The cucumber (Cucumis sativus) CS and CR were used as materials in this study, which were preserved at Institute of Facility Agriculture, Guangdong Academy of Agricultural Sciences. CS (EA background) was identified from North South China ecotype cucumber varieties, which is commonly used in modern cucumber breeding with high-quality genome and very rich omics data (Huang et al., 2009; Li et al., 2019). CR (EA background) was identified from the South China ecotype cucumber varieties. Plants were grown in plug trays in a plant incubator that maintained a temperature of (25 ± 1) °C and a 14-hour light/10-hour dark cycle. The relative humidity was 60%-80%. To determine the optimal low-light-stress condition, six distinct white LED light intensity treatments (0, 10, 40, 80, 120, 160 μmol m^-2 s^-1) were applied to the CS and CR plants, respectively. The effect of light quality on the growth of cucumber seedlings (CS) was evaluated by four different wavelengths LED light treatment, including 40 μmol m^-2 s^-1 white light LED (WL40), 160 μmol m^-2 s^-1 white light LED (WL160), 160 μmol m^-2 s^-1 LED light composed of blue light (16 μmol m^-2 s^-1) and red light (144 μmol m^-2 s^-1) (RB91), 160 μmol m^-2 s^-1 LED light composed of blue light (144 μmol m^-2 s^-1) and red light (16 μmol m^-2 s^-1) (RB19) ( Figure 1A ). After two weeks, the growth characteristics of cucumber seedlings were measured.

To identify the photoreceptor genes (PHYs, CRYs, UVRs, PHOTs) involved in low-light-stress, the protein sequences of Arabidopsis thaliana were retrieved from the The Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/), and these sequences were used as queries to identify the photoreceptors in cucumber by conducting BLASTP searches in the CuGenDBv2 database (http://cucurbitgenomics.org/v2/) (Yu et al., 2023). Then, the Pfam (https://www.ebi.ac.uk/Tools/hmmer/search/phmmer) and MEME suite (http://meme-suite.org/) were utilized to analyze and validate the conserved motifs of these photoreceptor proteins.

The CRY protein sequences of Cucumis sativus, Citrullus lanatus, Cucumis melo, Cucurbita moschata, Momordica charantia, Lagenaria siceraria, Benincasa hispida, Sechium edule, Luffa cylindrical, Trichosanthes anguina, and Cucurbita pepo were obtained from CuGenDB (http://cucurbitgenomics.org/v2/), and those of Solanum lycopersicum were from the Sol Genomics Network (https://solgenomics.net/), and those of Arabidopsis thaliana were from TAIR (https://www.arabidopsis.org/). The phylogenetic tree was constructed using MEGA 7 software (Institute of Molecular Evolutionary Genetics, USA). The gene structure of CsCRY1 was predicted using GSDS 2.0 (http://gsds.gao-lab.org/). The protein length, molecular weight (Mw), and theoretical isoelectric point (pI) of CRY1 were analyzed by the ExPASy ProtParam (https://web.expasy.org/protparam/). The subcellular location of CRY1 was predicted by INSP (http://www.csbio.sjtu.edu.cn/bioinf/INSP/).

To analyze the gene expression of these photoreceptor genes, we retained the expression data from the Cucurbit Expression Atlas (http://cucurbitgenomics.org/v2/). Among them, tissue expression data of cucumber photoreceptor genes was obtained from the transcriptome atlas of cucumber (PRJNA312872), and tissue expression data of melon was obtained from gene expression atlas of melon (PRJDB6414) (Yano et al., 2018). Tissue expression data of watermelons was obtained from transcriptome profiling of watermelon fruit development (PRJNA543725) (Guo et al., 2019). Then, the heatmaps were constructed using TBtools (Chen et al., 2020).

Total RNA was extracted from different cucumber tissue of CS (Rt: root, Sm: stem, Tl: tendril, Ap: apical point, Yl: young leaves, Ml: young leaves, Ol: young leaves, Pe: petal, St: stigma, Pi: pistil, Ov: ovary), using an OminiPlant RNA Kit (DNAaseI) from CWBIO (www.cwbio.com). The PrimeScriptTM RT reagent kit (TakaRa, Dalian, China) was using to produce reverse transcribed cDNA. The TSINGKE TSE030 T3 Super PCR Mix was used for RT-PCR assays. We selected CsACTIN as the reference gene. All the primer pairs in this study were listed in Supplementary Table 1 . Three biological replicates were performed. To analyze the expression of CsCRY1 gene under low-light-stress, RNA-seq of cucumber was conducted using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). The hypocotyls of CS and CR cucumber were sampled for RNA-seq and RT-PCR after 40 μmol m^-2 s^-1 low-light-stress (LL) and 160 μmol m^-2 s^-1 normal light (CK) treatments 72h. The software rMATS (version 4.0.1) (http://rnaseq-mats.sourceforge.net/index.html) was used to identify significant AS events with a false discovery rate (FDR) <0.05 (Shen et al., 2014). The CsCRY1 transcripts structure information was analyzed using IGV-GSAman software (https://gitee.com/CJchen/IGV-sRNA).

The full-length CDS of CsCRY1.1 from CS was amplified by PCR using 2 × High-Fidelity Master Mix (Tsingke, Inc., Beijing, China), and the PCR fagments were inserted in the KpnI and XbaI site of the pCambia1301-35s-EGFP vector by using ClonExperess II one Step cloning Kits (Vazyme, Piscataway, NJ, United States). The 35S::CsCRY1.1-GFP fusion protein was subsequently generated, controlled by the Cauliflower mosaic virus (CaMV) 35S promoter. The vector and the empty vector were each transformed into Agrobacterium strain GV3101. Subsequently, the positive strains were infiltrated into the leaves of tobacco (Nicotiana benthamiana) using the Agrobacterium-mediated transformation method (Sheludko et al., 2007). Finally, laser scanning confocal microscope (CarlZeiss LSM710) was used to take the GFP fluorescence signal pictures.

Statistical analysis of the experimental data was performed using SAS statistical package. Results were expressed as means ± standard deviation (SD). Differences in data from treatments were analyzed by one-way ANOVA and the analysis results were corrected with Turkey’s multiple comparison tests at a significance level of P<0.05.

The low-light-stress has a significantly negative impact on the quality of seedlings. However, there is a lack of optimal parameters for evaluating low-light-stress, thus resulting in limited understanding of the mechanism underlying low stress tolerance in cucumber. Therefore, we evaluated the growth characteristics of cucumber (CS) under various light intensities, including 0, 10, 40, 80, 120, and 160 μmol m^-2 s^-1, respectively. The hypocotyls elongation was observed to be rapidly promoted, while the leaf size was significantly inhibited by light treatments below 40 μmol m^-2 s^-1 ( Figures 1A, C ; Supplementary Figure 1 ). Furthermore, the elongation of cucumber hypocotyl (CS) was evaluated at the whole seedling stage under 160 μmol m^-2 s^-1. The results indicated that the elongation process of cucumber hypocotyl can be categorized into two distinct stages: stage I (0-4 days) characterized by rapid elongation of hypocotyl, and stage II characterized by slower elongation of hypocotyl (5-16 days) ( Figure 1B ). Additionally, we also compared the growth of two cultivars (CS and CR) under different light intensities. Firstly, we found that elongation of both material hypocotyls gradually stop after stage I. Secondly, there was a significant difference in the hypocotyls length of CS and CR under 40 μmol m^-2 s^-1, while the difference gradually became smaller in other light intensities treatments ( Figure 1D ; Supplementary Figure 1 ). Therefore, we selected 40μmol m^-2 s^-1 white LED light treatment for 7 days as the optimal parameters for evaluating low-light-stress. This approach allowed us to quickly and efficiently identify a low-light tolerant cucumber line (CR) and a low-light-sensitive cucumber line (CS) ( Figure 1D ). Notably, CR had a significantly shorter hypocotyl, better photosynthesis capacity, and superior resistance to lodging under low-light-stress compared with CS ( Figure 1D ; Supplementary Figure 2 ).

To assess the effect of different light qualities under low-light-stress, we cultivated cucumber seedlings for two weeks under four LED conditions with different intensity of blue and red light: WL40 (40 μmol m^-2 s^-1 total light intensity, containing 10 μmol m^-2 s^-1 blue light intensity), WL160 (160 μmol m^-2 s^-1 total light intensity, containing 40 μmol m^-2 s^-1 blue light intensity), RB91 (160 μmol m^-2 s^-1 total light intensity, containing 16 μmol m^-2 s^-1 blue light intensity) and RB19 (160 μmol m^-2 s^-1 total light intensity, containing 144 μmol m^-2 s^-1 blue light intensity) ( Figure 2A ). The results revealed that under the WL40 condition, the hypocotyl length was the longest, whereas the SPAD value and total root length were the smallest. When the light intensity was the same, the hypocotyl of treatment RB91 was the longest, followed by treatment WL160, and the shortest was treatment RB19. The results indicated that the hypocotyl length was totally negatively correlated with the intensity of blue light ( Figures 2B, C ). Additionally, our findings revealed that CR exhibited significantly shorter hypocotyls compared to CS when exposed to blue light treatment. However, no significant difference was observed in hypocotyl length between CS and CR under red light treatment ( Supplementary Figure 3 ). Furthermore, the supplementary LED experiment demonstrated that the elongation of CS hypocotyls can also be most effectively inhibited by supplement of blue light, whereas red light exhibited the least effective inhibitory effect ( Supplementary Figure 4 ). The research findings indicated that the reduction in blue light intensity is the primary factor responsible for inducing low-light-stress in cucumber.

Photoreceptors play a critical role in regulating low-light-stress tolerance of plants. However, in cucurbit crops, photoreceptor genes have not been clearly explained in detail. Here, we identified the major classes of photoreceptors (CRYs, UVRs, PHYs, PHOTs) and analyzed their expression patterns in different cucurbit crops. A total of 11 and 13 photoreceptor genes were identified in cucumber and melon, respectively. The gene ID information of these factors was shown in Supplementary Table 2 . Chromosome mapping results revealed that the 11 photoreceptor genes in cucumber were each mapped to five distinct chromosomes. In the photoreceptors, the PHYA and PHOT2 genes have an additional copy in almost all cucurbit crops. Furthermore, a segmental duplication gene pair (PHOT2) was identified through syntenic analysis ( Figure 3A ). It is well-recognized that analyzing the expression of these genes across different tissues is pivotal in deciphering their function. The heatmaps, created using expression data from the Cucurbit Expression Atlas, revealed that CsaV3_3G047490.1 consistently exhibited a higher expression level than other photoreceptor genes in various cucurbit crops, including cucumber, melon, watermelon, and bitter melon ( Figures 3B, C ; Supplementary Figure 5 ). Additionally, CRY1 was also highly expressed in the hypocotyls of cucumber seedlings (Figure 3B).

CsaV3_3G047490 was annotated as the cryptochrome blue light receptors in the cucumber genome. To further identify the CsCRY genes, we used 3 CRYs (AtCRY1, AtCRY2 and AtCRY3) protein sequences from Arabidopsis as queries to carry out a Blastp search. As a result, only two CRYs genes were identified in the Chinese Long v3 genome of cucumber. To investigate the evolutionary relationships among the CRY proteins in cucurbit crops, a phylogenetic tree was constructed using MEGA 7. All the CRYs proteins in cucurbits crops could be divided into three subfamilies: CRY1, CRY2, and CRY3 according to the classification of CRYs in Arabidopsis. The CRY1 subfamily has the highest number of members, with a total of 17. Conversely, the CRY2 subfamily is the smallest with only 13 members ( Figure 4A ).

The physical and chemical properties of CsCRY1 and CsCRY3 were shown in Table 1 . The amino acid sequence lengths of CsCRY1 and CsaCRY3 are 613 amino acids and 592 amino acids, respectively, while their pI values are 5.46 and 9.4. Both CRYs genes are mapped to almost the same region of chromosome 3 ( Figure 3A ). Interestingly, the CRY2 gene is absent in the cucumber 9930 genome, but not in other cucurbit crops such as melon, watermelon, pumpkin, and bitter melon ( Table 1 ; Supplementary Table 2 ). Therefore, we identified the cucumber CRY2 gene in the remaining four reference cucumber genomes within CuGenDBv2 database. Interestingly, we identified a completely intact CRY2 gene (636aa) in Cucumis hystrix var (2n = 2x = 24), a wild cucumber species that can hybridize with cultivated cucumber varieties (C. sativus L., 2n =2x = 14). The CRY2 is incomplete (229 amino acids) in the genome of wild/semi-wild varieties Cucumis sativus var. hardwickii cv. PI 183967. All cultivated cucumbers (Cucumis sativus L. var. sativus cv. Chinese Long, Cucumis sativus L. var. sativus cv. Gy14, and Cucumis sativus L. var. sativus cv B10) lack the CRY2 gene ( Supplementary Table 3 ). The findings suggest that the CsCRY2 gene has been explicitly lost over a long evolution from wild to cultivated varieties.

To investigate the responses of the CsCRY genes to low stress, RNA-seq analysis was conducted on tolerant and sensitive cucumber hypocotyls. The hypocotyls were sampled after 72 hours of treatment under 40μmol m^-2 s^-1 (LL) and 160μmol m^-2 s^-1 (CK) white light LED. During this period, the hypocotyl length of the tolerant cucumber CR was significantly shorter than that of the sensitive cucumber CS under low-light-stress ( Supplementary Figure 6 ). The expression result of CsCRY1 and CsCRY3 indicated that the expression level of CsCRY1 was much higher than CsCRY3. Additionally, the expression of CsCRY3 was significantly depressed by low-light-stress treatment in both tolerant and sensitive cucumber material. However, only in the sensitive cucumber material, the expression of CsCRY1 was significantly induced by low-light-stress ( Figure 4B ). Therefore, the above results indicated CsCRY1 may play a critical role under low-light-stress.

To further understand the functions of CsCRY1, we aligned the amino acid of CRY1 protein from various cucurbit species. Our findings indicated that CRY1 is highly conserved, particularly in its N-terminal PHR domain. However, the length of all CRY1 proteins from various crops is almost 681 amino acids, except for cucumber ( Supplementary Table 4 ). The C-terminal of CCT domain in CsCRY1 of cucumber was short 70 aa ( Figure 5 ). Then, we cloned the CsCRY1 gene and verified the presence of CsCRY1 mRNA in the cucumber genome, which is capable of encoding a short CsCRY1 protein. It is well-established that both CRY1 and CRY2 regulate low-light-stress, albeit a relatively minor role for CRY2 compared with that of CRY1 in Arabidopsis. However, CsCRY2 was absent in cucumber. The absence of the CsCRY2 gene in cultivated cucumber leads to a significant role for CsCRY1 in low-light-stress conditions.

To further elucidate the distinct features of cucumber CsCRY1, we have successfully obtained 34 transcripts including 15 full-length transcripts of CsCRY1 through single-molecule long-read sequencing in cucumber CS ( Supplementary Figure 7 ). By utilizing the IGV-GSAman software to align these CsCRY1 transcripts to the cucumber genome, we discovered that the pre-mRNA of CsCRY1 undergoes alternative splicing (AS). Additionally, alternative 5′ splice sites (5’SS) in the last intron was the major AS events. Interestingly, there were five kinds of 5’SS events in intron four, one total intron retention event, three partial intron retention events, and one total intron splicing event. We predicted the open reading frames (ORFs) of these CsCRY1 transcripts. The findings indicated that intron 4 contains a crucial coding sequence, with the stop codon positioned at the midpoint of intron four. Therefore, the five splice variants can be translated into three kinds of splice proteins with different lengths of C-terminal domains ( Figure 6A ). The transcripts CsaV3_3G047490.3, CsaV3_3G047490.4, and CsaV3_3G047490.5 encode the full-length protein of CRY1, designated as CsCRY1.3. The transcripts CsaV3_3G047490.1 and CsaV3_3G047490.2 encode two different truncated C-terminal CRY1 proteins, named CsCRY1.1 and CsCRY1.2 ( Figure 6B ). We analyzed the conserved domains of these spliced proteins by HMMER. All the spliced protein contains an intact DNA photolyase domain and FAD binding domain. However, only CsaV3_3G047490.1 contained an impaired CCT domain ( Figure 6C ). Moreover, we did not find the 5’SS of CRY1 in other species including melon, pumpkin, Arabidopsis, etc. ( Supplementary Figure 8 ). Therefore, we concluded that 55’SS of CsCRY1 is responsible for the formation of various transcripts with diverse coding capabilities in cucumber. However, the precise role of CsCRY1 remains elusive.

To further validate the AS of CsCRY1, we conducted a transcriptome analysis of both CR and CS cucumber hypocotyls following 72 hours of exposure to low-light-stress (40 μmol m^-2 s^-1) and normal light (160 μmol m^-2 s^-1) treatment. The results reaffirmed the presence of a 5’SS in the last intron of CsCRY1 in both CR and CS ( Figure 7A ; Supplementary Figure 9 ). To unravel the expression pattern of CsCRY1 splicing variants in different tissues, we designed a pair of the primer crossing the intron four to perform RT-PCR assay. The results revealed that there were four spliced transcripts identified in almost all the samples. Among them, the expression level of CsaV3_3G047490.3 was the highest, followed by CsaV3_3G047490.4 and CsaV3_3G047490.1. In contrast, the expression of CsaV3_3G047490.5 was the lowest, while no expression of CsaV3_3G047490.2 was detected ( Figures 7B, C ). Furthermore, all four transcripts were present in hypocotyls under low-light-stress conditions. However, we only found that the expression of CsaV3_3G047490.1 was induced by low-light-stress, and the RNA-seq analysis confirmed these findings ( Figure 7 ; Supplementary Figure 9 ).

The CsCRY1.1 protein, a newly identified blue light receptor in plants, was cloned and its subcellular localization was evaluated. Here, we found that the subcellular localization of CsCRY1.1 was same to GFP. The fluorescence signal was widely observed in both the cytoplasm and nucleus ( Figure 8 ). The result indicated that loss of the last valine-proline-containing (VP) motif did not change subcellular localization of CsCRY1.1. But, whether CsCRY1.1 without the last VP motif can interact with the COP1/SPA complex is still unknown.