Green light reduces elongation when partially replacing sole blue light independently from cryptochrome 1a

Abstract Although green light is sometimes neglected, it can have several effects on plant growth and development. Green light is probably sensed by cryptochromes (crys), one of the blue light photoreceptor families. The aim of this study is to investigate the possible interaction between green and blue light and the involvement of crys in the green light response of plant photomorphogenesis. We hypothesize that green light effects on morphology only occur when crys are activated by the presence of blue light. Wild‐type Moneymaker (MM), cry1a mutant (cry1a), and two CRY2 overexpressing transgenic lines (CRY2‐OX3 and CRY2‐OX8) of tomato (Solanum lycopersicum) were grown in a climate chamber without or with green light (30 μmol m−2 s−1) on backgrounds of sole red, sole blue and red/blue mixture, with all treatments having the same photosynthetic photon flux density of 150 μmol m−2 s−1. Green light showed no significant effects on biomass accumulation, nor on leaf characteristics such as leaf area, specific leaf area, and chlorophyll content. However, in all genotypes, green light significantly decreased stem length on a sole blue background, whereas green light hardly affected stem length on sole red and red/blue mixture background. MM, cry1a, and CRY2‐OX3/8 plants all exhibited similar responses of stem elongation to green light, indicating that cry1a, and probably cry2, is not involved in this green light effect. We conclude that partially replacing blue light by green light reduces elongation and that this is independent of cry1a.


| INTRODUCTION
Leaves reflect a relatively large part of green light (G), causing the green appearance of plants. Green light was for a long time thought to be irrelevant for plant functioning. However, this perception is now fading (Smith et al. 2017). Although leaves appear green, the fraction of green light that is reflected is only about 10-15% (Paradiso et al. 2011;Smith 1986), while the major share (about 75-80%) is absorbed, and the rest transmitted. This suggests that there might very well be a role of green light in photomorphogenesis. Green light may play a major role in controlling plant development in orchestration with red light (R) and blue light (B) (Folta & Maruhnich 2007).  suggested that this role is particularly important at low light conditions, like a canopy with a high planting density. On the other hand, Terashima et al. (2009) reported that at high photosynthetic photon flux density (PPFD), G drives leaf photosynthesis more efficiently than R and B. This is related to the fact that G can penetrate deep into the mesophyll layers at the single-leaf level (Smith et al. 2017).
There is increasing evidence for the ability of green light to regulate plant photomorphogenesis. Supplementing G to white light (W) or a mixture of R and B (RB) increased hypocotyl and petiole length in Arabidopsis (Folta 2004;Wang et al. 2015;Zhang et al. 2011).
Hypocotyls were longer when G:B ratio was higher (Sellaro et al. 2010). Higher G intensity also increased the content of photosynthetic pigments in Arabidopsis seedlings, and biomass and photosynthetic parameters in leaves of lettuce (Efimova et al. 2013;Golovatskaya & Karnachuk 2008;Johkan et al. 2012;Muneer et al. 2014). Lettuce plants grown in a mixture of R, B and G (RBG) had larger specific leaf area (SLA) but lower stomatal conductance compared with RB alone, where the total light intensity of RBG was higher than that of RB (Kim 2005). Plant height and dry weight increased in cucumbers when adding 520 nm G to a mixture of B, R and far-red light (RBFrG) compared with RBFr alone of similar light intensity, whereas such effects were not found when adding 595 nm G (Brazaitytė et al. 2009). In a recent review on green light, Battle et al. (2020) indicated that short-wavelength green light (500-530 nm) may lead to different responses compared to long-wavelength green light (530-600 nm). Growing lettuce plants at different combinations of G with RB showed that growth increased when the fraction of green light was raised from 0 to 24%, but increasing its proportion from 24 to 86% decreased the growth of leaf area and shoot mass (Dougher & Bugbee 2001;Kim et al. 2004).
The nature of the green light receptor remains controversial, although most researchers proposed that green light is sensed by cryptochromes (crys) (Banerjee et al. 2007;Bouly et al. 2007;Sato et al. 2015). In higher plants, three crys have been described to date: CRY1 and CRY2, both localized predominantly in the nucleus and the cytoplasm (Lin & Shalitin 2003), and CRY3 in the organelles (Kleine et al. 2003). Two CRY1 (CRY1a and CRY1b), one CRY2 and one CRY3 (CRY-DASH) genes have been isolated in tomato (Facella et al. 2006;Perrotta et al. 2000Perrotta et al. , 2001. It has been suggested that green light reverses the action of blue light on the activity of crys, making them inactive for blue light (Banerjee et al. 2007;Bouly et al. 2007). This antagonistic blue-green interaction was supposed to be mediated through the interconversion of flavin redox states of crys. The authors concluded that the fully oxidized chromophore (FAD) absorbs blue light and is then converted to a semi-reduced chromophore (FADH), which is the biologically active green-absorbing form. However, there are some inconsistencies with this proposition.  found that G cannot reverse the cry-mediated B inhibition of early stem elongation but acts additively with B to drive cry-mediated inhibition. Sato et al. (2015) found that sole G or sole B during the night period inhibited hypocotyl elongation, which seemed to be mediated by cry2. The carotenoid zeaxanthin has been suggested as a photoreceptor for the stomatal blue light response, which could be reversed when adding G to B, indicating that zeaxanthin might absorb G (Frechilla et al. 1999(Frechilla et al. , 2000. Using different photoreceptor mutants of The aim of this study is to investigate the interaction between G and B and the involvement of crys in the green light response of plant photomorphogenesis. We hypothesize that the effect of G on stem elongation only occurs when crys are activated by the presence of B. Experiments in climate rooms were conducted where the effects of 525 nm G were studied by replacing 20% background light of sole B, sole R as well as red/blue mixture. To study the involvement of crys, we used a cryptochrome-deficient genotype and two genotypes overexpressing crys. In contrast to many other studies on G, we kept the PPFD as well as the ratio of other colors the same when G was added. Ten days after sowing, plants were transplanted in 11 Â 11 Â 12 cm black plastic pots filled with $6 mm expanded clay grid (4-8 mm; Jongkind hydrocorns) and light treatments started.
The treatments consisted of sole blue, sole red, red/blue mixture (red/blue ratio = 3/1) with or without green. Total PPFD was kept at 150 μmol m À2 s À1 at the top of plants in all treatments. When green was added, the red/blue ratio was kept the same as in the treatment without green light (Table 1)

| Measurements
Plants were measured 21 days after transplanting. Stem length was measured up to the apex. Total leaf area was measured using a leaf area meter (model LI-3000; LI-COR). Roots, stems and leaves were separated and dried in a ventilated oven at 105 C for 24 h to determine the dry weight (DW). From the above, the specific leaf area (m 2 of leaf area g À1 of leaf DW) was determined.
The fourth leaf counted from the top was used for measuring photosynthetic pigments. Photosynthetic pigments of fresh leaves were extracted in 100% N,N-Dimethylformamide (DMF) and then measured using Varian Cary 4000 spectrophotometer. The equations of Wellburn (1994) were used to determine concentrations of chlorophyll a (Chla) and b (Chlb) as well as total carotenoids (Car) in μg ml À1 DMF.  T A B L E 1 Total PPFD (photosynthetic photon flux density) and PPFD of red (R; 600-700 nm), blue (B; 400-500 nm), and green (G; 500-600 nm) for the six spectral treatments as well as the phytochrome photostationary state (PSS)

| No significant effect of green light on biomass accumulation
The total dry weight was not significantly affected by partially replacing the different colors (R, B, or RB) by green light, nor was there a significant difference among the genotypes and other spectra ( Figure 5). Similarly, the contents of chlorophylls (chls, chl a + b) and total carotenoids (car), as well as the ratio of chl a to chl b and chl a + b/car ratio were mostly not influenced by the genotypes and light treatments ( Figure S4). However, partially replacing sole R Through blue light, the neutral FAD chromophore in crys is converted into an active state (FADH) absorbing green light, which converts the crys into a fully reduced and inactive state (Lin & Shalitin 2003;Banerjee et al. 2007;Bouly et al. 2007). Green light partially inhibits cry2 oxidation by blue light (Banerjee et al. 2007;Bouly et al. 2007;Frechilla et al. 2000;Zeugner et al. 2005), contributing to reduced levels of FADH. However, this photocycle model could not explain all interactions between blue and green light on stem length, like the finding that G could also act additively to B to inhibit cry-mediated stem elongation in Arabidopsis . In contrast to several other studies on the role of crys, where Arabidopsis seedlings (including cry-null mutants) received light for very short periods (e.g. 30 min in the works of Banerjee et al. and Bouly et al. 2007), our study was conducted with larger tomato plants that were exposed to different light spectra for a number of weeks. In such long-term experiments, the responses of the measured parameters (leaf expansion, stem growth, etc.) can be under the control of many photoreceptors and many cellular pathways (Hammad et al. 2020). Therefore, apart from the direct effects of green light on crys, indirect effects can also play a role. In our study G induced a similar response of stem elongation in CRY2 overexpressing lines than  (Figure 2), confirming that the mechanism underlying crys activation during plant growth has not been elucidated.
Another interpretation of the G-reduced elongation when partially replacing sole B is that G may activate phytochromes, as also suggested by the increase in PSS value (Table 1). Partially replacing sole B by R also remarkably reduced elongation (Figures 1 and 2), suggesting a potent cry-phy interaction. Battle et al. (2020) summarized the reported interactions between blue and green light, indicating that green light could act to complement or antagonize blue lightinduced responses dependent on the wavelength of the green light, either through the direct repression of cryptochrome signaling or via a phytochrome-dependent mechanism.

| The involvement of CRY2 in regulating plant photomorphogenesis
In contrast with MM and cry1a mutant, stem length was reduced in CRY2-OX3 and CRY2-OX8 when partly replacing sole R by G (Figure 2), while partly replacing B by G induced a lower shoot: root ratio and smaller leaf area (not significant in CRY2-OX3; Figures 3 and 4). These results indicate the involvement of CRY2 in green light effects on stem length, shoot: root ratio and leaf area. However, it is not easy to interpret why G did not affect the stem length of CRY2 overexpressors when partially replacing RB mixture. Maybe this effect was absent because these genotypes had quite short stems when grown under RB compared to sole R or B.
Comparing the tomato CRY2 overexpressing lines with wild-type plants, CRY2 may control vegetative development and photosynthesis as suggested by high-throughput transcriptomic and proteomic analyses by Lopez et al. (2012), and by the overproduction of chlorophylls in CRY2 overexpressors (Giliberto et al. 2005). However, we did not observe significant differences in SLA and chlorophyll content between CRY2-OX3/OX8 and MM ( Figures S3 and S4). We conclude that the effects of CRY2 on phenotype are limited, which might result from its redundant role with CRY1a.

| PHYs play a role in blue light effects on elongation
Besides mediation by CRYs, the blue light effects might also be mediated by PHYs. The PSS value, which is an indicator of phytochrome status, was lower under sole blue than that under all other light treatments; green light had little effect on the PSS value (Table 1). CRYs and PHYs converge blue and red light signals at different levels to co-regulate physiological responses, such as root greening, de-etiolation, shade avoidance symptoms, photoperiodic flowering, etc . Although many studies report that an increasing fraction of blue light reduces stem length (e.g. Kalaitzoglou et al. 2021)  In tomato, cryptochrome 1, phytochromes A, B1, and B2 are all capable of mediating responses to B under some circumstances (Weller et al. 2001). In Arabidopsis, CRYs may act in a blue-light independent manner to affect PHY regulation of gene expression and development, resulting in different protein expression between the WT and cry1cry2 mutant in red light as well as in blue light (Lopez et al. 2012;Yang et al. 2008 (Sun et al. 1998), even more efficiently than R or B (Nishio 2000), because it could penetrate deep into the mesophyll layers (Smith et al. 2017). In our study, where the light contained 0 or 20% green, the plant biomass production rate was not significantly affected by green light (Figure 5).
Similarly, the contents of chlorophyll a and b and carotenoids, as well as their ratios, were hardly affected by green light ( Figure S4). resulted in the highest growth rate (Kim et al. 2004). However, in our study, 20% G did not induce such effects, which is comparable to the study of Hernández and Kubota (2015), who analyzed the effect of 28% G in cucumber. Kaiser et al. (2019) found that replacing 32% of a red/blue mixture spectrum by green light significantly increased plant biomass and yield. These different observations among studies suggest that G effects might be genotype-specific and dependent on and/or interact with other environmental conditions.
Although the effects of light spectrum on biomass production were limited in this study, there were profound effects on plant shoot architecture (e.g. stem length). This can be of practical relevance in horticulture to manipulate shoot architecture. M. Marcelis and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

SUPPORTING INFORMATION
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