A Mutation in CsHY2 Encoding a Phytochromobilin (PΦB) Synthase Leads to an Elongated Hypocotyl 1(elh1) Phenotype in Cucumber (Cucumis sativus L.)

doi:10.21203/rs.3.rs-182705/v1

Download PDF

Original Article

A Mutation in CsHY2 Encoding a Phytochromobilin (PΦB) Synthase Leads to an Elongated Hypocotyl 1(elh1) Phenotype in Cucumber (Cucumis sativus L.)

https://doi.org/10.21203/rs.3.rs-182705/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Hypocotyl length is a critical determinant in establishing high quality seedlings for successful cucumber production, but knowledge on the molecular regulation of hypocotyl growth in cucumber is very limited. Here we reported identification and characterization of a cucumber elongated hypocotyl 1 (elh1) mutant. We found that the longer hypocotyl in elh1 was due to longitudinal growth of hypocotyl cells. With fine mapping, the elh1 locus was delimited to a 20.9-kb region containing three annotated genes; only one polymorphism was identified in this region between two parental lines, which was a non-synonymous SNP (G28153633A) in the third exon of CsHY2 (CsGy1G030000) that encodes a phytochromobilin (PΦB) synthase. Uniqueness of the mutant allele at CsHY2 was verified in 515 cucumber lines. Ectopic expression of CsHY2 in Arabidopsis hy2-1 mutant led to reduced hypocotyl length. The PΦB protein was targeted to chloroplasts. The expression levels of CsHY2 and five phytochrome genes, CsPHYA1, CsPHYA2, CsPHYB, CsPHYC and CsPHYE were all significantly down-regulated while several cell elongation related genes were up-regulated in elh1 mutant compared to wild type cucumber, which are correlated with dynamic hypocotyl elongation in the mutant. RNA-seq analysis in the WT and mutant revealed differentially expressed genes involved in porphyrin and chlorophyll metabolisms, cell elongation, and plant hormone signal transduction pathways. This is the first report to characterize and clone the CsHY2 gene in cucumber. This work reveals the important of CsHY2 in regulating hypocotyl length and extends our understanding of the roles of CsHY2 in cucumber.

Molecular Genetics

Cucumber

hypocotyl growth

map-based cloning

phytochrome

light signaling

Plant development not only depends on endogenous genetic manipulation, but also their modulation in response to environmental factors (Song et al. 2019). Light is among the most important environmental cues controlling plant growth and development such as seed germination, cotyledon expansion, hypocotyl elongation, root growth, flowering and fruit set (Pham et al. 2018; Seo et al.2019; Huai et al. 2020; Liu et al. 2021). Plant perceives lights by a suite of photoreceptors including phytochromes (for red and far-red light) (Oh et al. 2020; Kahle et al. 2020), cryptochromes and phototropins (for blue/UVA light) (Sztatelman et al. 2016; Fantini et al. 2019; Lin et al. 2019) and UVR8 (for UVB) (Zhao et al. 2016; Tavridou et al. 2020; Yang et al. 2020). Among them, the best characterized are the phytochromes (phys), which regulate a wide range of light responses from seed germination to flowering (Josse et al. 2008; Montgomery. 2009; Song and Choi. 2019)

In Arabidopsis, five phytochromes, PHYA to PHYE, have been identified (Quail. 2002). A phytochrome consists of an apoprotein covalently attached to the linear tetrapyrrole chromophore, (3E)-phytochromobilin (PΦB) (Montgomery. 2008). Phytochrome apoproteins in all plants are encoded by a small multigene family. The apoproteins are synthesized in the cytosol, where they assemble autocatalytically with the plastid-derived chromophore PΦB (Li et al. 2011). The synthesis of PΦB is accomplished by an enzymatic cascade in the plastid that begins with the 5-aminolevulinic acid (ALA) which is a common precursor for the biosynthesis of tetrapyrrole compounds such as PΦB and chlorophyll (Li et al. 2011). The first committed step in the synthesis of PΦB is the conversion of the heme to biliverdin (BV) IXα by a ferredoxin-dependent heme oxygenase HO1 encoding by the HY1 gene (Kraepiel et al. 1994; Terry and Kendrick. 1996; Izawa et al. 2000; Linley et al. 2006; Zhu et al. 2017). Subsequently, the BV IXα is reduced to 3Z-PΦB by the PΦB synthase or HY2 that is encoded by the HY2 gene, and is then isomerized to 3E-PΦB and assembled into the functional holo-phys (Tanaka et al. 2011).

Mutants that are unable to synthesize the PΦB have been instrumental in improving our understanding of the roles of phys in light-regulated plant development and morphogenesis (Linley et al. 2006). All plant phys bind the same PΦB (Muramoto et al. 2005). Therefore, the disruption of PΦB synthesis could inactivate the entire phytochrome system and impairs photomorphogenesis (Sawers et al. 2004). For example, all hy2 mutants in different plants, such as Arabidopsis hy2-1, tomato au, pea pcd2, tobacco pew2, and maize elm1 are characterized by an elongated hypocotyl and yellow-green phenotype due to a deficiency in photoactive phys (Weller et al. 1997; Kraepiel et al. 1994; Kohchi et al. 2001; Sawers et al. 2004; Muramoto et al. 2005; Wu et al. 2020).

Cucumber (Cucumis sativus L., 2n=14), an economically important vegetable crop worldwide (Li et al. 2013; Chen et al. 2017). In many regions, cucumbers are produced from transplanted seedlings in both open field and protected cultivation. The use of high quality seedlings is a key factor for the success of cucumber production. Hypocotyl length is a critical determinant in establishing strong seedlings for safe handling, seedling transportation, and survival rate after seedling transplanting, and eventually cucumber yield (Ming et al. 2011). Further, cucumber hypocotyl is an ideal system for understanding the mechanism of light-regulated cell elongation and seedling morphogenesis (Bo et al. 2016; Song et al. 2019). However, little is known about the genetic and molecular control of hypocotyl growth in cucumber. So far, only one SHORT HYPOCOTYL1 (Sh1) gene has been cloned and characterized (Bo et al. 2016), but the molecular mechanisms of sh1-regulated hypocotyl growth is unknown. In addition, several long hypocotyl mutants have been characterized in a number of papers (Robinson and Shail. 1981; Koornneef and van der knaap. 1983; López-Juez et al. 1992) However, the gene responsible for the long hypocotyl have not been cloned and the molecular mechanisms of hypocotyl growth is also unclear.

In the present study, we reported and characterized a novel elongated hypocotyl mutant (elh1) in cucumber. Map-based cloning of elh1 revealed that this mutant phenotype was due to single nucleotide polymorphism in CsHY2, a homolog of Arabidopsis HY2 gene for the PΦB synthase. This mutant provides a valuable tool to understand the role of phys signaling in cucumber development and to explore the degree, and potential agronomic importance, of light-induced phenotypic plasticity.

Plant materials and mapping populations

The long hypocotyl mutant C1238 was identified from an EMS-induced mutagenesis population of the cucumber inbred line CCMC with normal hypocotyl and green leaves (Chen et al. 2017). The mutant C1238 exhibits apparently elongated hypocotyl and yellow-green leaves at the seedling stage (Figs. 1A-1D). We designated this mutant locus as elh1 (elongated hypocotyl-1) in C1238.

To study the inheritance of elh1, several segregating populations were employed by crossing of the C1238 with the original parental line CCMC, the American pickling cucumber line Gy14 and the North China fresh marker type cucumber line 9930, respectively (Table 1). For linkage mapping and cloning of the elh1 gene, F₂ populations were derived from the C1238 x 9930 F₁and C1238 x Gy14 F₁plants, respectively. Since the polymorphism of molecular markers was very low between C1238 and 9930, only the C1238 x Gy14 population was employed in molecular mapping of elh1. Recombinants among F₂ plants of C1238 x Gy14 were defined by flanking molecular markers. All the F₂ plants used for primary mapping and recombinants were self-pollination to produce F₃ offspring. At least 50 plants of each F₃ family were examined for segregation of hypocotyl to determine F₂genotype at the elh1 locus. Segregation was tested against expected ratio with Chi-square (χ²) tests. All individuals were grown in plastic greenhouses under natural sunlight at the Northwest A&F University (Yangling, China). The length of hypocotyl of all plants was visually scored at the cotyledon stage as either WT or mutant.

Microscopic observation of the hypocotyl cells

Hypocotyls of 10d-old seedlings of C1238 and CCMC were cut into small sections and fixed for 24h at 4℃ in a 1:1:18 solution mixed of acetic acid, formaldehyde and 70% ethanol, respectively. The hypocotyls were stained overnight at 42℃ with 1% safranin in 75% ethanol, subsequently dehydrated using a graded ethanol series. And then the samples were treated with xylene and embedded in paraffin. The samples were then sectioned using the vibrating microtome and washed with 100% ethanol for 3min, after which they were stained with toluidine blue for 20min. Paraffin sections were visualized using the BX63 microscope (Olympus, Tokyo, Japan). The cell length and cell number of longitudinal sections from elh1 mutant and WT samples were measured using the software ImageJ after photographing (https://imagej.nih.gov/ij/).

The dynamic change of hypocotyl elongation under the white light was also evaluated. Hypocotyl length and its cell elongation rate were evaluated every two days for 13d.

Effects of light quality on hypocotyl elongation

To investigate the effect of light quality on hypocotyl elongation, the seedlings from mutant C1238 and WT were cultured in the growth chambers with red (R), far red (FR) and blue LED light sources with a peak wavelength of 620, 740 and 420nm, respectively. The 28℃day and 18℃ night with a 14 h photoperiod were set in the growth chambers. Dark treatment was also employed in parallel with these experiments. Hypocotyl length was measured at 13 d after germination.

Fine genetic mapping of elh1 gene

Bulked segregation analysis (BSA) was used for the initial mapping of the elh1 locus with the C1238 x Gy14 F₂ population. Two DNA pools, LH-bulk (long hypocotyl bulk) and NH-bulk (normal hypocotyl bulk), were constructed by pooling equal amount of DNA from five mutant and five WT plants, respectively. SSR markers (1680 total) evenly distributed across the seven cucumber chromosomes were screened for polymorphism between the parental lines C1238 and Gy14 (Ren et al. 2009; Cavagnaro et al. 2010). The polymorphic markers in the two parental lines were used to the two pools, and then applied to 96 F₂individuals for linkage analysis.

For fine mapping of the elh1 gene, additional markers were developed from SNPs (single nucleotide polymorphisms) and indels (insertion or deletion of base) in the target region by aligning resequencing reads of C1238 against the Gy14 draft genome (v2.0) with DNAMAN V10.0 (http://www.lynnon.com/). The SNPs were converted to CAPS or dCAPS markers using dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html). Primers were designed with Primer Premier 5.0 (http://www.premierbiosoft.com/ primerdesign/). Information of all linked markers and primers for various purposes in the present study is provided in Supplemental Table S1.

DNA extraction, PCR amplification of molecular markers, and polyacrylamide gel electrophoresis was executed as described by Li et al. (2011). Linkage analysis of lh1 locus with molecular markers was performed using JoinMap 4.0 at an LOD threshold of 5.0.

Gene prediction and candidate gene identification

The gene prediction was performed with the online program FGENESH from the SoftBerry (http://sunl.softberry.com/). Gene function prediction was performed using the BLASTP tool at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All predicted genes in the candidate gene region were Sanger sequenced from the C1238 and WT, to confirm the polymorphisms.

dCAPS assay with the causal SNP of elh1 candidate gene

A causal SNP inside the elh1 candidate gene was identified between C1238 and WT. A dCAPS marker dCAPS1238-1 was developed from this SNP and used in dCAPS assays among plants from the 5317 F₂ individuals of C1238 x Gy14 and 1249 F₂ plants of C1238 x 9930. The uniqueness of this causal SNP was checked among 115 re-sequenced cucumber lines by sequences alignments (Qi et al. 2013). This dCAPS marker was also used to examine allelic diversity among 400 cucumber lines according to Bo et al (2016). For dCAPS assay, the PCR amplicons were digested with the restriction enzyme Taq I for 65℃ for 14h, separated with 9% polyacrylamide gel electrophoresis and visualized with silver staining. In the WT, the 253bp target fragment was cut into two bands with 233bp and 20bp in size by Taq I, but there was only one 253-bp band in the mutant.

Cloning and sequencing of elh1 candidate gene

Total RNA was extracted from the hypocotyls of C1238 and WT plants using the BioFast BIOZOL Total RNA Extraction Reagent (Bioer, China). The first-strand cDNA was synthesized using the 5x All-In-One RT MasterMix (ABM, Canada). The full length cDNA of elh1 candidate gene (CsHY2) was amplified by gene-specific primers (Table S1) as described in Wang et al. (2017) and sequenced by TsingKe Biological Technology (China).

Expression analysis of the elh1 and other functionally related genes

We examined the time-course expression level of the elh1 candidate gene and other genes in the candidate gene region (20.9kb) with the quantitative real-time PCR (qPCR). The hypocotyl samples from C1238 and WT were collected 3, 5, 7, 9, 11, and 13d days after germination. We also examined the expression pattern of elh1 gene in the roots, stem, cotyledon, true leaf, female flowers, male flowers and fruit. Total RNA extraction and the first-strand cDNA synthesis followed Wang et al. (2017).

To investigate the effect of the mutation in CsHY2 on the function of phys, the expression dynamics of cucumber PHYs genes (CsPHYA1, CsPHYA2, CsPHYB, CsPHYC and CsPHYE) were analyzed. We also conducted qPCR on six cell elongation-related genes including CsEXT3 (EXTENSIN3), CsEXPA8 (EXPANSIN-A8), CsDWF4 (DWARF4), CsXTH22 (XYLOGLUCAN ENDOTRANSGLUCOSYLASE /HYDROLASE PROTEIN22), CsXTR6 (XYLOGLUCAN ENDOTRANSGLYCOSYLASE6), and CsIAA29 (AUXIN-RESPONSIVE PROTEIN IAA29). Relative expression level of the target genes was calculated using the 2^-ΔΔCt method (Livak and Schmittgen. 2001) with UBI-ep (ubiquitin extension protein) as the internal reference (Wan et al. 2010).There were three biological and three technical replications for each sample. Information of all the primers used in qPCR in the study is provided in the Supplemental Table S3.

Phylogenetic analysis of CsHY2 homologs from different plants

The phylogenetic relationships of HY2 proteins from 11 species were investigated. The NCBI accession numbers of these sequences are listed in the Supplemental Table S4. Multiple protein sequence alignment was performed with Clustal W and the phylogentic tree was constructed with neighbor-joining method (Saitou and Nei. 1987) based on distance calculation with 1000 bootstrap replications in MEGA 7.0 (https://www.megasoftware.net/).

Subcellular localization of CsHY2 protein

The coding region of the CsHY2 gene, without the stop codon, was cloned with the gene-specific primers 35S-CsHY2-L and 35S-CsHY2-R (Table S5). The fragment was then cloned into the Nco I/Bgl II site of the overexpression vector pCAMBIA3301-EGFP. The empty pCAMBIA3301-EGFP (35S:EGFP) vector was used as the negative control. And then the constructed plasmid 35S:CsHY2-EGFP and the 35S:EGFP were used for transient expression in tobacco (N. benthamiana) leaves following Li (2008). Fluorescence was observed using an Olympus BX63 fluorescence microscope.

Functional complementation of hy2 mutant in Arabidopsis

The above-mentioned 35S:CsHY2-EGFP plasmid construct was also transformed into Agrobacterium tumefaciens GV3101. Arabidopsis hy2-1 recessive homozygous mutants were transformed by the floral dip method (Clough and Bent. 1998). To identify the transgenic lines, T1 generation seedlings were selected by spraying Basta solution (0.0015%) (Bayer, Germany) every three days. The survived plants were further confirmed by PCR and used for further charactization.

RNA-Seq analysis

Total RNA was extracted from hypocotyls of seven 5d-old seedlings of C1238 and WT using the Trizol. The cDNA libraries were constructed with the TruSeq™ RNA sample prep kit (Illumina) and sequenced on the Illumina HiSeq-2500 machine by Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). After removing low-quality and poly-N reads, the clean reads were mapped to the 9930 v3.0 reference genome using TopHat version 2.0.13. FPKM (fragments per kilobase of transcript sequence per million base pairs sequenced) were calculated by Stringtie (https://ccb.jhu.edu/software/stringtie/) to estimate transcript abundance. Differentially expressed genes (DEGs) were identified using cutoff of FDR (false discovery rate) <0.05 and log2(fold change)≥2. Meanwhile, we also performed GO- (gen ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway-enrichment analyses on identified DGEs at the Majorbio Cloud Platform (www.majorbio.com).

Phenotypic characterization of elh1 mutant

Under natural light conditions, as compared with the WT (CCMC), the elh1 mutant showed a much longer hypocotyl, more elongated internode and yellow-green leaf (Figs. 1A-1D, S1, S2), indicating that this mutant might be defective in light perception or signal transduction due to the similar morphological phenotypes of elh1 with some previously characterized mutants in Arabidopsis (Kohchi et al. 2001). However, under field conditions, the elh1 mutant also exhibited fewer numbers of internodes than the WT. Therefore, there was no significant difference in plant height between the mutant and WT in adult plants (Figs. S2A-S2D).

Under a microscope, the hypocotyl cells of the mutant were significantly longer than those of the WT (Figs. 1E-1G), but the total cell numbers of the whole hypocotyl longitudinal section were not significantly different between elh1 and WT (Fig. 1H). These results suggested that the longer hypocotyl in the mutant is due primarily to increased cell elongation.

Hypocotyl elongation of mutant under different light conditions

We examined hypocotyl growth dynamics of the mutant and WT under white light. The hypocotyl length of both elh1 and WT increased gradually until 13d after germination (Figs. S3A, S3B). The hypocotyl elongation rate in elh1 was significantly higher than that of WT at any time point, which reached its peak at 5d after germination (Fig. S3C). We further investigated hypocotyl growth of the mutant and WT under continuous monochromic red (R), far-red (FR), blue lights, as well as in the dark. The hypocotyl length of elh1 was significantly longer than that of WT under both R and FR (Figs. 2A, 2B). In other words, elongation was strongly inhibited by both R and FR in WT seedlings, suggesting that the elh1 plants were blind to both R and FR. In contrast, the WT and elh1 seedlings showed similar elongation of the hypocotyl in the dark or blue light (Figs. 2A, 2B). These results suggest that elh1 is defective in the repression of the photomorphogenesis during development in either R or FR light, indicating that light responses mediated by phytochromes are missing in elh1.

Inheritance analysis of the elh1 locus

We examined segregation of hypocotyl length in different F₂ populations from crosses of the C1238 mutant with CCMC, Gy14 and 9930 (Table 1). All F₁ plants from different crosses had the same hypocotyl length as the WT parents. Among 5317 C1238 x Gy14 F₂ plants, 4012 and 1305 had normal and long hypocotyl, respectively, which was consistent with 3:1 segregation (p=0.442 in χ² test) (Table 1). Of 1249 F₂ plants from C1238 x 9930, 949 had normal hypocotyl and 300 had long hypocotyl (p=0.423 in χ² test fit 3:1) (Table 1). These results indicated that the long hypocotyl mutation in C1238 was controlled by a single recessive gene, elh1.

Linkage mapping of the elh1 locus

Among 1680 SSR markers screened between C1238 and Gy14, 113 were polymorphic, 9 of which were also polymorphic between the LH-bulk and NH-bulk. All the nine polymorphic SSRs were located on cucumber chromosome (Chr) 1. Indeed, linkage analysis in 96 F₂ plants of C1238 x Gy14 revealed that all the nine SSR markers were linked with the elh1 locus with the two closest markers. UW032400 and UW031473 being 4.7 and 0.5cM away from the elh1 locus, respectively (Fig. 3A).

To narrow down the mapping region, 14 new SSR markers were developed in this region, and five UW032647, UW033042, UW009754, UW069958, and SSR17922 exhibited polymorphisms between C1238 and Gy14 and the two bulks. All five markers were linked with the elh1 locus (Fig. 3B). We further phenotyped 5317 F₂ plants from C1238 × Gy14 cross using the two flanking markers SSR17922 and UW009754, and two recombinants were identified between them. Physically, the elh1 region flanked by SSR17922 and UW009754 was 82.1kb (Table S1).

From sequence alignment of the CCMC re-sequencing reads against the Gy14 v2.0 draft genome assembly, we identified three NSPs, which were converted to CAPS or dCAPS markers (dCAPS1238-3, CAPS1238-1 and dCAPS1238-1). Genotyping of the recombinants and linkage analysis allowed establishment of the genetic and physical orders of all linked markers in relation to the elh1 locus (Fig. 3B). The elh1 locus was finally delimited to the 20.9kb interval flanked by SSR17922 and CAPS1238-1 in the Gy14 scaffold00598 (Table S1).

Cloning and identification of elh1 candidate gene

In the 20.9kb genomic DNA region, three genes were predicted using the FGENESH program. The predicted functions and relevant information of the three genes are presented in Table S2. We compared the genomic DNA sequences and expression of the three genes between the mutant and WT. No sequence or expression level differences were found in the first and third genes (CsGy1G029990 and CsGy1G030010) between C1238 and WT (Figs. 4B, 4C); in contrast, a non-synonymous G to A mutation (G28153633A) was identified in the CDS (coding sequence) region of the second gene, CsGy1G030000 (CsHY2). We further checked the uniqueness of the mutant allele of CsHY2 by sequence alignment in this region between C1238 and 115 resequenced cucumber lines (Qi et al. 2013). We found that the mutant A allele at this SNP locus only occurred in C1238, while all the 115 lines carried the WT G allele.

Based on this SNP, a dCAPS marker, dCAPS1238-1 was developed and used to genotype the 5317 C1238 × Gy14 F₂plants and 1249 C1238 × 9930 F₂ plants, which confirmed the co-segregation of phenotypes and the two SNP alleles in both populations (Figs. 3B, S5). To further verify the uniqueness of the SNP, we performed dCAPS assay with dCAPS1238-1 among 400 cucumber lines, and found that all the 400 WT lines shared the same G SNP allele as Gy14 and CCMC (Fig. S6). These results all supported the conclusion that this SNP in the elh1 gene is responsible for the long hypocotyl phenotype in mutant C1238.

To confirm the identity between elh1 and CsHY2, we performed a complementation test by introducing the CsHY2 cDNA driven by the CaMV35S promoter into Arabidopsis hy2-1 mutant. Ten independent transgenic plants carrying the 35S:CsHY2 construct were analyzed for hypocotyl length. All of them exhibited normal hypocotyl length similar to those of the wild-type Arabidopsis plants (Figs. 5A, 5B) further confirming that elh1 encodes CsHY2, a cucumber homologue of Arabidopsis HY2.

We cloned the full-length cDNA sequence of CsHY2 from WT and C1238. Alignments of cDNA sequences and deduced amino acid sequences between WT and mutant are shown in Supplemental files 1 and 2, respectively. There was a SNP at position 28153633 (G in WT to A in mutant) in third exon of CsHY2 in elh1, which results in an amino acid substitution from Asp¹⁰⁴ (D) in WT to Asn¹⁰⁴(N) (Fig. 3D; Supplemental file 2). Annotation of CsHY2 gene showed that it was 5701bp in length and consisted of eight exons and seven introns (Fig. 3D), which was predicted to encode a PΦB synthase with 322 amino acid residues (Supplemental file 2).

Phylogenetic relation among CsHY2 and its homologs in other sepecies

To better understand the phylogenetic relationships of CsHY2 protein with its orthologs in other species, a phylogenic tree was constructed by use of the amino acid sequences of HY2 from 11 species (Table S4; Fig. S7). The results manifested that the mutation site occurred in the CsHY2 was highly conserved in different species (Supplementary file 3). Sequences similarity between the cucumber CsHY2 and its homologs suggested that CsHY2 may serve conserved functions like in these species (Supplemental file 3). This could be reflected from the very similar phenotypes of mutant of the HY2 gene in different plant species such as long hypocotyl and yellow-green leaves (e.g., Parks and Quail. 1991; Terry and Kendrick. 1996; Kohchi et al. 2001; Muramoto et al. 2005; Fig. 1 of this study).

Spatiotemporal expression patterns of CsHY2

We investigated the expression dynamics of CsHY2 with qPCR in elh1 and WT hypocotyls at different time points after germination (Fig. 4A). We found that the expression level of CsHY2 was highly consistent with the hypocotyl elongation rate in the two lines (Figs. 4A, S3C). In the mutant, the fastest elongation rate occurred around 3-5d after germination (Fig. S3C). This was consistent with the highest expression of CsHY2 at this time (Fig. 4A).

We further examined the expression of CsHY2 in seven organs from elh1 and WT (Fig. 4D). The expression level of CsHY2 in mutant was the highest in male and female flowers, followed in order by stem, true leaves, root, cotyledon and fruit. However, there was no significant difference in CsHY2 expression in all these organs except the cotyledon, true leaves and stem between the WT and elh1 (Fig. 4D). These results suggest that the long hypocotyl, elongated internode (stem) and yellow-green leaves of elh1 may be the result of reduced expression of CsHY2.

Expression analysis of phys and cell elongation-related genes

We also compared time-course expression of five phytochrome genes including CsPHYA1, CsPHYA2, CsPHYB, CsPHYC and CsPHYE in the mutant and WT. We found that each of the expression of all five genes was peaked at fifth day after germination (Figs. 6A-6E). This was consistent with hypocotyl elongation rate (Fig. S3C), supporting early findings that phys genes play an important role in the regulation of hypocotyl cell elongation. We also observed coordinate changes in the levels of CsHY2 and five cucumber phys in mutant indicating that CsHY2 affects the expression levels of all the phys.

In order to explore the underlying molecular mechanism of CsHY2-regulated hypocotyl elongation, time-course expression of several cell-elongation-related genes was tested including CsDWF4, CsEXT3, CsEXPA8, CsXTH22, CsXTR6 and CsIAA29. All of them showed their highest expression at the fifth day, which was consistent with the hypocotyl elongation rate (Figs. 7A-7F, S3C). These data suggested that these genes are involved in hypocotyl cell elongation and the functional CsHY2 gene suppress their expression in cucumber.

Comparative transcriptome profiling analysis

To understand the CsHY2-mediated gene network for hypocotyl growth, we conducted RNA-seq analysis with hypocotyls of 5d-old seedlings of the elh1 mutant and WT. We identified 1251 DEGs, of which 414 and 837 were up- and down-regulated, respectively in the mutant as compared with the WT. Go enrichment analysis showed these DEGs were mainly involved in tetrapyrrole binding, chlorophyll binding and photoreceptor activities in the molecular function category, chloroplast structure (chloroplast thylakoid membrane and chloroplast envelope) in the cellular component and tetrapyrrole metabolic, chloropyll biosynthesis and response to red or far-red light in the biological process category (Fig. S8). KEGG pathway enrichment analysis indicated that these DEGs were enriched in pathways involved of porphyrin and chlorophyll metabolism, photosynthesis, as well as phytohormone signal transduction (Fig. S9). Thus, RNA-seq data strongly supported that the CsHY2 involves in the tetrapyrrole chromophore biosynthesis through the porphyrin and chlorophyll metabolism pathway.

In the mutant and WT transcriptomes, all five phytochrome genes including CsPHYA1, CsPHYA2, CsPHYB, CsPHYC and CsPHYE were down-regulated, whereas many cell elongation related genes such as CsDWF4, CsEXT3, CsXTH22, CsEXPA8, CsXTR6 and CsIAA29 were up-regulated in the mutant. The expression trend of these 11 genes in RNA-seq analysis was consistent with the RT-qPCR results (Figs 6 and 7) further supporting their important roles in CsHY2-regulated hypocotyl elongation in cucumber.

Subcellular localization of CsHY2 protein

The subcellular location of a protein is pivotal to understand its function and associated biological processes (Briesemeister et al. 2010). We performed subcellular localization of the CsHY2 protein in tobacco leaves cells. We found that, while the negative control (EGFP fluorescence) was present throughout the cytomembrane and nucleus, the fusion protein 35s:CsHY2-EGFP was localized to chloroplast (Fig. S10). These findings confirmed that the CsHY2 protein played a role in the chloroplast, which was consistent with the plastid localization of PΦB synthesis and the RNA-seq data suggesting that tetrapyrrole chromophore biosynthesis and chlorophyll metabolism were mainly in the chloroplast.

In this report, we cloned elh1 by fine mapping for the elh1 mutant and provided evidence to support CsHY2 as the candidate gene which encodes the PΦB synthase known to function in chromophore biosynthesis. First, like Arabidopsis hy2 mutant, the cucumber elh1 mutant is deficient in phytochrome responses such as hypocotyl elongation under R and FR (Figs. 2A, 2B). Second, in the 20.9 kb region harboring the elh1 locus, there was only one non-synonymous SNP between the mutant and WT, which was located in the third exon of CsHY2 resulting in an amino acid substitution from Asp¹⁰⁴ to Asn¹⁰⁴ (Fig. 3C-D; Supplemental file 1-3). Third, this SNP-derived dCAPS marker (dCAPS1238-1) showed complete co-segregation with the long hypocotyl mutant phenotype in two large F₂ segregating populations (Figs. 3B, S5B). Allelic analysis among 515 cucumber lines revealed the unique A allele in elh1 (Fig. S6). Further, ectopic expression of CsHY2 in Arabidopsis can rescue the phenotype of hy2 mutant phenotype (short hypocotyl) in transgenic plants (Fig. 5). Finally, the expression pattern of CsHY2 well matches its role in hypocotyl elongation in elh1 mutant (Figs. 4A, S3). Taken together, these results provided convincing evidence in support of the CsHY2 as the best candidate gene of elh1.

The PΦB synthase/HY2 is characterized by a conserved Fe_bilin_red (FBR) domain with catalytic activity (Frankenberg et al. 2001; Rockwell and Lagarias. 2017; Zhang et al. 2019). The FBR domain is crucial for catalytic performance, and mutations in the domain would lead to loss-of-function of HY2 and thus severely deficient in photoreversible phys (Terry and Kendrick. 1996; Kohchi et al. 2001; Sawers et al. 2002; Sawers et al. 2004; Muramoto et al. 2005; Wu et al. 2020; Reig-Valiente et al. 2020). For example, all reported mutants with mutations in the FBR domain, such as Arabidopsis hy2-1, hy2-104, hy2-101, and hy2-103 (Kohchi et al. 2001), tomato au, au^ls and au^w (Terry and Kendrick. 1996; Muramoto et al. 2005; Wu et al. 2020), maize elm1, and rice se13 (Sawers et al. 2002; Sawers et al. 2004; Saito et al. 2011; Reig-Valiente et al. 2020), display common defective phenotypes with long hypocotyl and yellow green leaves as in the cucumber elh1 mutant identified herein that also carried an amino acid substitution (D104N) in the FBR domain (Figs. 1A-1D, 3D, S1; Supplemental file 3). These data further support the cucumber CsHY2 with conserved function as other plant species for hypocotyl growth.

However, unlike most hy2 mutants with early flowering phenotype in other species such as maize elm1 and rice se13 (Sawers et al. 2002; Saito et al. 2011; Reig-Valiente et al. 2020), mature elh1 plants had normal flowering time under field condition (data not shown). This might be due to the relatively small contributions of phys on the control of flowering time in cucumber plants. Instead, unexpectedly, we found that phys might be involved in the regulation of sex determination in cucumber (Figs. S2A, S2B). CCMC is monoecious with both separate male and female flowers on the same plant. We found that elh1 mutant had fewer male and more female flowers compared with the WT plants (Figs. S2A, S2B) which may suggest involvement of phys in regulating cucumber sexual development. The basis for such phenomenon is unknown, but one possibility concerns the genotype used in this study, CCMC, which like most of cucumber germplasms (Lai et al. 2018), is sensitive to seasonal or photoperiodic change in sex expression (i.e.different female/male flower ratios). Interestingly, in this study, we found the elh1 mutant has higher female/male flower ratios than WT either the summer (Fig. S2E) or autumn (Fig. S2F) under the field condition. It has been verified that phyB can regulates gynoecium formation in Arabidopsis (Foreman et al. 2011). Based on the role of phys in regulating flower development, together with the phenomenon of the photoperiodic response of sex type in cucumber, we speculated that phys might involve with the photoperiod-mediated sex determination in cucumber. More evidence is still needed to elucidate this.

It is worth mentioning that despite of the long hypocotyl and elongated internodes in the elh1 mutant, the plant height was normal due to the reduced internode number under field condition (Fig. S2), indicating that phys participate in the control of internode number in cucumber. The internode number, little mentioned in chomophore deficient mutants in other species, is an important determining factor for cucumber plant height. Although it is unknown how phys regulate node number of cucumber, the result presented here might extend our understanding in stem development and plant height of cucumber.

Consistently with the reduced chlorophyll (Chl) levels (Fig. S4), the transcriptome analysis revealed that porphyrin and chlorophyll metabolism was profusely altered in elh1 (Figs. S7, S8). It has been proposed that the PΦB and chlorophyll were synthesized from the common precursor-5-aminolevulinic acid (ALA) (Li et al. 2011). Meanwhile, previous studies have shown the Chl biosynthesis was feedback-inhibited by the branch of the tetrapyrrole biosynthetic pathway in phytochrome chromophore-deficient mutants (Terry and Kendrick. 1999). Therefore, this is not surprising that the role of CsHY2 in the synthesis of PΦB and Chl could explain the yellow green phenotype (Figs. 1C, 1D) in the elh1 plants, a phenotypic trait observed in most chromophore-deficient mutants (Weller et al. 1997; Kraepiel et al. 1994; Kohchi et al. 2001; Sawers et al.2002; Sawers et al. 2004; Muramoto et al. 2005; Zhu et al. 2017; Wu et al. 2020).

Here, similar to hy2 mutants in Arabidopsis (Derbyshire et al. 2007; Boron and Vissenberg. 2014), the long hypocotyl phenotype of elh1 mutant is due primarily to increased cell length rather than cell numbers (Figs. 1E-1H). This is consistent with the by time-course expression patterns of six cell-elongation-related genes from both qPCR (Fig. 7) and transcriptome data from RNA-Seq analysis where all these genes were significantly up-regulated in the mutant (Table S6).

The mechanism underlying hypocotyl elongation also involves endogenous phytohormones, especially the gibberellin (GA) and auxin which are known to be involved in promoting cell elongation in excised stem and hypocotyl segments (Qu et al. 2017; Du et al. 2018; Liu et al.2018; Bawa et al. 2020; Jiang et al. 2020). In this study, transcriptome analysis showed that the CsKAO and CsGA20ox2, two key genes responsible for bioactive GA biosynthesis, were dramatically up-regulated in the mutant elh1 (Table S6). On the contrary, CsGA2ox-1 encoding GA-inactivating enzyme (Serrani et al. 2007), and CsGAI functioning signal transduction repressors of GA (Eckardt. 2007), were down-regulated in the mutant (Table S6). Meanwhile, some auxin responsive (IAA4, IAA14, IAA29, IAA32, SAUR23, SAUR32) and induced (AUX22, AUX28,) genes were also up-regulated. These results suggested that GA and auxin might regulate photomorphogenesis-related hypocotyl elongation in cucumber through modulating the biosynthesis or signaling of auxin and GA.

In Arabidopsis, it has been well documented that a number of transcription factors (TFs) promoting photomorphogenesis, such as PIFs (phytochrome-interacting factors), HY5 (elongated hypocotyl 5), HFR1 (long hypocotyl in far-red), LAF1 (long after far-red light 1) and HYH (hy5 homolog), can physically interact with phytochromes to regulate hypocotyl growth (Castillon et al. 2007; Jang et al. 2013; Dong et al. 2020). But none of the TFs was present in the DEG list of the mutant and WT transcriptomes. Meanwhile, many other TFs like members of TCP, AFR, ERF and WRKY gene families, were up-regulated in the mutant (Table S6); some of them have been shown to be involve in regulation of the hypocotyl elongation. For example, Zhou et al (2018) reported overexpression of TCP17 leads to a long hypocotyl phenotype in Arabidopsis. TCP14 and TCP15 are required for optimal elongation and for the appropriate gene expression responses to a group of auxin inducible genes and the GA biosynthetic gene GA20ox1 in hypocotyls of Arabidopsis (Ferrero et al. 2019; Ferrero et al. 2021). Based on the transcriptome data, combined the knowledge from scientific literature, we speculate that the hypocotyl elongation in elh1 is mainly due to the interaction and cooperative regulation of phys, GA/Auxin, cell cell-elongation-related genes and these differential TFs. However, whether and how they make interactions and crosstalk to control hypocotyl elongation remains to be investigated in the future.

Author Contribution Statement

LH performed the research, prepared a draft of the manuscript. PL, ZJ and JS participated in the research. PC participated in data analysis and provided technical help. YW participated in data analysis and manuscript writing. AW and YL designed the experiments, supervised this study and wrote the manuscript. All authors have read and approved the manuscript.

Acknowledgments

Work in AW’s lab was supported by the National Key Research and Development Program of China (2016YFD0101900).Work in PC’s and YL’slab were supported by the National Natural Science Foundation of China under Project #31860557 and 31772300, respectively. YW’s lab was supported by the Agriculture and Food Research Initiative competitive grant no. 2017-67013-26195 of the USDA National Institute of Food and Agriculture.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Bawa G, Feng LY, Chen GP, Chen H, Hu Y et al. (2020) Gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature interaction. Physiol Plant 170:345-356.

Bo KL, Wang H, Pan YP, Behera TK, Pandey S, Wen CL, Wang YH, Simon PW, Li YH, Chen JF, Weng YQ (2016) SHORT HYPOCOTYL1 encodes a SMARCA3-like chromatin remodeling factor regulating elongation. Plant Physiol 172(2):1273-1292.

Boron AK, Vissenberg K (2014) The Arabidopsis thaliana hypocotyl, a model to identify and study control mechanisms of cellular expansion. Plant Cell Rep 33: 697–706.

Briesemeister S, Rahnenführer J, Kohlbacher O (2010) Going from where to why-interpretable prediction of protein subcellular localization. Bioinformatics 26:1232-1238.

Castillon A, Shen H, Huq E (2007) Phytochrome interacting factors: central players in phytochrome-mediated light signaling networks. Trends Plant Sci 12:514-521.

Cavagnaro PF, Senalik DA, Yang LM, Simon PW, Harkins TT, Kodira CD, Huang SW, Weng Y (2010) Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.) BMC Genom 11:569.

Chen FF, Fu BB, Pan YP, Zhang CW, Wen HF, Weng YQ, Chen P, Li YH (2017) Fine mapping identifies CsGCN5 encoding a histone acetyltransferase as putative candidate gene for tendril-less1 mutation (td-1) in cucumber. Theor Appl Genet 130:1549-1558.

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-743.

Derbyshire P, McCann MC, Roberts K (2007) Restricted cell elongation in Arabidopsis hypocotyls is associated with a reduced average pectin esterification level. BMC Plant Biology 7:31.

Dong J, Chen HD, Deng XW, Irish VF, Wei N (2020) Phytochrome B induces intro retention and translational inhibition of PHYTOCHROME-INTERACTING FACTOR3. Plant Physiol 182:159-166.

Du JB, Jiang HK, Sun X, Li Y, Liu Y, Sun MY, Fan Z et al. (2018) Auxin and Gibberellins are required for the receptor-like kinase ERECTA regulated hypocotyl elongation in shade avoidance in Arabidopsis. Front Plant Sci 9:124.

Eckardt NA (2007) GA perception and signal transduction: molecular interactions of the GA receptor GID1 with GA and the DELLA protein SLR1 in rice. Plant Cell 19:2095-2097.

Fantini E, Sulli M, Zhang L, Aprea G, Jiménez-Gómez JM, Bendahmane A, Perrotta G, Giuliano G, Facella P (2019) Pivotal roles of cryptochromes 1a and 2 in tomato development and physiology. Plant Physiol 179:732-748.

Ferrero LV, Viola IL Ariel FD, Gonzalez DH (2019) Class I TCP transcription factors target the gibberellin biosynthesis gene GA20ox1 and the growth-promoting genes HBI1 and PRE6 during thermomorphogenic growth in Arabidopsis. Plant Cell Physiol 60:1633-1645.

Ferrero LV, Gastaldi V, Ariel FD, Viola IL, Gonzalez DH (2021) Class I TCP proteins TCP14 and TCP15 are required for elongation and gene expression responses to auxin. Plant Mol Biol 105:147-159.

Foreman J, White JN, Graham IA, Halliday KJ, Josse EM (2011) Shedding light on flower development: Phytochrome B regulates gynoecium formation in association with the transcription factor SPATULA. Plant Signal Behav 6:471-476.

Frankenberg N, Mukougawa K, Kohchi T, Lagarias JC (2001) Functioal genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms. Plant Cell 13:965-978.

Huai JL, Jing YJ, Lin RC (2020) Functional analysis of ZmCOP1 and ZmHY5 reveals conserved light signaling mechanism in maize and Arabidopsis. Physiol Plant 169:369-379.

Izawa T, Oikawa T, Tokutomi S, Okuno K, Shimamoto K (2000) Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant). Plant J 22:391-399.

Jang IC, Henriques R, Chua NH (2013) Three transcription factors, HFR1, LAF1 and HY5, regulate largely independent signaling pathways downstream of phytochrome A. Plant Cell Physiol 54:907-916.

Jiang HK, Shui ZW, Xu L, Yang YH, Li Y et al. (2020) Gibberellins modulate shade-induced soybean hypocotyl elongation downstream of the mutual promotion of auxin and brassinosteroids. Plant Physiol Bioch 150:209-221.

Josse EM, Halliday KJ (2008) Skotomorphogenesis: the dark side of light signalling. Curr Biol 18:R1144-R1146.

Koornneef M, van der Knaap BJ (1983) A second long hypocotyl mutant at the lh Locus. Cucurbit Genet Coop Rpt 6:13.

Kahle N, Sheerin DJ, Fischbach P, Koch LA, Schwenk P, Lambert D, Rodriguez R, Kerner K, Hoecker U, Zurbriggen MD, Hiltbrunner A (2020) COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling. Plant J 104:1038-1053.

Kohchi T, Mukougawa K, Frankenberg N, Masuda M, Yokota A, Lagarias J (2001) The arabidopsis HY2 gene encodes phytochromobilin synthase, a ferredoxin-dependent biliverdin reductase. Plant Cell 13: 425-436.

Kraepiel Y, Jullien M, Cordonnier-Pratt MM, Pratt L (1994) Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant. Mol Gen Genet 242:559-565.

López-Juez E, Nagatani A, Tomizawa KI, Deak M, Kern R, Kendrick RE, Furuya M (1992) The cucumber long hypocotyl mutant lacks a light-stable PHYB-like phytochrome. Plant Cell 4:241-251.

Lai YS, Shen D, Zhang W, Zhang XH, Qiu Y, Wang HP et al. (2018) Temperature and photoperiod changes affect cucumber sex expression by different epigenetic regulations. BMC Plant Biol 18:268.

Li XY (2008) Infiltration of Nicotiana benthamiana protocol for transient expression via Agrobacterium. Bio-Protocols 1:1-3.

Li JG, Li G, Wang HY, Deng XW (2011) Phytochrome signaling mechanisms. Arabidopsis Book 9:e0148.

Li YH, W CL, W YQ (2013) Fine mapping of the pleiotropic locus B for black spine and orange mature fruit color in cucumber identifies a 50 kb region containing a R2R3-MYB transcription factor. Theor Appl Genet 126:2187-2196.

Lin YJ, Chen YC, Tseng KC, Chang WC, Ko SS (2019) Phototropins mediate chloroplast movement in phalaenopsis aphrodite (Moth Orchid). Plant Cell Physiol 60:2243-2254.

Linley PJ, Landsberger M, Kohchi T, Cooper JB, Terry MJ (2006) The molecular basis of heme oxyngenase deficiency in the pcd1 mutant of pea. FEBS J 273:2594-2606.

Liu K, Li YH, Chen XN, Li LJ, Liu K, Zhao HP, Wang YD, Han SC (2018) ERF72 interacts with ARF6 and BZR1 to regulate hypocotyl elongation in Arabidopsis. J Exp Bot 69:3933-3947.

Liu B, Long H, Yan J, Ye LL, Zhang Q, Chen HM et al. (2021) A HY5-COL3-COL13 regulatory chain for controlling hypocotyl elongation in Arabidopsis. Plant Cell Environ 44:130-142.

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods 25:402-408.

Ming CH, Jiang FL, Hu HM, Zhou XC, Zhan FH, Wu Z (2011) Effects of different leggy extent seedling on cucumber growth, yield and quality. China Vegetables 4:29-34. (In Chinese)

Montgomery BL (2008) Right place, right time: Spatiotemporal light regulation of plant growth and development. Plant Signal Behav 3:1053-1060.

Montgomery BL (2009) Spatial-specific phytochrome responses during de-etiolation in Arabidopsis thaliana. Plant Signal Behav 4:47-49.

Muramoto T, Kami C, Kataoka H, Iwata N, Linley PJ, Mukougawa K, Yokota A, Kohchi T (2005) The tomato photomorphogenetic mutant, aurea, is defect in phytochromobilin synthase for phytochrome chromophore biosynthesis. Plant Cell Physiol 46:661-665.

Oh J, Park E, Song K, Bae G, Choi G (2020) Phytochrome interacting factor8 inhibits phytochrome a-mediated far-red light responses in Arabidopsis. The Plant Cell 32:186-205.

Parks BM, Quail PH (1991) Phytochrome-defect hy1 and hy2 long hypocotyl mutants of Arabidopsis are defective in phytochrome chromophore biosynthesis. Plant Cell 3:1177-1186.

Pham VN, Kathare PK, Huq E (2018) Phytochromes and phytochrome interacting factors. Plant Physiol 176:1025-1038.

Qi JJ, Liu X, Shen D, Miao H, Xie BY, Li XX, Zeng P et al (2013) A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nat Genet 45(12):1510-1515.

Qu XY, Zhao Z, Tian ZX (2017) ERECTA regulates cell elongation by activating auxin biosynthesis in Arabidopsis thaliana. Front Plant Sci 8:1688.

Quail PH (2002) Phytochrome photosensory signalling networks. Nat Rev Mol Cell Biol 3:85-93.

Ren Y, Zhang Z, Liu J, Staub JE, Han Y, Cheng Z, Li X et al (2009) An integrated genetic and cytogenetic map of the cucumber genome. PLoS ONE 4:e5795.

Reig-valiente J, Borredá C, Talón M, Domingo C (2020) The G123 rice mutant, carrying a mutation in SE13, presents alterations in the expression patterns of photosynthetic and major flowering regulatory. PLoS One 15:e0233120.

Robinson RW, Shail JW (1981) A cucumber mutant with increased hypocotyl and internode length. Cucurbit Genet Coop Rpt 4:19-20.

Rockwell NC, Lagarias JC (2017) Ferredoxin-dependent bilin reductases in eukaryotic algae: Ubiquity and diversity. J Plant Physiol 217:57-67.

Saito H, Okumoto Y, Yoshitake Y, Inoue H, Yuan Q, Teraishi M, Tsukiyama T, Nishida H, Tanisaka T (2011) Complete loss of photoperiodic response in the rice mutant line X61 is caused by deficiency of phytochrome chromophore biosynthesis gene. Theor Appl Genet 122:109-118.

Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425.

Sawers RJ, Linley PJ, Farmer PR, Hanley NP, Costich DE, Terry MJ, Brutnell TP (2002) Elongated mesocotyl1, a phytochrome-defect mutant of maize. Plant Physiol 130: 155-163.

Sawers RJ, Linley PJ, Gutierrez-Marcos JF, Delli-Bovi T, Farmer PR, Kohchi T, Terry M, Brutnell TP (2004) The Elm1 (ZmHy2) gene of maize encodes a phytochromobilin synthase. Plant Physiol 136: 2771-2781.

Serrani JC, Sanjuán R, Ruiz-Rivero O, Fos M, García-Martínez JL (2007) Gibberellin regulation of fruit set and growth in tomato. Plant Physiol 145:246-257.

Seo DH, Yoon GM (2019) Light-induced stabilization of ACS contributes to hypocotyl elongation during the dark-to-light transition in Arabidopsis seedlings. Plant J 98:898-911.

Song JL, Cao K, Hao YW, Song SW, Wei S, Liu HC (2019) Hypocotyl elongation is regulated by supplemental blue and red light in cucumber seedling. Gene 707:117-125.

Song K, Choi G (2019) Phytochrome regulation of seed germination. Methods Mol Biol 2026:149-156.

Sztatelman O, Łabuz J, Hermanowicz P, Banaś AK, Banaś A, Zgłobicki P, Aggarwal C, Nadzieja M, Krzeszowiec W, Strzałka W, Gabryś H (2016) Fine tuning chloroplast movements through physical interactions between phototropins. J Exp Bot 67:4963-4978.

Tanaka R, Kobayashi K, Masuda T (2011) Tetrapyrrole metabolism in Arabidopsis thaliana. Arabidopsis Book 9:e0145.

Tavridou E, Pireyre M, Ulm R (2020) Degradation of the transcription factors PIF4 and PIF5 under UV-B promotes UVR8-mediated inhibition of hypocotyl growth in Arabidopsis. Plant J 101:507-517.

Terry MJ, Kendrick RE (1996) The aurea and yellow-green-2 mutants of tomato are defect in phytochrome chromophore synthesis. J Biol Chem 271: 21681-21686.

Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257-261.

Wang H, Li WQ, Qin YG, Pan YP, Wang XF, Weng YQ, Chen P, Li YH (2017) The cytochrome P450 gene CsCYP85A1 is a putative candidate for super compact-1 (Scp-1) plant architecture mutation in cucumber (Cucumis sativus L.) Front Plant Sci 8:266.

Weller JL, Terry MJ, Reid JB, Kendrick RE (1997) The phytochrome-defect pcd2 mutant of pea is unable to convert biliverdin IXα to 3 (Z)-phytochromobilin. Plant J 11: 1177-1186.

Wu JQ, Cheng J, Xu CM, Qi SL, Sun WR, Wu S (2020) AUREA maintains the balance between chlorophyll synthesis and adventitious root formation in tomato. Hortic Res 7:166.

Yang Y, Zhang LB, Chen P, Liang T, Li X, Liu HT (2020) UV-B photoreceptor UVR8 interacts with MYB73/MYB77 to regulate auxin responses and lateral root development. EMBO J 39:e101928.

Zhao C, Mao K, You CX, Zhao XY, Wang SH, Li YY, Hao YJ (2016) Molecular cloning and functional analysis of a UV-B photoreceptor gene, MdUVR8 (UV Resistance Locus 8), from apple. Plant Sci 247:115-126.

Zhang WQ, Zhong H, Lu H, Zhang YX, Deng X et al. (2019) Characterization of Ferredoxin-dependent biliverdin reductase PCYA1 reveals the dual function in retrograde bilin biosynthesis and interaction with light-dependent protochlorophyllide oxidoreductase LPOR in Chlamydomonas reinhardtii. Front Plant Sci 9:676.

Zhou Y, Zhang DZ, An JX, Yin HJ, Fang S et al. (2018) TCP transcription factors regulate shade avoidance via directly mediating the expression of both phytochrome interacting factors and auxin biosynthetic genes. Plant Physio 176:1850-1861.

Zhu LX, Yang ZH, Zeng XH, Gao J, Liu J, Yi B et al. (2017) Heme oxygenase 1 defects lead to reduced chlorophyll in Brassica napus. Plant Mol Biol 93:579-592.

Table 1. Segregation analysis of elongated hypocotyl-1(elh1) in F₁ and F₂ populations from various crosses.

Populations	Total	Wild Type	Mutant	Expected ratio	P in χ2 tests
C1238 × CCMC F₁	63	63	0	1:0	/
C1238 × Gy14 F₁	72	72	0	1:0	/
C1238 × 9930 F₁	56	56	0	1:0	/
C1238 × 9930 F₂	1249	949	300	3:1	0.423
C1238 × Gy14 F₂	5317	4012	1305	3:1	0.442

SupplementalFiles.pdf
SupplementalTables.xlsx
Fig.S1.pptx
The mutant C1238 displays apparently elongated hypocotyl at the (a) cotyledon stage and (b) seedling stage.
Fig.S2.pptx
The mutant C1238 exhibits elongated internode at the adult stage under field conditions. As compared with the wild type CCMC (b), the mutant C1238 (a) exhibits elongated internode. (c) Internode number of the mutant C1238 and wild type CCMC. (d) Internode length of mutant C1238 and wild type CCMC. (e) The female/male flower ratios of mutant C1238 and wild type CCMC during the summer season. (f) The female/male flower ratios of mutant C1238 and wild type CCMC during the autumn season. Inset in A and B are the enlarged hypocotyl of mutant C1238 and wild type CCMC, respectively. Asterisks indicate statistically significant differences compared with the wild type at **p < 0.01 by Student’s t-test.
Fig.S3.pptx
Hypocotyl elongation dynamics of C1238 and CCMC under white light condition within 13d after germination. Seedling images under investigation at different time points are shown in (a), and hypocotyl length and elongation rate C1238 and CCMC are graphed in (b) and (c), respectively. Asterisks indicate statistically significant differences compared with the wild type at **p < 0.01 and *p < 0.05 by Student’s t-test.
Fig.S4.pptx
Chlorophyll contents of mutant C1238 and wild type CCMC at the cotyledon (a) and first true leaf stage (b). For chlorophyll content, data are mean ± SD (n = 3). Error bars represent SD of three independent replications.
Fig.S5.pptx
Genetic mapping of elh1 loci in cucumber. (a) Preliminary mapping with 96 F2 plants of C1238×9930 placed elh1 in Chromosome 1, which was narrowed down to a region of 537.9kb through fine mapping (b) with 1249 C1238×9930 F2 plants.
Fig.S6.pptx
dCAPS assay of 403 cucumber lines including C1238 with dCAPS marker dCAPS1238-1. Among all tested lines, only C1238 has the long hypocotyl phenotype. The marker patterns are completely consistent with the SNP alleles.
Fig.S7.pptx
Neighbor-joining phylogenetic tree of HY2 proteins of cucumber and 10 other species. The numbers represent the bootstrap values, which are shown next to the branching points.
Fig.S8.pptx
Gene Ontology (GO) terms (P<0.05) significantly enriched in the up-regulated genes between the hypocotyl of elongated hypocotyl-1 (elh1) and WT CCMC. Go terms belong to molecular function (MF), cellular component (CC) and biological process (BP) are shown in red, blue and purple, respectively. Go terms are sorted based on p-values.
Fig.S9.pptx
Scatterplot of top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Rich factor represents the ratio of the number of DEGs and the number of all genes in the pathways. The larger a bubble is, the more DEGs it contains. The smaller of the enrichment P value, the more significant it is.
Fig.S10.pptx
Subcellular localization of the CsHY2 protein in epidermal cells of Nicotiana benthamiana leaves. Scale bar=100um.

Download PDF

First submitted to journal
21 Jan, 2021

You are reading this latest preprint version

A Mutation in CsHY2 Encoding a Phytochromobilin (PΦB) Synthase Leads to an Elongated Hypocotyl 1(elh1) Phenotype in Cucumber (Cucumis sativus L.)

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Tables

Supplementary Files

Status:

Version 1