Red-light mediated inhibition of hypocotyl elongation in Brassica napus was less effective than in other tested species and other light conditions.
Although the effects of light quality on photomorphogenesis and the underlying molecular mechanism has been well studied in Arabidopsis [16–18], it is poorly understood in other plant species. We tested the effects of light quality on the hypocotyl elongation in eight eudicot species. All tested plant species showed longest and shortest hypocotyl under dark and white light (Fig. S1). However, the extent in inhibition of hypocotyl elongation showed wide variations among plant species and among light quality. The red light induced the inhibition of hypocotyl elongation was in a less extent in B. napus (cultivar ZD622) than other tested plant species, especially Brassica rapa (Fig. S1), even though they shared the most closely kinship. Moreover, ZD622 showed longer hypocotyl under red light than other light quality (Fig. 1). We also tested the effect of different light quality on hypocotyl length in 267 rapeseed genotypes collected from worldwide, and we found that the overall hypocotyl length was longer in red light than that in blue or far-red light (data not shown). These results suggested that red light has less influence on promotion of photomorphogenesis in B. napus.
The overview of transcriptomic profiles in B. napus under different light quality.
Transcriptional reprograming, including signal transduction and activation or inhibition of downstream pathways (phytohormone and plant growth), plays a vital role in photomorphogenesis [1, 5]. In order to investigate the transcriptional regulation in response to different light quality, we performed transcriptome analysis of seedling under dark and four light quality. To validate the reliability of transcriptome data, we performed qRT-PCR analysis of 12 genes. The qRT-PCR result was in accordance with that in RNA-seq result, indicating the high reliability of transcriptome data (Fig. S2). Our transcriptional data shows that there are 9748 genes, approximately 15% portion of total genes, differential expressed in seedling under different light quality. To visualize the overall transcriptional patterns in different light conditions, we conducted the principle cluster analysis (PCA) of all differentially expression genes (DEGs). The transcriptional profiles under dark/red light and blue/far-red light was clearly separated by principle component 1 (PC1), which explained 49.6% of variation. This result indicates that dark/red light regulated transcriptional profiles was distinct from that in blue/far-red light conditions. In addition, strong negative correlations were found in gene expression under dark vs blue light (r=-0.77), dark vs far-red light (r=-0.76), red light vs blue light (r=-0.7) and red light vs far-red light (r=-0.71), which support the opinion above from another perspective (Fig. 2B). Furthermore, significant positive correlations were observed between gene expression under far-red and blue light (r=0.71), and between dark and red light (r=0.38). It is consistent with the observed phenotypes that red light treated plants showed longer hypocotyl than blue and far-red light treated plants.In a word, red light mediated transcriptional profile exhibited an analogous pattern to dark to some extent, and displayed a distinct pattern with that under blue and far-red light.
Hierarchical cluster and gene ontology (GO) analysis of DEGs
Hierarchical cluster analysis of DEGs divided all DEGs into 12 clusters. It is worthy noted that approximately 75% of DEGs exhibiting the analogous tendency between dark and red light, or between blue and far-red light. These genes includes 2789 genes in Group Ⅰ (high expression in dark/red and low expression in blue/far-red), 4480 genes in Group Ⅱ and Group Ⅲ (low expression in dark/red and high expression in blue/far-red). We performed gene ontology (GO) analysis of these DEGs. Group Ⅰ associated GO terms include “circadian rhythm”, “photoperiodism”, “shade avoidance”, “plant growth”, “cell wall” (Fig. 4A). Group Ⅱ and Ⅲ associated GO terms include “red/far-red/blue/UV-A/UV-B light signal response”, “circadian rhythm”, “de-etiolation”, “photomorphogenesis”, “photoperiodism”, “shade avoidance”, “chloroplast movement” and “photosynthesis” (Fig. 4C). Additionally, 23.67% of blue and far-red light activated genes were directly associated with light signaling and light-dependent biological processes, while only 3.8% genes induced by dark and red light were enriched in those processes (Fig. 4B, 4D).
Key regulators in light signaling pathway
Light signaling and its downstream pathway was displayed according to our knowledge on photomorphogenesis in plants, mainly in Arabidopsis. PHYA and PHYB perceive far-red and red light, respectively, to be converted to active Pfr form. Previous studies showed that PHYA, as a type I phytochrome, accumulates abundantly in the dark, and it is degraded or repressed under both red and far-red light exposure. PHYB belongs to Type II phytochrome, and it is stable in the light [1, 5]. In this study, two PHYA transcripts were accumulated in dark, and were significantly decreased in all light conditions (Fig. 5). This result indicates that light not only represses PHYA in protein level, but also inhibits its gene expression. Although the active Pfr form of PHYB can be activated by red light, the gene expression of PHYB didn’t change in response to red light, indicating the influence of red light on PHYB was in protein level rather than in transcriptional level.
HY5 is one of the key positive regulators in seedling photomorphogenesis [10] and plays central role in integrating branches of all photoreceptors in light signaling pathway. In Arabidopsis, light enhances both HY5 expression and HY5 protein level, thereby promoting photomorphogenesis [19]. HYH, a homolog of HY5, interacts with HY5 to promote photomorphogenesis [20]. Our result showed that the HY5 and HYH was in low expression under red light whereas their expressions were upregulated in response to blue and far-red light (Fig. 5 and S2C). HY5 physically interacts with HFR1 to promote PHYA-mediated photomorphogenesis [21]. In this study, HFR1 expression was strongly repressed in dark and red light, while it was highly upregulated in blue and far-red light. We speculated that the low expression of these three positive regulators may lead to enhance hypocotyl elongation under red light.
On the contrary, COP1 is regarded as a central negative factor on plant photomorphogenesis [22, 23]. It has been reported that COP1-SPAs complex not only target positive regulators (HY5, HFR1, etc) for ubiquitination and degradation, but also promote the accumulation of negative factors such as PIFs [17, 24, 25]. We found four COP1 transcripts, among which only one transcript had relative high expression. Due to conserved and fundamental function of COP1 from animals to plants [26], we speculated that single homologous gene may function properly, and two or more COP1s may cause confusion in regulation of downstream pathway. In dark and red light condition, hypocotyl length was relative long, while COP1 was in relative low expression (Fig. 5 and S2D). The result indicated that the gene expression level of COP1 can’t explain phenotypic difference of hypocotyl elongation. Actually, COP1 functions in post-translational level [27], rather than in transcriptional level.
PIFs and BBXs transcription factors
PIFs, the basic helix-loop-helix (bHLH) transcription factor, are negative regulators of light responses by repressing photomorphogenesis [2]. All of PIF members contains APB domain, while only PIF1 and PIF3 contain APA motifs (PHYA-Pfr interacting site). Intriguingly, PIF1 and PIF3 had analogous transcriptional pattern, in which their expressions were repressed by dark and red light, and were upregulated by blue and far-red light (Fig. 5 and S3A). PIF3 is a major negative regulator, and it is deduced that its high expression and following accumulation in protein level helps to repress photomorphogenesis. PIF1 was reported as a key factor regulating red/far-red light mediated seed germination. Although PIF1 had a similar transcriptional pattern with PIF3, whether PIF1 have a putative role in regulation of photomorphogenesis remains elusive.
PIF4 is responsible for high temperature-induced hypocotyl elongation [28], and both PIF4 and PIF5 function in shade avoidance-mediated hypocotyl elongation. Overexpression of PIF4 or PIF5 caused constitutively long hypocotyl [29]. In this study, we found the PIF4 and PIF5 were highly expressed in red light (Fig. S4), which may partially explain the long hypocotyl under red light.
BBXs, B-BOX domain proteins, are one of the most important groups of transcription factors related with light signal. It has been identified BBX21/22/23 promotes photomorphogenesis [30–32], whereas BBX24/25/28/30/31/32 negatively regulate light signaling [33, 34]. These BBXs interact with HY5 via distinct regulatory ways in regulation of photomorphogenesis [34]. However, unlike PIFs, the transcriptional pattern of many BBXs genes can’t explain the hypocotyl phenotypes under different light conditions (Fig. S3B). Previous study suggested that BBXs function in post-translational level in regulation of HY5 expression [32, 35].
Phytohormone
Previous studies reported that abscisic acid (ABA) promotes photomorphogenesis whereas gibberellin (GA), brassinosteroid (BR), ethylene, and auxin promote skotomorphogenesis [20, 36–41]. These multiple hormonal pathways interact with a key signal integration center HY5 to regulate photomorphogenesis [9]. ABA INSENSITIVE 5 (ABI5), a positive regulator in ABA signaling, is activated through binding with HY5, thereby promoting photomorphogenesis [9]. Both of INDOLE ACETIC ACID 7 (IAA7) and INDOLE ACETIC ACID 14 (IAA14), auxin signaling inhibitors, are promoted by HY5 and negatively regulate hypocotyl elongation [9]. GA2ox2, an important GA catabolic gene, is induced via HY5 to negatively regulate GA level and inhibited hypocotyl elongation [9]. Overexpression of GA2ox8 reduces bioactive GA level and causes growth retardation, flowing delay and lignification decrease [42]. Taken together, we can conclude that ABI5, IAA7/IAA14, GA2ox2/GA2ox8 serves as positive regulators which indirectly promotes photomorphogenesis. In this study, the gene expression of ABI5, IAA7/IAA14, GA2ox2/GA2ox8 was promoted by blue/far-red light rather than red light (Fig 5), which can explain the phenotypic difference between blue/far-red light and red light. BZR1 (BRASSINAZOLE RESISTANT 1), a positive regulator in BR signaling pathway, suppressed by HY5 to promote photomorphogenesis [9]. Our results showed the expression of BZR1 was induced in dark while it was repressed in light condition (Fig 5), which can explain the light regulated photomorphogenesis in transcriptional level.
It has been reported that EIN3 plays an important role in the balance of ethylene and light signaling in hypocotyl growth [43]. ETHYLENE INSENSITIVE 3 (EIN3) has been shown to repress cotyledon development and promote hypocotyl elongation in the dark, and EIN protein is rapidly degraded by light exposure. Consistently, our result showed EIN3 showed higher expression level in dark, which helps to promote skotomorphogenesis [18]. Previous study showed that ethylene can promote hypocotyl growth in high intensity of red light via activation of PIF3 expression by EIN3/EIL1 [44]. It can be an explanation for the observation of high PIF3 expression and long hypocotyl in red light (Fig. 5).
Genes related with cell elongation and cell wall modification
In dark, PIFs activate the gene expressions of many genes responsible for cell elongation/division to promote hypocotyl elongation [37, 38]. For instance, Xyloglucan endotransglycosylase/hydrolase (XTH) and expansin (EXP) are two classes of vital enzymes in cell wall loosening and cell expansion [45, 46]. Pectin methylesterases (PME) plays pivotal role in plant cell wall formation [47]. In this study, EXP3/EXP9, XTH9 and PME31 exhibited high expression in dark/red light and low expression in blue/far-red light. These genes may function in dark/red light mediated hypocotyl elongation via promoting cell elongation and cell wall development.