absence of either phot1 or phot2. For rosette canopy height, phototropin mutants also differed from wild type in patterns of response to the light treatments (Fig. 2B). For the wild type genotype, the rosette canopy was the highest under B, followed by R and BRF, and was the lowest under BR. For phototropin mutants, B resulted in rosette canopy heights similar to only R for P1 and P2 plants, and similar to both R and BRF for P1P2 plants, although BR still led to the shortest rosette canopy for P2 and P1P2 plants. Under R, the phototropin mutants showed rosette canopy heights similar to wild types, but rosette canopy under B was shorter for phototropin mutants than wild types. This suggests that, in the absence of phototropins (either phot1 or phot2), the promotion effects of B vs. R on rosette canopy height disappeared. All genotypes showed similar patterns of response of leaf number to light treatments. Total leaf number did not change under B and BRF vs. R, but deceased under BR vs. R (Fig. 3A). With the only exception that P1P2 mutant plants under BR developed fewer leaves than the wild-type plants, there were similar total leaf numbers between phototropin mutant and wild type under R, B, or BRF. Generally, rosette leaf number showed a similar pattern of response to light treatments between phototropin mutants and the wild type; B, BR, and BRF, compared to R, reduced rosette leaf number

ro of

(Fig. 3B). For blue lights (B, BR, and BRF), plants had more rosette leaves under BR than B and BRF for wild-type plants, and only than BRF for phototropin mutants. Under BR, P1P2 mutants had fewer rosette leaves than wild-type plants. This suggests that BR inhibited leaf development (both total and rosette leaves) in the absence of both phot1 and phot2. Flowering index showed a light response pattern similar to main stem length (Fig. 3C). For both wild type and phototropin mutant, B and BRF increased flowering index compared to R and BR, and BR showed a similar inhibitory effect as R, and BRF showed a greater promotion effect on flowering than B except for P2 plants. However, all the phototropin mutants, compared to wild type, reduced the flowering promotion effects of B relative to R and BR, and P2 and P1P2 plants, compared to wild type, reduced the promotion effects of BRF relative to R and BR. Flowering index was reduced under B for all the phototropin mutants, and under BRF for P2 and P1P2 mutants compared to wild types. This suggests that B or BRF, compared to R or BR, promoted flowering, and the promotion effect was reduced under B in the absence of either phot1 or phot2, and under BRF in the absence of phot2.

Jo

ur

na

lP

re

-p

Generally, petiole length showed similar patterns of response to light treatments between wild type and phototropin mutant, and the shortest petioles were found under BR treatment (Fig. 4A). There were no differences in petiole length between B and R treatments, and petiole length decreased under BR vs. R. There were also no differences in petiole length under BRF vs. R treatments except for P1P2 mutant, which had shorter petioles. Under each light treatment, petiole length was similar between phototropin mutant and wild type, suggesting that phototropin contributed little to petiole elongation mediated by the tested light treatments. For leaf area and maximum blade length, wild type differed from P2 and P1P2 mutants in the pattern of response to light treatments (Fig. 4B and C). For plants from wild type and P1 mutant, B and BRF, compared to R and BR, increased leaf area and maximum blade length, but there were no differences between B and BRF. For P2 mutants, the promotion effects of BRF vs. R on the two traits disappeared. For P1P2 mutants, the promotion effects of both B and BRF vs. R on the two traits disappeared. Compared with wild-type plants, these two traits were reduced in P1 and P1P2 mutants under R or B, in the P1P2 mutant under BR, and in all the three phototropin mutants under BRF. This suggests that leaf expansion had the involvement of phot1 under R and B, either phot1 or phot2 under BRF, and both phot1 and phot2 under BR. For leaf color, wild type showed a different pattern of response to light treatments than P1 and P2 mutants (Fig. 4D). For wild type and P1P2 mutants, there were no differences in leaf hue angle among the four light treatments. For the P1 mutant, leaf hue angle was the smallest under R, and greater under B than BR, but BRF showed no difference from B and BR. For the P2 mutant, B and BR compared to R had increased leaf hue angle, but BRF showed no difference from other light treatments. Leaf hue angle was reduced under R and BR for the P1 mutant and under B and BRF for P1P2 mutants, compared to wild-type plants. This suggests that leaves reduced greenness under R and BR in the absence of phot1, and under B and BRF in the absence of phot1 and phot2. 4. Discussion 4.1. Phototropins are partly involved in blue-light-mediated stem elongation Phytochromes have a small peak of absorption in the region of blue-light, and it can mediate plant responses to blue light in addition to red light (Casal, 2012). In the present study, for wild-type

Jo

ur

na

lP

re

-p

ro of

Arabidopsis, B and BRF, compared to R and BR, had lower PPS values, and promoted main stem elongation. This indicated that the promotion effect of B and BRF was related to lower phytochrome activity, which supported the results reported in our previous study on bedding plants (Kong et al., 2018). However, the promotion effects on stem elongation cannot be explained only by low phytochrome activity, since BRF promoted stem elongation to a larger degree than B, despite having a higher PPS value (0.60 vs. 0.50). A recent study indicated that blue LED light affected the transcript levels of phytochromes, cryptochromes and phototropins (Pashkovskiy et al., 2016). The involvement of other photoreceptors cannot be ruled out. Based on the results of the present study, phototropins appear to be one of the photoreceptors involved in the elongation promoted by blue light in association with low phytochrome activity. Main stem elongation was reduced under B in the absence of phot2, and under BRF in the absence of either phot1 or phot2 or both. However, the absence of phototropins did not affect stem elongation under BR or R. Apparently, at least phot2 was involved in the promotion of main stem elongation by blue lights in association with low phytochrome activity (i.e., B and BRF). For rosette canopy height, the promotion effect of B vs. R also disappeared in the absence of phototropin (either phot1 or phot2 or both), because this trait was reduced under B, but not under R for all the phototropindeficient mutants relative to wild-type plants. Possibly, phototropin was also involved in the promotion of leaf hyponasty and/or hypocotyl length by B, which resulted in the increased rosette canopy height. It appears that when associated with lower, rather than higher, phytochrome activity, phototropin(s), as a blue light receptor, can promote plant elongation. Our recent study on ornamental bedding plants indicated that the stem elongation promoted by blue light was a shadeavoidance response (Kong et al., 2018). A recent study showed that foliar shade also promoted elongation growth toward light (i.e., phototropism), which involves a crosstalk between two different photoreceptor systems: phototropins and phytochromes (Goyal et al., 2016). PHYTOCHROME KINASE SUBSTRATE 1 (PKS1) proteins are required for blue-light-induced phototropism in Arabidopsis (Lariguet et al., 2006). The PKS1 can interact with both blue and red light photoreceptors, phototropin and phytochrome, and may link the two different photoreceptor families with each other (Lariguet et al., 2006; Pashkovskiy et al., 2016). The crosstalk process needs a further study at the molecular level. Cryptochrome, another blue light photoreceptor, can also mediate plant elongation; showing inhibitory effects when it is activated (Huche-Thelier et al., 2016). In the present study, regardless of genotype, BR reduced rosette canopy height relative to R, despite having a lower PPS value (0.69 vs. 0.89). This suggests that cryptochrome plays an important role in the elongation inhibition mediated by blue light in association with higher phytochrome activity (i.e., BR). However, the inhibitory effect on elongation disappeared under blue lights in association with lower phytochrome activity (i.e., B and BRF) in the present study. In this case, low-activity phytochrome might reduce cryptochrome activity, and induce phototropin-triggering phototropism. Also, efficient phototropism relies on the mediation of cryptochromes and phytochromes, since the two photoreceptor systems control the expression of key regulators of phototropin signaling (Goyal et al., 2013). It appears that plant elongation mediated by blue light might result from the co-action of at least cryptochrome, phototropin, and phytochrome. A model about the crosstalk between the three photoreceptors could be proposed as follows (Fig. 5). 4.2. Phototropins contribute to flowering promotion by blue light in association with low

ur

na

lP

re

-p

ro of

phytochrome activity An acceleration of flowering is a typical shade-avoidance responses, seen clearly in Arabidopsis, but readily observable in many shade-avoiding plants (Smith and Whitelam, 1997). In our previous study on bedding plants, B or BRF, compared to R or BR, promoted flowering, and BRF showed a promotion effect similar to B (Kong et al., 2018). Likewise, in the present study on wild-type Arabidopsis, B or BRF initiated earlier flowering than R or BR. However, differing from bedding plants, wild-type Arabidopsis plants showed a greater promotion effect under BRF than B. Previous studies have indicated that both blue light and far-red light can promote plant flowering, but in a separate way, and these two spectra can show an additive effect, in some cases (Eskins, 1992; Guo et al., 1998; Lin, 2000). The additive promotion effect on flower development appeared to be species-dependent, and was more obvious in Arabidopsis than the tested bedding plants. For the Arabidopsis phototropin mutants, B and BRF vs. R and BR also promoted flowering, but the promotion effect was reduced under B in the absence of either phot1 or phot2, and under BRF in the absence of phot2. This suggests that at least one of the two phototropins (i.e., phot2) was involved in B- or BRF-promoted flowering. However, the absence of either phot1 or phot2 did not affect BR-mediated flowering. BR had a higher PPS value than B or BRF (Fig. 1), indicating a higher phytochrome activity. This suggests that phototropin may contribute to blue-light-mediated flowering when phytochrome activity is lower rather than higher. Further study is required to elucidate the underlying mechanism involved with the co-action of phototropin and phytochrome on flowering. In the absence of phototropin(s), B and BRF, compared to R and BR, still promoted flowering although the promotion effect was reduced, relative to wild-type plants. This suggests that phototropin may contribute only partly to flowering promotion by blue light in association with low phytochrome activity. Previous studies have indicated that cryptochrome and phytochrome are two key photoreceptors mediating plant flowering (Guo et al., 1998; Lin, 2000). However, recently, it has been found that there are surprising levels of crosstalk between photoreceptors, circadian clock, and sugar signaling in regulating floral signal transduction (Matsoukas, 2017). As blue light photoreceptors, phototropins, interact directly with circadian clock genes and proteins (Matsoukas, 2017). Also under low blue light condition, phototropins can increase light use efficiency and promote photosynthesis, thereby increasing accumulation of sugars as photo-assimilates (Takemiya et al., 2005; Christie, 2007; Boccalandro et al., 2012). Therefore, phototropins may exert an indirect effect on flowering through circadian clock and sugar signaling.

Jo

4.3. Phototropins play a role in blue-light-mediated leaf expansion Plants produce larger, thinner leaves in response to vegetated shade (Smith and Whitelam, 1997). Phototropin can function on leaf expansion that enlarges the effective area of leaves for light interception (Matsuoka et al., 2007). Both phototropins (i.e., phot1 and phot2) induce leaf expansion, independently of the other blue light receptors, cryptochromes, at a PPFD of around 100 μmol m-2 s-1 (Ohgishi et al., 2004). In the present study, for wild-type plants, B and BRF resulted in larger individual leaf area and greater blade length than R and BR, showing typical shade-avoidance responses. However, the promotion effects of B and BRF on leaf expansion were reduced in absence of phot1, and even disappeared in the further absence of phot2. In the absence of both phototropins, although R and BR also reduced leaf area and blade length, relative to wild-type plants, the reduction magnitudes were much smaller than B and BRF. It appeared that phototropins were more actively

ur

na

lP

re

-p

ro of

involved in leaf expansion mediated by blue light in association with lower vs. higher phytochrome activity. The co-action between phototropin and phytochrome was also supported by a previous study on Arabidopsis wherein the two photoreceptors co-regulated leaf flattening through a phototropin signaling transducer, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) (Kozuka et al., 2012). The development of longer petioles is another important leaf morphology response to vegetated shade (Smith and Whitelam, 1997). In the present study, wild-type plants had similar petiole lengths under B and BRF as R, indicating no shade-avoidance response. Also, in some previous studies, the shade signal from lowering blue light increased little petiole length (Djakovic-Petrovic et al., 2007; Pierik et al., 2009). Furthermore, in the present study, the absence of phototropins did not change petiole length under any light treatments, leading to similar patterns of response to light treatments between wild type and phototropin mutants. Possibly, the promotion of petiole growth by vegetated shade signals are mediated primarily by phytochrome, especially phyB, and secondarily by cryptochrome, mainly cry1 (Casal, 2012). This was also supported by the present study, where BR showed shorter petiole length than other light treatments in either wild type or phototropin mutants, possibly due to both activated phytochrome and cryptochrome. Substantial reductions in chlorophyll production is another, readily-observable, aspect of shadeavoidance response in leaf development (Smith and Whitelam, 1997). For wild-type plants in the present study, B and BRF caused similar leaf greenness as R and BR, without showing the typical shade-avoidance response such as stem elongation. The varying responses of different plant traits in the same species might result from different threshold levels of blue light required to induce shade-avoidance responses in different cells, and even organelles (Mishra and Khurana, 2017). However, leaves reduced greenness under R and BR in the absence of phot1, and under B and BRF in the absence of phot1 and phot2. Possibly, chlorophyll biosynthesis process involves phot1 when phytochrome activity is high, and both phot1 and phot2 when phytochrome activity is low. This suggests that phototropin was partly involved in chlorophyll biosynthesis, and phototropin had a coaction with phytochrome during this process. Using a phot2 mutant in Arabidopsis, previous study indicated that chlorophyll biosynthesis may be controlled by co-action between phytochromes and phototropins through regulating the formation of different protochlorophyllide forms (MysliwaKurdziel et al., 2013). Another study on rice showed that total chlorophyll concentration was twice as high in wild-type plants vs. phot1 mutants (Goh et al., 2009).

Jo

In summary, for wild-type Arabidopsis plants, B or BRF promoted stem elongation, plant flowering, and leaf expansion, relative to R or BR. However, in the absence of the phototropin(s), plants had reduced stem elongation and delayed flower initiation under B or BRF rather than R or BR, or reduced leaf expansion to a larger degree under B or BRF than R or BR. This suggests that phototropin is partly involved in the shade-avoidance responses mediated by blue light in association with low phytochrome activity in Arabidopsis. Author contributions YK designed and performed the experiments, collected and analyzed the data, and wrote and revised the manuscript. YZ was the principal investigator who participated in experimental design, data collection and analyzing, manuscript preparation and editing. Both authors approved the final manuscript.

Conflict of interest The authors declare that the study was conducted without any commercial or financial relationships which could be construed as a potential conflict of interest.

Jo

ur

na

lP

re

-p

ro of

Acknowledgements This research was supported by Natural Sciences and Engineering Research Council of Canada. We thank Heliospectra AB (Gothenburg, Sweden) for providing LED lighting systems for this study. We are grateful to Professor John M. Christie (University of Glasgow, Glasgow, Scotland, United Kingdom) for the donation of phototropin mutant seeds, and instructions for seed germination. Thanks also go to Katherine Schiestel for her excellent technical support during the trial and Dave Llewellyn for editing this manuscript during the revision.

References Ballaré, C. L., Pierik, R. 2017. The shade-avoidance syndrome: Multiple signals and ecological consequences. Plant Cell Environ. 40, 2530-2543. Boccalandro, H. E., Giordano, C. V., Ploschuk, E. L., Piccoli, P. N., Bottini, R., Casal, J. J. 2012. Phototropins but not cryptochromes mediate the blue light-specific promotion of stomatal conductance, while both enhance photosynthesis and transpiration under full sunlight. Plant Physiol. 158, 1475-1484. Briggs, W. R., Christie, J. M. 2002. Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Sci. 7, 204-210. Casal, J. J. 2012. Shade avoidance. The Arabidopsis book. 10, e0157. Christie, J. M. 2007. Phototropin blue-light receptors. Annu. Rev. Plant Biol. 58, 21-45. Christie, J. M., Murphy, A. S. 2013. Shoot phototropism in higher plants: new light through old concepts. Am. J. Bot. 100, 35-46. growth responses to canopy signals. Plant J. 51, 117-126.

ro of

Djakovic-Petrovic, T., Wit, M. d., Voesenek, L. A. C. J., Pierik, R. 2007. DELLA protein function in

Eskins, K. 1992. Light quality effects on Arabidopsis development: Red, blue and far-red regulation of flowering and morphology. Physiol. Plant. 86, 439-444.

Fraser, D. P., Hayes, S., Franklin, K. A. 2016. Photoreceptor crosstalk in shade avoidance. Curr. Opin.

-p

Plant Biol. 33, 1-7.

Goh, C.-H., Jang, S., Jung, S., Kim, H.-S., Kang, H.-G., Park, Y.-i., Bae, H.-J., Lee, C.-H., An, G. 2009. Rice phot1a mutation reduces plant growth by affecting photosynthetic responses to light during

re

early seedling growth. Plant Mol. Biol. 69, 605-619.

Goyal, A., Karayekov, E., Galvão, V. C., Ren, H., Casal, J. J., Fankhauser, C. 2016. Shade promotes phototropism through phytochrome B-controlled auxin production. Curr. Biol. 26, 3280-3287.

lP

Goyal, A., Szarzynska, B., Fankhauser, C. 2013. Phototropism: at the crossroads of light-signaling pathways. Trends Plant Sci. 18, 393-401.

Guo, H., Yang, H., Mockler, T. C., Lin, C. 1998. Regulation of flowering time by Arabidopsis photoreceptors. Science. 279, 1360-1363.

na

Huche-Thelier, L., Crespel, L., Le Gourrierec, J., Morel, P., Sakr, S., Leduc, N. 2016. Light signaling and plant responses to blue and UV radiations—perspectives for applications in horticulture. Environ. Exp. Bot. 121, 22-38. doi:10.1016/j.envexpbot.2015.06.009.

ur

Karcher, D. E., Richardson, M. D. 2003. Quantifying turfgrass color using digital image analysis. Crop Sci. 43, 943-951.

Kong, Y., Schiestel, K., Zheng, Y. 2019. Pure blue light effects on growth and morphology are slightly

Jo

changed by adding low-level UVA or far-red light: a comparison with red light in four microgreen species. Environ. Exp. Bot. 157, 58-68.

Kong, Y., Stasiak, M., Dixon, M. A., Zheng, Y. 2018. Blue light associated with low phytochrome activity can promote elongation growth as shade-avoidance response: a comparison with red light in four bedding plant species. Environ. Exp. Bot. 155, 345-359. https://doi.org/10.1016/j.envexpbot.2018.07.021. Kozuka, T., Suetsugu, N., Wada, M., Nagatani, A. 2012. Antagonistic regulation of leaf flattening by phytochrome B and phototropin in Arabidopsis thaliana. Plant Cell Physiol. 54, 69-79. Lariguet, P., Schepens, I., Hodgson, D., Pedmale, U. V., Trevisan, M., Kami, C., de Carbonnel, M., Alonso, J. M., Ecker, J. R., Liscum, E. 2006. PHYTOCHROME KINASE SUBSTRATE 1 is a

phototropin 1 binding protein required for phototropism. Proc. Natl. Acad. Sci. 103, 10134-10139. Lin, C. 2000. Photoreceptors and regulation of flowering time. Plant Physiol. 123, 39-50. Liu, B., Yang, Z., Gomez, A., Liu, B., Lin, C., Oka, Y. 2016. Signaling mechanisms of plant cryptochromes in Arabidopsis thaliana. J. Plant Res. 129, 137-148. doi:10.1007/s10265-015-0782z. Matsoukas, I. G. 2017. Crosstalk between photoreceptor and sugar signaling modulates floral signal transduction. Front. Physiol. 8, 382. Matsuoka, D., Iwata, T., Zikihara, K., Kandori, H., Tokutomi, S. 2007. Primary processes during the light-signal transduction of phototropin. Photochem. Photobiol. 83, 122-130. Mishra, S., Khurana, J. P. 2017. Emerging roles and new paradigms in signaling mechanisms of plant cryptochromes. Crit. Rev. Plant Sci. 36, 89-115. doi:10.1080/07352689.2017.1348725. Mysliwa-Kurdziel, B., Turek, E., Malec, P. 2013. Protochlorophyllide forms in etiolated seedlings of photoreceptor mutants of Arabidopsis thalianais chlorophyll biosynthesis controlled by

ro of

cooperation between phytochromes and phototropins?, p. 381-384. Photosynthesis Research for Food, Fuel and the Future. Springer.

Ohgishi, M., Saji, K., Okada, K., Sakai, T. 2004. Functional analysis of each blue light receptor, cry1,

cry2, phot1, and phot2, by using combinatorial multiple mutants in Arabidopsis. Proc. Natl. Acad. Sci. 101, 2223-2228.

-p

Pashkovskiy, P. P., Kartashov, A. V., Zlobin, I. E., Pogosyan, S. I., Kuznetsov, V. V. 2016. Blue light alters miR167 expression and microRNA-targeted auxin response factor genes in Arabidopsis thaliana plants. Plant Physiol. Biochem. 104, 146-154.

re

Pierik, R., Djakovic-Petrovic, T., Keuskamp, D. H., de Wit, M., Voesenek, L. A. C. J. 2009. Auxin and ethylene regulate elongation responses to neighbor proximity signals independent of gibberellin and DELLA proteins in Arabidopsis. Plant Physiol. 149, 1701-1712.

lP

Sager, J. C., Smith, W. O., Edwards, J. L., Cyr, K. L. 1988. Photosynthetic efficiency and phytochrome photoequilibria determination using spectral data. Trans. ASAE. 31, 1882-1889. Smith, H., Whitelam, G. C. 1997. The shade avoidance syndrome: multiple responses mediated by multiple phytochromes. Plant Cell Environ. 20, 840-844. doi:10.1046/j.1365-3040.1997.d01-104.x.

na

Sullivan, S., Hart, J. E., Rasch, P., Walker, C. H., Christie, J. M. 2016. Phytochrome A mediates bluelight enhancement of second-positive phototropism in Arabidopsis. Front. Plant Sci. 7, 290. Takemiya, A., Inoue, S.-i., Doi, M., Kinoshita, T., Shimazaki, K.-i. 2005. Phototropins promote plant

ur

growth in response to blue light in low light environments. Plant Cell. 17, 1120-1127. Woitzik, F., Mohr, H. 1988. Control of hypocotyl phototropism by phytochrome in a dicotyledonous

Jo

seedling (Sesamum indicum L.). Plant Cell Environ. 11, 653-661.

-1

A R

-2 -1

4

5 4

Light intensity (µmol m s nm )

-1 -2 -1

Light intensity (µmol m s nm )

5

0.89

3 2 1 0

B B

0.50

3 2 1 0

300 400 500 600 700 800 300 400 500 600 700 800

BR

0.69

3 2 1 0

D 4

BRF6

3 2 1 0

0.60

ro of

-1

4

-2 -1

C

Wavelength (nm)

5

-p

Light intensity (µmol m s nm )

-2 -1

-1

Light intensity (µmol m s nm )

Wavelength (nm) 5

300 400 500 600 700 800 300 400 500 600 700 800

Wavelength (nm)

re

Wavelength (nm)

na

lP

Fig. 1 The spectral distribution of four light quality treatments delivered by light emitting diodes (LEDs). R: pure red light; B: pure blue light; BR: unpure blue light created by mixing B with lowlevel (6%) R; and BRF: unpure blue light created by mixing BR with low-level far-red light (red/farred ≈ 1). The numbers inside the figures are PPS (phytochrome photostationary state) values, which are the estimated phytochrome photoequilibrium according to the method by Sager et al. (1988).

12

a

A

R BR

Jo

B BRF b

10

L: ***, G: **, L × G: *

8 6

d

de

4 2

fg

c

c

ur

Main stem length (cm)

14

e g

fg

g

fg

efg g

g

g

0 WT

P1

P2

P1+2

Rosette canopy height (cm)

1.2

B

R BR

a

1.0

B BRF

L: ***, G: ***, L × G: **

b 0.8

cde

cd

0.6

bc

bc

b efg

fg

fg gh

0.4

def

def

fg h

h

0.2 0.0

WT WT

P1

P2

P1P2

-p

ro of

Fig. 2. Elongation growth in wild-type and phototropin-deficient mutants of Arabidopsis under different light treatments. For the X-axis labels, WT: wild type; P1: phot1 mutant; P2: phot2 mutant; P1P2: phot1phot2 double mutant. For the figure legends, R: pure red light; B: pure blue light; BR: unpure blue light created by mixing B with low-level (6%) R; and BRF: unpure blue light created by mixing BR with low-level far-red light (red/far-red ≈ 1). For each plant trait (i.e., each chart), symbols for light quality (L), plant genotype (G), or the interaction of light quality and plant genotype (L × G) followed by ns, *, **, or *** denote that treatment effects are not significant or significant at P ≤ 0.05, 0.01, or 0.001, respectively. Within the same plant trait (i.e., inside each chart), data bearing the same letter are not significantly different at P ≤ 0.05, according to Duncan’s new multiple range test.

9

L: ***, G: **, L × G: ns

Jo

ab

ab ab

ab

ab

abc

cd

8 7 6

a

ab

abc cd

cd

na

5 12

B

10

re

10

A

lP

11

WT

8

P1 B BRF

R BR

a

ur

Rosette leaves (no.plant -1)

Total leaves (no.plant-1)

12

a

de

6

ab abc bcd

P2 P1+2 L: ***, G: ***, L × G: ns a bc

bc de

B BRF

e

a

b

R BR

cd

cd de

ef

ef de f

4 WT

P1

P2

P1+2

Flowering stage index

4.0

C

a

a

3.0

R

B

b

2.0

BRF

b

bc c

1.0 d

d

BR

L: ***, G: ***, L × G: ***

c

c

d

d

d

d

d

d

0.0

WT WT

P1

P2

P1P2

1.0 ab abc

0.6

Jo

a

abcd

abc

abc

bcde bcde

0.4 0.2

fg

na

0.0 1.5

B

1.2

WT

abc cdef def

P1 P2 L: ***, G: ***, L × G: **

P1+2 R B BR BRF

ab bc

bc

c

B BRF

g

a

a

abcd

R BR

ef

lP

efg

0.9

re

L: ***, G: ***, L × G: ns

ur

Individual leaf area (cm2)

Petiole length (cm)

A 0.8

-p

ro of

Fig. 3. Leaf development (A-B) and flowering development (C) in wild-type and phototropindeficient mutants of Arabidopsis under different light treatments. For the X-axis labels, WT: wild type; P1: phot1 mutant; P2: phot2 mutant; P1P2: phot1phot2 double mutant. For the figure legends, R: pure red light; B: pure blue light; BR: unpure blue light created by mixing B with low-level (6%) R; and BRF: unpure blue light created by mixing BR with low-level far-red light (red/far-red ≈ 1). For each plant trait (i.e., each chart), symbols for light quality (L), plant genotype (G), or the interaction of light quality and plant genotype (L × G) followed by ns, *, **, or *** denote that treatment effects are not significant or significant at P ≤ 0.05, 0.01, or 0.001, respectively. Within the same plant trait (i.e., inside each chart), data bearing the same letter are not significantly different at P ≤ 0.05, according to Duncan’s new multiple range test.

bc

bc

bc

cd

0.6

de

de

ef

0.3

f

ef f

0.0 WT

P1

P2

P1+2

Max. blade length (cm)

2.4

C

L: ***, G: ***, L × G: ***

2.0 1.6

a

ab

abc bcd

de gh

1.2

R BR

cd def

fgh

B BRF

de efg ghi

hi

hi

ij

0.8

j

0.4 WT

73 71

P1 R BR

D abc abc abc

P2 B BRF

L: ***, G: ***, L × G: *

a a ab abc bcd de bcde

bcde

f 69 67

WT WT

P1+2

P1

P2

cde de de

ef

ro of

Leaf hue angle (°)

75

P1P2

Jo

ur

na

lP

re

-p

Fig 4. Leaf morphology in wild-type and phototropin-deficient mutants of Arabidopsis under different light treatments. For the X-axis labels, WT: wild type; P1: phot1 mutant; P2: phot2 mutant; P1P2: phot1phot2 double mutant. For the figure legends, R: pure red light; B: pure blue light; BR: unpure blue light created by mixing B with low-level (6%) R; and BRF: unpure blue light created by mixing BR with low-level far-red light (red/far-red ≈ 1). For each plant trait (i.e., each chart), symbols for light quality (L), plant genotype (G), or the interaction of light quality and plant genotype (L × G) followed by ns, *, **, or *** denote that treatment effects are not significant or significant at P ≤ 0.05, 0.01, or 0.001, respectively. Within the same plant trait (i.e., inside each chart), data bearing the same letter are not significantly different at P ≤ 0.05, according to Duncan’s new multiple range test.

Fig. 5. A blue light action model about the crosstalk between phytochrome, cryptochrome, and phototropin in terms of elongation growth. PPS: phytochrome photostationary state.

A

B

Top view

Side view

WT

P1

P2

R

B BR Light treatments

BRF

R

ro of

P1P2

B BR BRF Light treatments

Jo

ur

na

lP

re

-p

Fig. S1. Plant morphology in wild-type and phototropin-deficient mutants of Arabidopsis under different light treatments. The length of horizontal reference bars in the upper right corners of the images is 1.6 cm. For Arabidopsis genotypes, WT: wild type; P1: phot1 mutant; P2: phot2 mutant; P1P2: phot1phot2 double mutant. For the four light treatments, R: pure red light; B: pure blue light; BR: unpure blue light created by mixing B with low-level (6%) R; and BRF: unpure blue light created by mixing BR with low-level far-red light (red/far-red ≈ 1). The pictures were taken after 20 days of light treatments.