Green means go: Green light promotes hypocotyl elongation via brassinosteroid signaling

Abstract

Although many studies have elucidated the mechanisms by which different wavelengths of light (blue, red, far-red, or ultraviolet-B [UV-B]) regulate plant development, whether and how green light regulates plant development remains largely unknown. Previous studies reported that green light participates in regulating growth and development in land plants, but these studies have reported conflicting results, likely due to technical problems. For example, commercial green light-emitting diode light sources emit a little blue or red light. Here, using a pure green light source, we determined that unlike blue, red, far-red, or UV-B light, which inhibits hypocotyl elongation, green light promotes hypocotyl elongation in Arabidopsis thaliana and several other plants during the first 2–3 d after planting. Phytochromes, cryptochromes, and other known photoreceptors do not mediate green-light-promoted hypocotyl elongation, but the brassinosteroid (BR) signaling pathway is involved in this process. Green light promotes the DNA binding activity of BRI1-EMS-SUPPRESSOR 1 (BES1), a master transcription factor of the BR pathway, thus regulating gene transcription to promote hypocotyl elongation. Our results indicate that pure green light promotes elongation via BR signaling and acts as a shade signal to enable plants to adapt their development to a green-light-dominant environment under a canopy.

In contrast to blue, red, far-red or UV-B light, which inhibit hypocotyl elongation, green light promotes hypocotyl elongation via promoting brassinosteroid signaling.

IN A NUTSHELL.

Background: Green leaves reflect substantial amounts of green light, which gives them their pleasing green color and might give the impression that green light is of little importance to plants. However, although green leaves reflect more green light than red or blue light, green leaves still absorb around 10–50% of green light. Previous studies reported that green light participates in regulating growth and development in land plants. However, due to technical limitations, especially the wide range of green light wavelengths and limitations in light-emitting diode technology, some of these studies reported conflicting results.

Question: Whether and how does green light regulate growth and development? Which photoreceptor mediates green light responses?

Findings: Other wavelengths of light (red, blue, and ultraviolet) inhibit hypocotyl elongation. By contrast, we find that green light promotes hypocotyl elongation in Arabidopsis thaliana and several other plants. Green light-promoted hypocotyl elongation does not require known photoreceptors, indicating that these photoreceptors may not function as green light receptors that mediate green light-promoted hypocotyl elongation. However, the brassinosteroid (BR) signaling pathway is involved in this process. Green light promotes the DNA binding activity of BRI1-EMS-SUPPRESSOR 1, a master transcription factor of BR signaling, thus regulating gene transcription to promote hypocotyl elongation.

Next steps: Find the green light receptor and identify the members of the green light signaling pathway.

Introduction

Light is a critical environmental factor for plant growth and development (Sullivan et al. 2003). Plants undergo dramatic changes in developmental patterns in the presence and absence of light. For example, the stem elongates rapidly in darkness, but the elongation is robustly repressed by blue light, red light, far-red light, and UV-B light (Chen et al. 2004). Plants have evolved sensitive signaling systems to fine-tune photomorphogenesis in response to changing light environments. Light is perceived by multiple photoreceptors, including blue/UV-A light receptor cryptochromes (cry1 and cry2) (Wang and Lin 2020); phototropins (phots) (Briggs and Christie 2002); the LOV-domain/F-box proteins ZEITLUPE (ZTL), FLAVIN BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (FKF), and LOV KELCH PROTEIN2 (LKP2) (Demarsy and Fankhauser 2009); the red/far-red light photoreceptors phytochromes (phys) (Quail 2002) and the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) (Podolec et al. 2021).

Green light accounts for more than half the energy of visible light. The pleasant green color of plants is caused by their reflectance of green light; however, although green leaves reflect more green light than red or blue light, green leaves still absorb around 10–50% of green light (between the wavelengths of 500 and 600 nm) (Smith et al. 2017). Green light was reported to participate in regulating growth and development in green land plants, including seed germination, root growth, stem elongation, and flowering (Torrey 1952; Goldthwaite et al. 1971; Klein 1992). However, due to technological limitations, especially the broad spectrum of green light, some of those results conflicted with each other.

In terms of green-light-regulated hypocotyl elongation, different research groups have provided conflicting descriptions. Green light was reported to inhibit hypocotyl elongation in many species including Arabidopsis thaliana, tomato (Solanum lycopersicum), pea (Pisum sativum), Cucumber (Cucumis sativus), and lettuce (Lactuca sativa Linn) (Koornneef et al. 1980; Klein 1992; Ahmad and Cashmore 1993; Lin et al. 1995a; Bouly et al. 2007; Battle and Jones 2020). Another study reported that green light was involved in mesocotyl repression and coleoptile stimulation in oat (Avena sativa) seedlings (Mandoli and Briggs 1981). However, analysis of seedling growth during the first 120 min after germination showed that green light induced a transient increase in stem growth rate, and red light or blue light plus green light led to a longer stem than that grown under only red or blue light (Folta 2004). The red/far-red light photoreceptors phytochromes and the blue light photoreceptors cryptochromes were reported to function as green light photoreceptors that mediate green-light-inhibited hypocotyl elongation (Lin et al. 1995a; Dhingra et al. 2006; Banerjee et al. 2007; Bouly et al. 2007; Battle and Jones 2020; Battle et al. 2020). Arabidopsis cry1 protein absorbed green light in some redox states, and the cry1 mutant exhibited impaired hypocotyl inhibition in green light (Lin et al. 1995b). Cry2 was reported to be degraded under green light (Li et al. 2011), similar to that under blue light, but another study showed that cry2 protein remains stable under green light (Banerjee et al. 2007). The reason for these contradictory findings, and whether and how green light regulates plant development, remains largely unknown.

Here we obtained pure 550 nm green light and found that it promotes hypocotyl elongation, while commercial green light-emitting diode (LED) lights with a 525 nm wavelength repress hypocotyl elongation because they also produce some blue light. The green light-promoted hypocotyl elongation persists in known photoreceptor mutant lines, and cry2 protein stability is not regulated by pure green light. However, the brassinosteroid (BR) signaling pathway is required for this process. Therefore, green light is an important photomorphogenic signal that promotes hypocotyl elongation via the BR signaling pathway.

Results

Green light promotes hypocotyl elongation

It has been reported that in green leaves, around 10–50% of green light (between the wavelengths of 500 and 600 nm) is reflected by chloroplasts and the rest is absorbed (Murchie and Horton 1998; Murchie et al. 2002; Smith et al. 2017). To further check the green light absorption in A. thaliana leaves, we used a transmission integrating sphere and a reflection integrating sphere (Supplemental Fig. S1, A and B) to measure how much green light penetrates into and is reflected from leaves (Fig. 1A). Although green leaves reflected more green light than blue or red light, the transmission plus reflection rate was only 50% (Fig. 1B). The transmission plus reflection rate of red or blue light was approximately 20% and that of far-red light was nearly 100% (Fig. 1B). The transmission of blue, green, red, and far-red light was 0.83%, 23.96%, 2.9%, and 54.55% (Fig. 1C), indicating that the leaves in the bottom of the canopy get mainly far-red light and green light. Therefore, the effects of green light on early de-etiolation and shade avoidance need to be further investigated.

Figure 1.

Green light promotes hypocotyl elongation. A) The spectrum of the light source generated with a transmission integrating sphere and a reflection integrating sphere. To measure the transmission of light, light is transmitted by an optical fiber to the transmission integrating sphere through a glass slide or an Arabidopsis leaf pressed with a glass slide and the spectrum measured using a spectrophotometer (Ocean Optics). To measure reflected light, light is transmitted with a fiber to the reflection integrating sphere and reflected with a whiteboard or an Arabidopsis leaf. The reflected light was also measured with a spectrophotometer (Ocean Optics). B) Quantification of the percentage of transmission and reflection of Arabidopsis leaves under different light conditions. Values under blue light (450 nm), green light (550 nm), red light (665 nm), and far-red light (730 nm) were calculated. C) Quantification of the percentage of the transmission of Arabidopsis leaves under different light conditions. Values under blue light (450 nm), green light (550 nm), red light (665 nm), and far-red light (730 nm) were calculated. D) The light spectra of the green light source with (peak 550 nm) and without (peak 525 nm) a 500 nm filter measured with a spectrograph. E) Phenotypes of Col-0 seedlings grown for 3 d in green light with (Green) or without (Green-) a filter at different fluence rates. Scale bar = 10 mm. F) Quantification of hypocotyl lengths of Col-0 grown for 3 d in green light with or without a filter at different fluence rates. Error bars represent standard deviation (n = 30). The letters “a” to “g” indicate statistically significant differences between hypocotyl length of the indicated treatment, as determined by Tukey's least significant difference (LSD) test (P ≤ 0.05). G) Quantification of cell lengths of Col-0 grown under the indicated conditions for the indicated number of days. Error bars represent standard deviation (n = 10). The letters “a” to “d” indicate statistically significant differences between cell lengths of the indicated treatment, as determined by Tukey's LSD test (P ≤ 0.05). H) Cell morphologies of representative Col-0 seedlings grown under the indicated conditions for 3 d. I) Quantification of hypocotyl lengths (n = 30) of Col-0 grown for 3 d in dark and green light (Green 550) with indicated concentrations of sucrose or mannitol control.

Due to the lack of appropriate green LEDs, engineers use blue or red LEDs to generate green LED light (Maur et al. 2015). This green LED light always contains a small amount of blue or red light, termed a “tail” because of its appearance on the emission spectrum. Commercial green LED lights with a 525 nm wavelength generate a blue light tail of less than 500 nm, whereas green LED lights with a wavelength of 550 nm generate a red light tail of more than 600 nm (Ahmad and Cashmore 1993; Folta 2004; Battle and Jones 2020). It is difficult to investigate the role of green light in regulating plant development because of the blue or red light tails from these commercial green LED lights. Plants are highly sensitive to both blue and red light, with strong effects on plant development. Here, we utilized filters that eliminated all wavelengths below 500 nm to eliminate the blue light tail from a 525 nm green light LED source and thus obtain pure 550 nm green light without blue and red light tails, which is the same as the transmission and reflection peak of leaves (Fig. 1D).

We examined hypocotyl length in Arabidopsis seedlings grown in the dark or in green light without a filter (Green 525 nm, 20 μmol·m^–2·s⁻¹) or green light with a filter (pure green light, Green 550 nm, 20 μmol·m⁻²·s⁻¹). Pure green light (Green 550) promoted hypocotyl elongation during the early stage of growth (the first 3 d). The hypocotyls of wild-type (WT) plants grown in Green (550 nm) were even longer than those grown in the dark (Supplemental Fig. S2, A and B), contrasting with the inhibitory effects of blue and red light on hypocotyl elongation (Somers et al. 1991; Ahmad and Cashmore 1993). Seedlings grown under the green light source without a filter (Green 525 nm) had shorter hypocotyls than those grown in the dark, even in the first 3 d of growth (Supplemental Fig. S2, A and B), indicating that the blue light tail in standard green light sources inhibits hypocotyl elongation. Pure green light promoted the opening of the apical hook and separation of the cotyledons (Supplemental Fig. S2A), the same as blue light, red light, far-red light, and UV-B light.

We also examined hypocotyl length under different intensities of green light. All fluence rates of pure green light (Green 550) tested promoted hypocotyl elongation, which increased with increasing green light intensity (Fig. 1, E and F). By contrast, all fluence rates of green light with a blue light tail inhibited hypocotyl elongation (Fig. 1, E and F). These results indicate that the blue light tail of the green LED light rather than the green light itself inhibits hypocotyl elongation. Thus, green light promotes rather than inhibits hypocotyl elongation, which is different from the conclusion of most previous studies that used a blue- or red-light-contaminated green light source (Lin et al. 1995b; Dhingra et al. 2006; Banerjee et al. 2007; Bouly et al. 2007; Battle and Jones 2020; Battle et al. 2020). A study by Folta (2004), in which hypocotyl elongation was measured during the first 120 min after germination indicated that green light stimulates hypocotyl elongation, which is consistent with our findings.

It is reported that green light promotes germination, and our results confirmed that the germination rate was higher under pure green light than in the dark (Supplemental Fig. S2C). To exclude the possibility that green-light-promoted early hypocotyl elongation is due to green-light-promoted germination rather than hypocotyl elongation, we germinated seeds in the dark or pure green light (Green 550) and transferred them to pure green light (Green 550) or the dark. The hypocotyl length was similar regardless of whether the seeds were germinated in the dark or green light (Supplemental Fig. S2D), which indicated that the longer hypocotyl phenotype in pure green light is mainly due to green light-promoted hypocotyl elongation rather than green light-promoted germination. To get rid of the influence of green light on germination, in this study, all seeds were germinated under white light for 36 h before being put into the dark or pure green light.

To check whether the green-light-promoted hypocotyl elongation depends on photosynthesis, we examined the hypocotyl length in the dark or green light with or without sucrose or mannitol. The hypocotyl length was longer in pure green light (Green 550) than in dark with or without sucrose (Fig. 1I), indicating that green light-promoted hypocotyl elongation was not sucrose dependent. Interestingly, a high concentration of sucrose inhibited hypocotyl elongation in dark, and the inhibition was more significant than mannitol, while the inhibition induced by sucrose and mannitol was similar in green light.

We also examined mutants that affect photosynthesis. TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 33 (TOC33) and TOC75 are involved in the import of photosynthetic proteins into chloroplasts (Kubis et al. 2003; Huang et al. 2011). The toc33 (Kubis et al. 2003) and toc75 (Huang et al. 2011) mutants showed yellow leaves phenotype and defects in photosynthesis. Similarly to WT plants, both toc33 and toc75 mutants showed elongated hypocotyl phenotype under pure green light compared with those in darkness (Supplemental Fig. S2, E and F). PHYTOENE DESATURASE 3 (PDS3) is involved in carotenoid biosynthesis and pds3 mutants have white leaves and cannot grow to maturity. The seeds from pds3 heterozygous mutants were planted and the bleached pds3 seedlings still showed elongated hypocotyl phenotype under pure green light compared with those in darkness, similar to those segregated green seedlings (Supplemental Fig. S2, G and H). These results indicate that the green-light-promoted hypocotyl elongation is not photosynthesis dependent.

To further confirm this observation, we measured cell length in the elongation zones of hypocotyls. The hypocotyl cells of WT plants grown in pure green light (Green 550) light were longer than those grown in the dark. The cells of WT plants grown in green light without a filter, white light, blue, or red light were all significantly shorter than those grown in the dark (Fig. 1, G and H).

We also grew various crop plants in pure green light, including soybean (Glycine max), sorghum (Sorghum bicolor), quinoa (Chenopodium quinoa), millet (Setaria italica), rice (Oryza sativa), and wheat (Triticum aestivum). Soybean, sorghum, quinoa, and millet showed much longer hypocotyls or mesocotyls in green light than in the dark. Rice and wheat did not show elongated mesocotyls, but the plants were much taller under pure green light than in the dark (Supplemental Fig. S2, I–P). Therefore, the green-light-promoted growth during the early stage of the plant's lifecycle is conserved in Arabidopsis and various crops.

Green-light-regulated hypocotyl elongation persists in known photoreceptor mutants

To examine whether known photoreceptors are green light receptors, including PHYs(Quail 2002), CRYs (Wang and Lin 2020), ZTL (ZEITLUPE)/FKF1 (FLAVIN-BINDING KELCH REPEAT F-box1)/LKP2 (LOV KELCH PROTEIN2) (Demarsy and Fankhauser 2009), PHOTs (Phototropins) (Briggs and Christie 2002), and UVR8 (Rizzini et al. 2011), we planted the cry1, cry2, cry1 cry2, cry1 cry2 phya, phya, phyb, phya phyb, phyabde, ztl lkp2 fkf, phot1 phot2, and uvr8 mutants in the dark and in pure green light conditions. All these mutants showed more elongated hypocotyls in green light than in dark, similar to the WT (Fig. 2, A and B). WT showed shorter hypocotyls under green light without a filter (Green 525) than in the dark, while cry1 cry2 mutants showed similar longer hypocotyl elongation under green light with or without filter and longer than in dark (Supplemental Fig. S3, A and B), indicating that the shorter phenotype of WT under green light without filter (Green 525) is caused by the blue light tail. These results indicate that green-light-promoted hypocotyl elongation persists in known photoreceptors.

Figure 2.

Known photoreceptors are not involved in green-light-regulated hypocotyl elongation. A) Phenotypes of seedlings of the indicated genotypes grown in the dark D) and pure green light with a 500 nm filter G) for 3 d. Scale bars = 10 mm. B) Quantification hypocotyl lengths of the plants shown in A). Error bars represent standard deviation (n = 30). The letters “a” to “b” indicate statistically significant differences between hypocotyl length of the indicated genotype and treatment, as determined by Tukey's LSD test (P ≤ 0.05). C–F) Immunoblots showing the change in native CRY1, CRY2, and actin (ACT) protein levels in response to green light without a filter (Green-) (10 μmol·m⁻²·s⁻¹), green light with a filter (Green) (10 μmol·m⁻²·s⁻¹), or blue light (10 μmol·m⁻²·s⁻¹). Col-0 seedlings were grown in the dark C–E) or green light F) for 3 d and transferred to green light without a filter C), blue light D), green light with a filter E), or the dark F), for 0.5, 1, 1.5, or 2 h before sample collection. All samples were fractionated by 10% SDS-PAGE, blotted, and probed with anti-CRY2, anti-CRY1, and anti-actin antibodies. G–J) Immunoblots showing the change in native PhyA, PhyB, and actin (ACT) protein levels in response to green light without a filter (Green-: Green 525) (10 μmol·m⁻²·s⁻¹), green light with a filter (Green: Green 550) (10 μmol·m⁻²·s⁻¹), red light (10 μmol·m⁻²·s⁻¹), or blue light (10 μmol·m⁻²·s⁻¹). Col-0 seedlings were grown in the dark or pure green light (Green) for 3 d and transferred to green light with a filter G), green light without a filter H), blue light I), or red light J) for 0.5, 1, 1.5, or 2 h before sample collection. All samples were fractionated by 10% SDS-PAGE, blotted, and probed with anti-PhyA, anti-PhyB, and anti-actin antibodies.

The mRNA accumulation of CRY1, CRY2, PHYA, and PHYB was not significantly affected when WT plants were moved from the dark to pure green light (Green 550) or from pure green light to the dark (Supplemental Fig. S3, D–G). The cry2 protein levels decreased significantly within 0.5 h of treatment with blue light (10 μmol·m⁻²·s⁻¹) or green light without a filter (Fig. 2, C and D), but cry2 protein levels remained constant when the plants were moved from the dark to pure green light or from pure green light to the dark (Fig. 2, E and F), indicating that pure green light does not affect the protein stability of cry2. The cry2 protein levels decreased in response to green light without a filter, indicating that the degradation of cry2 protein in response to green light without a filter occurred due to the blue light tail in this green light source. The phyB protein levels were not affected by green light, red light, or blue light (Fig. 2, G–J). The phyA protein levels decreased significantly when plants were moved from dark to red light (10 μmol·m⁻²·s⁻¹), blue light (10 μmol·m⁻²·s⁻¹), green light without a filter (10 μmol·m⁻²·s⁻¹), or pure green light (10 μmol·m⁻²·s⁻¹) (Fig. 2, G–J), indicating that the degradation of phyA was regulated by green light though phyA was not responsible for the green light-promoted hypocotyl elongation.

It has been reported before that monochromatic 300–780 nm lights of very low fluence induced germination in a phyA-dependent manner (Shinomura et al. 1996). To investigate whether phyA participated in green light-induced germination, we germinated Col-0 and phyA mutants in dark and green light (Supplemental Fig. S3C). We found there was no significant difference between the germination rate of Col-0 and phyA mutants, indicating that the green light-promoted germination still persists in phyA mutants.

Green light regulates the transcript accumulation of BR-regulated genes

We then performed deep sequencing of the transcriptome (RNA-seq) to identify downstream genes affected by green light. To this end, 3-d-old dark-grown seedlings were kept in the dark or moved to pure green light (Green 550) for 1 h before collection; 1,287 differentially expressed genes (−2 < Log₂ (Fold Change) <2) were identified between treatments (Fig. 3A, Supplemental Data Set S1). Hypocotyl elongation is mainly regulated by external light signals and by endogenous signaling by auxin and BRs (Yang et al. 2011; Reed et al. 2018; Nakano 2019). Our RNA-seq datasets of genes differentially expressed in response to green light significantly overlapped with RNA-seq datasets of genes regulated by BRs (Fig. 3, A–D, Oh et al. 2014). For 37.99% of these genes, the effects of pure green light (Green 550) on gene expression were the same as the effects of BRs (compare Col-0 green vs. Col-0 dark and Col-0 with 24-epibrassinolide [eBL, the most active BR] vs. Col-0 without eBL in Fig. 3, C and D), including genes involved in photosynthesis, cell wall formation, light responses, and the circadian clock (Fig. 3D). These transcriptomic data provide direct evidence for a role for green light in hypocotyl elongation, particularly in promoting transcription of cell elongation genes regulated by BRs.

Figure 3.

Green light regulates the expression of BR-regulated genes. A) Venn diagram showing the overlap between the sets of differentially expressed genes in dark vs. pure green light (Green) (10 μmol·m⁻²·s⁻¹) and eBL-treated vs. untreated Col-0 (BR). B) Heatmap of BR- and green-light-regulated genes. The scale bar shows fold changes (log₂ value). C) Distribution of genes regulated by both BRs and green light. D) GO analysis of the 4 categories of co-regulated genes depicted in C. E) RT-qPCR analysis of gene expression in 3-d-old dark-grown Col-0 seedlings treated with 1 h of pure green light (Green 550). The U-box gene was used as an internal control. Gene expression in green light was normalized to dark-grown Col-0 (i.e. the expression level in Col-0 was defined as “1”). Error bars represent the standard deviation of 3 biological replicates (*P < 0.05, **P < 0.01, ***P < 0.001, paired sample t-test).

We verified the transcriptomic data using RT-qPCR. We selected PHYTOCHROME-INTERACTING FACTOR 5 (PIF5), Expansin 1 (EXPA1), Expansin 2 (EXPA2), Expansin 10 (EXPA10), REVEILLE 1 (RVE1), LIGHT-HARVESTING CHLOROPHYLL B-BINDING 1.1 (LHCB1.1), and LIGHT-HARVESTING CHLOROPHYLL B-BINDING 2.1 (LHCB2.1) from the list of genes regulated by both green light and BRs (Fig. 3E). All these genes were upregulated by green light treatment, consistent with the transcriptome data, suggesting that green light promotes hypocotyl elongation via promoting the transcript accumulation of cell elongation genes regulated by BRs.

We also performed RNA-seq of BES1-RNAi plants, with downregulated transcript accumulation of BRI1-EMS-SUPPRESSOR 1 (BES1; encoding a master transcription factor of the BR signaling pathway) and its homologs (Yin et al. 2005; Yang et al. 2011), under pure green light and identified 654 genes regulated by green light (−2 < Log₂ (Fold Change) <2) in WT but not in BES1-RNAi plants (Supplemental Fig. S4A, Supplemental Data Set S2). Gene ontology (GO) analysis showed that these genes were enriched in the following categories: light signaling, photosynthesis, cell wall formation, and response to hormone (Supplemental Fig. S4B). These results are similar to the findings for genes co-regulated by BR and green light, further indicating the important role of BR signaling in green-light-promoted hypocotyl elongation.

Surprisingly, unlike BR-related genes, auxin response genes were downregulated by green light based on GO analysis (Fig. 3, C and D). To verify this surprising finding of elongated hypocotyls coupled with repressed auxin signaling, we performed RT-qPCR to measure auxin-related gene transcript levels after pure green light (Green 550) treatment and found that these genes were indeed repressed by green light (Supplemental Fig. S4C).

BR biosynthesis and signaling are necessary for green-light-induced hypocotyl elongation

BR signaling promotes hypocotyl elongation (Oh et al. 2012). To determine whether the BR signaling pathway is involved in green-light-induced hypocotyl elongation, we analyzed the response of BR biosynthesis and signaling mutants to green light. The BR-deficient de-etiolated 2-1 (det2-1) (Li et al. 1996) mutants and the BR receptor mutants bri1-301(Greene et al. 2003) were both insensitive to green light. Green-light-promoted hypocotyl elongation did not occur in these mutants (Fig. 4, A–C). The BES1-RNAi line (Yin et al. 2005), in which BES1 and its homologs were downregulated, exhibited much shorter hypocotyl in green light than in dark compared with the WT (the hypocotyl length ratio of green/dark is 1.37, WT is 1.49), indicating that BES1-RNAi was also insensitive to green light. Moreover, the bes1 bzr1 (brassinazole-resistant 1) beh1 (bes1/bzr1 homolog 1) beh2 (bes1/bzr1 homolog 2) beh3 (bes1/bzr1 homolog 3) beh4 (bes1/bzr1 homolog 4) hextuple mutant (Chen et al. 2019) was also insensitive to green light (Fig. 4D–F). We also examined the auxin biosynthetic mutant yucca1246 (23) and found that it produced elongated hypocotyls in pure green light, like WT plants (Supplemental Fig. S5A), indicating that not all mutants that show shorter hypocotyls than the WT are insensitive to green light.

Figure 4.

The BR signaling pathway is involved in green-light-induced hypocotyl elongation. A) Phenotypes of seedlings of the indicated genotypes grown in the dark D) and green light with a filter G). Bar = 10 mm. B) Quantification of hypocotyl lengths of the indicated plants grown in the dark or pure green light (Green 550) for 3 d. Error bars represent standard deviation (n = 30). The letters “a” to “f” indicate statistically significant differences between hypocotyl length of the indicated genotype and treatment, as determined by Tukey's LSD test (P ≤ 0.05). C) Hypocotyl length ratios (Green/Dark) of quantified hypocotyl length in B. The letters “a” to “c” indicate statistically significant differences between ratios of the indicated genotype, as determined by Tukey's LSD test (P ≤ 0.05). D) Phenotypes of seedlings of the Col-0 and bes1 bzr1 beh1 beh2 beh3 beh4 grown in the dark D) and green light with a filter (pure green light) G). Bar = 10 mm. E) Quantification of hypocotyl lengths of the Col-0 and bes1 bzr1 beh1 beh2 beh3 beh4 grown in the darkness or pure green light (Green 550) for 3 d. Error bars represent standard deviation (n = 15). The letters “a” to “c” indicate statistically significant differences between hypocotyl length of the indicated genotype and treatment, as determined by Tukey's LSD test (P ≤ 0.05). F) Hypocotyl length ratios (Green/Dark) of quantified hypocotyl length in E. The letters “a” to “b” indicate statistically significant differences between ratios of the indicated genotype, as determined by Tukey’s LSD test (P ≤ 0.05). G and H) Col-0 was grown in a series of eBL (c) or BRZ (d) concentrations in the presence or absence of pure green light. The hypocotyl lengths of plants under the indicated treatments are shown. Standard deviation (n = 15) is indicated. I) RT-qPCR analysis of CPD and DWF4 gene expression in 3-d-old Col-0 and BES1-RNAi seedlings treated with 1 h of pure green light (Green 550) or darkness. The U-box gene was used as an internal control. Gene expression in green light was normalized to that of WT in the dark (i.e. the expression level in Col-0 was defined as “1”). Error bars represent the standard deviation of 3 biological replicates. The letters “a” to “c” indicate statistically significant differences between ratios of the expression levels of each gene, as determined by Tukey’s LSD test (P ≤ 0.05). J) The effect of green light on the DNA binding activity of BES1. ChIP-qPCR assays were performed using 10-d-old BES1-Flag transgenic plants, which were transferred to the dark for 1 d and treated with 1 h of pure green light (Green: Green 550), green light without a filter (Green-: Green 525), or dark before harvesting. Chromatin fragments (∼500 bp) were immunoprecipitated by anti-Flag-Agarose beads (IP). The precipitated DNA was analyzed by qPCR using primer pairs for DWF4 and for CNX5 and UBC as negative controls. The level of binding was calculated as the ratio between IP and MOCK, normalized to that of IP/MOCK of UBC as an internal control. Error bars represent the standard deviation of 3 biological replicates. The letters “a” to “d” indicate statistically significant differences between ratios of the expression levels of each gene, as determined by Tukey’s LSD test (P ≤ 0.05).

We then examined the sensitivity of bri1-301 and WT to eBL and the BR biosynthesis inhibitor brassinazole (BRZ, which promotes the formation of physiologically inactive phosphorylated BES1) in the dark and pure green light (Green 550). The sensitivity of WT plants was reduced in pure green light (Green 550) compared with that in darkness. WT plants were also hyposensitive to BRZ treatment in pure green light (Green 550) compared to that in darkness, suggesting that the long-hypocotyl phenotype of WT in pure green light (Green 550) is dependent on both BR biosynthesis and BR signaling. The eBL insensitive and BRZ hypersensitive phenotypes under pure green light were abolished in the BR receptor mutant bri-301 (Fig. 4, G and H). These results demonstrate that BR biosynthesis and signaling are indeed involved in green-light-regulated hypocotyl elongation.

To further explore the relationship between green light and BR biosynthesis and signaling, we analyzed the transcript accumulation of the BR signaling marker genes CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM (CPD) and DWARF4 (DWF4), which encode BR biosynthesis enzymes and are target genes of BES1. Both CPD and DWF4 were downregulated after 1 h of green light treatment (Fig. 4I). These results indicate that BR signaling plays a dominant role in regulating the green light response, since BR activates BES1-mediated feedback regulation to repress the transcription of CPD and DWF4 (Yang et al. 2011).

Both CRY1 and UVR8 interact with BES1 to inhibit its DNA binding activity, thus inhibiting the transcription of genes involved in hypocotyl elongation (Liang et al. 2018; Wang et al. 2018). As green light does not affect the transcript or protein levels of BES1 (Supplemental Fig. S5B and C), we reasoned that the putative green light receptor might also interact with BES1 to regulate the transcript accumulation of its target genes. We performed chromatin immunoprecipitation (ChIP) qPCR assays to determine whether pure green light (Green 550) affects the DNA binding activity of BES1 to its previously reported targets. BES1 has been associated with the DWF4 promoter, but not the UBIQUITIN or MOLYBDOPTERIN SYNTHASE SULFURYLASE (CNX5, At5g55130) promoters (He et al. 2005) (controls) (Fig. 4J). These 2 genes are not responsive to BR and contain no BRRE elements in their promoters; BRRE elements are frequently enriched in the promoters of BR-regulated target genes (Sun et al. 2010). Pure green light (Green 550) promoted the DNA binding activity of BES1 to the DWF4 promoter thus repressing its transcription, whereas green light without a filter (Green 525) did not (Fig. 4J). These data demonstrate that green light promotes the DNA binding activity of BES1 to its target genes, thus regulating their transcript accumulation and hypocotyl elongation.

Discussion

The advancement of lighting technology makes it possible to investigate more narrow light wavebands. However, the “Green Gap” is still a difficult problem in lighting technology. The green LED light sources used in previous studies had a tail of either blue or red light to which plants are highly sensitive. As a result, the inhibitory effect of green light on hypocotyl elongation observed in previous studies was mainly because of the blue or red light tail in the green light sources. The inappropriate usage of contaminated green light sources led to conflicting results in previous studies. In the current study, we created a pure green light source by filtering out the blue light tail and found that unlike red, far-red, blue, or UV-B light, green light promotes hypocotyl elongation (Supplemental Fig. S5D). Thus red, far-red, blue, and UV-B light all are negative regulators of hypocotyl elongation (Jiao et al. 2007; Teixeira 2020), whereas green light is a positive regulator of this process; this likely ensures shade avoidance (Klein 1992; Sellaro et al. 2010; Zhang et al. 2011; Smith et al. 2017).

In nature, leaves are usually present in a structurally complex canopy. The spectral distribution at different levels of a tobacco (Nicotiana tabacum) canopy was measured in 1971, and the sharp decline in the red/far-red ratio from the top to the bottom layers was noticed. Green light at 543 and 576 nm was also transmitted through the canopy much more readily than blue and red light (Kasperbauer 1971; Smith et al. 2017). More than 20% far-red light and up to 6.5% green light reached leaves at the base, while only <0.5% of blue and <2.1% red light from above the canopy reached the bottom layer (Kasperbauer 1971; Smith et al. 2017). It has been found that the addition of green light to a background of constant red and blue light-induced shade avoidance symptoms, including the elongation of petioles and the upward orientation of leaves, even though the addition of green light increased the intensity of the growth light (Zhang et al. 2011). Arabidopsis hypocotyl length increased proportionally to decreases in the blue to green light ratio, indicating that blue:green light ratio could act as a shade signal within a canopy (Sellaro et al. 2010). Measurements of spectra were made within mature oat (A. sativa) canopies in the field, and the proportion of green light compared with blue light increased from the top to the bottom of the canopy (Smith et al. 2017). Far-red light inhibits hypocotyl elongation by activating phyA during de-etiolation. This activity of phyA is important to favor young seedling survival (Yanovsky et al. 1995). While an increased proportion of far-red light is a critical signal for shade avoidance, far-red light promotes hypocotyl elongation by inhibiting phyB. Green light may be an additional signal for activating shade avoidance (Smith et al. 2017). Here we show that green light promotes rather than inhibits hypocotyl elongation in A. thaliana and several other plants during de-etiolation. Green light is the only known light signal that promotes hypocotyl elongation during de-etiolation; green-light-mediated growth promotion is expected to enhance the survival of de-etiolating seedlings under a canopy that is rich in FR light leading to phyA-mediated inhibition of hypocotyl elongation. Our results also consistent with that green light acts as a shade signal to enable plants to adapt their development in a low-light environment within a canopy.

Light signals influence endogenous hormone signaling to regulate hypocotyl elongation (Lau and Deng 2010) and regulate almost all hormone pathways at various levels. Auxin and BRs play critical roles in regulating hypocotyl elongation (Yang et al. 2011; Reed et al. 2018; Nakano 2019). Phys and crys interact with AUX/IAAs to release their inhibition of downstream signaling (Xu et al. 2018). UVR8 interacts with MYB73/77 to inhibit auxin responses and lateral root development (Yang et al. 2020). COP1, an important negative regulator of light signaling, suppresses PIN-FORMED (PIF) proteins to regulate auxin polar transport (Lin et al. 2017). PIF4 regulates auxin biosynthesis and signaling in response to high temperature and shade (Ma et al. 2016; Pedmale et al. 2016). Cry1 and UVR8 interact with BES1 to inhibit its DNA binding activity, thus inhibiting hypocotyl elongation (Liang et al. 2018; Wang et al. 2018). Light also regulates the levels of the SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA (SINAT) E3 ubiquitin ligases to control dephosphorylated BES1 levels (Nolan et al. 2017; Yang et al. 2017; Hu et al. 2021). Moreover, PIF4 and BZR1 interact with each other to regulate plant growth (Oh et al. 2012). All of these findings demonstrate the tight cooperation between light signaling and endogenous auxin or BR signaling. Multiple light signals act together to regulate BES1. Interestingly, green light acts in a manner opposite to that of blue or UV-B light: green light promotes the DNA binding activity of BES1, thus promoting hypocotyl elongation (Supplemental Fig. S5D).

There were several interpretations for a Phot1-mediated green light reversal of red-light-induced inhibition, green light treatment antagonized red light, and far-red light-mediated hypocotyl inhibition, and it has been shown that the green light reversal of red light response was actually a low-fluence rate blue light response revealed by a green light environment (Wang et al. 2013). Our results indicate that green-light-promoted hypocotyl elongation persists in known photoreceptor mutants (Fig. 2, A and B), so green-light-promoted hypocotyl elongation might not be mediated by any known photoreceptor. Thus, plants might perceive green light with a novel receptor, a concept that requires further investigation. It is interesting that phyA protein levels decrease significantly when plants were moved from dark to pure green light (Fig. 2G), indicating that the degradation of phyA is regulated by green light though phyA is not responsible for the green light-promoted hypocotyl elongation. PhyA is involved in regulating germination (Cheng et al. 2021), and the green light-promoted germination still persists in phyA mutants, demonstrating that phyA might not be responsible for the green light-promoted germination. The function of phyA in green light needs to be further studied.

Materials and methods

Plant materials and growth conditions

Except where indicated, the Columbia (Col-0) ecotype of A. thaliana was used. The mutants cry1-304 (Ma et al. 2016), cry2-201 (Guo et al. 1998), cry1 cry2 (Mockler et al. 1999), phya-211 (Lariguet et al. 2003), phyb-9 (Oh et al. 2004), phya phyb (Mockler et al. 2003), phyabde (Franklin et al. 2003), cry1 cry2 phya (Mockler et al. 2003), phot1 phot2 (Mao et al. 2005), ztl-3 lkp2 fkf1 (Liu et al. 2013a), uvr8-6 (Yang et al. 2018), bri1-301 (Greene et al. 2003), det2-1 (Li et al. 1996), BES1-RNAi (Yin et al. 2005), bes1 bzr1 beh1 beh2 beh3 beh4 (Chen et al. 2019), and toc33, toc75 (Ling et al. 2021) were previously described. pds3 mutant is a Crispr cas9 mutant from Jirong Huang's lab.

For hypocotyl elongation and immunoblot, seeds were planted on 1/2 MS medium, stratified for 3 d at 4°C, and germinated under white light for 36 h and then transferred to the indicated light environment for 3 d at 22°C. For Q-PCR, RNA-seq, and ChIP assays, seeds were planted on soil, stratified for 3 d at 4°C and grown in the dark for 3 d and then transferred to the indicated light environment at 22°C. Soybeans (G. max), sorghums (S. bicolor), quinoas (C. quinoa), millets (S. italica), and wheats (T. aestivum) were planted on soil, stratified for 3 d at 4°C, and then transferred to dark and pure green light at 22°C for 3 d before measurement. Rice (O. sativa) were soaked in water in dark at 37°C and planted in water in dark or pure green light at 22°C for 3 d before measurement.

For pure green light condition, 525 nm green light was generated in Muqing Liu's lab and a specific cutoff filter was used to modulate green light wavelength, filtering out <500 and >580 nm light. The filtered green light peak was at 550 nm.

Light transmission and reflection of leaves

To measure the green light transmission through Arabidopsis leaves, light is transmitted to the transmission integral sphere (Supplemental Fig. S1A) (Guangzhou Changhui Electronic Technology Co., Ltd) using an optical fiber, and light transmitted through a glass slide or an Arabidopsis leaf under a glass slide was measured using a spectrophotometer (Ocean Optics). To measure the reflected light, light is transmitted to the reflection integral sphere (Supplemental Fig. S1B) (Guangzhou Changhui Electronic Technology Co., Ltd) using an optical fiber, and light reflected with a whiteboard or an Arabidopsis leaf was measured using a spectrophotometer (Ocean Optics).

Cell morphology and microscopy

To measure the cell length, Arabidopsis seedlings were placed on glass slides and observed on Leica SP8 microscope (Leica, Germany) with DIC optics. The length of the 10th cell of the hypocotyl was measured in each seedlings. Cell length measurements were done using the Leica SP8 software.

RNA-seq and transcriptome analysis

For RNA-seq, Col-0 and BES1-RNAi seedlings were grown for 3 d in the dark and then exposed to green light (10 μmol·m⁻²·s⁻¹) for 1 h; 1 batch of etiolated seedlings was kept in darkness for an additional 1 h as a dark control. The seedlings were harvested, and total RNA was isolated using RNAprep pure Plant Kit (Tiangen, DP432). Three biological replicates were independently prepared throughout the processes, from the induction of seed germination to the preparation of mRNA-seq libraries. The RNA library generation process followed the manufacturer's protocol for the Illumina Truseq RNA sample prep Kit. The average RNA fragment was about 300 bp, and a 15-cycle PCR amplification was carried out with the primer mixture provided in the kit. Library preparation and sequencing using an Illumina Hiseq4000 instrument with 2 × 150 bp paired-end reads were performed by Majorbio (Shanghai).

Majorbio cloud (https://cloud.majorbio.com/) was used to analyze differentially expressed genes. Genes with a corrected P-value <0.05 and absolute log₂FoldChange >2 were taken as differentially expressed. BR-regulated genes are downloaded from Oh et al. (2014). Venn diagrams, heatmaps, and GO enrichment analysis are conducted by R packages.

mRNA expression analyses

Total RNA isolation and RT-qPCR were previously described (Hao et al. 2016; Yang et al. 2020). Total RNAs were isolated using the RNAprep pure Plant Kit (Tiangen, DP432). cDNA was synthesized from 500 ng total RNA using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). TB Green Premix Ex Taq (Takara) was used for qPCR, on the MX3000 System (Stratagene). The level of U-box mRNA (At5g15400; Supplemental Table S1) was used as the internal control. Primers are provided in Supplemental Table S1.

Immunoblot analysis

Seeds were grown for 3 d in dark or green light before being moved to a defined light treatment (light to dark or dark to light for the indicated times). After treatment, we collected seedlings and prepared whole protein extracts with extraction buffer (25.2 g Glycerol, 0.02 g Bromophenol Blue, 4 g SDS, 20 ml 1 M Tris-HCl [pH6.8], 3.1 g DTT, to 50 ml ddH₂O). Equal protein amounts were loaded and separated on a 10% or 10% SDS-PAGE gel and transferred to Pure Nitrocellulose Blotting Membrane (P/N66485, PALL, USA). We then probed the membrane with anti-cry1 antibody (produced by Youke, China, 1:5,000 dilution for immunoblot), anti-cry2 antibody (produced by Youke, China, 1:5,000 dilution for immunoblot), anti-phyA antibody (from Jigang Li Lab [Dong et al. 2020], 1:5,000 dilution for immunoblot), anti-phyB antibody (from Jigang Li Lab [Dong et al. 2020], 1:5,000 dilution for immunoblot), and anti-ACTIN antibody (AC009, Abclonal, China, 1:5,000 dilution for immunoblot) as a loading control.

Chromatin immunoprecipitation assays

We performed ChIP experiments as described previously (Liu et al. 2008, 2013b; Ma et al. 2016; Liang et al. 2018; Yang et al. 2018), using 10 d-old BES1-FLAG seedlings grown in LD conditions. We harvested 2 g plant material, which we then cross-linked with 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min under a vacuum. We stopped cross-linking by the addition of glycine to the solution, to a final concentration of 0.125 M. We rinsed seedlings with water, froze them in liquid nitrogen, and ground them into a fine powder. We immunoprecipitated chromatin fragments (∼500 bp) which were sonicated by a bioruptor (Bioruptor Plus, Diagenode SA, Belgium, at program 30 s on and 30 s off for 15 min) with anti-FLAG Affinity gel (D111139, Sangon, China) and analyzed the precipitated DNA by qPCR with the indicated primer pairs (Supplemental Table S2). The level of binding was calculated as the ratio between the IP and Input proteins.

Statistical analysis

Statistical data are provided in Supplemental Data Set S3.

Accession numbers

Sequence data from this work can be found in the Arabidopsis Information Resource or GenBank databases under the following accession numbers: CRY1 (At4g08920), CRY2 (At1g04400), PHYA (AT1G09570), PHYB (AT2G18790), BES1 (AT1G19350), BRI1 (AT4G39400), DET2 (AT2G38050), TOC33 (AT1G02280), TOC75 (AT3G46740), PDS3 (AT4G14210). RNA-seq data are available from National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) https://www.cncb.ac.cn/gsa) under the series entries GSE196648.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1 . Green light promotes hypocotyl elongation.

Supplemental Figure S2 . Known photoreceptors are not green light photoreceptors.

Supplemental Figure S3 . Green light regulates the transcript accumulation of BR-regulated genes.

Supplemental Figure S4 . The BR signaling pathway is involved in green light-induced hypocotyl elongation.

Supplemental Table S1 . Primers used for RT-qPCR.

Supplemental Table S2 . Primers used for ChIP-qPCR.

Supplemental Data Set S1 . List of genes regulated by green light.

Supplemental Data Set S2 . List of genes regulated by green light in WT but not in BES1-RNAi plants.

Supplemental Data Set S3 . Statistical data.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32150007, 31825004, 31721001, 31730009, and 31701231), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27030000), China Postdoctoral Science Foundation grants (2018M690478 and 2019T120361), and the Program of Shanghai Academic Research Leader. Dr Y. H. and X. L are supported by the foundation of Youth Innovation Promotion Association of CAS.