Introduction

Unlike animals, plants have a sessile lifestyle and maintain life by responding to environmental changes without moving location. Therefore, plants use light not only as a source of energy, but also as a signal for morphogenesis to acclimate to the surrounding environment and to carry out their own growth, development and differentiation (McNellis and Deng 1995). After germination in the soil, plants elongate their hypocotyls out of the ground to receive light for the first time and begin morphogenesis, including de-etiolation, suppression of hypocotyl elongation and cotyledon expansion. This is called photomorphogenesis (Paik and Huq 2019). Dramatic changes in gene expression occur during this first exposure to light, but many of the details, especially the translational control governing light reception, remain unknown.

Of the various wavelengths of light, plants mainly receive blue light through specific photoreceptors, cryptochromes (CRYs) and phototropins, and red and far-red light through phytochromes (Kami et al. 2010; Paik and Huq 2019). CRYs are widespread, found in algae to seed plants, and two CRYs, CRY1 and CRY2, act as blue-light receptors in Arabidopsis thaliana (L.) Heynh. (Arabidopsis) (Ponnu and Hoecker 2022). CRYs are involved not only in de-etiolation, but also in stomatal opening and closing, flowering time, shading response, inhibition of hypocotyl elongation, anthocyanin synthesis, and photoperiodic regulation (Ahmad et al. 1998; Cashmore et al. 1999; Yu et al. 2010).

The ELONGATED HYPOCOTYL 5 (HY5) bZIP-type transcription factor is responsible for transduction following photoreception by each photoreceptor (Xiao et al. 2022). HY5 functions to inhibit hypocotyl elongation, induce lateral root formation and promote light-dependent pigment accumulation in Arabidopsis through transcriptional modulation of downstream genes (Oyama et al. 1997). In hy5 loss-of-function mutants, optimization of the gene expression profile and light signaling, started at photoreception, is strongly inhibited (Kurihara et al. 2018; Singh et al. 2012).

The environmental change from dark to light just after germination also affects organelle morphogenesis. When etioplasts present in germinated seedlings receive light, they develop into chloroplasts via etio-chloroplasts (Solymosi and Schoefs 2010). The chloroplasts harvest light independently of photoreceptors and perform photosynthesis (Nelson and Yocum 2006). There is signaling back and forth between the mature chloroplasts and the nucleus. Retrograde signals from chloroplasts to the nucleus regulate the expression of nuclear genes (Calderon and Strand 2021). However, the impact of these signals on nuclear gene expression, especially at the translational level, still remains to be determined.

Previously, we studied changes in transcription and translation of nuclear genes when Arabidopsis seedlings germinated in the dark were exposed to blue light, and showed that chloroplast-related and microRNA biosynthesis genes were up-regulated at the translational level by the exposure (Kurihara et al. 2020). This result suggests that the dramatic changes in gene expression during blue-light reception are regulated not only at the transcriptional level but also at the translational level. In this study, we report the results of ribosome profiling analysis of light-signaling mutants and plants treated with chloroplast inhibitors to further investigate the signaling pathways involved in gene expression changes at the translational level during blue-light reception.

Materials and methods

Plant growth conditions

Arabidopsis wild type (Col-0), cry1cry2 (Ong et al. 2017) and hy5 (Kurihara et al. 2018) mutants were used in this work. Seeds were sterilized and then vernalized for more than 3 days at 4 °C in the dark. The seeds were sowed onto filter paper (Toyo Roshi Kaisha) placed on 1× Murashige and Skoog (MS) germination medium [0.8% agar (Nacalai Tesque Co., Ltd.), pH 5.8 (Murashige and Skoog 1962). The plants were grown for 3 days in the dark at 22 °C, and then exposed to blue light (6.316 µmol m− 2 s− 1) for 3 h. For Ribo-seq and RNA-seq analyses, the seedlings before and after blue-light exposure were harvested and frozen in liquid nitrogen.

Chloroplast inhibitor treatment

DMSO (FUJIFILM Wako), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (Nacalai Tesque) and norflurazon (FUJIFILM Wako) were each added to 1× Murashige and Skoog (MS) agar medium [0.8% agar (Nakalai Tesque Co., Ltd.) pH 5.8 (Murashige and Skoog 1962)] at a final concentration of 15 µM. Plant growth conditions and harvesting are described above.

RNA extraction and computational analysis for Ribo-seq and RNA-seq

For Ribo-seq and RNA-seq analyses, 400 µL of lysis buffer [100 mM Tris-HCl pH 7.5, 40 mM KCl, 20 mM MgCl2, 1 mM DTT, 1% Triton X100, 100 µg/mL cycloheximide, 10 µg/mL actinomycin D] was dispensed into 400 mg of the sample frozen in liquid nitrogen and the liquid nitrogen was evaporated in a deep freezer. The sample in the lysis buffer was crushed and powdered using a Shake Master (Biomedical), and thawed at 4 °C. After thawing, 2.5 µL 2 U uL− 1 DNase I (ThermoFisher Scientific) were added into the crude lysate followed by centrifugation. The supernatant was extracted as a pure lysate. The RNA concentration of the lysate was measured using a Qubit RNA BR Assay Kit (ThermoFisher Scientific). Ribo-seq library construction from the lysate has been described previously (Kurihara et al. 2018). Libraries were sequenced using HiSeq X (Illumina). Ribo-seq reads were mapped to the TAIR10 genome using Tophat version 2.1.1 after rRNA/tRNA removal. The mapped data derived from WT, cry1cry2, hy5, and plants treated with DMSO, DCMU and norflurazon were integrated into a dataset and translated ORFs were predicted by RiboTaper v1.2 (Calviello et al. 2016) using 27–29 nt ribosome footprint (RF) reads of the integrated datasets. The read count for each predicted ORF was normalized using DESeq (Trapnell et al. 2012).

For RNA-seq, total RNA was extracted from the lysate using TRizol LS reagent (ThermoFisher Scientific) and a Direct-zol RNA Kit (ZYMO RESEARCH) and its concentration was measured with a NANODROP 2000c spectrophotometer (ThermoFisher Scientific). RNA-seq libraries were prepared using a TruSeq Stranded Total RNA Library Prep Kit (Illumina, Inc.). The libraries were sequenced using HiSeq X (Illumina). RNA-seq reads were mapped to the Arabidopsis TAIR10 genome using STAR (Dobin et al. 2013) after rRNA/tRNA removal. The read count for each gene was calculated using DESeq (Trapnell et al. 2012). Two biological replicates were performed for all samples. Significant differences were defined by 1.5-fold change (light vs. dark) and determined by the Student’s t-test (p-value < 0.01).

Translational efficiency (TE) for each gene was calculated by dividing the read counts from Ribo-seq by the read counts from RNA-seq using DESeq (Trapnell et al. 2012). Significant differences in TE were defined by the Benjamini-Hochberg method (q < 0.05).

The dataset of sequenced reads produced by next-generation sequencing is deposited in the DDBJ/EMBL/GenBank BioProject under accession number DRA014541.

Gene ontology (GO) enrichment analysis

GO enrichment analysis was performed using a TAIR tool (https://www.arabidopsis.org/tools/go_term_enrichment.jsp). GO terms were obtained from the GO ontology database released on August 18, 2021 by the Binomial test using the Bonferroni correction for multiple testing (p < 0.05).

Optical microscope

An IX73 inverted microscope (OLYMPUS) was used to observe Arabidopsis cotyledons when exposed to blue light for 3 days. The exposure time mode was manual 30 ms and ISO sensitivity 400.

Cycloheximide (CHX) treatment

Seeds of WT, cry1cry2 and hy5 were sown on 1× MS medium with DMSO, grown for 2 days, and then transferred to medium supplemented with 100 µM CHX (Sigma) and grown for an additional 2 days.

Results

Gene expression analysis in cry1cry2 and hy5 mutants

To identify a set of genes that shows blue-light-dependent expression, we performed expression analysis in WT, cry1cry2, which lacks two blue-light-receptor genes CRY1 and CRY2, and hy5, which lacks a key transcription factor gene for light-response signaling. Young seedlings grown in the dark for 3 days after sowing were exposed to blue monochromatic light. RNA-seq and ribosome profiling (Ribo-seq) analyses were carried out on the seedlings before and after 3 h of blue-light exposure. Our investigation in the cry1cry2 and hy5 mutants was performed on 20,293 translated protein-coding ORFs (genes) detected by Ribo-seq analysis. The lengths of the RFs obtained were predominantly 28 nt and the RFs showed 3-nt periodicity in the ORFs, confirming the robustness of the Ribo-seq analysis (Fig. S1a–l). In addition to changes in accumulation of mRNAs and RFs, changes in translation efficiency (TE: number of ribosomes per mRNA) upon blue-light exposure were investigated.

Comparison of differentially expressed genes upon blue-light exposure between wild type, cry1cry2 and hy5

From the data obtained by RNA-seq and Ribo-seq, we calculated the number of differentially expressed genes whose mRNA accumulation, RF accumulation, and TE significantly changed when exposed to blue light after darkness (Fig. 1a–f; Table 1, Table S1). In all the genotypes, changes in mRNA accumulation were relatively correlated with those in RF accumulation, while significant changes in TE were detected in some genes (Fig. 1a–c). The number of genes elevated in mRNA accumulation, RF accumulation, and TE were reduced in both cry1cry2 and hy5 mutants compared to those in the wild type (Fig. 1d–f; Table 1). These results indicate that, similar to a previous report (Kurihara et al. 2020), there is regulation of gene expression not only at the transcriptional level but also at the translational level in response to blue-light reception.

Fig. 1
figure 1

Comparison of gene expression upon blue-light exposure between WT, cry1cry2 and hy5. a–c Scatter plots showing the correlation between mRNA and RF amounts for each gene, with fold change (FC) of mRNA accumulation in RNA-seq (Blue/Dark) plotted on the x-axis and FC of ribosomal footprint (RF) accumulation on the y-axis. Orange or gray dots indicate the number of genes whose TE was predominantly increased or decreased, respectively, by exposure to blue light (q-value < 0.01). a WT, b cry1cry2 double mutant, c hy5 mutant. d–f Venn diagrams for the overlap of the number of genes with increased mRNA and RF accumulation and increased TE between wild type, cry1cry2 and hy5. d increased mRNA accumulation, e increased RF accumulation, f increased TE

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Table 1 Number of differentially expressed genes upon blue-light exposure in RNA-seq and ribosome profiling analysis
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Gene ontology analysis for differentially expressed genes upon blue-light exposure

Gene ontology (GO) enrichment analysis was performed to determine what categories the genes with increased mRNA accumulation, RF accumulation, and TE belong to (Fig. S2a–f, Fig. 2a–c). In all lines, WT, cry1cry2 and hy5, genes up-regulated in mRNA and RF accumulation were predominantly classified into the photosynthesis-related and light-response-related GO categories (Fig. S2a–f). Interestingly, in the genes with elevated TE, ribosome-related GO categories were seen only in WT (Fig. 2a), and not in cry1cry2 and hy5 (Fig. 2b, c).

Fig. 2
figure 2

GO enrichment analysis of genes whose TE was predominantly increased by blue-light exposure. The number of enriched genes out of the total number of genes is shown in each bar graph. a WT, b cry1cry2 double mutant, c hy5 mutant. red: photosynthesis-related GO category, blue: light-response-related GO category, green: ribosome-related GO category. The top 15 categories or all categories obtained from GO analysis are shown

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Comparison of expression changes in ribosome-related genes upon blue-light exposure

From the results of the GO analysis, ribosome-related GO categories were not represented in the genes with elevated TE in cry1cry2 and hy5 mutants. Therefore, in WT, cry1cry2 and hy5 we compared mRNA and RF accumulation and TE of 84 ribosome-related genes that, in WT, showed elevated TE upon blue-light exposure (Fig. 3a–c, Fig. S3a–c). Changes in mRNA accumulation upon blue-light exposure were not significantly different between WT and the two mutants (Fig. 3a, Fig. S3a), but RF accumulation were less increased in both mutants than those in WT (Fig. 3b, Fig. S3b), as there was in TE (Fig. 3c, Fig. S3c). These results are consistent with the GO analysis, where the genes with elevated TE were not enriched in ribosome-associated GO categories in both mutants.

Fig. 3
figure 3

Expression comparison of 84 ribosome-related genes with elevated TE in WT between WT, and the cry1cry2 and hy5 mutants. a–c Fold changes (FCs, blue/dark) are shown in box plots. a mRNA accumulation, b RF, c TE. p-values were calculated by the Student’s t-test

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Effects of translation inhibitors on young seedlings

Ribosome-associated GO categories were not represented in cry1cry2 and hy5 (Fig. 2). Therefore, to determine whether translation is required for early development, the translation inhibitor cycloheximide was added to the germination medium and WT, cry1cry2, and hy5 were sown and grown under blue light (Fig. S4a, b). The results showed that growth after germination was severely affected by cycloheximide in all three lines; they all showed shorter hypocotyls compared to the control.

Gene expression analysis under chloroplast inhibitors

GO analysis showed that genes with elevated mRNA and RF accumulation upon blue-light exposure were enriched in photosynthesis-related categories not only in WT but also in the cry1cry2 and hy5 mutants (Fig. S2a–f). Genes with elevated TE in hy5, where suppression of light signaling occurs in the nucleus, were also in photosynthesis-related categories (Fig. 2c). This led us to hypothesize that chloroplast-mediated signals through photoharvesting may affect expression of nuclear genes. Therefore, we investigated how suppression of chloroplast function affects gene expression upon blue-light exposure. For this purpose, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and norflurazon were each added to the growth medium. DCMU reversibly inhibits electron transfer reactions in photosystem II, an early stage of photosynthesis (Petrillo et al. 2014; Szczepaniak et al. 2009), while norflurazon inhibits the carotenoid synthesis system, leading to a depletion of chloroplasts (Hernández-Verdeja et al. 2020; Park et al. 2017). Indeed, long-term treatment with norflurazon led to photobleaching (Fig. S5a–d). As a control, the solvent DMSO was added to the growth medium. To analyze gene expression by Ribo-seq, WT seeds were sown on germination media containing these additives and grown in the dark for 3 days. The young germinated seedlings were exposed to blue light for 3 h. This investigation using chloroplast inhibitors was performed on 19,579 translated protein-coding ORFs (genes) detected by Ribo-seq analysis. The lengths of the obtained RFs were predominantly 28 nt and the RFs showed 3-nt periodicity on the ORFs, confirming the robustness of the Ribo-seq analysis (Fig. S6a–l).

Comparison of gene expression changes upon blue-light exposure between DMSO and chloroplast inhibitors

The number of differentially expressed genes was calculated from the data obtained by RNA-seq and Ribo-seq on exposure to blue light after growth in the dark in the presence of each chemical (Fig. 4a–f; Table 2, Table S2). Compared to the control with DMSO, there was no significant difference in the number of differentially expressed genes under the chloroplast inhibitors (Table 2).

Fig. 4
figure 4

Comparison of gene expression upon blue-light exposure between DMSO, DCMU and norflurazon treatments. a–c Scatter plots showing the correlation between mRNA and RF amounts for each gene, with FC of mRNA accumulation in RNA-seq (blue/dark) plotted on the x-axis and FC of ribosomal footprint (RF) accumulation on the y-axis. Orange or gray dots indicate the number of genes whose TE was predominantly increased or decreased, respectively, by exposure to blue light (q-value < 0.01). a DMSO, b DCMU, c norflurazon. d–f Venn diagrams for the overlap of the number of genes with increased mRNA and RF accumulation and increased TE between DMSO, DCMU and norflurazon. d increased mRNA accumulation, e increased RF accumulation, f increased TE

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Table 2 Number of differentially expressed genes when exposed to blue light from dark under DMSO, DCMU and norflurazon
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GO analysis for differentially expressed genes upon blue-light exposure in the presence of the chemicals

GO enrichment analysis was applied to genes up-regulated when exposed to blue light after darkness (Fig. S7a–f, Fig. 5a–c). The results showed that the genes with increased mRNA and RF accumulation are mainly classified into photosynthesis- and light-response-related GO categories when any of the chemicals are present (Fig. S7a–f). Importantly, in the genes with increased TE, ribosome-related GO categories were not represented after treatment with chloroplast inhibitors, as was the case in the cry1cr2 and hy5 mutants (Fig. 5a–c).

Fig. 5
figure 5

GO enrichment analysis of genes whose TE was predominantly increased by blue-light exposure. The number of enriched genes out of the total number of genes is shown in each bar graph. a DMSO, b DCMU, c norflurazon; red: photosynthesis-related GO category, blue: light response-related GO category, green: ribosome-related GO category. The top 15 categories obtained from GO analysis are shown

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Comparison of ribosome-related genes

In the GO analysis shown in Fig. 5, ribosome-related GO categories were also not seen in the presence of chloroplast inhibitors, whereas they were in the DMSO control. Subsequently, mRNA accumulation, RF accumulation, and TE of 53 ribosome-related genes, which showed increased TE in the DMSO control, were compared between the DMSO and inhibitor treatments (Fig. 6a–c, Fig. S8a–c). After blue-light exposure, accumulation of their mRNAs and RFs was more increased under the chloroplast inhibitors than the DMSO control (Fig. 6a, b, Fig. S8a, b). However, TE elevation seen in the DMSO control was significantly suppressed under the chloroplast inhibitors (Fig. 6c, Fig. S8c). The suppression was particularly strong in the norflurazon treatment compared with DCMU. This result is consistent with GO enrichment analysis where ribosome-associated categories were not represented in the genes with increased TE under the chloroplast inhibitors. Taken together with the result in Fig. 5, these results suggest that signals derived from chloroplasts are also required for translational activation of ribosome-related genes.

Fig. 6
figure 6

Expression comparison of 53 ribosome-related genes with elevated TE in the DMSO control between DMSO and chloroplast inhibitor treatments. a–c Fold changes (FCs, blue/dark) are shown in box plots. p values were calculated by the Student’s t-test

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Discussion

It has already been reported in our previous study that TE of some genes fluctuates when young Arabidopsis seedlings germinated in the dark are exposed to blue light (Kurihara et al. 2020). In this study, we investigated which signaling pathways are responsible for these changes in gene expression at the translational level. We analyzed the responses of Arabidopsis cry1cry2 and hy5 mutants and plants treated with chloroplast inhibitors to blue-light irradiation after darkness. The analyses showed that upregulation of TE of ribosome-related genes were suppressed both in the mutants and under reduced chloroplast function. Thus, we conclude that translational activation of ribosome-related genes upon blue-light perception is dependent on signals from both the nucleus and the chloroplasts.

Changes in gene expression in photoreceptor and light-signaling mutants

It is generally believed that mRNA and RF levels are proportional. In fact, the two are strongly correlated, but changes in translation efficiency were observed for some genes (Fig. 1a–c). The results are consistent with a previous report (Kurihara et al. 2020). Thus, our work confirms that gene expression upon blue-light exposure is regulated not only at the transcriptional level but also at the translational level.

In the genes whose mRNA and RF levels were increased by blue-light exposure, photosynthesis-related GO categories were enriched in all the genotypes examined, WT, cry1cry2 and hy5 (Fig. S2). On the other hand, in the genes with increased TE, there were a variety of enriched GO categories among the genotypes (Fig. 2). This suggests that a signaling cascade, which starts at the point of blue-light reception by CRYs and passes through HY5, regulates dynamic expression changes at the translational level as well as at the transcriptional level.

Previous GO analysis for the genes whose TE increased after 1 h of blue-light irradiation in WT showed that the photosynthesis-related categories were predominant (Kurihara et al. 2020), but, in this study, after 3 h of irradiation, the ribosome-related categories and not the photosynthesis-related categories were predominant. Thus, it is suggested that the types of genes regulated at the translational level changes over time. The result of GO analysis after 3 h of irradiation in hy5 (Fig. 2c) was very similar to that of previous GO analysis after 1 h of blue-light irradiation (Kurihara et al. 2020). Therefore, we speculate that hy5 mutants show a signaling failure and delayed activation of the translation of targeted genes. HY5 Homolog (HYH) cooperatively functions with HY5 (Singh et al. 2012). We previously reported that expression of HY5 reaches a maximum 1 h after blue-light irradiation but that of HYH reaches a maximum at 3 h (Kurihara and Makita 2020). Furthermore, we speculate that HYH partially assists in the possible delayed translational activation in the hy5 mutant.

Changes in gene expression under chloroplast inhibitors

The number of genes whose TE increased upon exposure to blue light did not differ significantly between the control and the chloroplast inhibitors, DCMU and norflurazon (Fig. 4; Table 2). This may be due to the fact that under DCMU, fewer genes are activated by signals from chloroplasts, and therefore, little difference was observed compared to the control. However, under norflurazon, the cotyledons remained white even after 3 days of exposure to blue light because the chloroplasts themselves cannot develop (Fig. S5a–d). Nevertheless, the fact that there is little difference in the number of TE-up-regulated genes between the control and norflurazon-treated seedlings suggests that translation of only a small number of genes is driven by signals from chloroplasts. However, importantly, these few genes include ribosome-related genes and it is suggested that a certain signal from chloroplasts is necessary to increase their TE upon blue-light irradiation.

GO enrichment analysis for the genes with increased TE upon exposure to blue light showed that the ribosome-related GO categories were seen only in the DMSO control (Fig. 5a–c). Conversely, ribosome-related GO categories were not represented in the presence of chloroplast inhibitors, suggesting that chloroplast function is also required for the translational activation of the ribosome-related genes in addition to nuclear light reception. Photosynthesis-related GO categories still came out on top even under DCMU (Fig. 5b). In angiosperms, there are two types of electron transport: linear electron transport from photosystem (PS) II to PS I and cyclic electron transport in PS I. The electrons transferred by cyclic electron transport in PS I could eventually produce ATP through ATP synthase (Yamori and Shikanai 2016). Since the electron transport is possible without PS II, it is supposed that activation of photosynthesis-related genes is reasonable, even if the linear electron flow from PS II to PS I is inhibited by DCMU. Due to this reason, it is inferred that photosynthesis-related GO categories are still seen under DCMU application. On the other hand, the GO categories related to beta-D-glucan came out on top under norflurazon (Fig. 5c). Beta-D-glucan is an important component of the cell wall (Boex-Fontvieille et al. 2014; Nevins et al. 1977). We speculate that inhibition of carotenoid biosynthesis by norflurazon leads to increased biosynthesis of beta-D-glucan in an attempt to reduce photooxidative stress by thickening the cell wall.

It is possible that DMSO treatment negatively affects the responsiveness of gene expression to blue light exposure. Certain ribosome-related GO categories such as ribosome maturation and large subunit biogenesis, which were seen in the GO category with elevated TE in WT (Fig. 2a), were not seen in the DMSO result (Fig. 5a). However, even under the DMSO background, our work demonstrated that the translation of ribosome-related genes was significantly affected by the effect of photosynthesis inhibitors.

Based on the results derived from the analyses of the mutants and the chloroplast inhibition treatments, we suggest that signals from the nucleus and chloroplasts are required for activation of translation of ribosome-related genes during blue-light reception. We believe that this study will be useful in understanding the changes in gene expression at the translational level upon perception of blue light for photomorphogenesis.