Effects of different diel light cycle on phenotype of lettuce
To investigate the sensitivity of lettuce to different diel light cycle, we measured plant shoot, canopy diameter and fresh and dry weight of lettuce grown at 1C, 1.3C and 2C. We found that the phenotype of lettuce changed significantly under different diel light cycle (Fig. 1A ). Lettuce that experienced diel change twice a day (2C) were significantly taller than lettuce that experienced diel change only once (1C) (Fig. 1B), indicating that different diel light cycle could affect lettuce growth (p < 0.05). Canopy diameter plays a crucial role in plant growth and fruit bearing. From the results, it was found that the diameter of the canopy of lettuce in 2C scenario was significantly larger than that of lettuce in 1.3C scenario (Fig. 1C) (p < 0.05). In addition, in terms of fresh weight and dry weight, lettuce in 2C scenario was not much different from lettuce in 1C scenario, but both were heavier than lettuce in 1.3C scenario (Fig. 1D and 1E) (p < 0.05). These results suggested that different diel light cycle had significant impacts on the lettuce phenotype. Moreover, contrary to previous understanding of the diel light cycle of plants, lettuce grown in 2C scenario grew better.
Lettuce physiological index by diel light cycle
To investigate the impact of different diel light cycle on lettuce quality, some physiological indexes of lettuce leaves were measured. In the lettuce leaves, the chlorophyll a, b, and a + b exhibited a greater rise in the 1C and 1.3C scenarios compared to the 2C scenario (Fig. 2A) (p < 0.05). On the contrary, the lettuce leaves exhibited a greater rise in carotenoid content under the 2C scenario compared to the 1C and 1.3C scenarios (Fig. 2B) (p < 0.05). Carotenoid, as auxiliary pigments in plant photosynthesis, restored the growth of lettuce mediated by chlorophyll reduction in the 2C scenario by compensating for the use of visible light radiation. In addition, we found that diel light cycle increased the quality of lettuce. The contents of soluble protein and soluble sugar in lettuce leaves were notably higher under the 2C scenario compared to the 1.3C scenario, indicating a increase of 3% and 5%, respectively (Fig. 2C and 2D) (p < 0.05). Moreover, it experienced a slight decrease under the 1C scenario compared to the 2C scenario, yet it did not attain statistical significance. There was no significant difference in total free amino acid content among the three scenarios (Fig. 2E). Nevertheless, when comparing the nitrate levels in lettuce leaves, we found that there was a significant decrease of approximately 50% in 2C scenario comparing that under 1C and 1.3C scenarios (p < 0.05) (Fig. 2F).
Transcriptome analysis of lettuce leaves
RNA-seq analysis was conducted on the lettuce leaves to investigate the underlying mechanism behind the different phenotypes observed in lettuce grown at 1C, 1.3C, and 2C. For transcriptome sequencing, a total of 9 RNA samples were sent, with each experiment comprising three biological replicates. Biological replicates displaying a strong correlation (R2 > 0.8) in gene expression levels (Fig. S1). The RNA-Seq data of 9 cDNA libraries (1C_1, 1C_2, 1C_3, 1.3C_1, 1.3C_2, 1.3C _3, 2C_1, 2C_2, and 2C_3) were summarized in Table S1. Following the completion of quality control, we obtained a total of 66.94 Gb of clean data. The content of Q30 reached 95.83% throughout the 9 samples. A total of 123,569 transcripts were obtained in the filtered assembly. Most of the sequence lengths were concentrated below 20000bp (Fig. S2).
There were 42,796 non-redundant high-quality unigenes annotated by searching a database of common functions. From the COG, EC, GO, KEGG, and Pfam databases, a total of 40,332 (94.24%), 9097 (21.26%), 20,882 (48.79%), 20,052 (46.85%), and 39,527 (92.36%) annotated unigenes were obtained, respectively(Fig. S3). The PCA analysis showed an obvious differentiation between the three scenarios, suggesting that the differences among the nine sample bioreplicates aligned with the anticipated experimental design (Fig. S4).
Analysis of differential expression genes responding to different diel light cycle
The fragments per kilobase of transcript per million reads mapped (FPKM) method were used to predict gene transcriptional accumulation. By comparing the expression level under different diel light cycle (1C vs 1.3C; 1C vs 2C; 1.3C vs 2C), genes that differentially up-regulated and down-regulated were screened out (Log2FoldChange > 1, adjust padj < 0.05). The results showed that 2270 DEGs were found between the 1C and 1.3C scenarios. And there were 2131 upregulated genes and 139 downregulated genes (Fig. 3A). Furthermore, 3284 DEGs were found between the 1C and 2C scenarios; among these genes, 1338 were found to be upregulated, while 1946 were downregulated (Fig. 3B). The numbers of DEGs between 1.3C and 2C scenarios were 5159. The upregulated genes were 2523, and downregulated genes were 2636 (Fig. 3C). In addition, venn diagram were used to summarize DEG counts between all combinations of 1C, 1.3C, and 2C scenarios. And there were 167 common DEGs between the three comparison scenarios (Fig. 3D)
By Go annotations, we discovered that the DEGs exhibited enrichment in the categories of “cellular process”, “cellular metabolic process”, and “metabolic process” within the GO classification of “biological process”. The DEGs were found to be enriched in terms such as “chloroplast”, “plastid”, and “intracellular membrane-bound organelle” within the “cellular component” categories. In the “molecular function” categories, the DEGs were enriched in “binding” and “catalytic activity” terms (Fig. 4A). By conducting KEGG pathway analysis, we enriched the top 10 DEGs and observed their significant enrichment in various signal transduction and metabolic pathways, such as “photosynthesis”, “plant hormone signal transduction”, and “carbon metabolism” (Fig. 4B). These results laid a solid foundation for the study of different diel light cycle adaptation mechanisms of lettuce.
Analysis of Weighted gene Co-expression Network (WGCNA) for genes associated with sensitivity to diel light cycles in lettuce
In order to identify the genes associated with the diel light cycle in lettuce, we performed a WGCNA analysis. When the scale-free fit index is 0.8, the minimum soft threshold for constructing scale-free networks is 9. Hence, the value of 9 can be chosen as the most suitable soft threshold for further analysis (Fig. S5). The co-expression network was constructed using the optimal soft threshold, dividing the genes into various modules, and conducting the cluster dendrogram(Fig. 5A). From the results of WGCNA, 36 gene modules were identified, and marked with different colors. The member number in modules ranged from 47 to 18221 (Fig. 5B). By constructing cluster tree and correlation heatmap, the interaction between modules was explored. These 36 modules could be aggregated into two clusters with a high degree of interactive connectivity (Fig. 5C).
Module identification and functional enrichment analysis
The modules with the highest potential associations with traits and phenotypes were identified by analyzing the correlation between module eigenvalues and specific trait and phenotype data. We were interested in the correlation analysis of soluble sugar (represent for quality), chlorophyll content (represent for photosynthesis) and fresh weight (represent for yield) with gene modules. From the results, we found a positive correlation between the turquoise module and chlorophyll content, while a negative correlation with fresh weight and soluble sugar. The green module showed a positive correlation with fresh weight, while showing a negative correlation with soluble sugar and chlorophyll content. Moreover, the greenyellow module exhibited a positive correlation with soluble sugar, while showing a negative correlation with fresh weight and chlorophyll content (Fig. 6A). These results demonstrated that the eigengenes in turquoise modules, green modules, and greenyellow modules were most likely involved in photosynthesis, growth, and metabolism pathways in lettuce, respectively.
By Go annotation, we found that the DEGs of three modules in green, greenyellow, and turquoise exhibited enrichment in the categories of “regulation of transcription”, “DNA-templated” and “translation” within the GO “biological process” terms. The DEGs were found to be enriched in the terms “cytosol” and “membrane” within the “cellular component” categories. The DEGs in the GO categories of “molecular function” were found to be enriched in terms such as “ribosome structural constituent” and “protein kinase activity” (Fig. 6B). The KEGG enrichment analysis revealed that the green module was enriched in the pathway of “sphingolipid metabolism”, the greenyellow module was enriched in the pathway of “plant hormone signal transduction”, and the turquoise module was enriched in the pathway of “nucleocytoplasmic transport”(Fig. 6C). These three pathways directly related to plant cell growth, differentiation and signal transduction. Overall, the eigengenes of these three modules played important roles in promoting the growth and improving the quality of lettuce.
Screening of hub genes in the related modules
In order to explore the key gene involved in lettuce's diel light cycle, we analyzed the top 30 nodes in the green, greenyellow, and turquoise modules with the highest connectivity through cytoscape 3.7.2 version. From the results, it was found that CIP1 and SCL34 were hub genes in green module. ROPGEF1 and ACD6 were located at the center of the greenyellow network. Furthermore, CcmB and Rps4 were hub genes in turquoise module (Fig. 7). As previously reported, CIP1 can be ubiquitinated by COP1, resulting in suppression of light signal transduction and light morphogenesis (Han et al. 2020). ROPGEF1 is associated with ABA-induced stomatal closure in plants (Li et al. 2012). In Arabidopsis, CcmB has the ability to form an ABC transporter within the mitochondria, playing a role in the maturation of cytochrome c (Rayapuram et al. 2007). Therefore, it is probable that these genes functioned as crucial switches in the alterations of plant growth and development induced by different diel light cycles.
qRT-PCR validation of hub gene
In the WGCNA assay, we have found 6 hub genes that related to the growth of lettuce under different diel light cycle. By qRT-PCR analysis, we examined the expression levels of LsCIP1, LsSCL34, LsROPGEF1, LsACD6, LsCcmB, and LsRps4 in different scenarios during this investigation. It was showed that LsCIP1 and LsACD6 expressed highest in the 2C scenario compared with others. The expression levels of LsSCL34 and LsROPGEF1 peaked at 1.3C, followed by down-regulation in 2C. Additionally, the expression levels of LsCcmB and LsRps4 were elevated in 1C compared to other scenarios (Fig. 8). Together, our findings collectively indicated that the diel light cycle of lettuce involves the participation of these six hub genes.