Short internode in the mutant is due to less cell numbers longitudinally
WI7201 displays compact phenotype with significantly reduced internode length with the petiole, flowers and tendrils all clustered on the main stem (Li et al.2011; Fig. 1A). When the cotyledons were fully expanded, the hypocotyl of compact plants (~ 2.44 cm) was significantly shorter than that of WT (~ 3.76 cm). The pedicel of male and female flowers (Figs. 1A1 and A2), and seed size (Fig. S1) of the mutant was also shorter or smaller than the WT. At adult stage, the difference of plant height between WT and mutant was even more obvious since the mutant had extremely short internode but the fruit set of WI 7201 was not affected (Figs. 1A3 -A5).
We counted cell numbers and measured cell size of WT and cp mutant stem by histological analysis (Figs. 1C-E). Longitudinally, the most notable difference was that WT had more cortex cells than the mutant while the average cell size was only ~ 50% that of the mutant. Given the fact that WT was much taller with more cells per unit area than the mutant, the much-reduced internode length must be due to reduced longitudinal cell proliferation. The larger cortex cells in the mutant were probably the result of lower division rate which potentially provided some compensation of cell elongation because of longer cell cycle.
Fine mapping placed the cp locus into 8.8-kb region harboring only one gene
Using segregating populations derived from WI7200 (CpCp) × WI7201 (cpcp), we previously mapped the cp locus into a 178-kb region on Chr4 (Li et al. 2011; Yong et al. 2013). Due to severe segregation distortion of markers in this region in this population, we developed a new F2population from the cross between WI7201 and WI7123 (WT) for fine mapping and cloning of the cp locus. The F1 plants had the same vine length as WI7123. Among 9545 F2 plants, 7302 were WT and 2243 were mutant, which was consistent with the expected 3:1 ratio (P = 0.026 in χ2 test). We genotyped the 2,243 mutant F2 plants with UW084680 and UW084870 that were flanking the cp locus, which were physically 340 kb apart (Gy14v2.0; Fig. 2A). Sixty recombinants were identified between the two markers. Based on resequencing reads alignment of the two parental lines against the Gy14v2.0 draft genome, 10 new polymorphic markers located in this region were developed to genotype the 60 recombinants. Combined genotypic and phenotypic data of these recombinants allowed to delimit the cp locus into a 13.2kb region flanked by markers NWSTS009 and NWSTS014-indel while NWSTS012-dCAPS was co-segregating with the cp locus (Fig. 2B). Since there were still 6 recombinants between the two flanking markers, 4 new CAPS/dCAPS markers were developed which further narrowed down the cp locus into an 8.8-kb region defined by markers NWSTS30-dCAPS and NWSTS035-CAPS. Interestingly, NWSTS035-CAPS was located inside the cp candidate gene indicating intragenic recombination (Fig. 2C).
The LRR receptor-like serine/threonine-protein kinase gene (CsERECTA) is a candidate for cp
In the Gy14v2.0 genome, only one gene, CsGy4G024070 was annotated in this 8.8kb region (Fig. 2c). It was predicted to encode a leucine-rich repeat (LRR) receptor-like serine/threonine-protein kinase. CsGy4G024070 has the highest homology to the Arabidopsis ERECTA (ER) gene (AT2G26330). As such, the cp candidate gene will be referred to CsERECTA (CsER) hereinafter. CsER was predicted to have 27 exons (Fig. 2D; Fig. S2A). We cloned the full-length CDS of CsER from WI7200 (WT), WI7201 (cpcp) and WI7123 (WT). Sanger sequencing indicated that CsER from the two WT lines had the same length (2976 bp), but it was 72-bp shorter in the mutant (2904-bp) (Fig. 2E). Alignment of the CDS between WT and cp indicated that the 22nd exon was missing in the mutant. Besides, there were 15 SNPs inside CsER among the three lines, only three of which were unique but synonymous in the mutant compared to the two WT lines (Fig. S2B) suggesting that the deletion of the 22nd exon in the mutant is the causal polymorphism for mutant phenotype. The DNA and deduced amino acid sequences of CsER have been deposited in the GenBank (accession numbers: MT505429-MT505433). The alignment of CDS and amino acid sequence between WI7200 and WI7201 are in Figs. S3 and S4, respectively.
Loss of the 22nd exon in the mutant is due to an LTR retrotransposon insertion
To find the reason why there was a 72-bp deletion in the mutant cDNA, we tried to clone the CsER genomic DNA (gDNA) sequences from the two WT and cp plants. Cloning from the two WT resulted in the expected sequences, which, however, was unsuccessful in the mutant. We thus designed different combinations of PCR primers to clone CsER gDNA from cp plants. All PCR amplifications were successful expect for the one using the primer pair targeting the 22nd exon. We performed genome walking from one side toward the unknown region, which revealed a 5548-bp insertion in the 22nd exon of CsER in the mutant. Annotation of this 5548-bp insertion suggested that it belongs to a Copia-type long terminal repeat-retrotransposon (LTR-RT) (Wicker et al. 2007) that had 5 typical conserved domains: RNase_HI_RT_Ty1, RVT_2, rve, gag_pre-integrs and Retrotran_gag 2 and 3 super families (Fig. S2A). The sequence “GTATCT” in CsER was the target site duplication (TSD) and the LTR-RT was located between the two duplicates.
To check if this LTR-RT insertion in the CsER gene also occurred in natural cucumber populations, two pairs of primers targeting the insertion sequences were designed (Table S1). The forward primer of “cp-locus” primer pair was within the LTR-RT and the reverse primer in the 23rd exon. Primer pair “cp-CK” was used to amplify a band as the positive control. The cp-CK band was 889 bp and the amplicon of the “cp-locus” was 633 bp in length. Thus, any cucumber lines carrying this insertion would have two PCR bands in agarose gel electrophoresis while the WT will only have the cp-CK band (Fig. S5). The two markers were used to genotype 300 cucumber accessions (Bo et al. 2016) with the two compact lines, PI 308915 (WI7201), PI 308916 (WI7202), and WI7123 × WI7201 F1 as the controls. Only the three controls amplified the LTR-RT-specific band and the rest only had cp-CK bands (Fig. S5). In addition to Gy14v2.0 and 9930v3.0, draft genomes of 10 other cucumber lines are also publicly available (https://www.cucurbitgenomics.org/). We examined the promoter, gene body and 3’ UTR sequences of CsER from these 10 genome assemblies. None of the 10 lines (all WT at CP locus) carried the LTR-RT insertion. These observations further confirmed that the LTR-RT insertion was responsible for the compact vining type.
In addition to the missing 24 amino acids in the cp mutant, the alignment of the WT and mutant protein sequences suggested that the 72bp deletion also resulted in an amino acid change because of the open reading frame (ORF) shift (Fig. 2E). The deletion of the amino acids did not seem to affect its conserved domains such as the leucine rich repeat (LRR) and the protein kinase domain (Fig. S2C). It is well known that ERECTA is a transmembrane protein with extra-cellular receptor domain and intra-cellular protein kinase domain (Torii et al. 1996; Shpak et al. 2003). We predicted the topological structure of WT and mutant proteins using Protter and Phobius (Käll et al. 2007; Omasits et al. 2013). It was interesting to see that the WT protein was predicted to have typical uni-membrane structure to allocate the extra- and intra-cellular parts, while the mutant protein had two transmembrane structures to locate the kinase domain from intra-cellular to extra-cellular (Fig. S6). This suggests that cp may be a loss-of-function mutation of CsER.
To characterize CsER as a typical LRR receptor-like serine/threonine-protein kinase, subcellular localization of CsER was carried out by co-infiltration of A. tumefaciens strain GV3101 carrying 35S::gCsER::eGFP plasmid into tobacco (Nicotiana benthamiana) leaves. Since the introns in CsER gDNA are essential for stabilizing its expression (Karve et al. 2011), the construct contained the whole length gDNA from the start codon to the stop codon (but without the stop codon) including all the introns. The 35S::gCsER-eGFP protein was found to be located on the plasma membrane (Fig. S7), which is consistent with its predicted function of transporting extracellular signals into cell.
ER seems to have conserved functions in plant development
The LRR receptor-like kinase proteins are a large family that have been reported to regulate cell division and differentiation, in which the proteins with similar structures often have redundant or overlapping functions (Shpak et al. 2004; Hord et al. 2006; Chen et al. 2013). We conducted phylogenetic analysis of ER proteins, and other structurally or functionally similar proteins with ER including ER-like (ERLs), CLAVATA1, and BAMs from nine plant species (Table S2). The resulting phylogram is presented in supplemental Fig. S8. ER proteins are highly conserved and are clustered among the selected species despite ERL proteins are the closest members of ER protein family, implying that ER protein may remain its unique function during evolution. In Arabidopsis, the ER protein family has three members: AtER, AtERL1 and AtERL2, while there were two annotated in cucumber so far: CsER and CsERL1. To find more potential ER family proteins in cucumber, we used the protein sequence of CsER to do BLASTP against the 9930v3.0 genome. Based on the similarity of protein sequences, CsaV3_6G044540 was the closest protein to CsER and CsERL1, which, however, belonged to the CLAVATA protein family. This suggested that there are only two members in the CsER protein family. Recently, CsCLAVATA1encoding an LRR receptor-like kinase protein was suggested as a putative candidate gene for a dwarf mutation Csdw in cucumber (Xu et al. 2018), which is homologous to BAM proteins in Arabidopsis. Interestingly, although the authors did not mention that loss-of-function of AtBAM proteins resulting in dwarf plant, the bam1bam2 double mutant and the bam1bam2bam3 triple mutant were significantly shorter than the wild-type Ler-1 (DeYoung et al. 2006). These results suggest that LRR receptor-like kinase family genes may perform similar functions that are important to plant growth especially stem elongation.
CsER is predominantly expressed in shoot apical meristem (SAM)
We compared spatial-temporal expression of CsER in WT and the mutant. We first performed qPCR in different organs (cotyledon, hypocotyl, true leaf, main stem) and SAM (from growing tips) at 7 and 21 days after germination (DAG) (Fig. 3A). At 7 DAG, the expression of CsER in SAM was slightly higher than that in the cotyledon or hypocotyl. At 21 DAG, CsER expression exhibited a significant increase in the SAM. Using the expression level in the cotyledon at 7 DAG as the reference, CsER expression in SAM at 21 DAG was the highest among all the organs examined, which was 66 folds higher compared to the reference. However, there was no difference in the expression level CsER in any organs examined between WT and mutant. mRNA in situ hybridization showed that transcripts of CsER strongly and peripherally accumulated in young organs such as leaf primordia, floral buds and especially in SAM (Fig. 3B). These observations suggest that CsER plays a very important role in development of meristematic tissue, which is critical for morphogenesis of aerial organs. To confirm whether the CsER protein level is different in SAM between WT and mutant, we conducted western blotting to detect abundance of CsER protein in SAM of the WT and mutant. The result revealed the accumulation of CsER protein in the mutant was much weaker than that in the WT (Fig. 3C).
To further characterize the expression pattern of CsER, we perform GUS assay using CsER promoter-driven GUS gene in Arabidopsis. We cloned the promoter (-2053 to -243 bp upstream the start codon) of CsER from WT plants and inserted it to pCambia1391Z to express a GUS reporter gene. To visualize promoter activities, whole transgenic Arabidopsis plants carrying PROERECTA::GUS were stained at cotyledon (Fig. 3D1), 4-true-leaf leaf (Fig. 3D2), and inflorescence (Fig. 3D3) stages. The GUS gene was expressed at leaf veins, stomata (Fig. 3D4-5), stem, flower, and most remarkably at SAM at all the three stages. In general, the GUS activity at the cotyledon stage was weaker than that of two later stages. Taken together, these data confirmed that CsER may predominantly control SAM development, where stem cells were proliferated and differentiated.
Ectopic expression CsER in Arabidopsis rescues er105 dwarf phenotype
The Arabidopsis er105 mutant carries loss-of-function mutations in the AtER gene (AT2G26330, homolog of CsER), which exhibits reduced plant height and pedicel, smaller rosette radius and more compact inflorescence than the WT (Col background) (https://www.arabidopsis.org/). Since the cucumber cp mutant exhibits stronger compact phenotype than er105, we asked if CsER and AtER perform similar functions, and WT CsER could rescue the er105 phenotype. Two gDNA sequences of CsER with 9812 bp and 8940 bp were ligated into the binary vector pCambia3301, respectively. One (PROERECTA-CsER) included the sequence from the 2053-bp promoter to the 896-bp of 3’UTR, and the second (PROERECTA-CsERf) was fused with a flag tag but without the native 3’UTR. The two constructs were transformed into Arabidopsis Col-0 (WT) and er105, respectively. Two representative lines (L1 and L2) were phenotypically characterized. CsERer105 (L2) could completely rescue the plant height of er105 (the same as Col-0), while that of CsERer105 (L1) and CsERfer105 (L1 and L2) was only partially recovered (Fig. 4A-B). We examined the expression of both endogenous AtER and exogenous CsER genes in non-transgenic and transgenic Arabidopsis (Fig. 4C). The plant height and expression level among these plants showed a positive correlation. No significant difference was observed between non-transgenic Col-0 and transgenic Col-0. However, the total expression of ER (AtER + CsER) was the highest in transgenic Col-0 plants (Fig. 4C). This might imply that there is an expression cap of ER, beyond which could not increase the effect of ER. CsER could also partially rescue the smaller rosette radius and increase the length of pedicels (Fig. S9) in er105. In addition, the PROERECTA-CsER and PROERECTA-CsERf transgenic plants did not show any significant differences (Fig. 4A-C) which may suggest that the effect of CsER protein is not affected by the fused flag tag. We also overexpressed CsER in er105 and Col-0 using constructs driving by the 35S promoter. The resulting transgenic plants had a similar phenotype of the CsER plants driven by CsER promoter (Fig. S10). Given the fact that the CsER promoter shows strong activity in almost all organs except in the root, it may have a great potential to be used for driving other genes regulating above ground organs in cucumber instead of using the exogenous 35S promoter.
Mutated Cser does not affect homo-dimerization of ER proteins
Like most mammalian receptor kinases, an ER protein forms dimers to perform its functions (Lee et al. 2012), we asked if the mutation in CsER would impact the self-association of CsER protein. The full length CDS of CsER from WT and WI 7201 was cloned into BiFC vectors to test the self-interaction in tobacco leaves. We found that CsER protein could associate with itself on cytomembrane. The mutated CsER protein from WI 7201 did not seem to affect such self-association with both WT CsER and mutant CsER proteins, though (Fig. 5).
Transcriptome profiling reveals hormone biosynthesis/signaling and photosynthesis pathways associated with CsER-dependent regulatory network
Since CsER showed the highest expression in SAM, to explore the CsER involved regulatory network in cucumber plant development, we performed BSR-Seq with pooled SAM tissues for the WT (CpCp) and mutant (cpcp) F2 plants. The complete transcriptome datasets have been deposited into NCBI under BioProject accession PRJNA858107. Differentially expressed genes (DEGs) in the mutant vs WT were identified using P value ≤ 0.05 and | log2 (Fold Change) | ≥ 0.5 as the cutoffs. Among 1796 DEGs detected, 1042 and 754 were up- and down-regulated in the mutant, respectively (Table S3). Nearly 11% (193 of 1796) of these DEGs were transcription factors which are known to play important roles in plant growth and development (Fig. 6A; Table S3). KEGG enrichment analysis revealed that the DEGs for basic and secondary metabolic pathways were the most enriched (Fig. 6B). In addition, GO enrichment analysis also identified highly enriched biological processes (response to stimulus, hormone-related pathways, photosynthesis pathways), cellular components (nucleus, cell periphery, cell membrane, extra-cellular region and photosystem), and molecular functions (DNA binding, transporter activity, transcription regulators chlorophyll binding) in the mutant as compared with the WT (Fig. S11). Many DEGs involved in major hormone biosynthesis/signaling pathways such as auxin/IAA, ethylene (ET), cytokinin (CK), BR, and GA. The DEGs for the auxin/IAA, ET and CK biosynthesis/signaling were particularly enriched. Cyclin-dependent kinase genes that controlling cell cycle transition and progression were also down-regulated and enriched. Some representative DEGs are illustrated in Fig. S12.
Since RNA-seq clearly indicated involvement of major phytohormone biosynthesis/signaling pathways by CsER, we investigated effects of exogeneous application of phytohormones on plant growth in both WT and mutant plants. Both plants were treated with IAA, 6-BA, GA and BR. While each hormone could significantly increase plant height in WT, such effect was not found in the mutant (Fig. S13) suggesting that the signaling transduction of these hormones may be impaired in the mutant. We also observed that 6-benzylaminopurine (6-BA) could induce branch development in WT plants (Fig. S13). Interestingly, the cp mutant was hyper-sensitive to high concentration of BR treatment. Although the WT and the mutants both showed defects on root development after BR treatment, the mutant did not show any root development at all, suggesting that CsER might play an important role in BR signaling pathway in a dominant-negative manner.
Many photosynthetic pathway genes in the mutant were up-regulated including those encoding chlorophyll a-b binding proteins (Fig. S12C; Table S3). This seems consistent with the dark green leaf of the mutant as seen in the field (Fig. 1A4). We speculate that the photosynthesis might be enhanced in the mutant. We measured the chlorophyll contents and the photosynthetic parameters in the WT and mutant (Table S4). Indeed, the mutant had higher chlorophyll a and chlorophyll b content than the WT. The mutant had a photosynthetic rate of 8.76 ± 0.40 µmol·m− 2·s− 1, which was significantly higher than that in the WT (3.97 ± 0.30µmol·m− 2·s− 1). The stomatal conductance and transpiration rate were also higher in the mutant (Table S4). We further noticed that the stoma density was higher in the mutant (283 ± 13.47 / mm2) than that in the WT (130 ± 11.54 / mm2) (Fig. S14). Collectively, these data may suggest that CsER has a negative effect on chlorophyll content and stoma density, which further affects photosynthesis in cucumber.